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CA 02401868 2002-08-22
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MOLECULES FOR DIAGNOSTICS AND THERAPEUTICS
TECHNICAL FIELD
The present invention relates to human molecules and to the use of these
sequences in the
s diagnosis, study, prevention, and treatment of diseases associated with, as
well as effects of exogenous
compounds on, the expression of human molecules.
BACKGROUND OF THE INVENTION
The human genome is comprised of thousands of genes, many encoding gene
products that
1 o function in the maintenance and growth of the various cells and tissues in
the body. Aberrant
expression or mutations in these genes and their products is the cause of, or
is associated with, a variety
of human diseases such as cancer and other cell proliferative disorders,
autoimmune/inflaW matory
disorders, infections, developmental disorders, endocrine disorders, metabolic
disorders; neurological
disorders, gastrointestinal disorders, transport disorders, and connective
tissue disorders. The
1 s identification of these genes and their products is the basis of an ever-
expanding effort to find markers
for early detection of diseases, and targets for their prevention and
treatment. Therefore, these genes
and their products are useful as diagnostics and therapeutics. These genes may
encode, for example,
enzyme molecules, molecules associated with growth and development,
biochemical pathway molecules,
extracellular information transmission molecules, receptor molecules,
intracellular signaling molecules,
2 o membrane transport molecules, protein modification and maintenance
molecules, nucleic acid synthesis
and modification molecules, adhesion molecules, antigen recognition molecules,
secreted and
extracellular matrix molecules, cytoskeletal molecules, ribosomal molecules,
electron transfer
associated molecules, transcription factor molecules, chromatin molecules,
cell membrane molecules,
and organelle associated molecules.
2 s For example, cancer represents a type of cell proliferative disorder that
affects nearly every
tissue in the body. A wide variety of molecules, either aberrantly expressed
or mutated, can be the
cause of, or involved with, various cancers because tissue growth involves
complex and ordered
patterns of cell proliferation, cell differentiation, and apoptosis. Cell
proliferation must be regulated to
maintain both the number of cells and their spatial organization. This
regulation depends upon the
3 o appropriate expression of proteins which control cell cycle progression in
response to extracellular
signals such as growth factors and other mitogens, and intracellular cues such
as DNA damage or.
nutrient starvation. Molecules which directly or indirectly modulate cell
cycle progression fall into
several categories, including growth factors and their receptors, second
messenger and signal
transduction proteins, oncogene products, tumor-suppressor proteins, and
mitosis-promoting factors.
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Aberrant expression or mutations in any of these gene products can result in
cell proliferative disorders
such as cancer. Oncogenes are genes generally derived from normal genes that,
through abnormal
expression or mutation, can effect the transformation of a normal cell to a
malignant one (oncogenesis).
Oncoproteins, encoded by oncogenes, can affect cell proliferation in a variety
of ways and include
growth factors, growth factor receptors, intracellular signal transducers,
nuclear transcription factors,
and cell-cycle control proteins. In contrast, tumor-suppressor genes are
involved in inhibiting cell
proliferation. Mutations which cause reduced function or loss of function in
tumor-suppressor genes
result in aberrant cell proliferation and cancer. Although many different
genes and their products have
been found to be associated with cell proliferative disorders such as cancer,
many more may exist that
are yet to be discovered.
DNA-based arrays can provide a simple way to explore the expression of a
single polymorphic
' gene or a large number of genes. When the expression of a single gene is
explored, DNA-based arrays
are employed to detect the expression of specific gene variants. For example,
a p53 tumor suppressor
gene array is used'to determine whether individuals are carrying mutations
that predispose them to
cancer. A cytochrome p450 gene array is useful to determine whether
individuals have one of a number
of specific mutations that could result in increased drug metabolism, drug
resistance or drug toxicity.
DNA-based array technology is especially relevant for the rapid screening of
expression of a
large number of genes. There is a growing awareness that gene expression is
affected in a global
fashion. A genetic predisposition, disease or therapeutic treatment may
affect, directly or indirectly, the
2 o expression of a large number of genes. In some cases the interactions may
be expected, such as when
the genes are part of the same signaling pathway. In other cases, such as when
the genes participate im
separate signaling pathways, the interactions may be totally unexpected.
Therefore, DNA-based arrays
can be used to investigate how genetic predisposition, disease, or therapeutic
treatment affects the : . ,
expression of a large number of genes.
Enzyme Molecules .
The cellular processes of biogenesis and biodegradation involve a number of
key enzyme
classes including oxidoreductases, transferases, hydrolases, lyases,
isomerases, and ligases. These'
enzyme classes are each comprised of numerous substrate-specific enzymes
having precise and well
3 0 regulated functions. These enzymes function by facilitating metabolic
processes such as glycolysis,
the tricarboxylic cycle, and fatty acid metabolism; synthesis or degradation
of amino acids, steroids,
phospholipids, alcohols, etc.; regulation of cell signalling, proliferation,
inflamation, apoptosis, etc.,
and through catalyzing critical steps in DNA replication and repair, and the
process of translation.
Oxidoreductases
2
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Many pathways of biogenesis and biodegradation require oxidoreductase
(dehydrogenase or
reductase) activity, coupled to the reduction or oxidation of a donor or
acceptor cofactor. Potential
cofactors include cytochromes, oxygen, disulfide, iron-sulfur proteins, flavin
adenine dinucleotide
(FAD), and the nicotinamide adenine dinucleotides NAD and NADP (Newsholme,
E.A. and A.R.
s Leech (1983) Biochemistry for the Medical Sciences, John Wiley and Sons,
Chichester, U.K., pp.
779-793). Reductase activity catalyzes the transfer of electrons between
substrates) and cofactors)
with concurrent oxidation of the cofactor. The reverse dehydrogenase reaction
catalyzes the reduction
of a cofactor and consequent oxidation of the substrate. Oxidoreductase
enzymes are a broad
superfamily of proteins that catalyze numerous reactions in all cells of
organisms ranging from
to bacteria to.plants to humans. These reactions include metabolism of sugar,
certain detoxification
reactions.in the liver, and the synthesis or degradation of fatty acids, amino
acids, glucocoiticoids,
estrogens.r androgens, and prostaglandins. Different family.members are named
according to the
direction in which their reactions are typically catalyzed; thus they may be
referred to as .
oxidoreductases, oxidases, reductases, or dehydrogenases. In addition, family
members often have
1 s distinct cellular localizations, including the cytosol, the plasma
membrane, mitochondrial inner or
outer membrane, and peroxisomes.
Short-chain alcohol dehydrogenases (SCADs) are a family of dehydrogenases that
only share
15 % to 30% sequence identity, with similarity predominantly in the coenzyme
binding domain and
the substrate binding domain. In addition to the well-known role in
detoxification of ethanol, SCADs
2 o are also involved in synthesis and degradation of fatty acids, steroids,
and some prostaglandins, and
are therefore implicated in a variety of disorders such as lipid storage
disease, myopathy, SCAD
deficiency, and certain genetic disorders. For example, retinol dehydrogenase
is, a SCAD-family
member (Simon, A. et al. (1995) J. Biol. Chem. 270:1107-1112) that converts
retinol to retinal, the
precursor of,retinoic acid. Retinoic acid, a regulator of differentiation and
apoptosis, has-been shown
2s to down-regulate genes involved in cell proliferation and inflammation
(Chaff, X. et al: (1995) J. Biol.
Chem. 270:3900-3904). In addition, retinol dehydrogenase has been linked to
hereditary eye diseases
such as autosomal recessive childhood-onset severe retinal dystrophy (Simon,
A. et al: (1996)
Genomics 36:424-430).
Propagation of nerve impulses, modulation of cell proliferation and
differentiation, induction
3 0 of the immune response, and tissue homeostasis involve neurotransmitter
metabolism (Weiss, B.
(1991) Neurotoxicology 12:379-386; Collins, S.M. et al. (1992) Ann. N.Y. Acad.
Sci. 664:415-424;
Brown, J.K. and H. Imam (1991) J. Inherit. Metab. Dis. 14:436-458). Many
pathways of
neurotransmitter metabolism require oxidoreductase activity, coupled to
reduction or oxidation of a
cofactor, such as NAD+/NADH (Newsholme, E.A. and A.R. Leech (1983)
Biochemistry for the
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Medical Sciences, John Wiley and Sons, Chichester, U.K. pp. 779-793).
Degradation of
catecholamines (epinephrine or norepinephrine) requires alcohol dehydrogenase
(in the brain) or
aldehyde dehydrogenase (in peripheral tissue). NAD+-dependent aldehyde
dehydrogenase oxidizes 5-
hydroxyindole-3-acetate (the product of 5-hydroxytryptamine (sexotonin)
metabolism) in the brain,
s . blood platelets, liver and pulmonary endothelium (Newsholme, supra, p.
786). Other
neurotransmitter degradation pathways that utilize NAD~/NADH-dependent
oxidoreductase activity
include those of L-DOPA (precursor of dopamine, a neuronal excitatory
compound), glycine (an
inhibitory neurotransmitter in the brain and spinal. cord), histamine
(liberated from mast cells during
the inflammatory response), and taurine (an inhibitory neurotransmitter of the
brain stem, spinal cord
1.o , and retina) (Newsholme, supra, pp. 790, 792). Epigenetic or genetic
defects in neurotransmitter
metabolic. pathways .can result in a spectrum of disease states in different
tissues including Parkinson
disease and inherited myoclonus (McCance, K.L. and S.E. Huether (1994)
Pathophvsiolo~y, Mosby-
Year Book, Inc., St. Louis MO, pp. 402-404; Gundlach, A.L. (1990) FASEB J.
4:2761-2766).
Tetrahydrofolate is a derivatized glutamate molecule that acts as a carrier,
providing activated
15 , one-carbon units to a wide variety of biosynthetic reactions, including
synthesis of purines,
pyrimidines, and the amino acid methionine. Tetrahydrofolate is generated by
the activity of a
holoenzyme complex called tetrahydrofolate synthase, which includes three
enzyme activities:
tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase, and
tetrahydrofolate synthetase.
Thus, tetrahydrofolate dehydrogenase plays an important role in generating
building blocks for
2 o nucleic and amino acids, crucial to proliferating cells.
3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acid metabolism.
It
catalyzes the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA, with
concomitant oxidation of
NAD to NADH, in the mitochondria and peroxisomes of eukaryotic cells. In
peroxisomes, fHACD
and enoyl-CoA hydratase form an enzyme complex called bifunctional enzyme,
defects in which are
2 s associated with peroxisomal bifunctional enzyme deficiency. This
interruption in fatty 'acid
metabolism produces accumulation of very-long chain fatty acids, disrupting
development of the
brain, bone, and adrenal glands. Infants born with this deficiency typically
die within 6 months
(Watkins, P. et al. (1989) J. Clin. Invest. 83:771-777; Online Mendelian
Inheritance in Man (OMIM);
#261515). The neurodegeneration that is characteristic of Alzheimer's disease
involves development
3 0 of extracellular plaques in certain brain regions. A major protein
component of these plaques is the
peptide amyloid-(3 (A(3), which is one of several cleavage products of amyloid
precursor protein
(APP). 3HACD has been shown to bind the A(3 peptide, and is overexpressed in
neurons affected in
Alzheimer's disease. In addition, an antibody against 3HACD can block the
toxic effects of A(3 in a
cell culture model of Alzheimer's disease (Yan, S. et al. (1997) Nature
389:689-695; OMIM,
4
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#602057).
Steroids, such as estrogen, testosterone, corticosterone, and others, are
generated from a
common precursor, cholesterol, and are interconverted into one another. A wide
variety of enzymes
act upon cholesterol, including a number of dehydrogenases. Steroid
dehydrogenases, such as the
s hydroxysteroid dehydrogenases, are involved in hypertension, fertility, and
cancer (Duax, W.L. and
D. Ghosh (1997) Steroids 62:95-100). One such dehydrogenase is 3-oxo-5-a-
steroid dehydrogenase
(DASD), a microsomal membrane protein highly expressed in prostate and other
androgen-responsive
tissues. DASD catalyzes the conversion of testosterone into
dihydrotestosterone, which is the most
potent androgen. Dihydrotestosterone is essential for the formation of the
male phenotype during
1 o embryogenesis, as well as for proper androgen-mediated growth of tissues
such as the prostate and
male genitalia. A defect in DASD that prevents the conversion of testosterone
into
dihydrotestosterone leads to a rare form of male pseudohermaphroditis,
characterized by defective
formation of the external genitalia (Andersson, S. et al. (1991) Nature
354:159-161; Labrie, F. et al.
(1992) Endocrinology 131:1571-1573; OMIM #264600). Thus, OASD plays a central
role in sexual
15 differentiation and androgen physiology.
17(3-hydroxysteroid dehydrogenase (17(3HSD6) plays an important role in the
regulation of
the male reproductive hormone, dihydrotestosterone (DHTT). 17(3HSD6 acts to
reduce levels of
DHTT by oxidizing a precursor of DHTT, 3a-diol, to androsterone which is
readily glucuronidated
and removed from tissues. 17 (3HSD6 is active with both androgen and estrogen
substrates when
2 o expressed in embryonic kidney 293 cells. At least five other isozymes of
17 (3HSD have been
identified that catalyze oxidation and/or reduction reactions in various
tissues with preferences for
different steroid substrates (Biswas, M.G. and D.W. Russell (1997) J. Biol.
Chem. 272:15959-
15966). For example, 17(3HSD1 preferentially reduces estradiol and is abundant
in the ovary and
placenta. 17(3HSD2 catalyzes oxidation of androgens and is present in the
endometrium and placenta.
2 s 17(3HSD3 is exclusively a reductive enzyme in the testis (Geissler, W.M.
et al. (1994) Nat. Genet.
7:34-39). An excess of androgens such as DHTT can contribute to certain
disease states such as
benign prostatic hyperplasia and prostate cancer.
Oxidoreductases are components of the fatty acid metabolism pathways in
mitochondria and
peroxisomes. The main beta-oxidation pathway degrades both saturated and
unsaturated fatty acids,
3 o while the auxiliary pathway performs additional steps required for the
degradation of unsaturated fatty
acids. The auxiliary beta-oxidation enzyme 2,4-dienoyl-CoA reductase catalyzes
the removal of
even-numbered double bonds from unsaturated fatty acids prior to their entry
into the main beta-
oxidation pathway. The enzyme may also remove odd-numbered double bonds from
unsaturated
fatty acids (Koivuranta, K.T. et al. (1994) Biochem. J. 304:787-792; Smeland,
T.E. et al. (1992) Proc.
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Natl. Acad. Sci. USA 89:6673-6677). 2,4-dienoyl-CoA reductase is located in
both mitochondria and
peroxisomes. Inherited deficiencies in mitochondria) and peroxisomal beta-
oxidation enzymes are
associated with severe diseases, some of which manifest themselves soon after
birth and lead to death
within a few years. Defects in beta-oxidation are associated with Reye's
syndrome, Zellweger
s syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, acyl-
CoA oxidase deficiency,
and bifunctional protein deficiency (Suzuki, Y. et al. (1994) Am. J. Hum.
Genet. 54:36-43; Hoefler,
supra; Cotran, R.S. et al. (1994) Robbins Pathologic Basis of Disease, W.B.
Saunders Co.,
Philadelphia PA, p.866). Peroxisomal beta-oxidation is impaired in cancerous
tissue. Although
neoplastic human breast epithelial cells have the same number of peroxisomes
as do normal cells,
to fatty acyl-CoA oxidase activity is lower than in control tissue (e)
Bouhtoury, F. et al. (1992) J. Pathol.
1.66:27-35). Human colon carcinomas have fewer peroxisomes than normal colon
tissue and have
lower fatty-acyl-CoA oxidase and bifunctional enzyme (including enoyl-CoA
hydratase) activities
than normal tissue (Cable, S. et al. (1992) Virchows Arch. B Cell Pathol.
Incl. Mol. Pathol. 62:221-
226). Another important oxidoreductase is isocitrate dehydrogenase, which
catalyzes the conversion
s5 of isocitrate to a-ketoglutarate, a substrate of the citric acid cycle.
Isocitrate dehydrogenase can be
either NAD or NADP dependent, and is found in the cytosol, mitochondria, and
peroxisomes.
Activity of isocitrate dehydrogenase is regulated developmentally, and by
hormones,
neurotransmitters, and growth factors.
Hydroxypyruvate reductase (HPR), a peroxisomal 2-hydroxyacid dehydrogenase in
the
2 o glycolate pathway, catalyzes the conversion of hydroxypyruvate to
glycerate with the oxidation of
both NADH and NADPH. The reverse dehydrogenase reaction reduces NAD+ and
NADP+. HPR
recycles nucleotides and bases back into pathways leading to the synthesis of
ATP and GTP. ATP
and GTP are used to produce DNA and RNA and to control various aspects of
signal transduction and
energy metabolism. Inhibitors of purine nucleotide biosynthesis have long been
employed as
2 s antiproliferative agents to treat cancer and viral diseases. HPR also
regulates biochemical synthesis
of serine and cellular serine levels available for protein synthesis.
The mitochondria) electron transport (or respiratory) chain is a series of
oxidoreductase-type
enzyme complexes in the mitochondria) membrane that is responsible for the
transport of electrons
from NADH through a series of redox centers within these complexes to oxygen,
and the coupling of
3 o this oxidation to the synthesis of ATP (oxidative phosphorylation). ATP
then provides the primary
source of energy for driving a cell's many energy-requiring reactions. The key
complexes in the
respiratory chain are NADH:ubiquinone oxidoreductase (complex I),
succinate:ubiquinone
oxidoreductase (complex II), cytochrome cl-b oxidoreductase (complex III),
cytochrome c oxidase
(complex IV), and ATP synthase (complex V) (Alberts, B. et al. (1994)
Molecular Biolo~y of the
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Cell, Garland Publishing, Inc., New York NY, pp. 677-678). All of these
complexes are located on
the inner matrix side of the mitochondrial membrane except complex II, which
is on the cytosolic
side. Complex II transports electrons generated in the citric acid cycle to
the respiratory chain. The
electrons generated by oxidation of succinate to fumarate in the citric acid
cycle are transferred
s through electron carriers in complex II to membrane bound ubiquinone (~.
Transcriptional
regulation of these nuclear-encoded genes appears to be the predominant means
for controlling the
biogenesis of respiratory enzymes. Defects and altered expression of enzymes
in the respiratory chain
are associated with a variety of disease conditions.
Other dehydrogenase activities using NAD as a cofactor are also important in
mitochondrial
1 o function. 3-hydroxyisobutyrate dehydrogenase (3HBD), important in valine
catabolism, catalyzes the
NAD-dependent oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde
within
mitochondria. Elevated levels of 3-hydroxyisobutyrate have been reported in a
number of disease
states, including ketoacidosis, methylmalonic acidemia, and other disorders
associated with
deficiencies in methylmalonate semialdehyde dehydrogenase (Rougraff, P.M. et
a1. (1989) J. Biol.
15 Chem.264:5899-5903).
Another mitochondrial dehydrogenase important in amino acid metabolism is the
enzyme
isovaleryl-CoA-dehydrogenase (IVD). IVD is involved in leucine metabolism and
catalyzes the
oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA. Human IVD is a tetrameric
flavoprotein that
is encoded in the nucleus and synthesized in the cytosol as a 45 kDa precursor
with a mitochondrial
2 o import signal sequence. A genetic deficiency, caused by a mutation in the
gene encoding IVD, results
in the condition known as isovaleric acidemia. This mutation results in
inefficient mitochondrial
import and processing of the IVD precursor (Vockley, J. et al. (1992) J. Biol.
Chem. 267:2494-2501).
Transferases
Transferases are enzymes that catalyze the transfer of molecular groups. The
reaction may
2 s involve an oxidation, reduction, or cleavage of covalent bonds, and is
often specific to a substrate or
to particular sites on a type of substrate. Transferases participate in
reactions essential to such
functions as synthesis and degradation of cell components, regulation of cell
functions including cell
signaling, cell proliferation, inflamation, apoptosis, secretion and
excretion. Transferases are
involved in key steps in disease processes involving these functions.
Transferases are frequently
3 o classified according to the type of group transferred. For example, methyl
transferases transfer one-
carbon methyl groups, amino transferases transfer nitrogenous amino groups,
and similarly
denominated enzymes transfer aldehyde or ketone, acyl, glycosyl, alkyl or
aryl, isoprenyl, saccharyl,
phosphorous-containing, sulfur-containing, or selenium-containing groups, as
well as small
enzymatic groups such as Coenzyme A.
7
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Acyl transferases include peroxisomal carnitine octanoyl transferase, which is
involved in the
fatty acid beta-oxidation pathway, and mitochondria) carnitine palmitoyl
transferases, involved in
fatty acid metabolism and transport. Choline O-acetyl transferase catalyzes
the biosynthesis of the
neurotransmitter acetylcholine.
s Amino transferases play key roles in protein synthesis and degradation, and
they contribute to
other processes as well. For example, the amino transferase 5-aminolevulinic
acid synthase catalyzes
the addition of succinyl-CoA to glycine, the first step in heme biosynthesis.
Other amino transferases
participate in pathways important for neurological function and metabolism.
For example, glutamine
phenylpyruvate amino transferase, also known as glutamine transaminase K
(GTK), catalyzes several
1 o reactions with a pyridoxal phosphate cofactor. GTK catalyzes the
reversible conversion of L
glutamine and phenylpyruvate to 2-oxoglutaramate and L-phenylalanine. Other
amino acid substrates
for GTK include L-methionine, L-histidine, and L-tyrosine. GTK also catalyzes
the conversion of
kynurenine to kynurenic acid, a tryptophan metabolite that is an antagonist of
the N-methyl-D-
aspartate (NMDA) receptor in the brain and may exert a neuromodulatory
function. Alteration of the
1 s kynurenine metabolic pathway may be associated with several neurological
disorders. GTK also
plays a role in the metabolism of halogenated xenobiotics conjugated to
glutathione, leading to
nephrotoxicity in rats and neurotoxicity in humans. GTK is expressed in
kidney, liver, and brain.
Both human and rat GTKs contain a putative pyridoxal phosphate binding site
(ExPASy ENZYME:
EC 2.6.1.64; Perry, S.J. et al. (1993) Mol. Pharmacol. 43:660-665; Perry, S.
et al. (1995) FEBS Lett.
20 360:277-280; and Alberati-Giani, D. et al. (1995) J. Neurochem. 64:1448-
1455). A second amino
transferase associated with this pathway is kynureninela-aminoadipate amino
transferase (AadAT).
AadAT catalyzes the reversible conversion of a-aminoadipate and a-
ketoglutarate to a-ketoadipate
and L-glutamate during lysine metabolism. AadAT also catalyzes the
transamination of kynurenine
to kynurenic acid. A cytosolic AadAT is expressed in rat kidney, liver, and
brain (Nakatani, Y, et al.
2s (1970) Biochim. Biophys. Acta 198:219-228; Buchli, R. et al. (1995) J.
Biol. Chem. 270:29330-
29335).
Glycosyl transferases include the mammalian UDP-glucouronosyl transferases, a
family of
membrane-bound microsomal enzymes catalyzing the transfer of glucouronic acid
to lipophilic
substrates in reactions that play important roles in detoxification and
excretion of drugs, carcinogens,
3 o and other foreign substances. Another mammalian glycosyl transferase,
mammalian UDP-galactose-
ceramide galactosyl transferase, catalyzes the transfer of galactose to
ceramide in the synthesis of
galactocerebrosides in myelin membranes of the nervous system. The UDP-
glycosyl transferases
share a conserved signature domain of about 50 amino acid residues (PROSITE:
PDOC00359,
http://expasy.hcuge.ch/sprot/prosite.html).
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Methyl transferases are involved in a variety of pharmacologically important
processes.
Nicotinamide N-methyl transferase catalyzes the N-methylation of nicotinamides
and other pyridines,
an important step in the cellular handling of drugs and other foreign
compounds.
Phenylethanolamine N-methyl transferase catalyzes the conversion of
noradrenalin to adrenalin. 6-O-
s methylguanine-DNA methyl transferase reverses DNA methylation, an important
step in
carcinogenesis. Uroporphyrin-III C-methyl transferase, which catalyzes the
transfer of two methyl
groups from S-adenosyl-L-methionine to uroporphyrinogen III, is the first
specific enzyme in the
biosynthesis of cobalamin, a dietary enzyme whose uptake is deficient in
pernicious anemia. Protein-
arginine methyl transferases catalyze the posttranslational methylation of
arginine residues in
~. o proteins, resulting in the mono- and dimethylation of arginine on the
guanidino group. Substrates
include histones, myelin basic protein, and heterogeneous nuclear
ribonucleoproteins involved in
mRNA processing, splicing, and transport. Protein-arginine methyl transferase
interacts with proteins
upregulated by mitogens, with proteins involved in chronic lymphocytic
leukemia, and with
interferon, suggesting an important role for methylation in cytokine receptor
signaling (Lin, W.-J. et
15 al. (1996) J. Biol. Chem. 271:15034-15044; Abramovich, C. et al. (1997)
EMBO J. 16:260-266; and
Scott, H.S. et al. (1998) Genomics 48:330-340).
Phosphotransferases catalyze the transfer of high-energy phosphate groups and
are important
in energy-requiring and -releasing reactions. The metabolic enzyme creatine
kinase catalyzes the
reversible phosphate transfer between creatine/creatine phosphate and ATP/ADP.
Glycocyamine
2 o kinase catalyzes phosphate transfer from ATP to guanidoacetate, and
arginine kinase catalyzes
phosphate transfer from ATP to arginine. A cysteine-containing active site is
conserved in this family
(PROSITE: PDOC00103).
Prenyl transferases are heterodimers, consisting of an alpha and a beta
subunit, that catalyze
the transfer of an isoprenyl group. An example of a prenyl transferase is the
mammalian protein
2 s farnesyl transferase. The alpha subunit of farnesyl transferase consists
of 5 repeats of 34 amino acids
each, with each repeat containing an invariant tryptophan (PROSITE:
PDOC00703).
Saccharyl transferases are glycating enzymes involved in a variety of
metabolic processes.
Oligosacchryl transferase-48, for example, is a receptor for advanced
glycation endproducts.
Accumulation of these endproducts is observed in vascular complications of
diabetes, macrovascular
3 o disease, renal insufficiency, and Alzheimer's disease (Thornalley, P.J.
(1998) Cell Mol. Biol. (Noisy-
Le-Grand) 44:1013-1023).
Coenzyme A (CoA) transferase catalyzes the transfer of CoA between two
carboxylic acids.
Succinyl CoA:3-oxoacid CoA transferase, for example, transfers CoA from
succinyl-CoA to a
recipient such as acetoacetate. Acetoacetate is essential to the metabolism of
ketone bodies, which
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accumulate in tissues affected by metabolic disorders such as diabetes
(PROSITE: PDOC00980).
Hvdrolases
Hydrolysis is the breaking of a covalent bond in a substrate by introduction
of a molecule of
water. The reaction involves a nucleophilic attack by the water molecule's
oxygen atom on a target
s . bond in the substrate. The water molecule is split across the target bond,
breaking the bond and
generating two product molecules. Hydrolases participate in reactions
essential to such functions as
synthesis and degradation of cell components, and for regulation of cell
functions including cell
signaling, cell proliferation, inflamation, apoptosis, secretion and
excretion. Hydrolases are involved
in key steps in disease processes involving these functions. Hydrolytic
enzymes, or hydrolases, may
1 o be grouped by substrate specificity into classes including phosphatases,
peptidases,
lysophospholipases, phosphodiesterases, glycosidases, and glyoxalases.
Phosphatases hydrolytically remove phosphate groups from proteins, an energy-
providing
step that regulates many cellular processes, including intracellular signaling
pathways that in turn
control cell growth and differentiation, cell-cell contact, the cell cycle,
and oncogenesis.
15 Lysophospholipases (LPLs) regulate intracellular lipids by catalyzing the
hydrolysis of ester
bonds to remove an acyl group, a key step in lipid degradation. Small LPL
isoforms, approximately
15-30 kD, function as hydrolases; larger isoforms function both as hydrolases
and transacylases. A.
particular substrate for LPLs, lysophosphatidylcholine, causes lysis of cell
membranes. LPL activity
is regulated by signaling molecules important in numerous pathways, including
the inflammatory
2 o response.
Peptidases, also called proteases, cleave peptide bonds that form the backbone
of peptide or
protein chains. Proteolytic processing is essential to cell growth,
differentiation, remodeling, and
homeostasis as well as inflammation and immune response. Since typical protein
half-lives range
from hours to a few days, peptidases are continually cleaving precursor
proteins to their active form,
2 s removing signal sequences from targeted proteins, and degrading aged or
defective proteins.
Peptidases function in bacterial, parasitic, and viral invasion and
replication within a host. Examples
of peptidases include trypsin and chymotrypsin (components of the complement
cascade and the
blood-clotting cascade) lysosornal cathepsins, calpains, pepsin, renin, and
chymosin (Beynon, R.J.
and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford
University Press, New
3 o York NY, pp. 1-5).
The phosphodiesterases catalyze the hydrolysis of one of the two ester bonds
in a
phosphodiester compound. Phosphodiesterases are therefore crucial to a variety
of cellular processes.
Phosphodiesterases include DNA and RNA endo- and exo-nucleases, which are
essential to cell
growth and replication as well as protein synthesis. Another phosphodiesterase
is acid
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sphingomyelinase, which hydrolyzes the membrane phospholipid sphingomyelin to
ceramide and
phosphorylcholine. Phosphorylcholine is used in the synthesis of
phosphatidylcholine, which is
involved in numerous intracellular signaling pathways. Ceramide is an
essential precursor for the
generation of gangliosides, membrane lipids found in high concentration in
neural tissue. Defective
s acid sphingomyelinase phosphodiesterase leads to a build-up of sphingomyelin
molecules in
lysosomes, resulting in Niemann-Pick disease.
Glycosidases catalyze the cleavage of hemiacetyl bonds of glycosides, which
are compounds
that contain one or more sugar. Mammalian lactase-phlorizin hydrolase, for
example, is an intestinal
enzyme that splits lactose. Mammalian beta-galactosidase removes the terminal
galactose from
to gangliosides, glycoproteins, and glycosaminoglycans, and deficiency of this
enzyme is associated
with a gangliosidosis known as Morquio disease type B. Vertebrate lysosomal
alpha-glucosidase,
which hydrolyzes glycogen, maltose, and isomaltose, and vertebrate intestinal
sucrase-isomaltase,
which hydrolyzes sucrose, maltose, and isomaltose, are widely distributed
members of this family
with highly conserved sequences at their active sites.
15 The glyoxylase system is involved in gluconeogenesis, the production of
glucose from
storage compounds in the body. It consists of glyoxylase I, which catalyzes
the formation of S-D-
lactoylglutathione from methyglyoxal, a side product of triose-phosphate
energy metabolism, and
glyoxylase II, which hydrolyzes S-D-lactoylglutathione to D-lactic acid and
reduced glutathiorie.
Glyoxylases are involved in hyperglycemia, non-insulin-dependent diabetes
mellitus, the
2 o detoxification of bacterial toxins, and in the control of cell
proliferation and microtubule assembly.
Leases
Lyases are a class of enzymes that catalyze the cleavage of C-C, C-O, C-N, C-
S, C-(halide),
P-O or other bonds without hydrolysis or oxidation to form two molecules, at
least one of which
contains a double bond (Stryer, L. (1995) Biochemistry W.H. Freeman and Co.
New York, NY
2 s p.620). Lyases are critical components of cellular biochemistry with roles
in metabolic energy
production including fatty acid metabolism, as well as other diverse enzymatic
processes. Further
classification of lyases reflects the type of bond cleaved as well as the
nature of the cleaved group.
The group of C-C lyases include carboxyl-lyases (decarboxylases), aldehyde-
lyases
(aldolases), oxo-acid-lyases and others. The C-O lyase group includes hydro-
lyases, lyases acting on
3 o polysaccharides and other lyases. The C-N lyase group includes ammonia-
lyases, amidine-lyases,
amine-lyases (deaminases) and other lyases.
Proper regulation of lyases is critical to normal physiology. For example,
mutation induced
deficiencies in the uroporphyrinogen decarboxylase can lead to photosensitive
cutaneous lesions in
the genetically-linked disorder familial porphyria cutanea tarda (Mendez, M.
et al. ( 1998) Am. J.
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Genet. 63:1363-1375). It has also been shown that adenosine deaminase (ADA)
deficiency stems
from genetic mutations in the ADA gene, resulting in the disorder severe
combined
immunodeficiency disease (SCID) (Hershfield, M.S. (1998) Semin. Hematol.
35:291-298).
Isomerases
s Isomerases are a class of enzymes that catalyze geometric or structural
changes within a
molecule to form a single product. This class includes racemases and
epimerases, cis-trans-
isomerases, intramolecular oxidoreductases, intramolecular transferases
(mutases) and intramolecular
lyases. Isomerases are critical components of cellular biochemistry with roles
in metabolic energy
production including glycolysis, as well as other diverse enzymatic processes
(Stryer, L. (1995)
to Biochemistrv, W.H. Freeman and Co., New York NY, pp.483-507).
Racemases are a subset of isomerases that catalyze inversion of a molecules
configuration
around the asymmetric carbon atom in a substrate having a single center of
asymmetry, thereby
interconverting two racemers. Epimerases are another subset of isomerases that
catalyze inversion of
configuration around an asymmetric carbon atom in a substrate with more than
one center of
15 symmetry, thereby interconverting two epimers. Racemases and epimerases can
act on amino acids
and derivatives, hydroxy acids and derivatives, as well as carbohydrates and
derivatives. The
interconversion of UDP-galactose and UDP-glucose is catalyzed by UDP-galactose-
4'-epimerase.
Proper regulation and function of this epimerase is essential to the synthesis
of glycoproteins and
glycolipids. Elevated blood galactose levels have been correlated with UDP-
galactose-4'-epimerase
2o deficiency in screening programs of infants (Gitzelmann, R. (1972) Helv.
Paediat. Acta 27:125-130).
Oxidoreductases can be isomerases as well. Oxidoreductases catalyze the
reversible transfer
of electrons from a substrate that becomes oxidized to a substrate that
becomes reduced. This class of
enzymes includes dehydrogenases, hydroxylases, oxidases, oxygenases,
peroxidases, and reductases.
Proper maintenance of oxidoreductase levels is physiologically important. For
example, genetically-
2 s linked deficiencies in lipoamide dehydrogenase can result in lactic
acidosis (Robinson, B.H. et al.
(1977) Pediat. Res. 11:1198-1202).
Another subgroup of isomerases are the transferases (or mutases). Transferases
transfer a
chemical group from one compound (the donor) to another compound (the
acceptor). The types of
groups transferred by these enzymes include acyl groups, amino groups,
phosphate groups
3 0 (phosphotransferases or phosphomutases), and others. The transferase
carnitine palmitoyltransferase
is an important component of fatty acid metabolism. Genetically-linked
deficiencies in this
transferase can lead to myopathy (Scriver, C.R. et al. (1995) The Metabolic
and Molecular Basis of
Inherited Disease, McGraw-Hill, New York NY, pp.1501-1533).
Yet another subgroup of isomerases are the topoisomersases. Topoisomerases are
enzymes
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that affect the topological state of DNA. For example, defects in
topoisomerases or their regulation
can affect normal physiology. Reduced levels of topoisomerase II have been
correlated with some of
the DNA processing defects associated with the disorder ataxia-telangiectasia
(Singly S.P. et al.
(1988) Nucleic Acids Res. 16:3919-3929).
Li_~ases
Ligases catalyze the formation of a bond between two substrate molecules. The
process
involves the hydrolysis of a pyrophosphate bond in ATP or a similar energy
donor. Ligases are
classified based on the nature of the type of bond they form, which can
include carbon-oxygen,
carbon-sulfur, carbon-nitrogen, carbon-carbon and phosphoric ester bonds.
1 o Ligases forming carbon-oxygen bonds include the aminoacyl-transfer RNA
(tRNA)
synthetases which are important RNA-associated enzymes with roles in
translation. Protein
biosynthesis depends on each amino acid forming a linkage with the appropriate
tRNA. The
aminoacyl-tRNA synthetases are responsible for the activation and correct
attachment of an amino
acid with its cognate tRNA. The 20 aminoacyl-tRNA synthetase enzymes can be
divided into two
structural classes, and each class is characterized by a distinctive topology
of the catalytic domain.
Class I enzymes contain a catalytic domain based on the nucleotide-binding
Rossman fold. Class II
enzymes contain a central catalytic domain, which consists of a seven-stranded
antiparallel 13-sheet
motif, as well as N- and C- terminal regulatory domains. Class II enzymes are
separated into two
groups based on the heterodimeric or homodimeric structure of the enzyme; the
latter group is further
2 o subdivided by the structure of the N- and C-terminal regulatory domains
(Hartlein, M. and S. Cusack
(1995) J. Mol. Evol. 40:519-530). Autoantibodies against aminoacyl-tRNAs are
generated by
patients with dermatomyositis and polymyositis, and correlate strongly with
complicating interstitial
lung disease (ILD). These antibodies appear to be generated in response to
viral infection, and
coxsackie virus has been used to induce experimental viral myositis in
animals.
2 5 Ligases forming carbon-sulfur bonds (Acid-thiol ligases) mediate a large
number of cellular
biosynthetic intermediary metabolism processes involve intermolecular transfer
of carbon
atom-containing substrates (carbon substrates). Examples of such reactions
include the tricarboxylic
acid cycle, synthesis of fatty acids and long-chain phospholipids, synthesis
of alcohols and aldehydes,
synthesis of intermediary metabolites, and reactions involved in the amino
acid degradation
3 o pathways. Some of these reactions require input of energy, usually in the
form of conversion of ATP
to either ADP or AMP and pyrophosphate.
In many cases, a carbon substrate is derived from a small molecule containing
at least two
carbon atoms. The carbon substrate is often covalently bound to a larger
molecule which acts as a
carbon substrate carrier molecule within the cell. In the biosynthetic
mechanisms described above,
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the carrier molecule is coenzyme A. Coenzyme A (CoA) is structurally related
to derivatives of the
nucleotide ADP and consists of 4'-phosphopantetheine linked via a
phosphodiester bond to the alpha
phosphate group of adenosine 3',5'-bisphosphate. The terminal thiol group of
4'-phosphopantetheine
acts as the site for carbon substrate bond formation. The predominant carbon
substrates which utilize
s CoA as a carrier molecule during biosynthesis and intermediary metabolism in
the cell are acetyl,
succinyl, and propionyl moieties, collectively referred to as acyl groups.
Other carbon substrates
include enoyl lipid, which acts as a fatty acid oxidation intermediate, and
carnitine, which acts as an
acetyl-CoA flux regulator/ mitochondrial acyl group transfer protein. Acyl-CoA
and acetyl-CoA are
synthesized in the cell by acyl-CoA synthetase and acetyl-CoA synthetase,
respectively.
s o Activation of fatty acids is mediated by at least three forms of acyl-CoA
synthetase activity:
i) acetyl-CoA synthetase, which activates acetate and several other low
molecular weight carboxylic
acids and is found in muscle mitochondria and the cytosol of other tissues;
ii) medium-chain
acyl-CoA synthetase, which activates fatty acids containing between four and
eleven carbon atoms
(predominantly from dietary sources), and is present only in liver
mitochondria; and iii) aryl CoA
s s synthetase, which is specific for long chain fatty acids with between six
and twenty carbon atoms, and
is found in ~microsomes and the mitochondria. Proteins associated with acyl-
CoA synthetase activity
have been identified from many sources including bacteria, yeast, plants,
mouse, and man. The
activity of acyl-CoA synthetase may be modulated by phosphorylation of the
enzyme by
cAMP-dependent protein kinase.
2 o Ligases forming carbon-nitrogen bonds include amide synthases such as
glutamine synthetase
(glutamate-ammonia ligase) that catalyzes the amination of glutamic acid to
glutamine by ammonia
using the energy of ATP hydrolysis. Glutamine is the primary source for the
amino group in various
amide transfer reactions involved in de novo pyrimidine nucleotide synthesis
and in purine and
pyrimidine ribonucleotide interconversions. Overexpression of glutamine
synthetase has been
2 s observed in primary liver cancer (Christa, L. et al. (1994) Gastroent.
106:1312-1320).
Acid-amino-acid ligases (peptide synthases) are represented by the ubiquitin
proteases which
are associated with the ubiquitin conjugation system (UCS), a major pathway
for the degradation of
cellular proteins in eukaryotic cells and some bacteria. The UCS mediates the
elimination of
abnormal proteins and regulates the half-lives of important regulatory
proteins that control cellular
3 o processes such as gene transcription and cell cycle progression. In the
UCS pathway, proteins
targeted for degradation are conjugated to a ubiquitin (Ub), a small heat
stable protein. Ub is first
activated by a ubiquitin-activating enzyme (E1), and then transferred to one
of several Ub-
conjugating enzymes (E2). E2 then links the Ub molecule through its C-terminal
glycine to an
internal lysine (acceptor lysine) of a target protein. The ubiquitinated
protein is then recognized and
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degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and
ubiquitin is released
for reutilization by ubiquitin protease. The UCS is implicated in the
degradation of mitotic cyclic
kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins,
cell surface receptors
associated with signal transduction, transcriptional regulators, and mutated
or damaged proteins
s (Ciechanover, A. (1994) Cell 79:13-21). A murine proto-oncogene, Unp,
encodes a nuclear ubiquitin
protease whose overexpression leads to oncogenic transformation of NIH3T3
cells, and the human
homolog of this gene is consistently elevated in small cell tumors and
adenocarcinomas of the lung
(Gray, D.A. (1995) Oncogene 10:2179-2183).
Cyclo-ligases and other carbon-nitrogen ligases comprise various enzymes and
enzyme
1 o complexes that participate in the de novo pathways to purine and
pyrimidine biosynthesis. Because
these pathways are critical to the synthesis of nucleotides for replication of
both RNA and DNA,
many of these enzymes have been the targets of clinical agents for the
treatment of cell proliferative
disorders such as cancer and infectious diseases.
Purine biosynthesis occurs de novo from the amino acids glycine and glutamine,
and other .
1 s small molecules. Three of the key reactions in this process are catalyzed
by a trifunctional enzyme
composed of glycinamide-ribonucleotide synthetase (GARS), aminoimidazole
ribonucleotide
synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART).
Together these three
enzymes combine ribosylamine phosphate with glycine to yield phosphoribosyl
aminoimidazole, a
precursor to both adenylate and guanylate nucleotides. This trifunctional
protein has been implicated
a o in the pathology of Downs syndrome (Aimi, J. et al. (1990) Nucleic Acid
Res. 18:6665-6672).
Adenylosuccinate synthetase catalyzes a later step in purine biosynthesis that
converts inosinic acid to
adenylosuccinate, a key step on the path to ATP synthesis. This enzyme is also
similar to another
carbon-nitrogen ligase, argininosuccinate synthetase, that catalyzes a similar
reaction in the urea cycle
(Powell, S.M. et al. (1992) FEBS Lett. 303:4-10).
2 5 Like the de novo biosynthesis of purines, de novo synthesis of the
pyrimidine nucleotides
uridylate and cytidylate also arises from a common precursor, in this instance
the nucleotide
orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP).
Again a trifunctional
enzyme comprising three carbon-nitrogen ligases plays a key role in the
process. In this case the
enzymes aspartate transcarbamylase (ATCase), carbamyl phosphate synthetase II,
and dihydroorotase
3 0 (DHOase) are encoded by a single gene called CAD. Together these three
enzymes combine the
initial reactants in pyrimidine biosynthesis, glutamine, CO2, and ATP to form
dihydroorotate, the
precursor to orotate and orotidylate (Iwahana, H. et al. (1996) Biochem.
Biophys. Res. Commun.
219:249-255). Further steps then lead to the synthesis of uridine nucleotides
from orotidylate.
Cytidine nucleotides are derived from uridine-5'-triphosphate (UTP) by the
amidation of UTP using
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glutamine as the amino donor and the enzyme CTP synthetase. Regulatory
mutations in the human
CTP synthetase are believed to confer multi-drug resistance to agents widely
used in cancer therapy
(Yamauchi, M. et al. (1990) EMBO J. 9:2095-2099).
Ligases forming carbon-carbon bonds include the carboxylases acetyl-CoA
carboxylase and
s pyruvate carboxylase. Acetyl-CoA carboxylase catalyzes the carboxylation of
acetyl-CoA from COZ
and H20 using the energy of ATP hydrolysis. Acetyl-CoA carboxylase is the rate-
limiting step in the
biogenesis of long-chain fatty acids. Two isoforms of acetyl-CoA carboxylase,
types I and types II,
are expressed in human in a tissue-specific manner (Ha, J. et al. (1994) Eur.
J. Biochem. 219:297-
306). Pyruvate carboxylase is a nuclear-encoded mitochondria) enzyme that
catalyzes the conversion
of pyruvate to oxaloacetate, a key intermediate in the citric acid cycle.
Ligases forming phosphoric ester bonds include the DNA ligases involved in
both DNA
replication and repair. DNA ligases seal phosphodiester bonds between two
adjacent nucleotides in a
DNA chain using the energy from ATP hydrolysis to first activate the free 5'-
phosphate of one
nucleotide and then react it with the 3'-OH group of the adjacent nucleotide.
This resealing reaction
is used in both DNA replication to join small DNA fragments called Okazaki
fragments that are
transiently formed in the process of replicating new DNA, and in DNA repair.
DNA repair is the
process by which accidental base changes, such as those produced by oxidative
damage, hydrolytic
attack, or uncontrolled methylation of DNA, are corrected before replication
or transcription of the
DNA can occur. Bloom's syndrome is an inherited human disease in which
individuals are partially
2 o deficient in DNA ligation and consequently have an increased incidence of
cancer (Alberts, B. et al.
(1994) The Molecular Biolo~y of the Cell, Garland Publishing Inc., New York
NY, p. 247).
Molecules Associated with Growth and Development
Human growth and development requires the spatial and temporal regulation of
cell
2 s differentiation, cell proliferation, and apoptosis. These processes
coordinately control reproduction,
aging, embryogenesis, morphogenesis, organogenesis, and tissue repair and
maintenance. At the
cellular level, growth and development is governed by the cell's decision to
enter into or exit from the
cell division cycle and by the cell's commitment to a terminally
differentiated state. These decisions
are made by the cell in response to extracellular signals and other
environmental cues it receives. The
3 o following discussion focuses on the molecular mechanisms of cell division,
reproduction, cell
differentiation and proliferation, apoptosis, and aging.
Cell Division
Cell division is the fundamental process by which all living things grow and
reproduce. In
unicellular organisms such as yeast and bacteria, each cell division doubles
the number of organisms,
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while in multicellular species many rounds of cell division are required to
replace cells lost by wear or
by programmed cell death, and for cell differentiation to produce a new tissue
or organ. Details of the
cell division cycle may vary, but the basic process consists of three
principle events. The first event,
interphase, involves preparations for cell division, replication of the DNA,
and production of essential
s proteins. In the second event, mitosis, the nuclear material is divided and
separates to opposite sides of
the cell. The final event, cytokinesis, is division and fission of the cell
cytoplasm. The sequence and
timing of cell cycle transitions is under the control of the cell cycle
regulation system which controls the
process by positive or negative regulatory circuits at various check points.
Regulated progression of the cell cycle depends on the integration of growth
control pathways
s o with the basic cell cycle machinery. Cell cycle regulators have been
identified by selecting for human
and yeast cDNAs that block or activate cell cycle arrest signals in the yeast
mating pheromone pathway
when they are overexpressed. Known regulators include human CPR (cell cycle
progression
restoration) genes, such as CPR8 and CPR2, and yeast CDC (cell division
control) genes, including
CDC91, that block the arrest signals. The CPR genes express a variety of
proteins including cyclins,
s s tumor suppresser binding proteins, chaperones, transcription factors,
translation factors, and
RNA-binding proteins (Edwards, M.C. et a1.(1997) Genetics 147:1063-1076).
Several cell cycle transitions, including the entry and exit of a cell from
mitosis, are dependent
upon the activation and inhibition of cyclin-dependent kinases (Cdks). The
Cdks are composed of a
kinase subunit, Cdk, and an activating subunit, cyclin, in a complex that is
subject to many levels of
2 o regulation. There appears to be a single Cdk in Saccharomyces cerevisiae
and Saccharomyces pombe
whereas mammals have a variety of specialized Cdks. Cyclins act by binding to
and activating
cyclin-dependent protein kinases which then phosphorylate and activate
selected proteins involved in the
mitotic process. The Cdk-cyclin complex is both positively and negatively
regulated by
phosphorylation, and by targeted degradation involving molecules such as CDC4
and CDC53. In
2 s addition, Cdks are further regulated by binding to inhibitors and other
proteins such as Suc1 that
modify their specificity or accessibility to regulators (Patra, D. and W.G.
Dunphy (1996) Genes Dev.
10:1503-1515; and Mathias, N. et al. (1996) Mol. Cell Biol. 16:6634-6643).
Reproduction
The male and female reproductive systems are complex and involve many aspects
of growth
s o and development. The anatomy and physiology of the male and female
reproductive systems are
reviewed in (Guyton, A.C. (1991) Textbook of Medical Physiolo~y, W.B. Saunders
Co., Philadelphia
PA, pp. 899-928).
The male reproductive system includes the process of spermatogenesis, in which
the sperm are
formed, and male reproductive functions are regulated by various hormones and
their effects on
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accessory sexual organs, cellular metabolism, growth, and other bodily
functions.
Spermatogenesis begins at puberty as a result of stimulation by gonadotropic
hormones
released from the anterior pituitary. Immature sperm (spermatogonia) undergo
several mitotic cell
divisions before undergoing meiosis and full maturation. The testes secrete
several male sex hormones,
s the most abundant being testosterone, that is essential for growth and
division of the immature sperm,
and for the masculine characteristics of the male body. Three other male sex
hormones, gonadotropin-
releasing hormone (GnRH), luteinizing hormone (LH), and follicle-stimulating
hormone (FSH) control
sexual function.
The uterus, ovaries, fallopian tubes, vagina, and breasts comprise the female
reproductive
s o system. The ovaries and uterus are the source of ova and the location of
fetal development,
respectively. The fallopian tubes and vagina are accessory organs attached to
the top and bottom of the
uterus, respectively. Both the uterus and ovaries have additional roles in the
development and loss of
reproductive capability during a female's lifetime. The primary role of the
breasts is lactation.
Multiple endocrine signals from the ovaries, uterus, pituitary, hypothalamus,
adrenal glands, and other
s s tissues coordinate reproduction and lactation. These signals vary during
the monthly menstruation
cycle and during the female's lifetime. Similarly, the sensitivity of
reproductive organs to these
endocrine signals varies during the female's lifetime.
A combination of positive and negative feedback to the ovaries, pituitary and
hypothalamus
glands controls physiologic changes during the monthly ovulation and
endometrial cycles. The anterior
2 o pituitary secretes two major gonadotropin hormones, follicle-stimulating
hormone (FSH) and luteinizing
hormone (LH), regulated by negative feedback of steroids, most notably by
ovarian estradiol. If
fertilization does not occur, estrogen and progesterone levels decrease. This
sudden reduction of the
ovarian hormones leads to menstruation, the desquamation of the endometrium.
Hormones further govern all the steps of pregnancy, parturition, lactation,
and menopause.
2 s During pregnancy large quantities of human chorionic gonadotropin (hCG),
estrogens, progesterone,
and human chorionic somatomaxnmotropin (hCS) are formed by the placenta. hCG,
a glycoprotein
similar to luteinizing hormone, stimulates the corpus luteum to continue
producing more progesterone
and estrogens, rather than to involute as occurs if the ovum is not
fertilized. hCS is similar to growth
hormone and is crucial for fetal nutrition.
3 o The female breast also matures during pregnancy. Large amounts of estrogen
secreted by the
placenta trigger growth and branching of the breast milk ductal system while
lactation is initiated by the
secretion of prolactin by the pituitary gland.
Parturition involves several hormonal changes that increase uterine
contractility toward the end
of pregnancy, as follows. The levels of estrogens increase more than those of
progesterone. Oxytocin
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is secreted by the neurohypophysis. Concomitantly, uterine sensitivity to
oxytocin increases. The fetus
itself secretes oxytocin, cortisol (from adrenal glands), and prostaglandins.
Menopause occurs when most of the ovarian follicles have degenerated. The
ovary then
produces less estradiol, reducing the negative feedback on the pituitary and
hypothalamus glands.
s Mean levels of circulating FSH and LH increase, even as ovulatory cycles
continue. Therefore, the
ovary is less responsive to gonadotropins, and there is an increase in the
time between menstrual cycles.
Consequently, menstrual bleeding ceases and reproductive capability ends.
Cell Differentiation and Proliferation
Tissue growth involves complex and ordered patterns of cell proliferation,
cell differentiation,
s o and apoptosis. Cell proliferation must be regulated to maintain both the
number of cells and their
spatial organization. This regulation depends upon the appropriate expression
of proteins which control
cell cycle progression in response to extracellular signals, such as growth
factors and other mitogens,
and intracellular cues, such as DNA damage or nutrient starvation. Molecules
which directly or
indirectly modulate cell cycle progression fall into several categories,
including growth factors and their
is receptors, second messenger and signal transduction proteins, oncogene
products, tumor-suppressor
proteins, and mitosis-promoting factors.
Growth factors were originally described as serum factors required to promote
cell
proliferation. Most growth factors are large, secreted polypeptides that act
on cells in their local
environment. Growth factors bind to and activate specific cell surface
receptors and initiate
2 o intracellular signal transduction cascades. Many growth factor receptors
are classified as receptor
tyrosine kinases which undergo autophosphorylation upon ligand binding.
Autophosphorylation
enables the receptor to interact with signal transduction proteins
characterized by the presence of SH2
or SH3 domains (Src homology regions 2 or 3). These proteins then modulate the
activity state of
small G-proteins, such as Ras, Rab, and Rho, along with GTPase activating
proteins (GAPs), guanine
2 s nucleotide releasing proteins (GNRPs), and other guanine nucleotide
exchange factors. Small G
proteins act as molecular switches that activate other downstream events, such
as mitogen-activated
protein kinase (MAP kinase) cascades. MAP kinases ultimately activate
transcription of mitosis-
promoting genes.
In addition to growth factors, small signaling peptides and hormones also
influence cell
3 o proliferation. These molecules bind primarily to another class of
receptor, the trimeric G-protein
coupled receptor (GPCR), found predominantly on the surface of immune,
neuronal and neuroendocrine
cells. Upon ligand binding, the GPCR activates a trimeric G protein which in
turn triggers increased
levels of intracellular second messengers such as phospholipase C, Ca2+, and
cyclic AMP. Most
GPCR-mediated signaling pathways indirectly promote cell proliferation by
causing the secretion or
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breakdown of other signaling molecules that have direct mitogenic effects.
These signaling cascades
often involve activation of kinases and phosphatases. Some growth factors,
such as some members of
the transforming growth factor beta (TGF-~3) family, act on some cells to
stimulate cell proliferation
and on other cells to inhibit it. Growth factors may also stimulate a cell at
one concentration and inhibit
s the same cell at another concentration. Most growth factors also have a
multitude of other actions
besides the regulation of cell growth and division: they can control the
proliferation, survival,
differentiation, migration, or function of cells depending on the
circumstance. For example, the tumor
necrosis factor/nerve growth factor (TNF/NGF) family can activate or inhibit
cell death, as well as
regulate proliferation and differentiation. The cell response depends on the
type of cell, its stage of
s o differentiation and transformation status, which surface receptors are
stimulated, and the types of
stimuli acting on the cell (Smith, A. et al. (1994) Cell 76:959-962; and
Nocentini, G. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:6216-6221).
Neighboring cells in a tissue compete for growth factors, and when provided
with "unlimited"
quantities in a perfused system will grow to even higher cell densities before
reaching density-dependent
is inhibition of cell division. Cells often demonstrate an anchorage
dependence of cell division as well.
This anchorage dependence may be associated with the formation of focal
contacts linking the
cytoskeleton with the extracellular matrix (ECM). The expression of ECM
components can be
stimulated by growth factors. For example, TGF-(3 stimulates fibroblasts to
produce a variety of ECM
proteins, including fibronectin, collagen, and tenascin (Pearson, C.A. et al.
(1988) EMBO J. 7:2677-
20 2981). In fact, for some cell types specific ECM molecules, such as laminin
or fibronectin, may act as
growth factors. Tenascin-C and -R, expressed in developing and lesioned neural
tissue, provide
stimulatory/anti-adhesive or inhibitory properties, respectively, for axonal
growth (Faissner, A. (1997)
Cell Tissue Res. 290:331-341).
Cancers are associated with the activation of oncogenes which are derived from
normal cellular
2 s genes. These oncogenes encode oncoproteins which convert normal cells into
malignant cells. Some
oncoproteins are mutant isoforms of the normal protein, and other oncoproteins
are abnormally
expressed with respect to location or amount of expression. The latter
category of oncoprotein causes
cancer by altering transcriptional control of cell proliferation. Five classes
of oncoproteins are known
to affect cell cycle controls. These classes include growth factors, growth
factor receptors, intracellular
3 o signal transducers, nuclear transcription factors, and cell-cycle control
proteins. Viral oncogenes are
integrated into the human genome after infection of human cells by certain
viruses. Examples of viral
oncogenes include v-src, v-abl, and v-fps.
Many oncogenes have been identified and characterized. These include sis,
erbA, erbB, her-2,
mutated GS, src, abl, ras, crk, jun, fos, myc, and mutated tumor-suppressor
genes such as RB, p53,
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mdm2, Cipl, p16, and cyclin D. Transformation of normal genes to oncogenes may
also occur by
chromosomal translocation. The Philadelphia chromosome, characteristic of
chronic myeloid leukemia
and a subset of acute lymphoblastic leukemias, results from a reciprocal
translocation between
chromosomes 9 and 22 that moves a truncated portion of the proto-oncogene c-
abl to the breakpoint
s cluster region (bcr) on chromosome 22.
Tumor-suppressor genes are involved in regulating cell proliferation.
Mutations which cause
reduced or loss of function in tumor-suppressor genes result in uncontrolled
cell proliferation. For
example, the retinoblastoma gene product (RB), in a non-phosphorylated state,
binds several early-
response genes and suppresses their transcription, thus blocking cell
division. Phosphorylation of RB
1 o causes it to dissociate from the genes, releasing the suppression, and
allowing cell division to proceed.
Al~Optosls
Apoptosis is the genetically controlled process by which unneeded or defective
cells undergo
programmed cell death. Selective elimination of cells is as important for
morphogenesis and tissue
remodeling as is cell proliferation and differentiation. Lack of apoptosis may
result in hyperplasia and
1 s other disorders associated with increased cell proliferation. Apoptosis is
also a crifiical component of
the immune response. Immune cells such as cytotoxic T-cells and natural killer
cells prevent the spread
of disease by inducing apoptosis in tumor cells and virus-infected cells. In
addition, immune cells that
fail to distinguish self molecules from foreign molecules must be eliminated
by apoptosis to avoid an
autoimmune response.
2 o Apoptotic cells undergo distinct morphological changes. Hallmarks of
apoptosis include cell
shrinkage, nuclear and cytoplasmic condensation, and alterations in plasma
membrane topology.
Biochemically, apoptotic cells are characterized by increased intracellular
calcium concentration,
fragmentation of chromosomal DNA, and expression of novel cell surface
components.
The molecular mechanisms of apoptosis are highly conserved, and many of the
key protein
2 s regulators and effectors of apoptosis have been identified. Apoptosis
generally proceeds in response to
a signal which is transduced intracellularly and results in altered patterns
of gene expression and protein
activity. Signaling molecules such as hormones and cytokines are known both to
stimulate and to
inhibit apoptosis through interactions with cell surface receptors.
Transcription factors also play an
important role in the onset of apoptosis. A number of downstream effector
molecules, particularly
3 o proteases such as the cysteine proteases called caspases, have been
implicated in the degradation of
cellulax components and the proteolytic activation of other apoptotic
effectors.
A~in~ and Senescence
Studies of the aging process or senescence have shown a number of
characteristic cellular and
molecular changes (Fauci et al. (1998) Harrison's Principles of Internal
Medicine, McGraw-Hill, New
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York NY, p.37). These characteristics include increases in chromosome
structural abnormalities, DNA
cross-linking, incidence of single-stranded breaks in DNA, losses in DNA
methylation, and degradation
of telomere regions. In addition to these DNA changes, post-translational
alterations of proteins
increase including, deamidation, oxidation, cross-linking, and nonenzymatic
glycation. Still further
s molecular changes occur in the mitochondria of aging cells through
deterioration of structure. These
changes eventually contribute to decreased function in every organ of the
body.
Biochemical Pathway Molecules
Biochemical pathways are responsible for regulating metabolism, growth and
development,
1 o protein secretion and trafficking, environmental responses, and ecological
interactions including
immune response and response to parasites.
DNA replication
Deoxyribonucleic acid (DNA), the genetic material, is found in both the
nucleus and
mitochondria of human cells. The bulk of human DNA is nuclear, in the form of
linear chromosomes,
1 s while mitochondrial DNA is circular. DNA replication begins at specific
sites called origins of
replication. Bidirectional synthesis occurs from the origin via two growing
forks that move in opposite
directions. Replication is semi-conservative, with each daughter duplex
containing 'one old strand and
its newly synthesized complementary partner. Proteins involved in DNA
replication include DNA
polymerases, DNA primase, telomerase, DNA helicase, topoisomerases, DNA
ligases, replication
2 o factors, and DNA-binding proteins.
DNA Recombination and Repair
Cells are constantly faced with replication errors and environmental assault
(such as ultraviolet
irradiation) that can produce DNA damage. Damage to DNA consists of any change
that modifies the
structure of the molecule. Changes to DNA can be divided into two general
classes, single base
2 s changes and structural distortions. Any damage to DNA can produce a
mutation, and the mutation may
produce a disorder, such as cancer.
Changes in DNA are recognized by repair systems within the cell. These repair
systems act to
correct the damage and thus prevent any deleterious affects of a mutational
event. Repair systems can
be divided into three general types, direct repair, excision repair, and
retrieval systems. Proteins
3 o involved in DNA repair include DNA polymerase, excision repair proteins,
excision and cross link
repair proteins, recombination and repair proteins, RAD51 proteins, and BLN
and WRN proteins that
are homologs of RecQ helicase. When the repair systems are eliminated, cells
become exceedingly
sensitive to environmental mutagens, such as ultraviolet irradiation. Patients
with disorders associated
with a loss in DNA repair systems often exhibit a high sensitivity to
environmental mutagens.
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Examples of such disorders include xeroderma pigmentosum (XP), Bloom's
syndrome (BS), and
Werner's syndrome (WS) (Yamagata, K. et al. (1998) Proc. Natl. Acad. Sci. USA
95:8733-8738),
ataxia telangiectasia, Cockayne's syndrome, and Fanconi's anemia.
Recombination is the process whereby new DNA sequences are generated by the
movements of
s large pieces of DNA. In homologous recombination, which occurs during
meiosis and DNA repair,
parent DNA duplexes align at regions of sequence similarity, and new DNA
molecules form by the
breakage and joining of homologous segments. Proteins involved include RAD51
recombinase. In site-
specific recombination, two specific but not necessarily homologous DNA
sequences are exchanged. In
the immune system this process generates a diverse collection of antibody and
T cell receptor genes.
s o Proteins involved in site-specific recombination in the immune system
include recombination activating
genes 1 and 2 (RAGl and RAG2). A defect in immune system site-specific
recombination causes
severe combined immunodeficiency disease in mice.
RNA Metabolism
Ribonucleic acid (RNA) is a linear single-stranded polymer of four
nucleotides, ATP, CTP,
15 UTP, and GTP. In most organisms, RNA is transcribed as a copy of DNA, the
genetic material of the
organism. In retroviruses RNA rather than DNA serves as the genetic material.
RNA copies of the
genetic material encode proteins or serve various structural, catalytic, or
regulatory roles in organisms.
RNA is classified according to its cellular localization and function.
Messenger RNAs (mRNAs)
encode polypeptides. Ribosomal RNAs (rRNAs) are assembled, along with
ribosomal proteins, into
2 o ribosomes, which are cytoplasmic particles that translate mRNA into
polypeptides. Transfer RNAs
(tRNAs) are cytosolic adaptor molecules that function in mRNA translation by
recognizing both an
mRNA codon and the amino acid that matches that codon. Heterogeneous nuclear
RNAs (hnRNAs)
include mRNA precursors and other nuclear RNAs of various sizes. Small nuclear
RNAs (snRNAs)
are a paxt of the nuclear spliceosome complex that removes intervening, non-
coding sequences (introns)
2 s and rejoins exons in pre-mRNAs.
RNA Transcription
The transcription process synthesizes an RNA copy of DNA. Proteins involved
include multi-
subunit RNA polymerases, transcription factors IIA, IIB, IID, IIE, IIF, IIH,
and IIJ. Many
transcription factors incorporate DNA-binding structural motifs which comprise
either a-helices or (3-
3 o sheets that bind to the major groove of DNA. Four well-characterized
structural motifs are helix-turn-
helix, zinc forger, leucine zipper, and helix-loop-helix.
RNA Processing
Various proteins are necessary for processing of transcribed RNAs in the
nucleus. Pre-mRNA
processing steps include capping at the 5' end with methylguanosine,
polyadenylating the 3' end, and
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splicing to remove introns. The spliceosomal complex is comprised of five
small nuclear
ribonucleoprotein particles (snRNPs) designated Ul, U2, U4, U5, and U6. Each
snRNP contains a
single species of snRNA and about ten proteins. The RNA components of some
snRNPs recognize and
base-pair with intron consensus sequences. The protein components mediate
spliceosome assembly and
s the splicing reaction. Autoantibodies to snRNP proteins are found in the
blood of patients with
systemic lupus erythematosus (Stryer, L. (1995) Biochemistry W.H. Freeman and
Company, New
York NY, p. 863).
Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that
have roles in
splicing, exporting of the mature RNAs to the cytoplasm, and mRNA translation
(Biamonti, G. et al.
to (1998) Clin. Exp. Rheumatol. 16:317-326). Some examples of hnRNPs include
the yeast proteins
Hrplp, involved in cleavage and polyadenylation at the 3' end of the RNA;
Cbp80p, involved in
capping the 5' end of the RNA; and Npl3p, a homolog of mammalian hnRNP Al,
involved in export of
mRNA from the nucleus (Shen, E.C. et al. (1998) Genes Dev. 12:679-691). HnRNPs
have been shown
to be important targets of the autoimmune response in rheumatic diseases
(Biamonti, supra).
~. s Many snRNP proteins, hnRNP proteins, and alternative splicing factors are
characterized by~
an RNA recognition motif (RRM). (Reviewed in Birney, E. et al. (1993) Nucleic
Acids Res. 21:5803-
5816.) The RRM is about 80 amino acids in length and forms four (3-strands and
two a-helices
arranged in an a/(3 sandwich. The RRM contains a core RNP-1 octapeptide motif
along with
surrounding conserved sequences.
2 0 RNA Stability and Degradation
RNA helicases alter and regulate RNA conformation and secondary structure by
using energy
derived from ATP hydrolysis to destabilize and unwind RNA duplexes. The most
well-characterized
and ubiquitous family of RNA helicases is the DEAD-box family, so named for
the conserved B-type
ATP-binding motif which is diagnostic of proteins in this family. Over 40 DEAD-
box helicases have
2 s been identified in organisms as diverse as bacteria, insects, yeast,
amphibians, mammals, and plants.
DEAD-box helicases function in diverse processes such as translation
initiation, splicing, ribosome
assembly, and RNA editing, transport, and stability. Some DEAD-box helicases
play tissue- and stage-
specific roles in spermatogenesis and embryogenesis. (Reviewed in Linder, P.
et al. (1989) Nature
337:121-122.)
3 o Overexpression of the DEAD-box 1 protein (DDX1) may play a role in the
progression of
neuroblastoma (Nb) and retinoblastoma (Rb) tumors. Other DEAD-box helicases
have been implicated
either directly or indirectly in ultraviolet light-induced tumors, B cell
lymphoma, and myeloid
malignancies. (Reviewed in Godbout, R. et al. (1998) J. Biol. Chem. 273:21161-
21168.)
Ribonucleases (RNases) catalyze the hydrolysis of phosphodiester bonds in RNA
chains, thus
24
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cleaving the RNA. For example, RNase P is a ribonucleoprotein enzyme which
cleaves the 5' end of
pre-tRNAs as part of their maturation process. RNase H digests the RNA strand
of an RNA/DNA
hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an
important enzyme in
the retroviral replication cycle. RNase H domains are often found as a domain
associated with reverse
s transcriptases. RNase activity in serum and cell extracts is elevated in a
variety of cancers and
infectious diseases (Schein, C.H. (1997) Nat. Biotechnol. 15:529-536).
Regulation of RNase activity is
being investigated as a means to control tumor angiogenesis, allergic
reactions, viral infection and
replication, and fungal infections.
Protein Translation
1 o The eukaryotic ribosome is composed of a 60S (large) subunit and a 40S
(small) subunit,
which together form the 80S ribosome. In addition to the 18S, 28S, SS, and
5.85 rRNAs, the ribosome
also contains more than fifty proteins. The ribosomal proteins have a prefix
which denotes the subunit
to which they belong, either L (large) or S (small). Three important sites are
identified on the ribosome.
The aminoacyl-tRNA site (A site) is where charged tRNAs (with the exception of
the initiator-tRNA)
1 s bind on arrival at the ribosome. The peptidyl-tRNA site (P site) is where
new peptide bonds are
formed, as well as where the initiator tRNA binds. The exit site (E site) is
where deacylated tRNAs
bind prior to their release from the ribosome. (Translation is reviewed in
Stryer, L. (1995)
Biochemistry, W.H. Freeman and Company, New York NY, pp. 875-908; and Lodish,
H. et al. (1995)
Molecular Cell Biolo~y, Scientific American Books, New York NY, pp. 119-138.)
2 o tRNA Charein~
Protein biosynthesis depends on each amino acid forming a linkage with the
appropriate tRNA.
The aminoacyl-tRNA synthetases are responsible for the activation and correct
attachment of an amino
acid with its cognate tRNA. The 20 aminoacyl-tRNA synthetase enzymes can be
divided into two
structural classes, Class I and Class II. Autoantibodies against aminoacyl-
tRNAs are generated by
2 s patients with dermatomyositis and polymyositis, and correlate strongly
with complicating interstitial
lung disease (ILD). These antibodies appear to be generated in response to
viral infection, and
coxsackie virus has been used to induce experimental viral myositis in
animals.
Translation Initiation
Initiation of translation can be divided into three stages. The first stage
brings an initiator
3 o transfer RNA (Met-tRNAf) together with the 40S ribosomal subunit to form
the 43S preinitiation
complex. The second stage binds the 43S preinitiation complex to the mRNA,
followed by migration of
the complex to the correct AUG initiation codon. The third stage brings the
60S ribosomal subunit to
the 40S subunit to generate an 80S ribosome at the initiation codon.
Regulation of translation primarily
involves the first and second stage in the initiation process (Pain, V.M.
(1996) Eur. J. Biochem.
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236:747-771).
Several initiation factors, many of which contain multiple subunits, are
involved in bringing an
initiator tRNA and 40S ribosomal subunit together. elF2, a guanine nucleotide
binding protein, recruits
the initiator tRNA to the 40S ribosomal subunit. Only When eIF2 is bound to
GTP does it associate
s with the initiator tRNA. eIF2B, a guanine nucleotide exchange protein, is
responsible for converting
eIF2 from the GDP-bound inactive form to the GTP-bound active form. Two other
factors, eIFlA and
eIF3 bind and stabilize the 40S subunit by interacting with 18S ribosomal RNA
and specific ribosomal
structural proteins. eIF3 is also involved in association of the 40S ribosomal
subunit with mRNA: The
Met-tRNAf, eIFlA, eIF3, and 40S ribosomal subunit together make up the 43S
preinitiation complex
i o (Pain, supra).
Additional factors are required fox binding of the 43S preinitiation complex
to an mRNA
molecule, and the process is regulated at several levels. eIF4F is a complex
consisting of three proteins:
eIF4E, eIF4A, and eIF4G. eIF4E recognizes and binds to the mRNA 5'-terminal
m'GTP cap, eIF4A is
a bidirectional RNA-dependent helicase, and elF4G is a scaffolding
polypeptide. eIF4G has three
15 binding domains. The N-terminal third of eIF4G interacts with eIF4E, the
central third interacts with
eIF4A, and the C-terminal third interacts with elF3 bound to the 43S
preinitiation complex. Thus,
eIF4G acts as a bridge between the 40S ribosomal subunit and the mRNA (Hentze,
M.W. (1997)
Science 275:500-501).
The ability of elF4F to initiate binding of the 43S preinitiation complex is
regulated by
2 o structural features of the mRNA. The mRNA molecule has an untranslated
region (UTR) between the
5' cap and the AUG start colon. In some mRNAs this region forms secondary
structures that impede
binding of the 43S preinitiation complex. The helicase activity of eIF4A is
thought to function in
removing this secondary structure to facilitate binding of the 43S
preinitiation complex (Pain, supra).
Translation Elongation
2 s Elongation is the process whereby additional amino acids are joined to the
initiator methionine
to form the complete polypeptide chain. The elongafiion factors EFl a, EFl (3
y, and EF2 are involved
in elongating the polypeptide chain following initiation. EFla is a GTP-
binding protein. In EFla's
GTP-bound form, it brings an aminoacyl-tRNA to the ribosome's A site. The
amino acid attached to
the newly arrived aminoacyl-tRNA forms a peptide bond with the initiator
methionine. The GTP on
3 o EF1 a is hydrolyzed to GDP, and EFl a-GDP dissociates from the ribosome.
EF1 (3 'y binds EF1 a -GDP
and induces the dissociation of GDP from EFla, allowing EFla to bind GTP and a
new cycle to begin.
As subsequent aminoacyl-tRNAs are brought to the ribosome, EF-G, another GTP-
binding
protein, catalyzes the translocation of tRNAs from the A site to the P site
and finally to the E site of the
ribosome. This allows the processivity of translation.
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Translation Termination
The release factor eRF carries out termination of translation. eRF recognizes
stop codons in
the mRNA, leading to the release of the polypeptide chain from the ribosome.
Post-Translational Pathways
Proteins may be modified after translation by the addition of phosphate,
sugar, prenyl, fatty
acid, and other chemical groups. These modifications axe often required for
proper protein activity.
Enzymes involved in post-translational modification include kinases,
phosphatases,
glycosyltransferases, and prenyltransferases. The conformation of proteins may
also be modified after
translation by the introduction and rearrangement of disulfide bonds
(rearrangement catalyzed by
1 o protein disulfide isomerase), the isomerization of proline sidechains by
prolyl isomerase, and by
interactions with molecular chaperone proteins.
Proteins may also be cleaved by proteases. Such cleavage may result in
activation,
inactivation, or complete degradation of the protein. Proteases include serine
proteases, cysteine
proteases, aspartic proteases, and metalloproteases. Signal peptidase in the
endoplasmic reticulum
(ER) lumen cleaves the signal peptide from membrane or secretory proteins that
are imported into the
ER. Ubiquitin proteases are associated with the ubiquitin conjugation system
(UCS), a major
pathway for the degradation of cellular proteins in eukaryotic cells and some
bacteria. The UCS
mediates the elimination of abnormal proteins and regulates the half lives of
important regulatory
proteins that control cellular processes such as gene transcription and cell
cycle progression. In~the
2 o UCS pathway, proteins targeted for degradation are conjugated to a
ubiquitin, a small heat stable
protein. Proteins involved in the UCS include ubiquitin-activating enzyme,
ubiquitin-conjugating
enzymes, ubiquitin-ligases, and ubiquitin C-terminal hydrolases. The
ubiquitinated protein is then
recognized and degraded by the proteasome, a large, multisubunit proteolytic
enzyme complex, and
ubiquitin is released for reutilization by ubiquitin protease.
2 5 Lipid Metabolism
Lipids are water-insoluble, oily or greasy substances that are soluble in
nonpolar solvents such
as chloroform or ether. Neutral fats (triacylglycerols) serve as major fuels
and energy stores. Polar
lipids, such as phospholipids, sphingolipids, glycolipids, and cholesterol,
are key structural components
of cell membranes.
3 o Lipid metabolism is involved in human diseases and disorders. In the
arterial disease
atherosclerosis, fatty lesions form on the inside of the arterial wall. These
lesions promote the loss of
arterial flexibility and the formation of blood clots (Guyton, A.C. Textbook
of Medical Phvsiologyr
(1991) W.B. Saunders Company, Philadelphia PA, pp.760-763). In Tay-Sacks
disease, the GMZ
ganglioside (a sphingolipid) accumulates in lysosomes of the central nervous
system due to a lack of the
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enzyme N-acetylhexosaminidase. Patients suffer nervous system degeneration
leading to early death
(Fauci, A.S. et al. (1998) Harrison's Principles of Internal Medicine McGraw-
Hill, New York NY, p.
2171). The Niemann Pick diseases are caused by defects in lipid metabolism.
Niemann-Pick diseases
types A and B are caused by accumulation of sphingomyelin (a sphingolipid) and
other lipids in the
s central nervous system due to a defect in the enzyme sphingomyelinase,
leading to neurodegeneration
and lung disease. Niemann-Pick disease type C results from a defect in
cholesterol transport, leading to
the accumulation of sphingomyelin and cholesterol in lysosomes and a secondary
reduction in
sphingomyelinase activity. Neurological symptoms such as grand mal seizures,
ataxia, and loss of
previously learned speech, manifest 1-2 years after birth. A mutation in the
NPC protein, which
1 o contains a putative cholesterol-sensing domain, was found in a mouse model
of Niemann-Pick disease
type C (Fauci, supra, p. 2175; Loftus, S.K. et al. (1997) Science 277:232-
235). (Lipid metabolism is
reviewed in Stryer, L. (1995) Biochemistry, W.H. Freeman and Company, New York
NY; Lehninger,
A. (1982) Principles of Biochemistry Worth Publishers, Inc., New York NY; and
ExPASy
"Biochemical Pathways" index of Boehringer Mannheim World Wide Web site.)
15 Fatty Acid Synthesis
Fatty acids are long-chain organic acids with a single carboxyl group and a
long non-polar
hydrocarbon tail. Long-chain fatty acids are essential components of
glycolipids, phospholipids~ and
cholesterol, which are building blocks for biological membranes, and of
triglycerides, which are '
biological fuel molecules. Long-chain fatty acids are also substrates for
eicosanoid production, and are
2 o important in the functional modification of certain complex carbohydrates
and proteins. 16-carbon and
18-carbon fatty acids are the most common.
Fatty acid synthesis occurs in the cytoplasm. In the first step, acetyl-
Coenzyme A (CoA)
carboxylase (ACC) synthesizes malonyl-CoA from acetyl-CoA and bicarbonate. The
enzymes which
catalyze the remaining reactions axe covalently linked into a single
polypeptide chain, referred to as the
2 s multifunctional enzyme fatty acid synthase (FAS). FAS catalyzes the
synthesis of palmitate from
acetyl-CoA and malonyl-CoA. FAS contains acetyl transferase, malonyl
transferase, (3-ketoacetyl
synthase, acyl carrier protein, (3-ketoacyl reductase, dehydratase, enoyl
reductase, and thioesterase
activities. The final product of the FAS reaction is the 16-carbon fatty acid
palmitate. Further
elongation, as well as unsaturation, of palmitate by accessory enzymes of the
ER produces the variety
3 0 of long chain fatty acids required by the individual cell. These enzymes
include a NADH-cytochrome
b5 reductase, cytochrome b5, and a desaturase.
Phospholipid and Triacyl~lycerol Synthesis
Triacylglycerols, also known as triglycerides and neutral fats, are major
energy stores in
animals. Triacylglycerols are esters of glycerol with three fatty acid chains.
Glycerol-3-phosphate is
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produced from dihydroxyacetone phosphate by the enzyme glycerol phosphate
dehydrogenase or from
glycerol by glycerol kinase. Fatty acid-CoA's are produced from fatty acids by
fatty acyl-CoA
synthetases. Glyercol-3-phosphate is acylated with two fatty acyl-CoA's by the
enzyme glycerol
phosphate acyltransferase to give phosphatidate. Phosphatidate phosphatase
converts phosphatidate to
s diacylglycerol, which is subsequently acylated to a triacylglyercol by the
enzyme diglyceride
acyltransferase. Phosphatidate phosphatase and diglyceride acyltransferase
form a triacylglyerol
synthetase complex bound to the ER membrane.
A major class of phospholipids are the phosphoglycerides, which are composed
of a glycerol
backbone, two fatty acid chains, and a phosphorylated alcohol.
Phosphoglycerides are components of
1 o cell membranes. Principal phosphoglycerides are phosphatidyl choline,
phosphatidyl ethanolamine,
phosphatidyl serine, phosphatidyl inositol, and diphosphatidyl glycerol. Many
enzymes involved in
phosphoglyceride synthesis are associated with membranes (Meyers, R.A.
(1995).Molecular Biolo~y
and Biotechnolo~y, VCH Publishers Inc., New York NY, pp. 494-501).
Phosphatidate is converted to
CDP-diacylglycerol by the enzyme phosphatidate cytidylyltransferase (ExPASy
ENZYME EC
is 2.7.7.41). Transfer of the diacylglycerol group from CDP-diacylglycerol to
serine to yield phosphatidyl
serine, or to inositol to yield phosphatidyl inositol, is catalyzed by the
enzymes CDP-diacylglycerol-
serine O-phosphatidyltransferase and CDP-diacylglycerol-inositol 3-
phosphatidyltransferase,
respectively (ExPASy ENZYME EC 2.7.8.8; ExPASy ENZYME EC 2.7.8.11). The enzyme
phosphatidyl serine decarboxylase catalyzes the conversion of phosphatidyl
serine to phosphatidyl
20 ethanolamine, using a pyruvate cofactor (Voelker, D.R. (1997) Biochim.
Biophys. Acta 1348:236-244).
Phosphatidyl choline is formed using diet-derived choline by the reaction of
CDP-choline with 1,2-
diacylglycerol, catalyzed by diacylglycerol cholinephosphotransferase (ExPASy
ENZYME 2.7.8.2).
Sterol, Steroid, and Isoprenoid Metabolism
Cholesterol, composed of four fused hydrocarbon rings with an alcohol at one
end, moderates
2 s the fluidity of membranes in which it is incorporated. In addition,
cholesterol is used in the synthesis of
steroid hormones such as cortisol, progesterone, estrogen, and testosterone.
Bile salts derived from
cholesterol facilitate the digestion of lipids. Cholesterol in the skin forms
a barrier that prevents excess
water evaporation from the body. Farnesyl and geranylgeranyl groups, which are
derived from
cholesterol biosynthesis intermediates, are post-translationally added to
signal transduction proteins
3 o such as ras and protein-targeting proteins such as rab. These
modifications are important for the
activities of these proteins (Guyton, su ra; Stryer, su ra, pp. 279-280, 691-
702, 934).
Mammals obtain cholesterol derived from both de novo biosynthesis and the
diet. The liver is
the major site of cholesterol biosynthesis in mammals. Two acetyl-CoA
molecules initially condense to
form acetoacetyl-CoA, catalyzed by a thiolase. Acetoacetyl-CoA condenses with
a third acetyl-CoA to
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form hydroxymethylglutaryl-CoA (HMG-CoA), catalyzed by HMG-CoA synthase.
Conversion of
HMG-CoA to cholesterol is accomplished via a series of enzymatic steps known
as the mevalonate
pafihway. The rate-limiting step is the conversion of HMG-CoA to mevalonate by
HMG-CoA
reductase. The drug lovastatin, a potent inhibitor of HMG-CoA reductase, is
given to patients to
s reduce their serum cholesterol levels. Other mevalonate pathway enzymes
include mevalonate kinase,
phosphomevalonate kinase, diphosphomevalonate decarboxylase,
isopentenyldiphosphate isomerase,
dimethylallyl transferase, geranyl transferase, faxnesyl-diphosphate
farnesyltransferase, squalene
monooxygenase, lanosterol synthase, lathosterol oxidase, and 7-
dehydrocholesterol reductase.
Cholesterol is used in the synthesis of steroid hormones such as cortisol,
progesterone,
~. o aldosterone, estrogen, and testosterone. First, cholesterol is converted
to pregnenolone by cholesterol
monooxygenases. The other steroid hormones are synthesized from pregnenolone
by a series of
enzyme-catalyzed reactions including oxidations, isomerizations,
hydroxylations, reductions, and
demethylations. Examples of these enzymes include steroid 4-isomerase, 3(3-
hydroxy-~5-steroid
dehydrogenase, steroid 21-monooxygenase, steroid 19-hydroxylase, and 3(3-
hydroxysteroid
1 s dehydrogenase. Cholesterol is also the precursor to vitamin D.
Numerous compounds contain 5-carbon isoprene units derived from the mevalonate
pathway
intermediate isopentenyl pyrophosphate. Isoprenoid groups are found in vitamin
K, ubiquinone, retinal,
dolichol phosphate (,a carrier of oligosaccharides needed for N-linked
glycosylation), and farnesyl and
geranylgeranyl groups that modify proteins. Enzymes involved include farnesyl
transferase, polyprenyl
2 o transferases, dolichyl phosphatase, and dolichyl kinase.
Sphin~olipid Metabolism
Sphingolipids are an important class of membrane lipids that contain
sphingosine, a long chain
amino alcohol. They are composed of one long-chain fatty acid, one polar head
alcohol, and
sphingosine or sphingosine derivative. The three classes of sphingolipids are
sphingomyelins,
2 s cerebrosides, and gangliosides. Sphingomyelins, which contain
phosphocholine or
phasphoethanolamine as their head group, are abundant in the myelin sheath
surrounding nerve cells.
Galactocerebrosides, which contain a glucose or galactose head group, are
characteristic of the brain.
Other cerebrosides are found in nonneural tissues. Gangliosides, whose head
groups contain multiple
sugar units, are abundant in the brain, but are also found in nonneural
tissues.
3 o Sphingolipids are built on a sphingosine backbone. Sphingosine is acylated
to ceramide by the
enzyme sphingosine acetyltransferase. Ceramide and phosphatidyl choline are
converted to
sphingomyelin by the enzyme ceramide choline phosphotransferase. Cerebrosides
are synthesized by
the linkage of glucose or galactose to ceramide by a transferase. Sequential
addition of sugar residues
to ceramide by transferase enzymes yields gangliosides.
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Eicosanoid Metabolism
Eicosanoids, including prostaglandins, prostacyclin, thromboxanes, and
leukotrienes, are 20-
carbon molecules derived from fatty acids. Eicosanoids are signaling molecules
which have roles in
pain, fever, and inflammation. The precursor of all eicosanoids is
arachidonate, which is generated
s from phospholipids by phospholipase AZ and from diacylglycerols by
diacylglycerol lipase.
Leukotrienes are produced from arachidonate by the action of lipoxygenases.
Prostaglandin synthase,
reductases, and isomerases are responsible for the synthesis of the
prostaglandins. Prostaglandins have
roles in inflammation, blood flow, ion transport, synaptic transmission, and
sleep. Prostacyclin and the
thromboxanes are derived from a precursor prostaglandin by the action of
prostacyclin synthase and
1 o thromboxane synthases, respectively.
Ketone Bodv Metabolism
Pairs of acetyl-CoA molecules derived from fatty acid oxidation in the liver
can condense to
form acetoacetyl-CoA, which subsequently forms acetoacetate, D-3-
hydroxybutyrate, and acetone.
These three products are known as ketone bodies. Enzymes involved in ketone
body metabolism
is include HMG-CoA synthetase, HMG-CoA cleavage enzyme, D-3-hydroxybutyrate
dehydrogenase,
acetoacetate decarboxylase, and 3-ketoacyl-CoA transferase. Ketone bodies are
a normal fuel supply
of the heart and xenal cortex. Acetoacetate produced by the liver is
transported to cells where the
acetoacetate is converted back to acetyl-CoA and enters the citaric acid
cycle. In times of starvation,
ketone bodies produced from stored triacylglyerols become an important fuel
source, especially for the
2 o brain. Abnormally high levels of ketone bodies are observed in diabetics.
Diabetic coma can result if
ketone body levels become too great.
Lipid Mobilization
Within cells, fatty acids are transported by cytoplasmic fatty acid binding
proteins (Online
Mendelian Inheritance in Man (OMIM) *134650 Fatty Acid-Binding Protein l,
Liver; FABPl).
2 s Diazepam binding inhibitor (DBI), also known as endozepine and acyl CoA-
binding protein, is an
endogenous y-aminobutyric acid (GABA) receptor ligand which is thought to down-
regulate the effects
of GABA. DBI binds medium- and long-chain acyl-CoA esters with very high
affinity and may
function as an intracellular carrier of acyl-CoA esters (OMIM *125950 Diazepam
Binding Inhibitor;
DBI; PROSITE PDOC00686 Acyl-CoA-binding protein signature).
3 o Fat stored in liver and adipose triglycerides may be released by
hydrolysis and transported in
the blood. Free fatty acids are transported in the blood by albumin.
Triacylglycerols and cholesterol
esters in the blood are transported in lipoprotein particles. The particles
consist of a core of
hydrophobic lipids surrounded by a shell of polar lipids and apolipoproteins.
The protein components
serve in the solubilization of hydrophobic lipids and also contain cell-
targeting signals. Lipoproteins
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include chylomicrons, chylomicron remnants, very-low-density lipoproteins
(VLDL), intermediate-
density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density
lipoproteins (HDL).
There is a strong inverse correlation between the levels of plasma HDL and
risk of premature coronary
heart disease.
s Triacylglycerols in chylomicrons and VLDL are hydrolyzed by lipoprotein
lipases that line
blood vessels in muscle and other tissues that use fatty acids. Cell surface
LDL receptors bind LDL
particles which are then internalized by endocytosis. Absence of the LDL
receptor, the cause of the
disease familial hypercholesterolemia, leads to increased plasma cholesterol
levels and ultimately to
atherosclerosis. Plasma cholesteryl ester transfer protein mediates the
transfer of cholesteryl esters
1 o from HDL to apolipoprotein B-containing lipoproteins. Cholesteryl ester
transfer protein is important
in the reverse cholesterol transport system and may play a role in
atherosclerosis (Yamashita, S. et al.
(1997) Curr. Opin. Lipidol. 8:101-110). Macrophage scavenger receptors, which
bind and internalize
modified lipoproteins, play a role in lipid transport and may contribute to
atherosclerosis (Greaves,
D.R. et al. (1998) Curr. Opin. Lipidol. 9:425-432).
15 Proteins involved in cholesterol uptake and biosynthesis are tightly
regulated in response to
cellular cholesterol levels. The sterol regulatory element binding protein
(SREBP) is a sterol-responsive
transcription factor. Under normal cholesterol conditions, SREBP resides in
the ER membrane. When
cholesterol levels are low, a regulated cleavage of SREBP occurs which
releases the extracellular
domain of the protein. This cleaved domain is then transported to the nucleus
where it activates the
2 o transcription of the LDL receptor gene, and genes encoding enzymes of
cholesterol synthesis, by
binding the sterol regulatory element (SRE) upstream of the genes (Yang, J. et
al. (1995) J. Biol. Chem.
270:12152-12161). Regulation of cholesterol uptake and biosynthesis also
occurs via the oxysterol-
binding protein (OSBP). OSBP is a high-affinity intracellular receptor for a
variety of oxysterols that
down-regulate cholesterol synthesis and stimulate cholesterol esterification
(Lagace, T.A. et al. (1997)
2 5 Biochem. J. 326:205-213).
Beta-oxidation
Mitochondrial and peroxisomal beta-oxidation enzymes degrade saturated and
unsaturated fatty
acids by sequential removal of two-carbon units from CoA-activated fatty
acids. The main beta-
oxidation pathway degrades both saturated and unsaturated fatty acids while
the auxiliary pathway
3 o performs additional steps required for the degradation of unsaturated
fatty acids.
The pathways of mitochondrial and peroxisomal beta-oxidation use similar
enzymes, but have
different substrate specificities and functions. Mitochondria oxidize short-,
medium-, and long-chain
fatty acids to produce energy for cells. Mitochondrial beta-oxidation is a
major energy source for
cardiac and skeletal muscle. In liver, it provides ketone bodies to the
peripheral circulation when
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glucose levels are low as in starvation, endurance exercise, and diabetes
(Eaton, S. et al. (1996)
Biochem. J. 320:345-357). Peroxisomes oxidize medium-, long-, and very-long-
chain fatty acids,
dicarboxylic fatty acids, branched fatty acids, prostaglandins, xenobiotics,
and bile acid intermediates.
The chief roles of peroxisomal beta-oxidation axe to shorten toxic lipophilic
carboxylic acids to
s facilitate their excretion and to shorten very-long-chain fatty acids prior
to mitochondrial beta-oxidation
(Mannaerts, G.P. and P.P. van Veldhoven (1993) Biochimie 75:147-158).
Enzymes involved in beta-oxidation include acyl CoA synthetase, carnitine
acyltransferase,
acyl CoA dehydrogenases, enoyl CoA hydratases, L-3-hydroxyacyl CoA
dehydrogenase, (3-ketothiolase,
2,4-dienoyl CoA reductase, and isomerase.
1 o Lipid Cleavage and Degradation
Triglycerides are hydrolyzed to fatty acids and glycerol by lipases.
Lysophospholipases
(LPLs) are widely distributed enzymes that metabolize intracellular lipids,
and occur in numerous
isoforms. Small isoforms, approximately 15-30 kD, function as hydrolases;
large isoforms, those
exceeding 60 kD, function both as hydrolases and transacylases. A particular
substrate for LPLs,
15 lysophosphatidylcholine, causes lysis of cell membranes when it is formed
or imported into a cell.
LPLs are regulated by lipid factors including acylcarnitine, axachidonic acid,
and phosphatidic acid.
These lipid factors are signaling molecules important in numerous pathways,
including the
inflammatory response. (Anderson, R. et al. (1994) Toxicol. Appl. Pharmacol.
125:176-183; Selle, H.
et al. (1993); Eur. J. Biochem. 212:411-416.)
2 o The secretory phospholipase A2 (PLA2) superfamily comprises a number of
heterogeneous
enzymes whose common feature is to hydrolyze the sn-2 fatty acid acyl ester
bond of
phosphoglycerides. Hydrolysis of the glycerophospholipids releases free fatty
acids and
lysophospholipids. PLA2 activity generates precursors for the biosynthesis of
biologically active lipids,
hydroxy fatty acids, and platelet-activating factor. PLA2 hydrolysis of the sn-
2 ester bond in
2 s phospholipids generates free fatty acids, such as arachidonic acid and
lysophospholipids.
Carbon and Carbohydrate Metabolism
Carbohydrates, including sugars or saccharides, starch, and cellulose, are
aldehyde or ketone
compounds with multiple hydroxyl groups. The importance of carbohydrate
metabolism is
demonstrated by the sensitive regulatory system in place for maintenance of
blood glucose levels. Two
3 o pancreatic hormones, insulin and glucagon, promote increased glucose
uptake and storage by cells, and
increased glucose release from cells, respectively. Carbohydrates have three
important roles in
mammalian cells. First, carbohydrates are used as energy stores, fuels, and
metabolic intermediates.
Carbohydrates are broken down to form energy in glycolysis and are stored as
glycogen for later use.
Second, the sugars deoxyribose and ribose form part of the structural support
of DNA and RNA,
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respectively. Third, carbohydrate modifications are added to secreted and
membrane proteins and lipids
as they traverse the secretory pathway. Cell surface carbohydrate-containing
macromolecules,
including glycoproteins, glycolipids, and transmembrane proteoglycans, mediate
adhesion with other
cells and with components of the extracellular matrix. The extracellular
matrix is comprised of diverse
s glycoproteins, glycosaminoglycans (GAGs), and carbohydrate-binding proteins
which are secreted from
the cell and assembled into an organized meshwork in close association with
the cell surface. The
interaction of the cell with the surrounding matrix profoundly influences cell
shape, strength, flexibility,
motility, and adhesion. These dynamic properties are intimately associated
with signal transduction
pathways controlling cell proliferation and differentiation, tissue
construction, and embryonic
1. o development.
Carbohydrate metabolism is altered in several disorders including diabetes
mellitus,
hyperglycemia, hypoglycemia, galactosemia, galactokinase deficiency, and UDP-
galactose-4-epimerase
deficiency (Fauci, A.S. et al. (1998) Harrison's Principles of Internal
Medicine, McGraw-Hill, New
York NY, pp. 2208-2209). Altered carbohydrate metabolism is associated with
cancer. Reduced GAG
15 and proteoglycan expression is associated with human lung carcinomas
(Nackaerts, K. et al. (1997) Int.
J. Cancer 74:335-345). The carbohydrate determinants sialyl Lewis A and sialyl
Lewis X are
frequently expressed on human cancer cells (Kannagi, R. (1997) Glycoconj. J.
14:577-584).
Alterations of the N-linked carbohydrate core structure of cell surface
glycoproteins are linked to colon
and pancreatic cancers (Schwarz, R.E. et al. (1996) Cancer Lett. 107:285-291).
Reduced expression
2 0 of the Sda blood group carbohydrate structure in cell surface glycolipids
and glycoproteins is observed
in gastrointestinal cancer (Dohi, T. et al. (1996) Int. J. Cancer 67:626-663).
(Carbon and
carbohydrate metabolism is reviewed in Stryer, L. (1995) Biochemistry W.H.
Freeman and Company,
New York NY; Lehninger, A.L. (1982) Principles of Biochemistry Worth
Publishers Inc., New York
NY; and Lodish, H. et al. (1995) Molecular Cell Biolo~y Scientific American
Books, New York NY.)
2 s Glycolvsis
Enzymes of the glycolytic pathway convert the sugar glucose to pyruvate while
simultaneously
producing ATP. The pathway also provides building blocks for the synthesis of
cellular components
such as long-chain fatty acids. After glycolysis, pyrvuate is converted to
acetyl-Coenzyme A, which, in
aerobic organisms, enters the citric acid cycle. Glycolytic enzymes include
hexokinase, phosphoglucose
3 o isomerase, phosphofructokinase, aldolase, triose phosphate isomerase,
glyceraldehyde 3-phosphate
dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase, and
pyruvate kinase. Of
these, phosphofructokinase, hexokinase, and pyruvate kinase are important in
regulating the rate of
glycolysis.
Gluconeo~enesis
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Gluconeogenesis is the synthesis of glucose from noncarbohydrate precursors
such as lactate
and amino acids. The pathway, which functions mainly in times of starvation
and intense exercise,
occurs mostly in the liver and kidney. Responsible enzymes include pyruvate
carboxylase,
phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose-6-
phosphatase.
Pentose Phosphate Pathway
Pentose phosphate pathway enzymes are responsible for generating the reducing
agent
NADPH, while at the same time oxidizing glucose-6-phosphate to ribose-5-
phosphate. Ribose-5-
phosphate and its derivatives become part of important biological molecules
such as ATP, Coenzyme
A, NAD+, FAD, RNA, and DNA. The pentose phosphate pathway has both oxidative
and non-
oxidative branches. The oxidative branch steps, which are catalyzed by the
enzymes glucose-6-
phosphate dehydrogenase, lactonase, and 6-phosphogluconate dehydrogenase,
convert glucose-6-
phosphate and NADP+ to ribulose-6-phosphate and NADPH. The non-oxidative
branch steps, which
are catalyzed by the enzymes phosphopentose isomerase, phosphopentose
epimerase, transketolase, and
transaldolase, allow the interconversion of three-, four-, five-, six-, and
seven-carbon sugars.
Glucouronate Metabolism
Glucuronate is a monosaccharide which, in the form of D-glucuronic acid, is
found in the
GAGs chondroitin and dermatan. D-glucuronic acid is also important in the
detoxification and
excretion of foreign organic compounds such as phenol. Enzymes involved in
glucuronate metabolism
include UDP-glucose dehydrogenase and glucuronate reductase.
2 o Disaccharide Metabolism
Disaccharides must be hydrolyzed to monosaccharides to be digested. Lactose, a
disaccharide
found in milk, is hydrolyzed to galactose and glucose by the enzyme lactase.
Maltose is derived from
plant starch and is hydrolyzed to glucose by the enzyme maltase. Sucrose is
derived from plants and is
hydrolyzed to glucose and fructose by the enzyme sucrase. Trehalose, a
disaccharide found mainly in
2 5 insects and mushrooms, is hydrolyzed to glucose by the enzyme trehalase
(OMIM *275360 Trehalase;
Ruf, J. et al. (1990) J. Biol. Chem. 265:15034-15039). Lactase, maltase,
sucrase, and trehalase are
bound to mucosal cells lining the small intestine, where they participate in
the digestion of dietary
disaccharides. The enzyme lactose synthetase, composed of the catalytic
subunit galactosyltransferase
and the modifier subunit a-lactalbumin, converts UDP-galactose and glucose to
lactose in the mammary
3 o glands.
Glycogen> Starch> and Chitin Metabolism
Glycogen is the storage form of carbohydrates in mammals. Mobilization of
glycogen
maintains glucose levels between meals and during muscular activity. Glycogen
is stored mainly in the
liver and in skeletal muscle in the form of cytoplasmic granules. These
granules contain enzymes that
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catalyze the synthesis and degradation of glycogen, as well as enzymes that
regulate these processes.
Enzymes that catalyze the degradation of glycogen include glycogen
phosphorylase, a transferase, a-
1,6-glucosidase, and phosphoglucomutase. Enzymes that catalyze the synthesis
of glycogen include
UDP-glucose pyrophosphorylase, glycogen synthetase, a branching enzyme, and
nucleoside
s diphosphokinase. The enzymes of glycogen synthesis and degradation are
tightly regulated by the
hormones insulin, glucagon, and epinephrine. Starch, a plant-derived
polysaccharide, is hydrolyzed to
maltose, maltotriose, and a-dextrin by a-amylase, an enzyme secreted by the
salivary glands and
pancreas. Chitin is a polysaccharide found in insects and crustacea. A
chitotriosidase is secreted by
macrophages and may play a role in the degradation of chitin-containing
pathogens (Boot, R.G. et al.
to (1995) J. Biol. Chem. 270:26252-26256).
Peptido~lvcans and Glycosamino~lycans
Glycosaminoglycans (GAGs) are anionic linear unbranched polysaccharides
composed of
repetitive disaccharide units. These repetitive units contain a derivative of
an amino sugar, either
glucosamine or galactosamine. GAGs exist free or as part of proteoglycans,
large molecules composed
s ~ of a core protein attached to one or more GAGs. GAGs are found on the cell
surface, inside cells, and
in the extracellular matrix. Changes in GAG levels are associated with several
autoimmune diseases
including autoimmune thyroid disease, autoimmune diabetes mellitus, and
systemic lupus
erythematosus (Hansen, C. et al. (1996) Clin. Exp. Rheum. 14 (Suppl. 15):559-
S67). GAGs include
chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, dermatan
sulfate, and hyaluronan.
2 o The GAG hyaluronan (HA) is found in the extracellular matrix of many
cells, especially in soft
connective tissues, and is abundant in synovial fluid (Pitsillides, A.A. et
al. (1993) Int. J. Exp. Pathol.
74:27-34). HA seems to play important roles in cell regulation, development,
and differentiation
(Laurent, T.C. and J.R. Fraser (1992) FASEB J. 6:2397-2404). Hyaluronidase is
an enzyme that
degrades HA to oligosaccharides. Hyaluronidases may function in cell adhesion,
infection,
2 s angiogenesis, signal transduction, reproduction, cancer, and inflammation.
Proteoglycans, also known as peptidoglycans, are found in the extracellular
matrix of
connective tissues such as cartilage and are essential for distributing the
load in weight-bearing joints.
Cell-surface-attached proteoglycans anchor cells to the extracellular matrix.
Both extracellular and
cell-surface proteoglycans bind growth factors, facilitating their binding to
cell-surface receptors and
3 o subsequent triggering of signal transduction pathways.
Amino Acid and Nitrogen Metabolism
NH4+ is assimilated into amino acids by the actions of two enzymes, glutamate
dehydrogenase and glutamine synthetase. The carbon skeletons of amino acids
come from the
intermediates of glycolysis, the pentose phosphate pathway, or the citric acid
cycle. Of the twenty
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amino acids used in proteins, humans can synthesize only thirteen
(nonessential amino acids). The
remaining nine must come from the diet (essential amino acids). Enzymes
involved in nonessential
amino acid biosynthesis include glutamate kinase dehydrogenase, pyrroline
carboxylate reductase,
asparagine synthetase, phenylalanine oxygenase, methionine
adenosyltransferase,
s adenosylhomocysteinase, cystathionine [3-synthase, cystathionine 'y-lyase,
phosphoglycerate
dehydrogenase, phosphoserine transaminase, phosphoserine phosphatase, serine
hydroxylmethyltransferase, and glycine synthase.
Metabolism of amino acids takes place almost entirely in the liver, where the
amino group is
removed by aminotransferases (transaminases), for example, alanine
aminotransferase. The amino
1 o group is transferred to a-ketoglutarate to form glutamate. Glutamate
dehydrogenase converts
glutamate to NH4+ and a-ketoglutarate. NH4+ is converted to urea by the urea
cycle which is
catalyzed by the enzymes arginase, ornithine transcarbamoylase,
arginosuccinate synthetase, and
arginosuccinase. Carbamoyl phosphate synthetase is also involved in urea
formation. Enzymes
involved in the metabolism of the carbon skeleton of amino acids include
serine dehydratase,
1 s asparaginase, glutaminase, propionyl CoA carboxylase, methylmalonyl CoA
mutase, branched-chain
a-keto dehydrogenase complex, isovaleryl CoA dehydrogenase, (3-methylcrotonyl
CoA carboxylase,
phenylalanine hydroxylase, p-hydroxylphenylpyruvate hydroxylase, and
homogentisate oxidise.
Polyanunes, which include spermidine, putrescine, and spermine, bind tightly
to nucleic acids
and are abundant in rapidly proliferating cells. Enzymes involved in polyamine
synthesis include
2 0 ornithine decarboxylase.
Diseases involved in amino acid and nitrogen metabolism include
hyperammonemia,
carbamoyl phosphate synthetase deficiency, urea cycle enzyme deficiencies,
methylmalonic aciduria,
maple syrup disease, alcaptonuria, and phenylketonuria.
Energy Metabolism
2 s Cells derive energy from metabolism of ingested compounds that may be
roughly categorized
as carbohydrates, fats, or proteins. Energy is also stored in polymers such as
triglycerides (fats) and
glycogen (carbohydrates). Metabolism proceeds along separate reaction pathways
connected by key
intermediates such as acetyl coenzyme A (acetyl-CoA). Metabolic pathways
feature anaerobic and
aerobic degradation, coupled with the energy-requiring reactions such as
phosphorylation of
3 o adenosine diphosphate (ADP) to the triphosphate (ATP) or analogous
phosphorylations of guanosine
(GDPIGTP), uridine (UDPIUTP), or cytidine (CDP/CTP). Subsequent
dephosphorylation of the
triphosphate drives reactions needed for cell maintenance, growth, and
proliferation.
Digestive enzymes convert carbohydrates and sugars to glucose; fructose and
galactose are
converted in the liver to glucose. Enzymes involved in these conversions
include galactose-1-
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phosphate uridyl transferase and UDP-galactose-4 epimerase. In the cytoplasm,
glycolysis converts
glucose to pyruvate in a series of reactions coupled to ATP synthesis.
Pyruvate is transported into the mitochondria and converted to acetyl-CoA for
oxidation via
the citric acid cycle, involving pyruvate dehydrogenase components,
dihydrolipoyl transacetylase, and
s dihydrolipoyl dehydrogenase. Enzymes involved in the citric acid cycle
include: citrate synthetase,
aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase
complex including
transsuccinylases, succinyl CoA synthetase, succinate dehydrogenase,
fumarases, and malate
dehydrogenase. Acetyl CoA is oxidized to COZ with concomitant formation of
NADH, FADH2, and
GTP. In oxidative phosphorylation, the transport of electrons from NADH and
FADHZ to oxygen by
~. o dehydrogenases is coupled to the synthesis of ATP from ADP and P; by the
FoFl ATPase complex in
the mitochondrial inner membrane. Enzyme complexes responsible for electron
transport and ATP
synthesis include the FoFI ATPase complex, ubiquinone(Co~-cytochrome c
reductase, ubiquinone
reductase, cytochrome b, cytochrome c1, FeS protein, and cytochrome c oxidase.
Triglycerides are hydrolyzed to fatty acids and glycerol by lipases. Glycerol
is then
15 phosphorylated to glycerol-3-phosphate by glycerol kinase and glycerol
phosphate dehydrogenase,
and degraded by the glycolysis. Fatty acids are transported into the
mitochondria as fatty acyl-
carnitine esters and undergo oxidative degradation.
In addition to metabolic disorders such as diabetes and obesity, disorders of
energy
metabolism are associated with cancers (Dorward, A. et al. (1997) J. Bioenerg.
Biomembr. 29:385-
20 392), autism (Lombard, J. (1998) Med. Hypotheses 50:497-500),
neurodegenerative disorders (Alexi,
T. et al. (1998) Neuroreport 9:857-64), and neuromuscular disorders (DiMauro,
S. et al. (1998)
Biochim. Biophys. Acta 1366:199-210). The myocardium is heavily dependent on
oxidative
metabolism, so metabolic dysfunction often leads to heart disease (DiMauro, S.
and M. Hirano (1998)
Curr. Opin. Cardiol. 13:190-197).
2 s For a review of energy metabolism enzymes and intermediates, see Stryer,
L. et a1. (1995)
Biochemistry, W.H. Freeman and Co., San Francisco CA, pp. 443-652. For a
review of energy
metabolism regulation, see Lodish, H. et al. (1995) Molecular Cell Bioloay,
Scientific American
Books, New York NY, pp. 744-770.
Cofactor Metabolism
3 o Cofactors, including coenzymes and prosthetic groups, are small molecular
weight inorganic
or organic compounds that are required for the action of an enzyme. Many
cofactors contain vitamins
as a component. Cofactors include thiamine pyrophosphate, flavin adenine
dinucleotide, flavin
mononucleotide, nicotinamide adenine dinucleotide, pyridoxal phosphate,
coenzyme A,
tetrahydrofolate, lipoamide, and heme. The vitamins biotin and cobalamin are
associated with
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enzymes as well. Heme, a prosthetic group found in myoglobin and hemoglobin,
consists of
protoporphyrin group bound to iron. Porphyrin groups contain four substituted
pyrroles covalently
joined in a ring, often with a bound metal atom. Enzymes involved in porphyrin
synthesis include 8-
aminolevulinate synthase, 8-aminolevulinate dehydrase, porphobilinogen
deaminase, and cosynthase.
s Deficiencies in heme formation cause porphyrias. Heme is broken down as a
part of erythrocyte
turnover. Enzymes involved in heme degradation include heme oxygenase and
biliverdin reductase.
Iron is a required cofactor for many enzymes. Besides the heme-containing
enzymes, iron is
found in iron-sulfur clusters in proteins including aconitase, succinate
dehydrogenase, and NADH-Q
reductase. Iron is transported in the blood by the protein transferrin.
Binding of transferrin to the
s o transferrin receptor on cell surfaces allows uptake by receptor mediated
endocytosis. Cytosolic iron is
bound to ferritin protein.
A molybdenum-containing cofactor (molybdopterin) is found in enzymes including
sulfite
oxidase, xanthine dehydrogenase, and aldehyde oxidase. Molybdopterin
biosynthesis is performed by
two molybdenum cofactor synthesizing enzymes. Deficiencies in these enzymes
cause mental
1 s retardation and lens dislocation. Other diseases caused by defects in
cofactor metabolism include
pernicious anemia and methylmalonic aciduria.
Secretion and Trafficking
Eukaxyotic cells are bound by a lipid bilayer membrane and subdivided into
functionally
distinct, membrane bound compartments. The membranes maintain the essential
differences between
2 o the cytosol, the extracellular environment, and the lumenal space of each
intracellular organelle. As
lipid membranes are highly impermeable to most polar molecules, transport of
essential nutrients,
metabolic waste products, cell signaling molecules, macromolecules and
proteins across lipid
membranes and between organelles must be mediated by a variety of transport-
associated molecules.
Protein Trafficking
2 s In eukaryotes, some proteins are synthesized on ER-bound ribosomes, co-
translationally
imported into the ER, delivered from the ER to the Golgi complex for post-
translational processing and
sorting, and transported from the Golgi to specific intracellular and
extracellular destinations. All cells
possess a constitutive transport process which maintains homeostasis between
the cell and its
environment. In many differentiated cell types, the basic machinery is
modified to carry out specific
3 o transport functions. For example, in endocrine glands, hormones and other
secreted proteins are
packaged into secretory granules for regulated exocytosis to the cell
exterior. In macrophage, foreign
extracellular material is engulfed (phagocytosis) and delivered to lysosomes
for degradation. In fat and
muscle cells, glucose transporters are stored in vesicles which fuse with the
plasma membrane only in
response to insulin stimulation.
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The Secretory Pathway
Synthesis of most integral membrane proteins, secreted proteins, and proteins
destined for the
lumen of a particular organelle occurs on ER-bound ribosomes. These proteins
are co-translationally
imported into the ER. The proteins leave the ER via membrane-bound vesicles
which bud off the ER at
specific sites and fuse with each other (homotypic fusion) to form the ER-
Golgi Intermediate
Compartment (ERGIC). The ERGIC matures progressively through the cis,
f~zedi.al, and trains cisternal
stacks of the Golgi, modifying the enzyme composition by retrograde transport
of specific Golgi
enzymes. In this way, proteins moving through the Golgi undergo post-
translational modification, such
as glycosylation. The final Golgi compartment is the Trans-Golgi Network
(TGN), where both
s o membrane and lumenal proteins are sorted for their final destination.
Transport vesicles destined for
intracellular compartments, such as the lysosome, bud aff the TGN. What
remains is a secretory
vesicle which contains proteins destined for the plasma membrane, such as
receptors, adhesion
molecules, and ion channels, and secretory proteins, such as hormones,
neurotransmitters, and digestive
enzymes. Secretory vesicles eventually fuse with the plasma membrane (Glick,
B.S. and V. Malhotra
(1998) Cell 95:883-889).
The secretory process can be constitutive or regulated. Most cells have a
constitutive pathway
for secretion, whereby vesicles derived from maturation of the TGN require no
specific signal to fuse
with the plasma membrane. In many cells, such as endocrine cells, digestive
cells, and neurons, vesicle
pools derived from the TGN collect in the cytoplasm and do not fuse with the
plasma membrane until
2 o they are directed to by a specific signal.
Endocytosis
Endocytosis, wherein cells internalize material from the extracellular
environment, is essential
for transmission of neuronal, metabolic, and proliferative signals; uptake of
many essential nutrients;
and defense against invading organisms. Most cells exhibit two forms of
endocytosis. The first,
2 5 phagocytosis, is an actin-driven process exemplified in macrophage and
neutrophils. Material to be
endocytosed contacts numerous cell surface receptors which stimulate the
plasma membrane to extend
and surround the particle, enclosing it in a membrane-bound phagosome. In the
mammalian immune
system, IgG-coated particles bind Fc receptors on the surface of phagocytic
leukocytes. Activation of
the Fc receptors initiates a signal cascade involving src-family cytosolic
kinases and the monomeric
3 o GTP-binding (G) protein Rho. The resulting actin reorganization leads to
phagocytosis of the particle.
This process is an important component of the humoral immune response,
allowing the processing and
presentation of bacterial-derived peptides to antigen-specific T-lymphocytes.
The second form of endocytosis, pinocytosis, is a more generalized uptake of
material from the
external milieu. Like phagocytosis, pinocytosis is activated by ligand binding
to cell surface receptors.
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Activation of individual receptors stimulates an internal response that
includes coalescence of the
receptor-ligand complexes and formation of clathrin-coated pits. Invagination
of the plasma membrane
at clathrin-coated pits produces an endocytic vesicle within the cell
cytoplasm. These vesicles undergo
homotypic fusion to form an early endosomal (EE) compartment. The
tubulovesicular EE serves as a
s sorting site for incoming material. ATP-driven proton pumps in the EE
membrane lowers the pH of the
EE lumen (pH 6.3-6.8). The acidic environment causes many ligands to
dissociate from their receptors.
The receptors, along with membrane and other integral membrane proteins, are
recycled back to the
plasma membrane by budding off the tubular extensions of the EE in recycling
vesicles (RV). This
selective removal of recycled components produces a carrier vesicle containing
ligand and other
1 o material from the external environment. The carrier vesicle fuses with TGN-
derived vesicles which
contain hydrolytic enzymes. The acidic environment of the resulting late
endosome (LE) activates the
hydrolytic enzymes which degrade the ligands and other material. As digestion
takes place, the LE
fuses with the lysosome where digestion is completed (Mellman, I. (1996) Annu.
Rev. Cell Dev. Biol.
12:575-625).
15 Recycling vesicles may return directly to the plasma membrane. Receptors
internalized and
returned directly to the plasma membrane have a turnover rate of 2-3 minutes.
Some RVs undergo
microtubule-directed relocation to a perinuclear site, from which they then
return to the plasma
membrane. Receptors following this route have a turnover rate of 5-10 minutes.
Still other RVs are
retained within the cell until an appropriate signal is received (Mellman,
supra; and James; D.E. et al.
a o (1994) Trends Cell Biol. 4:120-126).
Vesicle Formation
Several steps in the transit of material along the secretory and endocytic
pathways require the
formation of transport vesicles. Specifically, vesicles form at the
transitional endoplasmic reticulum
(tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN),
the plasma membrane
2 s (PM), and tubular extensions of the endosomes. The process begins with the
budding of a vesicle out of
the donor membrane. The membrane-bound vesicle contains proteins to be
transported and is
surrounded by a protective coat made up of protein subunits recruited from the
cytosol. The initial
budding and coating processes are controlled by a cytosolic ras-like GTP-
binding protein, ADP-
ribosylating factor (Arf), and adapter proteins (AP). Different isoforms of
both Arf and AP are
3 o involved at different sites of budding. Another small G-protein, dynamin,
forms a ring complex around
the neck of the forming vesicle and may provide the mechanochemical force to
accomplish the final step
of the budding process. The coated vesicle complex is then transported through
the cytosol. During the
transport process, Arf bound GTP is hydrolyzed to GDP and the coat dissociates
from the transport
vesicle (West, M.A. et al. (1997) J. Cell Biol. 138:1239-1254). Two different
classes of coat protein
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have also been identified. Clathrin coats form on the TGN and PM surfaces,
whereas coatomer or COP
coats form on the ER and Golgi. COP coats can further be distinguished as
COPI, involved in
retrograde traffic through the Golgi and from the Golgi to the ER, and COPII,
involved in anterograde
traffic from the ER to the Golgi (Mellman, supra). The COP coat consists of
two major components, a
s G-protein (Arf or Sar) and coat protomer (coatomer). Coatomer is an
equimolar complex of seven
proteins, termed alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP.
(Harter, C. and F.T.
Wieland (1998) Proc. Natl. Acad. Sci. USA 95:11649-11654.)
Membrane Fusion
Transport vesicles undergo homotypic or heterotypic fusion in the secretory
and endocytotic
1 o pathways. Molecules required for appropriate targeting and fusion of
vesicles with their target
membrane include proteins incorporated in the vesicle membrane, the target
membrane, and proteins
recruited from the cytosol. During budding of the vesicle from the donor
compartment, an integral
membrane protein, VAMP (vesicle-associated membrane protein) is incorporated
into the vesicle. Soon
after the vesicle uncoats, a cytosolic prenylated GTP-binding protein, Rab (a
member of the Ras
1 s superfamily), is inserted into the vesicle membrane. GTP-bound Rab
proteins are directed into nascent
transport vesicles where they interact with VAMP. Following vesicle transport,
GTPase activating
proteins (GAPS) in the target membrane convert Rab proteins to the GDP-bound
form. A cytosolic
protein, guanine-nucleotide dissociation inhibitor (GDI) helps return GDP-
bound Rab proteins to their
membrane of origin. Several Rab isoforms have been identified and appear to
associate with specific
2 o compartments within the cell. Rab proteins appear to play a role in
mediating the function of a viral
gene, Rev, which is essential for replication of HIV-1, the virus responsible
for AIDS (Flavell, R.A. et
al. (1996) Proc. Natl. Acad. Sci. USA 93:4421-4424).
Docking of the transport vesicle with the target membrane involves the
formation of a complex
between the vesicle SNAP receptor (v-SNARE), target membrane (t-) SNAREs, and
certain other
2 s membrane and cytosolic proteins. Many of these other proteins have been
identified although their
exact functions in the docking complex remain uncertain (Tellam, J.T. et al.
(1995) J. Biol. Chem.
270:5857-5863; and Hata, Y. and T.C. Sudhof (1995) J. Biol. Chem. 270:13022-
13028).
N-ethylmaleimide sensitive factor (NSF) and soluble NSF-attachment protein (a-
SNAP and (3-SNAP)
axe two such proteins that are conserved from yeast to man and function in
most intracellular membrane
3 o fusion reactions. Sec1 represents a family of yeast proteins that function
at many different stages in the
secretory pathway including membrane fusion. Recently, mammalian homologs of
Secl, called
Munc-18 proteins, have been identified (Katagiri, H. et al. (1995) J. Biol.
Chem. 270:4963-4966; Hata
et al. su~a).
The SNARE complex involves three SNARE molecules, one in the vesicular
membrane and
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two in the target membrane. Synaptotagmin is an integral membrane protein in
the synaptic vesicle
which. associates with the t-SNARE syntaxin in the docking complex.
Synaptotagmin binds calcium in
a complex with negatively charged phospholipids, which allows the cytosolic
SNAP protein to displace
synaptotagmin from syntaxin and fusion to occur. Thus, synaptotagmin is a
negative regulator of
s fusion in the neuron (Littleton, J.T. et al. (1993) Cell 74:1125-1134). The
most abundant membrane
protein of synaptic vesicles appears to be the glycoprotein synaptophysin, a
38 kDa protein with four
transmembrane domains.
Specificity between a vesicle and its target is derived from the v-SNARE, t-
SNAREs, and
associated proteins involved. Different isoforms of SNARES and Rabs show
distinct cellular and
1o subcellular distributions. VAMP-1/synaptobrevin, membrane-anchored
synaptosome-associated
protein of 25 kDa (SNAP-25), syntaxin-1, Rab3A, RablS, and Rab23 are
predominantly expressed in
the brain and nervous system. Different syntaxin, VAMP, and Rab proteins are
associated with
distinct subcellular compartments and their vesicular carriers.
Nuclear Transport
i 5 Transport of proteins and RNA between the nucleus and the cytoplasm occurs
through nuclear
pore complexes (NPCs). NPC-mediated transport occurs in both directions
through the nuclear
envelope. All nuclear proteins are imported from the cytoplasm, their site of
synthesis: tRNA and
mRNA are exported from the nucleus, their site of synthesis, to the cytoplasm,
their site of function.
Processing of small nuclear RNAs involves export into the cytoplasm, assembly
with proteins and
2 o modifications such as hypermethylation to produce small nuclear
ribonuclear proteins (snRNPs), and
subsequent import of the snRNPs back into the nucleus. The assembly of
ribosomes requires the initial
import of ribosomal proteins from the cytoplasm, their incorporation with RNA
into ribosomal
subunits, and export back to the cytoplasm. (Gorlich, D. and LW. Mattaj (1996)
Science 271:1513-
1518.)
2 s The transport of proteins and mRNAs across the NPC is selective, dependent
on nuclear
localization signals, and generally requires association with nuclear
transport factors. Nucleax
localization signals (NLS) consist of short stretches of amino acids enriched
in basic residues. NLS are
found on proteins that are targeted to the nucleus, such as the glucocorticoid
receptor. The NLS is
recognized by the NLS receptor, importin, which then interacts with the
monomeric GTP-binding
3 o protein Ran. This NLS protein/receptor/Ran complex navigates the nuclear
pore with the help of the
homodimeric protein nuclear transport factor 2 (NTF2). NTF2 binds the GDP-
bound form of Ran and
to multiple proteins of the nuclear pore complex containing FXFG repeat
motifs, such as p62.
(Paschal, B. et al. (1997) J. Biol. Chem. 272:21534-21539; and Wong, D.H. et
al. (1997) Mol. Cell
Biol. 17:3755-3767). Some proteins are dissociated before nuclear mRNAs are
transported across the
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NPC while others are dissociated shortly after nuclear mRNA transport across
the NPC and are
reimported into the nucleus.
Disease Correlation
The etiology of numerous human diseases and disorders can be attributed to
defects in the
s transport or secretion of proteins. For example, abnormal hormonal secretion
is linked to disorders
such as diabetes insipidus (vasopressin), hyper- and hypoglycemia (insulin,
glucagon), Grave's disease
and goiter (thyroid hormone), and Cushing's and Addison's diseases
(adrenocorticotropic hormone,
ACTH). Moreover, cancer cells secrete excessive amounts of hormones or other
biologically active
peptides. Disorders related to excessive secretion of biologically active
peptides by tumor cells include
1 o fasting hypoglycemia due to increased insulin secretion from insulinoma-
islet cell tumors; hypertension
due to increased epinephrine and norepinephrine secreted from
pheochromocytomas of the adrenal
medulla and sympathetic paraganglia; and carcinoid syndrome, which is
characterized by abdominal
cramps, diarrhea, and valvular heart disease caused by excessive amounts of
vasoactive substances
such as serotonin, bradykinin, histamine, prostaglandins, and polypeptide
hormones, secreted from
1 s intestinal tumors. Biologically active peptides that are ectopically
synthesized in and secreted from
tumor cells include ACTH and vasopressin (lung and pancreatic cancers);
parathyroid hormone (lung
and bladder cancers); calcitonin (lung and breast cancers); and thyroid-
stimulating hormone (medullary
thyroid carcinoma). Such peptides may be useful as diagnostic markers for
tumorigenesis (Schwartz,
M.Z. (1997) Semin. Pediatr. Surg. 3:141-146; and Said, S.I. and G.R. Faloona
(1975) N. Engl. J. Med.
2 0 293:155-160).
Defective nuclear transport may play a role in cancer. The BRCA1 protein
contains three
potential NLSs which interact with importin alpha, and is transported into the
nucleus by the
importin/NPC pathway. In breast cancer cells the BRCAl protein is aberrantly
localized in the
cytoplasm. The mislocation of the BRCAl protein in breast cancer cells may be
due to a defect in the
2s NPC nuclear import pathway (Chen, C.F. et al. (1996) J. Biol. Chem.
271:32863-32868).
It has been suggested that in some breast cancers, the tumor-suppressing
activity of p53 is
inactivated by the sequestration of the protein in the cytoplasm, away from
its site of action in the cell
nucleus. Cytoplasmic wild-type p53 was also found in human cervical carcinoma
cell lines. (Moll,
U.M. et al. (1992) Proc. Natl. Acad. Sci. USA 89:7262-7266; and Liang, X.H. et
al. (1993) Oncogene
30 8:2645-2652.)
Environmental Res op nses
Organisms respond to the environment by a number of pathways. Heat shock
proteins,
including hsp 70, hsp60, hsp90, and hsp 40, assist organisms in coping with
heat damage to cellular
proteins.
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Aquaporins (AQP) are channels that transport water and, in some cases,
nonionic small solutes
such as urea and glycerol. Water movement is important for a number of
physiological processes
including renal fluid filtration, aqueous humor generation in the eye,
cerebrospinal fluid production in
the brain, and appropriate hydration of the lung. Aquaporins are members of
the major intrinsic protein
s (MIP) family of membrane fransporters (King, L.S. and P. Agre (1996) Annu.
Rev. Physiol. 58:619-
648; Ishibashi, K. et al. (1997) J. Biol. Chem. 272:20782-20786). The study of
aquaporins may have
relevance to understanding edema formation and fluid balance in both normal
physiology and disease
states (King, supra). Mutations in AQP2 cause autosomal recessive nephrogenic
diabetes insipidus
(OMIM *107777 Aquaporin 2; AQP2). Reduced AQP4 expression in skeletal muscle
may be
to associated with Duchenne muscular dystrophy (Frigeri, A. et al. (1998) J.
Clin. Invest. 102:695-703).
Mutations in AQPO cause autosomal dominant cataracts in the mouse (OMIM
*154050 Major Intrinsic
Protein of Lens Fiber; MIP).
The metallothioneins (MTs) are a group of small (61 amino acids), cysteine-
rich proteins that
bind heavy metals such as cadmium, zinc, mercury, lead, and copper and are
thought to play a role in
i s metal detoxification or the metabolism and homeostasis of metals. Arsenite-
resistance proteins have
been identified in hamsters that are resistant to toxic levels of arsenite
(Rossman, T.G. et al. (1997)
Mutat. Res. 386:307-314).
Humans respond to light and odors by specific protein pathways. Proteins
involv ed in light
perception include rhodopsin, transducin, and cGMP phosphodiesterase. Proteins
involved in odor
2 o perception include multiple olfactory receptors. Other proteins are
important in human Circadian
rhythms and responses to wounds.
Immunity and Host Defense
All vertebrates have developed sophisticated and complex immune systems that
provide
protection from viral, bacterial, fungal and parasitic infections. Included in
these systems are the
2 s processes of humoral immunity, the complement cascade and the inflammatory
response (Paul, W.E.
(1993) Fundamental Immunolo~y, Raven Press, Ltd., New York NY, pp.l-20).
The cellular components of the humoral immune system include six different
types of
leukocytes: monocytes, lymphocytes, polymorphonuclear granulocytes (consisting
of neutrophils,
eosinophils, and basophils) and plasma cells. Additionally, fragments of
megakaryocytes, a seventh
3 o type of white blood cell in the bone marrow, occur in large numbers in the
blood as platelets.
Leukocytes are formed from two stem cell lineages in bone marrow. The myeloid
stem cell
line produces granulocytes and monocytes and, the lymphoid stem cell produces
lymphocytes.
Lymphoid cells travel to the thymus, spleen and lymph nodes, where they mature
and differentiate
into lymphocytes. Leukocytes are responsible for defending the body against
invading pathogens.
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Neutrophils and monocytes attack invading bacteria, viruses, and other
pathogens and destroy them
by phagocytosis. Monocytes enter tissues and differentiate into macrophages
which are extremely
phagocytic. Lymphocytes and plasma cells are a part of the immune system which
recognizes
specific foreign molecules and organisms and inactivates them, as well as
signals other cells to attack
s the invaders.
Granulocytes and monocytes are formed and stored in the bone marrow until
needed.
Megakaryocytes are produced in bone marrow, where they fragment into platelets
and are released
into the bloodstream. The main function of platelets is to activate the blood
clotting mechanism.
Lymphocytes and plasma cells are produced in various lymphogenous organs,
including the lymph
1 o nodes, spleen, thymus, and tonsils.
Both neutrophils and macrophages exhibit chemotaxis towards sites of
inflammation. Tissue
inflammation in response to pathogen invasion results in production of chemo-
attractants for
leukocytes, such as endotoxins or other bacterial products, prostaglandins,
and products of leukocytes
or platelets.
15 Basophils participate in the release of the chemicals involved in the
inflammatory process.
The main function of basophils is secretion of these chemicals to such a
degree that they have been
referred to as "unicellular endocrine glands." A distinct aspect of basophilic
secretion is that the
contents of granules go directly into the extracellular environment, not into
vacuoles as occurs with
neutrophils, eosinophils and monocytes. Basophils have receptors for the Fc
fragment of
2 o immunoglobulin E (IgE) that are not present on other leukocytes.
Crosslinking of membrane IgE
with anti-IgE or other ligands triggers degranulation.
Eosinophils are bi- or mufti-nucleated white blood cells which contain
eosinophilic granules.
Their plasma membrane is characterized by Ig receptors, particularly IgG and
IgE. Generally,
eosinophils are stored in the bone marrow until recruited for use at a site of
inflammation ox invasion.
2 s They have specific functions in parasitic infections and allergic
reactions, and are thought to detoxify
some of the substances released by mast cells and basophils which cause
inflammation. Additionally,
they phagocytize antigen-antibody complexes and further help prevent spread of
the inflammation.
Macrophages are monocytes that have left the blood stream to settle in tissue.
Once
monocytes have migrated into tissues, they do not re-enter the bloodstream.
The mononuclear
3 o phagocyte system is comprised of precursor cells in the bone marrow,
monocytes in circulation, and
macrophages in tissues. The system is capable of very fast and extensive
phagocytosis. A
macrophage may phagocytize over 100 bacteria, digest them and extrude
residues, and then survive
for many more months. Macrophages are also capable of ingesting large
particles, including red
blood cells and malarial parasites. They increase several-fold in size and
transform into macrophages
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that are characteristic of the tissue they have entered, surviving in tissues
for several months.
Mononuclear phagocytes are essential in defending the body against invasion by
foreign
pathogens, particularly intracellular microorganisms such as M. tuberculosis,
listeria, leishmania and
toxoplasma. Macrophages can also control the growth of tumorous cells, via
both phagocytosis and
s secretion of hydrolytic enzymes. Another important function of macrophages
is that of processing
antigen and presenting them in a biochemically modified form to lymphocytes.
The immune system responds to invading microorganisms in two major ways:
antibody
production and cell mediated responses. Antibodies are immunoglobulin proteins
produced by
B-lymphocytes which bind to specific antigens and cause inactivation or
promote destruction of the
1 o antigen by other cells. Cell-mediated immune responses involve T-
lymphocytes (T cells) that react
with foreign antigen on the surface of infected host cells. Depending on the
type of T cell, the
infected cell is either killed or signals are secreted which activate
macrophages and other cells to
destroy the infected cell (Paul, supra).
T-lymphocytes originate in the bone marrow or liver in fetuses. Precursor
cells migrate via
1 s the blood to the thymus, where they are processed to mature into T-
lymphocytes. This processing is
crucial because of positive and negative selection of T cells that will react
with foreign antigen and
not with self molecules. After processing, T cells continuously circulate in
the blood and secondary
lymphoid tissues, such as lymph nodes, spleen, certain epithelium-associated
tissues in the
gastrointestinal tract, respiratory tract and skin. When T-lymphocytes are
presented with the
2 o complementary antigen, they are stimulated to proliferate and release
large numbers of activated T
cells into the lymph system and the blood system. These activated T cells can
survive and circulate
for several days. At the same time, T memory cells are created, which remain
in the lymphoid tissue
for months or years. Upon subsequent exposure to that specific antigen, these
memory cells will
respond more rapidly and with a stronger response than induced by the original
antigen. This creates
2 s an "immunological memory" that can provide immunity for years.
There are two major types of T cells: cytotoxic T cells destroy infected host
cells, and helper
T cells activate other white blood cells via chemical signals. One class of
helper cell, TH1, activates
macrophages to destroy ingested microorganisms, while another, TH2, stimulates
the production of
antibodies by B cells.
3 o Cytotoxic T cells directly attack the infected target cell. In virus-
infected cells, peptides
derived from viral proteins are generated by the proteasome. These peptides
are transported into the
ER by the transporter associated with antigen processing (TAP) (Pamer, E. and
P. Cresswell (1998)
Annu. Rev. Immunol. 16:323-358). Once inside the ER, the peptides bind MHC I
chains, and the
peptide/MHC I complex is transported to the cell surface. Receptors on the
surface of T cells bind to
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antigen presented on cell surface MHC molecules. Once activated by binding to
antigen, T cells
secrete 'y-interferon, a signal molecule that induces the expression of genes
necessary for presenting
viral (or other) antigens to cytotoxic T cells. Cytotoxic T cells kill the
infected cell by stimulating
programmed cell death.
s Helper T cells constitute up to 75 % of the total T cell population. They
regulate the immune
functions by producing a variety of lymphokines that act on other cells in the
immune system and on
bone marrow. Among these lymphokines are: interleukins-2,3,4,5,6; granulocyte-
monocyte colony
stimulating factor, and 'y-interferon.
Helper T cells are required for most B cells to respond to antigen. When an
activated helper
1 o cell contacts a B cell, its centrosome and Golgi apparatus become oriented
toward the B cell, aiding
the directing of signal molecules, such as transmembrane-bound protein called
CD40 ligand, onto the
B cell surface to interact with the CD40 transmembrane protein. Secreted
signals also help B cells to
proliferate and mature and, in some cases, to switch the class of antibody
being produced.
B-lymphocytes (B cells) produce antibodies which react with specific antigenic
proteins
1 s presented by pathogens. Once activated, B cells become filled with
extensive rough endoplasmic
reticulum and are known as plasma cells. As with T cells, interaction of B
cells with antigen
stimulates proliferation of only those B cells which produce.antibody specific
to that antigen. There
are five classes of antibodies, known as immunoglobulins, which together
comprise about 20% of
total plasma protein. Each class mediates a characteristic biological response
after antigen binding.
2 0 Upon activation by specific antigen B cells switch from making membrane-
bound antibody to
secretion of that antibody.
Antibodies, or immunoglobulins (Ig), are the founding members of the Ig
superfamily and the
central components of the humoral immune response. Antibodies are either
expressed on the surface
of B cells or secreted by B cells into the circulation. Antibodies bind and
neutralize blood-borne
2 s foreign antigens. The prototypical antibody is a tetramer consisting of
two identical heavy
polypeptide chains (H-chains) and two identical light polypeptide chains (L-
chains) interlinked by
disulfide bonds. This arrangement confers the characteristic Y-shape to
antibody molecules.
Antibodies are classified based on their H-chain composition. The five
antibody classes, IgA, IgD,
IgE, IgG and IgM, are defined by the a, 8, e, 'y, and ~ H-chain types. There
are two types of L-
3 o chains, K and ~., either of which may associate as a pair with any H-chain
pair. IgG, the most
common class of antibody found in the circulation, is tetrameric, while the
other classes of antibodies
are generally variants or multimers of this basic structure.
H-chains and L-chains each contain an N-terminal variable region and a C-
terminal constant
region. Both H-chains and L-chains contain repeated Ig domains. For example, a
typical H-chain
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contains four Ig domains, three of which occur within the constant region and
one of which occurs
within the variable region and contributes to the formation of the antigen
recognition site. Likewise,
a typical L-chain contains two Ig domains, one of which occurs within the
constant region and one of
which occurs within the variable region. In addition, H chains such as ~ have
been shown to
s associate with other polypeptides during differentiation of the B cell.
Antibodies can be described in terms of their two main functional domains.
Antigen
recognition is mediated by the Fab (antigen binding fragment) region of the
antibody, while effector
functions are mediated by the Fc (crystallizable fragment) region. Binding of
antibody to an antigen,
such as a bacterium, triggers the destruction of the antigen by phagocytic
white blood cells such as
1 o macrophages and neutrophils. These cells express surface receptors that
specifically bind to the
antibody Fc region and allow the phagocytic cells to engulf, ingest, and
degrade the antibody-bound
antigen. The Fc receptors expressed by phagocytic cells are single-pass
transmembrane glycoproteins
of about 300 to 400 amino acids (Sears, D.W, et al. (1990) J. Immunol. 144:371-
378). The
extracellular portion of the Fc receptor typically contains two or three Ig
domains.
15 Diseases which cause over- or under-abundance of any one type of leukocyte
usually result in
the entire immune defense system becoming involved. A well-known autoimmune
disease is AIDS
(Acquired Immunodeficiency Syndrome) where the number of helper T cells is
depleted, leaving the
patient susceptible to infection by microorganisms and parasites. Another
widespread medical
condition attributable to the immune system is that of allergic reactions to
certain antigens. Allergic
2 o reactions include: hay fever, asthma, anaphylaxis, and urticaria (hives).
Leukemias are an excess
production of white blood cells, to the point where a major portion of the
body's metabolic resources
are directed solely at proliferation of white blood cells, leaving other
tissues to starve. Leukopenia or
agranulocytosis occurs when the bone marrow stops producing white blood cells.
This leaves the
body unprotected against foreign microorganisms, including those which
normally inhabit skin,
2 s mucous membranes, and gastrointestinal tract. If all white blood cell
production stops completely;
infection will occur within two days and death may follow only 1 to 4 days
later.
Impaired phagocytosis occurs in several diseases, including monocytic
leukemia, systemic
lupus, and granulomatous disease. In such a situation, macrophages can
phagocytize normally, but
the enveloped organism is not killed. A defect in the plasma membrane enzyme
which converts
3 0 oxygen to lethally reactive forms results in abscess formation in liver,
lungs, spleen, lymph nodes,
and beneath the skin. Eosinophilia is an excess of eosinophils commonly
observed in patients with
allergies (hay fever, asthma), allergic reactions to drugs, rheumatoid
arthritis, and cancers (Hodgkin's
disease, lung, and liver cancer) (Isselbacher, K.J. et al. (1994) Harrison's
Principles of Internal
Medicine, McGraw-Hill, Inc., New York NY).
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Host defense is further augmented by the complement system. The complement
system
serves as an effector system and is involved in infectious agent recognition.
It can function as an
independent immune network or in conjunction with other humoral immune
responses. The
complement system is comprised of numerous plasma and membrane proteins that
act in a cascade of
s reaction sequences whereby one component activates the next. The result is a
rapid and amplified
response to infection through either an inflammatory response or increased
phagocytosis.
The complement system has more than 30 protein components which can be divided
into
functional groupings including modified serine proteases, membrane-binding
proteins and regulators
of complement activation. Activation occurs through two different pathways the
classical and the
1 o alternative. Both pathways serve to destroy infectious agents through
distinct triggering mechanisms
that eventually merge with the involvement of the component C3.
The classical pathway requires antibody binding to infectious agent antigens.
The antibodies
serve to define the target and initiate the complement system cascade,
culminating in the destruction
of the infectious agent. In this pathway, since the antibody guides initiation
of the process, the
1 s complement can be seen as an effector arm of the humoral immune system.
The alternative pathway of the complement system does not require the presence
of pre-
existing antibodies for targeting infectious agent destruction. Rather, this
pathway, through low
levels of an activated component, remains constantly primed and provides
surveillance in the non-
immune host to enable targeting and destruction of infectious agents. In this
case foreign material
2 o triggers the cascade, thereby facilitating phagocytosis or lysis (Paul,
supra, pp.918-919).
Another important component of host defense is the process of inflammation.
Inflammatory
responses are divided into four categories on the basis of pathology and
include allergic
inflammation, cytotoxic antibody mediated inflammation, immune complex
mediated inflammation
and monocyte mediated inflammation. Inflammation manifests as a combination of
each of these
2 s forms with one predominating.
Allergic acute inflammation is observed in individuals wherein specific
antigens stimulate
IgE antibody production. Mast cells and basophils are subsequently activated
by the attachment of
antigen-IgE complexes, resulting in the release of cytoplasmic granule
contents such as histamine.
The products of activated mast cells can increase vascular permeability and
constrict the smooth
3 o muscle of breathing passages, resulting in anaphylaxis or asthma. Acute
inflammation is also
mediated by cytotoxic antibodies and can result in the destruction of tissue
through the binding of
complement-fixing antibodies to cells. The responsible antibodies are of the
IgG or IgM types.
Resultant clinical disorders include autoimmune hemolytic anemia and
thrombocytopenia as
associated with systemic lupus erythematosis.
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Immune complex mediated acute inflammation involves the IgG or IgM antibody
types
which combine with antigen to activate the complement cascade. When such
immune complexes
bind to neutrophils and macrophages they activate the respiratory burst to
form protein- and vessel-
damaging agents such as hydrogen peroxide, hydroxyl radical, hypochlorous
acid, and chloramines.
s Clinical manifestations include rheumatoid arthritis and systemic lupus
erythematosus.
In chronic inflammation or delayed-type hypersensitivity, macrophages are
activated and
process antigen for presentation to T cells that subsequently produce
lymphokines and monokines.
This type of inflammatory response is likely important for defense against
intracellular parasites and
certain viruses. Clinical associations include, granulomatous disease,
tuberculosis, leprosy, and
to sarcoidosis (Paul, W.E., supra, pp.1017-1018).
Extracellular Information Transmission Molecules
Intercellular communication is essential for the growth and survival of
multicellular
organisms, and in particular, for the function of the endocrine, nervous, and
immune systems. In
1 s addition, intercellular communication is critical for developmental
processes such as tissue
construction and organogenesis, in which cell proliferation, cell
differentiation, and morphogenesis
must be spatially and temporally regulated in a precise and coordinated
manner. Cells communicate
with one another through the secretion and uptake of diverse types of
signaling molecules such as
hormones, growth factors, neuropeptides, and cytokines.
2 o Hormones
Hormones are signaling molecules that coordinately regulate basic
physiological processes
from embryogenesis throughout adulthood. These processes include metabolism,
respiration,
reproduction, excretion, fetal tissue differentiation and organogenesis,
growth and development,
homeostasis, and the stress response. Hormonal secretions and the nervous
system are tightly
2 s integrated and interdependent. Hormones are secreted by endocrine glands,
primarily the hypothalamus
and pituitary, the thyroid and parathyroid, the pancreas, the adrenal glands,
and the ovaries and testes.
The secretion of hormones into the circulation is tightly controlled. Hormones
are often
secreted in diurnal, pulsatile, and cyclic patterns. Hormone secretion is
regulated by perturbations in
blood biochemistry, by other upstream-acting hormones, by neural impulses, and
by negative feedback
3 0 loops. Blood hormone concentrations are constantly monitored and adjusted
to maintain optimal,
steady-state levels. Once secreted, hormones act only on those target cells
that express specific
receptors.
Most disorders of the endocrine system are caused by either hyposecretion or
hypersecretion of
hormones. Hyposecretion often occurs when a hormone's gland of origin is
damaged or otherwise
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impaired. Hypersecretion often results from the proliferation of tumors
derived from hormone-secreting
cells. Inappropriate hormone levels may also be caused by defects in
regulatory feedback loops or in
the processing of hormone precursors. Endocrine malfunction may also occur
when the target cell fails
to respond to the hormone.
Hormones can be classified biochemically as polypeptides, steroids,
eicosanoids, or amines.
Polypeptides, which include diverse hormones such as insulin and growth
hormone, vary in size and
function and are often synthesized as inactive precursors that are processed
intracellularly into mature,
active forms. Amines, which include epinephrine and dopamine, are amino acid
derivatives that
function in neuroendocrine signaling. Steroids, which include the cholesterol-
derived hormones
s o estrogen and testosterone, function in sexual development and
reproduction. Eicosanoids, which .
include prostaglandins and prostacyclins, are fatty acid derivatives that
function in a variety of
processes. Most polypeptides and some amines are soluble in the circulation
where they are highly
susceptible to proteolytic degradation within seconds after their secretion.
Steroids and lipids are
insoluble and must be transported in the circulation by carrier proteins. The
following discussion will
s 5 focus primarily on polypeptide hormones.
Hormones secreted by the hypothalamus and pituitary gland play a critical role
in endocrine
function by coordinately regulating hormonal secretions from other endocrine
glands in response to
neural signals. Hypothalamic hormones include thyrotropin-releasing hormone,
gonadotropin-releasing
hormone, somatostatin, growth-hormone releasing factor, corticotropin-
releasing hormone, substance P,
2 o dopamine, and prolactin-releasing hormone. These hormones directly
regulate the secretion of
hormones from the anterior lobe of the pituitary. Hormones secreted by the
anterior pituitary include
adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone,
somatotropic hormones such
as growth hormone and prolactin, glycoprotein hormones such as thyroid-
stimulating hormone,
luteinizing hormone (LH), and follicle-stimulating hormone (FSH), (3-
lipotropin, and (3-endorphins.
2 s These hormones regulate hormonal secretions from the thyroid, pancreas,
and adrenal glands, and act
directly on the reproductive organs to stimulate ovulation and
spermatogenesis. The posterior pituitary
synthesizes and secretes antidiuretic hormone (ADH, vasopressin) and oxytocin.
Disorders of the hypothalamus and pituitary often result from lesions such as
primary brain
tumors, adenomas, infarction associated with pregnancy, hypophysectomy,
aneurysms, vascular
3 o malformations, thrombosis, infections, immunological disorders, and
complications due to head trauma.
Such disorders have profound effects on the function of other endocrine
glands. Disorders associated
with hypopituitarism include hypogonadism, Sheehan syndrome, diabetes
insipidus, Kallman's disease,
Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty
sella syndrome, and
dwarfism. Disorders associated with hyperpituitarism include acromegaly,
giantism, and syndrome of
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inappropriate ADH secretion (SIADH), often caused by benign adenomas.
Hormones secreted by the thyroid and parathyroid primarily control metabolic
rates and the
regulation of serum calcium levels, respectively. Thyroid hormones include
calcitonin, somatostatin,
and thyroid hormone. The parathyroid secretes parathyroid hormone. Disorders
associated with
s hypothyroidism include goiter, myxedema, acute thyroiditis associated with
bacterial infection,
subacute thyroiditis associated with viral infection, autoimmune thyroiditis
(Hashimoto's disease), and
cretinism. Disorders associated with hyperthyroidism include thyrotoxicosis
and its various forms,
Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid
carcinoma, and Plummer's
disease. Disorders associated with hyperparathyroidism include Conn disease
(chronic hypercalemia)
s o leading to bone resorption and parathyroid hyperplasia.
Hormones secreted by the pancreas regulate blood glucose levels by modulating
the rates of
carbohydrate, fat, and protein metabolism. Pancreatic hormones include
insulin, glucagon, amylin, y-
aminobutyric acid, gastrin, somatostatin, and pancreatic polypeptide. The
principal disorder associated
with pancreatic dysfunction is diabetes mellitus caused by insufficient
insulin activity. Diabetes
~ s mellitus is generally classified as either Type I (insulin-dependent,
juvenile diabetes) or Type II (non-
insulin-dependent, adult diabetes). The treatment of both forms by insulin
replacement therapy is well
known. Diabetes mellitus often leads to acute complications such as
hypoglycemia (insulin shock),
coma, diabetic ketoacidosis, lactic acidosis, and chronic complications
leading to disorders of the eye,
kidney, skin, bone, joint, cardiovascular system, nervous system, and to
decreased resistance to
a o infection.
The anatomy, physiology, and diseases related to hormonal function are
reviewed in McCance,
K.L. and S.E. Huether (1994) Path~hysiolo~y: The Biological Basis for Disease
in Adults and
Children, Mosby-Year Book, Inc., St. Louis MO; Greenspan, F.S. and J.D. Baxter
(1994) Basic and
Clinical Endocrinolo~y, Appleton and Lange, East Norwalk CT.
2 s Growth Factors
Growth factors are secreted proteins that mediate intercellular communication.
Unlike
hormones, which travel great distances via the circulatory system, most growth
factors are primarily
local mediators that act on neighboring cells. Most growth factors contain a
hydrophobic N-terminal
signal peptide sequence which directs the growth factor into the secretory
pathway. Most growth
s o factors also undergo post-translational modifications within the secretory
pathway. These
modifications can include proteolysis, glycosylation, phosphorylation, and
intramolecular disulfide bond
formation. Once secreted, growth factors bind to specific receptors on the
surfaces of neighboring
target cells, and the bound receptors trigger intracellular signal
transduction pathways. These signal
transduction pathways elicit specific cellular responses in the target cells.
These responses can include
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the modulation of gene expression and the stimulation or inhibition of cell
division, cell differentiation,
and cell motility.
Growth factors fall into at least two broad and overlapping classes. The
broadest class
includes the large polypeptide growth factors, which are wide-ranging in their
effects. These factors
s include epidermal growth factor (EGF), fibroblast growth factor (FGF),
transforming growth factor-(3
(TGF-(3), insulin-like growth factor (IGF), nerve growth factor (NGF), and
platelet-derived growth
factor (PDGF), each defining a family of numerous related factors. The large
polypeptide growth
factors, with the exception of NGF, act as mitogens on diverse cell types to
stimulate wound healing,
bone synthesis and remodeling, extracellular matrix synthesis, and
proliferation of epithelial,
~. o epidermal, and connective tissues. Members of the TGF-(3, EGF, and FGF
families also function as
inductive signals in the differentiation of embryonic tissue. NGF functions
specifically as a
neurotrophic factor, promoting neuronal growth and differentiation.
Another class of growth factors includes the hematopoietic growth factors,
which are narrow in
their target specificity. These factors stimulate the proliferation and
differentiation of blood cells such
s s as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils,
basophils, neutrophils,
macrophages, and their stem cell precursors. These factors include the colony-
stimulating factors (G-
CSF, M-CSF, GM-CSF, and CSF1-3), erythropoietin, and the cytokines. The
cytokines are specialized
hematopoietic factors secreted by cells of the immune system and are discussed
in detail below.
Growth factors play critical roles in neoplastic transformation of cells in
vitro and iri tumor
2 o progression in vivo. Overexpression of the large polypeptide growth
factors promotes the
proliferation and transformation of cells in culture. Inappropriate expression
of these growth factors
by tumor cells in vivo may contribute to tumor vascularization and metastasis.
Inappropriate activity
of hematopoietic growth factors can result in anemias, leukemias, and
lymphomas. Moreover, growth
factors are both structurally and functionally related to oncoproteins, the
potentially cancer-causing
2 s products of proto-oncogenes. Certain FGF and PDGF family members are
themselves homologous to
oncoproteins, whereas receptors for some members of the EGF, NGF, and FGF
families are encoded
by proto-oncogenes. Growth factors also affect the transcriptional regulation
of both proto-oncogenes
and oncosuppressor genes (Pimentel, E. (1994) Handbook of Growth Factors, CRC
Press, Ann Arbor
MI; McKay, I. and I. Leigh, eds. (1993) Growth Factors: A Practical Approach,
Oxford University
3 o Press, New York NY; Habenicht, A., ed. (1990) Growth Factors,
Differentiation Factors> and
Cytokines, Springer-Verlag, New York NY).
In addition, some of the large polypeptide growth factors play crucial roles
in the induction of
the primordial germ layers in the developing embryo. This induction ultimately
results in the formation
of the embryonic mesoderm, ectoderm, and endoderm which in turn provide the
framework for the
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entire adult body plan. Disruption of this inductive process would be
catastrophic to embryonic
development.
Small Peptide Factors - Neuropeptides and Vasomediators
Neuropeptides and vasomediators (NP/VM) comprise a family of small peptide
factors,
s typically of 20 amino acids or less. These factors generally function in
neuronal excitation and
inhibition of vasoconstriction/vasodilation, muscle contraction, and hormonal
secretions from the
brain and other endocrine tissues. Included in this family are neuropeptides
and neuropeptide
hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N,
melanocortins, opioids,
galanin, somatostatin, tachykinins, urotensin II and related peptides involved
in smooth muscle
1 o stimulation, vasopressin, vasoactive intestinal peptide, and circulatory
system-borne signaling
molecules such as angiotensin, complement, calcitonin, endothelins, formyl-
methionyl peptides,
glucagon, cholecystokinin, gastrin, and many of the peptide hormones discussed
above. NP/VMs can
transduce signals directly, modulate the activity or release of other
neurotransmitters and hormones, 'and
act as catalytic enzymes in signaling cascades. The effects of NP/VMs range
from extremely brief to
15 long-lasting. (Reviewed in Martin, C.R. et al. (1985) Endocrine Physiology,
Oxford University Press,
New York NY, pp. 57-62.)
C okines
Cytokines comprise a family of signaling molecules that modulate the immune
system and the
inflammatory response. Cytokines are usually secreted by leukocytes, or white
blood cells, in response
2 o to injury or infection. Cytokines function as growth and differentiation
factors that act primarily on
cells of the immune system such as B- and T-lymphocytes, rnonocytes,
macrophages, and granulocytes.
Like other signaling molecules, cytokines bind to specific plasma membrane
receptors and trigger
intracellular signal transduction pathways which alter gene expression
patterns. There is considerable
potential for the use of cytokines in the treatment of inflammation and immune
system disorders.
2 s Cytokine structure and function have been extensively characterized in
vitro. Most cytokines
are small polypeptides of about 30 kilodaltons or less. Over 50 cytokines have
been identified from
human and rodent sources. Examples of cytokine subfamilies include the
interferons (IFN-a, -(3, and -
y), the interleukins (IL1-IL13), the tumor necrosis factors (TNF-a and -(3),
and the chemokines. Many
cytokines have been produced using recombinant DNA techniques, and the
activities of individual
3 o cytokines have been determined in vitro. These activities include
regulation of leukocyte proliferation,
differentiation, and motility.
The activity of an individual cytokine in vitro may not reflect the full scope
of that cytokine's
activity in vivo. Cytokines are not expressed individually in vivo but are
instead expressed in
combination with a multitude of other cytokines when the organism is
challenged with a stimulus.
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Together, these cytokines collectively modulate the immune response in a
manner appropriate for that
particular stimulus. Therefore, the physiological activity of a cytokine is
determined by the stimulus
itself and by complex interactive networks among co-expressed cytokines which
may demonstrate both
synergistic and antagonistic relationships.
s Chemokines comprise a cytokine subfamily with over 30 members. (Reviewed in
Wells, T.
N.C. and M.C. Peitsch (1997) J. Leukoc. Biol. 61:545-550.) Chemokines were
initially identified as
chemotactic proteins that recruit monocytes and macrophages to sites of
inflammation. Recent evidence
indicates that chemokines may also play key roles in hematopoiesis and HIV-1
infection. Chemokines
are small proteins which range from about 6-15 kilodaltons in molecular
weight. Chemokines are
1 o further classified as C, CC, CXC, or CX3C based on the number and position
of critical cysteine
residues. The CC chemokines, for example, each contain a conserved motif
consisting of two
consecutive cysteines followed by two additional cysteines which occur
downstream at 24- and 16-
residue intervals, respectively (ExPASy PROSITE database, documents PS00472
and PDOC00434).
The presence and spacing of these four cysteine residues are highly conserved,
whereas the intervening
1 s residues diverge significantly. However, a conserved tyrosine located
about 15 residues downstream of
the cysteine doublet seems to be important for chemotactic activity. Most of
the human genes encoding
CC chemokines are clustered on chromosome 17, although there are a few
examples of CC chemokine
genes that map elsewhere. Other chemokines include lymphotactin (C chemokine);
macrophage
chemotactic and activating factor (MCAF/MCP-l; CC chemokine); platelet factor
4 and IL-8 (CXC
2 o chemokines); and fractalkine and neurotractin (CX3C chemokines). (Reviewed
in Luster, A.D. (1998)
N. Engl. J. Med. 338:436-445.)
Receptor Molecules
The term receptor describes proteins that specifically recognize other
molecules. The category
2 5 is broad and includes proteins with a variety of functions. The bulk of
receptors are cell surface
proteins which bind extracellular ligands and produce cellular responses in
the areas of growth,
differentiation, endocytosis, and immune response. Other receptors facilitate
the selective transport of
proteins out of the endoplasmic reticulum and localize enzymes to particular
locations in the cell. The
term may also be applied to proteins which act as receptors for ligands with
known or unknown
3 o chemical composition and which interact with other cellular components.
For example, the steroid
hormone receptors bind to and regulate transcription of DNA.
Regulation of cell proliferation, differentiation, and migration is important
for the formation
and function of tissues. Regulatory proteins such as growth factors
coordinately control these cellular
processes and act as mediators in cell-cell signaling pathways. Growth factors
are secreted proteins
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that bind to specific cell-surface receptors on target cells. The bound
receptors trigger intracellular
signal transduction pathways which activate various downstream effectors that
regulate gene
expression, cell division, cell differentiation, cell motility, and other
cellular processes.
Cell surface receptors are typically integral plasma membrane proteins. These
receptors
s recognize hormones such as catecholamines; peptide hormones; growth and
differentiation factors;
small peptide factors such as thyrotropin-releasing hormone; galanin,
somatostatin, and tachykinins;
and circulatory system borne signaling molecules. Cell surface receptors on
immune system cells
recognize antigens, antibodies, and major histocompatibility complex (MHC)-
bound peptides. Other
cell surface receptors bind ligands to be internalized by the cell. This
receptor-mediated endocytosis
1 o functions in the uptake of low density lipoproteins (LDL), transferrin,
glucose- or mannose-terminal
glycoproteins, galactose-terminal glycoproteins, immunoglobulins,
phosphovitellogenins, fibrin,
proteinase-inhibitor complexes, plasminogen activators, and thrombospondin
(Lodish, H. et al. (1995)
Molecular Cell Biology, Scientific American Books, New York NY, p. 723;
Mikhailenko, I. et al.
(1997) J. Biol. Chem. 272:6784-6791).
1 s Receptor Protein Kinases
Many growth factor receptors, including receptors for epidermal growth factor,
platelet-derived growth factor, fibroblast growth factor, as well as the
growth modulator a=thrombin,
contain intrinsic protein kinase activities. When growth factor binds to the
receptor, it triggers the
autophosphorylation of a serine, threonine, or tyrosine residue on the
receptor. These phosphorylated
2 o sites are recognition sites for the binding of other cytoplasmic signaling
proteins. These proteins
participate in signaling pathways that eventually link the initial receptor
activation at the cell surface to
the activation of a specific intracellular target molecule. In the case of
tyrosine residue
autophosphorylation, these signaling proteins contain a common domain referred
to as a Src homology
(SH) domain. SH2 domains and SH3 domains are found in phospholipase C-'y, PI-3-
K p85 regulatory
25 subunit, Ras-GTPase activating protein, and pp60'-Sr° (Lowenstein,
E.J. et al. (1992) Cel170:431-442).
The cytokine family of receptors share a different common binding domain and
include transmembrane
receptors for growth hormone (GH), interleukins, erythropoietin, and
prolactin.
Other receptors and second messenger-binding proteins have intrinsic
serinelthreonine protein
kinase activity. These include activinlTGF-(iIBMP-superfamily receptors,
calcium- and diacylglycerol-
3 o activatedlphospholipid-dependant protein kinase (PK C), and RNA-dependant
pxotein kinase (PK-R).
In addition, other serine/threonine protein kinases, including nematode
Twitchin, have fibronectin-like,
immunoglobulin C2-like domains.
G-Protein Coupled Receptors
G-protein coupled receptors (GPCRs) are integral membrane proteins
characterized by the
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presence of seven hydrophobic transmembrane domains which span the plasma
membrane and form a
bundle of antiparallel alpha (a) helices. These proteins range in size from
under 400 to over 1000
amino acids (Strosberg, A.D. (1991) Eur. J. Biochem. 196:1-10; Coughlin, S.R.
(1994) Curr. Opin.
Cell Biol. 6:191-197). The amino-terminus of the GPCR is extracellular, of
variable length and often
s glycosylated; the carboxy-terminus is cytoplasmic and generally
phosphorylated. Extracellular loops of
the GPCR alternate with intracellular loops and link the transmembrane
domains. The most conserved
domains of GPCRs are the transmembrane domains and the first two cytoplasmic
loops. The
transmembrane domains account for structural and functional features of the
receptor. In most cases,
the bundle of a helices forms a binding pocket. In addition, the extracellular
N-terminal segment or one
l o or more of the three extracellular loops may also participate in ligand
binding. Ligand binding activates
the receptor by inducing a conformational change in intracellular portions of
the receptor. The
activated receptor, in turn, interacts with an intracellular heterotrimeric
guanine nucleotide binding (G)
protein complex which mediates further intracellular signaling activities,
generally the production of
second messengers such as cyclic AMP (CAMP), phospholipase C, inositol
triphosphate, or interactions
15 with ion channel proteins (Baldwin, J.M. (1994) Curr. Opin. Cell Biol.
6:180-190).
GPCRs include those for acetylcholine, adenosine, epinephrine and
norepinephrine, bombesin
bradykinin, chemokines, dopamine, endothelin, ~y-aminobutyric acid (GABA),
follicle-stimulating
hormone (FSH), glutamate, gonadotropin-releasing hormone (GnRH), hepatocyte
growth factor,
histamine, leukotrienes, melanocortins, neuropeptide Y, opioid peptides,
opsins, prostanoids, serotonin,
2 o somatostatin, tachykinins, thrombin, thyrotropin-releasing hormone (TRH),
vasoactive intestinal
polypeptide family, vasopressin and oxytocin, and orphan receptors.
GPCR mutations, which may cause loss of function or constitutive activation,
have been
associated with numerous human diseases (Coughlin, su ra). For instance,
retinitis pigmentosa may
arise from mutations in the rhodopsin gene. Rhodopsin is the retinal
photoreceptor which is located
2 s within the discs of the eye rod cell. Parma, J. et al. (1993, Nature
365:649-651) report that somatic
activating mutations in the thyrotropin receptor cause hyperfunctioning
thyroid adenomas and suggest
that certain GPCRs susceptible to constitutive activation may behave as
protooncogenes.
Nuclear Receptors
Nuclear receptors bind small molecules such as hormones or second messengers,
leading to
s o increased receptor-binding affinity to specific chromosomal DNA elements.
In addition the affinity for
other nuclear proteins may also be altered. Such binding and protein-protein
interactions may regulate
and modulate gene expression. Examples of such receptors include the steroid
hormone receptors
family, the retinoic acid receptors family, and the thyroid hormone receptors
family.
Li~and-Gated Receptor Ion Channels
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Ligand-gated receptor ion channels fall into two categories. The first
category, extracellular
ligand-gated receptor ion channels (ELGs), rapidly transduce neurotransmitter-
binding events into
electrical signals, such as fast synaptic neurotransmission. ELG function is
regulated by post-
translational modification. The second category, intracellular ligand-gated
receptor ion channels
s (ILGs), are activated by many intracellular second messengers and do not
require post-translational
modifications) to effect a channel-opening response.
ELGs depolarize excitable cells to the threshold of action potential
generation. In non-excitable
cells, ELGs permit a limited calcium ion-influx during the presence of
agonist. ELGs include channels
directly gated by neurotransmitters such as acetylcholine, L-glutamate,
glycine, ATP, serotonin,
s o GABA, and histamine. ELG genes encode proteins having strong structural
and functional similarities.
ILGs are encoded by distinct and unrelated gene families and include receptors
for cAMP, cGMP,
calcium ions, ATP, and metabolites of arachidonic acid.
Macrophage Scavenger Receptors
Macrophage scavenger receptors with broad ligand specificity may participate
in the binding of
1 s low density lipoproteins (LDL) and foreign antigens. Scavenger receptors
types I and II are trimeric
membrane proteins with each subunit containing a small N-terminal
intracellular domain, a
transmembrane domain, a large extracellular domain, and a C-terminal cysteine-
rich domain: The
extracellular domain contains a short spacer domain, an a-helical Boiled-coil
domain, and a triple helical
collagenous domain. These receptors have been shown to bind a spectrum of
ligands, including
2 o chemically modified lipoproteins and albumin, polyribonucleotides,
polysaccharides, phospholipids, and
asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9133-9137;
Elomaa, O. et al.
(1995) Cell 80:603-609). The scavenger receptors are thought to play a key
role in atherogenesis by
mediating uptake of modified LDL in arterial walls, and in host defense by
binding bacterial
endotoxins, bacteria, and protozoa.
2 s T-Cell Receptors
T cells play a dual role in the immune system as effectors and regulators,
coupling antigen
recognition with the transmission of signals that induce cell death in
infected cells and stimulate
proliferation of other immune cells. Although a population of T cells can
recognize a wide range of
different antigens, an individual T cell can only recognize a single antigen
and only when it is presented
3 o to the T cell receptor (TCR) as a peptide complexed with a major
histocompatibility molecule (MHC)
on the surface of an antigen presenting cell. The TCR on most T cells consists
of immunoglobulin-like
integral membrane glycoproteins containing two polypeptide subunits, a and [3,
of similar molecular
weight. Both TCR subunits have an extracellular domain containing both
variable and constant
regions, a transmembrane domain that traverses the membrane once, and a short
intracellular domain
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(Saito, H. et al. (1984) Nature 309:757-762). The genes for the TCR subunits
are constructed through
somatic rearrangement of different gene segments. Interaction of antigen in
the proper MHC context
with the TCR initiates signaling cascades that induce the proliferation,
maturation, and function of
cellular components of the immune system (Weiss, A. (1991) Annu. Rev. Genet.
25:487-510).
s Rearrangements in TCR genes and alterations in TCR expression have been
noted in lymphomas,
leukemias, autoimmune disorders, and immunodeficiency disorders (Aisenberg,
A.C. et al. (1985) N.
Engl. J. Med. 313:529-533; Weiss, su ra).
Intracellular Signaling Molecules
~. o Intracellular signaling is the general process by which cells respond to
extracellular signals
(hormones, neurotransmitters, growth and differentiation factors, etc.)
through a cascade of
biochemical reactions that begins with the binding of a signaling molecule to
a cell membrane
receptor and ends with the activation of an intracellular target molecule.
Intermediate steps in the
process involve the activation of various cytoplasmic proteins by
phosphorylation via protein kinases,
is and their deactivation by protein phosphatases, and the eventual
translocation of some of these
activated proteins to the cell nucleus where the transcription of specific
genes is triggered. The
intracellular signaling process regulates all types of cell functions
including cell proliferation, cell
differentiation, and gene transcription, and involves a diversity of molecules
including protein kinases
and phosphatases, and second messenger molecules, such as cyclic nucleotides,
calcium-calmodulin,
2 o inositol, and various mitogens, that regulate protein phosphorylation.
Protein Phosphorylation
Protein kinases and phosphatases play a key role in the intracellular
signaling process by
controlling the phosphorylation and activation of various signaling proteins.
The high energy
phosphate for this reaction is generally transferred from the adenosine
triphosphate molecule (ATP) to
2 s a particular protein by a protein kinase and removed from that protein by
a protein phosphatase.
Protein kinases are roughly divided into two groups: those that phosphorylate
tyrosine residues
(protein tyrosine kinases, PTK) and those that phosphorylate serine or
threonine residues
(serine/threonine kinases, STK). A few protein kinases have dual specificity
for serine/threonine and
tyrosine residues. Almost all kinases contain a conserved 250-300 amino acid
catalytic domain
3 o containing specific residues and sequence motifs characteristic of the
kinase family (Hardie, G. and S.
Hanks (1995) The Protein Kinase Facts Books, Vol I:7-20, Academic Press, San
Diego CA).
STKs include the second messenger dependent protein kinases such as the cyclic-
AMP
dependent protein kinases (PKA), involved in mediating hormone-induced
cellular responses;
calcium-calmodulin (CaM) dependent protein kinases, involved in regulation of
smooth muscle
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contraction, glycogen breakdown, and neurotransmission; and the mitogen-
activated protein kinases
(MAP) which mediate signal transduction from the cell surface to the nucleus
via phosphorylation
cascades. Altered PKA expression is implicated in a variety of disorders and
diseases including
cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular
disease (Isselbacher, K.J. et al.
s (1994) Harrison's Principles of Internal Medicine McGraw-Hill, New York NY,
pp. 416-431, 1887).
PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-
receptor
PTKs. Transmembrane PTKs are receptors for most growth factors. Non-receptor
PTKs lack
transmembrane regions and, instead, form complexes with the intracellular
regions of cell surface
receptors. Receptors that function through non-receptor PTKs include those for
cytokines and
1 o hormones (growth hormone and prolactin) and antigen-specific receptors on
T and B lymphocytes.
Many of these PTKs were first identified as the products of mutant oncogenes
in cancer cells in which
their activation was no longer subject to normal cellular controls. In fact,
about one third of the
known oncogenes encode PTKs, and it is well known that cellular transformation
(oncogenesis) is
often accompanied by increased tyrosine phosphorylation activity (Charbonneau,
H. and N.K. Tonks
15 (1992) Annu. Rev. Cell Biol. 8:463-493).
An additional family of protein kinases previously thought to exist only in
procaryotes is the
histidine protein kinase family (HPK). HPKs bear little homology with
mammalian STKs or PTKs
but have distinctive sequence motifs of their own (Davie, J.R. et al. (1995)
J. Biol. Chem.
270:19861-19867). A histidine residue in the N-terminal half of the molecule
(region I) is an
2 o autophosphorylation site. Three additional motifs located in the C-
terminal half of the molecule
include an invariant asparagine residue in region II and two glycine-rich
loops characteristic of
nucleotide binding domains in regions III and IV. Recently a branched chain
alpha-ketoacid
dehydrogenase kinase has been found with characteristics of HPK in rat (Davie,
supra).
Protein phosphatases regulate the effects of protein kinases by removing
phosphate groups
2 s from molecules previously activated by kinases. The two principal
categories of protein phosphatases
are the protein (serine/threonine) phosphatases (PPs) and the protein tyrosine
phosphatases (PTPs).
PPs dephosphorylate phosphoserine/threonine residues and are important
regulators of many
cAMP-mediated hormone responses (Cohen, P. (1989) Annu. Rev. Biochem. 58:453-
508). PTPs
reverse the effects of protein tyrosine kinases and play a significant role in
cell cycle and cell
3 o signaling processes (Charbonneau, supra). As previously noted, many PTKs
are encoded by
oncogenes, and oncogenesis is often accompanied by increased tyrosine
phosphorylation activity. It
is therefore possible that PTPs may prevent or reverse cell transformation and
the growth of various
cancers by controlling the levels of tyrosine phosphorylation in cells. This
hypothesis is supported by
studies showing that overexpression of PTPs can suppress transformation in
cells, and that specific
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inhibition of PTPs can enhance cell transformation (Charbonneau, supra).
Phospholipid and Inositol-Phosphate Si~nalin~
Inositol phospholipids (phosphoinositides) are involved in an intracellular
signaling pathway
that begins with binding of a signaling molecule to a G-protein linked
receptor in the plasma
s membrane. This leads to the phosphorylation of phosphatidylinositol (PI)
residues on the inner side
of the plasma membrane to the biphosphate state (PIP2) by inositol kinases.
Simultaneously, the G-
protein linked receptor binding stimulates a trimeric G-protein which in turn
activates a
phosphoinositide-specific phospholipase C-(3. Phospholipase C-(3 then cleaves
PIP2 into two
products, inositol triphosphate (IP3) and diacylglycerol. These two products
act as mediators for
1 o separate signaling events. IP3 diffuses through the plasma membrane to
induce calcium release from
the endoplasmic reticulum (ER), while diacylglycerol remains in the membrane
and helps activate
protein kinase C, an STK that phosphorylates selected proteins in the target
cell. The calcium
response initiated by IP3 is terminated by the dephosphorylation of IP3 by
specific inositol
phosphatases. Cellular responses that are mediated by this pathway are
glycogen breakdown in the
1 s liver in response to vasopressin, smooth muscle contraction in response to
acetylcholine, and
thrombin-induced platelet aggregation.
Cyclic Nucleotide Signaling
Cyclic nucleotides (CAMP and cGMP) function as intracellular second messengers
to
transduce a variety of extracellular signals including hormones, light, and
neurotransmitters. In
a o particular, cyclic-AMP dependent protein kinases (PKA) are thought to
account for all of the effects
of cAMP in most mammalian cells, including various hormone-induced cellular
responses. Visual
excitation and the phototransmission of light signals in the eye is controlled
by cyclic-GMP regulated,
Ca2+-specific channels. Because of the importance of cellular levels of cyclic
nucleotides in
mediating these various responses, regulating the synthesis and breakdown of
cyclic nucleotides is an
2 s important matter. Thus adenylyl cyclase, which synthesizes CAMP from AMP,
is activated to
increase cAMP levels in muscle by binding of adrenaline to (3-andrenergic
receptors, while activation
of guanylate cyclase and increased cGMP levels in photoreceptors leads to
reopening of the
Ca2+-specific channels and recovery of the dark state in the eye. In contrast,
hydrolysis of cyclic
nucleotides by cAMP and cGMP-specific phosphodiesterases (PDEs) produces the
opposite of these
3 o and other effects mediated by increased cyclic nucleotide levels. PDEs
appear to be particularly
important in the regulation of cyclic nucleotides, considering the diversity
found in this family of
proteins. At least seven families of mammalian PDEs (PDE1-7) have been
identified based on
substrate specificity and affinity, sensitivity to cofactors, and sensitivity
to inhibitory drugs (Beavo,
J.A. (I995) Physiological Reviews 75:725-748). PDE inhibitors have been found
to be particularly
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useful in treating various clinical disorders. Rolipram, a specific inhibitor
of PDE4, has been used in
the treatment of depression, and similar inhibitors are undergoing evaluation
as anti-inflammatory
agents. Theophylline is a nonspecific PDE inhibitor used in the treatment of
bronchial asthma and
other respiratory diseases (Banner, I~.H. and C.P. Page (1995) Eur. Respir. J.
8:996-1000).
s G-Protein Si~nalin~
Guanine nucleotide binding proteins (G-proteins) are critical mediators of
signal transduction
between a particular class of extracellular receptors, the G-protein coupled
receptors (GPCR), and
intracellular second messengers such as cAMP and Ca2+. G-proteins are linked
to the cytosolic side
of a GPCR such that activation of the GPCR by ligand binding stimulates
binding of the G-protein to
1 o GTP, inducing an "active" state in the G-protein. In the active state, the
G-protein acts as a signal to
trigger other events in the cell such as the increase of cAMP levels or the
release of Ca2+ into the
cytosol from the ER, which, in turn, regulate phosphorylation and activation
of other intracellular
proteins. Recycling of the G-protein to the inactive state involves hydrolysis
of the bound GTP to
GDP by a GTPase activity in the G-protein. (See Alberts, B. et al. (1994)
Molecular Biolo~y of the
15 Cell, Garland Publishing, Inc., New York NY, pp.734-759.) Two structurally
distinct classes of G-
proteins are recognized: heterotrimeric G-proteins, consisting of three
different subunits, and
monomeric, low molecular weight (LMW), G-proteins consisting of a single
polypeptide chain.
The three polypeptide subunits of heterotrimeric G-proteins are the a, ~3, and
y subunits. The
a subunit binds and hydrolyzes GTP. The (3 and ~( subunits form a tight
complex that anchors the
2 o protein to the inner side of the plasma membrane. The (3 subunits, also
known as G-(3 proteins or (3
transducins, contain seven tandem repeats of the WD-repeat sequence motif, a
motif found in many
proteins with regulatory functions. Mutations and variant expression of (3
transducin proteins are
linked with various disorders (Neer, E.J. et al. (1994) Nature 371:297-300;
Margottin, F. et al. (1998)
Mol. Cell 1:565-574).
2 s LMW GTP-proteins are GTPases which regulate cell growth, cell cycle
control, protein
secretion, and intracellular vesicle interaction. They consist of single
polypeptides which, like the a
subunit of the heterotrimeric G-proteins, are able to bind and hydrolyze GTP,
thus cycling between an
inactive and an active state. At least sixty members of the LMW G-protein
superfamily have been
identified and are currently grouped into the six subfamilies of ras, rho,
arf, sarl, ran, and rab.
3 o Activated ras genes were initially found in human cancers, and subsequent
studies confirmed that ras
function is critical in determining whether cells continue to grow or become
differentiated. Other
members of the LMW G-protein superfamily have roles in signal transduction
that vary with the
function of the activated genes and the locations of the G-proteins.
Guanine nucleotide exchange factors regulate the activities of LMW G-proteins
by
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determining whether GTP or GDP is bound. GTPase-activating protein (GAP) binds
to GTP-ras and
induces it to hydrolyze GTP to GDP. In contrast, guanine nucleotide releasing
protein (GNRP) binds
to GDP-ras and induces the release of GDP and the binding of GTP.
Other regulators of G-protein signaling (RGS) also exist that act primarily by
negatively
s regulating the G-protein pathway by an unknown mechanism (Druey, K.M, et al.
(1996) Nature
379:742-746). Some 15 members of the RGS family have been identified. RGS
family members are
related structurally through similarities in an approximately 120 amino acid
region termed the RGS
domain and functionally by their ability to inhibit the interleukin (cytokine)
induction of MAP kinase
in cultured mammalian 293T cells (Druey, supra).
to Calcium Si~nalin~ Molecules
Ca+2 is another second messenger molecule that is even more widely used as an
intracellular
mediator than cAMP. Two pathways exist by which Ca+2 can enter the cytosol in
response to
extracellular signals: One pathway acts primarily in nerve signal transduction
where Ca+2 enters a
nerve terminal through a voltage-gated Ca+2 channel. The second is a more
ubiquitous pathway in
~ 5 which Ca+' is released from the ER into the cytosol in response to binding
of an extracellular
signaling molecule to a receptor. Ca2+ directly activates regulatory enzymes,
such as protein kinase C,
which trigger signal transduction pathways. Ca2+ also binds to specific Ca2+-
binding proteins (CBPs)
such as calmodulin (CaM) which then activate multiple target proteins in the
cell including enzyW es,
membrane transport pumps, and ion channels. CaM interactions are involved in a
multitude of cellular
2 o processes including, but not limited to, gene regulation, DNA synthesis,
cell cycle progression,
mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal
transduction, ion
homeostasis, exocytosis, and metabolic regulation (Celio, M.R. et al, (1996)
Guidebook to
Calcium-binding Proteins, Oxford University Press, Oxfoxd, UK, pp, 15-20).
Some CBPs can serve
as a storage depot for Ca2+ in an inactive state. Calsequestrin is one such
CBP that is expressed in
2 s isoforms specific to cardiac muscle and skeletal muscle. It is suggested
that calsequestrin binds Ca2+
in a rapidly exchangeable state that is released during Ca2+ -signaling
conditions (Celio, M.R. et al.
(1996) Guidebook to Calcium-binding Proteins, Oxford University Press, New
York NY, pp. 222-
224).
Cyclins
3 o Cell division is the fundamental process by which all living things grow
and reproduce. In
most organisms, the cell cycle consists of three principle steps; interphase,
mitosis, and cytokinesis.
Interphase, involves preparations for cell division, replication of the DNA
and production of essential
proteins. In mitosis, the nuclear material is divided and separates to
opposite sides of the cell.
Cytokinesis is the final division and fission of the cell cytoplasm to produce
the daughter cells.
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The entry and exit of a cell from mitosis is regulated by the synthesis and
destruction of a
family of activating proteins called cyclins. Cyclins act by binding to and
activating a group of
cyclin-dependent protein kinases (Cdks) which then phosphorylate and activate
selected proteins
involved in the mitotic process. Several types of cyclins exist. (Ciechanover,
A. (1994) Cell
s 79:13-21.) Two principle types are mitotic cyclin, or cyclin B, which
controls entry of the cell into
mitosis, and Gl cyclin, which controls events that drive the cell out of
mitosis.
Signal Complex Scaffolding Proteins
Ceretain proteins in intracellular signaling pathways serve to link or cluster
other proteins
involved in the signaling cascade. A conserved protein domain called the PDZ
domain has been
1 o identified in various membrane-associated signaling proteins. This domain
has been implicated in
receptor and ion channel clustering and in the targeting of multiprotein
signaling complexes to
specialized functional regions of the cytosolic face of the plasma membrane.
(For a review of PDZ
domain-containing proteins, see Ponting, C.P. et al. (1997) Bioessays 19:469-
479.) A large
proportion of PDZ domains are found in the eukaxyotic MAGUK (membrane-
associated guanylate
15 kinase) protein family, members of which.bind to the intracellular domains
of receptors and channels.
However, PDZ domains are also found in diverse membrane-localized proteins
such as protein
tyrosine phosphatases, serine/threonine kinases, G-protein cofactors, and
synapse-associated proteins
such as syntrophins and neuronal nitric oxide synthase (nNOS). Generally,
about one to three PDZ
domains are found in a given protein, although up to nine PDZ domains have
been identified in a
2 o single protein.
Membrane Transport Molecules
The plasma membrane acts as a barrier to most molecules. Transport between the
cytoplasm
and the extracellular environment, and between the cytoplasm and lumenal
spaces of cellular
2 5 organelles requires specific transport proteins. Each transport protein
carries a particular class of
molecule, such as ions, sugars, or amino acids, and often is specific to a
certain molecular species of
the class. A variety of human inherited diseases are caused by a mutation in a
transport protein. For
example, cystinuria is an inherited disease that results from the inability to
transport cystine, the
disulfide-linked dimer of cysteine, from the urine into the blood.
Accumulation of cystine in the
3 o urine leads to the formation of cystine stones in the kidneys.
Transport proteins are multi-pass transmembrane proteins, which either
actively transport
molecules across the membrane or passively allow them to cross. Active
transport involves
directional pumping of a solute across the membrane, usually against an
electrochemical gradient.
Active transport is tightly coupled to a source of metabolic energy, such as
ATP hydrolysis or an
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electrochemically favorable ion gradient. Passive transport involves the
movement of a solute down
its electrochemical gradient. Transport proteins can be further classified as
either carrier proteins or
channel pxoteins. Carrier proteins, which can function in active or passive
transport, bind to a specific
solute to be transported and undergo a conformational change which transfers
the bound solute across
s the membrane. Channel proteins, which only function in passive transport,
form hydrophilic pores
across the membrane. When the pores open, specific solutes, such as inorganic
ions, pass through the
membrane and down the electrochemical gradient of the solute.
Carrier proteins which transport a single solute from one side of the membrane
to the other
are called uniporters. In contrast, coupled transporters link the transfer of
one solute with
~. o simultaneous or sequential transfer of a second solute, either in the
same direction (symport) or in the
opposite direction (antiport). For example, intestinal and kidney epithelium
contains a variety of
symporter systems driven by the sodium gradient that exists across the plasma
membrane. Sodium
moves into the cell down its electrochemical gradient and brings the solute
into the cell with it. The
sodium gradient that provides the driving force for solute uptake is
maintained by the ubiquitous
15 Na+/K~ ATPase. Sodium-coupled transporters include the mammalian glucose
transporter (SGLT1),
iodide transporter (NIS), and multivitamin transporter (SMVT). All three
transporters have twelve
putative transmembrane segments, extracellular glycosylation sites, and
cytoplasmically-oriented N-
and C-termini. NIS plays a crucial role in the evaluation, diagnosis, and
treatment of various thyroid
pathologies because it is the molecular basis for radioiodide thyroid-imaging
techniques and for
2 o specific targeting of radioisotopes to the thyroid gland (Levy, O. et al.
(1997) Proc. Natl. Acad. Sci.
USA 94:5568-5573). SMVT is expressed in the intestinal mucosa, kidney, and
placenta, and is
implicated in the transport of the water-soluble vitamins, e.g., biotin and
pantothenate (Prasad, P.D. et
al. (1998) J. Biol. Chem. 273:7501-7506).
Transporters play a major role in the regulation of pH, excretion of drugs,
and the cellular
2 s K+/Na+ balance. Monocarboxylate anion transporters are proton-coupled
symporters with a broad
substrate specificity that includes L-lactate, pyruvate, and the ketone bodies
acetate, acetoacetate, and
beta-hydroxybutyrate. At least seven isoforms have been identified to date.
The isoforms are predicted
to have twelve transmembrane (TM) helical domains with a large intracellular
loop between TM6 and
TM7, and play a critical role in maintaining intracellular pH by removing the
protons that are produced
3 o stoichiometrically with lactate during glycolysis. The best characterized
H(+)-monocarboxylate
transporter is that of the erythrocyte membrane, which transports L-lactate
and a wide range of other
aliphatic monocarboxylates. Other cells possess H(+)-linked monocarboxylate
transporters with
differing substrate and inhibitor selectivities. In particular, cardiac muscle
and tumor cells have
transporters that differ in their Km values for certain substrates, including
stereoselectivity for L- over
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D-lactate, and in their sensitivity to inhibitors. There are Na(+)-
monocarboxylate cotransporters on the
luminal surface of intestinal and kidney epithelia, which allow the uptake of
lactate, pyruvate, and
ketone bodies in these tissues. In addition, there are specific and selective
transporters for organic
canons and organic anions in organs including the kidney, intestine and liver.
Organic anion
s transporters are selective for hydrophobic, charged molecules with electron-
attracting side groups.
Organic canon transporters, such as the ammonium transporter, mediate the
secretion of a variety of
drugs and endogenous metabolites, and contribute to the maintenance of
intercellular pH. (Poole, R.C.
and A.P. Halestrap (1993) Am. J. Physiol. 264:C761-C782; Price, N.T. et al.
(1998) Biochem. J.
329:321-328; and Martinelle, K. and I. Haggstrom (1993) J. Biotechnol. 30: 339-
350.)
s o The largest and most diverse family of transport proteins known is the ATP-
binding cassette
(ABC) transporters. As a family, ABC transporters can transport substances
that differ markedly in
chemical structure and size, ranging from small molecules such as ions,
sugars, amino acids, peptides,
and phospholipids, to lipopeptides, large proteins, and complex hydrophobic
drugs. ABC proteins
consist of four modules: two nucleotide-binding domains (NBD), which hydrolyze
ATP to supply the
15 energy required for transport, and two membrane-spanning domains (MSD),
each containing six
putative transmembrane segments. These four modules may be encoded by a single
gene, as is the case
for the cystic fibrosis transmembrane regulator (CFTR), or by separate genes.
When encoded by
separate genes, each gene product contains a single NBD and MSD. These "half
molecules" form
homo- and heterodimers, such as Tapl and Tap2, the endoplasmic reticulum-based
major
2 0 ~histocompatibility (MHC) peptide transport system. Several genetic
diseases are attributed to defects in
ABC transporters, such as the following diseases and their corresponding
proteins: cystic fibrosis
(CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy protein,
ALDP), Zellweger
syndrome (peroxisomal membrane protein-70, PMP70), and hyperinsulinemic
hypoglycemia
(sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR)
protein, another
2 s ABC transporter, in human cancer cells makes the cells resistant to a
variety of cytotoxic drugs used in
chemotherapy (Taglight, D. and S. Michaelis (1998) Meth. Enzymol. 292:131-
163).
Transport of fatty acids across the plasma membrane can occur by diffusion, a
high capacity,
low affinity process. However, under normal physiological conditions a
significant fraction of fatty
acid transport appears to occur via a high affinity, low capacity protein-
mediated transport process.
3 o Fatty acid transport protein (FATP), an integral membrane protein with
four transmembrane segments,
is expressed in tissues exhibiting high levels of plasma membrane fatty acid
flux, such as muscle, heart,
and adipose. Expression of FATP is upregulated in 3T3-L1 cells during adipose
conversion, and
expression in COS7 fibroblasts elevates uptake of long-chain fatty acids (Hui,
T.Y. et al. (1998) J.
Biol. Chem. 273:27420-27429).
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Ion Channels
The electrical potential of a cell is generated and maintained by controlling
the movement of
ions across the plasma membrane. The movement of ions requires ion channels,
which form an ion-
selective pore within the membrane. There are two basic types of ion channels,
ion transporters and
s gated ion channels. Ion transporters utilize the energy obtained from ATP
hydrolysis to actively
transport an ion against the ion's concentration gradient. Gated ion channels
allow passive flow of an
ion down the ion's electrochemical gradient under restricted conditions.
Together, these types of ion
channels generate, maintain, and utilize an electrochemical gradient that is
used in 1) electrical impulse
conduction down the axon of a nerve cell, 2) transport of molecules into cells
against concentration
1 o gradients, 3) initiation of muscle contraction, and 4) endocrine cell
secretion.
Ion transporters generate and maintain the resting electrical potential of a
cell. Utilizing the
energy derived from ATP hydrolysis, they transport ions against the ion's
concentration gradient.
These transmembrane ATPases are divided into three families. The
phosphorylated (P) class ion
transporters, including Na+-K+ ATPase, Ca2+-ATPase, and H+-ATPase, are
activated by a
15 phosphorylation event. P-class ion transporters are responsible for
maintaining resting potential
distributions such that cytosolic concentrations of Nay and Ca2+ are low and
cytosolic concentration of
K~ is high. The vacuolar (V) class of ion transporters includes H~ pumps on
intracellular organelles,
such as lysosomes and Golgi. V-class ion transporters are responsible for
generating the low pH within
the lumen of these organelles that is required for function. The coupling
factor (F) class consists of H+
2 o pumps in the mitochondria. F-class ion transporters utilize a proton
gradient to generate ATP from
ADP and inorganic phosphate (Pi).
The resting potential of the cell is utilized in many processes involving
carrier pxoteins and
gated ion channels. Carrier proteins utilize the resting potential to
transport molecules into and out of
the cell. Amino acid and glucose transport into many cells is linked to sodium
ion co-transport
2 s (symport) so that the movement of Na+ down an electrochemical gradient
drives transport of the other
molecule up a concentration gradient. Similarly, cardiac muscle links transfer
of Ca2+ out of the cell
with transport of Na+ into the cell (antiport).
Ion channels share common structural and mechanistic themes. The channel
consists of four or
five subunits or protein monomers that are axranged like a barrel in the
plasma membrane. Each
3 o subunit typically consists of six potential transmembrane segments (S1,
S2, S3, S4, S5, and S6). The
center of the barrel forms a pore lined by a-helices or (3-strands. The side
chains of the amino acid
residues comprising the a-helices or J3-strands establish the charge (canon or
anion) selectivity of the
channel. The degree of selectivity, or what specific ions axe allowed to pass
through the channel,
depends on the diameter of the narrowest paxt of the pore.
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Gated ion channels control ion flow by regulating the opening and closing of
pores. These
channels are categorized according to the manner of regulating the gating
function. Mechanically-gated
channels open pores in response to mechanical stress, voltage-gated channels
open pores in response to
changes in membrane potential, and ligand-gated channels open pores in the
presence of a specific ion,
s nucleotide, or neurotransmitter.
Voltage-gated Na+ and K+ channels are necessary for the function of
electrically excitable cells,
such as nerve and muscle cells. Action potentials, which lead to
neurotransmitter release and muscle
contraction, arise from large, transient changes in the permeability of the
membrane to Na+ and K+ ions.
Depolarization of the membrane beyond the threshold level opens voltage-gated
Na+ channels. Sodium
1 o ions flow into the cell, further depolarizing the membrane and opening
more voltage-gated Na+
channels, which propagates the depolarization down the length of the cell.
Depolarization also opens
voltage-gated potassium channels. Consequently, potassium ions flow outward,
which leads to
repolarization of the membrane. Voltage-gated channels utilize charged
residues in the fourth
transmembrane segment (S4) to sense voltage change. The open state lasts only
about 1 millisecond, at
1 s which time the channel spontaneously converts into an inactive state that
cannot be opened irrespective
of the membrane potential. Inactivation is mediated by the channel's N-
terminus, which acts as a plug
that closes the pore. The transition from an inactive to a closed state
requires a return to resting
potential.
Voltage-gated Na+ channels are heterotrimeric complexes composed of a 260 kDa
pore forming
2 o a subunit that associates with two smaller auxiliary subunits, (31 and
X32. The (32 subunit is an integral
membrane glycoprotein that contains an extracellular Ig domain, and its
association with a and (31
subunits correlates with increased functional expression of the channel, a
change in its gating
properties, and an increase in whole cell capacitance due to an increase in
membrane surface area.
(Isom, L.L. et al. (1995) Cell 83:433-442.)
2 s Voltage-gated Ca 2+ channels are involved in presynaptic neurotransmitter
release, and heart
and skeletal muscle contraction. The voltage-gated Ca 2+ channels from
skeletal muscle (L-type) and
brain (N-type) have been purified, and though their functions differ
dramatically, they have similar
subunit compositions. The channels are composed of three subunits. The al
subunit forms the
membrane pore and voltage sensor, while the a2& and /3 subunits modulate the
voltage-dependence,
3 o gating properties, and the current amplitude of the channel. These
subunits are encoded by at least six
al, one a28, and four (3 genes. A fourth subunit, 'y, has been identified in
skeletal muscle. (Walker, D.
et al. (1998) J. Biol. Chem. 273:2361-2367; and Jay, S.D. et al. (1990)
Science 248:490-492.)
Chloride channels are necessary in endocrine secretion and in regulation of
cytosolic and
organelle pH. In secretory epithelial cells, Cl- enters the cell across a
basolateral membrane through an
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Na+, K+/Cl- cotransporter, accumulating in the cell above its electrochemical
equilibrium concentration.
Secretion of Cl- from the apical surface, in response to hormonal stimulation,
leads to flow of Na+ and
water into the secretory lumen. The cystic fibrosis transmembrane conductance
regulator (CFTR) is a
chloride channel encoded by the gene for cystic fibrosis, a common fatal
genetic disorder in humans.
s Loss of CFTR function decreases transepithelial water secretion and, as a
result, the layers of mucus
that coat the respiratory tree, pancreatic ducts, and intestine are dehydrated
and difficult to clear. The
resulting blockage of these sites leads to pancreatic insufficiency, "meconium
ileus", and devastating
"chronic obstructive pulmonary disease" (Al-Awqati, Q. et al. (1992) J. Exp.
Biol. 172:245-266).
Many intracellular organelles contain H+-ATPase pumps that generate
transmembrane pH and
1 o electrochemical differences by moving protons from the cytosol to the
organelle lumen. If the
membrane of the organelle is permeable to other ions, then the electrochemical
gradient can be
abrogated without affecting the pH differential. In fact, removal of the
electrochemical barrier allows
more H+ to be pumped across the membrane, increasing the pH differential. C1-
is the sole counterion
of H+ translocation in a number of organelles, including chromaffin granules,
Golgi vesicles,
1 s lysosomes, and endosomes. Functions that require a low vacuolar pH include
uptake of small
molecules such as biogenic amines in chromaffin granules, processing of
vacuolar constituents such as
pro-hormones by proteolytic enzymes, and protein degradation in lysosomes (Al-
Awqati, supra).
Ligand-gated channels open their pores when an extracellular or intracellular
mediator binds to
the channel. Neurotransmitter-gated channels are channels that open when a
neurotransmitter binds to
2 o their extracellular domain. These channels exist in the postsynaptic
membrane of nerve or muscle cells.
There are two types of neurotransmitter-gated channels. Sodium channels open
in response to
excitatory neurotransmitters, such as acetylcholine, glutamate, and serotonin.
This opening causes an
influx of Na+ and produces the initial localized depolarization that activates
the voltage-gated channels
and starts the action potential. Chloride channels open in response to
inhibitory neurotransmitters, such
2 s as y-aminobutyric acid (GABA) and glycine, leading to hyperpolarization of
the membrane and the
subsequent generation of an action potential.
Ligand-gated channels can be regulated by intracellular second messengers.
Calcium-activated
K+ channels are gated by internal calcium ions. In nerve cells, an influx of
calcium during
depolarization opens K+ channels to modulate the magnitude of the action
potential (Ishi, T.M. et al.
3 0 (1997) Proc. Natl. Acad. Sci. USA 94:11651-11656). Cyclic nucleotide-gated
(CNG) channels are
gated by cytosolic cyclic nucleotides. The best examples of these are the cAMP-
gated Na+ channels
involved in olfaction and the cGMP-gated canon channels involved in vision.
Both systems involve
ligand-mediated activation of a G-protein coupled receptor which then alters
the level of cyclic
nucleotide within the cell.
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Ion channels are expressed in a number of tissues where they are implicated in
a variety of
processes. CNG channels, while abundantly expressed in photoreceptor and
olfactory sensory cells, are
also found in kidney, lung, pineal, retinal ganglion cells, testis, aorta, and
brain. Calcium activated K+
channels may be responsible for the vasodilatory effects of bradykinin in the
kidney and for shunting
s excess K+ from brain capillary endothelial cells into the blood. They are
also implicated in repolarizing
granulocytes after agonist-stimulated depolarization (Ishi, sunra)~ Ion
channels have been the target for
many drug therapies. Neurotransmitter-gated channels have been targeted in
therapies for treatment of
insomnia, anxiety, depression, and schizophrenia. Voltage-gated channels have
been targeted in
therapies for arrhythmia, ischemic stroke, head trauma, and neurodegenerative
disease (Taylor, C.P.
to andL.S. Narasimhan (1997) Adv. Pharmacol. 39:47-98).
Disease Correlation
The etiology of numerous human diseases and disorders can be attributed to
defects in the
transport of molecules across membranes. Defects in the trafFcking of membrane-
bound transporters
and ion channels are associated with several disorders, e.g. cystic fibrosis,
glucose-galactose
1 s malabsorption syndrome, hypercholesterolemia, von Gierke disease, and
certain forms of diabetes
mellitus. Single-gene defect diseases resulting in an inability to transport
small molecules across
membranes include, e.g., cystinuria, iminoglycinuria, Hartup disease, and
Fanconi disease (van't Hoff,
W.G. (1996) Exp. Nephrol. 4:253-262; Talente, G.M. et al. (1994) Ann. Intern.
Med. 120:218-226;
and Chillon, M. et al. (1995) New Engl. J. Med. 332:1475-1480).
Protein Modification and Maintenance Molecules
The cellular processes regulating modification and maintenance of protein
molecules
coordinate their conformation, stabilization, and degradation. Each of these
processes is mediated by
key enzymes or proteins such as proteases, protease inhibitors, transferases,
isomerases, and
molecular chaperones.
Proteases
Proteases cleave proteins and peptides at the peptide bond that forms the
backbone of the
peptide and.protein chain. Proteolytic processing is essential to cell growth,
differentiation,
remodeling, and homeostasis as well as inflammation and immune response.
Typical protein half-
3 0 lives range from hours to a few days, so that within all living cells,
precursor proteins are being
cleaved to their active form, signal sequences proteolytically removed from
targeted proteins, and
aged or defective proteins degraded by proteolysis. Proteases function in
bacterial, parasitic, and viral
invasion and replication within a host. Four principal categories of mammalian
proteases have been
identified based on active site structure, mechanism of action, and overall
three-dimensional structure.
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(Beynon, R.J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach,
Oxford University
Press, New York NY, pp. 1-5).
The serine proteases (SPs) have a serine residue, usually within a conserved
sequence, in an
active site composed of the serine, an aspartate, and a histidine residue. SPs
include the digestive
s enzymes trypsin and chymotrypsin, components of the complement cascade and
the blood-clotting
cascade, and enzymes that control extracellular protein degradation. The main
SP sub-families are
trypases, which cleave after arginine or lysine; aspartases, which cleave
after aspartate; chymases,
which cleave after phenylalanine or leucine; metases, which cleavage after
methionine; and serases
which cleave after serine. Enterokinase, the initiator of intestinal
digestion, is a serine protease found
1 o in the intestinal brush border, where it cleaves the acidic propeptide
from trypsinogen to yield active
trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7588-7592).
Prolylcarboxypeptidase, a lysosomal serine peptidase that cleaves peptides
such as angiotensin II and
III and [des-Arg9] bradykinin, shares sequence homology with members of both
the serine
carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J.
Biol. Chem. 268:16631-
15 16638).
Cysteine proteases (CPs) have a cysteine as the major catalytic residue at an
active site where
catalysis proceeds via an intermediate thiol ester and is facilitated by
adjacent histidine and aspartic
acid residues. CPs are involved in diverse cellular processes ranging from the
processing of precursor
proteins to intracellular degradation. Mammalian CPs include lysosomal
cathepsins and cytosolic
2 o calcium activated proteases, calpains. CPs are produced by monocytes,
macrophages and other cells
of the immune system which migrate to sites of inflammation and secrete
molecules involved in
tissue repair. Overabundance of these repair molecules plays a role in certain
disorders. In
autoimmune diseases such as rheumatoid arthritis, secretion of the cysteine
peptidase cathepsin C
degrades collagen, laminin, elastin and other structural proteins found in the
extracellular matrix of
2 s bones.
Aspartic proteases are members of the cathepsin family of lysosomal proteases
and include
pepsin A, gastricsin, chymosin, renin, and cathepsins D and E. Aspartic
proteases have a pair of
aspartic acid residues in the active site, and are most active in the pH 2 - 3
range, in which one of the
aspartate residues is ionized, the other un-ionized. Aspartic proteases
include bacterial
3 o penicillopepsin, mammalian pepsin, renin, chymosin, and certain fungal
proteases. Abnormal
regulation and expression of cathepsins is evident in various inflammatory
disease states. In cells
isolated from inflamed synovia, the mRNA for stromelysin, cytokines, TIMP-1,
cathepsin, gelatinase,
and other molecules is preferentially expressed. Expression of cathepsins L
and D is elevated in
synovial tissues from patients with rheumatoid arthritis and osteoarthritis.
Cathepsin L expression may
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also contribute to the influx of mononuclear cells which exacerbates the
destruction of the rheumatoid
synovium. (Keyszer, G.M. (1995) Arthritis Rheum. 38:976-984.) The increased
expression and
differential regulation of the cathepsins are linked to the metastatic
potential of a variety of cancers and
as such are of therapeutic and prognostic interest (Chambers, A.F. et al.
(1993) Crit. Rev. Oncog.
s 4:95-114).
Metalloproteases have active sites that include two glutamic acid residues and
one histidine
residue that serve as binding sites for zinc. Carboxypeptidases A and B are
the principal mammalian
metalloproteases. Both are exoproteases of similar structure and active sites.
Carboxypeptidase A,
like chymotrypsin, prefers C-terminal aromatic and aliphatic side chains of
hydrophobic nature,
1 o whereas carboxypeptidase B is directed toward basic arginine and lysine
residues. Glycoprotease
(GCP), or O-sialoglycoprotein endopeptidase, is a metallopeptidase which
specifically cleaves
O-sialoglycoproteins such as glycophorin A. Another metallopeptidase,
placental leucine
aminopeptidase (P-LAP) degrades several peptide hormones such as oxytocin and
vasopressin,
suggesting a role in maintaining homeostasis during pregnancy, and is
expressed in several tissues
15 (Rogi, T. et al. (1996) J. Biol. Chem. 271:56-61).
Ubiquitin proteases are associated with the ubiquitin conjugation system
(UCS), a major
pathway for the degradation of cellular proteins in eukaryotic cells and some
bacteria: The UCS
mediates the elimination' of abnormal proteins and regulates the half lives of
important regulatory
proteins that control cellular processes such as gene transcription and cell
cycle progression. In the
2 o UCS pathway, proteins targeted for degradation are conjugated to a
ubiquitin, a small heat stable
protein. The ubiquitinated protein is then recognized and degraded by
proteasome, a large,
multisubunit proteolytic enzyme complex, and ubiquitin is released for
reutilization by ubiquitin
protease. The UCS is implicated in the degradation of mitotic cyclic kinases,
oncoproteins, tumor
suppressor genes such as p53, viral proteins, cell surface receptors
associated with signal
25 transduction, transcriptional regulators, and mutated or damaged proteins
(Ciechanover, A. (1994)
Cell 79:13-21). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin
protease whose
overexpression leads to oncogenic transformation of NIH3T3 cells, and the
human homolog of this
gene is consistently elevated in small cell tumors and adenocarcinomas of the
lung (Gray, D.A.
(1995) Oncogene 10:2179-2183).
3 o Signal Peptidases
The mechanism for the translocation process into the endoplasmic reticulum
(ER) involves
the recognition of an N-terminal signal peptide on the elongating protein. The
signal peptide directs
the protein and attached ribosome to a receptor on the ER membrane. The
polypeptide chain passes
through a pore in the ER membrane into the lumen while the N-terminal signal
peptide remains
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attached at the membrane surface. The process is completed when signal
peptidase located inside the
ER cleaves the signal peptide from the protein and releases the protein into
the lumen.
Protease Inhibitors
Protease inhibitors and other regulators of protease activity control the
activity and effects of
s proteases. Protease inhibitors have been shown to control pathogenesis in
animal models of
proteolytic disorders (Murphy, G. (1991) Agents Actions Suppl. 35:69-76). Low
levels of the
cystatins, low molecular weight inhibitors of the cysteine proteases,
correlate with malignant
progression of tumors. (Catkins, C. et al (1995) Biol. Biochem. Hoppe Seyler
376:71-80). Serpins
are inhibitors of mammalian plasma serine proteases. Many serpins serve to
regulate the blood
s o clotting cascade and/or the complement cascade in mammals. Sp32 is a
positive regulator of the
mammalian acrosomal protease, acrosin, that binds the proenzyme, proacrosin,
and thereby aides in
packaging the enzyme into the acrosomal matrix (Baba, T. et al. (1994) J.
Biol. Chem. 269:10133-
10140). The Kunitz family of serine protease inhibitors are characterized by
one or more "Kunitz
domains" containing a series of cysteine residues that are regularly spaced
over approximately 50
~. s amino acid residues and form three intrachain disulfide bonds. Members of
this family include
aprotinin, tissue factor pathway inhibitor (TFPI-1 and TFPI-2), inter-a-
trypsin inhibitor, and bikunin.
(Marlor, C.W. et al. (1997) J. Biol. Chem. 272:12202-12208.) Members of this
family are potent
inhibitors (in the nanomolar range) against serine proteases such as
kallikrein and plasmin. Aprotinin
has clinical utility in reduction of perioperative blood loss.
2 o A major portion of all proteins synthesized in eukaryotic cells are
synthesized on the
cytosolic surface of the endoplasmic reticulum (ER). Before these immature
proteins are distributed
to other organelles in the cell or are secreted, they must be transported into
the interior lumen of the
ER where post-translational modifications are performed. These modifications
include protein folding
and the formation of disulfide bonds, and N-linked glycosylations.
2 s Protein Isomerases
Protein folding in the ER is aided by two principal types of protein
isomerases, protein
disulfide isomerase (PDI), and peptidyl-prolyl isomerase (PPI). PDI catalyzes
the oxidation of free
sulfhydryl groups in cysteine residues to form intramolecular disulfide bonds
in proteins. PPI, an
enzyme that catalyzes the isomerization of certain proline imidic bonds in
oligopeptides and proteins,
3 o is considered to govern one of the rate limiting steps in the folding of
many proteins to their final
functional conformation. The cyclophilins represent a major class of PPI that
was originally
identified as the major receptor for the immunosuppressive drug cyclosporin A
(Handschumacher,
R.E. et al. (1984) Science 226: 544-547).
Protein Glycosylation
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The glycosylation of most soluble secreted and membrane-bound proteins by
oligosaccharides linked to asparagine residues in proteins is also performed
in the ER. This reaction
is catalyzed by a membrane-bound enzyme, oligosaccharyl transferase. Although
the exact purpose
of this "N-linked" glycosylation is unknown, the presence of oligosaccharides
tends to make a
s glycoprotein resistant to protease digestion. In addition, oligosaccharides
attached to cell-surface
proteins called selectins are known to function in cell-cell adhesion
processes (Alberts, B. et al.
(1994) Molecular Biolog, ofd the Cell, Garland Publishing Co., New York NY,
p.608). "O-linked"
glycosylation of proteins also occurs in the ER by the addition of N-
acetylgalactosamine to the
hydroxyl group of a serine or threonine residue followed by the sequential
addition of other sugar
s o residues to the first. This process is catalysed by a series of
glycosyltransferases each specific for a
particular donor sugar nucleotide and acceptor molecule (Lodish, H. et al.
(1995) Molecular Cell
Biolo~v, W.H. Freeman and Co., New York NY, pp.700-708). In many cases, bath N-
and O-linked
oligosaccharides appear to be required for the secretion of proteins or the
movement of plasma
membrane glycoproteins to the cell surface.
1 s An additional glycosylation mechanism operates in the ER specifically to
target lysosomal
enzymes to lysosomes and prevent their secretion. Lysosomal enzymes in the ER
receive an N-linked
oligosaccharide, like plasma membrane and secreted proteins, but are then
phospharylated on one or
two mannose residues. The phosphorylation of mannose residues occurs in two
steps, the first step
being the addition of an N-acetylglucasamine phosphate residue by N-
acetylglucosamine
2 o phosphotransferase, and the second the removal of the N-acetylglucosamine
group by
phosphodiesterase. 'The phosphorylated mannose residue then targets the
lysosomal enzyme to a
mannose 6-phosphate receptor which transports it to a lysosome vesicle
(Lodish, supra, pp. 708-711).
Chaperones
Molecular chaperones are proteins that aid in the proper folding of immature
proteins and
2 s refolding of improperly folded ones, the assembly of protein subunits, and
in the transport of
unfolded proteins across membranes. Chaperones are also called heat-shock
proteins (hsp) because of
their tendency to be expressed in dramatically increased amounts following
brief exposure of cells to
elevated temperatures. This latter property most likely reflects their need in
the refolding of proteins
that have become denatured by the high temperatures. Chaperones may be divided
into several
3 o classes according to their location, function, and molecular weight, and
include hsp60, TCPI, hsp70,
hsp40 (also called DnaJ), and hsp90. For example, hsp90 binds to steroid
hormone receptors,
represses transcription in the absence of the ligand, and provides proper
folding of the ligand-binding
domain of the receptor in the presence of the hormone (Burston, S.G. and A.R.
Clarke (1995) Essays
Biochem. 29:125-136). Hsp60 and hsp70 chaperones aid in the transport and
folding of newly
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synthesized proteins. Hsp70 acts early in protein folding, binding a newly
synthesized protein before
it leaves the ribosome and transporting the protein to the mitochondria or ER
before releasing the
folded protein. Hsp60, along with hspl0, binds misfolded proteins and gives
them the opportunity to
refold correctly. All chaperones share an affinity for hydrophobic patches on
incompletely folded
s proteins and the ability to hydrolyze ATP. The energy of ATP hydrolysis is
used to release the hsp-
bound protein in its properly folded state (Alberts, supra, pp 214, 571-572).
Nucleic Acid Synthesis and Modification Molecules
Polymerases
Z o DNA and RNA replication are critical processes for cell replication and
function. DNA and
RNA replication are mediated by the enzymes DNA and RNA polymerase,
respectively, by a
"templating" process in which the nucleotide sequence of a DNA or RNA strand
is copied by
complementary base-pairing into a complementary nucleic acid sequence of
either DNA or RNA.
However, there are fundamental'differences between the two processes.
15 DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to
the 3'-OH end
of a polynucleotide strand (the primer strand) that is paired to a second
(template) strand. The new
DNA strand therefore grows in the 5' to 3' direction (Alberts, B. et al.
(1994)The Molecular Biolo~y
of the Cell, Garland Publishing Inc., New York NY, pp. 251-254). The
substrates for the
polymerization reaction are the corresponding deoxynucleotide triphosphates
which must base-pair
2 o with the correct nucleotide on the template strand in order to be
recognized by the polymerase. '
Because DNA exists as a double-stranded helix, each of the two strands may
serve as a template for
the formation of a new complementary strand. Each of the two daughter cells of
the dividing cell
therefore inherits a new DNA double helix containing one old and one new
strand. Thus, DNA is
said to be replicated "semiconservatively" by DNA polymerase. In addition to
the synthesis of new
2 s DNA, DNA polymerase is also involved in the repair of damaged DNA as
discussed below under
"Ligases."
In contrast to DNA polymerase, RNA polymerase uses a DNA template strand to
"transcribe"
DNA into RNA using ribonucleotide triphosphates as substrates. Like DNA
polymerization, RNA
polymerization proceeds in a 5' to 3' direction by addition of a
ribonucleoside monophosphate to the
3~0 3'-OH end of a growing RNA chain. DNA transcription generates messenger
RNAs (mRNA) that
carry information for protein synthesis, as well as the transfer, ribosomal,
and other RNAs that have
structural or catalytic functions. In eukaryotes, three discrete RNA
polymerases synthesize the three
different types of RNA (Alberts, su ra, pp. 367-368). RNA polymerase I makes
the large ribosomal
RNAs, RNA polymerase II makes the mRNAs that will be translated into proteins,
and RNA
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polymerase III makes a variety of small, stable RNAs, including SS ribosomal
RNA and the transfer
RNAs (tRNA). In all cases, RNA synthesis is initiated by binding of the RNA
polymerase to a
promoter region on the DNA and synthesis begins at a start site within the
promoter. Synthesis is
completed at a broad, general stop or termination region in the DNA where both
the polymerase and
s the completed RNA chain are released.
L_ i~ases
DNA repair is the process by which accidental base changes, such as those
produced by
oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA are
corrected before
replication or transcription of the DNA can occur. Because of the efficiency
of the DNA repair
1 o process, fewer than one in one thousand accidental base changes causes a
mutation (Alberts, sunia,
pp. 245-249). The three steps common to most types of DNA repair are (1)
excision of the damaged
or altered base or nucleotide by DNA nucleases, leaving a gap; (2) insertion
of the correct nucleotide
in this gap by DNA polymerase using the complementary strand as the template;
and (3) sealing the
break left between the inserted nucleotides) and the existing DNA strand by
DNA ligase. In the last
s s reaction, DNA ligase uses the energy from ATP hydrolysis to activate the
5' end of the broken
phosphodiester bond before forming the new bond with the 3'-OH of the DNA
strand. In Bloom's
syndrome, an inherited human disease, individuals are partially deficient in
DNA ligation and
consequently have an increased incidence of cancer (Alberts, supra, p. 247).
Nucleases
2 o Nucleases comprise both enzymes that hydrolyze DNA (DNase) and RNA
(RNase). They
serve different purposes in nucleic acid metabolism. Nucleases hydrolyze the
phosphodiester bonds
between adjacent nucleotides either at internal positions (endonucleases) or
at the terminal 3' or 5'
nucleotide positions (exonucleases). A DNA exonuclease activity in DNA
polymerase, for example,
serves to remove improperly paired nucleotides attached to the 3'-OH end of
the growing DNA strand
2 s by the polymerase and thereby serves a "proofreading" function. As
mentioned above, DNA
endonuclease activity is involved in the excision step of the DNA repair
process.
RNases also serve a variety of functions. For example, RNase P is a
ribonucleoprotein
enzyme which cleaves the 5' end of pre-tRNAs as part of their maturation
process. RNase H digests
the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by
retroviruses, and
3 o RNase H is an important enzyme in the retroviral replication cycle.
Pancreatic RNase secreted by the
pancreas into the intestine hydrolyzes RNA present in ingested foods. RNase
activity in serum and
cell extracts is elevated in a variety of cancers and infectious diseases
(Schein, C.H. (1997) Nat.
Biotechnol. 15:529-536). Regulation of RNase activity is being investigated as
a means to control
tumor angiogenesis, allergic reactions, viral infection and replication, and
fungal infections.
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Methvlases
Methylation of specific nucleotides occurs in both DNA and RNA, and serves
different
functions in the two macromolecules. Methylation of cytosine residues to form
5-methyl cytosine in
DNA occurs specifically at CG sequences which are base-paired with one another
in the DNA double-
s helix. This pattern of methylation is passed from generation to generation
during DNA replication by
an enzyme called "maintenance methylase" that acts preferentially on those CG
sequences that are
base-paired with a CG sequence that is already methylated. Such methylation
appears to distinguish
active from inactive genes by preventing the binding of regulatory proteins
that "turn on" the gene,
but permit the binding of proteins that inactivate the gene (Alberts, s_ upra,
pp. 448-451). In RNA
s o metabolism, "tRNA methylase" produces one of several nucleotide
modifications in tRNA that affect
the conformation and base-pairing of the molecule and facilitate the
recognition of the appropriate
mRNA codons by specific tRNAs. The primary methylation pattern is the
dimethylation of guanine
residues to form N,N-dimethyl guanine.
Helicases and Sinele-Stranded Binding Proteins
15 Helicases are enzymes that destabilize and unwind double helix structures
in both DNA and
RNA. Since DNA replication occurs more or less simultaneously on both strands,
the two strands
must first separate to generate a replication "fork" for DNA polymerase to act
on. Two types of
replication proteins contribute to this process, DNA helicases and single-
stranded binding proteins.
DNA helicases hydrolyze ATP and use the energy of hydrolysis to separate the
DNA strands. Single-
2 o stranded binding proteins (SSBs) then bind to the exposed DNA strands
without covering the bases,
thereby temporarily stabilizing them for templating by the DNA polymerase
(Alberts, su ra, pp. 255-
256).
RNA helicases also alter and regulate RNA conformation and secondary
structure. Like the
DNA helicases, RNA helicases utilize energy derived from ATP hydrolysis to
destabilize and unwind
2 s RNA duplexes. The most well-characterized and ubiquitous family of RNA
helicases is the DEAD-
box family, so named for the conserved B-type ATP-binding motif which is
diagnostic of proteins in
this family. Over 40 DEAD-box helicases have been identified in organisms as
diverse as bacteria,
insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function
in diverse processes
such as translation initiation, splicing, ribosome assembly, and RNA editing,
transport, and stability.
3 o Some DEAD-box helicases play tissue- and stage-specific roles in
spermatogenesis and
embryogenesis. Overexpression of the DEAD-box 1 protein (DDXl) may play a role
in the
progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R.
et al. (1998) J. Biol.
Chem. 273:21161-21168). These observations suggest that DDX1 may promote or
enhance tumor
progression by altering the normal secondary structure and expression levels
of RNA in cancer cells.
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Other DEAD-box helicases have been implicated either directly or indirectly in
tumorigenesis
(Discussed in Godbout, supra). For example, marine p68 is mutated in
ultraviolet light-induced
tumors, and human DDX6 is located at a chromosomal breakpoint associated with
B-cell lymphoma.
Similarly, a chimeric protein comprised of DDX10 and NUP98, a nucleoporin
protein, may be
s involved in the pathogenesis of certain myeloid malignancies.
Topoisomerases
Besides the need to separate DNA strands prior to replication, the two strands
must be
"unwound" from one another prior to their separation by DNA helicases. This
function is performed
by proteins known as DNA topoisomerases. DNA topoisomerase effectively acts as
a reversible
1 o nuclease that hydrolyzes a phosphodiesterase bond in a DNA strand,
permitting the two strands to
rotate freely about one another to remove the strain of the helix, and then
rejoins the original
phosphodiester bond between the two strands. Two types of DNA topoisomerase
exist, types I and II.
DNA Topoisomerase I causes a single-strand break in a DNA helix to allow the
rotation of the two
strands of the helix about the remaining phosphodiester bond in the opposite
strand. DNA
15 topoisomerase II causes a transient break in both strands of a DNA helix
where two double helices
cross over one another. This type of topoisomerase can efficiently separate
two interlocked DNA
circles (Alberts, su ra, pp.260-262). Type II topoisomerases are largely
confined to proliferating cells
in eukaryotes, such as cancer cells. For this reason they are targets for
anticancer dings.
Topoisomerase II has been implicated in multi-drug resistance (MDR) as it
appears to aid in the repair
2 0 of DNA damage inflicted by DNA binding agents such as doxorubicin and
vincristine.
Recombinases
Genetic recombination is the process of rearranging DNA sequences within an
organism's
genome to provide genetic variation for the organism in response to changes in
the environment.
DNA recombination allows variation in the particular combination of genes
present in an individual's
2 s genome, as well as the timing and level of expression of these genes (see
Alberts, supra, pp. 263-
273). Two broad classes of genetic recombination are commonly recognized,
general recombination
and site-specific recombination. General recombination involves genetic
exchange between any
homologous pair of DNA sequences usually located on two copies of the same
chromosome. The
process is aided by enzymes called recombinases that "nick" one strand of a
DNA duplex more or less
3 o randomly and permit exchange with the complementary strand of another
duplex. The process does
not normally change the arrangement of genes on a chromosome. In site-specific
recombination, the
recombinase recognizes specific nucleotide sequences present in one or both of
the recombining
molecules. Base-pairing is not involved in this form of recombination and
therefore does not require
DNA homology between the recombining molecules. Unlike general recombination,
this form of
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recombination can alter the relative positions of nucleotide sequences in
chromosomes.
Splicing Factors
Various proteins are necessary for processing of transcribed RNAs in the
nucleus. Pre-
mRNA processing steps include capping at the 5' end with methylguanosine,
polyadenylating the 3'
s end, and splicing to remove introns. The primary RNA transcript from DNA is
a faithful copy of the
gene containing both exon and intron sequences, and the latter sequences must
be cut out of fihe RNA
transcript to produce an mRNA that codes for a protein. This "splicing" of the
mRNA sequence takes
place in the nucleus with the aid of a large, multicomponent ribonucleoprotein
complex known as a
spliceosome. The spliceosomal complex is composed of five small nuclear
ribonucleoprotein
to particles (snRNPs) designated Ul, U2, U4, U5, and U6, and a number of
additional proteins. Each
snRNP contains a single species of snRNA and about ten proteins. The RNA
components of some
snRNPs recognize and base pair with intron consensus sequences. The protein
components mediate
spliceosome assembly and the splicing reaction. Autoantibodies to snRNP
proteins are found in the
blood of patients with systemic lupus erythematosus (Stryer, L. (1995)
Biochemistry, W.H. Freeman
is and Company, New York NY, p. 863).
Adhesion Molecules
The surface of a cell is rich in transmembrane proteoglycans, glycoproteins,
glycolipids, and
receptors. These macromolecules mediate adhesion with other cells and with
components of the
2 o extracellular matrix (ECM). The interaction of the cell with its
surroundings profoundly influences
cell shape, strength, flexibility, motility, and adhesion. These dynamic
properties are intimately
associated with signal transduction pathways controlling cell proliferation
and differentiation, tissue
construction, and embryonic development.
Cadherins
2 s Cadherins comprise a family of calcium-dependent glycoproteins that
function in mediating
cell-cell adhesion in virtually all solid tissues of multicellular organisms.
These proteins share
multiple repeats of a cadherin-specific motif, and the repeats form the
folding units of the cadherin
extracellular domain. Cadherin molecules cooperate to form focal contacts, or
adhesion plaques,
between adjacent epithelial cells. The cadherin family includes the classical
cadherins and
3 o protocadherins. Classical cadherins include the E-cadherin, N-cadherin,
and P-cadherin subfamilies.
E-cadherin is present on many types of epithelial cells and is especially
important for embryonic
development. N-cadherin is present on nerve, muscle, and lens cells and is
also critical for embryonic
development. P-cadherin is present on cells of the placenta and epidermis.
Recent studies report that
protocadherins are involved in a variety of cell-cell interactions (Suzuki,
S.T. (1996) J. Cell Sci.
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109:2609-2611). The intracellular anchorage of cadherins is regulated by their
dynamic association
with catenins, a family of cytoplasmic signal transduction proteins associated
with the actin
cytoskeleton. The anchorage of cadherins to the actin cytoskeleton appears to
be regulated by protein
tyrosine phosphorylation, and the cadherins are the target of phosphorylation-
induced functional
s disassembly (Aberle, H. et al. (1996) J. Cell. Biochem. 61:514-523).
nte rins
Integrins are ubiquitous transmembrane adhesion molecules that link the ECM to
the internal
cytoskeleton. Integrins are composed of two noncovalently associated
transmembrane glycoprotein
subunits called a and (3. Integrins function as receptors that play a role in
signal transduction. For
1 o example, binding of integrin to its extracellular ligand may stimulate
changes in intracellular calcium
levels or protein kinase activity (Sjaastad, M.D. and W.J. Nelson (1997)
BioEssays 19:47-55). At
least ten cell surface receptors of the integrin family recognize the ECM
component fibronectin,
which is involved in many different biological processes including cell
migration and embryogenesis
(Johansson, S. et al. (1997) Front. Biosci. 2:D126-D146).
~. s Lectins
Lectins comprise a ubiquitous family of extracellular glycoproteins which bind
cell surface
carbohydrates specifically and reversibly, resulting in the agglutination of
cells (reviewed in
Drickamer, K. and M.E. Taylor (1993) Annu. Rev. Cell Biol. 9:237-264). This
function is
particularly important for activation of the immune response. Lectins mediate
the agglutination and
2 o mitogenic stimulation of lymphocytes at sites of inflammation (Lasky, L.A.
(1991) J. Cell. Biochem.
45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857).
Lectins are further classified into subfamilies based on carbohydrate-binding
specificity and
other criteria. The galectin subfamily, in particular, includes lectins that
bind (3-galactoside
carbohydrate moieties in a thiol-dependent manner (reviewed in Hadaxi, Y.R. et
al. (1998) J. Biol.
2s Chem. 270:3447-3453). Galectins are widely expressed and developmentally
regulated. Because all
galectins lack an N-terminal signal peptide, it is suggested that galectins
are externalized through an
atypical secretory mechanism. Two classes of galectins have been defined based
on molecular weight
and oligomerization properties. Small galectins form homodimers and are about
14 to 16 kilodaltons
in mass, while large galectins are monomeric and about 29-37 kilodaltons.
3 o Galectins contain a characteristic carbohydrate recognition domain (CRD).
The CRD is
about 140 amino acids and contains several stretches of about 1 - 10 amino
acids which are highly
conserved among all galectins. A particular 6-amino acid motif within the CRD
contains conserved
tryptophan and arginine residues which are critical for carbohydrate binding.
The CRD of some
galectins also contains cysteine residues which may be important for disulfide
bond formation.
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Secondary structure predictions indicate that the CRD forms several ~3-sheets.
Galectins play a number of roles in diseases and conditions associated with
cell-cell and cell-
matrix interactions. For example, certain galectins associate with sites of
inflammation and bind to
cell surface immunoglobulin E molecules. In addition, galectins may play an
important role in cancer
s metastasis. Galectin overexpression is correlated with the metastatic
potential of cancers in humans
and mice. Moreover, anti-galectin antibodies inhibit processes associated with
cell transformation,
such as cell aggregation and anchorage-independent growth (See, for example,
Su, Z.-Z. et al. (1996)
Proc. Natl. Acad. Sci. USA 93:7252-7257).
Selectins
s o Selectins, or LEC-CAMS, comprise a specialized lectin subfamily involved
primarily in
inflammation and leukocyte adhesion (Reviewed in Lasky, su ra). Selectins
mediate the recruitment
of leukocytes from the circulation to sites of acute inflammation and are
expressed on the surface of
vascular endothelial cells in response to cytokine signaling. Selectins bind
to specific ligands on the
leukocyte cell membrane and enable the leukocyte to adhere to and migrate
along the endothelial
s s surface. Binding of selectin to its ligand leads to polarized
rearrangement of the actin cytoskeleton
and stimulates signal transduction within the leukocyte (Brenner, B. et al.
(1997) Biochem. Biophys.
Res. Commun. 231:802-807; Hidari, K.I. et al. (1997) J. Biol. Chem. 272:28750-
28756). Members
of the selectin family possess three characteristic motifs: a lectin or
carbohydrate recognition domain;
an epidermal growth factor-like domain; and a variable number of short
consensus repeats (scr or
2 0 "sushi" repeats) which are also present~.in complement regulatory
proteins. The selectins include
lymphocyte adhesion molecule-1 (Lam-1 or L-selectin), endothelial leukocyte
adhesion molecule-1
(ELAM-1 or E-selectin), and granule membrane protein-140 (GMP-140 or P-
selectin) (Johnston, G.I.
et al. (1989) Cell 56:1033-1044).
25 Antigen Recognition Molecules
A11 vertebrates have developed sophisticated and complex immune systems that
provide
protection from viral, bacterial, fungal, and parasitic infections. A key
feature of the immune system
is its ability to distinguish foreign molecules, or antigens, from "self"
molecules. This ability is
mediated primarily by secreted and transmembrane proteins expressed by
leukocytes (white blood
3 o cells) such as lymphocytes, granulocytes, and monocytes, Most of these
proteins belong to the
immunoglobulin (Ig) superfamily, members of which contain one or more repeats
of a conserved
structural domain. This Ig domain is comprised of antiparallel ~3 sheets
joined by a disulfide bond in
an arrangement called the Ig fold. Members of the Ig superfamily include T-
cell receptors, major
histocompatibility (MHC) proteins, antibodies, and immune cell-specific
surface markers such as
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CD4, CDB, and CD28.
MHC proteins are cell surface markers that bind to and present foreign
antigens to T cells.
MHC molecules are classified as either class I or class II. Class I MHC
molecules (MHC I) are
expressed on the surface of almost all cells and are involved in the
presentation of antigen to
s cytotoxic T cells. For example, a cell infected with virus will degrade
intracellular viral proteins and
express the protein fragments bound to MHC I molecules on the cell surface.
The MHC I/antigen
complex is xecognized by cytotoxic T-cells which destroy the infected cell and
the virus within.
Class II MHC molecules are expressed primarily on specialized antigen-
presenting cells of the
immune system, such as B-cells and macrophages. These cells ingest foreign
proteins from the
z o extracellular fluid and express MHC II/antigen complex on the cell
surface. This complex activates
helper T-cells, which then secrete cytokines and other factors that stimulate
the immune response.
MHC molecules also play an important role in organ rejection following
transplantation. Rejection
occurs when the recipient's T-cells respond to foreign MHC molecules on the
transplanted organ in
the same way as to self MHC molecules bound to foreign antigen. (Reviewed in
Alberts, B. et al.
1 s (1994) Molecular Biolo~y of the Cell, Garland Publishing, New York NY, pp.
1229-1246.)
Antibodies, or immunoglobulins, are either expressed on the surface of B-cells
or secreted by
B-cells into the circulation. Antibodies bind and neutralize foreign antigens
in the blood and other
extracellular fluids. The prototypical antibody is a tetramer consisting of
two identical heavy
polypeptide chains (H-chains) and two identical light polypeptide chains (L-
chains) interlinked by
2 o disulfide bonds. This arrangement confers the characteristic Y-shape to
antibody molecules.
Antibodies are classified based on their H-chain composition. The five
antibody classes, IgA, IgD,
IgE, IgG and IgM, are defined by the a, b, e, y, and ~ H-chain types. There
are two types of L-
chains, x and ~,, either of which may associate as a pair with any H-chain
pair. IgG, the most
common class of antibody found in the circulation, is tetrameric, while the
other classes of antibodies
2 s are generally variants or multimers of this basic structure.
H-chains and L-chains each contain an N-terminal variable region and a C-
terminal constant
region. The constant region consists of about 110 amino acids in L-chains and
about 330 or 440
amino acids in H-chains. The amino acid sequence of the constant region is
nearly identical among
H- or L-chains of a particular class. The variable region consists of about
110 amino acids in both H-
3 o and L-chains. However, the amino acid sequence of the variable region
differs among H- or L-chains
of a particular class. Within each H- or L-chain variable region are three
hypervariable regions of
extensive sequence diversity, each consisting of about 5 to 10 amino acids. In
the antibody molecule,
the H- and L-chain hypervariable regions come together to form the antigen
recognition site.
(Reviewed in Alberts, supra, pp. 1206-1213 and 1216-1217.)
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Both H-chains and L-chains contain repeated Ig domains. For example, a typical
H-chain
contains four Ig domains, three of which occur within the constant region and
one of which occurs
within the variable region and contributes to the formation of the antigen
recognition site. Likewise,
a typical L-chain contains two Ig domains, one of which occurs within the
constant region and one of
which occurs within the variable region.
The immune system is capable of recognizing and responding to any foreign
molecule that
enters the body. Therefore, the immune system must be armed with a full
repertoire of antibodies
against all potential antigens. Such antibody diversity is generated by
somatic rearrangement of gene
segments encoding variable and constant regions. These gene segments are
joined together by site-
1 o specific recombination which occurs between highly conserved DNA sequences
that flank each gene
segment. Because there are hundreds of different gene segments, millions of
unique genes can be
generated combinatorially. In addition, imprecise joining of these segments
and an unusually high
rate of somatic mutation within these segments further contribute to the
generation of a diverse
antibody population.
1 s T-cell receptors are both structurally and functionally related to
antibodies. (Reviewed in
Alberts, su ra, pp. 1228-1229.) T-cell receptors are cell surface proteins
that bind foreign antigens and
mediate diverse aspects of the immune response. A typical T-cell receptor is a
heterodimer comprised
of two disulfide-linked polypeptide chains called a and (3. Each chain is
about 280 amino acids in
length and contains one variable region and one constant region. Each variable
or constant region folds
2 o into an Ig domain. The variable regions from the a and /3 chains come
together in the heterodimer to
form the antigen recognition site. T-cell receptor diversity is generated by
somatic rearrangement of
gene segments encoding the a and J3 chains. T-cell receptors recognize small
peptide antigens that are
expressed on the surface of antigen-presenting cells and pathogen-infected
cells. These peptide antigens
are presented on the cell surface in association with major histocompatibility
proteins which provide the
2 s proper context for antigen recognition.
Secreted and Extracellular Matrix Molecules
Protein secretion is essential for cellular function. Protein secretion is
mediated by a signal
peptide located at the amino terminus of the protein to be secreted. The
signal peptide is comprised of
3 o about ten to twenty hydrophobic amino acids which target the nascent
protein from the ribosome to the
endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed
through the secxetory
pathway or remain in any of the secretory organelles such as the ER, Golgi
apparatus, or lysosomes.
Proteins that transit through the secretory pathway are either secreted into
the extracellular space or
retained in the plasma membrane. Secreted proteins are often synthesized as
inactive precursors that
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are activated by post-translational processing events during transit through
the secretory pathway.
Such events include glycosylation, proteolysis, and removal of the signal
peptide by a signal peptidase.
Other events that may occur during protein transport include chaperone-
dependent unfolding and
folding of the nascent protein and interaction of the protein with a receptor
or pore complex. Examples
s of secreted proteins with amino terminal signal peptides include receptors,
extracellulax matrix
molecules, cytokines, hormones, growth and differentiation factors,
neuropeptides, vasomediators, ion
channels, fransporters/pumps, and proteases. (Reviewed in Alberts, B. et al.
(1994) Molecular Biolo~y
of The Cell, Garland Publishing, New York NY, pp. 557-560, 582-592.)
The extracellular matrix (ECM) is a complex network of glycoproteins,
polysaccharides,
1 o proteoglycans, and other macromolecules that are secreted from the cell
into the extracellular space.
The ECM remains in close association with the cell surface and provides a
supportive meshwork that
profoundly influences cell shape, motility, strength, flexibility, and
adhesion. In fact, adhesion of a
cell to its surrounding matrix is required for cell survival except in the
case of metastatic tumor cells,
which have overcome the need for cell-ECM anchorage. This phenomenon suggests
that the ECM
15 plays a critical role in the molecular mechanisms of growth control and
metastasis. (Reviewed in
Ruoslahti, E, (1996) Sci. Am. 275:72-77.) Furthermore, the ECM determines the
structure and
physical properties of connective tissue and is particularly important for
morphogenesis and other
processes associated with embryonic development and pattern formation.
The collagens comprise a family of ECM proteins that provide structure to
bone, teeth, skin,
2 0 ligaments, tendons, cartilage, blood vessels, and basement membranes.
Multiple collagen proteins have
been identified. Three collagen molecules fold together in a triple helix
stabilized by interchain disulfide
bonds. Bundles of these triple helices then associate to form fibrils.
Collagen primary structure
consists of hundreds of (Gly-X-Y) repeats where about a third of the X and Y
residues are Pro.
Glycines are crucial to helix formation as the bulkier amino acid sidechains
cannot fold into the triple
2 s helical conformation. Because of these strict sequence requirements,
mutations in collagen genes have
severe consequences. Osteogenesis imperfecta patients have brittle bones that
fracture easily; in severe
cases patients die in utero or at birth. Ehlers-Danlos syndrome patients have
hyperelastic skin,
hypermobile joints, and susceptibility to aortic and intestinal rupture.
Chondrodysplasia patients have
short stature and ocular disorders. Alport syndrome patients have hematuria,
sensorineuxal deafness,
3 o and eye lens deformation. (Isselbacher, K.J. et al. (1994) Harrison's
Principles of Internal Medicine,
McGraw-Hill, Inc., New York NY, pp. 2105-2117; and Creighton, T.E. (1984)
Proteins, Structures
and Molecular Principles, W.H. Freeman and Company, New York NY, pp. 191-197.)
Elastin and related proteins confer elasticity to tissues such as skin, blood
vessels, and lungs.
Elastin is a highly hydrophobic protein of about 750 amino acids that is rich
in pxoline and glycine
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residues. Elastin molecules are highly cross-linked, forming an extensive
extracellular network of fibers
and sheets. Elastin fibers are surrounded by a sheath of microfibrils which
are composed of a number
of glycoproteins, including fibrillin. Mutations in the gene encoding
fibrillin are responsible for
Marfan's syndrome, a genetic disorder characterized by defects in connective
tissue. In severe cases,
s the aortas of afflicted individuals are prone to rupture. (Reviewed in
Alberts, supra, pp. 984-986.)
Fibronectin is a large ECM glycoprotein found in all vertebrates. Fibronectin
exists as a dimer
of two subunits, each containing about 2,500 amino acids. Each subunit folds
into a rod-like structure
containing multiple domains. The domains each contain multiple repeated
modules, the most common
of which is the type III fibronectin repeat. The type III fibronectin repeat
is about 90 amino acids in
s o length and is also found in other ECM proteins and in some plasma membrane
and cytoplasmic
proteins. Furthermore, some type III fibronectin repeats contain a
characteristic tripeptide consisting of
Arginine-Glycine-Aspartic acid (RGD). The RGD sequence is recognized by the
integrin family of cell
surface receptors and is also found in other ECM proteins. Disruption of both
copies of the gene
encoding fibronectin causes early embryonic lethality in mice. The mutant
embryos display extensive
1 s morphological defects, including defects in the formation of the
notochord, somites, heart, blood
vessels, neural tube, and extraembryonic structures. (Reviewed in Alberts, s-
upra, pp. 986-987.)
Laminin is a major glycoprotein component of the basal lamina which underlies
and supports
epithelial cell sheets. Laminin is one of the first ECM proteins synthesized
in the developing embryo.
Laminin is an 850 kilodalton protein composed of three polypeptide chains
joined in the shape of a
2 o cross by disulfide bonds. Laminin is especially important for angiogenesis
and in particular, for
guiding the formation of capillaries. (Reviewed in Alberts, supra, pp. 990-
991.)
There are many other types of proteinaceous ECM components, most of which can
be
classified as proteoglycans. Proteoglycans are composed of unbranched
polysaccharide chains
(glycosaminoglycans) attached to protein cores. Common proteoglycans include
aggrecan, betaglycan,
2 s decorin, perlecan, serglycin, and syndecan-1. Some of these molecules not
only provide mechanical
support, but also bind to extracellular signaling molecules, such as
fibroblast growth factor and
transforming growth factor (3, suggesting a role for proteoglycans in cell-
cell communication and cell
growth. (Reviewed in Alberts, su ra, pp. 973-978.) Likewise, the glycoproteins
tenascin-C and
tenascin-R are expressed in developing and lesioned neural tissue and provide
stimulatory and anti-
c o adhesive (inhibitory) properties, respectively, for axonal growth.
(Faissner, A. (1997) Cell Tissue Res.
290:331-341.)
Cytoskeletal Molecules
The cytoskeleton is a cytoplasmic network of protein fibers that mediate cell
shape, structure,
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and movement. The cytoskeleton supports the cell membrane and forms tracks
along which
organelles and other elements move in the cytosol. The cytoskeleton is a
dynamic structure that
allows cells to adopt various shapes and to carry out directed movements.
Major cytoskeletal fibers
include the microtubules, the microfilaments, and the intermediate filaments.
Motor proteins,
s including myosin, dynein, and kinesin, drive movement of or along the
fibers. The motor protein
dynamin drives the formation of membrane vesicles. Accessory or associated
proteins modify the
structure or activity of the fibexs while cytoskeletal membrane anchors
connect the fibers to the cell
membrane.
Tubulins
1 o Microtubules, cytoskeletal fibers with a diameter of about 24 nm, have
multiple roles in the
cell. Bundles of microtubules form cilia and flagella, which are whip-like
extensions of the cell
membrane that are necessary for sweeping materials across an epithelium and
for swimming of
sperm, respectively. Marginal bands of microtubules in red blood cells and
platelets are important for
these cells' pliability. Organelles, membrane vesicles, and proteins are
transported in the cell along
15 tracks of microtubules. For example, microtubules run through nerve cell
axons, allowing bi-
directional transport of materials and membrane vesicles between the cell body
and the nerve
terminal. Failure to supply the nerve terminal with these vesicles blocks the
transmission of neural
signals. Microtubules are also critical to chromosomal movement during cell
division. Both stable'
and short-lived populations of microtubules exist in the cell.
2 o Microtubules are polymers of GTP-binding tubulin protein subunits. Each
subunit is a
heterodimer of a- and (3- tubulin, multiple isoforms of which exist. The
hydrolysis of GTP is linked
to the addition of tubulin subunits at the end of a microtubule. The subunits
interact head to tail to
form protofilaments; the protofilaments interact side to side to form a
microtubule. A microtubule is
polarized, one end ringed with a-tubulin and the other with (3-tubulin, and
the two ends differ in their
2 s rates of assembly. Generally, each microtubule is composed of 13
protofilaments although 11 or 15
protofilament-microtubules are sometimes found. Cilia and flagella contain
doublet microtubules.
Mcrotubules grow from specialized structures known as centrosomes or
microtubule-organizing
centers (MTOCs). MTOCs may contain one or two centrioles, which are pinwheel
arrays of triplet
microtubules. The basal body, the organizing center located at the base of a
cilium or flagellum,
3 o contains one centriole. Gamma tubulin present in the MTOC is important for
nucleating the
polymerization of a- and (3- tubulin heterodimers but does not polymerize into
microtubules.
Microtubule-Associated Proteins
Microtubule-associated proteins (MAPs) have roles in the assembly and
stabilization of
microtubules. One major family of MAPs, assembly MAPs, can be identified in
neurons as well as
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non-neuronal cells. Assembly MAPs are responsible for cross-linking
microtubules in the cytosol.
These MAPS are organized into two domains: a basic microtubule-binding domain
and an acidic
projection domain. The projection domain is the binding site for membranes,
intermediate filaments, or
other microtubules. Based on sequence analysis, assembly MAPs can be further
grouped into two
s types: Type I and Type II. Type I MAPs, which include MAP1A and MAP1B, are
large, filamentous
molecules that co-purify with microtubules and are abundantly expressed in
brain and testes. Type I
MAPs contain several repeats of a positively-charged amino acid sequence motif
that binds and
neutralizes negatively charged tubulin, leading to stabilization of
microtubules. MAPlA and MAP1B
are each derived from a single precursor polypeptide that is subsequently
proteolytically processed to
1 o generate one heavy chain and one light chain.
Another light chain, LC3, is a 16.4 kDa molecule that binds MAP1A, MAP1B, and
microtubules. It is suggested that LC3 is synthesized from a source other than
the MAP1A or MAP1B
transcripts, and that the expression of LC3 may be important in regulating the
microtubule binding
activity of MAP1A and MAP1B during cell proliferation (Mann, S.S. et al.
(1994) J. Biol. Chem.
15 269:11492-11497).
Type II MAPs, which include MAP2a, MAP2b, MAP2c, MAP4, and Tau, are
characterized
by three to four copies of an 18-residue sequence in the microtubule-binding
domain. MAP2a, MAP2b,
and MAP2c are found only in dendrites, MAP4 is found in non-neuronal cells,
and Tau is found in
axons and dendrites of nerve cells. Alternative splicing of the Tau mRNA leads
to the existence of
2 o multiple forms of Tau protein. Tau phosphorylation is altered in
neurodegenerative disorders such as
Alzheimer's disease, Pick's disease, progressive supranuclear palsy,
corticobasal degeneration, and
familial frontotemporal dementia and Parkinsonism linked to chromosome 17. The
altered Tau
phosphorylation leads to a collapse of the microtubule network and the
formation of intraneuronal
Tau aggregates (Spillantini, M.G. and M. Goedert (1998) Trends Neurosci.
21:428-433).
2 5 The protein pericentrin is found in the MTOC and has a role in microtubule
assembly.
Actins
Microfilaments, cytoskeletal filaments with a diameter of about 7-9 nm, are
vital to cell
locomotion, cell shape, cell adhesion, cell division, and muscle contraction.
Assembly and
disassembly of the microfilaments allow cells to change their morphology.
Microfilaments are the
3 o polymerized form of actin, the most abundant intracellular protein in the
eukaryotic cell. Human cells
contain six isoforms of actin. The three a-actins are found in different kinds
of muscle, nonmuscle (3-
actin and nonmuscle 'y-actin are found in nonmuscle cells, and another 'y-
actin is found in intestinal
smooth muscle cells. G-actin, the monomeric form of actin, polymerizes into
polarized, helical F-
actin filaments, accompanied by the hydrolysis of ATP to ADP. Actin filaments
associate to form
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bundles and networks, providing a framework to support the plasma membrane and
determine cell
shape. These bundles and networks are connected to the cell membrane. In
muscle cells, thin
filaments containing actin slide past thick filaments containing the motor
protein myosin during
contraction. A family of actin-related proteins exist that are not part of the
actin cytoskeleton, but
s rather associate with microtubules and dynein.
Actin-Associated Proteins
Actin-associated proteins have roles in cross-linking, severing, and
stabilization of actin
filaments and in sequestering actin monomers. Several of the actin-associated
proteins have multiple
functions. Bundles and networks of actin filaments are held together by actin
cross-linking proteins.
s o These proteins have two actin-binding sites, one for each filament. Short
cross-linking proteins
promote bundle formation while longer, more flexible cross-linking proteins
promote network
formation. Calmodulin-like calcium-binding domains in actin cross-linking
proteins allow calcium
regulation of cross-linking. Group I cross-linking proteins have unique actin-
binding domains and
include the 30 kD protein, EF-1 a, fascin, and scrum. Group II cross-linking
proteins have a 7,000-
15 MW actin-binding domain and include villin and dematin. Group III cross-
linking proteins have
pairs of a 26,000-MW actin-binding domain and include fimbrin, spectrin,
dystrophin, ABP 120, and
filamin.
Severing proteins regulate the length of actin filaments by breaking them into
shoxt pieces or
by blocking their ends. Severing proteins include gCAP39, severin (fragmin),
gelsolin, and villin.
2 o Capping proteins can cap the ends of actin filaments, but cannot break
filaments. Capping proteins
include CapZ and tropomodulin. The proteins thymosin and profilin sequester
actin monomers in the
cytosol, allowing a pool of unpolymerized actin to exist. The actin-associated
proteins tropomyosin,
troponin, and caldesmon regulate muscle contraction in response to calcium.
Intermediate Filaments and Associated Proteins
2 s Intermediate filaments (IFs) are cytoskeletal fibers with a diameter of
about 10 nm,
intermediate between that of microfilaments and microtubules. IFs serve
structural roles in the cell,
reinforcing cells and organizing cells into tissues. IFs are particularly
abundant in epidermal cells and
in neurons. IFs are extremely stable, and, in contrast to microfilaments and
microtubules, do not
function in cell motility.
3 o Five types of IF proteins are known in mammals. Type I and Type II
proteins are the acidic
and basic keratins, respectively. Heterodimers of the acidic and basic
keratins are the building blocks
of keratin lFs. Keratins are abundant in soft epithelia such as skin and
cornea, hard epithelia such as
nails and hair, and in epithelia that Line internal body cavities. Mutations
in keratin genes lead to
epithelial diseases including epidermolysis bullosa simplex, bullous
congenital ichthyosiform
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erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and
epidermolytic palmoplantar
keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white
sponge nevus. Some of
these diseases result in severe skin blistering. (See, e.g., Wawersik, M. et
al. (1997) J. Biol. Chem.
272:32557-32565; and Corden L.D. and W.H. McLean (1996) Exp. Dermatol. 5:297-
307.)
s Type III IF proteins include desmin, filial fibrillary acidic protein,
vimentin, and peripherin.
Desmin filaments in muscle cells link myofibrils into bundles and stabilize
sarcomeres in contracting
muscle. Glial fibrillary acidic protein filaments are found in the filial
cells that surround neurons and
astrocytes. Vimentin filaments are found in blood vessel endothelial cells,
some epithelial cells, and
mesenchymal cells such as fibxoblasts, and are commonly associated with
microtubules. Vimentin
s o filaments may have roles in keeping the nucleus and other organelles in
place in the cell. Type IV IFs
include the neurofilaments and nestin. Neurofilaments, composed of three
polypeptides NF-L, NF-M,
and NF-H, axe frequently associated with microtubules in axons. Neurofilaments
are responsible for
the radial growth and diameter of an axon, and ultimately for the speed of
nerve impulse transmission.
Changes in phosphorylation and metabolism of neurofilaments are observed in
neurodegenerative
15 diseases including amyotrophic lateral sclerosis, Parkinson's disease, and
Alzheimer's disease (Julien,
J.P. and W.E. Mushynski (1998) Prog. Nucleic Acid Res. Mol. Biol. 61:1-23).
Type V IFs, the lamins,
are found in the nucleus where they support the nuclear membrane.
IFs have a central a-helical rod region interrupted by short nonhelical linker
segments. The rod
region is bracketed, in most cases, by non-helical head and tail domains. The
rod regions of
2 0 intermediate filament proteins associate to form a coiled-coil dimer. A
highly ordered assembly process
leads from the dimers to the IFs. Neither ATP nor GTP is needed for IF
assembly, unlike that of
microfilaments and microtubules.
IF-associated proteins (IFAPs) mediate the interactions of IFs with one
another and with other
cell structures. IFAPs cross-link IFs into a bundle, into a network, or to the
plasma membrane, and
2 s may cross-link IFs to the microfilament and microtubule cytoskeleton.
Microtubules and IFs are in
particular closely associated. IFAPs include BPAGl, plakoglobin, desmoplakin
I, desmoplakin II,
plectin, ankyrin, filaggrin, and lamin B receptor.
Cytoskeletal-Membrane Anchors
Cytoskeletal fibers are attached to the plasma membrane by specific proteins.
These
3 o attachments are important for maintaining cell shape and for muscle
contraction. In erythrocytes, the
spectrin-actin cytoskeleton is attached to cell membrane by three proteins,
band 4.1, ankyrin, and
adducin. Defects in this attachment result in abnormally shaped cells which
are more rapidly
degraded by the spleen, leading to anemia. In platelets, the spectrin-actin
cytoskeleton is also linked
to the membrane by ankyrin; a second actin network is anchored to the membrane
by filaxnin. In
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muscle cells the protein dystrophin links actin filaments to the plasma
membrane; mutations in the
dystrophin gene lead to Duchenne muscular dystrophy. In adherens junctions and
adhesion plaques
the peripheral membrane proteins a-actinin and vinculin attach actin filaments
to the cell membrane.
IFs are also attached to membranes by cytoskeletal-membrane anchors. The
nuclear lamina is
s attached to the inner surface of the nuclear membrane by the lamin B
receptor. Vimentin IFs are
attached to the plasma membrane by ankyrin and plectin. Desmosome and
hemidesmosome
membrane junctions hold together epithelial cells of organs and skin. These
membrane junctions
allow shear forces to be distributed across the entire epithelial cell layer,
thus providing strength and
rigidity to the epithelium. IFs in epithelial cells are attached to the
desmosome by plakoglobin and
s o desmoplakins. The proteins that link IFs to hemidesmosomes are not known.
Desmin IFs surround
the sarcomere in muscle and are linked to the plasma membrane by paranemin,
synemin, and ankyrin.
Myosin-related Motor Proteins
Myosins are actin-activated ATPases, found in eukaryotic cells, that couple
hydrolysis of
ATP with motion. Myosin provides the motor function for muscle contraction and
intracellular
15 movements such as phagocytosis and rearrangement of cell contents during
mitotic cell division
(cytokinesis). The contractile unit of skeletal muscle, termed the sarcomere,
consists of highly ordered
arrays of thin actin-containing filaments and thick myosin-containing
filaments. Crossbridges form
between the thick and thin filaments, and the ATP-dependent movement of myosin
heads within the
thick filaments pulls the thin filaments, shortening the sarcomere and thus
the muscle fiber.
2 o Myosins are composed of one or two heavy chains and associated light
chains. Myosin
heavy chains contain an amino-terminal motor or head domain, a neck that is
the site of light-chain
binding, and a carboxy-terminal tail domain. The tail domains may associate to
form an a-helical
coiled coil. Conventional myosins, such as those found in muscle tissue, are
composed of two
myosin heavy-chain subunits, each associated with two light-chain subunits
that bind at the neck
2 s region and play a regulatory role. Unconventional myosins, believed to
function in intracellular
motion, may contain either one or two heavy chains and associated light
chains. There is evidence for
about 25 myosin heavy chain genes in vertebrates, more than half of them
unconventional.
Dynein-related Motor Proteins
Dyneins are (-) end-directed motor proteins which act on microtubules. Two
classes of
3 o dyneins, cytosolic and axonemal, have been identified. Cytosolic dyneins
are responsible for
translocation of materials along cytoplasmic microtubules, for example,
transport from the nerve
terminal to the cell body and transport of endocytic vesicles to lysosomes.
Cytoplasmic dyneins are
also reported to play a role in mitosis. Axonemal dyneins are responsible for
the beating of flagella and
cilia. Dynein on one microtubule doublet walks along the adjacent microtubule
doublet. This sliding
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force produces bending forces that cause the flagellum or cilium to beat.
Dyneins have a native mass
between 1000 and 2000 kDa and contain either two or three force-producing
heads driven by the
hydrolysis of ATP. The heads are linked via stalks to a basal domain which is
composed of a highly
variable number of accessory intermediate and light chains.
s I~inesin-related Motor Proteins
Kinesins are (+) end-directed motor proteins which act on microtubules. The
prototypical
kinesin molecule is involved in the transport of membrane-bound vesicles and
organelles. This function
is particularly important for axonal transport in neurons. Kinesin is also
important in all cell types for
the transport of vesicles from the Golgi complex to the endoplasmic reticulum.
This role is critical for
1 o maintaining the identity and functionality of these secretory organelles.
Kinesins define a ubiquitous, conserved family of over 50 proteins that can be
classified into at
least 8 subfamilies based on primary amino acid sequence, domain structure,
velocity of movement, and
cellular function. (Reviewed in Moore, J.D. and S.A. Endow (1996) Bioessays
18:207-219; and Hoyt,
A.M. (1994) Curr. Opin. Cell Biol. 6:63-68.) The prototypical kinesin molecule
is a heterotetramer
is comprised of two heavy polypeptide chains (I~HCs) and two light polypeptide
chains (I~L,Cs). The
I~HC subunits are typically referred to as "kinesin." KHC is about 1000 amino
acids in length, and
KLC is about 550 amino acids in length. Two KHCs dimerize to form a rod-shaped
molecule with
three distinct regions of secondary structure. At one end of the molecule is a
globular motor domain
that functions in ATP hydrolysis and microtubule binding. Kinesin motor
domains are highly conserved
2 o and share over 70% identity. Beyond the motor domain is an a-helical
coiled-coil region which
mediates dimerization. At the other end of the molecule is a fan-shaped tail
that associates with
molecular cargo. The tail is formed by the interaction of the KHC C-termini
with the two KhCs.
Members of the more divergent subfamilies of kinesins are called kinesin-
related proteins
(KRPs), many of which function during mitosis in eukaryotes (Hoyt, su ra).
Some KRPs are required
2 s for assembly of the mitotic spindle. In vivo and in vitro analyses suggest
that these KRPs exert force
on microtubules that comprise the mitotic spindle, resulting in the separation
of spindle poles.
Phosphorylation of KRP is required for this activity. Failure to assemble the
mitotic spindle results in
abortive mitosis a.nd chromosomal aneuploidy, the latter condition being
characteristic of cancer cells.
In addition, a unique KRP, centromere protein E, localizes to the kinetochore
of human mitotic
3 o chromosomes and may play a role in their segregation to opposite spindle
poles.
Dynamin-related Motor Proteins
Dynamin is a large GTPase motor protein that functions as a "molecular
pinchase,"
generating a mechanochemical force used to sever membranes. This activity is
important in forming
clathrin-coated vesicles from coated pits in endocytosis and in the biogenesis
of synaptic vesicles in
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neurons. Binding of dynamin to a membrane leads to dynamin's self assembly
into spirals that may
act to constrict a flat membrane surface into a tubule. GTP hydrolysis induces
a change in
conformation of the dynamin polymer that pinches the membrane tubule, leading
to severing of the
membrane tubule and formation of a membrane vesicle. Release of GDP and
inorganic phosphate
s leads to dynamin disassembly. Following disassembly the dynamin may either
dissociate from the
membrane or remain associated to the vesicle and be transported to another
region of the cell. Three
homologous dynamin genes have been discovered, in addition to several dynamin-
related proteins.
Conserved dynamin regions are the N-terminal GTP-binding domain, a central
pleckstrin homology
domain that binds membranes, a central coiled-coil region that may activate
dynamin's GTPase
s o activity, and a C-terminal proline-rich domain that contains several
motifs that bind SH3 domains on
other proteins. Some dynamin-related proteins do not contain the pleckstrin
homology domain or the
proline-rich domain. (See McNiven, M.A. (1998) Cell 94:151-154; Scaife, R.M.
and R.L. Margolis
(1997) Cell. Signal. 9:395-401.)
The cytoskeleton is reviewed in Lodish, H. et al. (1995) Molecular Cell
Biolo~y, Scientific
15 American Books, New York NY.
Ribosomal Molecules
Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into
ribosomes,
which are cytoplasmic particles that translate messenger RNA into
polypeptides. The eukaryotic -
2 o ribosome is composed of a 60S (large) subunit and a 40S (small) subunit,
which together form the
80S ribosome. In addition to the 185, 285, 5 S, and 5.8S rRNAs, the ribosome
also contains more
than fifty proteins. The ribosomal proteins have a prefix which denotes the
subunit to which they
belong, either L (large) or S (small). Ribosomal protein activities include
binding rRNA and
organizing the conformation of the junctions between rRNA helices (Woodson,
S.A. and N.B.
25 Leontis (1998) Curr. Opin. Struct. Biol. 8:294-300; Ramakrishnan, V. and
S.W. White (1998) Trends
Biochem. Sci. 23:208-212.) Three important sites are identified on the
ribosome. The aminoacyl-
tRNA site (A site) is where charged tRNAs (with the exception of the initiator-
tRNA) bind on arrival
at the ribosome. The peptidyl-tRNA site (P site) is where new peptide bonds
are formed, as well as
where the initiator tRNA binds. The exit site (E site) is where deacylated
tRNAs bind prior to their
3 o release from the ribosome. (The ribosome is reviewed in Stryer, L. (1995)
Biochemistry W.H.
Freeman and Company, New York NY, pp. 888-908; and Lodish, H. et al. (1995)
Molecular Cell
Biolo~y Scientific American Books, New York NY. pp. 119-138.)
Chromatin Molecules
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The nuclear DNA of eukaryotes is organized into chromatin. Two types of
chromatin are
observed: euchromatin, some of which may be transcribed, and heterochromatin
so densely packed that
much of it is inaccessible to transcription. Chromatin packing thus serves to
regulate protein
expression in eukaryotes. Bacteria lack chromatin and the chromatin-packing
level of gene regulation.
The fundamental unit of chromatin is the nucleosome of 200 DNA base pairs
associated with
two copies each of histones H2A, H2B, H3, and H4. Adjascent nucleosomes are
linked by another
class of histones, H1. Low molecular weight non-histone proteins called the
high mobility group
(HMG), associated with chromatin, may function in the unwinding of DNA and
stabilization of single-
stranded DNA. Chromodomain proteins function in compaction of chromatin into
its transcriptionally
1 o silent heterochromatin form.
During mitosis, all DNA is compacted into heterochromatin and transcription
ceases.
Transcription in interphase begins with the activation of a region of
chromatin. Active chromatin is
decondensed. Decondensataon appears to be accompanied by changes in binding
coefficient,
phosphorylation and acetylation states of chromatin histones. HMG proteins
HMG13 and HMG17
selectively bind activated chromatin. Topoisomerases remove superhelical
tension on DNA. The
activated region decondenses, allowing gene regulatory proteins and
transcription factors to assemble
on the DNA.
Patterns of chromatin structure can be stably inherited, producing heritable
patterns of gene
expression. In mammals, one of the two X chromosomes in each female cell is
inactivated by
2 o condensation to heterochromatin during zygote development. The inactive
state of this chromosome is
inherited, so that adult females are mosaics of clusters of paternal-X and
maternal-X clonal cell groups.
The condensed X chromosome is reactivated in meiosis.
Chromatin is associated with disorders of protein expression such as
thalassemia, a genetic
anemia resulting from the removal of the locus control region (LCR) required
for decondensation of the
2 5 globin gene locus.
For a review of chromatin structure and function see Alberts, B. et al. (1994)
Molecular Cell
Biolo~y, third edition, Garland Publishing, Inc., New York NY, pp. 351-354,
433-439.
Electron Transfer Associated Molecules
3 o Electron carriers such as cytochromes accept electrons from NADH or FADIiz
and donate
them to other electron carriers. Most electron-transferring proteins, except
ubiquinone, are prosthetic
groups such as flavins, heme, FeS clusters, and copper, bound to inner
membrane proteins.
Adrenodoxin, for example, is an FeS protein that forms a complex with
NADPH:adrenodoxin
reductase and cytochrome p450. Cytochromes contain a heme prosthetic group, a
porphyrin ring
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containing a tightly bound iron atom. Electron transfer reactions play a
crucial role in cellular energy
production.
Energy is produced by the oxidation of glucose and fatty acids. Glucose is
initially converted
to pyruvate in the cytoplasm. Fatty acids and pyruvate are transported to the
mitochondria for
s complete oxidation to COZ coupled by enzymes to the transport of electrons
from NADH and FADH2
to oxygen and to the synthesis of ATP (oxidative phosphorylation) from ADP and
Pi.
Pyruvate is transported into the mitochondria and converted to acetyl-CoA for
oxidation via
the citric acid cycle, involving pyruvate dehydrogenase components,
dihydrolipoyl transacetylase, and
dihydrolipoyl dehydrogenase. Enzymes involved in the citric acid cycle
include: citrate synthetase,
i o aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase
complex including
transsuccinylases, succinyl CoA synthetase, succinate dehydrogenase,
fumarases, and malate
dehydrogenase. Acetyl CoA is oxidized to COZ with concomitant formation of
NADH, FADH2, and
GTP. In oxidative phosphorylation, the transfer of electrons from NADH and
FADH2 to oxygen by
dehydrogenases is coupled to the synthesis of ATP from ADP and P; by the FoFl
ATPase complex in
15 the mitochondria) inner membrane. Enzyme complexes responsible for electron
transport and ATP
synthesis include the FoFI ATPase complex, ubiquinone(CoQ)-cytochrome c
reductase, ubiquinone
reductase, cytochrome b, cytochrome c), FeS protein, and cytochrome c oxidase.
ATP synthesis requires membrane transport enzymes including the phosphate
transporter and
the ATP-ADP antiport protein. The ATP-binding casette (ABC) superfamily has
also been suggested
2 o as belonging to the mitochondria) transport group (Rogue, D.L. et al.
(1999) J. Mol. Biol. 285:379-
389). Brown fat uncoupling protein dissipates oxidative energy as heat, and
may be involved the fever
response to infection and trauma (Cannon, B, et a1. (1998) Ann. NY Acad. Sci.
856:171-187).
Mitochondria are oval-shaped organelles comprising an outer membrane, a
tightly folded
inner membrane, an intermembrane space between the outer and inner membranes,
and a matrix
2 s inside the inner membrane. The outer membrane contains many porin
molecules that allow ions and
charged molecules to enter the intermembrane space, while the inner membrane
contains a variety of
transport proteins that transfer only selected molecules. Mitochondria are the
primary sites of energy
production in cells.
Mitochondria contain a small amount of DNA. Human mitochondria) DNA encodes 13
3 o proteins, 22 tRNAs, and 2 rRNAs. Mitochondria)-DNA encoded proteins
include NADH-Q
reductase, a cytochrome reductase subunit, cytochrome oxidase subunits, and
ATP synthase subunits.
Electron-transfer reactions also occur outside the mitochondria in locations
such as the
endoplasmic reticulum, which plays a crucial role in lipid and protein
biosynthesis. Cytochrome b5
is a central electron donor for various reductive reactions occurring on the
cytoplasmic surface of liver
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endoplasmic reticulum. Cytochrome b5 has been found in Golgi, plasma,
endoplasmic reticulum
(ER), and microbody membranes.
For a review of mitochondria) metabolism and regulation, see Lodish, H, et al.
(1995)
Molecular Cell Biolo~y, Scientific American Books, New York NY, pp. 745-797
and Stryer (1995)
s Biochemistry, W.H. Freeman and Co., San Francisco CA, pp 529-558, 988-989.
The majority of mitochondria) proteins are encoded by nuclear genes, are
synthesized on
cytosolic ribosomes, and are imported into the mitochondria. Nuclear-encoded
proteins which are
destined for the mitochondria) matrix typically contain positively-charged
amino terminal signal
sequences. Import of these preproteins from the cytoplasm requires a
multisubunit protein complex
s o in the outer membrane known as the translocase of outer mitochondria)
membrane (TOM; previously
designated MOM; Pfanner, N. et al. (1996) Trends Biochem. Sci. 21:51-52) and
at least three inner
membrane proteins which comprise the translocase of inner mitochondria)
membrane (TIM;
previously designated MIM; Pfanner, supra). An inside-negative membrane
potential across the inner
mitochondria) membrane is also required for preprotein import. Preproteins are
recognized by surface
W receptor components of the TOM complex and are translocated through a
proteinaceous pore formed
by other TOM components. Proteins targeted to the matrix are then recognized
by the import
machinery of the TIM complex. The import systems of the outer and inner
membranes can function
independently (Segui-Real, B. et al. (1993) EMBO J. 12:2211-2218).
Once precursor proteins are in the mitochondria, the leader peptide is cleaved
by a signal
2 o peptidase to generate the mature protein. Most leader peptides are removed
in a one step process by a
protease termed mitochondria) processing peptidase (MPP) (Paces, V. et al.
(1993) Proc. Nat). Acad.
Sci. USA 90:5355-5358). In some cases a two-step process occurs in which MPP
generates an
intermediate precursor form which is cleaved by a second enzyme, mitochondria)
intermediate
peptidase, to generate the mature protein.
z s Mitochondria) dysfunction leads to impaired calcium buffering, generation
of free radicals
that may participate in deleterious intracellular and extracellulax processes,
changes in mitochondria)
permeability and oxidative damage which is observed in several
neurodegenerative diseases.
Neurodegenerative diseases linked to mitochondria) dysfunction include some
forms of Alzheimer's
disease, Friedreich's ataxia, familial amyotrophic lateral sclerosis, and
Huntington's disease (Beal,
3 o M.F. (1998) Biochim. Biophys. Acta 1366:211-213). The myocardium is
heavily dependent on
oxidative metabolism, so mitochondria) dysfunction often leads to heart
disease (DiMauro, S. and M.
Hirano (1998) Curr. Opin. Cardiol 13:190-197). Mitochondria are implicated in
disorders of cell
proliferation, since they play an important role in a cell's decision to
proliferate or self destruct
through apoptosis. The oncoprotein Bcl-2, for example, promotes cell
proliferation by stabilizing
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mitochondria) membranes so that apoptosis signals are not released (Susin,
S.A. (1998) Biochim.
Biophys. Acta 1366:151-165).
Transcription Factor Molecules
Multicellular organisms are comprised of diverse cell types that differ
dramatically both in
structure and function. The identity of a cell is determined by its
characteristic pattern of gene
expression, and different cell types express overlapping but distinctive sets
of genes throughout
development. Spatial and temporal regulation of gene expression is critical
for the control of cell
proliferation, cell differentiation, apoptosis, and other processes that
contribute to organismal
1 o development. Furthermore, gene expression is regulated in response to
extracellular signals that
mediate cell-cell communication and coordinate the activities of different
cell types. Appropriate
gene regulation also ensures that cells function efficiently by expressing
only those genes whose
functions are required at a given time.
Transcriptional regulatory proteins are essential for the control of gene
expression. Some'of
these proteins function as transcription factors that initiate, activate,
repress, or terminate gene
transcription. Transcription factors generally bind to the promoter, enhancer,
and upstream regulatory
regions of a gene in a sequence-specific manner, although some factors bind
regulatory elements
within or downstream of a gene's coding region. Transcription factors may bind
to a specific region
of DNA singly or as a complex with other accessory factors, (Reviewed in
Lewin, B. (1990) Genes
2 o IV, Oxford University Press, New York NY, and Cell Press, Cambridge MA,
pp. 554-570.)
The double helix structure and repeated sequences of DNA create topological
and chemical
features which can be recognized by transcription factors. These features are
hydrogen bond donor
and acceptor groups, hydrophobic patches, major and minor grooves, and
regular, repeated stretches
of sequence which induce distinct bends in the helix. Typically, transcription
factors recognize
2 5 specific DNA sequence motifs of about 20 nucleotides in length. Multiple,
adjacent transcription
factor-binding motifs may be required for gene regulation.
Many transcription factors incorporate DNA-binding structural motifs which
comprise either
a helices or 13 sheets that bind to the major groove of DNA. Four well-
characterized structural motifs
are helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix.
Proteins containing these
3 o motifs may act alone as monomers, or they may form homo- or heterodimers
that interact with DNA.
The helix-turn-helix motif consists of two a helices connected at a fixed
angle by a short
chain of amino acids. One of the helices binds to the major groove. Helix-turn-
helix motifs are
exemplified by the homeobox motif which is present in homeodomain proteins.
These proteins are
critical for specifying the anterior-posterior body axis during development
and are conserved
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throughout the animal kingdom. The Antennapedia and Ultrabithorax proteins of
Drosophila
melano~aster are prototypical homeodomain proteins (Patio, C.O. and R.T. Sauer
(1992) Annu. Rev.
Biochem. 61:1053-1095).
The zinc finger motif, which binds zinc ions, generally contains tandem
repeats of about 30
s amino acids consisting of periodically spaced cysteine and histidine
residues. Examples of this
sequence pattern, designated C2H2 and C3HC4 ("RING" finger), have been
described (Lewin, supra).
Zinc finger proteins each contain an a helix and an antiparallel 13 sheet
whose proximity and
conformation are maintained by the zinc ion. Contact with DNA is made by the
arginine prece ding
the a helix and by the second, third, and sixth residues of the a helix.
Variants of the zinc finger
1 o motif include poorly defined cysteine-rich motifs which bind zinc or other
metal ions. These motifs
may not contain histidine residues and are generally nonrepetitive.
The leucine zipper motif comprises a stretch of amino acids rich in leucine
which can form an
amphipathic a helix. This structure provides the basis for dimerization of two
leucine zipper
proteins. The region adjacent to the leucine zipper is usually basic, and upon
protein dimerization, is
15 optimally positioned for binding to the major groove. Proteins containing
such motifs are generally
referred to as bZIP transcription factors.
The helix-loop-helix motif (HLH) consists of a short a helix connected by a
loop to a longer
a helix. The loop is flexible and allows the two helices to fold back against
each other and to bind to
DNA. The transcription factor Myc contains a prototypical HLH motif.
2 o Most transcription factors contain characteristic DNA binding motifs, and
variations on the
above motifs and new motifs have been and are currently being characterized
(Faisst, S. and S. Meyer
(1992) Nucleic Acids Res. 20:3-26).
Many neoplastic disorders in humans can be attributed to inappropriate gene
expression.
Malignant cell growth may result from either excessive expression of tumor
promoting genes or
25 insufficient expression of tumor suppressor genes (Cleary, M.L. (1992)
Cancer Surv. 15:89-104).
Chromosomal translocations may also produce chimeric loci which fuse the
coding sequence of one
gene with the regulatory regions of a second unrelated gene. Such an
arrangement likely results in
inappropriate gene transcription, potentially contributing to malignancy.
In addition, the immune system responds to infection or trauma by activating a
cascade of
3 o events that coordinate the progressive selection, amplification, and
mobilization of cellular defense
mechanisms. A complex and balanced program of gene activation and repression
is involved in this
process. However, hyperactivity of the immune system as a result of improper
or insufficient
regulation of gene expression may result in considerable tissue or organ
damage. This damage is well
documented in immunological responses associated with arthritis, allergens,
heart attack, stroke, and
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infections (Isselbacher, K.J. et al. (1996) Harrison's Principles of Internal
Medicine, l3le, McGraw
Hill, Inc. and Teton Data Systems Software).
Furthermore, the generation of multicellular organisms is based upon the
induction and
coordination of cell differentiation at the appropriate stages of development.
Central to this process is
s differential gene expression, which confers the distinct identities of cells
and tissues throughout the
body. Failure to regulate gene expression during development can result in
developmental disorders.
Human developmental disorders caused by mutations in zinc finger-type
transcriptional regulators
include: urogenenital developmental abnormalities associated with WTI; Greig
cephalopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type
A (GLI3); and
to Townes-Brocks syndrome, characterized by anal, renal, limb, and ear
abnormalities (SALL1)
(Engelkamp, D. and V. van Heyningen (1996) Curr. Opin. Genet. Dev. 6:334-342;
Kohlhase, J. et al.
(1999) Am. J. Hum. Genet. 64:435-445).
CeII Membrane Molecules
15 Eukaryotic cells are surrounded by plasma membranes which enclose the cell
and maintain an
environment inside the cell that is distinct from its surroundings. In
addition, eukaryotic organisms
are distinct from prokaryotes in possessing many intracellular organelle and
vesicle structures. Many
of the metabolic reactions which distinguish eukaryotic. biochemistry from
prokaryotic biochemistry
take place within these structures. The plasma membrane and the membranes
surrounding organelles
2 o and vesicles are composed of phosphoglycerides, fatty acids, cholesterol,
phospholipids, glycolipids,
proteoglycans, and proteins. These components confer identity and
functionality to the membranes
with which they associate.
Integral Membrane Proteins
The majority of known integral membrane proteins are transmembrane proteins
(TM) which
2 s are characterized by an extracellular, a transmembrane, and an
intracellular domain. TM domains are
typically comprised of 15 to 25 hydrophobic amino acids which are predicted to
adopt an a-helical
conformation. TM proteins are classified as bitopic (Types I and II) and
polytopic (Types III and IV)
(Singer, S.J. (1990) Annu. Rev. Cell Biol. 6:247-296). Bitopic proteins span
the membrane once
while polytopic proteins contain multiple membrane-spanning segments. TM
proteins function as
a o cell-surface receptors, receptor-interacting proteins, transporters of
ions or metabolites, ion channels,
cell anchoring proteins, and cell type-specific surface antigens.
Many membrane proteins (MPs) contain amino acid sequence motifs that target
these proteins
to specific subcellular sites. Examples of these motifs include PDZ domains,
KDEL, RGD, NGR,
and GSL sequence motifs, von Willebrand factor A (vWFA) domains, and EGF-like
domains. RGD,
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NGR, and GSL motif containing peptides have been used as drug delivery agents
in targeted cancer
treatment of tumor vasculature (Arap, W. et al. (1998) Science 279:377-380).
Furthermore, MPs may
also contain amino acid sequence motifs, such as the carbohydrate recognition
domain (CRD), that
mediate interactions with extracellular or intracellular molecules.
s G-Protein Coupled Receptors
G-protein coupled receptors (GPCR) are a superfamily of integral membrane
proteins which
transduce extracellular signals. GPCRs include receptors for biogenic amines,
lipid mediators of
inflammation, peptide hormones, and sensory signal mediators. The structure of
these
highly-conserved receptors consists of seven hydrophobic transmembrane
regions, an extracellular
z o N-terminus, and a cytoplasmic C-terminus. Three extracellular loops
alternate with three intracellular
loops to link the seven transmembrane regions. Cysteine disulfide bridges
connect the second and
third extracellular loops. The most conserved regions of GPCRs are the
transmembrane regions and
the first two cytoplasmic loops. A conserved, acidic-Arg-aromatic residue
triplet present in the
second cytoplasmic loop may interact with G proteins. A GPCR consensus pattern
is characteristic of
s s most proteins belonging to this superfamily (ExPASy PROSITE document
PS00237; and Watson, S.
and S. Arkinstall (1994) The G-protein Linked Rector Facts Book, Academic
Press, San Diego CA,
pp. 2-6). Mutations and changes in transcriptional activation of GPCR-encoding
genes have been
associated with neurological disorders such as schizophrenia, Parkinson's
disease, Alzheimer's
disease, drug addiction, and feeding disorders.
2 o Scavenger Receptors
Macrophage scavenger receptors with broad ligand specificity may participate
in the binding
of low density lipoproteins (LDL) and foreign antigens. Scavenger receptors
types I and II are
trimeric membrane proteins with each subunit containing a small N-terminal
intracellular domain, a
transmembrane domain, a large extracellular domain, and a C-terminal cysteine-
rich domain. The
2 5 extracellular domain contains a short spacer region, an a-helical coiled-
coil region, and a triple helical
collagen-like region. These receptors have been shown to bind a spectrum of
ligands, including
chemically modified lipoproteins and albumin, polyribonucleotides,
polysacchaxides, phospholipids,
and asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9133-
9137; and Elomaa, O.
et al. (1995) Cell 80:603-609). The scavenger receptors are thought to play a
key role in
3 o atherogenesis by mediating uptake of modified LDL in arterial walls, and
in host defense by binding
bacterial endotoxins, bacteria, and protozoa.
Tetraspan Family Proteins
The transmembrane 4 superfamily (TM4SF) or tetraspan family is a multigene
family
encoding type III integral membrane proteins (Wright, M.D. and M.G. Tomlinson
(1994) Immunol.
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Today 15:588-594). The TM4SF is comprised of membrane proteins which traverse
the cell
membrane four times. Members of the TM4SF include platelet and endothelial
cell membrane
proteins, melanoma-associated antigens, leukocyte surface glycoproteins,
colonal carcinoma antigens,
tumor-associated antigens, and surface proteins of the schistosome parasites
(Jankowski, S.A. (2994)
Oncogene 9:1205-1211). Members of the TM4SF share about 25-30% amino acid
sequence identity
with one another.
A number of TM4SF members have been implicated in signal transduction, control
of cell
adhesion, regulation of cell growth and proliferation, including development
and oncogenesis, and
cell motility, including tumor cell metastasis. Expression of TM4SF proteins
is associated with a
1 o variety of tumors and the level of expression may be altered when cells
are growing or activated.
Tumor Antigens
Tumor antigens are cell surface molecules that are differentially expressed in
tumor cells
relative to normal cells. Tumor antigens distinguish tumor cells
immunologically from normal cells
and provide diagnostic arid therapeutic targets for human cancers (Takagi, S.
et al. ( 1995) Int. J.
Cancer 61:706-715; Liu, E. et al. (1992) Oncogene 7:1027-1032). .
Leukocyte Antigens
Other types of cell surface antigens include those identified on leukocytic
cells of the immune
system. These antigens have been identified using systematic, monoclonal
antibody (mAb)-based
"shot gun" techniques. These techniques have resulted in the production of
hundreds of mAbs
2 o directed against unknown cell surface Ieukocytic antigens. These antigens
have been grouped into
"clusters of differentiation" based on common immunocytochemical localization
patterns in various
differentiated and undifferentiated leukocytic cell types. Antigens in a given
cluster are presumed to
identify a single cell surface protein and are assigned a "cluster of
differentiation" or "CD"
designation. Some of the genes encoding proteins identified by CD antigens
have been cloned and
2 5 verified by standard molecular biology techniques. CD antigens have been
characterized as both
transmembrane proteins and cell surface proteins anchored to the plasma
membrane via covalent
attachment to fatty acid-containing glycolipids such as
glycosylphosphatidylinositol (GPI).
(Reviewed in Barclay, A.N. et al. (1995) The Leucocyte Antigen Facts Book,
Academic Press, San
Diego CA, pp. 17-20.)
3 o Ion Channels
Ion channels are found in the plasma membranes of virtually every cell in the
body. For
example, chloride channels mediate a variety of cellular functions including
regulation of membrane
potentials and absorption and secretion of ions across epithelial membranes.
Chloride channels also
regulate the pH of organelles such as the Golgi apparatus and endosomes (see,
e.g., Greger, R. (1988)
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Annu. Rev. Physiol. 50:111-122). Electrophysiological and pharmacological
properties of chloride
channels, including ion conductance, current-voltage relationships, and
sensitivity to modulators,
suggest that different chloride channels exist in muscles, neurons,
fibroblasts, epithelial cells, and
lymphocytes.
s Many ion channels have sites for phosphorylation by one or more protein
kinases including
protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, all
of which regulate ion
channel activity in cells. Inappropriate phosphorylation of proteins in cells
has been linked to
changes in cell cycle progression and cell differentiation. Changes in the
cell cycle have been linked
to induction of apoptosis or cancer. Changes in cell differentiation have been
linked to diseases and
to disorders of the reproductive system, immune system, skeletal muscle, and
other organ systems.
Proton Pumps
Proton ATPases comprise a large class of membrane proteins that use the energy
of ATP
hydrolysis to generate an electrochemical proton gradient across a membrane.
The resultant gradient
may be used to transport other ions across the membrane (Na+, K+, or Cf) or to
maintain organelle
15 pH. Proton ATPases are further subdivided into the mitochondrial F-ATPases,
the plasma membrane
ATPases, and the vacuolar ATPases. The vacuolar ATPases establish and maintain
an acidic pH
within various organelles involved in the processes of endocytosis and
exocytosis (Mellman, I. et aI.
(1986) Annu. Rev. Biochem. 55:663-700).
Proton-coupled, 12 membrane-spanning domain transporters such as PEPT l and
PEPT 2 are
2 0 , responsible for gastrointestinal absorption and for renal reabsorption
of peptides using an
electrochemical H+ gradient as the driving force. Another type of peptide
transporter, the TAP
transporter, is a heterodimer consisting of TAP 1 and TAP 2 and is associated
with antigen
processing. Peptide antigens are transported across the membrane of the
endoplasmic reticulum by
TAP so they can be expressed on the cell surface in association with MHC
molecules. Each TAP
2 s protein consists of multiple hydrophobic membrane spanning segments and a
highly conserved,
ATP-binding cassette (Boll, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:284-
289). Pathogenic
microorganisms, such as herpes simplex virus, may encode inhibitors of TAP-
mediated peptide
transport in order to evade immune surveillance (Marusina, K. and J.J Manaco
(1996) Curr. Opin.
Hematol. 3:19-26).
3o ABC Transporters
The ATP-binding cassette (ABC) transporters, also called the "traffic
ATPases", comprise a
superfamily of membrane proteins that mediate transport and channel functions
in prokaryotes and
eukaryotes (Higgins, C.F. (1992) Annu. Rev. Cell Biol. 8:67-113). ABC proteins
share a similar
overall structure and significant sequence homology. All ABC proteins contain
a conserved domain
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of approximately two hundred amino acid residues which includes one or more
nucleotide binding
domains. Mutations in ABC transporter genes are associated with various
disorders, such as
hyperbilirubinemia II/Dubin-Johnson syndrome, recessive Stargardt's disease, X-
linked
adrenoleukodystrophy, multidrug resistance, celiac disease, and cystic
fibrosis.
s Peripheral and Anchored Membrane Proteins
Some membrane proteins are not membrane-spanning but are attached to the
plasma
membrane via membrane anchors or interactions with integral membrane proteins.
Membrane
anchors are covalently joined to a protein post-translationally and include
such moieties as prenyl,
myristyl, and glycosylphosphatidyl inositol groups. Membrane localization of
peripheral and
1 o anchored proteins is important for their function in processes such as
receptor-mediated signal
transduction. For example, prenylation of Ras is required for its localization
to the plasma membrane
and for its normal and oncogenic functions in signal transduction.
Vesicle Coat Proteins
Intercellular communication is essential for the development and survival of
multicellular
~ s organisms. Cells communicate with one another through the secretion and
uptake of protein
signaling molecules. The uptake of proteins into the cell is achieved by the
endocytic pathway, in
which the interaction of extracellular signaling molecules with plasma
membrane receptors results in
the formation of plasma membrane-derived vesicles that enclose and transport
the molecules into the
cytosol. These transport vesicles fuse with and mature into endosomal and
lysosomal (digestive)
2 o compartments. The secretion of proteins from the cell is achieved by
exocytosis, in which molecules
inside of the cell proceed through the secretory pathway. In this pathway,
molecules transit from the
ER to the Golgi apparatus and finally to the plasma membrane, where they are
secreted from the cell.
Several steps in the transit of material along the secretory and endocytic
pathways require the
formation of transport vesicles. Specifically, vesicles form at the
transitional endoplasmic reticulum
2 s (tER), the rim of Golgi cisternae, the face of the Traps-Golgi Network
(TGN), the plasma membrane
(PM), and tubular extensions of the endosomes. Vesicle formation occurs when a
region of
membrane buds off from the donor organelle. The membrane-bound vesicle
contains proteins to be
transported and is surrounded by a proteinaceous coat, the components of which
are recruited from
the cytosol. Two different classes of coat protein have been identified.
Clathrin coats form on
3 o vesicles derived from the TGN and PM, whereas coatomer (COP) coats form on
vesicles derived
from the ER and Golgi. COP coats can be further classified as COPI, involved
in retrograde traffic
through the Golgi and from the Golgi to the ER, and COPII, involved in
anterograde traffic from the
ER to the Golgi (Mellman, supra).
In clathrin-based vesicle formation, adapter proteins bring vesicle cargo and
coat proteins
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together at the surface of the budding membrane. Adapter protein-1 and -2
select cargo from the
TGN and plasma membrane, respectively, based on molecular information encoded
on the
cytoplasmic tail of integral membrane cargo proteins. Adapter proteins also
recruit clathrin to the bud
site. Clathrin is a protein complex consisting of three large and three small
polypeptide chains
s arranged in a three-legged structure called a triskelion. Multiple
triskelions and other coat proteins
appear to self-assemble on the membrane to form a coated pit. This assembly
process may serve to
deform the membrane into a budding vesicle. GTP-bound ADP-ribosylation factor
(Arf) is also
incorporated into the coated assembly. Another small G-protein, dynamin, forms
a ring complex
around the neck of the forming vesicle and may provide the mechanochemical
force to seal the bud,
1 o thereby releasing the vesicle. The coated vesicle complex is then
transported through the cytosol.
During the transport process, Arf-bound GTP is hydrolyzed to GDP, and the coat
dissociates from the
transport vesicle (West, M.A. et al. (1997) J. Cell Biol. 138:1239-1254).
Vesicles which bud from the ER and the Golgi axe covered with a protein coat
similar to the
clathrin coat of endocytic and TGN vesicles. The coat protein (COP) is
assembled from cytosolic
15 precursor molecules at specific budding regions on the organelle. The COP
coat consists of two
major components, a G-protein (Arf or Sar) and coat protomer (coatomer).
Coatomer is an equimolar
complex of seven proteins, termed alpha-, beta-, beta'-, gamma-, delta-,
epsilon- and zeta-COP. The
coatomex complex binds to dilysine motifs contained on the cytoplasmic tails
of integral membrane
proteins. These include the KKXX retrieval motif of membrane proteins of the
ER and
2 o dibasic/diphenylamine motifs of members of the p24 family. The p24 family
of type I membrane
proteins represent the major membrane proteins of COPI vesicles (Harter, C.
and F.T. Wieland (1998)
Proc. Natl. Acad. Sci. USA 95:11649-11654).
Organelle Associated Molecules
2 5 Eukaryotic cells are organized into various cellular organelles which has
the effect of
separating specific molecules and their functions from one another and from
the cytosol. Within the
cell, various membrane structures surround and define these organelles while
allowing them to
interact with one another and the cell environment through both active and
passive transport
processes. Important cell organelles include the nucleus, the Golgi apparatus,
the endoplasmic
3 o reticulum, mitochondria, peroxisomes, lysosomes, endosomes, and secretory
vesicles.
Nucleus
The cell nucleus contains all of the genetic information of the cell in the
form of DNA, and
the components and machinery necessary for replication of DNA and for
transcription of DNA into
RNA. (See Alberts, B, et al. (1994) Molecular Biolo~y of the Cell, Garland
Publishing Inc., New
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York NY, pp. 335-399.) DNA is organized into compact structures in the nucleus
by interactions
with various DNA-binding proteins such as histones and non-histone chromosomal
proteins.
DNA-specific nucleases, DNAses, partially degrade these compacted structures
prior to DNA
replication or transcription. DNA replication takes place with the aid of DNA
helicases which
s unwind the double-stranded DNA helix, and DNA polymerases that duplicate the
separated DNA
strands.
Transcriptional regulatory proteins are essential for the control of gene
expression. Some of
these proteins function as transcription factors that initiate, activate,
repress, or terminate gene
transcription. Transcription factors generally bind to the promoter, enhancer,
and upstream
1 o regulatory regions of a gene in a sequence-specific manner, although some
factors bind regulatory
elements within or downstream of a gene's coding region. Transcription factors
may bind to a specific
region of DNA singly or as a complex with other accessory factors. (Reviewed
in Lewin, B. (1990)
Genes IV, Oxford University Press New York NY, and Cell Press, Cambridge MA,
pp. 554-570.)
Many transcription factors incorporate DNA-binding structural motifs which
comprise either a
15 helices or 13 sheets that bind to the major groove of DNA. Four well-
characterized structural motifs
are helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix.
Proteins containing these
motifs may act alone as monomers, or they may form homo- or heterodimers that
interact with DNA.
Many neoplastic disorders in humans can be attributed to inappropriate gene
expression.
Malignant cell growth may result from either excessive expression of tumor
promoting genes or
2 o insufficient expression of tumor suppressor genes (Cleary, M.L. (1992)
Cancer Surv. 15:89-104).
Chromosomal translocations may also produce chimeric loci which fuse the
coding sequence of one
gene with the regulatory regions of a second unrelated gene. Such an
arrangement likely results in
inappropriate gene transcription, potentially contributing to malignancy.
In addition, the immune system responds to infection or trauma by activating a
cascade of
2 s events that coordinate the progressive selection, amplification, and
mobilization of cellular defense
mechanisms. A complex and balanced program of gene activation and repression
is involved in this
process. However, hyperactivity of the immune system as a result of improper
or insufficient
regulation of gene expression may result in considerable tissue or organ
damage. This damage is well
documented in immunological responses associated with arthritis, allergens,
heart attack, stroke, and
3o infections (Isselbacher, K.J. et al. (1996) Harrison's Principles of
Internal Medicine, 13/e, McGraw
Hill, Inc. and Teton Data Systems Software).
Transcription of DNA into RNA also takes place in the nucleus catalyzed by RNA
polymerases. Three types of RNA polymerase exist. RNA polymerase I makes large
ribosomal
RNAs, while RNA polymerase III makes a variety of small, stable RNAs including
5S ribosomal
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RNA and the transfer RNAs (tRNA). RNA polymerise II transcribes genes that
will be translated
into proteins. The primary transcript of RNA polymerise II is called
heterogenous nuclear RNA
(hnRNA), and must be further processed by splicing to remove non-coding
sequences called introns.
RNA splicing is mediated by small nuclear ribonucleoprotein complexes, or
snRNPs, producing
s mature messenger RNA (mRNA) which is then transported out of the nucleus for
translation into
proteins.
Nucleolus
The nucleolus is a highly organized subcompartment in the nucleus that
contains high
concentrations of RNA and proteins and functions mainly in ribosomal RNA
synthesis and assembly
l o (Alberts, et al. supra, pp. 379-382). Ribosomal RNA (rRNA) is a structural
RNA that is complexed
with proteins to form ribonucleoprotein structures called ribosomes. Ribosomes
provide the platform
on which protein synthesis takes place.
Ribosomes are assembled in the nucleolus initially from a large, 45S rRNA
combined with a
variety of proteins imported from the cytoplasm, as well as smaller, 5S rRNAs.
Latex processing of
1 s the immature ribosome results in formation of smaller ribosomal subunits
which are'transported from
the nucleolus to the cytoplasm where they are assembled into functional
ribosomes.
Endoplasmic Reticulum
In eukaryotes, proteins are synthesized within the endoplasmic reticulum (ER),
delivered from
the ER to the Golgi apparatus for post-translational processing and sorting,
and transported from the
2 o Golgi to specific intracellular and extracellular destinations. Synthesis
of integral membrane proteins,
secreted proteins, and proteins destined for the lumen of a particular
organelle occurs on the rough
endoplasmic reticulum (ER). The rough ER is so named because of the rough
appearance in electron
micrographs imparted by the attached ribosomes an which protein synthesis
proceeds. Synthesis of
proteins destined for the ER actually begins in the cytosol with the synthesis
of a specific signal
2 s peptide which directs the growing polypeptide and its attached ribosome to
the ER membrane where
the signal peptide is removed and protein synthesis is completed. Soluble
proteins destined for the
ER lumen, for secretion, or for transport to the lumen of other organelles
pass completely into the ER
lumen. Transmembrane proteins destined for the ER or for other cell membranes
are translocated
across the ER membrane but remain anchored in the lipid bilayer of the
membrane by one or more
3 o membrane-spanning a-helical regions.
Translocated polypeptide chains destined for other organelles or for secretion
also fold and
assemble in the ER lumen with the aid of certain "resident" ER proteins.
Protein folding in the ER is
aided by two principal types of protein isomerases, protein disulfide
isomerase (PDI), and peptidyl-
prolyl isomerase (PPI). PDI catalyzes the oxidation of free sulfhydryl groups
in cysteine residues to
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form intramolecular disulfide bonds in proteins. PPI, an enzyme that catalyzes
the isomerization of
certain proline imide bonds in oligopeptides and proteins, is considered to
govern one of the rate
limiting steps in the folding of many proteins to their final functional
conformation. The cyclophilins
represent a major class of PPI that was originally identified as the major
receptor for the
s immunosuppressive drug cyclosporin A (Handschumacher, R.E. et al. (1984)
Science 226:544-S47).
Molecular "chaperones" such as BiP (binding protein) in the ER recognize
incorrectly folded proteins
as well as proteins not yet folded into their final form and bind to them,
both to prevent improper
aggregation between them, and to promote proper folding.
The "N-linked" glycosylation of most soluble secreted and membrane-bound
proteins by
oligosacchrides linked to asparagine residues in proteins is also performed in
the ER. This reaction is
catalyzed by a membrane-bound enzyme, oligosaccharyl transferase.
Golgi Apparatus
The Golgi apparatus is a complex structure that lies adj acent to the ER in
eukaryotic cells and
serves primarily as a sorting and dispatching station for products of the ER
(Alberts, et al. s-unra, pp.
600-610). Additional posttranslational processing, principally additional
glycosylation, also occurs in
the Golgi. Indeed, the Golgi is a major site of carbohydrate synthesis,
including most of the '
glycosaminoglycans of the extracellular matrix. N-linked oligosacchaxides,
added to proteins in the
ER, are also further modified in the Golgi by the addition of more sugar
residues to form complex N-
linked oligosaccharides. "O-linked" glycosylation of proteins also occurs in
the Golgi by he addition
2 0 of N-acetylgalactosamine to the hydroxyl group of a serine or threonine
residue followed by the
sequential addition of other sugar residues to the first. This process is
catalyzed by a series of
glycosyltransferases each specific for a particular donor sugar nucleotide and
acceptor molecule
(Lodish, H. et al. (1995) Molecular Cell Biolo~y, W.H. Freeman and Co., New
York NY, pp.700-
708). In many cases; both N- and O-linked oligosaccharides appear to be
required for the secretion of
2 s proteins or the movement of plasma membrane glycoproteins to the cell
surface.
The terminal compartment of the Golgi is the Trans-Golgi Network (TGN), where
both
membrane and lumenal proteins are sorted for their Bnal destination. Transport
(or secretory) vesicles
destined for intracellular compartments, such as lysosomes, bud off of the
TGN. Other transport
vesicles bud off containing proteins destined for the plasma membrane, such as
receptors, adhesion
s o molecules, and ion channels, and secretory proteins, such as hormones,
neurotransmitters, and digestive
enzymes.
Vacuoles
The vacuole system is a collection of membrane bound compartments in
eukaryotic cells that
functions in the processes of endocytosis and exocytosis. They include
phagosomes, lysosomes,
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endosomes, and secretory vesicles. Endocytosis is the process in cells of
internalizing nutrients, solutes
or small particles (pinocytosis) or large particles such as internalized
receptors, viruses, bacteria, or
bacterial toxins (phagocytosis). Exocytosis is the process of transporting
molecules to the cell surface.
It facilitates placement or localization of membrane-bound receptors or other
membrane proteins and
s secretion of hormones, neurotransmitters, digestive enzymes, wastes, etc.
A common property of all of these vacuoles is an acidic pH environment ranging
from
approximately pH 4.5-5Ø This acidity is maintained by the presence of a
proton ATPase that uses the
energy of ATP hydrolysis to generate an electrochemical proton gradient across
a membrane (Mellman,
I. et al. (1986) Annu. Rev. Biochem. 55:663-700). Eukaryotic vacuolar proton
ATPase (vp-ATPase) is
i o a multimeric enzyme composed of 3-10 different subunits. One of these
subunits is a highly
hydrophobic polypeptide of approximately 16 kDa that is similar to the
proteolipid component of vp-
ATPases from eubacteria, fungi, and plant vacuoles (Mandel, M. et al. (1988)
Proc. Natl. Acad. Sci.
USA 85:5521=5524). The 16 kDa proteolipid component is the major subunit of
the membrane portion
of vp-ATPase and functions in the transport of protons across the membrane.
1 s Lysosomes
Lysosomes are membranous vesicles containing various hydrolytic enzymes used
for the
controlled intracellular digestion of macromolecules. Lysosomes contain some
40 types of enzymes
including proteases, nucleases, glycosidases, lipases, phospholipases,
phosphatases, and sulfatases, all
of which are acid hydrolases that function at a pH of about 5. Lysosomes are
surrounded by a unique
2 o membrane containing transport proteins that allow the final products of
macromolecule degradation,
such as sugars, amino acids, and nucleotides, to be transported to the cytosol
where they may be
either excreted or reutilized by the cell. A vp-ATPase, such as that described
above, maintains the
acidic environment necessary for hydrolytic activity (Alberts, s_ upra, pp.
610-611).
Endosomes
2 s Endosomes are another type of acidic vacuole that is used to transport
substances from the
cell surface to the interior of the cell in the process of endocytosis. Like
lysosomes, endosomes have
an acidic environment provided by a vp-ATPase (Alberts et al. supra, pp. 610-
618). Two types of
endosomes are apparent based on tracer uptake studies that distinguish their
time of formation in the
cell and their cellular location. Early endosomes are found near the plasma
membrane and appear to
3 o function primarily in the recycling of internalized receptors back to the
cell surface. Late endosomes
appear later in the endocytic process close to the Golgi apparatus and the
nucleus, and appear to be
associated with delivery of endocytosed material to lysosomes or to the TGN
where they may be
recycled. Specific proteins are associated with particular transport vesicles
and their target
compartments that may provide selectivity in targeting vesicles to their
proper compartments. A
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cytosolic prenylated GTP-binding protein, Rab, is one such protein. Rabs 4, 5,
and 11 are associated
with the early endosome, whereas Rabs 7 and 9 associate with the late
endosome.
Mitochondria
Mitochondria are oval-shaped organelles comprising an outer membrane, a
tightly folded
s inner membrane, an intermembrane space between the outer and inner
membranes, and a matrix
inside the inner membrane. The outer membrane contains many porin molecules
that allow ions and
charged molecules to enter the intermembrane space, while the inner membrane
contains a variety of
transport proteins that transfer only selected molecules. Mitochondria are the
primary sites of energy
production in cells.
s o Energy is produced by the oxidation of glucose and fatty acids. Glucose is
initially converted
to pyruvate in the cytoplasm. Fatty acids and pyruvate are transported to the
mitochondria for
complete oxidation to COZ coupled by enzymes to the transport of electrons
from NADH and FADH2
to oxygen and to the synthesis of ATP (oxidative phosphorylation) from ADP and
Pi.
Pyruvate is transported into the mitochondria and converted to acetyl-CoA for
oxidation via
1 s the citric acid cycle, involving pyruvate dehydrogenase components,
dihydrolipoyl transacetylase, and
dihydrolipoyl dehydrogenase. Enzymes involved in the citric acid cycle
include: citrate synthetase,
aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase
complex including
transsuccinylases, succinyl CoA synthetase, succinate dehydrogenase,
fumarases, and malate
dehydrogenase. Acetyl CoA is oxidized to COZ with concomitant formation of
NADH, FADH2, and
2 o GTP. In oxidative phosphorylation, the transfer of electrons from NADH and
FADH2 to oxygen by
dehydrogenases is coupled to the synthesis of ATP from ADP and Pi by the FoFl
ATPase complex in
the mitochondria) inner membrane. Enzyme complexes responsible for electron
transport and ATP
synthesis include the FoFl ATPase complex, ubiquinone(Co~-cytochrome c
reductase, ubiquinone
reductase, cytochrome b, cytochrome c), FeS protein, and cytochrome c oxidase.
2 s Peroxisomes
Peroxisomes, like mitochondria, are a major site of oxygen utilization. They
contain one or
more enzymes, such as catalase and urate oxidase, that use molecular oxygen to
remove hydrogen
atoms from specific organic substrates in an oxidative reaction that produces
hydrogen peroxide
(Alberts, s_ upra, pp. 574-577). Catalase oxidizes a variety of substrates
including phenols, formic
3 o acid, formaldehyde, and alcohol and is important in peroxisomes of liver
and kidney cells for
detoxifying various toxic molecules that enter the bloodstream. Another major
function of oxidative
reactions in peroxisomes is the breakdown of fatty acids in a process called
~i oxidation. (3 oxidation
results in shortening of the alkyl chain of fatty acids by blocks of two
carbon atoms that are converted
to acetyl CoA and exported to the cytosol for reuse in biosynthetic reactions.
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Also like mitochondria, peroxisomes import their proteins from the cytosol
using a specific
signal sequence located near the C-terminus of the protein. The importance of
this import process is
evident in the inherited human disease Zellweger syndrome, in which a defect
in importing proteins
into perixosomes leads to a perixosomal deficiency resulting in severe
abnormalities in the brain,
liver, and kidneys, and death soon after birth. One form of this disease has
been shown to be due to a
mutation in the gene encoding a perixosomal integral membrane protein called
peroxisome assembly
factor-1.
The discovery of new human molecules satisfies a need in the art by providing
new
compositions which are useful in the diagnosis, study, prevention, and
treatment of diseases associated
s o with, as well as effects of exogenous compounds on, the expression of
human molecules.
SUMMARY OF THE INVENTION
The present invention relates to nucleic acid sequences comprising human
diagnostic and
therapeutic polynucleotides (dithp) as presented in the Sequence Listing. The
dithp uniquely identify
genes encoding human structural, functional, and regulatory molecules.
The invention provides an isolated polynucleotide comprising a polynucleotide
sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID NO:1-21 l; b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:1-21 l; c) a
2 0 polynucleotide sequence complementary to a); d) a polynucleotide sequence
complementary to b); and
e) an RNA equivalent of a) through d). In one alternative, the polynucleotide
comprises a
polynucleotide sequence selected from the group consisting of SEQ ID N0:1-211.
In another
alternative, the polynucleotide comprises at least 60 contiguous nucleotides
of a polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID NO:l-211; b) a naturally occurring polynucleotide
sequence having at least 90%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID NO:1-
211; c) a polynucleotide sequence complementary to a); d) a polynucleotide
sequence complementary to
b); and e) an RNA equivalent of a) through d). The invention further provides
a composition for the
detection of expression of human diagnostic and therapeutic polynucleotides
comprising at least one
3 o isolated polynucleotide comprising a polynucleotide sequence selected from
the group consisting of a) a
polynucleotide sequence selected from the group consisting of SEQ ID N0:1-21
l; b) a naturally
occurring polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence
selected from the group consisting of SEQ ID N0:1-211; c) a polynucleotide
sequence complementary
to a); d) a polynucleotide sequence complementary to b); and e) an RNA
equivalent of a) through d);
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and a detectable label.
The invention also provides a method for detecting a target polynucleotide in
a sample, said
target polynucleotide comprising a polynucleotide sequence selected from the
group consisting of a) a
polynucleotide sequence selected from the group consisting of SEQ ID N0:1-211;
b) a naturally
occurring polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence
selected from the group consisting of SEQ ID N0:1-211; c) a polynucleotide
sequence complementary
to a); d) a polynucleotide sequence complementary to b); and e) an RNA
equivalent of a) through d).
The method comprises a) amplifying said target polynucleotide or a fragment
thereof using polymerise
chain reaction amplification, and b) detecting the presence or absence of said
amplified target
s o polynucleotide or fragment thereof, and, optionally, if present, the
amount thereof.
The invention also provides a method for detecting a target polynucleotide in
a sample, said
target polynucleotide comprising a polynucleotide sequence selected from the
group consisting of a) a
polynucleotide sequence selected from the group consisting of SEQ ID NO:1-211;
b) a naturally
occurring polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence
selected from the group consisting of SEQ ID NO:1-211; c) a polynucleotide
sequence complementary
to a); d) a polynucleotide sequence complementary to b); and e) an RNA
equivalent of a) through d).
The method comprises a) hybridizing the sample with a probe comprising at
least 20 contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
2 o hybridization complex is formed between said probe and said target
polynucleotide, and b) detecting the
presence or absence of said hybridization complex, and, optionally, if
present, the amount thereof. In
one alternative, the probe comprises at Ieast 30 contiguous nucleotides. In
another alternative, the
probe comprises at least 60 contiguous nucleotides.
The invention further provides a recombinant polynucleotide comprising a
promoter sequence
2 s operably linked to an isolated polynucleotide comprising a polynucleotide
sequence selected from the
group consisting of a) a polynucleotide sequence selected from the group
consisting of SEQ ID NO:1-
211; b) a naturally occurring polynucleotide sequence having at least 90%
sequence identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:1-211;
c) a polynucleotide
sequence complementary to a); d) a polynucleotide sequence complementary to
b); and e) an RNA
3 o equivalent of a) through d). In one alternative, the invention provides a
cell transformed with the
recombinant polynucleotide. In another alternative, the invention provides a
transgenic organism
comprising the recombinant polynucleotide. In a further alternative, the
invention provides a method
for producing a human diagnostic and therapeutic polypeptide, the method
comprising a) culturing a
cell under conditions suitable for expression of the human diagnostic and
therapeutic polypeptide,
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wherein said cell is transformed with the recombinant polynucleotide, and b)
recovering the human
diagnostic and therapeutic polypeptide so expressed.
The invention also provides a purified human diagnostic and therapeutic
polypeptide (DITHP)
encoded by at least one polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ ID NO:1-211. Additionally, the invention provides an
isolated antibody which
specifically binds to the human diagnostic and therapeutic polypeptide. The
invention further provides
a method of identifying a test compound which specifically binds to the human
diagnostic and
therapeutic polypeptide, the method comprising the steps of a) providing a
test compound; b) combining
the human diagnostic and therapeutic polypeptide with the test compound fox a
sufficient time and
s o under suitable conditions for binding; and c) detecting binding of the
human diagnostic and therapeutic
polypeptide to the test compound, thereby identifying the test compound which
specifically binds the
human diagnostic and therapeutic polypeptide.
The invention further provides a microarray wherein at least one element of
the microarray is
an isolated polynucleotide comprising at least 60 contiguous nucleotides of a
polynucleotide comprising
s 5 a polynucleotide sequence selected from the group consisting of a) a
polynucleotide sequence selected
from the group consisting of SEQ ID N0:1-211; b) a naturally occurring
polynucleotide sequence
having at least 90% sequence identity to a polynucleotide sequence selected
from the group consisting
of SEQ ID N0:1-211; c) a polynucleotide sequence complementary to a); d) a
polynucleotide sequence
complementary to b); arid e) an RNA equivalent of a) through d). The invention
also provides- a method
2 o for generating a transcript image of a sample which contains
polynucleotides. The method comprises a)
labeling the polynucleotides of the sample, b) contacting the elements of the
microarray with the labeled
polynucleotides of the sample under conditions suitable for the formation of a
hybridization complex,
and c) quantifying the expression of the polynucleotides in the sample.
Additionally, the invention provides a method for screening a compound for
effectiveness in
2 5 altering expression of a target polynucleotide, Wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of a) a
polynucleotide sequence selected
from the group consisting of SEQ ID N0:1-211; b) a naturally occurring
polynucleotide sequence
having at least 90% sequence identity to a polynucleotide sequence selected
from the group consisting
of SEQ ID N0:1-211; c) a polynucleotide sequence complementary to a); d) a
polynucleotide sequence
3 o complementary to b); and e) an RNA equivalent of a) through d). The method
comprises a) exposing a
sample comprising the target polynucleotide to a compound, and b) detecting
altered expression of the
target polynucleotide, and c) comparing the expression of the target
polynucleotide in the presence of
varying amounts of the compound and in the absence of the compound.
The invention further provides a method for assessing toxicity of a test
compound, said method
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comprising a) treating a biological sample containing nucleic acids with the
test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID N0:1-
211; ii) a naturally occurring polynucleotide sequence having at least 90%
sequence identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:1-211;
iii) a polynucleotide
sequence complementary to i), iv) a polynucleotide sequence complementary to
ii), and v) an RNA
equivalent of i)-iv). Hybridization occurs under conditions whereby a specific
hybridization complex
is formed between said probe and a target polynucleotide in the biological
sample, said target
1 o polynucleotide comprising a polynucleotide sequence selected from the
group consisting of i) a
polynucleotide sequence selected from the group consisting of SEQ ID NO:1-211;
ii) a naturally
occurring polynucleotide sequence having at least 90% sequence identity to a
polynucleotide
sequence selected from the group consisting of SEQ ID N0:1-211; iii) a
polynucleotide sequence
complementary to i), iv) a polynucleotide sequence complementary to ii), and
v) an RNA equivalent
of i)-iv), and alternatively, the target polynucleotide comprises a fragment
of a polynucleotide sequence
selected from the group consisting of i-v above; c) quantifying the amount of
hybridization complex;
and d) comparing the amount of hybridization complex in the treated biological
sample with the amount
of hybridization complex in an untreated biological sample, wherein a
difference inthe'amount of
hybridization complex in the treated biological sample is indicative of
toxicity of the test compound.
2 o The invention further provides an isolated polypeptide comprising an amino
acid sequence
selected from the group consisting of a) an amino acid sequence selected from
the group consisting of
SEQ ID N0:212-422, b) a naturally occurring amino acid sequence having at
least 90% sequence
identity to an amino acid sequence selected from the group consisting of SEQ
ID N0:212-422, c) a
biologically active fragment of an amino acid sequence selected from the group
consisting of SEQ ID
2 s N0:212-422, and d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID N0:212-422. In one alternative, the invention provides an
isolated polypeptide
comprising the amino acid sequence of SEQ ID N0:212-422.
DESCRIPTION OF THE TABLES
3 o Table 1 shows the sequence identification numbers (SEQ ID NOa) and
template identification
numbers (template IDs) corresponding to the polynucleotides of the present
invention, along with their
GenBank hits (GI Numbers), probability scores, and functional annotations
corresponding to the
GenB auk hits.
Table 2 shows the sequence identification numbers (SEQ ID NOa) and template
identification
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numbers (template IDs) corresponding to the polynucleotides of the present
invention, along with
polynucleotide segments of each template sequence as defined by the indicated
"start" and "stop"
nucleotide positions. The reading frames of the polynucleotide segments and
the Pfam hits, Pfam
descriptions, and E-values corresponding to the polypeptide domains encoded by
the polynucleotide
s segments are indicated.
Table 3 shows the sequence identification numbers (SEQ ID NOa) and template
identification
numbers (template IDs) corresponding to the polynucleotides of the present
invention, along with
polynucleotide segments of each template sequence as defined by the indicated
"start" and "stop"
nucleotide positions. The reading frames of the polynucleotide segments are
shown, and the
z o polypeptides encoded by the polynucleotide segments constitute either
signal peptide (SP) or
fransmembrane (TM) domains, as indicated. The membxane topology of the encoded
polypeptide
sequence is indicated, the N-terminus (N) listed as being oriented to either
the cytosolic (in) or non-
cytosolic (out) side of the cell membrane or organelle.
Table 4 shows the sequence identification numbers (SEQ ID NOa) corresponding
to the
15 polynucleotides of the present invention, along with component sequence
identification numbers
(component IDs) corresponding to each template. The component sequences, which
were used to
assemble the template sequences, are defined by the indicated "start" and
"stop" nucleotide positions
along each template.
Table S shows the tissue distribution profiles for the templates of the
invention.
2 o Table 6 shows the sequence identification numbers (SEQ ID NOa)
corresponding to the
polypeptides of the present invention, along with the reading frames used to
obtain the polypeptide
segments, the lengths of the polypeptide segments, the "start" and "stop"
nucleotide positions of the
polynucleotide sequences used to define the encoded polypeptide segments, the
GenBank hits (GI
Numbers), probability scores, and functional annotations corresponding to the
GenBank hits.
2 s Table 7 summarizes the bioinformatics tools which are useful for analysis
of the
polynucleotides of the present invention. The first column of Table 7 lists
analytical tools, programs,
and algorithms, the second column provides brief descriptions thereof, the
third column presents
appropriate references, all of which are incorporated by reference herein in
their entirety, and the fourth
column presents, where applicable, the scores, probability values, and other
parameters used to evaluate
3 o the strength of a match between two sequences (the higher the score, the
greater the homology between
two sequences).
DETAILED DESCRIPTION OF THE INVENTION
Before the nucleic acid sequences and methods are presented, it is to be
understood that this
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invention is not limited to the particular machines, methods, and materials
described. Although
particular embodiments are described, machines, methods, and materials similar
or equivalent to these
embodiments may be used to practice the invention. The preferred machines,
methods, and materials
set forth are not intended to limit the scope of the invention which is
limited only by the appended
claims.
The singular forms "a", "an", and "the" include plural reference unless the
context clearly
dictates otherwise. All technical and scientific terms have the meanings
commonly understood by one
of ordinary skill in the art. All publications are incorporated by reference
for the purpose of describing
and disclosing the cell lines, vectors, and methodologies which are presented
and which might be used in
s o connection with the invention. Nothing in the specification is to be
construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
Definitions
As used herein, the lower case "dithp" refers to a nucleic acid sequence,
while the upper case
"DITHP" refers to an amino acid sequence encoded by dithp. A "full-length"
dithp refers to a nucleic
acid sequence containing the entire coding region of a gene endogenously
expressed in human tissue.
"Adjuvants" are materials such as Freund's adjuvant, mineral gels (aluminum
hydroxide), and
surface active substances (lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole
limpet hemocyanin, and dinitrophenol) which may be administered to increase a
host's immunological
2 o response.
"Allele" refers to an alternative form of a nucleic acid sequence. Alleles
result from a
"mutation," a change or an alternative reading of the genetic code. Any given
gene may have none, one,
or many allelic forms. Mutations which give rise to alleles include deletions,
additions, or substitutions
of nucleotides. Each of these changes may occur alone, or in combination with
the others, one or more
2 s times in a given nucleic acid sequence. The present invention encompasses
allelic dithp.
"Amino acid sequence" refers to a peptide, a polypeptide, or a protein of
either natural or
synthetic origin. The amino acid sequence is not limited to the complete,
endogenous amino acid
sequence and may be a fragment, epitope, variant, or derivative of a protein
expressed by a nucleic acid
sequence.
3 0 "Amplification" refers to the production of additional copies of a
sequence and is carried out
using polymerase chain reaction (PCR) technologies well known in the art.
"Antibody" refers to intact molecules as well as to fragments thereof, such as
Fab, F(ab')2, and
Fv fragments, which are capable of binding the epitopic determinant.
Antibodies that bind DITIiP
polypeptides can be prepared using intact polypeptides or using fragments
containing small peptides of
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interest as the immunizing antigen. The polypeptide or peptide used to
immunize an animal (e.g., a
mouse, a rat, or a rabbit) can be derived from the translation of RNA, or
synthesized chemically, and
can be conjugated to a carrier protein if desired. Commonly used carriers that
are chemically coupled
to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet
hemocyanin (KhH). The
s coupled peptide is then used to immunize the animal.
"Antisense sequence" refers to a sequence capable of specifically hybridizing
to a target
sequence. The antisense sequence may include DNA, RNA, or any nucleic acid
mimic or analog such
as peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
s o sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as S-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine.
"Antisense sequence" refers to a sequence capable of specifically hybridizing
to a target
sequence. The antisense sequence can be DNA, RNA, or any nucleic acid mimic or
analog.
"Antisense technology" refers to any technology which relies on the specific
hybridization of an
2 ~ antisense sequence to a target sequence.
A "bin" is a portion of computer memory space used by a computer program for
storage of
data, and bounded in such a manner that data stored in a bin may be retrieved
by the program.
"Biologically active" refers to an amino acid sequence having a structural,
regulatory, or
biochemical function of a naturally occurring amino acid sequence.
2 0 "Clone joining" is a process for combining gene bins based upon the bins'
containing sequence
information from the same clone. The sequences may assemble into a primary
gene transcript as well
as one or more splice variants.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing (5'-A-G-T-3' pairs with its complement
3'-T-C-A-5').
2 s A "component sequence" is a nucleic acid sequence selected by a computer
program such as
PHRED and used to assemble a consensus or template sequence from one or more
component
sequences.
A "consensus sequence" or "template sequence" is a nucleic acid sequence which
has been
assembled from overlapping sequences, using a computer program for fragment
assembly such as the
3 o GEL'VIEW fragment assembly system (Genetics Computer Group (GCG), Madison
WI) or using a
relational database management system (RDMS).
"Conservative amino acid substitutions" are those substitutions that, when
made, least interfere
with the properties of the original protein, i.e., the structure and
especially the function of the protein is
conserved and not significantly changed by such substitutions. The table below
shows amino acids
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which may be substituted for an original amino acid in a protein and which are
regarded as conservative
substitutions.
Original Residue Conservative Substitution
s Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
s o Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
15 Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
2 o Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Va1 Ile, Leu, Thr
Conservative substitutions generally maintain (a) the structure of the
polypeptide backbone in
the area of the substitution, fox example, as a beta sheet or alpha helical
conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side
chain.
"Deletion" refers to a change in either a nucleic or amino acid sequence in
which at least one
3 o nucleotide or amino acid residue, respectively, is absent.
"Derivative" refers to the chemical modification of a nucleic acid sequence,
such as by
replacement of hydrogen by an alkyl, acyl, amino, hydroxyl, or other group.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
3 5 "E-value" refers to the statistical probability that a match between two
sequences occurred by
chance.
A "fragment" is a unique portion of dithp or DITHP which is identical in
sequence to but
shorter in length than the parent sequence. A fragment may comprise up to the
entire length of the
defined sequence, minus one nucleotide/amino acid residue. For example, a
fragment may comprise
4 o from 10 to 1000 contiguous amino acid residues or nucleotides. A fragment
used as a probe, primer,
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antigen, therapeutic molecule, or for other purposes, may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50,
60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues or
nucleotides in length.
Fragments may be preferentially selected from certain regions of a molecule.
For example, a
polypeptide fragment may comprise a certain length of contiguous amino acids
selected from the first
s 250 or 500 amino acids (or first 25 % or 50%) of a polypeptide as shown in a
certain defined sequence.
Clearly these lengths are exemplary, and any length that is supported by the
specification, including the
Sequence Listing and the figures, may be encompassed by the present
embodiments.
A fragment of dithp comprises a region of unique polynucleotide sequence that
specifically
identifies dithp, for example, as distinct from any other sequence in the same
genome. A fragment of
1 o dithp is useful, for example, in hybridization and amplification
technologies and in analogous methods
that distinguish dithp from related polynucleotide sequences. The precise
length of a fragment of dithp
and the region of dithp to which the fragment corresponds are routinely
determinable by one of ordinary
skill in the art based on the intended purpose for the fragment.
A fragment of DITHP is encoded by a fragment of dithp. A fragment of DITHP
comprises a
1 s region of unique amino acid sequence that specifically identifies DITHP.
For example, a fragment of
DITHP is useful as an immunogenic peptide for the development of antibodies
that specifically
recognize DITHP. The precise length of a fragment of DITHP and the region of
DITHP to which the
fragment corresponds are routinely determinable by one of ordinary skill in
the art based on the intended
purpose for the fiagment.
2 o A "full length" nucleotide sequence is one containing at least a start
site for translation to a
protein sequence, followed by an open reading frame and a stop site, and
encoding a "full length"
polypeptide.
"Hit" refers to a sequence whose annotation will be used to describe a given
template. Criteria
for selecting the top hit are as follows: if the template has one or more
exact nucleic acid matches, the
2 s top hit is the exact match with highest percent identity. If the template
has no exact matches but has
significant protein hits, the top hit is the protein hit with the lowest E-
value. If the template has no
significant protein hits, but does have significant non-exact nucleotide hits,
the top hit is the nucleotide
hit with the lowest E-value.
"Homology" refers to sequence similarity either between a reference nucleic
acid sequence and
3 o at least a fragment of a dithp or between a reference amino acid sequence
and a fragment of a DITHP.
"Hybridization" refers to the process by which a strand of nucleotides anneals
with a
complementary strand through base pairing. Specific hybridization is an
indication that two nucleic
acid sequences share a high degree of identity. Specific hybridization
complexes form under defined
annealing conditions, and remain hybridized after the "washing" step. The
defined hybridization
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conditions include the annealing conditions and the washing step(s), the
latter of which is particularly
important in determining the stringency of the hybridization process, with
more stringent conditions
allowing less non-specific binding, i.e., binding between paws of nucleic acid
probes that are not
perfectly matched. Permissive conditions for annealing of nucleic acid
sequences are routinely
s determinable and may be consistent among hybridization experiments, whereas
wash conditions may be
varied among experiments to achieve the desired stringency.
Generally, stringency of hybridization is expressed with reference to the
temperature under
which the wash step is carried out. Generally, such wash temperatures are
selected to be about 5°C to
20°C lower than the thermal melting point (T,~ for the specific
sequence at a defined ionic strength and
s o pH. The Tm is the temperature (under defined ionic strength and pH) at
which 50% of the target
sequence hybridizes to a perfectly matched probe. An equation for calculating
Tm and conditions for
nucleic acid hybridization is well known and can be found in Sambrook et al.,
1989, Molecular
Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press,
Plainview NY; specifically
see volume 2, chapter 9,
~ s High stringency conditions for hybridization between polynucleotides of
the present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, or 55°C
may be used. SSC concentration may be
varied from about 0.2 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking reagents.
are used to block non-specific hybridization. Such blocking reagents include,
for instance, denatured
2 o salmon sperm DNA at about 100-200 ~ g/ml. Useful variations on these
conditions will be readily
apparent to those skilled in the art. Hybridization, particularly under high
stringency conditions, may
be suggestive of evolutionary similarity between the nucleotides. Such
similarity is strongly indicative
of a similar role for the nucleotides and their resultant proteins.
Other parameters, such as temperature, salt concentration; and detergent
concentration may be
2 5 varied to achieve the desired stringency. Denaturants, such as formamide
at a concentration of about
35-50%o v/v, may also be used under particular circumstances, such as RNA:DNA
hybridizations.
Appropriate hybridization conditions are routinely determinable by one of
ordinary skill in the art.
"Immunogenic" describes the potential for a natural, recombinant, or synthetic
peptide, epitope,
polypeptide, or protein to induce antibody production in appropriate animals,
cells, or cell lines.
3 0 "Insertion" or "addition" refers to a change in either a nucleic or amino
acid sequence in which
at least one nucleotide or residue, respectively, is added to the sequence.
"Labeling" refers to the covalent or noncovalent joining of a polynucleotide,
polypeptide, or
antibody with a reporter molecule capable of producing a detectable or
measurable signal.
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"Microarray" is any arrangement of nucleic acids, amino acids, antibodies,
etc., on a substrate.
The substrate may be a solid support such as beads, glass, paper,
nitrocellulose, nylon, or an
appropriate membrane.
"Linkers" are short stretches of nucleotide sequence which may be added to a
vector or a dithp
s to create restriction endonuclease sites to facilitate cloning.
"Polylinkers" are engineered to incorporate
multiple restriction enzyme sites and to provide for the use of enzymes which
leave 5' or 3' overhangs
(e.g., BamHI, EcoRI, and HindIII) and those which provide blunt ends (e.g.,
EcoRV, SnaBI, and StuI).
"Naturally occurring" refers to an endogenous polynucleotide or polypeptide
that may be
isolated from viruses or prokaryotic or eukaryotic cells.
z o "Nucleic acid sequence" refers to the specific order of nucleotides joined
by phosphodiester
bonds in a linear, polymeric arrangement. Depending on the number of
nucleotides, the nucleic acid
sequence can be considered an oligomer, oligonucleotide, or polynucleotide.
The nucleic acid can be
DNA, RNA, or any nucleic acid analog, such as PNA, may be of genomic or
synthetic origin, may be
either double-stranded or single-stranded, and can represent either the sense
or antisense
15 (complementary) strand.
"Oligomer" refers to a nucleic acid sequence of at least about 6 nucleotides
and as many as
about 60 nucleotides, preferably about 15 to 40 nucleotides, and most
preferably between about 20 and
30 nucleotides, that may be used in hybridization or amplification
technologies. Oligomers may be used
as, e.g., primers for PCR, and are usually chemically synthesized.
2 0 "Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with the second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Generally, operably linked DNA sequences may be in close proximity
or contiguous and,
where necessary to join two protein coding regions, in the same reading frame.
2 s "Peptide nucleic acid" (PNA) refers to a DNA mimic in which nucleotide
bases are attached to
a pseudopeptide backbone to increase stability. PNAs, also designated antigene
agents, can prevent
gene expression by targeting complementary messenger RNA.
The phrases "percent identity" and "% identity", as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
3 o standardized algorithm. Such an algorithm may insert, in a standardized
and reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence
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alignment program. This program is part of the LASERGENE software package, a
suite of molecular
biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in
Higgins, D.G.
and Sharp, P.M. (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
s Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
"percent similarity" between
aligned polynucleotide sequence pairs.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBn Basic
Local Alignment Search
l o Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to determine alignment between a
known polynucleotide
sequence and other sequences on a variety of databases. Also available is a
tool called "BLAST 2
1 s Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.govlgorflbl2/. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2 0 2Ø9 (May-07-1999) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOS UM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 atad Extensios2 Gap: 2 pe~aalties
2 s Gap x drop-off.' S0
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example, as
a o defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least S0, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
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shown herein, in figures or Sequence Listings, may be used to describe a
length over which percentage
identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
s nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases "percent identity" and "% identity", as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
i o methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the hydrophobicity and
acidity of the substituted
residue, thus preserving the structure (and therefore function) of the folded
polypeptide:
Percent identity between polypeptide sequences may be determined using the
default parameters
of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment
15 program (described and referenced above). For pairwise alignments of
polypeptide sequences using
CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3,
window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default residue
weight table. As with
polynucleotide alignments, the percent identity is reported by CLUSTAL V as
the "percent similarity"
between aligned polypeptide sequence pairs.
2 o Alternatively the NCBI BLAST software suite may be used. For example, for
a pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø9
(May-07-1999) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: Il and Extension Gap: 1 penalty
2 s Gap x drop-off. 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
3 o example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
a fragment of at least 15, at least 20, at least 30, at least 40, at least 50,
at least 70 or at least 150
contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment length
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supported by the sequences shown herein, in figures or Sequence Listings, may
be used to describe a
length over which percentage identity may be measured.
"Post-translational modification" of a DITHP may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
s art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary by
cell type depending on the enzymatic milieu and the DITHP.
"Probe" refers to dithp or fragments thereof, which are used to detect
identical, allelic or related
nucleic acid sequences. Probes are isolated oligonucleotides or
polynucleotides attached to a detectable
label or reporter molecule. Typical labels include radioactive isotopes,
figands, chemiluminescent
1 o agents, and enzymes. "Primers" are short nucleic acids, usually DNA
oligonucleotides, which may be
annealed to a target polynucleotide by complementary base-pairing. The primer
may then be extended
along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be
used for amplification
(and identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
i5 nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 30, 40, 50,
60, 70, 80, 90, 100, or
at least 150 consecutive nucleotides of the disclosed nucleic acid sequences.
Probes and primers may
be considerably longer than these examples, and it is understood that any
length supported by the '
specification, including the figures and Sequence Listing, may be used.
2 o Methods for preparing and using probes and primers are described in the
references, for
example Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel et al., 1987, Current Protocols in
Molecular Biolo~y,
Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis et al., 1990,
PCR Protocols, A Guide
to Methods and Applications, Academic Press, San Diego CA. PCR primer pairs
can be derived from
2 s a known sequence, for example, by using computer programs intended for
that purpose such as Primer
(Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
a o nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from
megabase sequences
and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection
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program (available to the public from the Whitehead Institute/MIT Center for
Genome Research,
Cambridge MA) allows the user to input a "mispriming library," in which
sequences to avoid as primer
binding sites are user-specified. Primer3 is useful, in particular, for the
selection of oligonucleotides for
microarrays. (The source code for the latter two primer selection programs may
also be obtained from
s their respective sources and modified to meet the user's specific needs.)
The PrimeGen program
(available to the public from the UK Human Genome Mapping Project Resource
Centre, Cambridge
UK) designs primers based on multiple sequence alignments, thereby allowing
selection of primers that
hybridize to either the most conserved or least conserved regions of aligned
nucleic acid sequences.
Hence, this program is useful for identification of both unique and conserved
oligonucleotides and
1 o polynucleotide fragments. The oligonucleotides and polynucleotide
fragments identified by any of the
above selection methods are useful in hybridization technologies, for example,
as PCR or sequencing
primers, microaxxay elements, or specific probes to identify fully or
partially complementar y
polynucleotides in a sample of nucleic acids. Methods of oligonucleotide
selection are not limited to
those described above.
~ 5 "Purified" refers to molecules, either polynucleotides or polypeptides
that are isolated or
separated from their natural environment and are at Ieast 60% free, preferably
at least 75% free, and
most preferably at least 90% free from other compounds with which they are
naturally associated.
A ''recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
2 o This artificial combination is often accomplished by chemical synthesis
or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
2 5 Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
"Regulatory element" refers to a nucleic acid sequence from nontranslated
regions of a gene,
3 o and includes enhancers, promoters, introns, and 3' untranslated regions,
which interact with host
proteins to carry out or regulate transcription or translation.
"Reporter" molecules are chemical or biochemical moieties used for labeling a
nucleic acid, an
amino acid, or an antibody. They include radionuclides; enzymes; fluorescent,
chemiluminescent, or
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chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and
other moieties known in
the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
s nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
"Sample" is used in its broadest sense. Samples may contain nucleic or amino
acids,
antibodies, or other materials, and may be derived from any source (e.g.,
bodily fluids including, but not
limited to, saliva, blood, and urine; chromosome(s), organelles, or membranes
isolated from a cell;
1 o genomic DNA, RNA, or cDNA in solution or bound to a substrate; and cleared
cells or tissues or blots
or imprints from such cells or tissues).
"Specific binding" or "specifically binding" refers to the interaction between
a protein or
peptide and its agonist, antibody, antagonist, or other binding partner. The
interaction is dependent
upon the presence of a particular structure of the protein, e.g., the
antigenic determinant or epitope,
1 s recognized by the binding molecule. For example, if an antibody is
specific for epitope "A," the
presence of a polypeptide containing epitope A, or the presence of free
unlabeled A, in a reaction
containing free labeled A and the antibody will reduce the amount of labeled A
that binds to the
antibody.
"Substitution" refers to the replacement of at least one nucleotide or amino
acid by a different
2 o nucleotide or amino acid.
"Substrate" refers to any suitable rigid or semi-rigid support including,
e.g., membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles or capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
2 s A "transcript image" refers to the collective pattern of gene expression
by a particular tissue or
cell type under given conditions at a given time.
"Transformation" refers to a process by which exogenous DNA enters a recipient
cell.
Transformation may occur under natural or artificial conditions using various
methods well known in
the art. Transformation may rely on any known method for the insertion of
foreign nucleic acid
3 o sequences into a prokaryotic or eukaryotic host cell. The method is
selected based on the host cell being
transformed.
"Transformants" include stably transformed cells in which the inserted DNA is
capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well as
cells which transiently express inserted DNA or RNA.
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A "transgenic organism," as used herein, is any organism, including but not
limited to animals
and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art. The
nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell,
s by way of deliberate genetic manipulation, such as by microinjection or by
infection with a recombinant
virus. The term genetic manipulation does not include classical cross-
breeding, or in vitro fertilization,
but rather is directed to the introduction of a recombinant DNA molecule. The
transgenic organisms
contemplated in accordance with the present invention include bacteria,
cyanobacteria, fungi, and plants
and animals. The isolated DNA of the present invention can be introduced into
the host by methods
1 o known in the art, for example infection, transfection, transformation or
transconjugation. Techniques
for transferring the DNA of the present invention into such organisms are
widely known and provided in
references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having at
least 25 % sequence identity to the particular nucleic acid sequence over a
certain length of one of the
15 nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-1999)
set at default parameters. Such a pair of nucleic acids may show, for example,
at least 30%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or
even at least 98% or
greater sequence identity over a certain defined length. The variant may
result in "conservative" amino
acid changes which do not affect structural and/or chemical properties. A
variant may be described as,
2 o for example, an ''allelic" (as defined above), "splice," "species," or
"polymorphic" variant. A splice
variant may have significant identity to a reference molecule, but will
generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during mRNA
processing. The
corresponding polypeptide may possess additional functional domains or lack
domains that are present
in the reference molecule. Species variants are polynucleotide sequences that
vary from one species to
2 s another. The resulting polypeptides generally will have significant amino
acid identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of
a particular gene between
individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base.
The presence of
SNPs may be indicative of, for example, a certain population, a disease state,
or a propensity for a
a o disease state.
In an alternative, variants of the polynucleotides of the present invention
may be generated
through recombinant methods. One possible method is a DNA shuffling technique
such as
MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
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Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of DITHI', such as its biological or enzymatic
activity or its ability to bind to
other molecules or compounds. DNA shuffling is a process by which a library of
gene variants is
produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
s selection or screening procedures that identify those gene variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selectionlscreening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular
evolution. For example, fragments of a single gene containing random point
mutations may be
recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
~ o fragments of a given gene may be recombined with fragments of homologous
genes in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
1 s the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60.%, at least 70%, at least 80%, at least 90%, at least 95%, or at
least 98% or greater sequence
identity over a certain defined length of one of the polypeptides.
2 o THE INVENTION
In a particular embodiment, cDNA sequences derived from human tissues and cell
lines were
aligned based on nucleotide sequence identity and assembled into "consensus"
or "template" sequences
which are designated by the template identification numbers (template IDs) in
column 2 of Table 1.
The sequence identification numbers (SEQ ID NOa) corresponding to the template
IDs are shown in
2 s column 1. The template sequences have similarity to GenBank sequences, or
"hits," as designated by
the GI Numbers in column 3. The statistical probability of each GenBank hit is
indicated by a
probability score in column 4, and the functional annotation corresponding to
each GenBank hit is listed
in column 5.
The invention incorporates the nucleic acid sequences of these templates as
disclosed in the
3 o Sequence Listing and the use of these sequences in the diagnosis and
treatment of disease states
characterized by defects in human molecules. The invention further utilizes
these sequences in
hybridization and amplification technologies, and in particular, in
technologies which assess gene
expression patterns correlated with specific cells or tissues and their
responses in vivo or in vitro to
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pharmaceutical agents, toxins, and other treatments. In this manner, the
sequences of the present
invention are used to develop a transcript image for a particular cell or
tissue.
Derivation of Nucleic Acid Sequences
s cDNA was isolated from libraries constructed using RNA derived from normal
and diseased
human tissues and cell lines. The human tissues and cell lines used for cDNA
library construction were
selected from a broad range of sources to provide a diverse population of
cDNAs representative of gene
transcription throughout the human body. Descriptions of the human tissues and
cell lines used for
cDNA libraxy construction are provided in the LIFESEQ database (Incyte
Genomics, Inc. (Incyte), Palo
1 o Alto CA). Human tissues were broadly selected from, for example,
cardiovascular, dermatologic,
endocrine, gastrointestinal, hematopoieticlimmune system, musculoskeletal,
neural, reproductive, and
urologic sources.
Cell lines used for cDNA library construction were derived from, for example,
leukemic cells,
teratocarcinomas, neuroepitheliomas, cervical carcinoma, lung fibroblasts, and
endothelial cells. Such
15 cell lines include, for example, THP-1, Jurkat, HUVEC, hNT2, WI38, HeLa,
and other cell lines
commonly used and available from public depositories (American Type Culture
Collection, Manassas
VA). Prior to mRNA isolation, cell lines were untreated, treated with a
pharmaceutical agent such as
5'-aza-2'-deoxycytidine, treated with an activating agent such as
lipopolysaccharide in the case of
leukocytic cell lines, or, in the case of endothelial cell lines, subjected to
shear stress.
Seauencin~ of the cDNAs
Methods for DNA sequencing are well known in the art. Conventional enzymatic
methods
employ the HIenow fragment of DNA polymerise I, SEQUENASE DNA polymerise (U.S.
Biochemical Corporation, Cleveland OH), Taq polymerise (Applied Biosystems,
Foster City CA),
2 s thermostable T7 polymerise (Amersham Pharmacia Biotech, Inc. (Amersham
Pharmacia Biotech),
Piscataway NJ), or combinations of polymerises and proofreading exonucleases
such as those found in
the ELONGASE amplification system (Life Technologies Inc. (Life Technologies),
Gaithersburg MD),
to extend the nucleic acid sequence from an oligonucleotide primer annealed to
the DNA template of
interest. Methods have been developed for the use of both single-stranded and
double-stranded
3 o templates. Chain termination reaction products may be electrophoresed on
urea-polyacrylamide gels
and detected either by autoradiography (for radioisotope-labeled nucleotides)
or by fluorescence (for
fluorophore-labeled nucleotides). Automated methods for mechanized reaction
preparation, sequencing,
and analysis using fluorescence detection methods have been developed.
Machines used to prepare
cDNAs for sequencing can include the MICROLAB 2200 liquid transfer system
(Hamilton Company
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(Hamilton), Reno NV), Peltier thermal cycler (PTC200; MJ Research, Inc. (MJ
Research), Watertown
MA), and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing can
be carried out
using, for example, the ABI 373 or 377 (Applied Biosystems) or MEGABACE 1000
(Molecular
Dynamics, Inc. (Molecular Dynamics), Sunnyvale CA) DNA sequencing systems, or
other automated
and manual sequencing systems well known in the art.
The nucleotide sequences of the Sequence Listing have been prepared by
current, state-of the-
art, automated methods and, as such, may contain occasional sequencing errors
or unidentified
nucleotides. Such unidentified nucleotides are designated by an N. These
infrequent unidentified bases
do not represent a hindrance to practicing the invention for those skilled in
the art. Several methods
1 o employing standard recombinant techniques may be used to correct errors
and complete the missing
sequence information. (See, e.g., those described in Ausubel, F.M. et al.
(1997) Short Protocols in
Molecular Biolo~y, John Wiley & Sons, New York NY; and Sambrook, J. et al.
(1989) Molecular
Cloning, A Laborator~Manual, Cold Spring Harbor Press, Plainview NY.)
Assembly of cDNA Seguences
Human polynucleotide sequences may be assembled using programs or algorithms
well known
in the art. . Sequences to be assembled are related, wholly or in part, and
may be derived from a single
or many different transcripts. Assembly of the sequences can be performed
using such programs as
PHRAP (Phils Revised Assembly Program) and the GELVIEW fragment assembly
system (GCG), or
2 0 other methods known in the art.
Alternatively, cDNA sequences are used as "component" sequences that are
assembled into
"template" or "consensus" sequences as follows. Sequence chromatograms are
processed, verified, and
quality scores are obtained using PHRED. Raw sequences are edited using an
editing pathway known
as Block 1 (See, e.g., the LIFESEQ Assembled User Guide, Incyte Genomics, Palo
Alto, CA). A series
of BLAST comparisons is performed and low-information segments and repetitive
elements (e.g.,
dinucleotide repeats, Alu repeats, etc.) are replaced by "n's", or masked, to
prevent spurious matches.
Mitochondrial and ribosomal RNA sequences are also removed. The processed
sequences are then
loaded into a relational database management system (RDMS) which assigns
edited sequences to
existing templates, if available. When additional sequences are added into the
RDMS, a process is
s o initiated which modifies existing templates or creates new templates from
works in progress (i.e.,
nonfmal assembled sequences) containing queued sequences or the sequences
themselves. After the new
sequences have been assigned to templates, the templates can be merged into
bins. If multiple templates
exist in one bin, the bin can be split and the templates reannotated.
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Once gene bins have been generated based upon sequence alignments; bins are
"clone joined"
based upon clone information. Clone joining occurs when the 5' sequence of one
clone is present in one
bin and the 3' sequence from the same clone is present in a different bin,
indicating that the two bins
should be merged into a single bin. Only bins which share at least two
different clones are merged.
s A resultant template sequence may contain either a partial or a full length
open reading frame,
or all or part of a genetic regulatory element. This variation is due in part
to the fact that the full length
cDNAs of many genes are several hundred, and sometimes several thousand, bases
in length. With
current technology, cDNAs comprising the coding regions of large genes cannot
be cloned because of
vector limitations, incomplete reverse transcription of the mRNA, or
incomplete "second strand"
~. o synthesis. Template sequences may be extended to include additional
contiguous sequences derived
from the parent RNA transcript using a variety of methods known to those of
skill in the art. Extension
may thus be used to achieve the full length coding sequence of a gene.
Analysis of the cDNA Sequences
15 The cDNA sequences are analyzed using a variety of programs and algorithms
which are well
known in the art. (See, e.g., Ausubel, 1997, supra, Chapter 7.7; Meyers, R.A.
(Ed.) (1995) Molecular
Biolo~y and Biotechnology, Wiley VCH, New York NY, pp. 856-853; and Table 7.)
These analyses
comprise both reading frame determinations, e.g., based on triplet codon
periodicity for particular
organisms (Fickett, J.W. (1982) Nucleic Acids Res. 10:5303-5318); analyses of
potential start and stop
2 o codons; and homology searches.
Computer programs known to those of skill in the art for performing computer-
assisted
searches for amino acid and nucleic acid sequence similarity, include, for
example, Basic Local
Alignment Search Tool (BLAST; Altschul, S.F. (1993) J. Mol. Evol. 36:290-300;
Altschul, S.F. et al.
(1990) J. Mol. Biol. 215:403-410). BLAST is especially useful in determining
exact matches and
2 s comparing two sequence fragments of arbitrary but equal lengths, whose
alignment is locally maximal
and for which the alignment score meets or exceeds a threshold or cutoff score
set by the user (Karlin,
S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:841-845). Using an appropriate
search tool (e.g.,
BLAST or HMM), GenBank, SwissProt, BLOCKS, PFAM and other databases may be
searched fox
sequences containing regions of homology to a query dithp or DITHP of the
present invention.
s o Other approaches to the identification, assembly, storage, and display of
nucleotide and
polypeptide sequences are provided in "Relational Database for Storing
Biomolecule Information,"
U.S.S.N. 08/947,845, filed October 9, 1997; "Project-Based Full-Length
Biomolecular Sequence
Database," U.S.S.N. 08/811,758, filed March 6, 1997; and "Relational Database
and System for
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Storing Information Relating to Biomolecular Sequences," U.S.S.N. 09/034,807,
filed March 4, 1998,
all of which are incorporated by reference herein in their entirety.
Protein hierarchies can be assigned to the putative encoded polypeptide based
on, e.g., motif,
BLAST, or biological analysis. Methods for assigning these hierarchies are
described, for example, in
s "Database System Employing Protein Function Hierarchies for Viewing
Biomolecular Sequence Data,"
U.S.S.N. 08/812,290, filed March 6, 1997, incorporated herein by reference.
Identification of Human Diagnostic and Therapeutic Molecules Encoded b
The identities of the DITHP encoded by the dithp of the present invention were
obtained by
1 o analysis of the assembled cDNA sequences.
SEQ ID N0:212, SEQ ID N0:213, SEQ ID NO:214, SEQ ID N0:215, SEQ ID N0:216,
SEQ ID N0:217, SEQ ID N0:218, SEQ ID N0:219, SEQ ID N0:220, SEQ ID N0:221, SEQ
ID
N0:222, and SEQ ID N0:223, encoded by SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3,
SEQ ID
NO:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID
N0:10,
is SEQ ID N0:11, and SEQ ID N0:12, respectively, are, for example, human
enzyme molecules.
SEQ ID N0:224, SEQ ID N0:225, SEQ ID N0:226, SEQ ID N0:227, and SEQ ID N0:228,
encoded by SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, and SEQ ID
N0:17,
respectively, are, for example, receptor molecules.
SEQ ID N0:229, SEQ ID N0:230, SEQ ID NO:231, SEQ ID N0:232, SEQ ID N0:233,
2 o SEQ ID N0:234, SEQ ID N0:235, SEQ ID N0:236, SEQ ID N0:237, SEQ ID N0:238,
SEQ ID
N0:239, SEQ ID N0:240, and SEQ ID NO:241, encoded by SEQ ID N0:18, SEQ ID
N0:19, SEQ
ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID NO:24, SEQ ID
N0:25, SEQ
ID N0:26, SEQ ID N0;27, SEQ ID N0:28, SEQ ID N0:29, and SEQ ID N0:30,
respectively, are,
for example, intracellular signaling molecules.
2s SEQ ID N0:242, SEQ ID N0:243, SEQ ID N0:244, SEQ ID N0:245, SEQ ID N0:246,
SEQ ID NO:247, SEQ ID N0:248, SEQ ID N0:249, SEQ ID N0:250, SEQ ID NO:251, SEQ
ID
N0:252, SEQ ID N0:253, SEQ ID N0:254, SEQ ID N0:255, SEQ ID N0:256, SEQ ID
N0:257,
SEQ ID N0:258, SEQ ID N0:259, SEQ ID N0:260, SEQ ID N0:261, SEQ ID N0:262, SEQ
ID
N0:263, SEQ ID N0:264, SEQ ID N0:265, SEQ ID N0:266, SEQ ID N0:267, SEQ ID
N0:268,
3 o SEQ ID N0:269, SEQ ID N0:270, SEQ ID N0:271, SEQ ID N0:272, SEQ ID N0:273,
SEQ ID
N0:274, SEQ ID N0:275, SEQ ID N0:276, SEQ ID N0:277, SEQ ID N0:278, SEQ ID
N0:279,
SEQ ID N0:280, SEQ ID N0:281, SEQ ID N0:282, SEQ ID N0:283, SEQ ID N0:284, SEQ
ID
N0:285, SEQ ID N0:286, SEQ ID N0:287, SEQ ID N0:288, SEQ ID N0:289, SEQ ID
N0:290,
SEQ ID N0:291, SEQ ID N0:292, SEQ ID N0:293, SEQ ID N0:294, SEQ ID N0:295, SEQ
ID
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N0:296, SEQ ID N0:297, SEQ ID N0:298, SEQ ID N0:299, SEQ ID N0:300, SEQ ID
N0:301,
SEQ ID N0:302, SEQ ID N0:303, SEQ ID N0:304, SEQ ID N0:305, SEQ ID N0:306, SEQ
ID
N0:307, SEQ ID N0:308, and SEQ ID N0:309, encoded by SEQ ID N0:31, SEQ ID
N0:32, SEQ
ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID
N0:38, SEQ
s ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID
N0:44, SEQ
ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID
N0:50, SEQ
ID N0:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID
N0:56, SEQ
ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
N0:62, SEQ
ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID
N0:68, SEQ
to ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID
N0:74, SEQ
ID N0;75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID
N0:80, SEQ
ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID
N0:86, SEQ
ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID
N0:92, SEQ
ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID NO:96, SEQ ID N0:97, and SEQ ID
N0:98,
s s respectively, are, for example, transcription factor molecules.
SEQ ID N0:310, SEQ ID N0:31 l, SEQ ID N0:312, SEQ ID N0:313, SEQ ID N0:314,
SEQ ID N0:315, SEQ ID N0:316, and SEQ ID N0:317, encoded by SEQ ID NO;99, SEQ
ID
NO:100, SEQ ID N0:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID
N0:105,
and SEQ ID N0:106, respectively, are, for example, membrane transport
molecules.
2 o SEQ ID NO:318, SEQ ID N0:319, SEQ ID N0:320, SEQ ID N0:321, SEQ ID NO:322,
SEQ ID N0:323, SEQ ID N0:324, SEQ ID N0:325, SEQ ID N0:326, and SEQ ID N0:327,
encoded
by SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID N0:110, SEQ ID N0:111,
SEQ ID
N0:112, SEQ ID N0:113, SEQ ID NO;114, SEQ ID N0:115, and SEQ ID N0:116,
respectively, are,
for example, protein modification and maintenance molecules.
2s SEQ ID N0:328, SEQ ID N0:329, SEQ ID N0:330, SEQ ID N0:331, SEQ ID N0:332,
SEQ ID N0:333, SEQ ID N0:334, SEQ ID N0:335, SEQ ID N0:336, SEQ ID N0:337, SEQ
ID
N0:338, SEQ ID N0:339, SEQ ID N0:340, and SEQ ID N0:341, encoded by SEQ ID
N0:117, SEQ
ID N0:118, SEQ ID N0:119, SEQ ID N0:120, SEQ ID N0:121, SEQ ID N0:122, SEQ ID
NO:123,
SEQ ID N0:124, SEQ ID N0:125, SEQ ID N0:126, SEQ ID NO:127, SEQ ID N0:128, SEQ
ID
3 o N0:129, and SEQ ID N0:130, respectively, are, for example, nucleic acid
synthesis and modification
molecules.
SEQ ID N0:342, encoded by SEQ ID N0:131 is, for example, an adhesion molecule.
SEQ ID N0:343, SEQ ID N0:344, SEQ ID N0:345, SEQ ID N0:346, SEQ ID N0:347,
SEQ ID N0:348, and SEQ ID N0:349, encoded by SEQ ID N0:132, SEQ ID N0:133, SEQ
ID
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N0:134, SEQ ID N0:135, SEQ ID N0:136, SEQ ID N0:137, and SEQ ID N0:138,
respectively, are,
for example, antigen recognition molecules.
SEQ ID N0:350, SEQ ID N0:351, SEQ ID N0:352, and SEQ ID N0:353, encoded by SEQ
ID N0:139, SEQ ID N0:140, SEQ ID N0:141, and SEQ ID N0:142, respectively, are,
for example,
s electron transfer associated molecules.
SEQ ID N0:354, SEQ ID N0:355, SEQ ID N0:356, SEQ ID N0:357, SEQ ID N0:358, and
SEQ ID N0:359, encoded by SEQ ID NO: 243, SEQ ID N0:144, SEQ ID N0:145, SEQ ID
N0:146,
SEQ ID NO:147, and SEQ ID N0:148, respectively, are, for example,
secreted/extracellular matrix
molecules.
so SEQ ID N0:360, SEQ ID N0:361, SEQ ID N0:362, SEQ ID N0:363, SEQ ID N0:364,
SEQ ID N0:365, SEQ ID N0:366, SEQ ID N0:367, SEQ ID N0:368, and SEQ ID N0:369,
encoded
by SEQ ID N0:149, SEQ ID NO:150, SEQ ID N0:151, SEQ ID N0:152, SEQ ID N0:153,
SEQ ID
N0:154, SEQ ID N0:155, SEQ ID N0:156, SEQ ID N0:157, and SEQ ID N0:158,
respectively,
are, for example, cytoskeletal molecules.
15 SEQ ID N0:370, SEQ ID N0:371, SEQ ID N0:372, and SEQ ID N0:373, encoded by
SEQ
ID N0:159, SEQ ID N0:160, SEQ ID N0:161, and SEQ ID N0:162, respectively, are,
for example,
cell membrane molecules.
SEQ ID N0:374, SEQ ID N0:375, SEQ ID N0:376, SEQ ID N0:377, SEQ ID N0:378,
SEQ ID N0:379, SEQ ID N0:380, SEQ ID N0:381; SEQ ID N0:382, SEQ ID N0:383, SEQ
ID
2 o N0:384, SEQ ID N0:38S, SEQ ID N0:386, SEQ ID N0:387, SEQ ID N0:388, SEQ ID
N0:389,
SEQ ID N0:390, SEQ ID N0:391, and SEQ ID N0:392, encoded by SEQ ID N0:163, SEQ
ID
N0:164, SEQ ID N0:165, SEQ ID N0:166, SEQ ID N0:167, SEQ ID NO:168, SEQ ID
N0:169,
SEQ ID N0:170, SEQ ID N0:171, SEQ ID N0:172, SEQ ID N0:173, SEQ ID N0:174, SEQ
ID
N0:175, SEQ ID NO:176, SEQ ID N0:177, SEQ ID N0:178, SEQ ID N0:179, SEQ ID
N0:180,
2 s and SEQ ID N0:181, respectively, are, for example, ribosomal molecules.
SEQ ID N0:393, SEQ ID N0:394, SEQ ID N0:395, SEQ ID N0:396, SEQ ID NO:397,
SEQ ID N0:398, SEQ ID N0:399, SEQ ID N0:400, SEQ ID N0:401, SEQ ID N0:402, and
SEQ ID
N0:403, encoded by SEQ ID N0:182, SEQ ID N0:183, SEQ ID N0:184, SEQ ID N0:185,
SEQ ID
N0:186, SEQ ID N0:187, SEQ ID N0:188, SEQ ID N0:189, SEQ ID N0:190, SEQ ID
N0:191,
s o and SEQ ID N0:192, respectively, are, for example, organelle associated
molecules.
SEQ ID N0:404, SEQ ID N0:40S, SEQ ID N0:406, SEQ ID N0:407, SEQ ID N0:408,
SEQ ID N0:409, SEQ ID N0:410, SEQ ID N0:411, SEQ ID N0:412, SEQ ID N0:413, and
SEQ ID
N0:414, encoded by SEQ ID N0:193, SEQ ID N0:194, SEQ ID N0:195, SEQ ID N0:196,
SEQ ID
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N0:197, SEQ ID N0:198, SEQ ID N0:199, SEQ ID NO:200, SEQ ID N0:201, SEQ ID
N0:202,
and SEQ ID N0:203, respectively, are; for example, biochemical pathway
molecules.
SEQ ID N0:415, SEQ ID N0:416, SEQ ID N0:417, SEQ ID N0:41$, SEQ ID N0:419,
SEQ ID N0:420, SEQ ID N0:421, and SEQ ID N0:422, encoded by SEQ ID N0:204, SEQ
ID
N0:205, SEQ ID N0:206, SEQ ID N0:207, SEQ ID N0:208, SEQ ID N0:209, SEQ ID
N0:210,
and SEQ ID N0:211, respectively, are, for example, molecules associated with
growth and
development. '
Sequences of Human Diagnostic and Therapeutic Molecules
t o The dithp of the present invention may be used for a variety of diagnostic
and therapeutic
purposes. For example, a dithp may be used to diagnose a particular condition,
disease, or disorder
associated with human molecules. Such conditions, diseases, and disorders
include, but are not limited
to, a cell proliferative disorder, such as actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus; an
2 o autoimmune/inflammatory disorder, such as inflammation, actinic keratosis,
acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome,
allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis,
cholecystitis, cirrhosis,
contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus,
2 5 emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis,
Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,
paroxysmal nocturnal
hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome,
episodic lymphopenia with
lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis, pancreatitis,
3 o polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic Iupus erythematosus,
systenuc sclerosis, primary
thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, trauma,
and hematopoietic
cancer including lymphoma, leukemia, and myeloma; an infection caused by a
viral agent classified
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as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus,
hepadnavirus, herpesvirus,
flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus,
picornavirus, poxvirus, reovirus,
retrovirus, rhabdovirus, or togavirus; an infection caused by a bacterial
agent classified as
pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium,
clostridium,
s meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus,
legionella, bordetella, gram-
negative enterobacterium including shigella, salmonella, or campylobacter,
pseudomonas, vibrio,
brucella, francisella, yersinia, bartonella, norcardium, actinomyces,
mycobacterium, spirochaetale,
rickettsia, chlamydia, or mycoplasma; an infection caused by a fungal agent
classified as aspergillus,
blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia,
histoplasma, or other mycosis-
s o causing fungal agent; and an infection caused by a parasite classified as
plasmodium or malaria-
causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma,
pneumocystis carinii, intestinal
protozoa such as giardia, trichomonas, tissue nematode such as trichinella,
intestinal nematode such
as ascaris, lymphatic filarial nematode, trematode such as schistosoma, and
cestrode such as
tapeworm; a developmental disorder such as renal tubular acidosis, anemia,
Cushing's syndrome,
15 achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation),
Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy,
2 o spina-bifida, anencephaly, craniorachischisis, congenital glaucoma,
cataract, and sensorineural hearing
loss; an endocrine disorder such as a disorder of the hypothalamus and/ox
pituitary resulting from
lesions such as a primaxy brain tumor, adenoma, infarction associated with
pregnancy,
hypophysectomy, aneurysm, vascular malformation, thrombosis, infection,
immunological disorder, and
complication due to head trauma; a disorder associated with hypopituitarism
including hypogonadism,
2 s Sheehan syndrome, diabetes insipidus, I~allman's disease, Hand-Schuller-
Christian disease, Letterer-
Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism; a disorder
associated with
hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate
antidiuretic hormone
(ADH) secretion (SIADH) often caused by benign adenoma; a disorder associated
with hypothyroidism
including goiter, myxedema, acute thyroiditis associated with bacterial
infection, subacute thyroiditis
3 o associated with viral infection, autoimmune thyroiditis (Hashimoto's
disease), and cretinism; a disorder
associated with hyperthyroidism including thyrotoxicosis and its various
forms, Grave's disease,
pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and
Plummer's disease; a disorder
associated with hyperparathyroidism including Conn disease (chronic
hypercalemia); a pancreatic
disorder such as Type I or Type II diabetes mellitus and associated
complications; a disorder associated
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with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal
cortex, hypertension
associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease,
Liddle's syndrome, and
Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease;
a disorder
associated with gonadal steroid hormones such as: in women, abnormal prolactin
production,
s infertility, endometriosis, perturbation of the menstrual cycle, polycystic
ovarian disease,
hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea,
galactorrhea, hermaphroditism,
hirsutism and virilization, breast cancer, and, in post-menopausal women,
osteoporosis; and, in men,
Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a
hypergonadal disorder
associated with Leydig cell tumors, androgen resistance associated with
absence of androgen receptors,
s o syndrome of 5 a-reductase, and gynecomastia; a metabolic disorder such as
Addison's disease,
cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin
resistance, cystic fibrosis,
diabetes, fatty hepatocirrhosis, fructose-2,6-diphosphatase deficiency,
galactosemia, goiter,
glucagonoma, glycogen storage diseases, hereditary fructose intolerance,
hyperadrenalism,
hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia,
hyperthyroidism,
15 hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid
myopathies, lipodystrophies,
lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity,
pentosuria
phenylketonuria, pseudovitamin D-deficiency rickets; disorders of carbohydrate
metabolism such as
congenital type II dyserythropoietic anemia, diabetes, insulin-dependent
diabetes mellitus,
non-insulin-dependent diabetes mellitus, fructose-1,6-diphosphatase
deficiency, galactosemia,
2 o glucagonoma, hereditary fructose intolerance, hypoglycemia, mannosidosis,
neuraminidase
deficiency, obesity, galactose epimerase deficiency, glycogen storage
diseases, lysosomal storage
diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate
metabolism; disorders of
lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis,
carnitine deficiency,
carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency,
hypertriglyceridemia,
2 s lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-
Pick's dssease,
metachromatic leukodystrophy, adrenoleukodystrophy, GMZ gangliosidosis, and
ceroid
lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia,
diabetes mellitus,
lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis,
adiposis dolorosa, lipoid
adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis,
hypercholesterolemia,
3 o hypercholesterolemia with hypertriglyceridemia, primary
hypoalphalipoproteinemia, hypothyroidism,
renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency,
cerebrotendinous
xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease,
Sandhoff's disease,
hyperlipidemia, hyperlipemia, lipid myopathies, and obesity; and disorders of
copper metabolism
such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX;
a neurological
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disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural muscular
atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases,
s bacterial and viral meningitis, brain abscess, subdural empyema, epidural
abscess, suppurative
intracranial thrombophlebitas, myelitis and radiculitis, viral central nervous
system disease, prion
diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-
Scheinker syndrome,
fatal familial insomnia, nutritional and metabolic diseases of the nervous
system, neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syncliome, mental
s o retardation and other developmental disorder of the central nervous
system, cerebral palsy, a
neuroskeletal disorder, an autonomic nervous system disorder, a cranial nerve
disorder, a spinal cord
disease, muscular dystrophy and other neuromuscular disorder, a peripheral
nervous system disorder,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathy, myasthenia
gravis, periodic paralysis, a mental disorder including mood, anxiety, and
schizophrenic disorders,
1 s seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic
neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder; a
gastrointestinal disorder including ulcerative colitis, gastric and duodenal
ulcers, cystinuria,
dibasicaminoaciduria, hypercystinuria, lysinuria, hartnup disease, tryptophan
malabsorption,
methionine malabsorptaon, histidinuria, iminoglycinuria,
dicarboxylicaminoaciduria, cystinosis, renal
2 o glycosuria, hypouricenua, familial hypophophatemic rickets, congenital
chloridorrhea, distal renal
tubular acidosis, Menkes' disease, Wilson's disease, lethal diarrhea, juvenile
pernicious anemia,
folate malabsorption, adrenoleukodystrophy, hereditary myoglobinuria, and
Zellweger syndrome; a
transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia
telangiectasia, cystic fibrosis,
Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease,
diabetes mellitus, diabetes
2 s insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic
periodic paralysis,
normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia,
multidrug resistance,
myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia,
dystonias, peripheral
neuropathy, cerebral neoplasms, prostate cancer, cardiac disorders associated
with transport, e.g.,
angina, bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome,
myocarditis,
3 o cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy,
mitochondrial
myopathy, thyrotoxic myopathy, ethanol myopathy, ldermatomyositis, inclusion
body myositis,
infectious myositis, and polymyositis, neurological disorders associated with
transport, e.g.,
Alzheimer's disease, amnesia, bipolar disorder, dementia, depression,
epilepsy, Tourette's disorder,
paranoid psychoses, and schizophrenia, and other disorders associated with
transport, e.g.,
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neurofibromatosis, postherpetic neuralgia, trigeminal neuropathy, sarcoidosis,
sickle cell anemia,
cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal
deafness, hyperglycemia,
hypoglycemia, Grave's disease, goiter, glucose-galactose malabsorption
syndrome,
hypercholesterolemia, Cushing's disease, and Addison's disease; and a
connective tissue disorder
such as osteogenesis imperfecta, Ehlers-Danlos syndrome, chondrodysplasias,
Marian syndrome,
Alport syndrome, familial aortic aneurysm, achondroplasia,
mucopolysaccharidoses, osteoporosis,
osteopetrosis, Paget's disease, rickets, osteomalacia, hyperparathyroidism,
renal osteodystrophy,
osteonecrosis, osteomyelitis, osteoma, osteoid osteoma, osteoblastoma,
osteosarcoma,
osteochondroma, chondroma, chondroblastoma, chondromyxoid fibroma,
chondrosarcoma, fibrous
to cortical defect, nonossifying fibroma, fibrous dysplasia, fibrosarcoma,
malignant fibrous
histiocytoma, Ewing's sarcoma, primitive neuroectodermal tumor, giant cell
tumor, osteoarthritis,
rheumatoid arthritis, ankylosing spondyloarthritis, Reiter's syndrome,
psoriatic arthritis, enteropathic
arthritis, infectious arthritis, gout, gouty arthritis, calcium pyrophosphate
crystal deposition disease,
ganglion, synovial cyst, villonodular synovitis, systemic sclerosis,
Dupuytren's contracture, hepatic .
i5 fibrosis, lupus erythematosus, mixed connective tissue disease,
epidermolysis bullosa simplex, bullous
congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-
epidermolytic and
epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens,
pachyonychia congenita, and
white sponge nevus. The dithp can be used to detect the presence of, or to
quantify the amount of, a
dithp-related polynucleotide in a sample. This information is then compared to
information obtained
2 o from appropriate reference samples, and a diagnosis is established.
Alternatively, a polynucleotide
complementary to a given dithp can inhibit or inactivate a therapeutically
relevant gene related to the
dithp.
Analysis of dithp Expression Patterns
2 5 The expression of dithp may be routinely assessed by hybridization-based
methods to
determine, for example, the tissue-specificity, disease-specificity, or
developmental stage-specificity of
dithp expression. For example, the level of expression of dithp may be
compared among different cell
types or tissues, among diseased and normal cell types or tissues, among cell
types or tissues at
different developmental stages, or among cell types or tissues undergoing
various treatments. This type
a o of analysis is useful, for example, to assess the relative levels of dithp
expression in fully or partially
differentiated cells or tissues, to determine if changes in dithp expression
levels are correlated with the
development or progression of specific disease states, and to assess the
response of a cell or tissue to a
specific therapy, for example, in pharmacological or toxicological studies.
Methods for the analysis of
dithp expression are based on hybridization and amplification technologies and
include membrane-
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based procedures such as northern blot analysis, high-throughput procedures
that utilize, for example,
microarrays, and PCR-based procedures.
Hybridization and Genetic Analysis
s The dithp, their fragments, or complementary sequences, may be used to
identify the presence
of andlor to determine the degree of similarity between two (or more) nucleic
acid sequences. The dithp
may be hybridized to naturally occurring or recombinant nucleic acid sequences
under appropriately
selected temperatures and salt concentrations. Hybridization with a probe
based on the nucleic acid
sequence of at least one of the dithp allows for the detection of nucleic acid
sequences, including
s o genomic sequences, which are identical or related to the dithp of the
Sequence Listing. Probes may be
selected from non-conserved or unique regions of at least one of the
polynucleotides of SEQ ID N0:1-
211 and tested for their ability to identify or amplify the target nucleic
acid sequence using standard
protocols.
Polynucleotide sequences that are capable of hybridizing, in particular, to
those shown in SEQ
15 ID NO:1-211 and fragments thereof, can be identified using various
conditions of stringency. (See,
e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel,
A.R. (1987)
Methods Enzymol. 152:507-511.) Hybridization conditions are discussed in
"Definitions."
A probe for use in Southern or northern hybridization may be derived from a
fragment of a
dithp sequence, or its complement, that is up to several hundred nucleotides
in length and is either
2 o single-stranded or double-stranded. Such probes may be hybridized in
solution to biological materials
such as plasmids, bacterial, yeast, or human artificial chromosomes, cleared
or sectioned tissues, or to
artificial substrates containing dithp. Microarrays are particularly suitable
for identifying the presence
of and detecting the level of expression for multiple genes of interest by
examining gene expression
correlated with, e.g., various stages of development, treatment with a drug or
compound, or disease
2 s progression. An array analogous to a dot or slot blot may be used to
arrange and link polynucleotides
to the surface of a substrate using one or more of the following: mechanical
(vacuum), chemical,
thermal, or UV bonding procedures. Such an array may contain any number of
dithp and may be
produced by hand or by using available devices, materials, and machines.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
3 o Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095135505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Probes may be labeled by either PCR or enzymatic techniques using a variety of
commercially
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available reporter molecules. For example, commercial kits are available for
radioactive and
chemiluminescent labeling (Amersham Pharmacia Biotech) and for alkaline
phosphatase labeling (Life
Technologies). Alternatively, dithp may be cloned into commercially available
vectors for the
production of RNA probes. Such probes may be transcribed in the presence of at
least one labeled
nucleotide (e.g., 32P-ATP, Amersham Pharmacia Biotech).
Additionally the polynucleotides of SEQ ID N0:1-211 or suitable fragments
thereof can be
used to isolate full length cDNA sequences utilizing hybridization andlor
amplification procedures well
known in the art, e.g., cDNA library screening, PCR amplification, etc. The
molecular cloning of such
full length cDNA sequences may employ the method of cDNA library screening
with probes using the
s o hybridization, stringency, washing, and probing strategies described above
and in Ausubel, su ra,
Chapters 3, 5, and 6. These procedures may also be employed with genomic
libraries to isolate
genomic sequences of dithp in order to analyze, e.g., regulatory elements.
Genetic Mapping
Gene identification and mapping are important in the investigation and
treatment of almost all
conditions, diseases, and disorders. Cancer, cardiovascular disease,
Alzheimer's disease, arthritis,
diabetes, and mental illnesses are of particular interest. Each of these
conditions is more complex than
the single gene defects of sickle cell anemia or cystic fibrosis, with select
groups of genes being
predictive of predisposition for a particular condition, disease, or disorder.
Fox example,
2 o cardiovascular disease may result from malfunctioning receptor molecules
that fail to clear cholesterol
from the bloodstream, and diabetes may result when a particular individual's
immune system is
activated by an infection and attacks the insulin-producing cells of
the,pancreas. In some studies,
Alzheimer's disease has been linked to a gene on chromosome 21; other studies
predict a different gene
and location. Mapping of disease genes is a complex and reiterative process
and generally proceeds
2 s from genetic linkage analysis to physical mapping.
As a condition is noted among members of a family, a genetic linkage map
traces parts of
a
chromosomes that are inherited in the same pattern as the condition.
Statistics link the inheritance of
particular conditions to particular regions of chromosomes, as defined by RFLP
or other markers.
(See, for example, Larder, E. S. and Botstein, D. (1986) Proc. Natl. Acad.
Sci. USA 83:7353-7357.)
3 o Occasionally, genetic markers and their locations are known from previous
studies. More often,
however, the markers are simply stretches of DNA that differ among
individuals. Examples of genetic
linkage maps can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site.
In another embodiment of the invention, dithp sequences may be used to
generate hybridization
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probes useful in chromosomal mapping of naturally occurring genomic sequences.
Either coding or
noncoding sequences of dithp may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a dithp coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
s chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific region
of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
l0 7:149-154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
mapping techniques and genetic map data. (See, e.g., Meyers, supra, pp. 965-
968.) Correlation
between the location of dithp on a physical chromosomal map and a specific
disorder, or a
predisposition to a specific disorder, may help define the region of DNA
associated with that disorder.
15 The dithp sequences may also be used to detect polymorphisms that are
genetically linked to the
inheritance of a particular condition, disease, or disorder.
In situ hybridization of chromosomal preparations and genetic mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending existing genetic
maps. Often the placement of a gene on the chromosome of another mammalian
species, such as
2 o mouse, may reveal associated markers even if the number or arm of the
corresponding human
chromosome is not known. These new marker sequences can be mapped to human
chromosomes and
may provide valuable information to investigators searching for disease genes
using positional cloning
or other gene discovery techniques. Once a disease or syndrome has been
crudely correlated by genetic
linkage with a particular genomic region, e.g., ataxia-telangiectasia to 11q22-
23, any sequences
2s mapping to that area may represent associated or regulatory genes for
further investigation. (See, e.g.,
Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequences of the
subject invention may
also be used to detect differences in chromosomal architecture due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
Once a disease-associated gene is mapped to a chromosomal region, the gene
must be cloned in
3 0 order to identify mutations or other alterations (e.g., translocations or
inversions) that may be correlated
with disease. This process requires a physical map of the chromosomal region
containing the disease-
gene of interest along with associated markers. A physical map is necessary
for determining the
nucleotide sequence of and order of marker genes on a particular chromosomal
region. Physical
mapping techniques are well known in the art and require the generation of
overlapping sets of cloned
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DNA fragments from a particular organelle, chromosome, or gename. These clones
are analyzed to
reconstruct arid catalog their order. Once the position of a marker is
determined, the DNA from that
region is obtained by consulting the catalog and selecting clones from that
region. The gene of interest
is located through positional cloning techniques using hybridization or
similar methods.
Diagnostic Uses
The dithp of the present invention may be used to design probes useful in
diagnostic assays.
Such assays, well known to those skilled in the art, may be used to detect or
confirm conditions,
disorders, or diseases associated with abnormal levels of ditlip expression.
Labeled probes developed
s o from dithp sequences are added to a sample under hybridizing conditions of
desired stringency. In some
instances, dithp, or fragments or oligonucleotides derived from dithp, may be
used as primers in
amplification steps prior to hybridization. The amount of hybridization
complex formed is quantified
and compared with standards for that cell or tissue. If dithp expression
varies significantly from the
standard, the assay indicates the presence of the condition, disorder, or
disease. Qualitative or
1 s quantitative diagnostic methods may include northern, dot blot, or other
membrane or dip-stick based
technologies or multiple-sample format technologies such as PCR, enzyme-linked
immunosorbent assay
(ELISA)-like, pin, or chip-based' assays.
The probes described above may also be used to monitor the progress of
conditions, disorders,
or diseases associated with abnormal levels of dithp expression, or to
evaluate the efficacy of a
z o particular therapeutic treatment. The candidate probe may be identified
from, the dithp that are specific
to a given human tissue and have not been observed in GenBank or other genome
databases. Such a
probe may be used in animal studies, preclinical tests, clinical trials, or in
monitoring the treatment of
an individual patient. In a typical process, standard expression is
established by methods well known in
the art for use as a basis of comparison, samples from patients affected by
the disorder or disease are
2 s combined with the probe to evaluate any deviation from the standard
profile, and a therapeutic agent is
administered and effects are monitored to generate a treatment profile.
Efficacy is evaluated by
determining whether the expression progresses toward or returns to the
standard normal pattern.
Treatment profiles may be generated over a period of several days or several
months. Statistical
methods well known to those skilled in the art may be use to determine the
significance of such
3 o therapeutic agents.
The polynucleotides are also useful for identifying individuals from minute
biological samples,
for example, by matching the RFLP pattern of a sample's DNA to that of an
individual's DNA. The
polynucleotides of the present invention can also be used to determine the
actual base-by-base DNA
sequence of selected portions of an individual's genome. These sequences can
be used to prepare PCR
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primers for amplifying and isolating such selected DNA, which can then be
sequenced. Using this
technique, an individual can be identified through a unique set of DNA
sequences. Once a unique ID
database is established for an individual, positive identification of that
individual can be made from
extremely small tissue samples.
In a particular aspect, oligonucleotide primers derived from the dithp of the
invention may be
used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions,
insertions and
deletions that are a frequent cause of inherited or acquired genetic disease
in humans. Methods of SNP
detection include, but are not limited to, single-stranded conformation
polymorphism (SSCP) and
fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived
from dithp are used to
s o amplify DNA using the polymerase chain reaction (PCR). The DNA may be
derived, for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and these
differences are detectable using gel electrophoresis in non-denaturing gels.
In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
? 5 throughput equipment such as DNA sequencing machines. Additionally,
sequence database analysis
methods, termed in silico SNP (isSNP), axe capable of identifying
polymorphisms by comparing the
sequences of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
~ o chromatograms. In the alternative, SNPs may be detected and characterized
by mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
DNA-based identification techniques are critical in forensic technology. DNA
sequences taken
from very small biological samples such as tissues, e.g., hair or skin, or
body fluids, e.g., blood, saliva,
semen, etc., can be amplified using, e.g., PCR, to identify individuals. (See,
e.g., Erlich, H. (1992)
2 5 PCR Technolo~y, Freeman and Co., New York, NY). Similarly, polynucleotides
of the present
invention can be used as polymorphic markers.
There is also a need for reagents capable of identifying the source of a
particular tissue.
Appropriate reagents can comprise, for example, DNA probes or primers prepared
from the sequences
of the present invention that are specific for particular tissues. Panels of
such reagents can identify
3 o tissue by species and/or by organ type. In a similar fashion, these
reagents can be used to screen tissue
cultures for contamination.
The polynucleotides of the present invention can also be used as molecular
weight markers on
nucleic acid gels or Southern blots, as diagnostic probes for the presence of
a specific mRNA in a
particular cell type, in the creation of subtracted cDNA libraries which aid
in the discovery of novel
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polynucleotides, in selection and synthesis of oligomers for attachment to an
array or other support, and
as an antigen to elicit an immune response.
Disease Model Stems Using dithp
The dithp of the invention or their mammalian homologs may be "knocked out" in
an animal
model system using homologous recombination in embryonic stem (ES) cells. Such
techniques are well
known in the art and are useful for the generation of animal models of human
disease. (See, e.g., U.S.
Patent Number 5,175,383 and U.S. Patent Number 5,767,337.) For example, mouse
ES cells, such as
the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown
in culture. The ES
1 o cells are transformed with a vector containing the gene of interest
disrupted by a marker gene, e.g., the
neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-
1292). The vector
integrates into the corresponding region of the host genome by homologous
recombination.
Alternatively, homologous recombination takes place using the Cre-loxP system
to knockout a gene of
interest in a tissue- or developmental stage-specific manner (Marth, J.D.
(1996) Clin. Invest. 97:1999-
2002; Wagner, I~.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
Transformed ES cells are
identiFied and microinjected into mouse cell blastocysts such as those from
the C57BL/6 mouse strain.
The blastocysts are surgically transferred to pseudopregnant dams, and the
resulting chimeric progeny
are genotyped and bred to produce heterozygous or homozygous strains.
Transgenic animals thus
generated may be tested with potential therapeutic or toxic agents.
2 o The dithp of the invention may also be manipulated in vitro in ES cells
derived from human
blastocysts. Human ES cells have the potential to differentiate into at least
eight separate cell lineages
including endoderm, mesoderm, and ectodermal cell types. These cell lineages
differentiate into, for
example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson,
J.A, et al. (1998) Science
282:1145-1147).
2 s The dithp of the invention can also be used to create "knockin" humanized
animals (pigs) or
transgenic animals (mice or rats) to model human disease. With knockin
technology, a region of dithp
is injected into animal ES cells, and the injected sequence integrates into
the animal cell genome.
Transformed cells are injected into blastulae, and the blastulae are implanted
as described above.
Transgenic progeny or inbred lines are studied and treated with potential
pharmaceutical agents to
3 0 obtain information on treatment of a human disease. Alternatively, a
mammal inbred to overexpress
dithp, resulting, e.g., in the secretion of DITHP in its milk, may also serve
as a convenient source of
that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74). ,
Screenin~Assays
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DITHP encoded by polynucleotides of the present invention may be used to
screen for
molecules that bind to or are bound by the encoded polypeptides. The binding
of the polypeptide and
the molecule may activate (agonist), increase, inhibit (antagonist), or
decrease activity of the
polypeptide or the bound molecule. Examples of such molecules include
antibodies, oligonucleotides,
s proteins (e.g., receptors), or small molecules.
Preferably, the molecule is closely related to the natural ligand of the
polypeptide, e.g., a ligand
or fragment thereof, a natural substrate, or a structural or functional
mimetic. (See, Coligan et al.,
(1991) Current Protocols in Immunolo~y 1(2): Chapter 5.) Similarly, the
molecule can be closely
related to the natural receptor to which the polypeptide binds, or to at least
a fragment of the receptor,
s o e.g., the active site. In either case, the molecule can be rationally
designed using lmown techniques.
Preferably, the screening for these molecules involves producing appropriate
cells which express the
polypeptide, either as a secreted protein or on the cell membrane. Preferred
cells include cells from
mammals, yeast, Droso~hila, or E. coli. Cells expressing the polypeptide or
cell membrane fractions
which contain the expressed polypeptide are then contacted with a test
compound and binding,
1s stimulation, or inhibition of activity of either the polypeptide or the
molecule is analyzed.
An assay may simply test binding of a candidate compound to the polypeptide,
wherein binding
is detected by a fluorophore, radioisotope, enzyme conjugate, or other
detectable label. Alternatively,
the assay may assess binding in the presence of a labeled competitor.
Additionally, the assay can be carried out using cell-free preparations,
polypeptideJmolecule
2 o affixed to a solid support, chenucal libraries, or natural product
mixtures. The assay may also. simply
comprise the steps of mixing a candidate compound with a solution containing a
polypeptide, measuring
polypeptide/molecule activity or binding, and comparing the
polypeptide/molecule activity or binding to
a standard.
Preferably, an ELISA assay using, e.g., a monoclonal or polyclonal antibody,
can measure
2 s polypeptide level in a sample. The antibody can measure polypeptide level
by either binding, directly or
indirectly, to the polypeptide or by competing with the polypeptide for a
substrate.
All of the above assays can be used in a diagnostic or prognostic context. The
molecules
discovered using fihese assays can be used to treat disease or to bring about
a particular result in a
patient (e.g., blood vessel growth) by activating or inhibiting the
polypeptidelmolecule. Moreover, the
3 o assays can discover agents which may inhibit or enhance the production of
the polypeptide from
suitably manipulated cells or tissues.
Transcript Imaging and Toxicological Testing
Another embodiment relates to the use of dithp to develop a transcript image
of a tissue or cell
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type. A transcript image represents the global pattern of gene expression by a
particular tissue or cell
type. Global gene expression patterns are analyzed by quantifying the number
of expressed genes and
their relative abundance under given conditions and at a given time. (See
Seilhamer et aL,
"Comparative Gene Transcript Analysis," U.S. Patent Number 5,840,484,
expressly incorporated by
s reference herein.) Thus a transcript image may be generated by hybridizing
the polynucleotides of the
present invention or their complements to the totality of transcripts or
reverse transcripts of a particular
tissue or cell type. In one embodiment, the hybridization takes place in high-
throughput format,
wherein the polynucleotides of the present invention or their complements
comprise a subset of a
plurality of elements on a microarray. The resultant transcript image would
provide a profile of gene
s o activity pertaining to human molecules for diagnostics and therapeutics.
Transcript images which profile dithp expression may be generated using
transcripts isolated
from tissues, cell lines, biopsies, or other biological samples. The
transcript image may thus reflect
dithp expression in vivo, as in the case of a tissue or biopsy sample, or in
vitro, as in the case of a cell
line.
15 Transcript images which profile dithp expression may also be used in
conjunction with in vitro
model systems and preclinical evaluation of pharmaceuticals, as well as
toxicological testing of
industrial and naturally-occurring environmental compounds. All compounds
induce characteristic
gene expression patterns, frequently termed molecular fingerprints or toxicant
signatures, which are
indicative of mechanisms of action and toxicity (Nuwaysir, E. F. et al. (1999)
Mol. Carcinog. 24:153-
20 159; Steiner, S. and Anderson, N.L. (2000) Toxicol. Lett. I 12-113:467-71,
expressly incorporated by
reference herein). If a test compound has a signature similar to that of a
compound with known
toxicity, it is likely to share those toxic properties. These fingerprints or
signatures are most useful and
refined when they contain expression information from a large number of genes
and gene families.
Ideally, a genome-wide measurement of expression provides the highest quality
signature. Even genes
2 5 whose expression is not altered by any tested compounds are important as
well, as the levels of
expression of these genes are used to normalize the rest of the expxession
data. The normalization
procedure is useful for comparison of expression data after treatment with
different compounds. While
the assignment of gene function to elements of a toxicant signature aids in
interpretation of toxicity
mechanisms, knowledge of gene function is not necessary for the statistical
matching of signatures
3 o which leads to prediction of toxicity. (See, for example, Press Release 00-
02 from the National
Institute of Environmental Health Sciences, released February 29, 2000,
available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
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containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the
present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of DITHP encoded by
polynucleotides of the
present invention to analyze the proteome of a tissue or cell type. The term
proteome refers to the
global pattern of protein expression in a particular tissue or cell type. Each
protein component of a
~ o proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating and
analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
1 s isoelectric focusing in the first dimension, and then according to
molecular weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins are
visualized in the gel as discrete and uniquely positioned spots, typically by
staining the geI with an
agent such as Coomassie Blue or silver or fluorescent stains. The optical
density of each protein spot is
generally proportional to the level of the protein in the sample. The optical
densities of equivalently
2 o positioned protein spots from different samples, for example, from
biological samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass spectrometry.
The identity of the protein in a spot may be determined by comparing its
partial sequence, preferably of
2 s at least 5 contiguous amino acid residues, to the polypeptide sequences of
the present invention. In
some cases, further sequence data may be obtained for definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for DITHP
to quantify the
levels of DITHP expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting the
3 0 levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-11;
Mendoze, L,G. et al. (1999) Biotechniques 27:778-88). Detection may be
performed by a variety of
methods known in the art, for example, by reacting the proteins in the sample
with a thiol- or amino-
reactive fluorescent compound and detecting the amount of fluorescence bound
at each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and should
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be analyzed in parallel with toxicant signatures at the transcript level.
There is a poor correlation
between transcript and protein abundances for some proteins in some tissues
(Anderson, N.L. and
Seilhamer, J. (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be useful in the
analysis of compounds which do not significantly affect the transcript image,
but which alter the
s proteomic profile. In addition, the analysis of transcripts in body fluids
is difficult, due to xapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated biological
sample are separated so that the amount of each protein can be quantified. The
amount of each protein
1 o is compared to the amount of the corresponding protein in an untreated
biological sample. A difference
in the amount of protein between the two samples is indicative of a toxic
response to the test compound
in the treated sample. Individual proteins are identified by sequencing the
amino acid residues of the
individual proteins and comparing these partial sequences to the DITHP encoded
by polynucleotides of
the present invention.
~. s In another embodiment, the toxicity of a test compound is assessed by
treating a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the DITHP encoded by polynucleotides of the
present invention. The amount
of protein recognized by the antibodies is quantified. The amount of protein
in the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
2 o protein between the two samples is indicative of a toxic response to the
test compound in the treated
sample.
Transcript images may be used to profile dithp expression in distinct tissue
types. This process
can be used to determine human molecule activity in a particular tissue type
relative to this activity in a
different tissue type. Transcript images may be used to generate a profile of
dithp expression
2 s characteristic of diseased tissue. Transcript images of tissues before and
after treatment may be used
for diagnostic purposes, to monitor the progression of disease, and to monitor
the efficacy of drug
treatments for diseases which affect the activity of human molecules.
Transcript images of cell lines can be used to assess human molecule activity
and/or to identify
cell lines that lack or misregulate this activity. Such cell lines may then be
treated with pharmaceutical
3 o agents, and a transcript image following treatment may indicate the
efficacy of these agents in restoring
desired levels of this activity. A similar approach may be used to assess the
toxicity of pharmaceutical
agents as reflected by undesirable changes in human molecule activity.
Candidate pharmaceutical
agents may be evaluated by comparing their associated transcript images with
those of pharmaceutical
agents of known effectiveness.
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Antisense Molecules
The polynucleotides of the present invention are useful in antisense
technology. Antisense
technology or therapy relies on the modulation of expression of a target
protein through the specific
binding of an antisense sequence to a target sequence encoding the target
protein or directing its
s expression. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc., Totawa
NJ; Alama, A. et al. (1997) Pharmacol. Res. 36(3):171-178; Crooke, S.T. (1997)
Adv. Pharmacol.
40:1-49; Sharma, H.W. and R. Narayanan (1995) Bioessays 17(12):1055-1063; and
Lavrosky, Y. et
al. (1997) Biochem. Mol. Med. 62(1):11-22.) An antisense sequence is a
polynucleotide sequence
capable of specifically hybridizing to at least a portion of the target
sequence. Antisense sequences
1 o bind to cellular mRNA and/or genomic DNA, affecting translation and/or
transcription. Antisense
sequences can be DNA, RNA, or nucleic acid mimics and analogs. (See, e.g.,
Rossi, J.J. et al. (1991)
Antisense Res. Dev. 1(3):285-288; Lee, R. et al. (1998) Biochemistry 37(3):900-
1010; Pardridge,
W.M. et al. (1995) Proc. Natl. Acad. Sci. USA 92(12):5592-5596; and Nielsen,
P. E. and Haaima, G.
(1997) Chem. Soc. Rev. 96:73-78.) Typically, the binding which results in
modulation of expression
1 s occurs through hybridization or binding of complementary base pairs.
Antisense sequences can also
bind to DNA duplexes through specific interactions in the major groove of the
double helix.
The polynucleotides of the present invention and fragments thereof can be used
as antisense
sequences to modify the expression of the polypeptide encoded by dithp. The
antisense sequences can
be produced ex vivo, such as by using any of the ABI nucleic acid synthesizer
series (Applied
2 o Biosystems) or other automated systems known in the art. Antisense
sequences can also be produced
biologically, such as by transforming an appropriate host cell with an
expression vector containing the
sequence of interest. (See, e.g., Agrawal, supra.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense sequences
into appropriate target cells can be used. Antisense sequences can be
delivered intracellularly in the
2 s form of an expression plasmid which, upon transcription, produces a
sequence complementary to at
least a portion of the cellular sequence encoding the target protein. (See,
e.g., Slater, J.E., et al. (1998)
J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J., et al. (1995)
9(13):1288-1296.)
Antisense sequences can also be introduced intracellularly through the use of
viral vectors, such as
retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990)
Blood 76:271; Ausubel,
3 o F.M. et al. (1995) Current Protocols in Molecular Biolo~y, John Wiley &
Sons, New York NY; Uckert,
W. and W. Walther (1994) Pharmacol. Ther. 63(3):323-347.) Other gene delivery
mechanisms include
Iiposome-derived systems, artificial viral envelopes, and other systems known
in the art. (See, e.g.,
Rossi, J.J. (1995) Br. Med. Bull. 51(1):217-225; Boado, R.J. et al. (1998) J.
Pharm. Sci. 87(11):1308-
1315; and Morris, M.C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736.)
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Expression
In order to express a biologically active DITHP, the nucleotide sequences
encoding DITHP or
fragments thereof may be inserted into an appropriate expression vector, i.e.,
a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in a
s suitable host. Methods which are well known to those skilled in the art may
be used to construct
expression vectors containing sequences encoding DITHP and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques, synthetic
techniques, and in vivo genetic recombination. (See, e.g., Sambrook, sue,
Chapters 4, 8, 16, and 17;
and Ausubel, supra, Chapters 9, 10, 13, and 16.)
1 o A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding DITHP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
15 tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal (mammalian) cell systems. (See, e.g., Sambrook, supra; Ausubel, 1995,
su ra, Van Heeke, G.
and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al.
(1987) Methods Enzymol.
153:516-544; Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard,
E.K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene
Ther: 7:1937-1945;
2 o Takamatsu, N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J.
3:1671-1680; Broglie,
R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105;
The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New
York NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retrovixuses, adenoviruses,
2 s or herpes or vaccinia viruses, or from various bacterial plasmids, may be
used for delivery of nucleotide
sequences to the targeted organ, tissue, or cell population. (See, e.g., Di
Nicola, M. et al. (1998)
Cancer Gen. Ther. 5(6):350-356; Yu, M. et al., (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344;
Buller, R.M. et aI. (1985) Nature 317(6040):813-8I5; McGregor, D.P. et al.
(1994) MoI. Immunol.
31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The
invention is not
3 0 limited by the host cell employed.
For long term production of recombinant proteins in mammalian systems, stable
expression of
DITHP in cell lines is preferred. For example, sequences encoding DITHP can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Any number of
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selection systems may be used to recover transformed cell lines. (See, e.g.,
Wigler, M. et al. (1977)
Cell 11:223-232; Lowy, I. et al. (1980) Cel122:817-823.; Wigler, M. et a1.
(1980) Proc. Natl. Acid.
Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-
14; Hartman, S.C. and
R.C.Mulligan (1988) Proc. Natl. Acid. Sci. USA 85:8047-8051; Rhodes, C.A.
(1995) Methods Mol.
s BioL 55:121-131.)
Therapeutic Uses of dithp
'The dithp of the invention may be used for somatic or germline gene therapy.
Gene therapy
may be performed to (i) correct a genetic deficiency (e.g., in the cases of
severe combined
to immunodeficiency (SCID)-Xl disease characterized by X-linked inheritance
(Cavazzana-Calvo, M. et
al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome
associated with an
inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995)
Science 270:475-480;
Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J.
et al. (1993) Cell 75:207-
216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et
al. (1995) Hum. Gene
15 Therapy 6:667-703), thalassemias, familial hypercholesterolemia, and
hemophilia resulting from Factor
VIII or Factor IX deficiencies (Crystal, R.G. (1995) Science 270:404-410;
Verma, LM. and Somia, N.
(1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product
(e.g., in the case of
cancers which result from unregulated cell proliferation), or (iii) express a
protein which affords
protection against intracellular parasites (e.g., against human retroviruses,
such as human
2o immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396;
Poeschla, E. et al. (1996)
Proc. Natl. Acid. Sci. USA. 93:11395-11399), hepatitis B or C virus (HBV,
HCV); fungal parasites,
such as Candida albicans and Paracoccidioides brasiliensis; and protozoan
parasites such as
Plasmodium falciparum and Trwanosoma cruzi). In the case where a genetic
deficiency in dithp
expression or regulation causes disease, the expression of dithp from an
appropriate population of
2 s transduced cells may alleviate the clinical manifestations caused by the
genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in dithp
are treated by constructing mammalian expression vectors comprising dithp and
introducing these
vectors by mechanical means into dithp-deficient cells. Mechanical transfer
technologies for use with
cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic gold
3 o particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and (v) the
use of DNA transposons (Morgan, R.A. and Anderson, W.F. (1993) Annu. Rev.
Biochem. 62:191-217;
Ivics, Z. (1997) CeII 91:501-510; Boulay, J-L. and Recipon, H. (1998) Curr.
Opin. Biotechnol. 9:445-
450).
Expression vectors that may be effective for the expression of dithp include,
but are not limited
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to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad
CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). The dithp of the
invention
may be expressed using (i) a constitutively active promoter, (e.g" from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and Bujard, H. (1992)
Proc. Natl. Acad. Sci.
U.S.A. 89:5547-5551; Gossen, M. et al., (1995) Science 268:1766-1769; Rossi,
F.M.V. and Blau,
H.M. (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the
T-REX plasmid
(Invitrogen); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and PIND;
1 o Invitrogen); the FK506lrapamycin inducible promoter; or the
RU486lmifepristone inducible promoter
(Rossi, F.M. V. and Blau, H.M. supra), or (iii) a tissue-specific promoter or
the native promoter of the
endogenous gene encoding DITHP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
~ s polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and Eb, A.J. (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
2 o In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to dithp expression are treated by constructing a retrovirus vector
consisting of (i) dithp under
the control of an independent promoter or the retrovirus long terminal repeat
(LTR) promoter, (ii)
appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE)
along with additional
retrovirus cis-acting RNA sequences and coding sequences required for
efficient vector propagation.
2 ~ Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available
(Stratagene) and are based on
published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. U.S.A.
92:6733-6737), incorporated by
reference herein. The vector is propagated in an appropriate vector producing
cell line (VPCL) that
expresses an envelope gene with a tropism for receptors on the target cells or
a promiscuous envelope
protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650;
Bender, M.A. et al. (1987)
3 o J. Virol. 61:1639-1646; Adam, M.A. and Miller, A.D. (1988) J. Virol.
62:3802-3806; Dull, T. et al.
(1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-
9880). U.S. Patent Number
5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines
producing high transducing
efficiency retroviral supernatant") discloses a method for obtaining
retrovirus packaging cell lines and
is hereby incorporated by reference. Propagation of retrovirus vectors,
transduction of a population of
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cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient
are procedures well known to
persons skilled in the art of gene therapy and have been well documented
(Ranga, U, et al. (1997) J.
Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi,
M.L. (1997) J. Virol.
71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:1201-
1206; Su, L. (1997)
s Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver dithp to
cells which have one or more genetic abnormalities with respect to the
expression of dithp. The
construction and packaging of adenovirus-based vectors are well known to those
with ordinary skill in
the art. Replication defective adenovirus vectors have proven to be versatile
for importing genes
1 o encoding immunoregulatory proteins into intact islets in the pancreas
(Csete, M.E. et al. (1995)
Transplantation 27:263-268). Potentially useful adenoviral vectors are
described in U.S. Patent
Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby
incorporated by
reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544
and Verma, LM. and Somia, N. (1997) Nature 18:389:239-242, both incorporated
by reference herein.
~ s In another alternative, a herpes-based, gene therapy delivery system is
used to deliver dithp to
target cells which have one or more genetic abnormalities with respect to the
expression of dithp. The
use of herpes simplex virus (HSV)-based vectors may be especially valuable for
introducing dithp to
cells of the central nervous system, for which HSV has a tropism. The
construction and packaging of
herpes-based vectors are well knownto those with ordinary skill in the art. A
replication-competent
2 o herpes simplex virus (HS V) type 1-based vector has been used to deliver a
reporter gene to the eyes of ,
primates (Liu, X. et al. (1999) Exp. Eye Res.169:385-395). The construction of
a HSV-1 virus vector
has also been disclosed in detail in U.S. Patent Number 5,804,413 to DeLuca
("Herpes simplex virus
strains for gene transfer"), which is hereby incorporated by reference. U.S.
Patent Number 5,804,413
teaches the use of recombinant HSV d92 which consists of a genome containing
at least one exogenous
2 s gene to be transferred to a cell under the control of the appropriate
promoter for purposes including
human gene therapy. Also taught by this patent are the construction and use of
recombinant HSV
strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.
F. et al, 1999 J.
Virol. 73:519-532 and Xu, H. et al., (1994) Dev. Biol. 163:152-161, hereby
incorporated by reference.
The manipulation of cloned herpesvirus sequences, the generation of
recombinant virus following the
3 o transfection of multiple plasmids containing different segments of the
large herpesvirus genomes, the
growth and propagation of herpesvirus, and the infection of cells with
herpesvirus are techniques well
known to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver dithp to target cells. The biology of the prototypic alphavirus,
Semliki Forest Virus (SFV), has
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been studied extensively and gene transfer vectors have been based on the SFV
genome (Garoff, H, and
Li, K-J. (1998) Curr. Opin. Biotech. 9:464-4.69). During alphavirus RNA
replication, a subgenomic
RNA is generated that normally encodes the viral capsid proteins. This
subgenomic RNA replicates to
higher levels than the full-length genomic RNA, resulting in the
overproduction of capsid proteins
relative to the viral proteins with enzymatic activity (e.g., protease and
polymerase). Similarly,
inserting dithp into the alphavirus genome in place of the capsid-coding
region results in the production
of a large number of dithp RNAs and the synthesis of high levels of DITHP in
vector transduced cells.
While alphavirus infection is typically associated with cell lysis within a
few days, the ability to
establish a persistent infection in hamster normal kidney cells (BHK-21) with
a variant of Sindbis virus
(SIN) indicates that the lytic replication of alphaviruses can be altered to
suit the needs of the gene
therapy application (Dryga, S.A. et aI. (1997) Virology 228:74-83). The wide
host range of
alphaviruses will allow the introduction of dithp into a variety of cell
types. The specific transduction
of a subset of cells in a population may require the sorting of cells prior to
transduction. The methods
of manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Antibodies
Anti-DTTHP antibodies may be used to analyze protein expression levels. Such
antibodies
2 o include, but are not limited to, polyclonal, monoclonal, chimeric, single
chain, and Fab fragments. For
descriptions of and protocols of antibody technologies see, e.g., Pound J.D.
(1998) Immunochemical
Protocols, Humana Press, Totowa, NJ.
The amino acid sequence encoded by the dithp of the Sequence Listing may be
analyzed by
appropriate software (e.g., LASERGENE NAVIGATOR software, DNASTAR) to
determine regions
2 s of high immunogenicity. The optimal sequences for immunization are
selected from the C-terminus, the
N-terminus, and those intervening, hydrophilic regions of the polypeptide
which are likely to be exposed
to the external environment when the polypeptide is in its natural
conformation. Analysis used to select
appropriate epitopes is also described by Ausubel (1997, su~xa, Chapter 11.7).
Peptides used for
antibody induction do not need to have biological activity; however, they must
be antigenic. Peptides
3 o used to induce specific antibodies may have an amino acid sequence
consisting of at least five amino
acids, preferably at least 10 amino acids, and most preferably at least 15
amino acids. A peptide which
mimics an antigenic fragment of the natural polypeptide may be fused with
another protein such as
keyhole limpet hemocyanin (KLH; Sigma, St. Louis MO) for antibody production.
A peptide
encompassing an antigenic region may be expressed from a dithp, synthesized as
described above, or
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purified from human cells.
Procedures well known in the art may be used for fine production of
antibodies. Various hosts
including mice, goats, and rabbits, may be immunized by injection with a
peptide. Depending on the
host species, various adjuvants may be used to increase immunological
response.
In one procedure, peptides about 15 residues in length may be synthesized
using an ABI 431A
peptide synthesizer (Applied Biosystems) using fmoc-chemistry and coupled to
KLH (Sigma) by
reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel, 1995,
supra). Rabbits are
immunized with the peptide-KhH complex in complete Freund's adjuvant. The
resulting antisera are
tested for antipeptide activity by binding the peptide to plastic, blocking
with 1 % bovine serum albumin
s o (BSA), reacting with rabbit antisera, washing, and reacting with
radioiodinated goat anti-rabbit IgG.
Antisera with antipeptide activity are tested for anti-DITHP activity using
protocols well known in the
art, including ELISA, radioimmunoassay (RIA), and immunoblotting.
In another procedure, isolated and purified peptide may be used to immunize
mice (about 100
~ g of peptide) or rabbits (about 1 mg of peptide). Subsequently, the peptide
is radioiodinated and used
to screen the immunized animals' B-lymphocytes for production of antipeptide
antibodies. Positive
cells are then used to produce hybridomas using standard techniques. About 20
mg of peptide is
sufFicient for labeling and screening several thousand clones. Hybridomas of
interest are detected by
screening with radioiodinated peptide to identify those fusions producing
peptide-specific monoclonal
antibody. In a typical protocol, wells of a multi-well plate (FAST, Becton-
Dickinson, Palo Alto, CA)
2 o are coated with affinity-purified, specific rabbit-anti-mouse (or suitable
anti-species IgG) antibodies at
10 mg/ml. The coated wells are blocked with 1 % BSA and washed and exposed to
supernatants from
hybridomas. After incubation, the wells are exposed to radiolabeled peptide at
1 m~ml.
Clones producing antibodies bind a quantity of labeled peptide that is
detectable above
background. Such clones are expanded and subjected to 2 cycles of cloning.
Cloned hybridomas are
2 5 injected into pristane-treated mice to produce ascites, and monoclonal
antibody is purified from the
ascitic fluid by affinity chromatography on protein A (Amersham Phaxmacia
Biotech). Several.
procedures for the production of monoclonal antibodies, including in vitro
production, are described in
Pound su ra). Monoclonal antibodies with antipeptide activity are tested for
anti-DITHP activity
using protocols well known in the art, including ELISA, RIA, and
immunoblotting.
3 o Antibody fragments containing specific binding sites for an epitope may
also be generated. For
example, such fragments include, but axe not limited to, the F(ab')2 fragments
produced by pepsin
digestion of the antibody molecule, and the Fab fragments generated by
reducing the disulfide bridges of
the F(ab')2 fragments. Alternatively, construction of Fab expression libraries
in filamentous
bacteriophage allows rapid and easy identification of monoclonal fragments
with desired specificity
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(Pound, supra, Chaps. 45-47). Antibodies generated against polypeptide encoded
by dithp can be used
to purify and characterize full-length DITHP protein and its activity, binding
partners, etc.
Assays Using Antibodies
s Anti-DITHP antibodies may be used in assays to quantify the amount of DITHP
found in a
particular human cell. Such assays include methods utilizing the antibody and
a label to detect
expression level under normal or disease conditions. The peptides and
antibodies of the invention may
be used with or without modification or labeled by joining them, either
covalently or noncovalently,
with a reporter molecule.
1 o Protocols for detecting and measuring protein expression using either
polyclonal or monoclonal
antibodies are well known in the art. Examples include ELISA, RIA, and
fluorescent activated cell
sorting (FAGS). Such immunoassays typically involve the formation of complexes
between the DITHP
and its specific antibody and the measurement of such complexes. These and
other assays are described
in Pound su ra).
1 s Without further elaboration, it is believed that one skilled in the art
can, using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
!imitative of the remainder of
the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
2o including U.S. Ser. No. 60/184,777, U.S. Ser. No. 60/184,797, U.S. Ser. No.
60/184,698, U.S. Ser.
No. 60/184,770, U.S. Ser. No. 60/184,774, U.S. Ser. No. 60/184,693, U.S. Ser.
No. 601184,771,U.S.
Ser. No. 60/184,813, U.S. Ser. No. 60/184,773, U.S. Ser. No. 60/184,776, U.S.
Ser. No. 60/184,769,
U.S. Ser. No. 60/184,768, U.S. Sex. No. 60/184,837, U.S. Ser. No. 60/184,697,
U.S. Sex. No.
60/184,841, U.S. Ser. No. 60/184,772, U.S. Ser. No. 60/185,213, U.S. Ser. No.
60/185,216, U.S. Ser.
25 No. 60/204,863, U.S. Ser. No. 60/205,221, U.S. Ser. No. 60/204,815, U.S.
Ser. No. 60/203,785, U.S.
Ser. No. 60/204,821, U.S. Ser. No. 60/204,908, U.S. Ser. No. 60/204,226, U.S.
Ser. No. 60/204,525,
U.S. Ser. No. 60/205,285, U.S. Ser. No. 60/205,232, U.S. Ser. No. 60/205,323,
U.S. Ser. No.
60/205,287, U.S. Ser. No. 60/205,324, and U.S. Ser. No. 60/205,286, are hereby
expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from CLONTECH Laboratories, Inc. (Palo Alto CA) or isolated
from
various tissues. Some tissues were homogenized and lysed in guanidinium
isothiocyanate, while others
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were homogenized and lysed in phenol or in a suitable mixture of denaturants,
such as TRIZOL (Life
Technologies), a monophasic solution of phenol and guanidine isothiocyanate.
The resulting lysates
were centrifuged over CsCl cushions or extracted with chloroform. RNA was
precipitated with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
s Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In most cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega Corporation
(Promega), Madison WI),
OLIGOTEX latex particles (QIAGEN, Inc. (QIAGEN), Valencia CA), or an OLIGOTEX
mRNA
purification kit (QIAGEN). Alternatively, RNA was isolated directly from
tissue lysates using other
~. o RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,
Inc., Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene Cloning Systems, Inc. (Stratagene), La Jolla CA) or
SUPERSCRIPT
plasmid system (Life Technologies), using the recommended procedures or
similar methods known in
15 the art. (See, e.g., Ausubel, 1997, supra, Chapters 5.1 through 6.6.)
Reverse transcription was
initiated using oligo d(T) or random primers. Synthetic oligonucleotide
adapters were ligated to double
stranded cDNA, and the cDNA was digested with the appropriate restriction
enzyme or enzymes: For
most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S
1000, SEPHAROSE
CLZB, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or
z o preparative agarose gel electrophoresis. cDNAs were ligated into
compatible restriction enzyme 'sites of
the polylinker of a suitable plasmid, e.g., PBLLTESCRIPT plasmid (Stratagene),
PSPORT1 plasmid
(Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV
plasmid (Stratagene),
ox pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including XLl-Blue, XL1-BlueMRF, or
SOLR from
2 s Stratagene or DHS a, DH 1 OB, or ElectroMAX DH 1 OB from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision using the UNIZAP
vector system
(Stratagene) or by cell lysis. Plasmids were purified using at least one of
the following: the Magic or
3 o WIZARD Minipreps DNA purification system (Promega); the AGTC Miniprep
purification kit (Edge
BioSystems, Gaithersburg MD); and the QIAWELL 8, QIAWELL 8 Plus, and QIAWELL 8
Ultra
plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit
(QIAGEN). Following
precipitation, plasmids were resuspended in 0.1 ml of distilled water and
stored, with or without
lyophilization, at 4°C.
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Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format. (Rao, V.B. (1994) Anal. Biochem. 216:1-14.) Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in 384-
well plates, and the concentration of amplified plasmid DNA was quantif ed
fluorometrically using
s PICOGREEN dye (Molecular Probes, Inc. (Molecular Probes), Eugene OR) and a
FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
cDNA sequencing reactions were processed using standard methods or high-
throughput
1 o instrumentation such as the ABI CATALYST 800 thermal cycler (Applied
Biosystems) or the PTC-
200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser
(Robbins Scientific
Corp., Sunnyvale CA) or the MICROLAB 2200 liquid transfer system (Hamilton),
cDNA sequencing
reactions were prepared using reagents provided by Amersham Pharmacia Biotech
or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready
reaction kit
15 (Applied Biosystems). Electxophoretic separation of cDNA sequencing
reactions and detection of
labeled polynucleotides were carried out using the MEGABACE 1000 DNA
sequencing system
(Molecular Dynamics}; the ABI PRISM 373 or 377 sequencing system (Applied
Biosystems} in
conjunction with standard ABI protocols and base calling software; or other
sequence analysis systems
known in the art. Reading frames within the cDNA sequences were identified
using standard methods
2 0 (reviewed in Ausubel, 1997, su ra, Chapter 7.7). Some of the cDNA
sequences were selected for
extension using the techniques disclosed in Example VIII.
IV. Assembly and Analysis of Sequences
Component sequences from chromatograms were subject to PHRED analysis and
assigned a
2 s quality score. The sequences having at least a required quality score were
subject to various pre-
processing editing pathways to eliminate, e.g., low quality 3' ends, vector
and linker sequences, polyA
tails, Alu repeats, mitochondrial and xibosomal sequences, bacterial
contamination sequences, and
sequences smaller than 50 base pairs. In particular, low-information sequences
and repetitive elements
(e.g., dinucleotide repeats, Alu repeats, etc.) were replaced by "n's", or
masked, to prevent spurious
3 o matches.
Processed sequences were then subject to assembly procedures in which the
sequences were
assigned to gene bins (bins). Each sequence could only belong to one bin.
Sequences in each gene bin
were assembled to produce consensus sequences (templates). Subsequent new
sequences were added to
existing bins using BLASTn (v.1.4 WashU) and CROSSMATCH. Candidate pairs were
identified as
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all BLAST hits having a quality score greater than or equal to 150. Alignments
of at least 82% local
identity were accepted into the bin. The component sequences from each bin
were assembled using a
version of PHRAP. Bins with several overlapping component sequences were
assembled using DEEP
PHRAP. The orientation (sense or antisense) of each assembled template was
determined based on the
s number and orientation of its component sequences. Template sequences as
disclosed in the sequence
listing correspond to sense strand sequences (the "forward" reading frames),
to the best determination.
The complementary (antisense) strands are inherently disclosed herein. The
component sequences
which were used to assemble each template consensus sequence are listed in
Table 4, along with their
positions along the template nucleotide sequences.
s o Bins were compared against each other and those having local similaxity of
at least 82% were
combined and reassembled. Reassembled bins having templates of insufficient
overlap (less than 95%
local identity) were re-split. Assembled templates were also subject to
analysis by STITCHER/EXON
MAPPER algorithms which analyze the probabilities of the presence of splice
variants, alternatively
spliced exons, splice junctions, differential expression of alternative
spliced genes across tissue types or
15 disease states, etc. These resulting bins were subject to several rounds of
the above assembly
procedures.
Once gene bins were generated based upon sequence alignments, bins were clone
joined based
upon clone information. If the 5' sequence of one clone was present in one bin
and the 3' sequence from
the same clone was present in a different bin, it was likely that the two bins
actually belonged together
2 o in a single bin. The resulting combined bins underwent assembly procedures
to regenerate the
consensus sequences.
The final assembled templates were subsequently annotated using the following
procedure.
Template sequences were analyzed using BLASTn (v2.0, NCBI) versus gbpri
(GenBank version 120).
"Hits" were defined as an exact match having from 95 % local identity over 200
base pairs through
25 100% local identity over 100 base pairs, or a homolog match having an E-
value, i.e. a probability
score, of s 1 x 10-$. The hits were subject to frameshift FASTx versus GENPEPT
(GenBank version
120). (See Table 7). In this analysis, a homolog match was defined as having
an E-value of s 1 x 10'8.
The assembly method used above was described in "System and Methods for
Analyzing Biomolecular
Sequences," U.S.S.N. 09/276,534, filed March 25, 1999, and the LIFESEQ Gold
user manual (Incyte)
3 o both incorporated by reference herein.
Following assembly, template sequences were subjected to motif, BLAST, and
functional
analyses, and categorized in protein hierarchies using methods described in,
e.g., "Database System
Employing Protein Function Hierarchies for Viewing Biomolecular Sequence
Data," U.S.S.N.
081812,290, filed March 6, 1997; "Relational Database for Storing Biomolecule
Information,"
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U.S.S.N, 08/947,845, filed October 9, 1997; "Project-Based Full-Length
Biomolecular Sequence
Database," U.S.S.N. 08!811,758, filed March 6, 1997; and "Relational Database
and System for
Storing Information Relating to Biomolecular Sequences," U.S.S.N. 09/034,807,
filed March 4, 1998,
all of which are incorporated by reference herein.
The template sequences were further analyzed by translating each template in
all three forward
reading frames and searching each translation against the Pfam database of
hidden Markov model-
based protein families and domains using the HMMER software package (available
to the public from
Washington University School of Medicine, St. Louis MO). Regions of templates
which, when
translated, contain similarity to Pfam consensus sequences are reported in
Table 2, along with
1 o descriptions of Pfam protein domains and families. Only those Pfam hits
with an E-value of s 1 x 10-3
are reported. (See also World Wide Web site http://pfam.wustl.edul for
detailed descriptions of Pfam
protein domains and families.)
Additionally, the template sequences were translated in all three forward
reading frames, and
each translation was searched against hidden Markov models for signal peptides
using the HMMER
s 5 software package. Construction of hidden Markov models and their usage in
sequence analysis has
been described. (See, for example, Eddy, S.R. (1996) Curr. Opin. Str. Biol.
6:361-365.) Only those
signal peptide hits with a cutoff scoxe of 11 bits or greater axe reported. A
cutoff score of 11 bits or
greater corresponds to at least about 91-94% true-positives in signal peptide
prediction. Template
sequences were also translated in all three forward reading frames, and each
translation was searched
2 o against TMAP, a program that uses weight matrices to delineate
transmembrane segments on protein
sequences and determine orientation, with respect to the cell cytosol
(Persson, B. and P, Argos (1994) J.
Mol. Biol. 237:182-192; Persson, B. and P. Argos (1996) Protein Sci. 5:363-
371). Regions of
templates which, when translated, contain similarity to signal peptide or
transmembrane consensus
sequences are reported in Table 3.
2 5 The results of HMMER analysis as reported in Tables 2 and 3 may support
the results of
BLAST analysis as reported in Table 1 or may suggest alternative or additional
properties of template-
encoded polypeptides not previously uncovered by BLAST or other analyses.
Template sequences are further analyzed using the bioinformatics tools listed
in Table 7, or
using sequence analysis software known in the art such as MACDNASIS PRO
software (Hitachi
3 o Software Engineering, South San Francisco CA) and LASERGENE software
(DNASTAR). Template
sequences may be further queried against public databases such as the GenBank
rodent, mammalian,
vertebrate, prokaryote, arid eukaryote databases.
The template sequences were translated to derive the corresponding longest
open reading frame
as presented by the polypeptide sequences. Alternatively, a polypeptide of the
invention may begin at
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any of the methionine residues within the fall length translated polypeptide.
Polypeptide sequences
were subsequently analyzed by querying against the GenBank protein database
(GENPEPT, (GenBank
version 121)). Full length polynucleotide sequences are also analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments are generated
using default
parameters specified by the CLUSTAL algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned sequences.
Table 6 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (GENPEPT) database. Column 1 shows
the polypeptide
~ o sequence identification number (SEQ ID NO:) for the polypeptide segments
of the invention. Column 2
shows the reading frame used in the translation of the polynucleotide
sequences encoding the
polypeptide segments. Column 3 shows the length of the translated polypeptide
segments. Columns 4
and 5 show the start and stop nucleotide positions of the polynucleotide
sequences encoding the
polypeptide segments. Column 6 shows the GenBank identification number (GI
Number) of the nearest
GenBank homolog. Column 7 shows the probability score for the match between
each polypeptide and
its GenBank homolog. Column 8 shows the annotation of the GenBank homolog.
V. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
a o particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel, 1995,
supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
2 5 search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the Iength
of the sequence match. The product score is a normalized value between 0 and
100, and is calculated
as follows: the BLAST score is multiplied by the percent nucleotide identity
and the product is divided
by (5 times the length of the shorter of the two sequences). The BLAST score
is calculated by
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assigning a score of +5 for every base that matches in a high-scoring segment
pair (HSP), and -4 for
every mismatch. Two sequences may share more than one HSP (separated by gaps).
If there is more
than one HSP, then the pair with the highest BLAST score is used to calculate
the product score. The
product score represents a balance between fractional overlap and quality in a
BLAST alignment. For
s example, a product score of 100 is produced only for 100% identity over the
entire length of the shorter
of the two sequences being compared. A product score of 70 is produced either
by 100% identity and
70% overlap at one end, or by 88% identity and 100% overlap at the other. A
product score of 50 is
produced either by 100% identity and 50% overlap at one end, or 79% identity
and 100% overlap:
to VI. Tissue Distribution Profiling
A tissue distribution profile is determined for each template by compiling the
cDNA library
tissue classifications of its component cDNA sequences. Each component
sequence, is derived from a
cDNA library constructed from a human tissue. Each human tissue is classified
into one of the
following categories: cardiovascular system; connective tissue; digestive
system; embryonic structures;
~ 5 endocrine system; exocrine glands; genitalia, female; genitalia, male;
germ cells; heroic and immune
system; liver; musculoskeletal system; nervous system; pancreas; respiratory
system; sense organs;
skin; stomatognathic system; unclassified/mixed; or urinary tract. Template
sequences, component
sequences, and cDNA library/tassue information are found in the LIFESEQ GOLD
database (Incyte
Genomics, Palo Alto CA).
2 o Table 5 shows the tissue distribution profile for the templates of the
invention. For each
template, the three most frequently observed tissue categories are shown in
column 3, along with the
percentage of component sequences belonging to each category. Only tissue
categories with
percentage values of > 10% are shown. A tissue distribution of "widely
distributed" in column 3
indicates percentage values of <10% in all tissue categories.
VII. Transcript Image Analysis
Transcript images are generated as described in Seilhamer et al., "Comparative
Gene
Transcript Analysis," U.S. Patent Number 5,840,484, incorporated herein by
reference.
3 o VIII. Extension of Poiynucleotide Sequences and Isolation of a Full-length
cDNA
Oligonucleotide primers designed using a dithp of the Sequence Listing are
used to extend the
nucleic acid sequence. One primer is synthesized to initiate 5' extension of
the template, and the other
primer, to initiate 3' extension of the template. The initial primers may be
designed using OLIGO 4.06
software (National Biosciences, Inc. (National Biosciences), Plymouth MN), or
another appropriate
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program, to be about 22 to 30 nucleotides in length, to have a GC content of
about 50% or more, and to
anneal to the target sequence at temperatures of about 68 °C to about
72°C. Any stretch of nucleotides
which would result in hairpin structures and primer-primer dimerizations are
avoided. Selected human
cDNA libraries are used to extend the sequence. If more than one extension is
necessary or desired,
s additional or nested sets of primers are designed.
High fidelity amplification is obtained by PCR using methods well known in the
art. PCR is
performed in 96-well plates using the PTC-200 thermal cycler (MJ Research).
The reaction mix
contains DNA template, 200 nmol of each primer, reaction buffer containing
Mg2~, (NH4)2S O4, and J3-
mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech), ELONGASE
enzyme (Life
s o Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer pair
PCI A and PCI B : Step 1: 94 ° C, 3 min; Step 2: 94 ° C, 15 sec;
Step 3: 60 ° C, 1 min; Step 4: 68 ° C, 2
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 ° C, 5
min; Step 7: storage at 4 ° C. In the
alternative, the parameters for primer pair T7 and SK+ are as follows: Step 1:
94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68 °C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
15 Step 6: 68 ° C, 5 min; Step 7: storage at 4 ° C.
The concentration of DNA in each well is determined by dispensing 100 ~l
PICOGREEN
quantitation reagent (0.25% (v/v); Molecular Probes) dissolved in 1X Tris-EDTA
(TE) and 0.5 ~1 of
undiluted PCR product into each well of an opaque fluorimeter plate (Corning
Incorporated (Corning),
Corning N~, allowing the DNA to bind to the reagent. The plate is scanned in a
FLUOROSKAN II
z o (Labsystems Oy) to measure the fluorescence of the sample and to quantify
the concentration of DNA.
A 5 ~1 to 10 ~1 aliquot of the reaction mixture is analyzed by electrophoresis
on a 1 % agarose mini-gel
to determine which reactions are successful in extending the sequence.
The extended nucleotides are desalted and concentrated, transferred to 384-
well plates, digested
with CviJI cholera virus endonuclease (Molecular Biology Research, Madison
WI), and sonicated or
2 5 sheared prior to religation into pUC 18 vector (Amersham Pharmacia
Biotech). For shotgun
sequencing, the digested nucleotides are separated on low concentration (0.6
to 0.8 %) agarose gels,
fragments are excised, and agar digested with AGAR ACE (Promega). Extended
clones are religated
using T4 ligase (New England Biolabs, Inc., Beverly MA) into pUC 18 vector
(Amersham Pharmacia
Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in restriction
site overhangs, and
3 o transfected into competent E. coli cells. Transformed cells are selected
on antibiotic-containing media,
individual colonies are picked and cultured overnight at 37 °C in 384-
well plates in LB/Zx carbenicillin
liquid media.
The cells are lysed, and DNA is amplified by PCR using Taq DNA polymerise
(Amersham
Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the following
parameters: Step 1:
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94°C, 3 min; Step 2: 94°C, 1S sec; Step 3: 60°C, 1 min;
Ste~4: 72°C, 2'min; Step S: steps 2, 3, and4
repeated 29 times; Step 6: 72°C, S min; Step 7: storage at 4°C.
DNA is quantified by PICOGREEN
reagent (Molecular Probes) as described above. Samples with low DNA recoveries
are reamplified
using the same conditions as described above. Samples are diluted with 20%
dimethysulfoxide (1:2,
s v/v), and sequenced using DYENAMIC energy transfer sequencing primers and
the DYENAMIC
DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle
sequencing ready reaction kit (Applied Biosystems). 'v
In like manner, the dithp is used to obtain regulatory sequences (promoters,
introns, and
enhancers) using the procedure above, oligonucleotides designed for such
extension, and an appropriate
1 o genomic library.
IX. Labeling of Probes and Southern Hybridization Analyses
Hybridization probes derived from the dithp of the Sequence Listing are
employed for screening
cDNAs, mRNAs, or genomic DNA. The labeling of probe nucleotides between 100
and 1000
15 nucleotides in length is specifically described, but,essentially the same
procedure may be used with
larger cDNA fragments. Probe sequences are labeled at room temperature for 30
minutes using a T4
polynucleotide kinase, ~ZP-ATP, and O.SX One-Phor-All Plus (Amersham Pharmacia
Biotech) buffer
and purified using a ProbeQuant G-50 Microcolumn (Amersham Pharmacia Biotech).
The probe
mixture is diluted to 10' dpm/~g/ml hybridization buffer and used in a typical
membrane-based
2 o hybridization analysis.
The DNA is digested with a restriction endonuclease such as Eco RV and is
electrophoresed
through a 0.7% agarose gel. The DNA fragments are transferred from the agarose
to nylon membrane
(NYTRAN Plus, Schleicher & Schuell, Inc., Keene NH) using procedures specified
by the
manufacturer of the membrane. Prehybridization is carried out for three or
more hours at 68 ° C, and
2 s hybridization is carried out overnight at 68 ° C. To remove non-
specific signals, blots are sequentially
washed at room temperature under increasingly stringent conditions, up to 0.1x
saline sodium citrate
(SSC) and O.S % sodium dodecyl sulfate. After the blots are placed in a
PHOSPHORIMAGER cassette
(Molecular Dynamics) or are exposed to autoradiography film, hybridization
patterns of standard and
experimental lanes are compared. Essentially the same procedure is employed
when screening RNA.
X. Chromosome Mapping of dithp
The cDNA sequences which were used to assemble SEQ ID NO:1-211 are compared
with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that match SEQ
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i
ID N0:1-211 are assembled into clusters of contiguous and overlapping
sequences using assembly
algorithms such as PHRAP (Table 7). Radiation hybrid and genetic mapping data
available from
public resources such as the Stanford Human Genome Center (SHGC), Whitehead
Institute for Genome
Research (WIGR), and Genethon are used to determine if any of the clustered
sequences have been
s previously mapped. Inclusion of a mapped sequence in a cluster will result
in the assignment of all
sequences of that cluster, including its particular SEQ ID NO:, to that map
location. The genetic map
locations of SEQ ID N0:1-211 are described as ranges, or intervals, of human
chromosomes. The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
1 o chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase
(Mb) of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters.
15 XI. Microarray Analysis
Probe Preparation from Tissue or Cell Samples
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
polyA+ RNA is purified using the oligo (dT) cellulose method. Each polyA+ RNA
sample is reverse
transcribed using MMLV reverse-transcriptase, 0.05 pg/~1 oligo-dT primer
(2lmer), 1X first strand
2 o buffer, 0.03 units/~l RNase inhibitor, 500 ~M dATP, 500 ~M dGTP, 500 ~M
dTTP, 40 ~M dCTP,
40 ~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse
transcription
reaction is performed in a 25 ml volume containing 200 ng polyA+ RNA with
GEMBRIGHT kits
(Incyte). Specific control polyA+ RNAs are synthesized by in vitro
transcription from non-coding yeast
genomic DNA (W. Lei, unpublished). As quantitative controls, the control mRNAs
at 0.002 ng, 0.02
2 s ng, 0.2 ng, and 2 ng are diluted into reverse transcription reaction at
ratios of 1:100,000, 1:10,000,
1:1000, 1:100 (w/w) to sample mRNA respectively. The control mRNAs are diluted
into reverse
transcription reaction at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to
sample mRNA differential
expression patterns. After incubation at 37° C for 2 hr, each reaction
sample (one with Cy3 and another
with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and
incubated for 20 minutes at
3 0 85° C to the stop the reaction and degrade the RNA. Probes are
purified using two successive
CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
(CLONTECH), Palo
Alto CA) and after combining, both reaction samples are ethanol precipitated
using 1 ml of glycogen (1
m~ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The probe is then
dried to completion
using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14
~1 SX SSC/0.2%
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SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element is
s amplified from bacterial cells containing vectoxs with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 fig.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
1 o slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR Scientific
Products Corporation (VWR), West Chester, PA), washed extensively in distilled
water, and coated
with 0.05 % aminopropyl silane (Sigma) in 95 % ethanol. Coated slides are
cured in a 110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
15 Patent No. 5,807,522, incorporated herein by reference. 1 ~1 of the array
element DNA, at an average
concentration of 100 n~~l, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATAL,INI~ER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
2, o Non-specific binding sites are blocked by incubation of microarrays in
0.2% casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford, MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
2 s Hybridization reactions contain 9 ~1 of probe mixture consisting of 0.2 p
g each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The probe mixture
is heated to 65° C for 5 minutes and is aliquoted onto the microarray
surface and covered with an 1.8
cm2 coverslip. The arrays are transferred to a waterproof chamber having a
cavity just slightly larger
than a microscope slide. The chamber is kept at 100% humidity internally by
the addition of 140 ~1 of
3 0 5x SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about 6.5
hours at 60° C. The arrays are washed for 10 min at 45° C in a
first wash buffer (1X SSC, 0.1 % SDS),
three times for 10 minutes each at 45° C in a second wash buffer (0.1X
SSC), and dried.
Detection
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Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
s containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophoxes sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
s. o Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
1 s The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the probe mix at a known concentration. A
specific location on the
array contains a complementary DNA sequence, allowing the intensity of the
signal at that location to
be correlated with a weight ratio of hybridizing species of 1:100,000. When
two probes from different
sources (e.g., representing test and control cells), each labeled with a
different fluorophore, are
~ o hybridized to a single array for the purpose of identifying genes that are
differentially expressed, the
calibration is done by labeling samples of the calibrating cDNA with the two
fluorophores and adding
identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood, MA) installed in an IBM-
compatible PC
2 s computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission spectrum.
a o A grid is superimposed over the fluorescence signal image such that the
signal from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated to
obtain a numerical value corresponding to the average intensity of the signal.
The software used for
signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
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XII. Complementary Nucleic Acids
Sequences complementary to the dithp are used to detect, decrease, or inhibit
expression of the
naturally occurring nucleotide. The use of oligonucleotides comprising from
about 1 S to 30 base pairs
is typical in the art. However, smaller or larger sequence fragments can also
be used. Appropriate
oligonucleotides are designed from the dithp using OLIGO 4.06 software
(National Biosciences) or
other appropriate programs and are synthesized using methods standard in the
art or ordered from a
commercial supplier. To inhibit transcription, a complementary oligonucleotide
is designed from the
most unique S' sequence and used to prevent transcription factor binding to
the promoter sequence. To
inhibit translation, a complementary oligonucleotide is designed to prevent
ribosomal binding and
s o processing of the transcript.
XIII. Expression of DITHP
Expression and purification of DITHP is accomplished using bacterial or virus-
based
expression systems. For expression of DITHP in bacteria, cDNA is subcloned
into an appropriate
~ s vector containing an antibiotic resistance gene and an inducible promoter
that directs high levels of
cDNA transcription. Examples of such promoters include, but are not limited
to, the trp-lac (tic)
hybrid promoter and the TS or T7 bacteriophage promoter in conjunction with
the lac operator .
regulatory element. Recombinant vectors are transformed into suitable
bacterial hosts, e.g.,
BL21 (DE3). Antibiotic resistant bacteria express DITHP upon induction with
isopropyl beta-D-
2 0 thiogalactopyranoside (IPTG). Expression of DITHP in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Auto~raphica califoxnica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding DITHP by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
2 s polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiQerda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See e.g., Engelhard,
supra; and Sandig, supra.)
In most expression systems, DITHP is synthesized as a fusion protein with,
e.g., glutathione S-
3 o transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma japonicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from DITHP at
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specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak
Company, Rochester N~. 6-His, a stretch of six consecutive histidine residues,
enables purification on
metal-chelate resins (QIAGEN). Methods for protein expression and purification
are discussed in
s Ausubel (1995, supra, Chapters 10 and 16). Purified DITHP obtained by these
methods can be used
directly in the following activity assay.
XIV. Demonstration of DITHP Activity
DITHP activity is demonstrated through a variety of specific assays, some of
which are
so outlined below.
Oxidoreductase activity of DITHP is measured by the increase in extinction
coefficient of
NAD(P)H coenzyme at 340 nmfor the measurement of oxidation activity, or the
decrease in extinction
coefficient of NAD(P)H coenzyme at 340 nmfor the measurement of reduction
activity (Dalziel, K.
(1963) J. Biol. Chem. 238:2850-2858). One of three substrates may be used: Asn-
~iGal, biocytidine, or
1 s ubiquinone-10. The respective subunits of the enzyme reaction, for
example, cytochtome cl-b
oxidoreductase and cytochrome c, are reconstituted. The reaction mixture
contains a)I-2 m~ml
DITHP; and b) 1S mM substrate, 2.4 mM NAD(P)+ in 0.1 M phosphate buffer, pH
7.1 (oxidation
reaction), or 2.0 mM NAD(P)H, in 0.1 M Na2HP04 buffer, pH 7.4 ( reduction
reaction); in a total
volume of 0.1 ml. Changes in absorbance at 340 nm (A34o) are measured at 23.5
° C using a recording
2 o spectrophotometer (Shimadzu Scientific Instruments, Inc., Pleasanton CA).
The amount of NAD(P)H
is stoichiometrically equivalent to the amount of substrate initially present,
and the change in A3ao is a
direct measure of the amount of NAD(P)H produced; DA340 = 6620[NADH].
Oxidoreductase activity
of DITHP activity is proportional to the amount of NAD(P)H present in the
assay.
Txansferase activity of DITHP is measured through assays such as a methyl
transferase assay
2 s in which the transfer of radiolabeled methyl groups between a donor
substrate and an acceptor
substrate is measured (Bokar, J.A. et al. (1994) J. Biol. Chem. 269:17697-
17704). Reaction mixtures
(SO girl final volume) contain 1S mM HEPES, pH 7.9, 1.S mM MgCl2, 10 mM
dithiothreitol, 3%
polyvinylalcohol, I.S ~Ci [methyl-3H]AdoMet (0.375 ~M AdoMet) (DuPont-NEN),
0.6 ~g DITHP,
and acceptor substrate (0.4 ~g [35S]RNA or 6-mercaptopurine (6-MP) to 1 mM
final concentration).
3o Reaction mixtures are incubated at 30°C for 30 minutes, then 6S
°C for S minutes. The products are
separated by chromatography or electrophoresis and the level of methyl
transferase activity is
determined by quantification of methyl-3H recovery.
DITHP hydrolase activity is measured by the hydrolysis of appropriate
synthetic peptide
substrates conjugated with various chromogenic molecules in which the degree
of hydrolysis is
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quantified by spectrophotometric (or fluorometric) absorption of the released
chromophore. (Beynon,
R.J. and J.S. Bond (1994) Proteo~tic Enzymes: A Practical Approach, Oxford
University Press, New
York NY, pp. 25-55) Peptide substrates are designed according to the category
of protease activity as
endopeptidase (serine, cysteine, aspartic proteases), animopeptidase (leucine
aminopeptidase), or
s carboxypeptidase (Carboxypeptidase A and B, procollagen C-proteinase).
DITHP isomerase activity such as peptidyl prolyl cisltrans isomerase activity
can be assayed
by an enzyme assay described by Rahfeld, J.U., et al. (1994) (FEBS Lett. 352:
180-184). The assay
is performed at 10°C in 35 mM HEPES buffer, pH 7.8, containing
chymotrypsin (0.5 mg/ml) and
DITHP at a variety of concentrations. Undex these assay conditions, the
substrate, Suc-Ala-Xaa-Pro-
1 o Phe-4-NA, is in equilibrium with respect to the prolyl bond, with 80-95 %
in traps and 5-20%a in cis
conformation. An aliquot (2 u1) of the substrate dissolved in dimethyl
sulfoxide (10 mg/ml) is added
to the reaction mixture described above. Only the cis isomer of the substrate
is a substrate for
cleavage by chymotrypsin. Thus, as the substrate is isomerized by DITHP, the
product is cleaved by
chymotrypsin to produce 4-nitroanilide, which is detected by it's absorbance
at 390 nm. 4-
1 s Nitroanilide appears in a time-dependent and a DITHP concentration-
dependent manner.
An assay for DITHP activity associated with growth and development measures
cell
proliferation as the amount of newly initiated DNA synthesis in Swiss mouse
3T3 cells. A plasmid
containing polynucleotides encoding DITHP is transfected into quiescent 3T3
culturedcells using
methods well known in the art. The transiently transfected cells are then
incubated in the presence of
2 0 [3H]thymidine, a radioactive DNA precursor. Where applicable, varying
amounts of DITHP ligand are
added to the transfected cells. Incorporation of [3H]thymidine into acid-
precipitable DNA is measured
over an appropriate time interval, and the amount incorporated is directly
proportional to the amount of
newly synthesized DNA.
Growth factor activity of DITHP is measured by the stimulation of DNA
synthesis in Swiss
2 s mouse 3T3 cells (McKay, I. and I. Leigh, eds. (1993) Growth Factors: A
Practical Approach, Oxford
University Press, New York NY). Initiation of DNA synthesis indicates the
cells' entry into the mitotic
cycle and their commitment to undergo later division. 3T3 cells are competent
to respond to most
growth factors, not only those that are mitogenic, but also those that are
involved in embryonic
induction. This competence is possible because the in vivo specificity
demonstrated by some growth
3 o factors is not necessarily inherent but is determined by the responding
tissue. In this assay, varying
amounts of DITHP are added to quiescent 3T3 cultured cells in the presence of
[3H]thymidine, a
radioactive DNA precursor. DITHP for this assay can be obtained by recombinant
means or from
biochemical preparations. Incorporation of [3H]thymidine into acid-
precipitable DNA is measured over
an appropriate time interval, and the amount incorporated is directly
proportional to fine amount of
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newly synthesized DNA. A linear dose-response curve over at least a hundred-
fold DITHP
concentration range is indicative of growth factor activity. One unit of
activity per milliliter is defined
as the concentration of DITHP producing a 50% response level, where 100%
represents maximal
incorporation of [3H]thymidine into acid-precipitable DNA.
Alternatively, an assay for cytokine activity of DITHP measures the
proliferation of
leukocytes. In this assay, the amount of tritiated thymidine incorporated into
newly synthesized DNA
is used to estimate proliferative activity. Varying amounts of DITHP are added
to cultured
leukocytes, such as granulocytes, monocytes, or lymphocytes, in the presence
of [3H]thymidine, a
radioactive DNA precursor. DITHP for this assay can be obtained by recombinant
means or from
s o biochemical preparations. Incorporation of [3H]thymidine into acid-
precipitable DNA is measured
over an appropriate time interval, and the amount incorporated is directly
proportional to the amount
of newly synthesised DNA. A linear dose-response curve over at least a hundred-
fold DITHP
concentration range is indicative of DITHP activity. One unit of activity per
milliliter is
conventionally defined as the concentration of DITHP producing a 50% response
level, where 100%
s s represents maximal incorporation of [3H]thymidine into acid-precipitable
DNA.
An alternative assay for DITHP cytokine activity utilizes a Boyden micro
chamber
(Neuroprobe, Cabin John MD) to measure leukocyte chemotaxis (Vicari, supra). '
In this assay, about
105 migratory cells such as macrophages or monocytes are placed in cell
culture media in the upper
compartment of the chamber. Varying dilutions of DITHP are placed in the lower
compartment. The
2 o two compartments are separated by a 5 or 8 nucron pore polycarbonate
filter (Nucleopore, Pleasanton
CA). After incubation at 37 ° C for 80 to 120 minutes, the filters are
fixed in methanol and stained
with appropriate labeling agents. Cells which migrate to the other side of the
filter are counted using
standard microscopy. The chemotactic index is calculated by dividing the
number of migratory cells
counted when DITHP is present in the lower compartment by the number of
migratory cells counted
2 5 when only media is present in the lower compartment. The chemotactic index
is proportional to the
activity of DITHP.
Alternatively, cell lines or tissues transformed with a vector containing
dithp can be assayed for
DITHP activity by immunoblotting. Cells are denatured in SDS in the presence
of [3-mercaptoethanol,
nucleic acids removed by ethanol precipitation, and proteins purified by
acetone precipitation. Pellets
3 o are resuspended in 20 mM tris buffer at pH 7.S and incubated with Protein
G-Sepharose pre-coated
with an antibody specific for DITHP. After washing, the Sepharose beads are
boiled in electrophoresis
sample buffer, and the eluted proteins subjected to SDS-PAGE. The SDS-PAGE is
transferred to a
nitrocellulose membrane for immunoblotting, and the DITHP activity is assessed
by visualizing and
quantifying bands on the blot using the antibody specific for DITHP as the
primary antibody and lasl-
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labeled IgG specific for the primary antibody as the secondary antibody.
DITHP kinase activity is measured by phosphorylation of a protein substrate
using y-labeled
[s2P]-ATP and quantitation of the incorporated radioactivity using a
radioisotope counter. DITHP is
incubated with the protein substrate, [32P]-ATP, and an appropriate kinase
buffer. The [32P]
s incorporated into the product is separated from free [32P]-ATP by
electrophoresis and the incorporated
[32P] is counted. The amount of [32P] recovered is proportional to the kinase
activity of DITHP in
the assay. A determination of the specific amino acid residue phosphorylated
is made by
phosphoamino acid analysis of the hydrolyzed protein.
In the alternative, DITHP activity is measured by the increase in cell
proliferation resulting
~. o from transformation of a mammalian cell line such as COS7, HeLa or CHO
with an eukaryotic
expression vector encoding DITHP. Eukaryotic expression vectors are
commercially available, and
the techniques to introduce them into cells are well known to those skilled in
the art. The cells are
incubated for 48-72 hours after transformation under conditions appropriate
for the cell line to allow
expression of DITHP. Phase micxoscopy is then used to compare the mitotic
index of transformed
s s versus control cells. An increase in the mitotic index indicates DITHP
activity.
In a further alternative, an assay for DITHP signaling activity is based upon
the ability of
GPCR family proteins to modulate G protein-activated second messenger signal
transduction
pathways (e.g., cAMP; Gaudin, P. et al. (1998) J. Biol. Chem. 273:4990-4996).
A plasmid encoding
fall length DITHP is transfected into a mammalian cell line (e.g., Chinese
hamster ovary (CHO) or
2 o human embryonic kidney (HEK-293) cell lines) using methods well-known in
the art. Transfected
cells are grown in 12-well bays in culture medium for 48 hours, then the
culture medium is discarded,
and the attached cells are gently washed with PBS. The cells are then
incubated in culture medium
with or without ligand for 30 minutes, then the medium is removed and cells
lysed by treatment with
1 M perchlouc acid. The cAMP levels in the lysate are measured by
radioimmunoassay using
2 s methods well-known in the art. Changes in the levels of cAMP in the lysate
from cells exposed to
ligand compared to those without ligand are proportional to the amount of
DITHP present in the
transfected cells.
Alternatively, an assay for DITHP protein phosphatase activity measures the
hydrolysis of P
nitrophenyl phosphate (PNPP). DITHP is incubated together with PNPP in HEPES
buffer pH 7.5, in
3 o the presence of 0.1 % (3-mercaptoethanol at 37 ° C for 60 min. The
reaction is stopped by the addition of
6 ml of 10 N NaOH, and the increase in light absorbance of the reaction
mixture at 410 nm resulting
from the hydrolysis of PNPP is measured using a spectrophotometer. The
increase in light absorbance
is proportional to the phosphatase activity of DITHP in the assay (Diamond,
R.H. et al (1994) Mol Cell
Biol 14:3752-3762).
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An alternative assay measures DITHP-mediated G-protein signaling activity by
monitoring
the mobilization of Ca++ as an indicator of the signal txansduction pathway
stimulation. (See, e.g.,
Grynkievicz, G. et al. (1985) J. Biol. Chem. 260:3440; McColl, S. et al.
(1993) J. Immunol.
150:4550-4555; and Aussel, C. et al. (1988) J. Immunol. 140:215-220). The
assay requires
preloading neutrophils or T cells with a fluorescent dye such as FURA-2 or
BCECF (Universal
Imaging Corp, Westchester PA) whose emission characteristics are altered by
Ca+* binding. When
the cells are exposed to one or more activating stimuli artificially (e.g.,
anti-CD3 antibody ligation of
the T cell receptor) or physiologically (e.g., by allogeneic stimulation),
Ca++ flux takes place. This
flux can be observed and quantified by assaying the cells in a fluorometer or
fluorescent activated cell
to sorter. Measurements of Ca++ flux are compared between cells in their
normal state and those
transfected with DITHP. Increased Ca++ mobilization attributable to increased
DITHP concentration
is proportional to DITHP activity.
DITHP transport activity is assayed by measuring uptake of labeled substrates
into Xeno~us
laevis oocytes. Oocytes at stages V and VI are injected with DITHP mRNA (10 ng
per oocyte) and
incubated for 3 days at 18°C in OR2 medium (82.SmM NaCl, 2.S mM KCl,
1mM CaCl2, 1mM MgCl2,
1mM NazHP04, 5 mM Hepes, 3.8 mM NaOH, SO~~ml gentamycin, pH 7.8) to allow
expression of
DITHP protein. Oocytes are then transferred to standard uptake medium (100mM
NaCI, 2 mM KCl,
1mM CaCl2, 1mM MgCl2, 10 mM Hepes/Tris pH 7.5). Uptake of various substrates
(e.g., amino
acids, sugars, drugs, ions, and neurotransmitters) is initiated by adding
labeled substrate (e.g.
2 o radiolabeled with 3H, fluorescently labeled with rhodamine, etc.) to the
oocytes. After incubating for 30
minutes, uptake is terminated by washing the oocytes three times in Na+-free
medium, measuring the
incorporated label, and comparing with controls. DITHP transport activity is
proportional to the level
of internalized labeled substrate.
DITHP transferase activity is demonstrated by a test for galactosyltransferase
activity. This
2 s can be determined by measuring the transfer of radiolabeled galactose from
UDP-galactose to a
GlcNAc-terminated oligosaccharide chain (Kolbinger, F. et al. (1998) J. Biol.
Chem. 273:58-65). The
sample is incubated with 14 p1 of assay stock solution (180 mM sodium
cacodylate, pH 6.5, 1 mg/ml
bovine serum albumin, 0.26 mM UDP-galactose, 2 ~1 of UDP-['H]galactose), 1 ~1
of MnCl2 (500
mM), and 2.5 ~l of GIcNAc(30-(CH_)$ COZMe (37 mp~ml in dimethyl sulfoxide) for
60 minutes at
3 0 37 ° C. The reaction is quenched by the addition of 1 ml of water
and loaded on a C 18 Sep-Pak
cartridge (Waters), and the column is washed twice with 5 ml of water to
remove unreacted UDP-
['H]galactose. The [3H]galactosylated GlcNAc(30-(CHZ)$ COZMe remains bound to
the column during
the water washes and is eluted with 5 ml of methanol. Radioactivity in the
eluted material is measured
by liquid scintillation counting and is proportional to galactosyltransferase
activity in the starting
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sample.
In the alternative, DITHP induction by heat or toxins may be demonstrated
using primary
cultures of human fibroblasts or human cell lines such as CCL-13, HEK293, or
HEP G2 (ATCC). To
heat induce DITHP expression, aliquots of cells are incubated at 42 °C
for 15, 30, or 60 minutes.
s Control aliquots are incubated at 37 °C for the same time periods. To
induce DITHP expression by
toxins, aliquots of cells are treated with 100 ~iM arsenite or 20 mM azetidine-
2-carboxylic acid for 0,
3, 6, or 12 hours. After exposure to heat, arsenite, or the amino acid
analogue, samples of the treated
cells are harvested and cell lysates prepared for analysis by western blot.
Cells are lysed in lysis
buffer containing 1 % Nonidet P-40, 0.15 M NaCl, 50 mM Tris-HCI, 5 mM EDTA, 2
mM
1 o N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin,
and 1 mg/ml pepstatin.
Twenty micrograms of the cell lysate is separated on an 8% SDS-PAGE gel and
transferred to a
membrane. After blocking with 5% nonfat dry milk/phosphate-buffered saline for
1 h, the membrane
is incubated overnight at 4°C or at room temperature fox 2-4 hours with
a 1: 2000 dilution of
anti-DITHP serum in 2% nonfat dry milk/phosphate-buffered saline. The membrane
is then washed
15 and incubated with a I :1000 dilution of horseradish peroxidase-conjugated
goat anti-rabbit IgG in 2%
dry milk/phosphate-buffexed saline. After washing with 0.1 % Tween 20 in
phosphate-buffered saline,
the DITHP protein is detected and compared to controls using
chemiluminescence.
Alternatively, DITHP protease activity is measured by the hydrolysis of
appropriate synthetic
peptide substrates conjugated with various chromogenic molecules in which the
degree of hydrolysis
2 o is quantified by spectrophotometric (or fluorometric) absorption of the
released chromophore
(Beynon, R.J. and J.S. Bond (1994) Proteolytic En~mes: A Practical Approach,
Oxford University
Press, New York, NY, pp.25-55). Peptide substrates are designed according to
the category of
protease activity as endopeptidase (serine, cysteine, aspartic proteases, or
metalloproteases),
aminopeptidase (leucine aminopeptidase), or carboxypeptidase
(carboxypeptidases A and B,
2s procollagen C-proteinase). Commonly used chromogens are 2-naphthylamine, 4-
nitroaniline, and
furylacrylic acid. Assays are performed at ambient temperature and contain an
aliquot of the enzyme
and the appropriate substrate in a suitable buffer. Reactions are carried out
in an optical cuvette, and
the increase/decrease in absorbance of the chromogen released during
hydrolysis of the peptide
substrate is measured. The change in absorbance is proportional to the DITHP
protease activity in the
3 o assay.
In the alternative, an assay for DITHP protease activity takes advantage of
fluorescence
resonance energy transfer (FRET) that occurs when one donor and one acceptor
fluorophore with an
appropriate spectral overlap are in close proximity. A flexible peptide linker
containing a cleavage
site specific for PRTS is fused between a red-shifted variant (RSGFP4) and a
blue variant (BFPS) of
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Green Fluorescent Protein. This fusion protein has spectral properties that
suggest energy transfer is
occurnng from BFPS to RSGFP4. When the fusion protein is incubated with DITHP,
the substrate is
cleaved, and the two fluorescent proteins dissociate. This is accompanied by a
marked decrease in
energy transfer which is quantified by comparing the emission spectra before
and after the addition of
DITHP (Mitra, R.D. et al (1996) Gene 173:13-17). This assay can also be
performed in living cells.
In this case the fluorescent substrate protein is expressed constitutively in
cells and DITHP is
introduced on an inducible vector so that FRET can be monitored in the
presence and absence of
DITHP (Sagot, I. et al (1999) FEBS Lett. 447:53-57).
A method to determine the nucleic acid binding activity of DITHP involves a
polyacrylamide
2 o gel mobility-shift assay. In preparation fox this assay, DITHP is
expressed by transforming a
mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression
vector containing
DITHP cDNA. The cells are incubated for 48-72 hours after transformation under
conditions
appropriate for the cell line to allow expression and accumulation of DITHP.
Extracts containing
solubilized proteins can be prepared from cells expressing DITHP by methods
well known in the art.
1 s Portions of the extract containing DITHP are added to [32P]-labeled RNA or
DNA. Radioactive
nucleic acid can be synthesized in vitro by techniques well known in the art.
The mixtures are
incubated at 25 °C in the presence of RNase- and DNase-inhibitors under
buffered conditions for 5-10
minutes. After incubation, the samples are analyzed by polyacxylamide gel
electrophoresis followed
by autoradiography. The presence of aband on the autoradiogram indicates the
formation of a
2 o complex between DITHP and the radioactive transcript. A band of similar
mobility will not be
present in samples prepared using control extracts prepared from untransformed
cells.
In the alternative, a method to determine the methylase activity of a DITHP
measures transfer
of radiolabeled methyl groups between a donor substrate and an acceptor
substrate. Reaction
mixtures (50 Ed final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl 2, 10
mM dithiothreitol,
2s 3% polyvinylalcohol, 1.5 ~Ci [methyl.-3H]AdoMet (0.375 ~M AdoMet) (DuPont-
NEN), 0.6 ~g
DITHP, and acceptor substrate (e.g., 0.4 ~g [35S]RNA, or 6-mercaptopurine (6-
MP) to 1 mM final
concentration). Reaction mixtures are incubated at 30°C for 30 minutes,
then 65 °C for 5 minutes.
Analysis of [methyl-3H]RNA is as follows: 1) 50 ~l of 2 x loading buffer (20
mM Tris-HCI, pH 7.6, 1
M LiCI, 1 mM EDTA, 1% sodium dodecyl sulphate (SDS)) and 50 ~..il oligo d(T)-
cellulose (10 mg/ml
3 o in 1 x loading buffer) are added to the reaction mixture, and incubated at
ambient temperature with
shaking fox 30 minutes. 2) Reaction mixtures are transferred to a 96-well
filtration plate attached to a
vacuum apparatus. 3) Each sample is washed sequentially with three 2.4 ml
aliquots of 1 x oligo d(T)
loading buffer containing 0.5% SDS, 0.1 % SDS, or no SDS. and 4) RNA is eluted
with 300 ftl of
water into a 96-well collection plate, transferred to scintillation vials
containing liquid scintillant, and
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radioactivity determined. Analysis of [methyl.-3H]6-MP is as follows: 1) 500
~.il 0.5 M borate buffer,
pH 10.0, and then 2.5 ml of 20% (v/v) isoamyl alcohol in toluene are added to
the reaction mixtures.
2) The samples mixed by vigorous vortexing for ten seconds. 3) After
centrifugation at 7008 for 10
minutes, 1.5 ml of the organic phase is transferred to scintillation vials
containing 0.5 ml absolute
s ethanol and liquid scintillant, and radioactivity determined. and 4) Results
are corrected for the
extraction of 6-MP into the organic phase (approximately 41 %).
An assay for adhesion activity of DITHP measures the disruption of
cytoskeletal filament
networks upon overexpression of DITHP in cultured cell lines (Rezniczek, G.A.
et al. (1998) J. Cell
Biol. 141:209-225). cDNA encoding DITHP is subcloned into a mammalian
expression vector that
~ o drives high levels of cDNA expression. This construct is transfected into
cultured cells, such as rat
kangaroo PtK2 or rat bladder carcinoma 8046 cells. Actin filaments and
intermediate filaments such
as keratin and vimentin are visualized by immunofluorescence microscopy using
antibodies and
techniques well known in the art. The configuration and abundance of
cytoskeletal filaments can be
assessed and quantified using confocal imaging techniques. In particular, the
bundling and collapse
25 Of cytoskeletal filament networks is indicative of DITHP adhesion activity.
Alternatively, an assay for DITHP activity measures the expression of DITHP on
the cell
surface. cDNA encoding DITHP is transfected into a non-leukocytic cell line.
Cell surface proteins
are labeled with biotin (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
Immunoprecipitations
are performed using DITHP-specific antibodies, and immunoprecipitated samples
are analyzed using
2 o SDS-PAGE and immunoblotting techniques. The ratio of labeled
immunoprecipitant to unlabeled
immunoprecipitant is proportional to the amount of DITHP expressed on the cell
surface.
Alternatively, an assay for DITHP activity measures the amount of cell
aggregation induced
by overexpression of DITHP. In this assay, cultured cells such as NIH3T3 are
transfected with cDNA
encoding DITHP contained within a suitable mammalian expression vector under
control of a strong
2 s promoter. Cotransfection with cDNA encoding a fluorescent marker protein,
such as Green
Fluorescent Protein (CLONTECH), is useful for identifying stable
transfectants. The amount of cell
agglutination, or clumping, associated with transfected cells is compared with
that associated with
untransfected cells. The amount of cell agglutination is a direct measure of
DITHP activity.
DITHP may recognize and precipitate antigen from serum. This activity can be
measured by
3 o the quantitative precipitin reaction (Golub, E.S. et al. (1987)
Immunology: A Synthesis, Sinauer
Associates, Sunderland MA, pages 113-115). DITHP is isotopically labeled using
methods known in
the art. Various serum concentrations are added to constant amounts of labeled
DITHP. DITHP-
antigen complexes precipitate out of solution and are collected by
centrifugation. The amount of
precipitable DITHP-antigen complex is proportional to the amount of
radioisotope detected in the
176
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
precipitate. The amount of precipitable DITHP-antigen complex is plotted
against the serum
concentration. For various serum concentrations, a-characteristic
precipitation curve is obtained, in
which the amount of precipitable DITHP-antigen complex initially increases
proportionately with
increasing serum concentration, peaks at the equivalence point, and then
decreases proportionately with
further increases in serum concentration. Thus, the amount of precipitable
DITHP-antigen complex is
a measure of DITHP activity which is characterized by sensitivity to both
limiting and excess quantities
of antigen.
A microtubule motility assay for DITHP measures motor protein activity. In
this assay,
recombinant DITHP is immobilized onto a glass slide or similar substrate.
Taxol-stabilized bovine
1 o brain microtubules (commercially available) in a solution containing ATP
and cytosolic extract are
perfused onto the slide. Movement of microtubules as driven by DITHP motor
activity can be
visualized and quantified using video-enhanced light microscopy and image
analysis techniques.
DITHP motor protein activity is directly proportional to the frequency and
velocity of microtubule
movement.
Alternatively, an assay for DITHP measures the formation of protein filaments
in vitro. A
solution of DITHP at a concentration greater than the "critical concentration"
for polymer assembly is
applied to carbon-coated grids. Appropriate nucleation sites may be supplied
in the solution. The
grids are negative stained with 0.7% (w/v) aqueous uranyl acetate and examined
by electron
microscopy. The appearance of filaments of approximately 25 nm (microtubules),
8 nm (actin), or 10
2 o nm (intermediate filaments) is a demonstration of protein activity.
DITHP electron transfer activity is demonstrated by oxidation or reduction of
NADP.
Substrates such as Asn-(3Gal, biocytidine, or ubiquinone-10 may be used. The
reaction mixture
contains 1-2 m~ml HORP, 15 mM substrate, arid 2.4 mM NAD(P)~ in 0.1 M
phosphate buffer, pH
7.1 (oxidation reaction), or 2.0 mM NAD(P)H, in 0.1 M Na2HP04 buffer, pH 7.4
(reduction reaction);
a 5 in a total volume of 0.1 ml. FAD may be included with NAD, according to
methods well known in fine
art. Changes in absorbance are measured using a recording spectrophotometer.
The amount of
NAD(P)H is stoichiometrically equivalent to the amount of substrate initially
present, and the change in
As4o is a direct measure of the amount of NAD(P)H produced; dA3øo =
6620[NADH]. DITHP activity
is proportional to the amount of NAD(P)H present in the assay. The increase in
extinction coefficient
3 0 of NAD(P)H coenzyme at 340 nm is a measure of oxidation activity, or the
decrease in extinction
coefficient of NAD(P)H coenzyme at 340 nm is a measure of reduction activity
(Dalziel, K. (1963) J.
Biol. Chem. 238:2850-2858).
DITHP transcription factor activity is measured by its ability to stimulate
transcription of a
reporter gene (Liu, H.Y, et al. (1997) EMBO J. 16:5289-5298). The assay
entails the use of a well
177
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WO 01/62927 PCT/USO1/06059
characterized reporter gene construct, LexAnp LacZ, that consists of LexA DNA
transcriptional control
elements (LexAbP) fused to sequences encoding the E. coli LacZ enzyme. The
methods for constructing
and expressing fusion genes, introducing them into cells, and measuring LacZ
enzyme activity, are well
known to those skilled in the art, Sequences encoding DITHP are cloned into a
plasmid that directs the
synthesis of a fusion protein, LexA-DITHP, consisting of DITHP and a DNA
binding domain derived
from the LexA transcription factor. The resulting plasmid, encoding a LexA-
DITHP fusion protein, is
introduced into yeast cells along with a plasmid containing the LexAop-LacZ
reporter gene. The amount
of LacZ enzyme activity associated with LexA-DITHP transfected cells, relative
to control cells, is
proportional to the amount of transcription stimulated by the DITHP.
1o Chromatin activity of DITHP is demonstrated by measuring sensitivity to
DNase I (Dawson,
B.A. et al. (1989) J. Biol. Chem. 264:12830-12837). Samples are treated with
DNase I, followed by
insertion of a cleavable biotinylated nucleotide analog, 5-[(N-
biotinamido)hexanoamido-ethyl-1,3-
thiopropionyl-3-aminoallyl]-2'-deoxyuridine S'-triphosphate using nick-repair
techniques well known
to those skilled in the art. Following purification and digestion with EcoRI
restriction endonuclease,
~ s biotinylated sequences are affinity isolated by sequential binding to
streptavidin and biotincellulose.
Another specific assay demonstrates the ion conductance capacity of DITHP
using an
electrophysiological assay. DITHP is expressed by transforming a mammalian
cell line such as
COS7, HeLa or CHO with a eukaryotic expression vector encoding DITHP.
Eukaryotic expression
vectors are commercially available, and the techniques to introduce them into
cells are well known to
2 o those skilled in the art. A small amount of a second plasmid, which
expresses any one of a number of
marker genes such as (3-galactosidase, is co-transformed into the cells in
order to allow rapid
identification of those cells which have taken up and expressed the foreign
DNA. 'The cells are
incubated for 48-72 hours after transformation under conditions appropriate
fox the cell line to allow
expression and accumulation of DITHP and (3-galactosidase. Transformed cells
expressing (3-
2 s galactosidase are stained blue when a suitable colorimetric substrate is
added to the culture media
under conditions that are well known in the art. Stained cells are tested for
differences in membrane
conductance due to various ions by electrophysiological techniques that are
well known in the art.
Untransformed cells, and/or cells transformed with either vector sequences
alone or j3-galactosidase
sequences alone, are used as controls and tested in parallel. The contribution
of DITHP to cation or
a o anion conductance can be shown by incubating the cells using antibodies
specific for either DITHP.
'The respective antibodies will bind to the extracellular side of DITHP,
thereby blocking the pore in
the ion channel, and the associated conductance.
XV. Functional Assays
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WO 01/62927 PCT/USO1/06059
DITHP function is assessed by expressing dithp at physiologically elevated
levels in
mammalian cell culture systems. cDNA is subcloned into a mammalian expression
vector containing a
strong promoter that drives high levels of eDNA expression. Vectors of choice
include pCMV SPORT
(Life Technologies) and pCR3.1 (Invitrogen Corporation, Carlsbad CA), both of
which contain the
cytomegalovirus promoter. 5-10 ~ g of recombinant vector are transiently
txansfected into a human cell
Line, preferably of endothelial or hematopoietic origin, using either liposome
formulations or
electxoporation. 1-2 ~.g of an additional plasmid containing sequences
encoding a marker protein are
co-transfected.
Expression of a marker protein provides a means to distinguish transfected
cells from
s o nontransfected cells and is a reliable predictor of cDNA expression from
the recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
CLONTECH), CD64, or a
CD64-GFP fusion protein. Flow cytometry (FCM), an automated laser optics-based
technique, is used
to identify transfected cells expressing GFP or CD64-GFP and to evaluate the
apoptotic state of the
cells and other cellular properties.
FCM detects and quantifies the uptake of fluorescent molecules that diagnose
events preceding
or coincident with cell death. These events include changes in nuclear DNA
content as measured by
staining of DNA with propidium iodide; changes in cell size and granularity as
measured by forward
light scatter and 90 degree side light scatter; down-regulation of DNA
synthesis as measured by
6
decrease in bromodeoxyuridine uptake; alterations in expression of cell
surface and intracellular
2 o proteins as measured by reactivity with specific antibodies; and
alterations in plasma membrane
composition as measured by the binding of fluorescein-conjugated Annexin V
protein to the cell
surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow
Cvtometrv, Oxford,
New York NY.
The influence of DITHP on gene expression can be assessed using highly
purified populations
2 s of cells transfected with sequences encoding DITHP and either CD64 or CD64-
GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Inc., Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
3 o Expression of mRNA encoding DITHP and other genes of interest can be
analyzed by northern analysis
or microarray techniques.
XVI. Production of Antibodies
DITHP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
179
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the DITHP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
peptide is synthesized
s and used to raise antibodies by means known to those of skill in the art.
Methods for selection of
appropriate epitopes, such as those near the C-terminus or in hydrophilic
regions are well described in
the art. (See, e.g., Ausubel, 1995, supra, Chapter 11.)
Typically, peptides 15 residues in length are synthesized using an ABI 431A
peptide
synthesizer (Applied Biosystems) using fmoc-chemistry and coupled to I~LH
(Sigma) by reaction with
to N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase
immunogenicity. (See, e.g.,
Ausubel, su ra.) Rabbits are immunized with the peptide-KLH complex in
complete Freund's
adjuvant. Resulting antisera are tested for antipeptide activity by, for
example, binding the peptide to
plastic, blocking with 1 % BSA, reacting with rabbit antisera, washing, and
reacting with radio-
iodinated goat anti-rabbit IgG. Antisera with antipeptide activity are tested
for anti-DITHP activity
1 s using protocols well known in the art, including ELISA, RIA, and
immunoblotting.
XVII. Purification of Naturally Occurring DITHP Using Specific Antibodies
Naturally occurring or recombinant DITHP is substantially purified by
immunoaffinity
chromatography using antibodies specific for DITHP. An immunoaffinity column
is constructed by
2 o covalently coupling anti-DITHP antibody to an activated chromatographic
resin, such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing DITHP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of DITHP (e.g.,
high ionic strength
2 s buffers in the presence of detergent). The column is eluted under
conditions that disrupt
antibody/DITHP binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such
as urea or thiocyanate ion), and DITHP is collected.
XVIII. Identification of Molecules Which Interact with DITHP
3 o DITHP, or biologically active fragments thereof, are labeled with 12s1
Bolton-Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled DITHP, washed, and
any wells with labeled DITHP complex are assayed. Data obtained using
different concentrations of
DITHP are used to calculate values for the number, affinity, and association
of DITHP with the
180
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
candidate molecules.
Alternatively, molecules interacting with DITHP are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246> or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(CLONTECH).
DITHP may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent
No. 6,057,101 ).
1 o All publications and patents mentioned in the above specification are
herein incorporated by
reference. Various modifications and variations of the described method and
system of the invention
wilt be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with specific
preferred embodiments, it should
be understood that the invention as claimed should not be unduly limited to
such specific embodiments.
1 s Indeed, various modifications of the above-described modes for carrying
out the invention which are
obvious to those skilled in the field of molecular biology or related fields
are intended to be within the
scope of the following claims.
181
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
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192
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
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1,_________193 -____-~~
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
TABLE 3
Template ID Start Stop Frame Domain Topology
SEQ ID Tvpe
NO:
1 LG:1040582.1:2000FEB1831 117 forward TM N in
1
1 LG:1040582.1:2000FEB18319 405 forward TM N in
1
1 LG:1040582.1:2000FEB18108 155 forward TM N out
3
2 LG:453570.1:2000FEB18361 447 forward TM N in
1
3 LG:408751.3:2000FEB181318 1404 forward TM N in
1
3 LG:408751.3:2000FEB181025 1099 forward TM N in
2
3 LG:408751.3:2000FEB181298 1360 forward TM N in
2
3 LG:408751.3:2000FEB181379 1441 forward TM N in
2
3 LG:408751.3:2000FEB181463 1537 forward TM N in
2
3 LG:408751.3:2000FE8181047 1133 forward TM N in
3
3 LG;408751.3:2000FEB181266 1352 forward TM N in
3
3 LG:408751.3:2000FEB181419 1469 forward TM N in
3
4 LI:090574.1:2000FEB0179 144 forward TM N in
1
4 LI:090574.1:2000FEB01607 678 forward TM N in
1
4 LI:090574.1:2000FEB011009 1080 forward TM N in
i
4 LI:090574.1:2000FEB01497 583 forward TM N out
2
4 LI:090574.1:2000FEB01743 829 forward TM N out
2
4 LI;090574.1:2000FEB011026 1085 forward TM N out
3
LI:229932.2:2000FEB0176 162 forward TM N out
1
5 LI:229932.2:2000FEB01190 276 forward TM N out
1
5 LI:229932.2:2000FEB011237 1323 forward TM N out
1
5 LI:229932.2:2000FEB0168 142 forward TM N in
2
5 LI:229932.2:2000FEB01335 412 forward TM N in
2
5 LI:229932.2:2000FEB01758 844 forward TM N in
2
5 LI:229932.2:2000FEB011229 1288 forward TM N in
2
5 LI:229932.2:2000FEB0160 146 forward TM N in
3
5 LI:229932.2:2000FEB01216 302 forward TM N in
3
5 LI:229932.2:2000FEB01690 752 forward TM N in
3
5 LI:229932.2:2000FEB01765 827 forward TM N in
3
5 LI:229932.2:2000FEB011209 1289 forward TM N in
3
6 LI:332176.1:2000FEB01343 399 forward TM N in
1
6 LI:332176.1:2000FEB011078 1131 forward TM N in
1
6 LI:332176.1:2000FEB011606 1692 forward TM N in
1
6 LI:332176.1:2000FEB012218 2274 forward TM N in
1
6 LI:332176.1:2000FEB012383 2433 forward TM N in
1
6 LI:332176.1:2000FEB01110 196 forward TM N in
2
6 LI:332176.1:2000FEB011307 1378 forward TM N in
2
6 LI:332176.1:2000FEB011640 1726 forward TM . N in
2
6 LI:332176.1:2000FEB011946 2005 forward TM N in
2
6 LI:332176.1:2000FEB01135 200 forward TM N in
3
6 L1:332176.1:2000FEB01693 752 forward TM N in
3
6 LI:332176.1:2000FEB01777 839 forward TM N in
3
6 LI:332176.1:2000FEB01867 929 forward TM N in
3
6 LI:332176.1:2000FEB011035 1118 forward TM N in
3
6 LI:332176.1;2000FEB011173 1253 forward TM N in
3
6 LI:332176.1:2000FEB011572 1658 forward TM N in
3
6 LI:332176.1:2000FEB012121 2180 forward TM N in
3
6 LI:332176.1:2000FEB012277 2363 forward TM N in
3
6 LI:332176.1:2000FEB012400 2456 forward TM N in
3
8 LG:220992.1:2000MAY19343 393 forward TM
1
8 LG:220992.1:2000MAY19646 732 forward TM
1
8 LG:220992.1:2000MAY191639 1725 forward TM
1
8 LG:220992.1:2000MAY191879 1965 forward TM
1
194 - _
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WO 01/62927 PCT/USO1/06059
8 LG:220992.1:2000MAY192005 2088 forward TM
1
8 LG:220992.1:2000MAY1917 76 forward TM N in
2
8 LG:220992.1:2000MAY191646 1732 forward TM N in
2
8 LG:220992.1:2000MAY191850 1933 forward TM N in
2
8 LG:220992.1:2000MAY191434 1484 forward TM N out
3
8 LG:220992.1:2000MAY191734 1820 forward TM N out
3
8 LG:220992.1:2000MAY191974 2036 forward TM N out
3
8 LG:220992.1:2000MAY192067 2129 forward TM N out
3
8 LG:220992.1:2000MAY192151 2237 forward TM N out
3
9 LG:1094571.1:2000MAY19781 867 forward TM N in
1
9 LG:1094571.1:2000MAY19419 505 forward TM N in
2
9 LG:1094571.1:2000MAY19767 853 forward TM N in
2
9 LG:1094571.1:2000MAY19756 842 forward TM N in
3
LI:350754.4:2000MAY01277 348 forward TM N in
1
10 LI:350754.4:2000MAY01583 651 forward TM N in
1
10 LI:350754.4:2000MAY01670 747 forward TM N in
1
10 LI:350754.4:2000MAY01381 467 forward TM N in
3
10 LI:350754.4:2000MAY012469 2555 forward TM N in
3
12 LI:1190263.1:2000MAY01664 735 forward TM N in
1
12 LI:1190263.1:2000MAY01787 861 forward TM N in
1
12 LI:1190263.1:2000MAY01901 954 forward TM N in
1
12 LI:1190263.1:2000MAY01188 274 forward TM N in
2
12 LI:1190263.1:2000MAY01455 508 forward TM N in
2
12 LI:1190263.1:2000MAY01809 895 forward TM N in
2
12 LI:1190263,1:2000MAY011616 1663 forward TM N in
2
12 LI:1190263.1:2000MAY01183 251 forward TM N in
3
12 L1:1190263.1:2000MAY01648 704 forward TM N in
3
12 LI:1190263.1:2000MAY011149 1235 forward TM N in
3
13 LG:270916.2:2000FEB18173 259 forward TM N out
2
14 LG:999414.3:2000FEB18109 195 forward TM N out
1
14 LG:999414.3:2000FEB18358 438 forward TM N out
1
14 LG:999414.3:2000FE818520 591 forward TM N out
1
14 LG:999414.3:2000FEB18661 744 forward TM N out
1
14 LG:999414.3:2000FEB18883 969 forward TM N out
1
14 LG:999414.3:2000FEB18976 1062 forward TM N out
1
14 LG:999414.3:2000FEB18302 388 forward TM N in
2
14 LG:999414.3:2000FEB18533 613 forward TM N in
2
14 LG:999414.3:2000FEB18992 1048 forward TM N in
2
14 LG:999414.3:2000FEB181169 1246 forward TM N in
2
14 LG:999414.3:2000FEB181307 1366 forward TM N in
2
14 LG:999414.3:2000FEB18207 284 forward TM N out
3
14 LG:999414.3:2000FEB18324 404 forward TM N out
3
i4 LG:999414,3:2000FEB18540 599 forward TM N out
3
14 LG:999414.3:2000FEB181029 1115 forward TM N out
3
14 LG:999414.3:2000FEB181167 1253 forward TM N out
3
14 LG:999414.3:2000FEB181314 1373 forward TM N out
3
LG:429446.1:2000FEB18628 699 forward TM N out
1
15 LG:429446.1:2000FEB18629 682 forward TM N in
2
15 LG:429446.1:2000FEB18627 713 forward TM N in
3
16 LI:057229.1:2000FEB0110 69 forward TM
1
16 LI:057229.1:2000FEB01118 198 forward TM
1
16 LI:057229.1:2000FEB01292 360 forward TM
1
16 LI:057229.1:2000FEB0111 67 forward TM
2
16 LI:057229.1:2000FEB01146 226 forward TM
2
16 LI:057229.1:2000FEB01290 355 forward TM
2
16 LI:057229.1:2000FEB0112 71 forward TM N out
3
_ _195 -___ _.._
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
16 LI:057229.1:2000FEB01114 176 forward TM N out
3
17 LI:351965.1:2000FEB01487 573 forward TM
1
17 L1:351965.1:2000FEB011036 1098 forward TM
1
17 LI:351965.1:2000FEB01492 578 forward TM N in
3
17 Lt:351965.1:2000FEB01969 1055 forward TM N in
3
17 LI:351965.1:2000FEB011098 1184 forward TM N in
3
18 LG:068682.1:2000FEB18707 793 forward TM N out
2
19 LG:242665.1:2000FEB1810 63 forward TM N out
1
19 LG:242665.1:2000FEB1812 62 forward TM N out
3
19 LG:242665.1:2000FEB18333 398 forward TM N out
3
20 LG:241743.1:2000FEB1843 99 forward TM N out
1
21 LI:034212.1:2000FEB011300 1365 forward TM N in
1
21 LI:034212.1:2000FEB011570 1647 forward TM N in
1
21 L1:034212.1:2000FEB012386 2472 forward TM N in
1
21 LI:034212.1:2000FE8012533 2598 forward TM N in
1
21 LI:034212.1;2000FEB012620 2706 forward TM N in
1
21 LI:034212.1:2000FEB012740 2826 forward TM N in
1
21 LI:034212.1:2000FEB01719 805 forward TM
2
21 LI:034212.1:2000FEB011205 1291 forward TM
2
21 LI:034212.1:2000FEB011460 1546 forward TM
2
21 LI:034212.1:2000FEB011685 1768 forward TM
2
21 LI:034212.1:2000FEB011814 1882 forward TM
2
21 LI:034212.1:2000FEB012066 2128 forward TM
2
21 LI:034212.1:2000FEB012156 2218 forward TM
2
21 LI:034212.1:2000FEB012540 2626 forward TM
2
21 LI:034212.1:2000FEB012657 2734 forward TM
2
21 LI:034212.1:2000FEB0112 62 forward TM N out
3
21 LI:034212.1:2000FEB011236 1301 forward TM N out
3
21 LI:034212.1:2000FEB011590 1646 forward TM N out
3
21 LI:034212.1:2000FEB011668 1721 forward TM N out
3
21 LI:034212.1:2000FEB012130 2216 forward TM N out
3
21 LI:034212.1:2000FEB012295 2381 forward TM N out
3
21 LI:034212.1:2000FEB012436 2513 forward TM N out
3
21 LI:034212.1:2000FEB012538 2624 forward TM N out
3
21 LI:034212.1:2000FEB012667 2735 forward TM N out
3
22 LG:344886.1:2000MAY19937 1002 forward TM N in
1
22 LG:344886.1:2000MAY191081 1155 forward TM N in
1
22 LG:344886.1:2000MAY191696 1782 forward TM N in
1
22 LG:344886.1:2000MAY19413 463 forward TM N in
2
22 LG:344886.1:2000MAY19551 637 forward TM N in
2
22 LG:344886.1;2000MAY19950 1012 forward TM N in
2
22 LG:344886.1:2000MAY191031 1093 forward TM N in
2
22 LG:344886.1:2000MAY191112 1183 forward TM N in
2
22 LG:344886.1:2000MAY191271 1348 forward TM N in
2
22 LG:344886:1:2000MAY191634 1720 forward TM N in
2
22 LG:344886.1:2000MAY19567 626 forward TM N in
3
22 LG:344886.1:2000MAY191011 1073 forward TM N in
3
22 LG:344886.1:2000MAY191089 1151 forward TM N in
3
22 LG:344886.1:2000MAY191707 1757 forward TM N in
3
23 LG:228930.1:2000MAY19111 167 forward TM N in
3
24 LG:338927.1:2000MAY19934 1020 forward TM N out
1
24 LG:338927.1;2000MAY191133 1219 forward TM N in
2
24 LG:338927.1;2000MAY191170 1250 forward TM N in
3
25 LG:898771.1:2000MAY191261 1314 forward TM N out
1
25 LG:898771.1:2000MAY191397 1450 forward TM N out
2
26 LI:257664.67:2000MAY01280 366 forward TM N in
1
__ _________________
196
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
26 LI:257664.67:2000MAY01421 498 forward TM N in
1
26 LI:257664.67:2000MAY0112 71 forward TM N out
3
27 LI:001496.2:2000MAY01399 473 forward TM
3
28 LI:1085273.2:2000MAY012188 2274 forward TM N in
1
28 LI:1085273.2:2000MAY01503 583 forward TM N out
2
28 LI:1085273.2:2000MAY012126 2194 forward TM N out
2
28 LI:1085273.2:2000MAY01897 968 forward TM N in
3
29 LI:333138.2:2000MAY011930 2016 forward TM N out
1
29 LI:333138.2:2000MAY0150 103 forward TM
2
29 LI:333138.2:2000MAY01884 940 forward TM
2
29 LI:333138.2:2000MAY01114 179 forward TM N out
3
29 LI:333138.2:2000MAY01273 356 forward TM N out
3
29 LI:333138.2:2000MAY01819 875 forward TM N out
3
29 LI:333138.2:2000MAY011581 1667 forward TM N out
3
30 LI:338927.1:2000MAY011069 1140 forward TM N in
1
30 LI:338927.1:2000MAY01968 1051 forward TM N in
2
30 LI:338927.1:2000MAY011056 1118 forward TM N out
3
30 LI:338927.1:2000MAY011155 1217 forward TM N out
3
31 LG:335558.1:2000FEB18518 604 forward TM N in
2
3i LG:335558.1:2000FEB18614 682 forward TM N in
2
31 LG:335558.1:2000FEB18761 829 forward TM N in
2
31 LG:335558.1:2000FEB18798 860 forward TM N in
3
31 LG:335558.1:2000FEB18882 944 forward TM N in
3
31 LG:335558.1:2000FEB18966 1028 forward TM N in
3
32 LG:998283.7:2000FEB181066 1146 forward TM N in
1
32 LG:998283.7:2000FEB1823 109 forward TM N in
2
32 LG:998283.7:2000FEB18194 280 forward TM N in
2
32 LG:998283.7:2000FEB18392 478 forward TM N in
2
32 LG:998283.7:2000FEB18527 613 forward TM N in
2
32 LG:998283.7:2000FEB18776 862 forward TM N in
2
32 LG;998283.7:2000FEB181064 1141 forward TM N in
2
32 LG;998283.7:2000FEB1812 65 forward TM N in
3
32 LG:998283.7:2000FEB18147 227 forward TM N in
3
32 LG:998283.7:2000FEB18684 770 forward TM N in
3
32 LG:998283.7:2000FEB181011 1097 forward TM N in
3
33 LI:402739.1:2000FEB01415 501 forward TM N in
1
35 LG:981076.2:2000MAY19388 450 forward TM N in
1
35 LG:981076.2:2000MAY1920 82 forward TM N out
2
35 LG:981076.2:2000MAY19389 451 forward TM N out
2
35 LG:981076.2:2000MAY19464 526 forward TM N out
2
35 LG:981076.2:2000MAY19539 604 forward TM N out
2
35 LG:981076.2:2000MAY19438 524 forward TM N in
3
37 LI:1190250.1:2000MAY01530 613 forward TM
2
37 LI:1190250.1:2000MAY01558 635 forward TM N out
3
38 LG:021371.3:2000FEB18122 208 forward TM N in
2
41 LG:410726.1:2000FEB1822 108 forward TM N in
1
41 LG:410726.1:2000FE818385 471 forward TM N in
1
42 LG:200005.1:2000FEB18166 222 forward TM N out
1
42 LG:200005.1:2000FEB18185 232 forward TM N out
2
42 LG:200005.1:2000FEB18162 248 forward TM N out
3
46 LG:1079203.1:2000FEB1811 70 forward TM N in
2
46 LG:1079203.1:2000FEB18125 196 forward TM N in
2
46 LG:1079203.1:2000FEB18965 1051 forward TM N in
2
47 LG:1082586.1:2000FEB18256 339 forward TM N in
1
47 LG:1082586.1:2000FEB18248 316 forward TM N out
2
49 LG:1082775.1:2000FEB18553 606 forward TM N in
1
__ _______________
I97
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
50 LG:1083120.1:2000FEB18214 291 forward TM N out
1
50 LG:1083120.1:2000FEB18233 319 forward TM N out
2
50 LG:1083120.1;2000FEB18252 320 forward TM N in
3
51 LG:1087707.1:2000FEB18367 453 forward TM N out
1
51 LG:1087707.1:2000FEB18469 531 forward TM N out
1
51 LG:1087707.1:2000FEB18667 729 forward TM N out
1
51 LG:1087707.1:2000FEB18742 804 forward TM N out
1
51 LG:1087707.1:2000FEB18407 481 forward TM N in
2
51 LG:1087707.1:2000FEB18671. 739 forward TM N in
2
51 LG:1087707.1:2000FEB18743 811 forward TM N in
2
51 LG:1087707.1:2000FEB18570 641 forward TM N out
3
51 LG:1087707.1:2000FEB18747 833 forward TM N out
3
52 LG:1090915.1:2000FEB1811 61 forward TM N out
2
53 LG:1094230.1:2000FEB18469 555 forward TM N out
1
53 LG:1094230.1:2000FEB18449 535 forward TM N out
2
54 LG:474848.3:2000FEB18445 531 forward TM N out
1
54 LG:474848.3:2000FEB18456 518 forward TM N out
3
58 LI:236654.2:2000FEB01221 307 forward TM N out
2
59 LI:200009.1:2000FEB011045 1131 forward TM N out
1
59 L1:200009.1:2000FEB011171 1233 forward TM N out
1
59 LI:200009.1:2000FEB011076 1162 forward TM N in
2
59 LI:200009.1:2000FEB011044 1130 forward TM N in
3
60 LI:758502.1:2000FEB01286 369 forward TM N out
1
60 LI:758502.1:2000FEB01755 805 forward TM N in
2
60 LI:758502.1:2000FEB01780 833 forward TM N in
3
62 LI:789445.1:2000FEB019 80 forward TM N out
3
63 LI:789657.1:2000FEB01854 937 forward TM N in
2 ~
64 LI:789808.1:2000FEB01347 400 forward TM N in
2
65 L1:792919.1:2000FEB01176 256 forward TM
2
65 LI:792919.1:2000FEB01371 427 forward TM
2
66 LI:793949.1:2000FEB01208 282 forward TM N out
1
66 LI:793949.1;2000FEB01472 558 forward TM N out
1
66 LI;793949.1;2000FEB01455 541 forward TM N out
2
67 LI:794389.1:2000FEB01265 333 forward TM N out
1
67 LI:794389.1:2000FEB01424 477 forward TM N out
i
67 LI:794389.1:2000FEB01384 455 forward TM N in
3
68 LI:796010.1:2000FEB01351 404 forward TM N in
3
69 LI:796324.1:2000FEB01365 418 forward TM N in
2
72 LI:798636.1:2000FEB01490 543 forward TM N in
1
73 LI:800045.1:2000FEB01627 701 forward TM N in
3
74 LI:800680.1:2000FEB01334 411 forward TM N out
1
74 LI:800680.1:2000FEB01359 421 forward TM N out
2
75 LI:800894.1:2000FEB01536 592 forward TM N in
2
75 LI:800894.1:2000FEB01300 374 forward TM N out
3
75 LI:800894.1:2000FEB01396 482 forward TM N out
3
77 LI:801236.1:2000FEB01262 318 forward TM N out
1
78 LI:803335.1:2000FEB01412 498 forward TM N out
1
78 LI:803335.1:2000FEB01423 485 forward TM N out
3
79 LI:803998.1:2000FEB01221 307 forward TM N out
2
81 LI;808532.1;2000FEB01472 558 forward TM N in
1
81 LI:808532.1:2000FEB01117 203 forward TM N in
3
81 LI:808532.1:2000FEB01363 443 forward TM N in
3
8i LI:808532.1:2000FEB01558 623 forward TM N in
3
82 LI:443073.1:2000FEB01293 379 forward TM N in
2
82 LI:443073.1:2000FEB0181 152 forward TM N in
3
82 LI:443073.1:2000FEB01189 260 forward TM N in
3
198
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
83 LI:479671.1:2000FEB01523 579 forward TM N out
1
85 LI:810224.1:2000FEB01246 299 forward TM
3
87 LG:892274.1:2000MAY1949 i 05 forward TM N out
1
87 LG:892274.1:2000MAY19613 681 forward TM N out
1
87 LG:892274.1:2000MAY19506 589 forward TM N in
2
91 LG:1084051.1:2000MAY19301 363 forward TM N in
i
92 LG:1076853.1:2000MAY19964 1050 forward TM N in
1
92 LG:1076853.1:2000MAY1956 130 forward TM N out
2
92 LG:1076853.1:2000MAY19741 818 forward TM N in
3
93 LG:481631.10:2000MAY19298 357 forward TM N out
1
93 LG:481631.10:2000MAY19598 654 forward TM N out
1
94 LG:1088431.2:2000MAY19379 441 forward TM N out
1
94 LG:1088431.2:2000MAY19354 431 forward TM N out
3
95 LI:401619.10:2000MAY01157 219 forward TM N out
1
95 LI:401619.10:2000MAY01232 294 forward TM N out
1
95 LI:401619.10:2000MAY01502 576 forward TM N out
1
95 LI:401619.10:2000MAY01146 232 forward TM N in
2
95 LI:401619.10:2000MAY01326 412 forward TM N in
2
95 LI:401619.10:2000MAY01440 490 forward TM N in
2
95 LI:401619.10:2000MAY01512 580 forward TM N in
2
95 LI:401619.10:2000MAY01186 257 forward TM N in
3
95 LI:401619.10:2000MAY01528 599 forward TM N in
3
96 LI:1144007.1:2000MAY012833 2910 forward TM N in
1
96 LI:1144007.1:2000MAY013301 3378 forward TM N in
1
96 LI:1144007.1:2000MAY013511 3597 forward TM N in
1
96 LI:1144007.i:2000MAY013634 3696 forward TM N in
1
96 LI:1144007.1:2000MAY013736 3801 forward TM N in
1
96 LI:1144007.1:2000MAY012645 2725 forward TM N out
2
96 LI:1144007.1:2000MAY012879 2965 forward TM N out
2
96 LI:1144007.1:2000MAY013356 3433 forward TM N out
2
96 LI:1144007.1:2000MAY013476 3523 forward TM N out
2
96 LI:1144007.1:2000MAY012772 2858 forward TM N in
3
96 LI:1144007.1:2000MAY013258 3332 forward TM N in
3
96 LI:1144007.1:2000MAY014017 4097 forward TM N in
3
97 LI:331074.1:2000MAY011264 1326 forward TM N in
1
97 LI:331074.1:2000MAY011357 1419 forward TM N in
1
97 LI:331074.1:2000MAY011450 1512 forward TM N in
1
97 LI:331074.1:2000MAY011540 1626 forward TM N in
1
97 L1:331074.1:2000MAY011433 1513 forward TM N in
2
97 LI:331074.1:2000MAY011574 1660 forward TM N in
2
97 LI:331074.1:2000MAY011461 1529 forward TM N in
3
97 LI:331074.1:2000MAY011560 1646 forward TM N in
3
98 LI:1170349.1:2000MAY0134 102 forward TM N in
1
99 LG:335097.1:2000FEB18601 672 forward TM N out
1
99 LG:335097.1:2000FEB18847 909 forward TM N out
1
99 LG:335097.1:2000FEB18928 981 forward TM N out
1
99 LG:335097.1:2000FEB18164 244 forward TM N out
2
99 LG:335097.1:2000FEB18623 682 forward TM N out
2
99 LG:335097.1:2000FEB1812 74 forward TM N in
3
99 LG:335097.1:2000FEBi8219 299 forward TM N in
3
99 LG:335097.1:2000FEB18594 680 forward TM N in
3
100 LG:1076451.1:2000FEB1894 156 forward TM N in
1
100 LG:1076451.1:2000FEB18101 187 forward TM N out
2
i LG:1076451.1:2000FEB1818 98 forward TM N out
00 3
100 LG:1076451.1:2000FEB1896 164 forward TM N out
3
100 LG:1076451.1:2000FEB18216 290 forward TM N out
3
i __ _ _ __ -i99 _ _ _____ _~
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
101 LI:805478.1:2000FEB0183 136 forward TM N out
2
101 LI:805478.1:2000FEB01212 298 forward TM N out
2
102 LG:101269.1:2000MAY19655 741 forward TM N in
1
102 LG:101269.1:2000MAY19650 736 forward TM N in
2
102 LG:101269.1:2000MAY1996 182 forward TM N in
3
102 LG:101269.1:2000MAY19249 335 forward TM N in
3
102 LG:101269.1:2000MAY19663 740 forward TM N in
3
103 LI:331087.1:2000MAY01251 298 forward TM N out
2 1
103 LI:331087.1:2000MAY01237 311 forward TM
3
104 LI:410188.1:2000MAY01520 591 forward TM N in
1
104 LI:410188.1:2000MAY01640 711 forward TM N in
1
104 LI:410188.1:2000MAY01724 810 forward TM N in
1
104 LI:410188.1:2000MAY01832 879 forward TM N in
1
104 LI:410188.1:2000MAY01883 969 forward TM N in
1
104 LI:410188.1:2000MAY011 i 1257 forward TM N in
71 1
104 LI:410188.1:2000MAY011303 1389 forward TM N in
1
104 LI:410188.1:2000MAY012290 2361 forward TM N in
1
104 LI:410188.1:2000MAY012389 2460 forward TM N in
1
104 LI:410188.1:2000MAY012470 2556 forward TM N in
1
104 LI:410188.1:2000MAY012635 2721 forward TM N in
1
104 LI:410188.1:2000MAY012794 2862 forward TM N in
1
104 LI:410188.1:2000MAY012878 2964 forward TM N in
1
104 LI:410188.1:2000MAY013757 3837 forward TM N in
1
104 LI:410188.1:2000MAY013871 3957 forward TM N in
1
104 Lf:410188.1:2000MAY013961 4047 forward TM N in
1
104 LI:410188.1:2000MAY014111 4194 forward TM N in
1
104 LI:410188.1:2000MAY014342 4428 forward TM N in
1 ~
104 LI:410188.1:2000MAY014492 4578 forward TM N in
1
104 LI:410188.1:2000MAY014714 4794 forward TM N in
1
104 LI:410188.1:2000MAY016439 6519 forward TM N in
1
104 LI:410188.1:2000MAY017492 7575 forward TM N in
1
104 LI:410188.1:2000MAY017783 7845 forward TM N in
1
104 LI:410188.1:2000MAY014673 4735 forward TM N in
2
104 LI:410188.1:2000MAY014766 4828 forward TM N in
2
104 LI:410188.1:2000MAY014928 5014 forward TM N in
2
104 LI:410188.1:2000MAY015231 5317 forward TM N in
2
104 LI:410188.1:2000MAY016341 6409 forward TM N in
2
104 LI:410188.1:2000MAY017655 7741 forward TM N in
2
104 LI:410188.1:2000MAY018060 8146 forward TM N in
2
104 LI:410188.1:2000MAY014776 4859 forward TM N in
3
104 LI:410188.1:2000MAY016309 6371 forward TM N in
3
104 LI:410188.1:2000MAY017704 7775 forward TM N in
3
105 LI:1188288.1:2000MAY01457 519 forward TM
1
105 LI:1188288.1:2000MAY01841 915 forward TM
1
105 LI:1188288.1:2000MAY01958 1038 forward TM
1
105 LI:1188288.1:2000MAY011072 1140 forward TM
1
105 LI:1188288.1:2000MAY011477 1539 forward TM
1
105 LI:1188288.1:2000MAY011564 1626 forward TM
1
105 LI:1188288.1:2000MAY011810 1896 forward TM
1
105 LI:1188288.1:2000MAY012134 2220 forward TM
1
105 LI:1188288.1:2000MAY012734 2820 forward TM
1
105 LI:1188288.1:2000MAY011067 1147 forward TM N out
2
105 LI:1188288.1:2000MAY011157 1243 forward TM N out
2
105 LI:1188288.1:2000MAY011313 1399 forward TM N out
2
105 LI:1188288.1:2000MAY011556 1618 forward TM N out
2
105 LI:1188288.1:2000MAY012294 2368 forward TM N out
2
200
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
105 LI:1188288.1:2000MAY01435 521 forward TM N in
3
105 LI:1188288.1:2000MAY01597 683 forward TM N in
3
.
105 LI:1188288.1:2000MAY012301 2354 forward TM N in
3
105 LI:1188288.1:2000MAY012700 2753 forward TM N in
3
106 LI:427997.4;2000MAY01148 222 forward TM N in
1
106 LI;427997.4:2000MAY01745 828 forward TM N in
1
106 LI;427997.4:2000MAY011192 1278 forward TM N in
1
106 LI:427997.4:2000MAY011351 1434 forward TM N in
1
106 LI:427997.4:2000MAY011450 1518 forward TM - N in
1
106 LI:427997.4:2000MAY011759 1845 forward TM N in
1
106 LI:427997.4:2000MAY01134 220 forward TM N in
2
106 LI:427997.4:2000MAY01749 832 forward TM N in
2
106 LI:427997.4:2000MAY011031 1087 forward TM N in
2
106 L1:427997.4:2000MAY011607 1693 forward TM N in
2
106 LI:427997.4:2000MAY011730 1816 forward TM N in
2
106 LI:427997.4:2000MAY012111 2191 forward TM N in
2
106 LI:427997.4:2000MAY01150 236 forward TM N in
3
106 LI:427997.4:2000MAY01681 767 forward TM N in
3
106 LI:427997.4:2000MAY01765 851 forward TM N in
3
106 LI:427997.4:2000MAY011068 1124 forward TM N in
3
106 LI:427997.4:2000MAY011665 1751 forward TM N in
3
106 LI:427997.4:2000MAY011782 1856 forward TM N in
3
107 LG:451682.1:2000FEB1893 155 forward TM
3
109 LG:481436.5:2000FEB18583 669 forward TM N in
1
109 LG:481436.5:2000FEB18769 834 forward TM N in
1
109 LG:481436.5:2000FEB181111 1176 forward TM N in
1
109 LG:481436.5:2000FEB18575 655 forward TM N out
2
109 LG:481436.5:2000FEB18764 826 forward TM N out
2
109 LG:481436.5:2000FEB181091 1153 forward TM N out
2
109 LG:481436.5:2000FEB181187 1249 forward TM N out
~ 2
109 LG:481436.5:2000FEB1884 170 forward TM N in
3
109 LG:481436.5:2000FEB18753 833 forward TM N in
3
109 LG:481436.5:2000FEB181164 1241 forward TM N in
3
110 LI:793701.1:2000FEB01352 405 forward TM N in
1
110 LI:793701.1;2000FEB01389 475 forward TM N in
2
111 LI:373637.1:2000FEB01412 498 forward TM
1
111 LI:373637.1:2000FEB01434 520 forward TM N out
2
111 LI:373637.1:2000FEB01866 919 forward TM N out
2
111 LI:373637.1:2000FEB01423 473 forward TM N in
3
111 LI:373637.1:2000FEB01867 920 forward TM N in
3
112 LG:239368.2:2000MAY19241 327 forward TM N out
1
113 LI;053826.1:2000MAY0131 117 forward TM N out
1
113 LI:053826.1:2000MAY011102 1188 forward TM N out
1
113 LI:053826.1:2000MAY011282 1350 forward TM N out
1
113 LI:053826.1:2000MAY0141 112 forward TM N out
2
113 LI:053826.1:2000MAY01164 238 forward TM N out
2
113 LI:053826.1:2000MAY01461 538 forward TM N out
2
113 LI:053826.1:2000MAY011130 1192 forward TM N out
2
113 LI:053826.1:2000MAY011214 1276 forward TM N out
2
113 LI:053826.1:2000MAY011307 1378 forward TM N out
2
113 LI:053826.1:2000MAY01126 200 forward TM N in
3
113 LI:053826.1:2000MAY01348 416 forward TM N in
3
113 LI:053826.1:2000MAY01624 683 forward TM N in
3
113 LI:053826.1:2000MAY011215 1277 forward TM N in
3
113 LI:053826.1:2000MAY011290 1352 forward TM N in
3
115 LI:1071427.96:2000MAY011072 1140 forward TM
1
_ _ _ _ -_ _- ___._ _ -_
201
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
115 LI:1071427.96:2000MAY011297 1383 forward TM
1
115 LI:1071427.96:2000MAY011459 1536 forward TM
1
1 LI:1071427.96:2000MAY011765 1851 forward TM
i 1
115 LI;107i427.96:2000MAY011909 1971 forward TM
1
115 LI:1071427.96:2000MAY012002 2064 forward TM
1
115 LI:1071427.96:2000MAY011562 1648 forward TM N out
2
115 LI:107i 427.96:2000MAY011706 i 792 forward TM N out
2
115 LI:1071427.96:2000MAY011823 1885 forward TM N out
2
115 LI:1071427.96;2000MAY011913 1975 forward TM N out
2
115 LI:1071427.96:2000MAY012045 2098 forward TM N out
2
115 LI:1071427.96:2000MAY01384 470 forward TM N out
3
115 LI:1071427.96:2000MAY01840 926 forward TM N out
3
115 LI:1071427.96:2000MAY01987 1049 forward TM N out
3
1 LI:1071427.96:2000MAY011092 1154 forward TM N out
i 3
5
115 LI:1071427.96:2000MAY011383 1454 forward TM N out
3
115 LI:1071427.96:2000MAY011599 1655 forward TM N out
3
115 LI:1071427.96:2000MAY011767 1844 forward TM N out
3
115 LI:1071427.96:2000MAY011884 1952 forward TM N out
3
115 L1:1071427.96:2000MAY012013 2099 forward TM N out
3
115 LI;1071427.96:2000MAY012127 2189 forward TM N out
3
116 LI:336338.8:2000MAY01100 186 forward TM N out
1
1 LI:336338.8:2000MAY01427 513 forward TM N out
i 1
6
116 LI:336338.8:2000MAY01110 196 forward TM
2
116 LI:336338.8:2000MAY01281 367 forward TM
2
116 LI:336338.8:2000MAY01422 508 forward TM
2
116 LI:336338.8:2000MAY01354 416 forward TM N out
3
116 LI:336338.8:2000MAY01432 494 forward TM N out
3
117 LG:345527.1:2000FEB1846 120 forward TM N out
1
117 LG:345527.1:2000FEB18917 979 forward TM N out
2
117 LG:345527.1:2000FEB1810i 1072 forward TM N out
0 2
117 LG:345527.1:2000FEB181112 1198 forward TM N out
2
117 LG:345527.1:2000FEB1896 182 forward TM N out
3
117 LG:345527.1:2000FEB18474 536 forward TM N out
3
117 LG:345527.1:2000FEB18552 614 forward TM N out
3
i LG:1089383.1:2000FEB1843 126 forward TM N out
18 1
118 LG:1089383.1:2000FEB1814 100 forward TM
2
118 LG:1089383.1:2000FEB18140 205 forward TM
2
1 LG:1089383.1:2000FEB1812 59 forward TM N out
i8 3
120 LG:1093216.1;2000FEB1831 117 forward TM N out
1
120 LG:1093216.1:2000FEB18151 234 forward TM N out
1
120 LG:1093216.1:2000FE818283 348 forward TM N out
1
120 LG:1093216.1:2000FEB1823 109 forward TM N in
2
120 LG:1093216.1:2000FEB18143 193 forward TM N in
2
120 LG:1093216.1:2000FEB1848 122 forward TM N out
3
120 LG:1093216.1:2000FEB18180 263 forward TM N out
3
122 LI:33567i .2:2000FEB0122 108 forward TM N out
1
122 LI:335671.2:2000FEB011048 1134 forward TM N out
1
122 LI:335671.2:2000FEB01854 916 forward TM N in
2
122 LI:33567i .2:2000FEBOi926 988 forward TM N in
2
122 LI:335671.2:2000FEB01998 1072 forward TM N in
2
122 LI:335671.2:2000FEB01399 461 forward TM N out
3
122 LI:335671.2:2000FEB01480 542 forward TM N out
3
122 LI:33567i .2:2000FEB01576 662 forward TM N out
3
122 LI:335671.2:2000FEB011023 1085 forward TM N out
3
122 LI:335671.2:2000FEB011098 1160 forward TM N out
3
122 LI:335671.2:2000FEBOi1173 i 235 forward TM N out
3
I_ _ _ - 202 _ _._,____
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
123 LI:793758.1:2000FEB0131 117 forward TM N out
1
123 LI:793758.1:2000FEB01151 234 forward TM N out
1
123 Li:793758.1:2000FEB01283 348 forward TM N out
1
123 LI:793758.1:2000FEB0123 109 forward TM N in
2
123 LI:793758.1:2000FEB01143 193 forward TM N in
2
123 LI:793758.1:2000FEB0148 i22 forward TM N out
3
123 LI:793758.1:2000FEB01180 263 forward TM N out
3
124 Li:803718.1:2000FEB0143 126 forward TM N out
1
124 LI:803718.1:2000FE80114 100 forward TM
2
124 LI:803718.1:2000FEB01140 205 forward TM
2
124 LI:803718.1:2000FEB0112 59 forward TM N out
3
125 LI:412179.1:2000FEB01328 414 forward TM
1
125 LI:412179.1:2000FEB01436 504 forward TM
1
125 LI:412179.1:2000FEB0156 115 forward TM N out
2
125 LI:412179.1:2000FEB01413 475 forward TM N out
2
125 LI:412179.1:2000FEB01512 574 forward TM N out
2
125 LI:412179.1:2000FEB0196 176 forward TM N out
3
125 LI:412179.1:2000FEB01384 446 forward TM N out
3
125 LI:412179.1:2000FEB01462 524 forward TM N out
3
126 LI:815679.1:2000FEB0110 84 forward TM N out
1
126 LI:815679.1:2000FEB01313 399 forward TM N out
1
126 LI:815679.1:2000FEB01946 1032 forward TM N out
1
126 LI:815679.1:2000FEB011171 1248 forward TM N out
1
i LI:815679.1:2000FEBOi323 409 forward TM N in
26 2
126 LI:815679.1:2000FEB01500 568 forward TM N in
2
126 LI:815679.1:2000FEB01971 1021 forward TM N in
2
126 LI:815679.1:2000FEB011493 1561 forward TM N in
2
126 LI:815679.1:2000FEB0115 92 forward TM N in
3
126 LI:815679.1:2000FEB01285 356 forward TM N in
3
126 LI:815679.1:2000FEB01690 764 forward TM N in
3
126 LI:815679.1:2000FEB01993 1076 forward TM N in
3
126 LI:815679.1:2000FEB011626 1712 forward TM N in
3
127 LI:481361.3:2000FEB01199 252 forward TM N out
1
128 LG:247388.1:2000MAY19190 240 forward TM N out
1
128 LG:247388.1:2000MAY19233 319 forward TM N out
2
128 LG:247388.1:2000MAY19446 532 forward TM N out
2
130 LI:787618.1:2000MAY0110 84 forward TM N in
1
130 LI:787618.1:2000MAY01313 399 forward TM N in
1
130 LI:787618.1:2000MAY01679 750 forward TM N in
1
130 L1:787618.1:2000MAY011018 1098 forward TM N in
1
130 LI:787618.1:2000MAY011189 1266 forward TM N in
1
130 LI:787618.1:2000MAY01323 409 forward TM N out
2
130 LI:787618.1:2000MAY01500 568 forward TM N out
2
130 LI:787618.1:2000MAY01944 1030 forward TM N out
2
130 LI:787618.1:2000MAY011508 1582 forward TM N out
2
130 LI:787618.1:2000MAY011616 1702 forward TM N out
2
130 LI:787618.1:2000MAY0115 92 forward TM N out
3
130 LI:787618.1:2000MAY01285 356 forward TM N out
3
131 LI:331610.2:2000MAY0191 156 forward TM
1
131 LI:331610.2:2000MAY01277 363 forward TM
1
131 LI:331610.2:2000MAY01682 744 forward TM
1
131 LI:331610.2:2000MAY014126 4212 forward TM
1
131 LI:331610.2:2000MAY014951 5001 forward TM
1
131 LI:331610.2:2000MAY015023 5109 forward TM
1
131 LI:331610.2:2000MAY015128 5190 forward TM
1
131 LI:331610.2:2000MAY015407 5469 forward TM
1
_.. _
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
131 LI:331610.2:2000MAY015485 5547 forward TM
1
131 LI:3316i 0.2:2000MAY015563 5625 forward TM
1
131 LI:331610.2:2000MAY015728 5805 forward TM
1
i LI:331610.2:2000MAY015896 5949 forward TM
31 1
131 LI:33i 610.2:2000MAY016268 6327 forward TM
1
131 LI:331610.2:2000MAY016454 6522 forward TM
1
131 LI:331610.2:2000MAY016559 6645 forward TM
i
131 LI:331610.2:2000MAY017477 7539 forward TM
1
13i LI:331610.2:2000MAY017552 7614 forward TM
1
131 LI:331610.2:2000MAY01671 724 forward TM N out
2
131 LI:331610.2:2000MAY014127 4213 forward TM N out
2
131 LI:331610.2:2000MAY014928 5011 forward TM N out
2
131 LI:331610.2:2000MAY015051 5113 forward TM N out
2
131 LI:331610.2:2000MAY015135 5197 forward TM N out
2
131 LI:331610.2:2000MAY015207 5269 forward TM N out
2
131 LI:331610.2;2000MAY015537 5611 forward TM N out
2
131 LI:331610.2:2000MAY015726 5797 forward TM N out
2
131 LI:331610.2:2000MAY015903 5989 forward TM N out
2
131 LI:331610.2:2000MAY016392 6478 forward TM N out
2
131 LI:331610.2:2000MAY016746 6814 forward TM N out
2
131 LI:33i 610.2:2000MAY017295 7381 forward TM N out
2
131 LI:331610.2:2000MAY017586 7633 forward TM N out
2
131 LI:331610.2:2000MAY012763 2849 forward TM
3
131 LI:331610.2:2000MAY014527 4595 forward TM
3
131 LI:331610.2:2000MAY015079 5165 forward TM
3
131 LI:3316i 0.2:2000MAY015445 5516 forward TM
3
131 LI:331610.2:2000MAY015676 5759 forward TM
3
131 LI:331610.2:2000MAY016255 6341 forward TM
3
131 LI:331610.2:2000MAYOi6378 6464 forward TM
3
13i L1:331610.2:2000MAY016624 6692 forward TM
3
131 LI:331610.2:2000MAY016705 6779 forward TM
3
131 LI:331610.2:2000MAY016810 6884 forward TM
3
131 LI:331610.2:2000MAY017062 7133 forward TM
3
131 LI:331610.2:2000MAY017677 7748 forward TM
3
131 LI:331610.2:2000MAY017833 7919 forward TM
3
132 LG;982697.1:2000FEB18355 441 forward TM N in
1
132 LG:982697.1:2000FEB18946 993 forward TM N in
1
132 LG:982697.1:2000FEB18897 983 forward TM N in
3
132 LG:982697.1:2000FEB181215 1301 forward TM N in
3
133 LG:1080896.1:2000FEB18367 426 forward TM N in
1
133 LG:1080896.1:2000FEB18476 562 forward TM N in
2
133 LG:1080896.1:2000FEB18815 901 forward TM N in
2
133 LG:1080896.1:2000FEB18342 395 forward TM N in
3
134 LI:811341.1:2000FEB01562 615 forward TM N out
1
134 LI:811341.1:2000FEB01691 777 forward TM N out
1
135 LI:903225.1:2000FEB0120 100 forward TM N out
2
135 LI:903225.1:2000FEB0112 83 forward TM N out
3
135 LI:903225.i:2000FEB01768 827 forward TM N out
3
137 LG:979580.1:2000MAY19298 354 forward TM N in
1
137 LG:979580.1:2000MAY19826 909 forward TM N in
1
137 LG:979580.1:2000MAY19934 1020 forward TM N in
i
137 LG:979580.1:2000MAY19233 289 forward TM N out
2
137 LG:979580.1:2000MAY19338 418 forward TM N out
2
137 LG:979580.1:2000MAY19201 272 forward TM N in
3
138 LI:1169865.1:2000MAY01197 283 forward TM N in
2
138 LI:1169865.1:2000MAY01863 949 forward TM N in
2
I _ _ . __204 _
-
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
139 LG:337818.2:2000FE81840 117 forward TM N out
1
139 LG:337818.2:2000FEB18532 618 forward TM N out
1
139 LG:337818.2:2000FEB18907 993 forward TM N out
1
139 LG:337818.2:2000FEB181372 1425 forward TM N out
1
140 LI:337818.1:2000FEB0140 114 forward TM N in
1
140 LI:337818.1:2000FEB0140i 466 forward TM N in
2
140 LI:337818.1:2000FEB01852 905 forward TM N in
3
141 LG:241577.4:2000MAY19496 582 forward TM N in
1
142 LG;344786.4:2000MAY1919 105 forward TM N out
1
142 LG:344786.4:2000MAY1914 88 forward TM N in
2
142 LG:344786.4:2000MAY19173 247 forward TM N in
2
142 LG:344786.4:2000MAY1921 107 forward TM
3
143 LI;414307.1:2000FEB01116 202 forward TM N in
2
144 LI:202943.2:2000FEB01166 237 forward TM N in
1
144 LI:202943.2:2000FEB01263 313 forward TM N out
2
144 LI:202943.2:2000FEB01276 326 forward TM N in
3
146 LI:815961.1:2000FEB01232 291 forward TM N out
1
146 LI:815961.1:2000FEB0181 167 forward TM N out
3
146 LI:815961.1:2000FEB01243 329 forward TM N out
3
146 LI:815961.1;2000FEB01354 422 forward TM N out
3
146 LI:815961.1;2000FEB01573 659 forward TM N out
3
146 LI:815961.1:2000FEB01741 803 forward TM N out
3
147 LG:120744.1:2000MAY19181 249 forward TM N out
1
147 LG:120744.1:2000MAY19188 256 forward TM
2
147 LG;120744.1:2000MAY19275 328 forward TM
2
148 LI:757520.1:2000MAY012140 2220 forward TM N in
1
148 LI:757520.1:2000MAY012293 2379 forward TM N in
1
148 LI:757520.1:2000MAY011988 2059 forward TM N in
2
148 LI;757520.1:2000MAY012285 2359 forward TM N in
2
148 LI;757520.1:2000MAY011677 1763 forward TM
3
148 LI:757520.1:2000MAY011995 2066 forward TM
3
149 LG:160570.1:2000FEB18345 413 forward TM N out
3
149 LG:160570.1:2000FEB18462 518 forward TM N out
3
151 LI:221285.1:2000FEB011375 1452 forward TM N out
1
152 L1:401605.2:2000FEB01235 321 forward TM N in
1
152 LI:401605.2:2000FEB01192 263 forward TM N in
3
152 LI:401605.2:2000FEB01489 563 forward TM N in
3
153 LI:329017.1:2000FEB01179 235 forward TM N in
2
153 LI:329017.1:2000FEB01359 433 forward TM N in
2
7 LI:329017.1:2000FEB01449 526 forward TM N in
53 2
153 LI:329017.1:2000FEB01617 703 forward TM N in
2
153 LI:329017.1:2000FEB01920 973 forward TM N in
2
155 LG:403409.1:2000MAY19136 222 forward TM N out
1
155 LG:403409.1:2000MAY19973 1029 forward TM N out
1
155 LG:403409.1:2000MAY191285 1371 forward TM N out
1
155 LG:403409.1;2000MAY19182 268 forward TM N in
2
156 LG:233933.5:2000MAY19148 234 forward TM N out
1
156 LG:233933.5:2000MAY1939 125 forward TM N out
3
157 LI:290344.1:2000MAY01232 312 forward TM N out
1
157 L1:290344.1:2000MAY011258 1311 forward TM N out
1
157 LI:290344.1:2000MAY013640 3714 forward TM N out
1
157 LI:290344.1:2000MAY014366 4449 forward TM N out
1
157 LI:290344.1:2000MAY014468 4548 forward TM N out
1
157 LI:290344.1:2000MAY01146 226 forward TM N out
2 .
157 LI:290344.1:2000MAY013122 3196 forward TM N out
2
157 LI:290344.1:2000MAY013833 3919 forward TM N out
2
__ _ ______________
205
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
157 LI:290344.1:2000MAY014457 4537 forward TM N out
2
157 LI:290344.1:2000MAY014760 4846 forward TM N out
2
157 LI:290344.1:2000MAY01432 503 forward TM N out
3
157 LI:290344.1:2000MAY011647 1733 forward TM N out
3
157 LI:290344.1:2000MAY013177 3248 forward TM N out
3
157 LI:290344.1:2000MAY013594 3680 forward TM N out
3
157 LI:290344.1:2000MAY013753 3815 forward TM N out
3
157 LI:290344.1:2000MAY013864 3926 forward TM N out
3
157 LI:290344.1:2000MAY014443 4526 forward TM N out
3
158 LI:410742.1:2000MAY01136 210 forward TM N out
1
158 LI:410742.1:2000MAY012200 2286 forward TM N out
1
158 LI:410742.1:2000MAY012437 2514 forward TM N out
1
158 LI:410742.1:2000MAY013149 3229 forward TM N in
2
158 LI:410742.1:2000MAY013437 3505 forward TM N in
2
158 LI:410742.1:2000MAY01510 578 forward TM N in
3
158 LI:410742.1:2000MAY011905 1991 forward TM N in
3
158 LI:410742.1:2000MAY012811 2897 forward TM N in
3
158 LI:410742.1:2000MAY013168 3254 forward TM N in
3
159 LG:406568.1:2000MAY19490 549 forward TM N in
1
159 LG:406568.1:2000MAY191732 1818 forward TM N in
1
159 LG:406568.1:2000MAY191825 1899 forward TM N in
1
159 LG:406568.1:2000MAY191918 2004 forward TM N in
1
159 LG:406568.1:2000MAY1912 59 forward TM N in
3
159 LG:406568.1:2000MAY191935 2018 forward TM N in
3
159 LG:406568.1:2000MAY192094 2174 forward TM N in
3
160 LI:283762.1:2000MAY011675 1746 forward TM
1
160 LI:283762.1:2000MAY012095 2181 forward TM
1
160 Lf:283762.1:2000MAY012632 2718 forward TM
1
160 LI:283762.1:2000MAY012830 2916 forward TM
1
160 LI:283762.1:2000MAY012941 3027 forward TM
1
160 LI:283762.1:2000MAY013235 3321 forward TM
1
160 LI:283762.1:2000MAY013328 3414 forward TM
1
160 LI:283762.1:2000MAY013592 3666 forward TM
1
160 LI:283762.1:2000MAY013682 3768 forward TM
1
160 LI:283762.1:2000MAY014153 4224 forward TM
1
160 LI:283762.1:2000MAY014360 4434 forward TM
1
160 LI:283762.1:2000MAY014594 4656 forward TM
1
160 LI:283762.1:2000MAY014681 4743 forward TM
1
160 LI:283762.1:2000MAY014885 4962 forward TM
1
160 LI:283762.1:2000MAY015011 5061 forward TM
1
160 LI:283762.1:2000MAY0192 178 forward TM N in
2
160 LI:283762.1:2000MAY01278 364 forward TM N in
2
160 LI:283762.1:2000MAY01995 1075 forward TM N in
2
160 LI:283762.1:2000MAY011523 1597 forward TM N in
2
160 LI:283762.1:2000MAY011817 1903 forward TM N in
2
160 LI:283762.1:2000MAY012522 2599 forward TM N in
2
160 LI:283762.1:2000MAY012666 2752 forward TM N in
2
160 LI:283762.1:2000MAY012837 2887 forward TM N in
2
160 LI:283762.1:2000MAY013038 3097 forward TM N in
2
160 LI:283762.1:2000MAY013563 3625 forward TM N in
2
160 LI:283762.1:2000MAY013638 3700 forward TM N in
2
160 LI:283762.1:2000MAY014067 4144 forward TM N in
2
160 LI:283762.1:2000MAY014439 4522 forward TM N in
2
160 LI:283762.1:2000MAY014685 4765 forward TM N in
2
160 LI:283762.1:2000MAY014784 4843 forward TM N in
2
160 LI:283762.1:2000MAY014973 5050 forward TM N in
2
I __ __. _ _ 206 _ _____ _
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
160 LI:283762.1:2000MAY015072 5125 forward TM N in
2
160 LI:283762.1:2000MAY01693 755 forward TM N out
3
160 LI:283762.1:2000MAY01765 827 forward TM N out
3
160 LI:283762.1:2000MAY01840 902 forward TM N out
3
160 LI:283762.1:2000MAY011623 1694 forward TM N out
3
160 LI:283762.1:2000MAY011800 1880 forward TM N out
3
160 LI:283762.1:2000MAY012622 2708 forward TM N out
3
160 LI:283762.1:2000MAYOi2778 2861 forward TM N out
3
160 LI:283762.1:2000MAY013144 3230 forward TM N out
3
160 LI:283762.1:2000MAY013276 3362 forward TM N out
3
160 LI:283762.1:2000MAY013441 3527 forward TM N out
3
160 LI:283762.1:2000MAY013666 3752 forward TM N out
3
160 LI:283762.1:2000MAY014077 4163 forward TM N out
3
160 LI:283762.1:2000MAY014245 4331 forward TM N out
3
160 LI:283762.1;2000MAY014395 4481 forward TM N out
3
160 LI:283762.1:2000MAY014584 4646 forward TM N out
3
160 LI:283762.1:2000MAY014662 4724 forward TM N out
3
160 LI:283762.1:2000MAY014845 4892 forward TM N out
3
161 LI:347687.113:2000MAY01319 405 forward TM N out
1
161 LI:347687.113:2000MAY01463 549 forward TM N out
1
161 LI:347687.113:2000MAY01733 819 forward TM N out
1
161 LI:347687.113:2000MAY011240 1293 forward TM N out
1
161 LI:347687.113:2000MAY011720 1797 forward TM N out
1
161 LI:347687.113:2000MAY011861 1908 forward TM N out
1
161 L1:347687.113:2000MAY011972 2034 forward TM N out
1
161 LI:347687.113:2000MAY012050 2112 forward TM N out
1
161 LI:347687.113:2000MAY012308 2394 forward TM N out
1
161 LI:347687.113:2000MAY01977 1057 forward TM N in
2
161 LI:347687.113:2000MAY011250 1309 forward TM N in
2
161 LI;347687.113:2000MAY011730 1792 forward TM N in
2
161 LI:347687.113:2000MAY011808 1870 forward TM N in
2
161 LI:347687.113:2000MAY011886 1948 forward TM N in
2
161 LI:347687.113:2000MAY01324 398 forward TM N in
3
161 LI:347687.113:2000MAY01948 1034 forward TM N in
3
161 LI:347687.113:2000MAY011686 1763 forward TM N in
3
161 LI:347687.113:2000MAY011791 1874 forward TM N in
3
161 LI:347687.113:2000MAY012025 2108 forward TM N in
3
163 LG:451710.1:2000FEB18502 588 forward TM N in
1
163 LG:451710.1:2000FEB18453 515 forward TM N in
3
164 LG:455771.1:2000FEB18199 285 forward TM N out
1
165 LG:452089.1:2000FEB18695 772 forward TM N out
2
165 LG:452089.1:2000FEB18708 764 forward TM N out
3
166 LG:246415.1:2000FEB18196 246 forward TM N in
1
167 LG:414144.10:2000FEB18589 672 forward TM N in
1
167 LG:414144.10:2000FEB18615 692 forward TM N out
3
168 LG:1101445.1:2000FEB18787 858 forward TM N out
1
168 LG:1101445.1:2000FEB18506 592 forward TM N out
2
169 LG:452134.1:2000FEB18276 326 forward TM N out
3
170 L1:903021.1:2000FEB01109 162 forward TM N out
1
172 LG:449404.1:2000MAY19163 219 forward TM N out
1
172 LG:449404.1:2000MAY19200 280 forward TM N out
2
173 LG:449413.1:2000MAY19353 439 forward TM N out
2
177 LG:1101153.1:2000MAY19520 600 forward TM N in
1
177 LG:1101153.1:2000MAY19585 671 forward TM N in
3
178 LI:257695.20:2000MAY01433 516 forward TM N in
1
179 LI:455771.1:2000MAY01199 285 forward TM N out
1
~____ _ __- _ _ __
207
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
180 LI:274551.1:2000MAY0181 152 forward TM N out
3
180 Li:274551.1:2000MAY01216 269 forward TM N out
3
181 LI:035973.1:2000MAY01622 708 forward TM , N
1 out
181 LI:035973.1:2000MAY01596 682 forward TM N out
2
181 LI:035973.1:2000MAY01588 674 forward TM N out
3
182 LG:978427.5:2000FEB18221 295 forward TM N out
2
182 LG:978427.5:2000FEB18365 433 forward TM N out
2
182 LG:978427.5:2000FEB18198 284 forward TM N out
3
183 LG:247781.2:2000FEB1822 108 forward TM N in
1
183 LG:247781.2:2000FE8181114 1200 forward TM N in
1
183 LG:247781.2:2000FEB181149 1235 forward TM N in
3
185 LI:333307.2:2000FEB0124 98 forward TM N out
3
187 LG:414732.1:2000MAY1940 93 forward TM N out
1
187 LG:414732.1:2000MAY19156 233 forward TM N out
3
188 LG:413910.6:2000MAY19385 441 forward TM N out
1
188 LG:413910.6:2000MAY19886 948 forward TM N out
1
188 LG:413910.6:2000MAY19104 190 forward TM N out
2
188 LG:413910.6:2000MAY19387 461 forward TM N out
3
188 LG:413910.6:2000MAY19921 1007 forward TM N out
3
189 LI:414732.2:2000MAY0134 93 forward TM N out
1
189 LI:414732.2:2000MAY0124 110 forward TM N out
3
189 LI:414732.2:2000MAY01159 236 forward TM N out
3
190 LI:900264.2:2000MAY01730 807 forward TM N in
1
190 LI:900264.2:2000MAY011018 1092 forward TM N in
1
190 LI:900264.2:2000MAY011294 1350 forward TM N in
1
190 LI:900264.2:2000MAY011519 1578 forward TM N in
1
190 LI:900264.2:2000MAY012311 2397 forward TM N in
1
190 ~LI:900264.2:2000MAY012509 2562 forward TM N in
1
190 LI;900264.2:2000MAY012752 2808 forward TM N in
1
190 LI:900264.2:2000MAY013103 3165 forward TM N in
1
190 LI:900264.2:2000MAY013178 3240 forward TM N in
1
190 LI:900264.2:2000MAY013253 3315 forward TM N in
1
190 LI:900264.2:2000MAY013424 3510 forward TM N in
1
190 LI:900264.2:2000MAY013520 3603 forward TM N in
1
190 LI:900264.2:2000MAY013883 3945 forward TM N in
1
190 LI:900264.2:2000MAY013982 4044 forward TM N in
1
190 LI:900264.2:2000MAY0168 154 forward TM
2
190 LI:900264.2:2000MAY01188 274 forward TM
2
190 LI:900264.2:2000MAY011079 1165 forward TM
2
190 LI:900264.2:2000MAY012285 2359 forward TM
2
190 L1:900264.2:2000MAY012732 2812 forward TM
2
190 LI:900264.2:2000MAY013095 3172 forward TM
2
190 LI:900264.2:2000MAY013260 3319 forward TM
2
190 LI:900264.2:2000MAY013434 3505 forward TM
2
190 LI:900264.2:2000MAY013515 3601 forward TM
2
190 LI:900264.2:2000MAY013662 3748 forward TM
2
190 LI:900264.2:2000MAY013842 3913 forward TM
2
190 LI:900264.2:2000MAY013992 4063 forward TM
2
190 LI:900264.2:2000MAY01198 248 forward TM N in
3
190 LI:900264.2:2000MAY011080 1133 forward TM N in
3
190 LI:900264.2:2000MAY011431 1517 forward TM N in
3
190 LI:900264.2:2000MAY011518 1571 forward TM N in
3
190 LI:900264.2:2000MAY011740 1814 forward TM N in
3
190 LI:900264.2:2000MAY012409 2480 forward TM N in
3
190 LI:900264.2:2000MAY012928 2993 forward TM N in
3
190 LI:900264.2:2000MAY013096 3161 forward TM N in
3
_ _ ___ __ _ ________
208
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
190 LI:900264.2;2000MAY013342 3404 forward TM N in
3
190 LI:900264.2:2000MAY013447 3509 forward TM N in
3 '
190 LI:900264.2:2000MAY013531 3614 forward TM N in
3
190 LI;900264.2:2000MAY013987 4064 forward TM N in
3
191 Lf:335593.1:2000MAY01685 771 forward TM N in
1
191 LI:335593.1:2000MAY011273 1335 forward TM N in
1
191 LI:335593.1:2000MAY011366 1428 forward TM N in
1
191 L1:335593.1:2000MAY01710 757 forward TM N in
2
191 LI:335593.1:2000MAY011250 1336 forward TM N in
2
191 LI:335593.1:2000MAY011358 1408 forward TM N in
2
191 LI:335593.1:2000MAY011448 1525 forward TM N in
2
191 L1:335593.1:2000MAY011604 1690 forward TM N in
2
191 LI:335593.1:2000MAY0181 128 forward TM N in
3
191 LI:335593.1:2000MAY01246 296 forward TM N in
3
191 LI:335593.1:2000MAY01807 866 forward TM N in
3
191 LI:335593.1:2000MAY01876 947 forward TM N in
3
191 LI:335593.1;2000MAY011155 1217 forward TM N in
3
191 L1:335593.1:2000MAY011233 1295 forward TM N in
3
191 LI:335593.1:2000MAY011359 1445 forward TM N in
3
192 LI:1189543.1:2000MAY011765 1842 forward TM
1
192 LI:1189543.1:2000MAY011861 1935 forward TM
1
192 LI:1189543.1:2000MAY012236 2307 forward TM
1
192 LI:1189543.1:2000MAY012356 2442 forward TM
1
192 LI:1189543.1:2000MAY012476 2544 forward TM
1
192 LI:1189543.1:2000MAY012659 2712 forward TM
1
192 L1:1189543.1:2000MAY013097 3174 torward TM
1
192 LI:1189543.1;2000MAY013217 3288 forward TM
1
192 LI:1189543.1:2000MAY013439 3492 forward TM
1
192 LI:1189543.1:2000MAY01860 946 forward TM
2
192 LI:1189543.1:2000MAY011016 1099 forward TM
2
192 LI:1189543.1:2000MAY011145 1216 forward TM
2
192 L1:1189543.1:2000MAY011601 1672 forward TM
2
192 LI:1189543.1:2000MAY011691 1768 forward TM
2
192 LI:1189543.1:2000MAY012411 2485 forward TM
2
192 LI:1189543.1:2000MAY012831 2917 forward TM
2
192 LI:1189543.1:2000MAYOi3080 3166 forward TM
2
192 LI:1189543.1:2000MAY013227 3310 forward TM
2
192 LI:1189543.1:2000MAY011155 1229 forward TM N out
3
192 LI:1189543.1:2000MAY011683 1766 forward TM N out
3
192 LI:1189543.1:2000MAY011770 1838 forward TM N out
3
192 LI:1189543.1:2000MAY012019 2069 forward TM N out
3
192 LI:1189543.1:2000MAY012352 2438 forward TM N out
3
192 LI;1189543.1:2000MAY012508 2594 forward TM N out
3
192 L1:1189543.1:2000MAY013030 3101 forward TM N out
3
192 Li;1189543.1:2000MAY013183 3263 forward TM N out
3
192 LI:1189543.1:2000MAY013360 3446 forward TM N out
3
193 LG:455450.1:2000FEB18422 490 forward TM N out
2
194 LG:1040978.1:2000FEB18500 586 forward TM N out
2
194 LG:1040978.1:2000FEB18276 332 forward TM N out
3
196 LG:132147.3:2000FEB18259 345 forward TM N out
1
196 LG:132147.3:2000FEB18418 504 forward TM N out
1
196 LG:132147.3:2000FEB18718 780 forward TM N out
1
196 LG:132147.3:2000FEB181477 1548 forward TM N out
1
196 LG:132147.3:2000FEBi81585 1647 forward TM N out
1
196 LG:132147.3:2000FEB181690 1752 forward TM N out
1
196 LG:132147.3:2000FEB182560 2637 forward TM N out
1
I _ ____ - 209 -_~_,
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
196 LG:132147.3:2000FEB182731 2790 forward TM N out
1
196 LG:132147.3:2000FEB182908 2976 forward TM N out
1
196 LG:132147.3:2000FEB183082 3168 forward TM N out
1
196 LG:132i 47.3:2000FEB183184 3243 forward TM N out
1
196 LG:132147.3:2000FEB183376 3462 forward TM N out
1
196 LG:132147.3;2000FEB181451 1531 forward TM N out
2
196 LG:132147.3:2000FEB181538 i6i5 forward TM N out
2
196 LG:132147.3:2000FEB182741 2827 forward TM N out
2
196 LG:132147.3:2000FEB182960 3031 forward TM N out
2
196 LG:132147.3:2000FEB183050 3112 forward TM N out
2
196 LG:132147.3:2000FEB181626 1703 forward TM N in
3
196 LG:132147.3:2000FEB182508 2594 forward TM N in
3
196 LG:132147.3:2000FEB182919 2987 forward TM N in
3
196 LG:132147.3:2000FEB183177 3263 forward TM N in
3
196 LG:132147.3:2000FEB183372 3422 forward TM N in
3
197 LI:036034.1:2000FEB01157 219 forward TM N out
1
197 LI:036034.1:2000FEB01395 457 forward TM N in
2
197 ' LI:036034.1:2000FEB01479 541 forward TM N in
2
197 LI:036034.1:2000FEB01563 625 forward TM N in
2
197 LI:036034.1:2000FEB01647 709 forward TM N in
2
198 LG:162161.1:2000MAY19372 458 forward TM N in
3
199 LG:407214.10:2000MAY1934 120 forward TM N out
1
199 LG:407214.10:2000MAY1944 124 forward TM N out
2
199 LG:407214.10:2000MAY19203 289 forward TM N out
2
200 LG:204626.1:2000MAY1919 99 forward TM N out
1
202 LI:476342.1:2000MAY0139 122 forward TM N out
3
203 LI;1072759.1:2000MAY01409 495 forward TM N in
1
203 LI:1072759.1:2000MAY01889 951 forward TM N in
1
203 L1:1072759.1:2000MAY017 387 1458 forward TM N in
1
203 LI:1072759.1:2000MAY011687 1770 forward TM N in
1
203 LI:1072759.1:2000MAY01392 478 forward TM N out
. 2
203 LI:1072759.1;2000MAY011055 1132 forward TM N out
2
203 LI:1072759.1:2000MAY011424 i 507 forward TM N out
2
203 LI:1072759.1:2000MAY011694 1768 forward TM N out
2
203 LI:1072759.1:2000MAY011191 1277 forward TM N out
3
203 LI:1072759.1:2000MAY011677 1760 forward TM N out
3
204 LG:998857.1:2000FEB181195 1281 forward TM N in
1
204 LG:998857.1:2000FEB18164 226 forward TM N out
2
204 LG:998857.1:2000FEB18344 400 forward TM N out
2
204 LG:998857.1:2000FEB18398 460 forward TM N out
2
204 LG:998857.1:2000FEB181478 1561 forward TM N out
2
205 LG:482261.1:2000FEB1819 93 forward TM N out
1
205 LG:482261.1:2000FEB18890 961 forward TM N out
2
205 LG:482261.1:2000FEB181070 1123 forward TM N out
2
205 LG:482261.1:2000FEB1821 89 forward TM N out
3
205 LG:482261.1:2000FEB181242 1292 forward TM N out
3
206 LG:480328.1:2000FEB18436 522 forward TM N out
1
206 LG:480328.1:2000FEB18568 642 forward TM N out
1
206 LG:480328.1:2000FEB18769 849 forward TM N out
1
206 LG:480328.1:2000FEB18967 1029 forward TM N out
1
206 LG:480328.1:2000FEBi856 130 forward TM N in
2
206 LG:480328.1:2000FEB18194 280 forward TM N in
2
206 LG:480328,1:2000FEB18396 482 forward TM N out
3
206 LG:480328.1;2000FEB18747 818 forward TM N out
3
207 LG:311197,1:2000MAY19241 315 forward TM N in
1
207 LG:311197.1:2000MAY19527 613 forward TM N out
2
210
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
208 LG:1054883.1:2000MAY19 76 129 forward 1 TM N out
208 LG:1054883.1:2000MAY19 83 145 forward 2 TM N out
209 LG:399395.1:2000MAY19 163 216 forward 1 TM N out
211 LI:272913.22:2000MAY01 37 123 forward 1 TM N in
___-______-_ ____,
211
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212
CA 02401868 2002-08-22
WO 01/62927 PCT/USO1/06059
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_-___213
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 3
~~ TTENANT LES PAGES 1 A 213
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