Note: Descriptions are shown in the official language in which they were submitted.
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HYDROLASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of hydrolases
and to the use
of these sequences in the diagnosis, treatment, and prevention of immune
system disorders, immune
deficiencies, neurological disorders, pulmonary disorders, and cell
proliferative disorders, including
cancer, and in the assessment of the effects of exogenous compounds on the
expression of nucleic
acid and amino acid sequences of hydrolases.
1o BACKGROUND OF THE INVENTION
Hydrolases are a class of enzymes that catalyze the cleavage of various
covalent bonds in a
substrate by the introduction of a molecule of water. The reaction involves a
nucleophilic attack by
the water molecule's oxygen atom on a target 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 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 contarol
cell growth and differentiation, cell-cell contact, the cell cycle, and
oncogenesis.
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 the immune response. Since typical
protein half lives range
from hours to a few days, peptidases are continually cleaving precursor
proteins to their active form,
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, lysosomal cathepsins, calpains, pepsin, renin, and
chymosin (Beynon, R.J, and
J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford TJnivexsity
Press, New York,
~~ pp~ 1-5).
Lysophospholipases (LPLs) regulate intracellular lipids by catalyzing the
hydrolysis of ester
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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
response.
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. Endonuclease V
(deoxyinosine 3'-endonuclease)
is au example of a type II site-specific deoxyribonuclease, a putative DNA
repair enzyme that cleaves
DNAs containing hypoxanthine, uracil, or mismatched bases. Escherichia coli
endonuclease V has
been shown to cleave DNA containing deoxyxanthosine at the second
phosphodiester bond 3' to
deoxyxanthosine, generating a 3'-hydroxyl and a 5'-phosphoryl group at the
nick site (He, B. et al.
(2000) Mutat. Res. 459:109-114). It has been suggested that Escherichia coli
endonuclease V plays a
role in the removal of deamunated guanine, i.e., xanthine, from DNA, thus
helping to protect the cell
against the mutagenic effects of nitrosative deamination (Schouten KA and
Weiss B (1999) Mutat.
Res. 435:245-254). In eukaryotes, the process of tRNA splicing requires the
removal of small tRNA
introns that interrupt the anticodon loop 1 base 3' to the anticodon. This
process requires the stepwise
action of an endonuclease, a ligase, and a phosphotransferase (Hong, L. et al.
(1998) Science 280:279-
284). Ribonuclease P (RNase P) is a ubiquitous RNA processing endonuclease
that is required for
generating the mature tRNA 5'-end during the tRNA splicing process. This is
accomplished through
the catalysis of the cleavage of P-3'O bonds to produce 5'-phosphate and 3'
hydroxyl end groups at
a specific site on pre-tRNA. Catalysis by RNase P is absolutely dependent on
divalent cations such
as Mg2+ or Mn2+(I~urz, J.C. et al. (2000) Curr. Opin. Chem. Biol. 4:553-558).
Substrate recognition
mechanisms of RNase P have been demonstrated to be well conserved among the
Eucarya, the
Archaea, and the Bacteria (Fabbri, S. et al. (1998) Science 280:284-286). In
S. cerevisiae, a gene
designated POP1 for 'processing of precursor RNAs', encodes a protein
component of both RNase P
and RNase MRP, another RNA processing protein. Mutations in yeast POP1 have
been shown to be
lethal (Lygerou, Z. et al. (1994) Genes Dev. 8:1423-1433). Another
phosphodiesterase is acid
sphingomyelinase, which hydrolyzes the membrane phospholipid sphingomye]in 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 acid
sphingomyelinase phosphodiesterase leads to a build-up of sphingomyelin
molecules in lysosomes,
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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
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.
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 glutathione.
Glyoxylases are involved in hyperglycemia, non-insulin-dependent diabetes
mellitus, the detoxification
of bacterial toxins, and in the control of cell proliferation and microtubule
assembly.
A small subclass of hydrolases acting on ether bonds includes the thioether
hydrolases. S-
adenosyl-L-homocysteine hydrolase, also known as AdoHcyase or SAHH (PROSITE
PDOC00603;
EC 3.3.1.1), is a thioether hydrolase first described in rat liver extracts as
the activity responsible for
the reversible hydrolysis of S-adenosyl-L-homocysteine (AdoHcy) to adenosine
and homocysteine
(Sganga, M.W. et al. (1992) PNAS 89:6328-6332). SAHH is a cytosolic enzyme
that has been found
in all cells that have been tested, with the exception of Escherichia coli and
certain related bacteria
(Walker, R.D. et al. (1975) Can. J. Biochem. 53:312-319; Shimizu, S. et al.
(1988) FEMS Microbiol.
Lett. 51:177-180; Shimizu, S. et al. (1984) Eur. J. Biochem. 141:385-392).
SAHH activity is
dependent on NAD+ as a cofactor. Deficiency of SAHH is associated with
hypermethioninemia
(Online Mendelian Inheritance in Man (OM1M) #180960 Hypermethioninemia), a
pathologic condition
characterized by neonatal cholestasis, failure to thrive, mental and motor
retardation, facial
dysmorphism with abnormal hair and teeth, and myocaridopathy (Labrune, P. et
al. (1990) J. Pediat.
117:220-226).
Another subclass of hydrolases includes those enzymes which act on carbon-
nitrogen (C-N)
bonds other than peptide bonds. To this subclass belong those enzymes
hydrolyzing amides, amidines,
and other C-N bonds. This subclass is further subdivided on the basis of the
substrate specificity such
as linear amides, cyclic amides, linear amidines, cyclic amidines, nitrites
and other compounds. A
hydrolase belonging to the sub-subclass of enzymes acting on the cyclic
amidines is adenosine
deaminase (ADA). ADA catalyzes the breakdown of adenosine to inosine. ADA is
present in many
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mammalian tissues, including placenta, muscle, lung, stomach, digestive
diverticulum, spleen,
erythrocytes, thymus, seminal plasma, thyroid, T-cells, bone marrow stem
cells, and liver. A subclass
of ADAs, ADAR, act on RNA and are classified as RNA editases. An ADAR from
Droso~hila,
dADAR, has been shown to be expressed iu the developing nervous system, making
it a candidate for
the editase that acts on para voltage-gated Na+ channel transcripts in the
central nervous system
(Palladino, M.J, et al. (2000) RNA 6:1004-1018). A deficiency of ADA causes
profound lymphopenia
with severe combined immunodeficiency (SCID). Cells from patients with ADA
deficiency contain
less than normal, and sometimes undetectable, amounts of ADA catalytic
activity and ADA protein.
It has been shown that ADA deficiency stems from genetic mutations in the ADA
gene, resulting in
SCID (Hershfield, M.S. (1998) Semin. Hematol. 4:291-298). Metabolic
consequences of ADA
deficiency in mice have been found to be associated with defects in
alveogenesis, pulmonary
inflammation, and airway obstruction (Blackburn, M.R, et al. (2000) J. Exp.
Med. 192:159-170).
Pancreatic ribonucleases (RNase) are pyrimidine-specific endonucleases found
in high
quantity in the pancreas of certain mammalian taxa and of some reptiles
(Beintema, J.J. et al (1988)
Prog. Biophys. Mol. Biol. 51:165-192). Proteins in the mammalian pancreatic
RNase superfamily are
noncytosolic endonucleases that degrade RNA through a two-step
transphosphorolytic-hydrolytic
reaction (Beintema, J.J. et al. (1986) Mol. Biol. Evol. 3:262-275).
Specifically, the enzymes are
involved in endonucleolytic cleavage of 3 =phosphomononucleotides and 3
=phosphooligonucleotides
ending in C-P or U-P with 2',3'-cyclic phosphate intermediates. Ribonucleases
can unwind the DNA
helix by complexing with single-stranded DNA; the complex arises by an
extended multi-site
cation-anion interaction between lysine and arginine residues of the enzyme
and phosphate groups of
the nucleotides. Some of the enzymes belonging to this family appear to play a
purely digestive role,
whereas others exhibit potent and unusual biological activities (D'Alessio, G.
(1993) Trends Cell Biol.
3:106-109). Proteins belonging to the pancreatic RNase family include: bovine
seminal vesicle and
brain ribonucleases; kidney non-secretory ribonucleases (Beintema, J.J. et al
(1986) FEBS Lett.
194:338-343); liver-type ribonucleases (Rosenberg, H.F. et al. (1989) PNAS
U.S.A. 86:4460-4464);
angiogenin, which induces vascularisation of normal and malignant tissues;
eosinophil cationic protein
(Hofsteenge, J. et al. (1989) Biochemistry 28:9806-9813), a cytotoxin and
helininthotoxin with
ribonuclease activity; and frog liver ribonuclease and frog sialic acid-
binding lectin. The sequences of
pancreatic RNases contain 4 conserved disulphide bonds and 3 amino acid
residues involved in the
catalytic activity.
Serine hydrolases are a functional class of hydrolytic enzymes that contain a
serine residue in
their active site. This class of enzymes contains proteinases, esterases, and
lipases which hydrolyze a
variety of substrates and, therefore, have different biological roles.
Proteins in this superfamily can be
4
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further grouped into subfamilies based on substrate specificity or amino acid
similarities (Puente, X.S.
and Lopez-Ont, C. (1995) J. Biol. Chem. 270(21): 12926-12932).
Carboxylesterases are proteins that hydrolyze carboxylic esters and are
classified into three
categories- A, B, and C. Most type-B carboxylesterases are evolutionarily
related and are considered
to comprise a family of proteins. The type-B carboxylesterase family of
proteins includes vertebrate
acetylcholinesterase, mammalian liver microsomal carboxylesterase, mammalian
bile-salt-activated
lipase, and duck fatty acyl-CoA hydrolase. Some members of this protein family
are not catalytically
active but contain a domain related evolutionarily to other type-B
carboxylesterases, such as
thyroglobulin and l~rosphila protein neuractin.
Nucleotidases catalyze the formation of free nucleosides from nucleotides. The
cytosolic
nucleotidase cN-I (5' nucleotidase-I) cloned from pigeon heart catalyzes the
formation of adenosine
from AMP generated during ATP hydrolysis (Sala-Newby, G.B. et al. (1999) J.
Biol. Chem.
274:17789-17793). Increased adenosine concentration is thought to be a signal
of metabolic stress,
and adenosine receptors mediate effects including vasodilation, decreased
stimulatory neuron firing
and ischemic preconditioning in the heart (Schrader, J. (1990) Circulation
81:389-391; Rubino, A. et al.
(1992) Eur. J. Pharmacol. 220:95-98; de Jong, J.W. et al. (2000) Pharmacol.
Ther. 87:141-149).
Deficiency of pyrimidine 5'-nucleotidase can result in hereditary hemolytic
anemia (OMIM Entry
266120).
ADP-ribosylation is a reversible post-translational protein modification in
which an ADP-
ribose moiety is transferred from (3-NAD to a target amino acid such as
arginine or cysteine. ADP-
xibosylarginine hydrolases regenerate arginine by removing ADP-ribose from the
protein, completing
the ADP-ribosylation cycle (Moss, J. et al. (1997) Adv. Exp. Med. Biol. 419:25-
33). ADP-
ribosylation is a well-known reaction among bacterial toxins. Cholera toxin,
for example, disrupts the
adenylyl cyclase system by ADP-ribosylating the a-subunit of the stimulatory G-
protein, causing an
increase in intracellular CAMP (Moss, J. and Vaughan, M. (eds) (1990) ADP-
ribosylating Toxins and
G-Proteins: Insights into Signal Transduction, American Society for
Microbiology, Washington, D.C.).
ADP-ribosylation may also have a regulatory function in eukaryotes, affecting
such processes as
cytoskeletal assembly (Zhou, H. et al. (1996) Arch. Biochem. Biophys. 334:214-
222) and cell
proliferation in cytotoxic T-cells (Wang, J. et al. (1996) J. Tm_m__unol.
156:2819-2827).
The discovery of new hydrolases, and the polynucleotides encoding them,
satisfies a need in
the art by providing new compositions which are useful in the diagnosis,
prevention, and treatment of
i_m_m__une system disorders, immune deficiencies, neurological disorders,
pulmonary disorders, and cell
proliferative disorders, including cancer, and in the assessment of the
effects of exogenous compounds
on the expression of nucleic acid and amino acid sequences of hydrolases.
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SUMMARY OF THE INVENTION
The invention features purified polypeptides, hydrolases, referred to
collectively as "IiYDR"
and individually as "HYDR-1," "HYDR-2," "HYDR-3," and "HYDR-4." In one aspect,
the invention
provides an isolated polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ll~ N0:1-4, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ >D N0:1-4, c) a
biologically active fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-4, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-4. In one alternative, the invention provides an
isolated polypeptide
comprising the amino acid sequence of SEQ ID N0:1-4.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
. the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ m N0:1-4, b) a polypeptide comprising a naturally occurring
amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ )D N0:1-
4, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-4, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ )D NO:1-4. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID N0:1-4. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ )D N0:5-8.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ll~ N0:1-4, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID N0:1-4, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ LD NO:1-4, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ LD N0:1-4. 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.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID N0:1-4, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID N0:1-4, c) a
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biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-4, and d) an i_mmunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID N0:1-4. The method
comprises a) culturing
a cell under conditions suitable for expression of the polypeptide, wherein
said cell is transformed with
a recombinant polynucleotide comprising a promoter sequence operably linked to
a polynucleotide
encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
au amino acid sequence
selected from the group consisting of SEQ m N0:1-4, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical to an amino acid sequence
selected from the
group consisting of SEQ m N0:1-4, c) a biologically active fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ ID N0:1-4, and d) an
immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-4.
The invention further provides an isolated polynucl~otide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
m N0:5-8, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:5-8, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). Iu one alternative,
the polynucleotide
comprises at Least 60 contiguous nucleotides.
Additionally, the invention provides a method fox detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
B7 N0:5-8, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:5-8, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-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 hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, 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 least 60 contiguous nucleotides.
7
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The invention further provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
NO:S-8, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ 1D NO:S-8, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
20 optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-4, b) a polypeptide comprising a
naturally occurring amino
acid sequence at least 90% identical to an amino acid sequence selected from
the group consisting of
SEQ 1D N0:1-4, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ )D N0:1-4, and d) an i_mmunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ )D N0:1-4, and
a pharmaceutically acceptable excipient. Iu one embodiment, the composition
comprises an amino
acid sequence selected from the group consisting of SEQ ID N0:1-4. The
invention additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional HYDR, comprising administering to a patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-4, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m NO:1-4, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ m N0:1-4, and
d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D N0:1-4. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional HYI~R, comprising
administering to a patient in need of such treatment the composition.
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Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ 1D N0:1-4, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID N0:1-4, c) a
biologically active fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-4, and
d) an imrnunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-4. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
HYDR, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ m N0:1-4, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-4, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ m N0:1-4, and
d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m NO:1-4. The method comprises a) combining the polypeptide
with at least one
test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID N0:1-4, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ~ N0:1-4, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ )D NO:1-4,
and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ >D N0:1-4. The method comprises a) combining the polypeptide
with at Least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
of the polypeptide in the presence of the test compound, and c) comparing the
activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence of
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the test compound, wherein a change in the activity of the polypeptide in the
presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ID N0:5-8,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, and b) detecting
altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method 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 selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:5-8, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ 117 N0:5-8,
iii) a polynucleotide
having a sequence complementary to i), iv) a polynucleotide complementary to
the polynucleotide of
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 polynucleotide selected from the group consisting of i) a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ ID N0:5-8,
ii) a polynucleotide
2o comprising a naturally occurring polynucleotide sequence at least 90%
identical to a polynucleotide
sequence selected from the group consisting of SEQ ID NO:S-8, iii) a
polynucleotide complementary
to the polynucleotide of i), iv) a polynucleotide complementary to the
polynucleotide of ii), and v) an
RNA equivalent of i)-iv). 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 in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
3o BRIEF DESCRIPTTON OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenB'ank
homolog for polypeptides of the invention. The probability score for the match
between each
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polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with the invention. Nothing herein is to be construed as an
admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"HYDR" refers to the amino acid sequences of substantially purified HYDR
obtained from
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any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
HYDR. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of HYDR either by
directly interacting with
HYDR or by acting on components of the biological pathway in which HYDR
participates.
An "allelic variant" is an alternative form of the gene encoding HYDR. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
1o many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Bach of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding HYDR include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as HYDR or a
polypeptide with at least one functional characteristic of HYDR. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding IiYDR, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding HYDR.
The encoded protein may also be "altered," and may contain deletions,
insertions, or substitutions of
amino acid residues which produce a silent change and result in a functionally
equivalent HYDR.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of HYDR is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains
having similar hydrophilicity values may include: leucine, isoleucine, and
valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
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"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of HYDR. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of HYDR either by
directly interacting with HYDR or by acting on components of the biological
pathway in which HYDR
participates.
The term "antibody' refers to intact i_mmunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind HYDR polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide 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 (KLI-~. The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
i_m_m__untze a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
1 Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules. The
nucleotide components of an aptamer may have modified sugar groups (e.g., the
2'-OH group of a
3o ribonucleotide may be replaced by 2'-F or 2'-NH2), which may improve a
desired property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e:g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
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The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus based RNA expression system has been used to express specific RNA
aptamers at high levels
in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci.
USA 96:3606-3610).
The term "spiegeliner" refers to an aptamer which includes L-DNA, L-RNA, or
other left
s handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic HYDR, or of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, S'-AGT-3' pairs with its
complement,
3'-TCA-S'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
.30 Compositions comprising polynucleotide sequences encoding HYDR or
fragments of HYDR may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCl), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
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"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to 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 which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
2o Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
CA 02423953 2003-03-26
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alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
iTrununologlcal function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of HYDR or the polynucleotide encoding HYDR
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 from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, 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
nucleotides or amino acid
residues 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 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, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:5-8 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ 1T7 NO:S-8, for example, as distinct from any
other sequence in the genome
from which the fragment was obtained. A fragment of SEQ ID N0:5-8 is useful,
for example, in
hybridization and amplification technologies and in analogous methods that
distinguish SEQ B7 N0:5-8
from related polynucleotide sequences. The precise length of a fragment of SEQ
ID NO:S-8 and the
region of SEQ m NO:S-8 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 SEQ m NO:1-4 is encoded by a fragment of SEQ m N0:5-8. A
fragment of
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SEQ ID N0:1-4 comprises a region of unique amino acid sequence that
specifically identifies SEQ 177
N0:1-4. For example, a fragment of SEQ ll~ N0:1-4 is useful as an immunogenic
peptide for the
development of antibodies that specifically recognize SEQ ID N0:1-4. The
precise length of a
fragment of SEQ ID N0:1-4 and the region of SEQ ID N0:1-4 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 "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "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
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 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 P.M. Sharp (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: 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 sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search 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.govBLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http:/lwww.ncbi.nlm.nih.gov/gorf/612.html. The
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"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
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for- match: 1
Penalty for mismatch: -2
Opefv. Gap: 5 eyed Extehsio~c Gap: 2 penalties
Gap x drop-off. 50
to Expect: l0
Word Size: 11
Filter': oa
Percent identity may be measured over the length of an entire defined
sequence, for 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 sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, 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 shown herein, in the tables, figures, or Sequence Listing,
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 anvno acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a 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
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
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 program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=l, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
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residue weight table. As with polynucleotide aligmnents, the percent identity
is reported by
CLUSTAL V as the "percent similarity' between aligned polypeptide sequence
pairs.
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
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62 .
Ope~i Gap: 11 and Exterisiofa Gap: 1 penalties
Gap x drop-off. SO
1o Expect: l0
Word Size: 3
Filter: o~i
Percent identity may be measured over the length of an entixe defined
polypeptide sequence,
for 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 supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
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varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 ~Cg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
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 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 are well known and can be found in
Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
specifically see volume 2, chapter 9.
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, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 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,
sheared and denatured salmon sperm DNA at about 100-200 ~,g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill 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 encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic~acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"hnmune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
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An "immunogenic fragment" is a polypeptide or oligopeptide fragment of HYDR
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fxagment of
HYDR which is useful in any of the antibody production methods disclosed
herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of HYDR. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or i_m_m__unological properties of HYDR.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. Far instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. 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.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an 1=IYDR may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary
by cell type depending on the enzymatic milieu of HYDR.
"Probe" refers to nucleic acid sequences encoding H~'DR, their complements, 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, ligands, chemiluminescent agents,
and enzymes. "Primers"
are short nucleic acids, usually DNA oligonucleotides, which may be annealed
to a target
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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
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, 25, 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 tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloni_u~: A Laboratory Manual,
2nd ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biology, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from 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
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 program (available to the public from the Whitehead Iustitute/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 their respective sources and modified to meet the user's
speci~tc 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
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oligonucleotides and 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, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
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.
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.
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.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and S' and 3'
untranslated regions
(ITTRs). Regulatory elements interact with host or viral proteins which
control transcription,
2o translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or 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 nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing HYDR,
nucleic acids encoding HYDR, or fragments thexeof may comprise a bodily fluid;
an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA,
in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
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synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if au antibody is specific for epitope "A," the presence of a
polypeptide comprising the
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.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60°Io free,
preferably at least 75°~o free, and most preferably at least
90°~o free from other components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid xesidues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of suxface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
2o well known in the art, and may rely on any known method for the insertion
of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or viral
infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed
cells" includes 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 transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
liix~ited 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,
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,
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fungi, plants and animals. 'The isolated DNA of the present invention can be
introduced into the host
by methods known in the art, fox 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), su ra.
' A "variant" of a particular nucleic acid sequence is defined as a nucleic
acid sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the 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 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, 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 vaxiants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally 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
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
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
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 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human hydrolases (HYDR), the
polynucleotides encoding HYDR, and the use of these compositions for the
diagnosis, treatment, or
prevention of immune system disorders, immune deficiencies, neurological
disorders, pulmonary
CA 02423953 2003-03-26
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disorders, and cell proliferative disorders, including cancer.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Iucyte Polypeptide ll~) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Iucyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Iucyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are hydrolases. For
example, SEQ ID N0:1 is 38%
identical to Arabidopsis thaliana putative adenosine deaminase (GenBank ZD
g7267246) as determined
by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score
is 2.6e-59, which indicates the probability of obtaining the observed
polypeptide sequence alignment by
chance. SEQ ID N0:1 also contains an adenosine/AMP deaminase domain as
determined by
searching for statistically significant matches in the hidden Markov model
(HIVINI)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from
BLllVIPS analysis provides .
further corroborative evidence that SEQ ID N0:1 is an adenosine deaminase. In
an alternative
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example, SEQ ll~ N0:2 is 40% identical to Xylella fastidiosa endonuclease V
(deoxyinosine 3'-
endonuclease) (GenBank ID g9106985) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability scoxe is 2.0e-28, which
indicates the probability of
obtaining the observed polypeptide sequence alignment by chance. Data from
PRODOM analysis
provides further corroborative evidence that SEQ ID N0:2 is an endonuclease.
In an alternative
example, SEQ 1D N0:3 fit 1is 46% identical to human ADP-ribosylar~io nine
hydrolase (GenBank ID
8402478) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) 'The
BLAST probability score is 1.2e-79, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID NO:3 also contains a domain
characteristic for
ADP-ribosylarginine hydrolases as found in the PRODOM database. (See Table 3.)
Similarly, SEQ
ID N0:4 is 85% identical to Columba livia 5' nucleotidase (GenBank 117
84902474). The BLAST
probability score is 3.1e-161. These data provide evidence that SEQ m N0:3 and
SEQ ID N0:4 are
hydrolases. The algorithms and parameters for the analysis of SEQ ID N0:1-4
are described in Table
7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ 117 NO:) and the corresponding
Incyte polynucleotide
consensus sequence number (Incyte Polynucleotide m) for each polynucleotide of
the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments of
the polynucleotide sequences which are useful, for example, in hybridization
or amplification
technologies that identify SEQ ID N0:5-8 or that distinguish between SEQ m
NO:S-8 and related
polynucleotide sequences. Column 5 shows identification numbers corresponding
to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and/or genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
3941090F8 is the
identification number of an Incyte cDNA sequence, and SCORNOT04 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.8., 71742519V1). Alternatively, the identification
numbers in column 5 may
refer to GenBank cDNAs or ESTs (e.8., 82165516) which contributed to the
assembly of the full
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length polynucleotide sequences. In addition, the identification numbers in.
column 5 may identify
sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UI~)
database (i.e., those
sequences including the designation "ENST"). Alternatively, the identification
numbers in column 5
may be derived from the NCBI RefSeq Nucleotide Sequence Records Database
(i.e., those
sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein
Sequence Records
(i.e., those sequences including the designation "NP"). Alternatively, the
identification numbers in
column 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, FL XXXXXXXX NI NZ YYYYY N3 N4
represents a
"stitched" sequence in which XX~,'XXX is the identification number of the
cluster of sequences to
which the algorithm was applied, and Yl'YYY is the number of the prediction
generated by the
algorithm, and N1,2,3...~ if present, represent specific exons that may have
been manually edited during
analysis (See Example V). Alternatively, the identification numbers in column
5 may refer to
assemblages of exons brought together by an "exon-stretching" algorithm. For
example,
FT .XXXXXX~g~4_gBBBBB_1 N is the identification number of a "stretched"
sequence, with
XXXXXX being the Incyte project identification number, gAAAAA, being the
GenBank identification
number of the human genomic sequence to which the "exon-stretching" algorithm
was applied,
gBBBBB being the GenBank identification number or NCBI RefSeq identification
number of the
nearest GenBank protein homolog, and N referring to specific exons (See
Example V). In instances
where a RefSeq sequence was used as a protein homolog for the "exon-
stretching" algorithm, a
RefSeq_identifier (denoted by "NM," "NP," or "NT") may be used in place of the
GenBank identifier
(i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
.
GNN, GFG,Exon prediction from genomic sequences using,
ENST for example,
GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
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INCY Full length transcript and exon prediction from mapping of EST
sequences to the genome. Genomic location and EST composition
data are combined to predict the exons and resulting transcript.
In some cases, Iucyte cDNA coverage redundant with the sequence coverage shown
in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library
is the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences
which were used to assemble and confirm the above polynucleotide sequences.
The tissues and
vectors which were used to construct the cDNA libraries shown in Table 5 are
described in Table 6.
The invention also encompasses HYDR variants. A preferred HYDR variant is one
which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the IiYDR amino acid sequence, and which contains at
least one functional or
structural characteristic of HYDR.
The invention also encompasses polynucleotides which encode HYDR. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID NO:S-8, which encodes HYDR. The
polynucleotide sequences
of SEQ ID N0:5-8, as presented in the Sequence Listing, embrace the equivalent
RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar
backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
HYDR. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at least
about 85%o, or even at least about 95% polynucleotide sequence identity to the
polynucleotide
sequence encoding HYDR. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:5-8
which has at least about 70%, or alternatively at least about 85%, or even at
least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ
ID N0:5-8. Any one of the polynueleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of HYDR.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding HYDR, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
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produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring HYDR, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode HYDR and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring HYDR under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding HYDR or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding HYDR and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater half life,
than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode HYDR
and
HYDR derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell systems
using reagents well known in the art. Moreover, synthetic chemistry may be
used to introduce
mutations into a sequence encoding HYDR or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
NO:S-8 and fragments thereof under 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, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
I~lenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carned out using either the ABI 373
ox 377 DNA
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sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms wluch are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biol~, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnolo~y, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding HYDR may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et
al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and
legations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFTNDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to
3.0 nucleotides in length,
to have a GC content of about 50% or more, and to anneal to the template at
temperatures of about
68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
31
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WO 02/26998 PCT/USO1/30310
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode HYDR may be cloned in recombinant DNA molecules that direct expression
of HYDR, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy
of the genetic code, other DNA sequences which encode substantially the same
or a functionally
equivalent amino acid sequence may be produced and used to express HYDR.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter HYDR-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of H~'DR, such as its biological or enaymatic
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
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
selection/screening. 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,
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.
In another embodiment, sequences encoding HYDR may be synthesized, in whole or
in part,
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WO 02/26998 PCT/USO1/30310
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
HYDR itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis maybe achieved
using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the
amino acid sequence
of HYDR, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, su ra, pp. 28-53.)
In order to express a biologically active HYDR, the nucleotide sequences
encoding HYDR or
derivatives 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 suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untxanslated regions in the vector and in
polynucleotide sequences
encoding HYDR. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
HYDR. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding HYDR and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals may
be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be provided
by the vector. Exogenous translational elements and initiation codons may be
of various origins, both
natural anal synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers
appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
Methods wluch are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding HYDR and appropriate transcriptional and
translational control
elements. These methods include in yitro recombinant DNA techniques, synthetic
techniques, and in
yiyo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning,, A Laboratory
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WO 02/26998 PCT/USO1/30310
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding HYDR. 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
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, su re; Ausubel, su re; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Aced. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; 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. Aced. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, 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 Nicole,
M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Aced. Sci. USA
90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994)
Mol. Tmmunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-
242.) The
invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding HYDR. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding HYDR can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding HYDR into the
vector's multiple cloning
site disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
3o Chem. 264:5503-5509.) When large quantities of HYDR are needed, e.g. for
the production of
antibodies, vectors which direct high level expression of HYDR may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HYDR. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
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CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
promoters, may be used in the yeast Saccharom~ces cerevisiae or Pichia
astoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of HYDR. Transcription of
sequences
encoding HYDR may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters maybe used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.) These
constructs can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw Hill,
New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding HYDR
may be ligated into
an adenovirus transcription/trauslation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses HYDR in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
HYDR in cell lines is preferred. For example, sequences encoding HYDR can be
transformed into
3o 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. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and Apr. cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example, dhfi-
confers resistance to
methotrexate; heo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and l2isD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferiu may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding HYDR is inserted within a marker gene sequence,
transformed cells
containing sequences encoding HYDR can be identified by the absence of marker
gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding
HYDR under the
control of a single promoter. Expression of the marker gene in response to
induction or selection
usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding HYDR
and that express
HYDR may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA ox DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
hnmunological methods for detecting and measuring the expression of HYDR using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on HYDR is
preferred, but a
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CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Tmmunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding HYDR
include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding HYDR, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerise
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding HYDR may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode HYDR may be designed to contain signal sequences
which direct
secretion of HYDR through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" or "pro" form of the
protein may also be used to specify protein targeting, folding, and/or
activity. Different host cells
which have specific cellular machinery and characteristic mechanisms for post-
translational activities
(e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type
Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and
processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding HYDR may be ligated to a heterologous sequence resulting in
translation of a
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CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
fusion protein in any of the aforementioned host systems. For example, a
chimeric HYDR protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of HYDR activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
trausferase (GST), maltose
binding protein (MBP), thioredoxiu (Trx), calmodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
1o proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the HYDR encoding sequence and the heterologous protein
sequence, so that
HYDR may be cleaved away from the heterologous moiety following purification.
Methods fox fusion
protein expression and purification are discussed in Ausubel (1995, supra, ch.
10). A variety of
commercially available kits may also be used to facilitate expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled HYDR may
be achieved in
yitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
pxecursor, for example, 35S-methionine.
HYDR of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to HYDR. At least one and up to a plurality of test
compounds may be screened
for specific binding to HYDR. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
HYDR, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Zinmunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which HYDR
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express HYDR,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing HYDR or cell membrane fractions which contain HYDR are
then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either HYDR or the
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CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example, the
assay may comprise the steps of combining at least one test compound with
HYDR, either in solution
or affixed to a solid support, and detecting the binding of HYDR to the
compound. Alternatively, the
assay may detect or measure binding of a test compound in the presence of a
labeled competitor.
Additionally, the assay may be carried out using cell-free preparations,
chemical libraries, or natural
product mixtures, and the test compounds) may be free in solution or affixed
to a solid support.
HYDR of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of HYDR. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for HYDR
activity, wherein HYDR is combined with at least one test compound, and the
activity of HYDR in the
presence of a test compound is compared with the activity of IIYDR in the
absence of the test
compound. A change in the activity of HYDR in the presence of the test
compound is indicative of a
compound that modulates the activity of HYDR. Alternatively, a test compound
is combined with au
in vitro or cell-free system comprising HYDR under conditions suitable for
HYDR activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of
HYDR may do so indirectly and need not come in direct contact with the test
compound. At least one
and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding HYDR 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 No. 5,175,383 and U.S. Patent No.
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 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) Cliu. Invest. 97:1999-2002; Wagner, K.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 trausferred to
pseudopregnant dams, and
the resulting chimeric progeny are genotyped and bred to produce heterozygous
or homozygous
strains. Trausgenic animals thus generated may be tested with potential
therapeutic or toxic agents.
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Polynucleotides encoding HYDR 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 a1.
S (1998) Science 282:1145-1147).
Polynucleotides encoding HYDR 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 a polynucleotide encoding HYDR 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 obtain information on treatment of a human
disease. Alternatively,
a mammal inbred to ovexexpress HYDR, e.g., by secreting HYDR in its milk, may
also serve as a
convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTICS
1S Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of HYDR and hydrolases. In addition, the expression of HYDR is closely
associated with
kidney, lung tumor, and cardiovascular tissues. Therefore, HYDR appears to
play a role in immune
system disorders, immune deficiencies, neurological disorders, pulmonary
disorders, and cell
proliferative disorders, including cancer. In the treatment of disorders
associated with increased
HYDR expression or activity, it is desirable to decrease the expression or
activity of HYDR. In the
treatment of disorders associated with decreased HYDR expression or activity,
it is desirable to
increase the expression or activity of HYDR.
Therefore, in one embodiment, HYDR or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of HYDR. Examples of such disorders include, but are not limited to,
an immune system
disorder, such as acquired immunodeficiency syndrome (AIDS), Addison's
disease, adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-candidiasis-
ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,
Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic
lymphopenia with
lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis, glomerulonephritis,
Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,
hypereosinophilia, irntable
bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's
syndrome, rheumatoid
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arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus,
systemic sclerosis, thrombocytopenic purpura, ulcerative colitis; uveitis,
Werner syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal, parasitic,
protozoal, and helminthic infections, and trauma; an immune deficiency, such
as acquired
immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common
variable
t_tn_m__unOdefIClenCy (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic
dysplasia, isolated IgA
deficiency, severe combined immunodeficiency disease (SCll~), immunodeficiency
with
thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi
syndrome, chronic
granulomatous diseases, hereditary angioneurotic edema, and immunodeficiency
associated with
Cushing's disease; a neurological 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, amyotropluc lateral sclerosis and
other motor neuron
disorders, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary ataxias, multiple
sclerosis and other demyelinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, 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 syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic nervous
system disorders, cranial nerve disorders, spinal cord diseases, muscular
dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskiuesia,dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia; a pulmonary disorder, such as congenital
lung anomalies,
atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary
hemorrhage, pulmonary
infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary
disease, restrictive
pulnonary disease, chronic obstructive pulmonary disease, emphysema, chronic
bronchitis, bronchial
asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia,
lung abscess,
pulnonary tuberculosis, diffuse interstitial diseases, pneumoconioses,
sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis,
pulnonary eosinophilia
bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage
syndromes,
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Goodpasture's syndromes, idiopathic puhnonary hemosiderosis, pulmonary
involvement in
collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors,
inflammatory and
nonin~lamrnatory pleural effusions, pneumothorax, pleural tumors, drug-induced
lung disease, radiation-
induced lung disease, and complications of lung transplantation; and 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, cancers 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, pxostate,
salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus.
In. another embodiment, a vector capable of expressing HYDR or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of HYDR including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
HYDR in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent
a disorder associated with decreased expression or activity of HYDR including,
but not limited to,
those provided above.
In still another embodiment, au agonist which modulates the activity of HYDR
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of HYDR including, but not limited to, those listed above.
In a further embodiment, an antagonist of HYDR may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of HYDR.
Examples of such
disorders include, but are not limited to, those immune system disorders,
immune deficiencies,
neurological disorders, pulmonary disorders, and cell proliferative disorders,
including cancer,
described above. In one aspect, an antibody which specifically binds HYDR may
be used directly as
an antagonist or indirectly as a targeting or delivery mechanism for bringing
a pharmaceutical agent to
cells or tissues which express HYDR.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding HYDR may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of HYDR including, but not limited to, those
described above.
In. other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
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by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment ox prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of HYDR may be produced using methods which are generally known
in the
art. In particular, purified HYDR may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind HYDR.
.Antibodies to HYDR may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans, and
others may be immunized by injection with HYDR or with any fragment or
oligopeptide thereof which
has immunogenic properties. Depending on the host species, various adjuvants
may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oiI emulsions, I~LH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
HYDR have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of HYDR amino acids may be fused with those of another protein, such as I~LH,
and antibodies to the
chimeric molecule may be produced.
Monoclonal antibodies to HYDR may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the BBB-
hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; I~ozbor,
D. et al. (1985) J.
T_mmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci.
USA 80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
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CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the axt, to produce
HYDR-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for HYDR may also be
generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries maybe
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using eithex
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
HYDR and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering HYDR epitopes is generally used, but a competitive
binding assay may also be
employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for HYDR. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of HYDR-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple HYDR epitopes, represents the average affinity, or avidity, of the
azrtibodies for HYDR. The
Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
HYDR epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka
ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in
which the HYDR-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka
ranging from about 10~ to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of HYDR, preferably in active
form, from the
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CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For°example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of HYDR-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding HYDR, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene
expression can be achieved by designing complementary sequences or antisense
molecules (DNA,
RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of
the gene encoding
HYDR. Such technology is well known in the art, and antisense oligonucleotides
or larger fragments
can be desired from various locations along the coding or control regions of
sequences encoding
HYDR. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press
Inc., Totawa NJ.)
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 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 Clip. T_m_m__unol. 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, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-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. (2998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et aI. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding HYDR may be
used for
somatic or gertnline gene therapy. Gene therapy may be performed to (i)
correct a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCm)-X1 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
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
(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 Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (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 retrovixuses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. 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
T.r~panosoma cruzi). In the
case where a genetic deficiency in HYDR expression or regulation causes
disease, the expression of
HYDR from au appropriate population of trausduced 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
HYDR are treated by constructing mammalian expression vectors encoding HYDR
and introducing
these vectors by mechanical means into HYDR-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 particle delivery, (iii) liposome-mediated trausfection, (iv) receptor-
mediated gene transfer, and
(v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu.
Rev. Biochem.
62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon
(1998) Curr. Opin.
Bioteehnol. 9:445-450).
Expression vectors that may be effective for the expression of HYDR include,
but are not
limited to, the PCDNA 3.1, EPTTAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTI~-HYG (Clontech, Palo Alto CA).
HYDR
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TIC), or (3-actin genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi,
F.M.V. and H.M. Blau
(1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Iuvitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible pxomoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and Blau, H.M. su ra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
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CA 02423953 2003-03-26
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gene encoding FiYDR from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION K1T, available from Invitrogen) allow one with ordinary skill in
the art to deliver
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 A.J. Eb (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.
Iu another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to HYDR expression are treated by constructing a retrovixus vector
consisting of (i) the
polynucleotide encoding HYDR 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. 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. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vectox producing cell line (VPCL) that expresses au 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) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (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 No. 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 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. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus based gene therapy delivery system is used
to deliver
polynucleotides encoding HYDR to cells which have one or more genetic
abnormalities with respect
to the expression of HYDR. 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 encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
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described in U.S. Patent No. 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 N. Somia (1997) Nature 18:389:239-242,
both incorporated by
reference herein.
Iu another alternative, a herpes based, gene therapy delivery system is used
to deliver
polynucleotides encoding HYDR to target cells which have one or more genetic
abnormalities with
respect to the expression of HYDR. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing HYDR to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
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 No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous 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 anal use of recombinant HSV strains deleted
for ICP4, ICP27 and
ICP22. For HSV vectors, see also Goius, 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
transfection of multiple
plasmids containing different segments of the large hexpesvirus 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 polynucleotides encoding HYDR to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). 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 the coding sequence for
HYDR into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
HYDR-coding RNAs and the synthesis of high levels of HYDR in vector transduced
cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
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CA 02423953 2003-03-26
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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 al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of HYDR 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.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and T_m_m__unolo i~c Approaches, Future Publishing, Mt. Kisco NY,
pp. 163-177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding HYDR.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanroing the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding HYDR. Such DNA sequences may be incorporated into a wide
variety of
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vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell
lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in aIt of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thyxnine, and uridine which are not as easily recognized by
endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding HYDR. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased HYDR
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding HYDR may be therapeutically useful, and in the treatment of disorders
associated with
decreased HYDR expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding HYDR may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding HYDR is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
HYDR are assayed
by any method commonly known in the art. Typically, the expression of
a~specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
CA 02423953 2003-03-26
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of the polynucleotide encoding HYDR. The amount of hybridization may be
quantified, thus forming
the basis for a comparison of the expression of the polynucleotide both with
and without exposure to
one or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to
a test compound indicates that the test compound is effective in altering the
expression of the
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carried out, for example, using a Schizosaccharomyces pombe gene
expression system (Atkins,
D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic
Acids Res. 28:E15) or a
human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem.
Biophys. Res. Commun.
268:8-13). A particular embodiment of the present invention involves screening
a combinatorial library
of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide
nucleic acids, and modified
oligonucleotides) for antisense activity against a specific polynucleotide
sequence (Bruice, T.W. et al.
(1997) U.S. Patent'No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No.
6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivexy by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15 :462-466. )
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin on's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of HYDR,
antibodies to HYDR, and mimetics, agonists, antagonists, or inhibitors of
HYDR.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivexy of fast-
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acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivexy via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising HYDR or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, HYDR or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes for
2o administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
HYDR or fragments thereof, antibodies of HYDR, and agonists, antagonists or
inhibitors of HYDR,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSp/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDso
with little or no toxicity.
'The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
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active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1,ug to 100,000 fig, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind HYDR may be used for
the
diagnosis of disorders characterized by expression of HYDR, or in assays to
monitor patients being
treated with HYDR or agonists, antagonists, or inhibitors of HYDR. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
assays for HYDR include methods which utilize the antibody and a label to
detect HYDR in human
body fluids or in extracts of cells or tissues. The antibodies may be used
with or without modification,
and may be labeled by covalent or non-covalent attachment of a reporter
molecule. A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring HYDR, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
HYDR expression. Normal
or standard values for HYDR expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to HYDR under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of HYDR
expressed in
subject, control, and disease samples from biopsied tissues are compared with
the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding HYDR may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of HYDR
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
HYDR, and to monitor regulation of HYDR levels during therapeutic
intervention.
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In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding HYDR or closely related
molecules may be used
to identify nucleic acid sequences which encode HYDR. The specificity of the
probe, whether it is
made from a highly speci~.c region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding HYDR, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the HYDR encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ )D
NO:S-8 or from
genomic sequences including promoters, enhancers, and introits of the HYDR
gene.
Means for producing specific hybridization probes for DNAs encoding HYDR
include the
cloning of polynucleotide sequences encoding HYDR or HYDR derivatives into
vectors for the
production of nnRNA probes. Such vectors are known in the art, are
commercially available, and may
be used to synthesize RNA probes in yitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding HYDR may be used for the diagnosis of
disorders
associated with expression of HYDR. Examples of such disorders include, but
are not limited to, an
immune system disorder, such as acquired immunodeficiency syndrome (A)DS),
Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
palyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinoplulia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and heltninthic infections, and trauma; an
immune deficiency, such as
acquired immunodeficiency syndrome (A)DS), X-linked agarnmaglobinemia of
Breton, common
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variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia),
thymic dysplasia,
isolated IgA deficiency, severe combined immunodeficiency disease (SCID),
immunodeficiency with
thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi
syndrome, chronic
granulomatous diseases, hereditary angioneurotic edema, and immunodeficiency
associated with
Cushing's disease; a neurologjcal 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, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, 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 syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic nervous
system disorders, cranial nerve disorders, spinal cord diseases, muscular
dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopatlues, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia,dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia; a pulmonary disorder, such as congenital
lung anomalies,
atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary
hemorrhage, pulmonary
infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary
disease, restrictive
pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic
bronchitis, bronchial
asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia,
lung abscess,
pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses,
sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis,
pulmonary eosinophilia
o bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage
syndromes,
Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary
involvement in
collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors,
inflammatory and
noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced
lung disease, radiation-
induced lung disease, and complications of lung transplantation; and a cell
proliferative disorder, such
as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective
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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, cancers 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. The polynucleotide sequences encoding
HYDR may be used in
Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies; in
dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing
fluids or tissues from
patients to detect altered HYDR expression. Such qualitative or quantitative
methods are well known
in the art.
In a particular aspect, the nucleotide sequences encoding HYDR may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding HYDR may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is quantified and
compared with a standard
value. If the amount of signal in the patient sample is significantly altered
in comparison to a control
sample then the presence of altered levels of nucleotide sequences encoding
HYDR in the sample
indicates the presence of the associated disorder. Such assays may also be
used to evaluate the
efficacy of a particular therapeutic treatment regiW en in animal studies, in
clinical trials, or to monitor
the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
HYDR, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding HYDR, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic fox a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
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With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding HYDR
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding HYDR, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
HYDR, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding HYDR 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 the polynucleotide sequences encoding HYDR are used to 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-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence 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
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).
Methods which may also be used to quantify the expression of HYDR include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Tm_m__unol. Methods
159:235-244; Duplaa, C.
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et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, HYDR, fragments of HYDR, or antibodies specific for
HYDR may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell 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 No.
5,840,484, expressly incorporated by 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 activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
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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. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, 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 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 expression data. The normalization procedure is useful fox
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 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.
2o In one embodiment, the toxicity of a test compound is assessed by treating
a biological sample
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 the polypeptide sequences
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 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 axe separated by
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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, su ra). The proteins
are visualized in the gel as discrete and uniquely positioned spots, typically
by staining the gel 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
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 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 HYDR
to quantify the
levels of HYDR 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 levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection maybe
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 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 J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures maybe
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
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 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
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residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
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 polypeptides 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 protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
1o 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; Shalon, D. et
al. (1995) PCT application W095/35505; Heller, R.A. et a1. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding HYDR
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during 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.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-IJlrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding HYDR on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
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region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11q22-23, any
sequences 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
sequence of the instant
invention may also be used to detect differences in the chromosomal location
due to translocation,
inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, HYDR, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening maybe free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between HYDR and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysers, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with HYDR, or
fragments thereof,
and washed. Bound HYDR is then detected by methods well known in the art.
Purified HYDR can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding HYDR specifically compete with a test compound
for binding HYDR.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with HYDR.
In additional embodiments, the nucleotide sequences which encode HYDR may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
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description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No. 60/237,093, U.S. Ser. No. 60/238/370, and U.S. Ser.
No. 60/241,284 are
expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
1o Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some
tissues were
homogenized and lysed in guanidinium isothiocyanate, while others 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 from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, 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) or SUPERSCRIPT plasmid system (Life
Technologies), using
the recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra,
units 5.1-6.6.) 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 SEPHA,CRYL 51000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBI~-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
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PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), or
pINCY (Incyte
Genomics), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli
cells including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DHSa, DH10B,
or
ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host calls by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
wexe resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at 4
°C.
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
3 84-well plates, and the concentration of amplified plasmid DNA was
quantified fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. 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 (Applied
Biosystems).
Electrophoretic 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
(reviewed in Ausubel,
1997, su ra, unit 7.7). Some of the cDNA sequences were selected for extension
using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
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vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (I~~IM)-based protein
family
databases such as PFAM. (HMM is a probabilistic approach which analyzes
consensus primary
structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.)
The queries were performed using programs based on BLAST, FASTA, BLIMPS, and
FilVEvIER.
The Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention may
begin at any of the methionine residues of the full length translated
polypeptide. Full length polypeptide
sequences were subsequently analyzed by querying against databases such as the
GenBank protein
databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and
hidden
Markov model (I~VIM)-based protein family databases such as PFAM. 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 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, 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 the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
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NO:S-8. Fragments from about 20 to about 4000 nucleotides which are useful in
hybridization and
amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative hydrolases were initially identified by running the Genscan gene
identification
program against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-
purpose gene identification program which analyzes genomic DNA sequences from
a variety of
organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and
Burge, C. and S. Karlin
(1998) C~rr. Opin. Struct. Biol. 8:346-354). The program concatenates
predicted exons to form an
assembled cDNA sequence extending from a methionine to a stop codon. The
output of Genscan is a
FASTA database of polynucleotide and polypeptide sequences. The maximum range
of sequence for
Genscan to analyze at once was set to 30 kb. To determine which of these
Genscan predicted cDNA
sequences encode hydrolases, the encoded polypeptides were analyzed by
querying against PFAM
models for hydrolases. Potential hydrolases were also identified by homology
to Incyte cDNA
sequences that had been annotated as hydrolases. These selected Genscan-
predicted sequences
were then compared by BLAST analysis to the genpept and gbpri public
databases. Where
necessary, the Genscan-predicted sequences were then edited by comparison to
the top BLAST hit
from genpept to correct errors in the sequence predicted by Genscan, such as
extra or omitted exons.
BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage
of the Genscan-
predicted sequences, thus providing evidence for transcription. When Incyte
cDNA coverage was
available, this information was used to correct or confirm the Genscan
predicted sequence. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences and/or public cDNA sequences using the assembly process
described in
Example III. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan. gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
)I! were mapped to genomic DNA and parsed into clusters containing related
eDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
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be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Seguences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank pxotein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Tnsertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
VT. ~ Chromosomal Mapping of HYDR Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:5-8 were 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 matched
SEQ ID N0:5-8 were 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 were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
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Map locations are represented by 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
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 wexe included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlm.nih.gov/genemap~, can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
VII. 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 particular cell type or tissue have been bound. (See, e.g., Sambrook,
su ra, ch. 7; Ausubel
(1995) su ra, 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 L1FESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
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
length 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 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 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
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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.
Alternatively, polynucleotide sequences encoding HYDR are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; 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. The number of libraries in each category is counted and divided
by the total number of
libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding HYDR. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of HYDR Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate 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 was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than.
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MT Research,
Ine.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH~)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
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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 SI~+
were 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; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each Well was determined by dispensing 100 ~.1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~tl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,u1 to 10 /c1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction
site overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37 °C in 384-
well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stxatagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min; Step
5: steps 2, 3, and 4 repeated 29 tunes; Step 6: 72°C, 5 min; Step 7:
storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide (1:2, 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).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
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designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes .
Hybridization probes derived from SEQ ID N0:5-8 are employed to screen cDNAs,
genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used with larger
nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ,uCi of [y-
32P] adenosine
triphosphate (Amersharn Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextran bead column (Amexsham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or
Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40 °C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
2o X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), su ra).
Suggested substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a
procedure analogous to a dot or slot blot may also be used to arrange and link
elements to the surface
of a substrate using thermal, W, chemical, or mechanical bonding procedures. A
typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain airy appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechuol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
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array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybxidized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~Cl oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/pl RNase inhibitor, 500 ~.M dATP, 500 ~,M dGTP, 500
~,M dTTP, 40 p.M
dCTP, 40 p.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. 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 85° C to the stop the reaction and degrade
the RNA. Samples are purified
20, using two successive CHROMA SPIN 30 geI 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 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and resuspended
in 14 ~Cl 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors 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 ~,g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound iu 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
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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 U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~1 of the array
element DNA, at an average
concentration of 100 ng/~,1, is loaded into the open capillary printing
element by a lugh-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
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
Hybridization reactions contain 9 ~.1 of sample mixture consisting of 0.2 ~,g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
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 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 %
2o SDS), three times for 10 minutes each at 45° C in a second wash
buffer (0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscbpe
equipped with an
Inuova 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
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
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
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,
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although the apparatus is capable of recording the spectra from both
fluorophores simultaueously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture 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 samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are 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, Iuc., Norwood MA) installed in an IBM-
compatible PC
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 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 obtaiu 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).
XI. Complementary Polynucleotides
Sequences complementary to the HYDR-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring HYDR.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of HYDR. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5'sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the HYDR-encoding
transcript.
3o XII. Expression of HYDR
Expression and purification of HYDR is achieved using bacterial or virus based
expression
systems. For expression of HYDR in bacteria, cDNA is subcloned into an
appropriate 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 (tac) hybrid
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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 HYDR upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of HYDR in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Autog-raphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding HYDR by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See 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.)
Tn most expression systems, HYDR is synthesized as a fusion protein with,
e.g., glutathione S-
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 HYDR at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive lustidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, supra,
ch. 10 and 16). Purified HYDR obtained by these methods can be used directly
in the assays shown
in Examples XVI and XVII where applicable.
XIII. Functional Assays '
HYDR function is assessed by expressing the sequences encoding HYDR 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 cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (lnvitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~cg of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
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transfected cells from 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 decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular 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 Cytometry, Oxford, New York NY.
The influence of HYDR on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding HYDR 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,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding HYDR and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XIV. Production of HYDR Specific Antibodies
HYDR substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
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 HYDR amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized 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, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
I~LH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysucciuimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are
immunized with the
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oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisexa are
tested for
antipeptide and anti-HYDR activity by, for example, binding the peptide or
HYDR to a substrate,
blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XV. Purification of Naturally Occurring HYDR Using Specific Antibodies
Naturally occurring or recombinant HYDR is substantially purified by
immunoafbnity
chromatography using antibodies specific for HYDR. An immunoafhnity column is
constructed by
covalently coupling anti-HYDR 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 HYDR are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of HYDR (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/HYDR 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 HYDR is collected.
XVI. Identification of Molecules Which Interact with HYDR
HYDR, or biologically active fragments thereof, are labeled with 1~I 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 mufti-well plate are incubated with the
labeled HYDR, washed,
and any wells with labeled HYDR complex are assayed. Data obtained using
different concentrations
of HYDR are used to calculate values for the number, affinity, and association
of HYDR with the
candidate molecules.
Alternatively, molecules interacting with HYDR 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).
HYDR 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).
3o XVII. Demonstration of HYDR Activity
HYDR activity is demonstrated through a variety of specific enzyme assays some
of which
are outlined below.
Protein phosphatase (PP) activity can be measured by the hydrolysis of P-
nitrophenyl
phosphate (PNPP). HYDR is incubated together with PNPP in HEPES buffer pH 7.5,
in the
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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 (Diamond, R.H. et al. (1994) Mol. Cell. Biol. 14:3752-62).
Alternatively, acid
phosphatase activity of HYDR is demonstrated by incubating HYDR containing
extract with 100 ~.l of
mM PNPP in 0.1 M sodium citrate, pH 4.5, and 50 ~.1 of 40 mM NaCl at 37
°C for 20 miu. The
5 reaction is stopped by the addition of 0.5 ml of 0.4 M glycine/NaOH, pH 10.4
(Saftig, P. et al. (1997)
J. Biol. Chem. 272:18628-18635). The increase in light absorbance at 410 nm
resulting from the
hydrolysis of PNPP is measured using a spectrophotometer. The increase in
Iight absorbance is
proportional to the activity of HYDR in the assay.
In the alternative, HYDR activity is determined by measuring the amount of
phosphate
1o removed from a phosphorylated protein substrate. Reactions are performed
with 2 or 4 nM HYDR in
a final volume of 30 p1 containing 60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA,
0.1 %
2-mercaptoethanol and 10 ~,M substrate, 32P-labeled on serine/threonine or
tyrosine, as appropriate.
Reactions are initiated with substrate and incubated at 30° C for 10-15
min. Reactions are quenched
with 450 p1 of 4% (w/v) activated charcoal in 0.6 M HCl, 90 mM Na4P~0~, and 2
rnM NaH2P0ø, then
centrifuged at 12,000 x g for 5 min. Acid-soluble 32Pi is quantified by liquid
scintillation counting
(Sinclair, C. et al. (1999) J. Biol. Chem. 274:23666-23672).
The adenosine deaminase activity of HYDR is determined by measuring the rate
of
deamination that occurs when adenosine substrate is incubated with HYDR.
Reactions are
performed with a predetermined amount of HYDR in a final volume of 3.0 ml
containing 53.3 mM
2o potassium phosphate and 0.045 mM adenosine. Assay reagents excluding HYDR
are mixed in a
quartz cuvette and equilibrated to 25° C. Reactions are initiated by
the addition of HYDR and are
mixed immediately by inversion. The decrease in light absorbance at 265 nm
resulting from the
hydrolysis of adenosine to inosine is measured usiug a spectrophotometer. The
decrease in the AZ~s "",
is recorded for approximately 5 minutes. The decrease in light absorbance is
proportional to the
activity of HYDR in the assay.
HYDR activity can be measured by determining the amount of free adenosine
produced by
the hydrolysis of AMP, as described by Sala-Newby, et aI. supra. Briefly, HYDR
is incubated with
AMP in a suitable buffer for 10 minutes at 37 °C. Free adenosine is
separated from AMP and
measured by reverse phase HPLC.
Alternatively, HYDR activity is measured by the hydrolysis of ADP-
ribosylarginine
(Konczalik, P. and J. Moss (1999) J. Biol. Chem. 274:16736-16740). 50 ng of
HYDR are incubated
with 100 p.M ADP-ribosyl-[14C]arginine (78,000 cpm) in 50 mM potassium
phosphate, pH 7,5, 5 mM
dithiothreitol, 10 mM MgCl2 in a final volume of 100 ~.1. After 1 h at
37° C, 90 ~,l of the sample is
applied to a column (0.5 x 4 cm) of Affi-Gel 601 (boronate) equilibrated and
eluted with five 1-ml
78
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
portions of 0.1 M glycine, pH 9.0, 0.1 M NaCl, and 10 mM MgClz. Free 14C-Arg
in the total eluate is
measured by liquid scintillation counting.
Various modifications and variations of the described methods and systems of
the invention
will 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 certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the
following claims.
79
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<110> INCYTE GENOMICS, INC.
YUE, HENRY
Baughn, Mariah R.
WARREN, Bridget A.
TRIBOULEY, Catherine M.
TANG, Y. Tom
KHAN, Farrah A.
YAO, Monique G.
LAL, Preeti
THORNTON, Michael
<120> HYDROLASES
<130> PI-0243 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/237,093; 60/238,370; 60/241,284
<151> 2000-09-29; 2000-10-06; 2000-10-17
<160> 8
<170> PERL Program
<210> 1
<211> 355
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3T31660CD.1
<400> 1
Met Ile Glu Ala Glu Glu Gln Gln Pro Cys Lys Thr Asp Phe Tyr
1 5 10 15
Ser Glu Leu Pro Lys Val Glu Leu His Ala His Leu Asn Gly Ser
20 25 30
Ile Ser Ser His Thr Met Lys Lys Leu Ile Ala Gln Lys Pro Asp
35 40 45
Leu Lys Ile His Asp Gln Met Thr Val Ile Asp Lys Gly Lys Lys
50 55 60
Arg Thr Leu Glu Glu Cys Phe Gln Met Phe Gln Thr Ile His Gln
65 TO 75
Leu Thr Ser Ser Pro Glu Asp Ile Leu Met Val Thr Lys Asp Val
80 85 90
Ile Lys Glu Phe Ala Asp Asp Gly Val Lys Tyr Leu Glu Leu Arg
95 100 105
Ser Thr Pro Arg Arg Glu Asn Ala Thr Gly Met Thr Lys Lys Thr
110 115 120
Tyr Val Glu Ser Ile Leu G1u Gly Ile Lys G1n Ser Lys Gln Glu
125 130 135
Asn Leu Asp Ile Asp Val Arg Tyr Leu Ile Ala Val Asp Arg Arg
140 145 150
Gly Gly Pro Leu Val Ala Lys Glu Thr Val Lys Leu Ala Glu Glu
155 160 165
Phe Phe Leu Ser Thr Glu Gly Thr Val Leu Gly Leu Asp Leu Ser
170 175 180
Gly Asp Pro Thr Val Gly Gln Ala Lys Asp Phe Leu GIu Pro Leu
185 190 195
Leu Glu Ala Lys Lys Ala Gly Leu Lys Leu Ala Leu His Leu Ser
1/7
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
200 205 210
Glu Ile Pro Asn Gln Lys Lys Glu Thr Gln Ile Leu Leu Asp Leu
215 220 225
Leu Pro Asp Arg Ile Gly His Gly Thr Phe Leu Asn Ser Gly Glu
230 235 240
Gly G1y Ser Leu Asp Leu Val Asp Phe Val Arg Gln His Arg Ile
245 250 255
Pro Leu Glu Leu Cys Leu Thr Ser Asn Val Lys Ser Gln Thr Val
260 265 270
Pro Ser Tyr Asp Gln His His Phe Gly Phe Trp Tyr Ser Ile Ala
275 280 285
His Pro Ser Val Ile Cys Thr Asp Asp Lys Gly Val Phe Ala Thr
290 295 300
His Leu Ser Gln Glu Tyr Gln Leu Ala Ala Glu Thr Phe Asn Leu
305 310 315
Thr Gln Ser Gln Val Trp Asp Leu Ser Tyr Glu Ser Ile Asn Tyr
320 325 330
Ile Phe Ala Ser Asp Ser Thr Arg Ser Glu Leu Arg Lys Lys Trp
335 340 345
Asn His Leu Lys Pro Arg Val Leu His Ile
350 355
<210> 2
<211> 308
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2656987CD1
<400> 2
Met Ala Leu Glu Ala Ala Gly Gly Pro Pro Glu Glu Thr Leu Ser
1 5 10 15
Leu Trp Lys Arg Glu Gln Ala Arg Leu Lys Ala His Val Val Asp
20 25 30
Arg Asp Thr Glu Ala Trp Gln Arg Asp Pro Ala Phe Ser Gly Leu
35 40 45
Gln Arg Val Gly Gly Val Asp Val Ser Phe Val Lys Gly Asp Ser
50 55 60
Val Arg Ala Cys Ala Ser Leu Val Val Leu Ser Phe Pro Glu Leu
65 70 75
Glu Val Val Tyr Glu Glu Ser Arg Met Val Ser Leu Thr Ala Pro
80 85 90
Tyr Val Ser Gly Phe Leu Ala Phe Arg Glu Val Pro Phe Leu Leu
95 100 105
Glu Leu Val Gln Gln Leu Arg Glu Lys Glu Pro Gly Leu Met Pro
110 115 120
Gln Val Leu Leu Val Asp Gly Asn Gly Val Leu His His Arg Gly
125 130 135
Phe Gly Val Ala Cys His Leu Gly Val Leu Thr Asp Leu Pro Cys
140 145 150
Val Gly Val Ala Lys Lys Leu Leu Gln Val Asp Gly Leu Glu Asn
155 160 165
Asn Ala Leu His Lys Glu Lys Ile Arg Leu Leu Gln Thr Arg Gly
170 175 180
Asp Ser Phe Pro Leu Leu Gly Asp Ser Gly Thr Val Leu Gly Met
185 190 195
Ala Leu Arg Ser His Asp Arg Ser Thr Arg Pro Leu Tyr Ile Ser
200 205 210
Val Gly His Arg Met her Leu Glu Ala Ala Val Arg Leu Thr Cys
2/7
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
215 220 225
Cys Cys Cys Arg Phe Arg Ile Pro Glu Pro Va1 Arg G1n Ala Asp
230 235 240
Ile Cys Ser Arg Glu His Ile Arg Lys Ser Leu Gly Leu Pro Gly
245 250 255
Pro Pro Thr Pro Arg Ser Pro Lys Ala Gln Arg Pro Val Ala Cys
260 265 270
Pro Lys Gly Asp Ser Gly Glu Ser Ser Gly Gly Ala Pro Ser Pro
275 280 285
Gln Arg Gln Ala Asp Arg Thr Thr Pro Gly Gly Arg Arg Ser Thr
290 295 300
Ala Gln His Gln Val Gly Gln Arg
305
<210> 3
<211> 354
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4009684CD1
<400> 3
Met Glu Lys Phe Lys Ala Ala Met Leu Leu Gly Ser Val Gly Asp
1 5 10 15
Ala Leu Gly Tyr Arg Asn Val Cys Lys Glu Asn Ser Thr Val Gly
20 25 30
Met Lys Ile Gln Glu Glu Leu Gln Arg Ser Gly Gly Leu Asp His
35 40 45
Leu Val Leu Ser Pro Gly Glu Trp Pro Val Ser Asp Asn Thr Ile
50 55 60
Met His Ile Ala Thr Ala Glu Ala Leu Thr Thr Asp Tyr Trp Cys
65 TO 75
Leu Asp Asp Leu Tyr Arg Glu Met Val Arg Cys Tyr Val Glu Ile
80 85 90
Val Glu Lys Leu Pro Glu Arg Arg Pro Asp Pro Ala Thr Ile Glu
95 100 105
Gly Cys Ala Gln Leu Lys Pro Asn Asn Tyr Leu Leu Ala Trp His
110 115 120
Thr Pro Phe Asn Glu Lys Gly Ser Gly Phe Gly Ala Ala Thr Lys
125 130 135
Ala Met Cys Ile Gly Leu Arg Tyr Trp Lys Pro Glu Arg Leu Glu
140 145 150
Thr Leu Ile Glu Val Ser Val Glu Cys Gly Arg Met'Thr His Asn
155 160 165
His Pro Thr Gly Phe Leu Gly Ser Leu Cys Thr Ala Leu Phe Val
170 175 180
Ser Phe Ala Ala Gln Gly Lys Pro Leu Val Gln Trp Gly Arg Asp
185 190 195
Met Leu Arg Ala Val Pro Leu AIa Glu Glu Tyr Cys Arg Lys Thr
200 205 210
Ile Arg His Thr Ala Glu Tyr Gln Glu His Trp Phe Tyr Phe Glu
215 220 225
Ala Lys Trp Gln Phe Tyr Leu Glu Glu Arg Lys Ile Ser Lys Asp
230 235 240
Ser Glu Asn Lys Ala Ile Phe Pro Asp Asn Tyr Asp Ala Glu Glu
245 250 255
Arg Glu Lys Thr Tyr Arg Lys Trp Ser Ser Glu Gly Arg Gly Gly
260 265 270
Arg Arg Gly His Asp Ala Pro Met Ile Ala Tyr Asp Ala Leu Leu
3/7
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
275 280 285
Ala Ala Gly Asn Ser Trp Thr Glu Leu Cys His Arg Ala Met Phe
290 295 300
His Gly Gly Glu Ser Ala Ala Thr Gly Thr I1e Ala Gly Cys Leu
305 310 315
Phe Gly Leu Leu Tyr Gly Leu Asp Leu Val Pro Lys Gly Leu Tyr
320 325 330
Gln Asp Leu Glu Asp Lys Glu Lys Leu Glu Asp Leu Gly Ala Ala
335 340 345
Leu Tyr Arg Leu Ser Thr Glu Glu Lys
350
<210> 4
<211> 366
<212> PRT
<213> Homo Sapiens
<220>
<221> mi.sc_feature
<223> Incyte ID No: 7473183CD1
<400> 4
Met Glu Pro Gly Gln Pro Arg Glu Pro Gln Glu Pro Arg Glu Pro
1 5 10 15
Gly Pro Gly Ala Glu Thr Ala Ala Ala Pro Val Trp Glu Glu Ala
20 25 30
Lys Ile Phe Tyr Asp Asn Leu Ala Pro Lys Lys Lys Pro Lys Ser
35 40 45
Pro Gln Asn Ala Val~Thr Ile Ala Val Ser Ser Arg Ala Leu Phe
50 55 60
Arg Met Asp Glu Glu Gln Gln Ile Tyr Thr Glu Gln Gly Val Glu
65 70 75
Glu Tyr Val Arg Tyr Gln Leu Glu His Glu Asn Glu Pro Phe Ser
80 85 90
Pro Gly Pro Ala Phe Pro Phe Val Lys Ala Leu Glu Ala Val Asn
95 100 105
Arg Arg Leu Arg Glu Leu Tyr Pro Asp Ser Glu Asp Val Phe Asp
110 115 120
Ile Val Leu Met Thr Asn Asn His Ala Gln Val Gly Val Arg Leu
125 130 135
Ile Asn Ser Ile Asn His Tyr Asp Leu Phe Ile Glu Arg Phe Cys
140 145 150
Met Thr Gly Gly Asn Ser Pro Ile Cys Tyr Leu Lys Ala Tyr His
155 160 165
Thr Asn Leu Tyr Leu Ser Ala Asp Ala Glu Lys Val Arg Glu Ala
170 175 180
Ile Asp Glu Gly Ile Ala Ala Ala Thr Ile Phe Ser Pro Ser Arg
185 190 195
Asp Val Val Val Ser Gln Ser Gln Leu Arg Val Ala Phe Asp Gly
200 205 210
Asp Ala Val Leu Phe Ser Asp Glu Ser Glu Arg Ile Val Lys Ala
215 220 225
His Gly Leu Asp Arg Phe Phe Glu His Glu Lys Ala His Glu Asn
230 235 240
Lys Pro Leu Ala Gln Gly Pro Leu Lys Gly Phe Leu Glu Ala Leu
245 250 255
Gly Arg Leu G1n Lys Lys Phe Tyr Ser Lys Gly Leu Arg Leu Glu
260 265 270
Cys Pro Ile Arg Thr Tyr Leu Val Thr Ala Arg Ser Ala Ala Ser
275 280 285
Ser Gly Ala Arg Ala Leu Lys Thr Leu Arg Ser Trp Gly Leu Glu
4/7
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
290 295 300
Thr Asp Glu Ala Leu Phe Leu Ala Gly Ala Pro Lys Gly Pro Leu
305 310 315
Leu G1u Lys Ile Arg Pro His Ile Phe Phe Asp Asp Gln Met Phe
320 325 330
His Val Ala Gly Ala Gln Glu Met Gly Thr Val Ala Ala His Val
335 340 345
Pro Tyr Gly Val Ala Gln Thr Pro Arg Arg Thr Ala Pro Ala Lys
350 355 360
Gln Ala Pro Ser Ala Gln
365
<210> 5
<211> 2269
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3731660CB1
<400> 5
gtggagaggg agaggttgaa ggaacagaaa aatgagaaga caactcactg gaggctgaag 60
tgggaggatc gcttgagtct ggaagcttga gactgcagca agctgtgatc gtgacactgc 120
actccagcct gggcaacaaa gtaagatcct gtctcaaaag aaaaaaaaaa aaaggtgaag 180
ctgaacccag atctacacat acagctaatc ttaccaaaat gtgtggaagt aaagcaatct 240
gaaggaaatt cagtaccata caatttactg actgaaatac atcatattgc tcatactaag 300
aataagagtg gagaagaatc atttttttcc tgctaaaatg atagaggcag aagagcaaca 360
gccttgcaag acagacttct attctgaatt gccaaaagtg gaacttcatg cccacttgaa 420
tggatccatt agttctcata ccatgaagaa attaatagcc cagaagccag atcttaaaat 480
ccacgatcag atgactgtga ttgacaaggg aaagaaaaga actttggaag aatgtttcca 540
gatgtttcaa actattcatc agcttactag tagccctgaa gatattctaa tggtcacaaa 600
agatgtcata aaagaatttg cagatgacgg cgtcaagtac ctggaactaa ggagcacacc 660
cagaagagaa aatgctactg gaatgactaa aaagacttat gtggaatcta tacttgaagg 720
tataaaacag tccaaacaag aaaacttgga cattgatgtt aggtatttga tagcagttga 780
cagaagaggt ggccctttag tagccaagga gactgtaaaa cttgccgagg agttcttcct 840
ttctactgag ggtacagttc ttggccttga cctcagtgga gaccctactg taggacaagc 900
aaaagacttc ttggaacctc ttttagaagc taagaaagca ggtctgaagt tagcattgca 960
tctttcagag attccaaacc aaaaaaaaga aacacaaata ctcctggatc tgcttcctga 1020
cagaatcggg catggaacat ttctcaactc cggtgaggga ggatccctgg atctggtgga 1080
ctttgtgagg caacatcgga taccactgga actctgtttg acctcaaacg tcaaaagtca 1140
gacagttcca tcttatgacc agcaccattt cggattctgg tacagcattg cccatccttc 1200
tgtgatctgt actgatgata agggtgtttt tgcaacacac ctttctcaag agtaccagct 1260
ggcagctgaa acatttaatt tgacccagtc tcaggtgtgg gatctgtctt atgaatccat 1320
caactacatc tttgcttctg acagcaccag atctgaactg aggaagaaat ggaatcacct 1380
gaagcccaga gtgttacata tttaagctat aatgaggtga actacttctg agtatgtgtt 1440
tcaatcaagt tcctgccata tcccacttag taaaacagtc caccactcct ttgaagcata 1500
gcaaccaagt tccttgggct ctatcaccag caccttacac atggcaggta ctcagtaaat 1560
acgtgtcttc aactgactca caagctctca ggtgcttact gggtgggact tgactgttgt 1620
tgctaattaa atccccattc caccagtgaa atctcctgtt gttctcatca cacagcacaa 1680
tgataacaat ggtaacaaca gctgactgag tgccttttat tggccatgca ctgtgctgag 1740
ttatttagac aaatcatttc acttcatctt catgacagcc cttaaaaggg aacctattta 1800
ggtcctaagg tcccaggaaa aatattacag atgtggaaac caaggctgag agcgggagtg 1860
acttgccagt aagtggctga tcccagacct catctcagac tttcctgact ctgaagccca 1920
cactcacggc accatgtcac actgaaactt tgccaggtag gagtcagacc acctgacttc 1980
tgggccttac ctagcctgcc agtgacatct atgtgtaact ttgggctcat caagcattct 2040
cttttgtgga cgaatgaaaa tgcctaccaa gcctacttcc tggggttagc ttgagcgagc 2100
acatgaactg ggaaaatgtg taacaccacc aaacacaaga gggcgaccag acacactagt 2160
gagcccgatc aacccgggga cataatcccg gaccggtact tgacagcggg ctgaggactc 2220
gcattcagct aatcgaaacg tcacccaaag agccgaaccc attgccaag 2269
5/7
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
<210> 6
<211> 1150
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2656987CB1
<400> 6
gcatggccct ggaggcggcg ggagggccgc cggaggaaac gctgtcactg tggaaacggg 60
agcaagctcg gctgaaggcc cacgtcgtag accgggacac cgaggcgtgg cagcgagacc 120
ccgccttctc gggtctgcag agggtcgggg gcgttgacgt gtccttcgtg aaaggggaca 180
gtgtccgcgc ttgtgcttcc ctggtggtgc tcagcttccc tgagctcgag gtggtgtatg 240
aggagagccg catggtcagc ctcacagccc cctacgtgtc gggcttcctg gccttccgag 300
aggtgccctt cttgctggag ctggtgcagc agctgcggga gaaggagccg ggcctcatgc 360
cccaggtcct tcttgtggat ggaaacgggg tactccacca ccgaggcttt ggggtggcct 420
gccaccttgg cgtccttaca gacctgccgt gtgttggggt ggccaagaaa cttctgcagg 480
tggatgggct ggagaacaac gccctgcaca aggagaagat ccgactcctg cagactcgag 540
gagactcatt ccctctgctg ggagactctg ggactgtcct gggaatggcc ctgaggagcc 600
acgaccgcag caccaggccc ctctacatct ccgtgggcca caggatgagc ctggaggccg 660
ctgtgcgcct gacttgctgc tgctgcaggt tccggatccc agagcccgtg cgccaggctg 720
acatctgctc ccgagagcac atccgcaagt cgctgggact ccccgggcca cccacaccga 780
ggagcccgaa ggcgcagagg ccagtggcat gccccaaagg agactccgga gagtcctcag 840
gtggagcacc cagtccccaa agacaggctg accgcaccac cccaggggga cgccgcagca 90-0
cagcccagca ccaggtgggg cagaggtgac cacggcccct ctttgctccg tcatcggctg 960
gtcagctgtg gtcacggtgc ctcagaggac agatctctat gggggcaagt gccagatcct 1020
gagagcgcat gagacgcttt cccggagccg acgaagggga ctcggagctg cagcctgcac 1080
gacccctgca gcctgtgctt tgcccacccc tttcaataga tggaacttgc ttgctcttta 1140
aaaaaaaaaa 1150
<210> 7
<211> 2000
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4009684CB1
<400> 7
ctccgcagct gttggggaag aggagctgcc tcctgggatg gagaaattta aggctgcgat 60
gttgctgggg agcgtcggcg atgctcttgg ctacagaaat gtctgcaagg agaacagcac 120
tgtaggcatg aagatccagg aggagctgca acgttccggg ggcctggacc acctcgtact 180
ctcgccagga gaatggcccg tgagtgacaa caccatcatg cacatcgcaa ccgccgaggc 240
cctcaccaca gactactggt gcctggatga tctgtaccgg gagatggtga gatgctatgt 300
ggaaatcgtt gagaagcttc cagaacgccg gccagaccca gctaccattg aaggctgtgc 360
tcagctaaag cccaataact accttctcgc ctggcacaca ccgttcaatg aaaaaggctc 420
agggtttgga gcggccacca aggccatgtg catcggcctg cggtactgga agcctgagcg 480
gctggagacc ctcatcgagg tcagcgtgga gtgcggccgg atgacccaca accatcccac 540
aggcttcctg ggctccctgt gcacggccct gtttgtgtcg ttcgccgcac aaggaaagcc 600
cctggtccag tgggggagag acatgctgcg ggcggtgcct ctggcagaag agtactgcag 660
gaagaccatc cggcacacgg cagaatacca ggagcactgg ttttactttg aagctaaatg T20
gcaattttat ttggaggaga ggaaaatcag taaagactca gaaaataaag ccatcttccc 780
cgacaattat gatgcagaag agagggaaaa gacctacagg aagtggagct cggaaggtcg 840
agggggaaga cgaggccacg atgcccccat gatagcctat gacgccctcc ttgcagcagg 900
aaacagctgg actgagctgt gtcaccgggc catgtttcat ggaggggaga gcgcggccac 960
gggcaccatt gcaggctgcc tgttcgggtt gctgtacggc ctggacctcg ttcccaaagg 1020
cttgtaccag gacctggagg acaaggagaa gctggaggac ctgggcgcgg ctctctaccg 1080
cctgtccaca gaggagaagt aaagccattt ctgccacttt ccccctagag agccgattcc 1140
accccggggc ccgtagggcc ctctcgcagc ccctgggtga gggtgtctct gtgaggctcc 1200
6/7
CA 02423953 2003-03-26
WO 02/26998 PCT/USO1/30310
actgcggtct gtgcctgact ggccacatct aactctctgt ttccaatttc agaatcctaa 1260
ctgttgcata aaatacattg tttgtcctgc gagaatattt tccgtcctcc accatcaaca 1320
ttgacactgc gtagatttgc cgcacttgga cctccatgcg tggcactcac ccgcagtctc 1380
ctggacaggc gctgtatttt attctgtcgc agagctaatg ctgtttactc actcacttca 1440
acaacactaa ctgcggtggt ggcctccagc aggccccccc gctgcagacc ctctgtcctg 1500
cctctgcctc caggcatgcg tttccccgtg agggccaatg cacctccccc caccccccac 1560
cctcccatgt ccacagtggg tcgtgtgttc ctggacagag aaacagtcca cactggggcc 1620
tgcgggacac atatagcagc atattttgct cttaacccca cccacctttt taatcacact 1680
agattttaag atcaatccct ttttgaaaca actcacggag aaaaccagaa cataaatggc 1740
ctcctgccag ctccggcgtc tctctgtggt ctgccttagt gggccaagtc caaatgcaga 1800
gaaggccttt cccttccgcg cctgccccat cgggctcgct gacgaggaag cgctgtccct 1860
gtgatgaggt tctctctcag agagtcttgg aaaagagacc acttgctctt gtttaaaata 1920
aatttggacg tgatttttcc atgcagcatc tggtgtgaat aaaacagcag ttgactgatg 1980
tttaaaaaaa aaaaaaaaaa 2000
<210> 8
<211> 1101
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473183CB1
<400> 8
atggaacctg ggcagccccg ggagccccag gagccccgcg agcccgggcc aggagcggag 60
accgctgcgg ccccggtctg ggaggaagcc aagattttct acgacaacct cgcgcccaag 120
aagaaaccca aatcgcctca gaatgcagtc accatcgctg tgtcctcccg agccttgttt 180
cgcatggacg aggagcagca gatctacacg gagcagggcg tggaggagta cgtgcgctac 240
cagctggaac atgagaacga acccttcagt eccgggccag ccttcccttt tgtgaaggct 300
ctggaggccg tgaacaggcg gctgcgggag ctgtaccctg atagtgagga cgtcttcgac 360
atcgtcctca tgactaacaa ccatgctcaa gtgggtgtcc gcctcatcaa cagtatcaac 420
cactatgacc tgttcatcga gaggttctgc atgacaggtg ggaacagccc gatctgctac 480
ctcaaggcct atcacaccaa cctctacttg tcagccgatg cggaaaaagt gcgagaagcc 540
attgatgagg ggatcgcagc tgccaccatc ttcagcccca gcagggatgt ggttgtgtcc 600
cagagtcagc tgcgcgtggc cttcgatggg gacgccgtgc tcttctcgga cgagtcggag 660
cgcatcgtca aggcccacgg gctggaccga ttcttcgagc atgagaaggc ccacgagaac 720
aaacctctgg ctcagggccc cttaaagggc tttctggagg cactgggtag gttgcagaag 780
aagttctact ccaaaggcct gcggctggag tgcccaattc gtacctactt ggtgacagca 840
cgcagtgcag ccagttccgg ggcccgggct ctcaagaccc tgcgcagctg gggcctggag 900
acagatgaag ccttgttcct tgctggagcg cccaagggcc ctctccttga gaagatccgc 960
ccacacatct tctttgatga ccagatgttc catgtggctg gggctcagga gatgggcact 1020
gtggccgccc atgtgcctta tggtgtggca cagacacccc ggcggactgc acctgcaaag 1080
caggccccat ctgcacagta g 1101
7/7
cttgtaccag gacctggagg acaaggagaa gctggaggac
