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CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
NUCLEIC ACID-ASSOCIATED PROTEINS
TECHNICAL FIELD
The invention relates to novel nucleic acids, nucleic acid-associated proteins
encoded by these
nucleic acids, and to the use of these nucleic acids and proteins in the
diagnosis, treatment, and
prevention of cell proliferative, neurological, developmental, and
autoimmune/inflammatory disorders,
and infections. The invention also relates to the assessment of the effects of
exogenous compounds
on the expression of nucleic acids and nucleic acid-associated proteins.
1o BACKGROUND OF THE INVENTION
Multicellular organisms are comprised of diverse cell types that differ
dramatically both in
structure and function. The identity of a cell is determined by its
characteristic pattern of gene
expression, and different cell types express overlapping but distinctive sets
of genes throughout
development. Spatial and temporal regulation of gene expression is critical
for the control of cell
proliferation, cell differentiation, apoptosis, and other processes that
contribute to organismal
development. Furthermore, gene expression is regulated in response to
extracellular signals that
mediate cell-cell communication and coordinate the activities of different
cell types. Appropriate gene
regulation also ensures that cells function efficiently by expressing only
those genes whose functions
are required at a given time.
2o Transcriution Factors
Transcriptional regulatory proteins are essential for the control of gene
expression. Some of
these proteins function as transcription factors that initiate, activate,
repress, or terminate gene
transcription. Transcription factors generally bind to the promoter, enhancer,
and upstream regulatory
regions of a gene in a sequence-specific manner, although some factors bind
regulatory elements
within or downstream of a gene coding region. Transcription factors may bind
to a specific region of
DNA singly or as a complex with other accessory factors. (Reviewed in Lewin,
B. (1990) Genes IV,
Oxford University Press, New York, NY, and Cell Press, Cambridge, MA, pp. 554-
570.)
The double hefix structure and repeated sequences of DNA create topological
and chemical
features which can be recognized by transcription factors. These features are
hydrogen bond donor
and acceptor groups, hydrophobic patches, major and minor grooves, and
regular, repeated stretches
of sequence which induce distinct bends in the helix. Typically, transcription
factors recognize specific
DNA sequence motifs of about 20 nucleotides in length. Multiple, adjacent
transcription factor-binding
motifs may be required for gene regulation.
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Many transcription factors incorporate DNA-binding structural motifs which
comprise either a
helices or B sheets that bind to the major groove of DNA. Four well-
characterized structural motifs
are helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix.
Proteins containing these motifs
may act alone as monomers, or they may form homo- or heterodimers that
interact with DNA.
The helix-turn-helix motif consists of two a helices connected at a fixed
angle by a short
chain of amino acids. One of the helices binds to the major groove. Helix-turn-
helix motifs are
exemplified by the homeobox motif which is present in homeodomain proteins.
These proteins are
critical for specifying the anterior-posterior body axis during development
and are conserved
throughout the animal kingdom. The Antennapedia and Ultrabithorax proteins of
Drosophila
l0 melanogaster are prototypical homeodomain proteins. (Patio, C.O. and R.T.
Sauer (1992) Annu.
Rev. Biochem. 61:1053-1095.)
Mouse HES-6 is a member of the Hairy/Enhancer-of split (HES) family of basic
helix-loop-helix transcription factors. HES genes act as nuclear effectors of
Notch signaling to
regulate the transcriptional activity of several Notch target genes. HES-6 is
expressed in all
15 neurogenic placodes and their derivatives and in the brain, where it is
patterned along both the
anteroposterior and dorsoventral axes. HES-6 is also expressed in embryonic
tissues where Notch
signaling controls cell-fate decisions, such as the trunk, the dorsal root
ganglia, myotomes, and thymus.
In the limb buds HES-6 is expressed in skeletal muscle and presumptive
tendons. It is also expressed
in epithelial cells of the embryonic respiratory, urinary and digestive
systems (Vasiliauskas, D. and
20 Stern C.D. (2000) Mech. Dev. 98:133-137; Pissarra, L. et al. (2000) Mech
Dev 95:275-278).
The zinc finger motif, which binds zinc ions, generally contains tandem
repeats of about 30
amino acids consisting of periodically spaced cysteine and histidine residues.
Examples of this
sequence pattern, designated C2H2 and C3HC4 ("RING" finger), have been
described. (Lewin,
supra.) Zinc forger proteins each contain an a helix and an antiparallel B
sheet whose proximity and
25 conformation are maintained by the zinc ion. Contact with DNA is made by
the arginine preceding
the a helix and by the second, third, and sixth residues of the a helix.
Variants of the zinc finger motif
include poorly defined cysteine-rich motifs which bind zinc or other metal
ions. These motifs may not
contain histidine residues and are generally nonrepetitive. The zinc finger
motif may be repeated in a
tandem array within a protein, such that the a helix of each zinc finger in
the protein makes contact
30 with the major groove of the DNA double helix. This repeated contact
between the protein and the
DNA produces a strong and specific DNA-protein interaction. The strength and
specificity of the
interaction can be regulated by the number of zinc finger motifs within the
protein. Though originally
identified in DNA-binding proteins as regions that interact directly with DNA,
zinc fingers occur in a
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variety of proteins that do not bind DNA (Lodish, H. et al. (1995) Molecular
Cell Biology, Scientific
American Books, New York, NY, pp. 447-451). For example, Galcheva-Gargova, Z.
et al. (1996)
Science 272:1797-1802) have identified zinc forger proteins that interact with
various cytokine
receptors.
The C2H2-type zinc forger signature motif contains a 28 amino acid sequence,
including 2
conserved Cys and 2 conserved His residues in a C-2-C-12-H-3-H type motif. The
motif generally
occurs in multiple tandem repeats. A cysteine-rich domain including the motif
Asp-His-His-Cys
(DHHC-CRD) has been identified as a distinct subgroup of zinc finger proteins.
The DHHC-CRD
region has been implicated in growth and development. One DHHC-CRD mutant
shows defective
to function of Ras, a small membrane-associated GTP-binding protein that
regulates cell growth and
differentiation, while other DHHC-CRD proteins probably function in pathways
not involving Ras
(Barrels, D.J. et al. (1999) Mol. Cell Biol. 19:6775-6787).
Zinc-finger transcription factors are often accompanied by modular sequence
motifs such as
the Kruppel-associated box (KRAB) and the SCAN domain. For example, the
hypoalphalipoproteinemia susceptibility gene ZNF202 encodes a SCAN box and a
KRAB domain
followed by eight C2H2 zinc-forger motifs (Hover, C. et al. (2001) Biochim.
Biophys. Acta
1517:441-448). The SCAN domain is a highly conserved, leucine-rich motif of
approximately 60
amino acids found at the amino-terminal end of zinc forger transcription
factors. SCAN domains are
most often linked to C2H2 zinc finger motifs through their carboxyl-terminal
end. Biochemical binding
studies have established the SCAN domain as a selective hetero- and homotypic
oligomerization
domain. SCAN domain-mediated protein complexes may function to modulate the
biological function
of transcription factors (Schumacher, C. et al. (2000) J. Biol. Chem.
275:17173-17179).
The KRAB (Kruppel-associated box) domain is a conserved amino acid sequence
spanning
approximately 75 amino acids and is found in almost one-third of the 300 to
700 genes encoding C2H2
zinc fingers. The KRAB domain is found N-terminally with respect to the finger
repeats. The KRAB
domain is generally encoded by two exons; the KRAB-A region or box is encoded
by one exon and
the KRAB-B region or box is encoded by a second exon. The function of the KRAB
domain is the
repression of transcription. Transcription repression is accomplished by
recruitment of either the
KRAB-associated protein-1, a transcriptional coreprescor, or the KRAB-A
interacting protein.
Proteins containing the KRAB domain are likely to play a regulatory role
during development
(Williams, A.J. et al. (1999) Mol. Cell Biol. 19:8526-8535). A subgroup of
highly related human
KRAB zinc finger proteins detectable in all human tissues is highly expressed
in human T lymphoid
cells (Bellefroid, E.J. et al. (1993) EMBO J. 12:1363-1374). The ZNF85 KRAB
zinc finger gene, a
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member of the human ZNF91 family, is highly expressed in normal adult testis,
in seminomas, and in
the NT2/D1 teratocarcinoma cell line (Poncelet, D.A. et al. (1998) DNA Cell
Bio1.17:931-943).
The Kruppel protein regulates Drosophila segmentation. There are approximately
300 genes
which encode such proteins in the whole human genome. In fact, more than 100
different mRNAs
encoding Kruppel multifingered proteins, most of them novel, have been found
in the human placenta.
The sequences of the 106 forger repeats present in nine of these proteins are
highly homologous.
There are a few positions located in the alpha-helical structure which show
variability. Research
implies that this variability impacts the DNA-binding specificity of the
proteins (Bellefroid, E.J. et al.
(1989) DNA 8:377-387).
ZNF143 is a human zinc finger Kruppel family protein of the GLI type. It is
84% identical to
the Xenopus laevis selenocysteine tRNA gene transcription activating factor
(Staf). Staf is implicated
in the enhanced transcription of small nuclear RNA (snRNA) and snRNA-type
genes by RNA
polymerises II (Pol II) and III (Pol III). Staf also possesses the capacity to
stimulate expression from
a Pol II mRNA promoter. ZNF143, along with the related ZNF138 and ZNF139, is
localized to
chromosome regions 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4. These regions are
involved in deletions
and/or translocations associated with Williams syndrome, split hand and foot
disease (SHFD1), and
Beckwith-Wiedemann syndrome, respectively, suggesting that ZNF143 gene is
involved in
developmental and malignant disorders. ZNF143 mRNAs are expressed in many
normal adult tissues,
including leukocytes, colon, small intestine, ovary, testis, prostate, thymus,
and spleen tissues. ..Further,
transcription of the mouse chaperone-encoding Ccta gene is regulated by ZNF143
and another Staf
family zinc-forger transcription factor, ZNF76, implying that these RNA and
chaperone genes are
coregulated to facilitate synthesis of mature proteins during active cell
growth (Tommerup,N. and
Vissing,H. (1995) Genomics 27: 259-264 ;Myslinski, E. et al. (1998) J. Biol.
Chem. 273:21998-2006;
Kubota, H. et al. (2000) J. Biol. Chem. 275:28641-28648).
The C4 motif is found in hormone-regulated proteins. The C4 motif generally
includes only 2
repeats. A number of eukaryotic and viral proteins contain a conserved
cysteine-rich domain of 40
to 60 residues (called C3HC4 zinc-finger or RING forger) that binds two atoms
of zinc, and is
probably involved in mediating protein-protein interactions. The 3D "cross-
brace" structure of the zinc
ligation system is unique to the RING domain. The spacing of the cysteines in
such a domain is
3o C-x(2)-C-x(9 to 39)-C-x(1 to 3)-H-x(2 to3)-C-x(2)-C-x(4 to 48)-C-x(2)-C.
The PHD forger is a
C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved
in chromatin-mediated
transcriptional regulation.
GATA-type transcription factors contain one or two zinc forger domains which
bind
4
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specifically to a region of DNA that contains the consecutive nucleotide
sequence GATA. NMR
studies indicate that the zinc forger comprises two irregular anti-parallel (3
sheets and an a helix,
followed by a long loop to the C-terminal end of the finger (Ominchinski, J.G.
(1993) Science
261:438-446). The helix and the loop connecting the two (3-sheets contact the
major groove of the
DNA, while the C-terminal part, which determines the specificity of binding,
wraps around into the
minor groove.
The LIM motif consists of about 60 amino acid residues and contains seven
conserved
cysteine residues and a histidine within a consensus sequence (Schmeichel,
K.L. and Beckerle, M.C.
(1994) Cell 79:211-219). The LIM family includes transcription factors and
cytoskeletal proteins
which may be involved in development, differentiation, and cell growth. One
example is actin-binding
LlM protein, which may play roles in regulation of the cytoskeleton and
cellular morphogenesis (Roof,
D.J. et al. (1997) J. Cell Biol. 138:575-588). The N-terminal domain of actin-
binding LIM protein has
four double zinc forger motifs with the LIM consensus sequence. The C-terminal
domain of actin-
binding LIM protein shows sequence similarity to known actin-binding proteins
such as dematin and
villin. Actin-binding LIM protein binds to F-actin through its dematin-like C-
terminal domain. The
L1M domain may mediate protein-protein interactions with other LIM-binding
proteins.
Myeloid cell development is controlled by tissue-specific transcription
factors. Myeloid zinc
finger proteins (MZF) include MZF-1 and MZF-2. MZF-1 functions in regulation
of the development
of neutrophilic granulocytes. A murine homolog MZF-2 is expressed in myeloid
cells, particularly in
the cells committed to the neutrophilic lineage. MZF-2 is down-regulated by G-
CSF and appears to
have a unique function in neutrophil development (Murai, K. et al. (1997)
Genes Cells 2:581-591).
The leucine zipper motif comprises a stretch of amino acids rich in leucine
which can form an
amphipathic a helix. This structure provides the basis for dimerization of two
leucine zipper proteins.
The region adjacent to the leucine zipper is usually basic, and upon protein
dimerization, is optimally
positioned for binding to the major groove. Proteins containing such motifs
are generally referred to as
bZIP transcription factors. The leucine zipper motif is found in the proto-
oncogenes Fos and Jun,
which comprise the heterodimeric transcription factor AP1 involved in cell
growth and the .
determination of cell fineage (Papavassiliou, A.G. (1995) N. Engl. J. Med.
332:45-47).
The mouse kreisler (kr) mutation causes segmentation abnormalities in the
caudal hindbrain
and defective inner ear development. The kr cDNA encodes a basic domain-
leucine zipper (bZIP)
transcription factor in which a serine is substituted for an asparagine
residue conserved in the
DNA-binding domain of all known bZIP family members. The identity, expression,
and mutant
phenotype of kr indicate an early role in axial patterning and provide
insights into the molecular and
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embryologic mechanisms that govern hindbrain and otic development (Cordes,
S.P. and Barsh, G.S.
(1994) Cel179:1025-1034).
The helix-loop-helix motif (HI.ITj consists of a short a helix connected by a
loop to a longer a
helix. The loop is flexible and allows the two helices to fold back against
each other and to bind to
DNA. The transcription factor Myc contains a prototypical HLH motif.
The NF-kappa-B/Rel signature defines a family of eukaryotic transcription
factors involved in
oncogenesis, embryonic development, differentiation and immune response. Most
transcription factors
containing the Rel homology domain (RHD) bind as dimers to a consensus DNA
sequence motif
termed kappa-B. Members of the Rel family share a highly conserved 300 amino
acid domain termed
the Rel homology domain. The characteristic Rel C-terminal domain is involved
in gene activation and
cytoplasmic anchoring functions. Proteins known to contain the RHD domain
include vertebrate
nuclear factor NF-kappa-B, which is a heterodimer of a DNA-binding subunit and
the transcription
factor p65, mammalian transcription factor RelB, and vertebrate proto-oncogene
c-rel, a protein
associated with differentiation and lymphopoiesis (Kabrun, N. and Enrietto,
P.J. (1994) Semin. Cancer
Bio1.5:103-112).
A DNA binding motif termed AR)D (AT-rich interactive domain) distinguishes an
evolutionarily conserved family of proteins. The approximately 100-residue
ARID sequence is present
in a series of proteins strongly implicated in the regulation of cell growth,
development, and
tissue-specific gene expression. ARID proteins include Bright (a regulator of
B-cell-specific gene
expression), dead ringer (involved in development), and MRF-2 (which represses
expression from the
cytomegalovirus enhancer) (Dallas, P.B. et al. (2000) Mol. Cell Biol. 20:3137-
3146).
The ELM2 (Egl-27 and MTA1 homology 2) domain is found in metastasis-associated
protein
MTA1 and protein ERI. The Caenorhabditis elegans gene egl-27 is required for
embryonic
patterning MTA1, a human gene with elevated expression in metastatic
carcinomas, is a component
of a protein complex with histone deacetylase and nucleosome remodelling
activities (Solari, F. et al.
(1999) Development 126:2483-2494). The ELM2 domain is usually found to the N
terminus of a
myb-like DNA binding domain. ELM2 is also found associated with an ARID DNA.
LEF-1 is a transcription factor that participates in the regulation of the T-
cell receptor alpha
(TCR alpha) enhancer by facilitating the assembly of multiple proteins into a
higher order
nucleoprotein complex. The function of LEF-1 is dependent, in part, on the HMG
domain. This
domain induces a sharp bend in the DNA helix and on an activation domain that
stimulates
transcription only in a specific context of other enhancer-binding proteins.
ALY is a LEF-1-interacting
protein which is a ubiquitously expressed, nuclear protein that specifically
associates with the
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activation domains of LEF-1 and AML-1 (acute myeloid leukemia 1). AML-1 is
another protein
component of the TCR alpha enhancer complex. ALY increases DNA binding by both
LEF-1 and
AML proteins. Overexpression of ALY stimulates the activity of the TCR alpha
enhancer complex in
transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-
sense oligonucleotides
eliminates TCR alpha enhancer activity in T cells. Similar to LEF-1, ALY can
stimulate transcription
in the context of the TCR alpha enhancer but apparently not when tethered to
DNA through an
heterologous DNA-binding domain. Research suggests that ALY mediates context-
dependent
transcriptional activation by facilitating the functional collaboration of
multiple proteins in the TCR
alpha enhancer complex (Bruhn, L. et al. (1997) Genes Dev. 11:640-653).
A family of nuclear proteins, designated SL3-3 enhancer factors 2 (SEF2),
interact with an
Ephrussi box-like motif within the glucocorticoid response element in the
enhancer of the murine
leukemia virus SL3-3. Mutation of the DNA sequence decreased the basal
enhancer activity in
various cell lines. The important nucleotides for binding of SEFZ are
conserved in most type C
retroviruses. Various cell types displayed differences both in the sets of
SEF2-DNA complexes
formed and in their amounts. A cDNA which encoded a protein, SEF2-lA, that
interacted specifically
with the SEF2-binding sequence has been isolated from human thymocytes. The
nucleotide sequence
specificity of the recombinant SER2-lA, expressed in Escherichia coli,
corresponds to that of one of
the nuclear SEF2 proteins (Corneliussen, B. et al. J(1991) J. Virol. 65:6084-
6093).
The Iroquois (Irx) family of genes are found in nematodes, insects and
vertebrates. Irx genes
usually occur in one or two genomic clusters of three genes each and encode
transcriptional
controllers that possess a characteristic homeodomain. The Irx genes function
early in development to
specify the identity of diverse territories of the body. Later in development
in both Drosophila and
vertebrates, the Irx genes function again to subdivide those terntories into
smaller domains. (For a
review of Iroquois genes, see Cavodeassi, F. et al. (2001) Development
128:2847-2855.) For
example, mouse and human Irx4 proteins are 83 % conserved and their 63-as
homeodomain is more
than 93% identical to that of the Drosophila Iroquois patterning genes. Irx4
transcripts are
predominantly expressed in the cardiac ventricles. The homeobox gene hx4
mediates ventricular
differentiation during cardiac development (Bruneau, B.G. et al. (2000) Dev.
Biol. 217:266-77).
Histidine triad (HIT) proteins share residues in distinctive dimeric, 10-
stranded half-barrel
structures that form two identical purine nucleotide-binding sites. Hint
(histidine triad
nucleotide-binding protein)-related proteins, found in all forms of life, and
fragile histidine triad
(Fhit)-related proteins, found in animals and fungi, represent the two main
branches of the HIT
superfamily. Fhit homologs bind and cleave diadenosine polyphosphates. Fhit-
Ap(n)A complexes
,~,
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appear to function in a proapoptotic tumor suppression pathway in epithelial
tissues (Brenner C. et al.
(1999) J. Cell Physio1.181:179-187).
Most transcription factors contain characteristic DNA binding motifs, and
variations on the
above motifs and new motifs have been and are currently being characterized.
(Faisst, S. and S.
Meyer (1992) Nucleic Acids Res. 20:3-26.)
Chromatin Associated Proteins
In the nucleus, DNA is packaged into chromatin, the compact organization of
which limits the
accessibility of DNA to transcription factors and plays a key role in gene
regulation. (Lewin, supra,
pp. 409-410.) The compact structure of chromatin is determined and influenced
by chromatin-
to associated proteins such as the histories, the high mobility group (HMG)
proteins, and the
chromodomain proteins. There are five classes of histories, H1, H2A, H2B, H3,
and H4, all of which
are highly basic, low molecular weight proteins. The fundamental unit of
chromatin, the nucleosome,
consists of 200 base pairs of DNA associated with two copies each of H2A, HZB,
H3, and H4. Hl
links adjacent nucleosomes. HMG proteins are low molecular weight, non-histone
proteins that may
play a role in unwinding DNA and stabilizing single-stranded DNA. Chromodomain
proteins play a
key role in the formation of highly compacted heterochromatin, which is
transcriptionally silent.
The SWI/SNF complex in yeast facilitates the function of transcriptional
activators by
opposing chromatin-dependent repression of transcription. In mammals SWI/SNF
complexes are
present in multiple forms made up of 9-12 proteins known as BRG1-associated
factors (BAFs)
2o ranging from 47 to 250 kD. BRG1-associated factors (BAFs) include the SWI2-
SNF2 homolog which
interacts with and ctivates human immunodeficiency virus integrase and is
homologous to the yeast
SNFS gene (Wang, W. et al. (1996) Genes Dev. 10:2117-2130).
Diseases and Disorders Related to Gene Regulation
Many neoplastic disorders in humans can be attributed to inappropriate gene
expression.
Malignant cell growth may result from either excessive expression of tumor
promoting genes or
insufficient expression of tumor suppressor genes. (Cleary, M.L. (1992) Cancer
Surv. 15:89-104.)
The zinc finger-type transcriptional regulator WT1 is a tumor-suppressor
protein that is inactivated in
children with Wilin's tumor. The oncogene bcl-6, which plays an important role
in large-cell
lymphoma, is also a zinc-finger protein (Papavassiliou, A.G. (1995) N. Engl.
J. Med. 332:45-47).
Chromosomal translocations may also produce chimeric loci that fuse the coding
sequence of one
gene with the regulatory regions of a second unrelated gene. Such an
arrangement likely results in
inappropriate gene transcription, potentially contributing to malignancy. In
Burkitt's lymphoma, for
example, the transcription factor Myc is translocated to the immunoglobulin
heavy chain locus, greatly
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enhancing Myc expression and resulting in rapid cell growth leading to
leukemia (Latchman, D.S.
(1996) N. Engl. J. Med. 334:28-33).
In addition, the immune system responds to infection or trauma by activating a
cascade of
events that coordinate the progressive selection, amplification, and
mobilization of cellular defense
mechanisms. A complex and balanced program of gene activation and repression
is involved in this
process. However, hyperactivity of the immune system as a result of improper
or insufficient
regulation of gene expression may result in considerable tissue or organ
damage. This damage is well-
documented in immunological responses associated with arthritis, allergens,
heart attack, stroke, and
infections. (Isselbacher et al. Harrison's Principles of Internal Medicine,
13/e, McGraw Hill, Inc. and
l0 Teton Data Systems Software, 1996.) The causative gene for autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED) was recently isolated and found to
encode a protein
with two PHD-type zinc forger motifs (Bjorses, P. et al. (1998) Hum. Mol.
Genet. 7:1547-1553).
Furthermore, the generation of multicellular organisms is based upon the
induction and
coordination of cell differentiation at the appropriate stages of development.
Central to this process is
differential gene expression, which confers the distinct identities of cells
and tissues throughout the
body. Failure to regulate gene expression during development could result in
developmental disorders.
Human developmental disorders caused by mutations in zinc finger-type
transcriptional regulators
include: urogenital developmental abnormalities associated with WT1; Greig
cephalopolysyndactyly,
Pallister-Hall syndrome, and postaxial polydactyly type A (GLI3), and Townes-
Brocks syndrome,
characterized by anal, renal, limb, and ear abnormalities (SALLl) (Engelkamp,
D. and V. van
Heyningen (1996) C~rr. Opin. Genet. Dev. 6:334-342; Kohlhase, J. et al. (1999)
Am. J. Hum. Genet.
64:435-445).
Human acute leukemias involve reciprocal chromosome translocations that fuse
the ALL-1
gene located at chromosome region l 1q23 to a series of partner genes
positioned on a variety of
human chromosomes. The fused genes encode chimeric proteins. The AF17 gene
encodes a protein
of 1093 amino acids, containing a leucine-zipper dimerization motif located 3'
of the fusion point and a
cysteine-rich domain at the N terminus that shows homology to a domain within
the protein Br140
(peregrin) (Prasad R. et al. (1994) Proc. Natl. Acid. Sci. U S A 91:8107-
8111).
SYNTHESIS OF NUCLEIC ACIDS
3o Polymerises
DNA and RNA replication are critical processes for cell replication and
function. DNA and
RNA replication are mediated by the enzymes DNA and RNA polymerise,
respectively, by a
"templating" process in which the nucleotide sequence of a DNA or RNA strand
is copied by
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complementary base-pairing into a complementary nucleic acid sequence of
either DNA or RNA.
However, there are fundamental differences between the two processes.
DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the
3'-OH end
of a polynucleotide strand (the primer strand) that is paired to a second
(template) strand. The new
DNA strand therefore grows in the 5' to 3' direction (Alberts, B. et al.
(1994) The Molecular BioloQy
of the Cell, Garland Publishing Inc., New York, NY, pp 251-254). The
substrates for the
polymerization reaction are the corresponding deoxynucleotide triphosphates
which must base-pair
with the correct nucleotide on the template strand in order to be recognized
by the polymerase.
Because DNA exists as a double-stranded helix, each of the two strands may
serve as a template for
the formation of a new complementary strand. Each of the two daughter cells of
a dividing cell
therefore inherits a new DNA double helix containing one old and one new
strand. Thus, DNA is said
to be replicated "semiconservatively" by DNA polymerase. In addition to the
synthesis of new DNA,
DNA polymerase is also involved in the repair of damaged DNA as discussed
below under "Ligases."
In contrast to DNA polymerase, RNA polymerase uses a DNA template strand to
"transcribe" DNA into RNA using ribonucleotide triphosphates as substrates.
Like DNA
polymerization, RNA polymerization proceeds in a 5' to 3' direction by
addition of a ribonucleoside
monophosphate to the 3'-OH end of a growing RNA chain. DNA transcription
generates messenger
RNAs (mRNA) that carry information for protein synthesis, as well as the
transfer, ribosomal, and
other RNAs that have structural or catalytic functions. In eukaryotes, three
discrete RNA
polymerases synthesize the three different types of RNA (Alberts, supra, pp.
367-368). RNA
polymerase I makes the large ribosomal RNAs, RNA polymerase II makes the mRNAs
that will be
translated into proteins, and RNA polymerase III makes a variety of small,
stable RNAs, including SS
ribosomal RNA and the transfer RNAs (tRNA). In all cases, RNA synthesis is
initiated by binding of
the RNA polymerase to a promoter region on the DNA and synthesis begins at a
start site within the
promoter. Synthesis is completed at a stop (termination) signal in the DNA
whereupon both the
polymerase and the completed RNA chain are released.
Li~ases
DNA repair is the process by which accidental base changes, such as those
produced by
oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA, are
corrected before
replication or transcription of the DNA can occur. Because of the efficiency
of the DNA repair
process, fewer than one in a thousand accidental base changes causes a
mutation (Alberts, supra, pp.
245-249). The three steps common to most types of DNA repair are (1) excision
of the damaged or
altered base or nucleotide by DNA nucleases, (2) insertion of the correct
nucleotide in the gap left by
to
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the excised nucleotide by DNA polymerase using the complementary strand as the
template and, (3)
sealing the break left between the inserted nucleotides) and the existing DNA
strand by DNA ligase.
In the last reaction, DNA ligase uses the energy from ATP hydrolysis to
activate the 5' end of the
broken phosphodiester bond before forming the new bond with the 3'-OH of the
DNA strand. In
Bloom's syndrome, an inherited human disease, individuals are partially
deficient in DNA ligation and
consequently have an increased incidence of cancer (Alberts, supra p. 247).
Nucleases
Nucleases comprise enzymes that hydrolyze both DNA (DNase) and RNA (Rnase).
They
serve different purposes in nucleic acid metabolism. Nucleases hydrolyze the
phosphodiester bonds
l0 between adjacent nucleotides either at internal positions (endonucleases)
or at the terminal 3' or 5'
nucleotide positions (exonucleases). A DNA exonuclease activity in DNA
polymerase, for example,
serves to remove improperly paired nucleotides attached to the 3'-OH end of
the growing DNA strand
by the polymerase and thereby serves a "proofreading" function. As mentioned
above, DNA
endonuclease activity is involved in the excision step of the DNA repair
process.
RNases also serve a variety of functions. For example, RNase P is a
ribonucleoprotein
enzyme which cleaves the S' end of pre-tRNAs as part of their maturation
process. RNase H digests
the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by
retroviruses, and
RNase H is an important enzyme in the retroviral replication cycle. Pancreatic
RNase secreted by
the pancreas into the intestine hydrolyzes RNA present in ingested foods.
RNase activity in serum
and cell extracts is elevated in a variety of cancers and infectious diseases
(Schein, C.H. (1997) Nat.
Biotechnol. 15:529-536). Regulation of RNase activity is being investigated as
a means to control
tumor angiogenesis, allergic reactions, viral infection and replication, and
fungal infections.
MODIFICATION OF NUCLEIC ACIDS
Methylases
Methylation of specific nucleotides occurs in both DNA and RNA, and serves
different
functions in the two macromolecules. Methylation of cytosine residues to form
5-methyl cytosine in
DNA occurs specifically in CG sequences which are base-paired with one another
in the DNA
double-helix. The pattern of methylation is passed from generation to
generation during DNA
replication by an enzyme called "maintenance methylase" that acts
preferentially on those CG
sequences that are base-paired with a CG sequence that is already methylated.
Such methylation
appears to distinguish active from inactive genes by preventing the binding of
regulatory proteins that
"turn on" the gene, but permiting the binding of proteins that inactivate the
gene (Alberts, supra pp.
448-451). In RNA metabolism, "tRNA methylase" produces one of several
nucleotide modifications
11
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in tRNA that affect the conformation and base-pairing of the molecule and
facilitate the recognition of
the appropriate mRNA codons by specific tRNAs. The primary methylation pattern
is the
dimethylation of guanine residues to form N,N-dimethyl guanine.
Helicases and Sinele-stranded Binding Proteins
Helicases are enzymes that destabilize and unwind double helix structures in
both DNA and
RNA. Since DNA replication occurs more or less simultaneously on both strands,
the two strands
must first separate to generate a replication "fork" for DNA polymerase to act
on. Two types of
replication proteins contribute to this process, DNA helicases and single-
stranded binding proteins.
DNA helicases hydrolyze ATP and use the energy of hydrolysis to separate the
DNA strands.
Single-stranded binding proteins (SSBs) then bind to the exposed DNA strands,
without covering the
bases, thereby temporarily stabilizing them for templating by the DNA
polymerase (Alberts, supra, pp.
255-256).
RNA helicases also alter and regulate RNA conformation and secondary
structure. Like the
DNA helicases, RNA helicases utilize energy derived from ATP hydrolysis to
destabilize and unwind
RNA duplexes. The most well-characterized and ubiquitous family of RNA
helicases is the DEAD-
box family, so named for the conserved B-type ATP-binding motif which is
diagnostic of proteins in
this family. Over 40 DEAD-box helicases have been identified in organisms as
diverse as bacteria,
insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function
in diverse processes
such as translation initiation, splicing, ribosome assembly, and RNA editing,
transport, and stability.
Examples of these RNA helicases include yeast Drs 1 protein, which is involved
in ribosomal RNA
processing; yeast TIFl and TIF2 and mammalian eIF-4A, which are essential to
the initiation of RNA
translation; and human p68 antigen, which regulates cell growth and division
(R.ipmaster, T.L. et al.
(1992) Proc. Natl. Acad. Sci. USA 89:11131-11135; Chang, T.-H. et al. (1990)
Proc. Natl. Acad. Sci.
USA 87:1571-1575). These RNA helicases demonstrate strong sequence homology
over a stretch of
some 420 amino acids. Included among these conserved sequences are the
consensus sequence for
the A motif of an ATP binding protein; the "DEAD box" sequence, associated
with ATPase activity;
the sequence SAT, associated with the actual helicase unwinding region; and an
octapeptide
consensus sequence, required for RNA binding and ATP hydrolysis (Pause, A. et
al. (1993) Mol. Cell
Biol. 13:6789-6798). Differences outside of these conserved regions are
believed to reflect
differences in the functional roles of individual proteins (Chang, T.H. et al.
(1990) Proc. Natl. Acad.
Sci. USA 87:1571-1575).
Some DEAD-box helicases play tissue- and stage-specific roles in
spermatogenesis and
embryogenesis. Overexpression of the DEAD-box 1 protein (DDX1) may play a role
in the
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progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R.
et al. (1998) J. Biol.
Chem. 273:21161-21168). These observations suggest that DDX1 may promote or
enhance tumor
progression by altering the normal secondary structure and expression levels
of RNA in cancer cells.
Other DEAD-box helicases have been implicated either directly or indirectly in
tumorigenesis.
(Discussed in Godbout, supra.) For example, murine p68 is mutated in
ultraviolet light-induced
tumors, and human DDX6 is located at a chromosomal breakpoint associated with
B-cell lymphoma.
Similarly, a chimeric protein comprised of DDX10 and NUP98, a nucleoporin
protein, may be involved
in the pathogenesis of certain myeloid malignancies.
Tonoisomerases
Besides the need to separate DNA strands prior to replication, the two strands
must be
'unwound" from one another prior to their separation by DNA helicases. This
function is performed
by proteins known as DNA topoisomerases. DNA topoisomerase effectively acts as
a reversible
nuclease that hydrolyzes a phosphodiesterase bond in a DNA strand, permits the
two strands to rotate
freely about one another to remove the strain of the helix, and then rejoins
the original phosphodiester
bond between the two strands. Topoisomerases are essential enzymes responsible
for the topological
rearrangement of DNA brought about by transcription, replication, chromatin
formation,
recombination, and chromosome segregation. Superhelical coils are introduced
into DNA by the
passage of processive enzymes such as RNA polymerase, or by the separation of
DNA strands by a
helicase prior to replication. Knotting and concatenation can occur in the
process of DNA synthesis,
storage, and repair. All topoisomerases work by breaking a phosphodiester bond
in the ribose-
phosphate backbone of DNA. A catalytic tyrosine residue on the enzyme makes a
nucleophilic attack
on the scissile phosphodiester bond, resulting in a reaction intermediate in
which a covalent bond is
formed between the enzyme and one end of the broken strand. A tyrosine-DNA
phosphodiesterase
functions in DNA repair by hydrolyzing this bond in occasional dead-end
topoisomerase I-DNA
intermediates (Pouliot, J.J. et al. (1999) Science 286:552-555).
Two types of DNA topoisomerase exist, types I and II. Type I topoisomerases
work as
monomers, making a break in a single strand of DNA while type II
topoisomerases, working as
homodimers, cleave both strands. DNA Topoisomerase I causes a single-strand
break in a DNA
hefix to allow the rotation of the two strands of the helix about the
remaining phosphodiester bond in
the opposite strand. DNA topoisomerase II causes a transient break in both
strands of a DNA helix
where two double helices cross over one another. This type of topoisomerase
can efficiently separate
two interlocked DNA circles (Alberts, supra, pp.260-262). Type II
topoisomerases are largely
confined to proliferating cells in eukaryotes, such as cancer cells. For this
reason they are targets for
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anticancer drugs. Topoisomerase II has been implicated in mufti-drug
resistance (1VIDR) as it appears
to aid in the repair of DNA damage inflicted by DNA binding agents such as
doxorubicin and
vincristine.
The topoisomerase I family includes topoisomerases I and III (topo I and topo
III). The
crystal structure of human topoisomerase I suggests that rotation about the
intact DNA strand is
partially controlled by the enzyme. In this "controlled rotation" model,
protein-DNA interactions limit
the rotation, which is driven by torsional strain in the DNA (Stewart, L. et
al. (1998) Science
379:1534-1541). Structurally, topo I can be recognized by its catalytic
tyrosine residue and a number
of other conserved residues in the active site region. Topo I is thought to
function during transcription.
Two topo Ills are known in humans, and they are homologous to prokaryotic
topoisomerase I, with a
conserved tyrosine and active site signature specific to this family. Topo III
has been suggested to
play a role in meiotic recombination. A mouse topo III is highly expressed in
testis tissue and its
expression increases with the increase in the number of cells in pachytene
(Seki, T. et al. (1998) J.
Biol. Chem. 273:28553-28556).
The topoisomerase II family includes two isozymes (IIa and II(3) encoded by
different genes.
Topo II cleaves double stranded DNA in a reproducible, nonrandom fashion,
preferentially in an AT
rich region, but the basis of cleavage site selectivity is not known.
Structurally, topo II is made up of
four domains, the first two of which are structurally similar and probably
distantly homologous to
similar domains in eukaryotic topo I. The second domain bears the catalytic
tyrosine, as well as a
highly conserved pentapeptide. The IIa isoform appears to be responsible for
unlinking DNA during
chromosome segregation. Cell lines expressing IIoc but not II~i suggest that
II(3 is dispensable in
cellular processes; however, II(3 knockout mice died perinatally due to a
failure in neural development.
That the major abnormalities occurred in predominantly late developmental
events (neurogenesis)
suggests that II(3 is needed not at mitosis, but rather during DNA repair
(Yang, X. et al. (2000)
Science 287:131-134).
Topoisomerases have been implicated in a number of disease states, and
topoisomerase
poisons have proven to be effective anti-tumor drugs for some human
malignancies. Topo I is
mislocalized in Fanconi's anemia, and may be involved in the chromosomal
breakage seen in this
disorder (blunder, E. (1984) Hum. Genet. 68:276-281). Overexpression of a
truncated topo IQ in
ataxia-telangiectasia (A-T) cells partially suppresses the A-T phenotype,
probably through a dominant
negative mechanism. This suggests that topo III is deregulated in A-T (Fritz,
E. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:4538-4542). Topo III also interacts with the Bloom's
Syndrome gene
product, and has been suggested to have a role as a tumor suppressor (Wu, L.
et al. (2000) J. Biol.
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Chem. 275:9636-9644). Aberrant topo II activity is often associated with
cancer or increased cancer
risk. Greatly lowered topo II activity has been found in some, but not all A-T
cell lines (Mohamed, R.
et al. (1987) Biochem. Biophys. Res. Commun. 149:233-238). On the other hand,
topo II can break
DNA in the region of the A-T gene (ATM), which controls all DNA damage-
responsive cell cycle
checkpoints (Kaufmann, W.K. (1998) Proc. Soc. Exp. Biol. Med. 217:327-334).
The ability of
topoisomerases to break DNA has been used as the basis of antitumor drugs.
Topoisomerase poisons
act by increasing the number of dead-end covalent DNA-enzyme complexes in the
cell, ultimately
triggering cell death pathways (Fortune, J.M. and N. Osheroff (2000) Prog.
Nucleic Acid Res. Mol.
Biol. 64:221-253; Guichard, S.M. and M.K. Danks (1999) C~rr. Opin. Oncol.
11:482-489). Antibodies
l0 against topo I are found in the serum of systemic sclerosis patients, and
the levels of the antibody may
be used as a marker of pulmonary involvement in the disease (Diot, E. et al.
(1999) Chest 116:715-
720). Finally, the DNA binding region of human topo I has been used as a DNA
delivery vehicle for
gene therapy (Chen, T.Y. et al. (2000) Appl. Microbiol. Biotechnol. 53:558-
567).
Recombinases
Genetic recombination is the process of rearranging DNA sequences within an
organism's
genome to provide genetic variation for the organism in response to changes in
the environment.
DNA recombination allows variation in the particular combination of genes
present in an individual's
genome, as well as the timing and level of expression of these genes. (See
Alberts, supra pp. 263-
273.) Two broad classes of genetic recombination are commonly recognized,
general recombination
and site-specific recombination. General recombination involves genetic
exchange between any
homologous pair of DNA sequences usually located on two copies of the same
chromosome. The
process is aided by enzymes, recombinases, that "nick" one strand of a DNA
duplex more or less
randomly and permit exchange with a complementary strand on another duplex.
The process does not
normally change the arrangement of genes in a chromosome. In site-specific
recombination, the
recombinase recognizes specific nucleotide sequences present in one or both of
the recombining
molecules. Base-pairing is not involved in this form of recombination and
therefore it does not require
DNA homology between the recombining molecules. Unlike general recombination,
this form of
recombination can alter the relative positions of nucleotide sequences in
chromosomes.
RNA METABOLISM
Ribonucleic acid (RNA) is a linear single-stranded polymer of four
nucleotides, ATP, CTP,
UTP, and GTP. In most organisms, RNA is transcribed as a copy of
deoxyribonucleic acid (DNA),
the genetic material of the organism. In retroviruses RNA rather than DNA
serves as the genetic
material. RNA copies of the genetic material encode proteins or serve various
structural, catalytic, or
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regulatory roles in organisms. RNA is classified according to its cellular
localization and function.
Messenger RNAs (mRNAs) encode polypeptides. Ribosomal RNAs (rRNAs) are
assembled, along
with ribosomal proteins, into ribosomes, which are cytoplasmic particles that
translate mRNA into
polypeptides. Transfer RNAs (tRNAs) are cytosolic adaptor molecules that
function in mRNA
translation by recognizing both an mRNA codon and the amino acid that matches
that codon.
Heterogeneous nuclear RNAs (hnRNAs) include mRNA precursors and other nuclear
RNAs of
various sizes. Small nuclear RNAs (snRNAs) are a part of the nuclear
spliceosome complex that
removes intervening, non-coding sequences (introns) and rejoins exons in pre-
mRNAs.
Proteins are associated with RNA during its transcription from DNA, RNA
processing, and
translation of mRNA into protein. Proteins are also associated with RNA as it
is used for structural,
catalytic, and regulatory purposes.
RNA Processing
Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into
ribosomes,
which are cytoplasmic particles that translate messenger RNA (mRNA) into
polypeptides. The
eukaryotic ribosome is composed of a 605 (large) subunit and a 40S (small)
subunit, which together
form the 80S ribosome. In addition to the 185, 28S, SS, and 5.8S rRNAs,
ribosomes contain from SO
to over 80 different ribosomal proteins, depending on the organism. Ribosomal
proteins are classified
according to which subunit they belong (i.e., L, if associated with the large
60S large subunit or S if
associated with the small 405 subunit). E. coli ribosomes have been the most
thoroughly studied and
2o contain 50 proteins, many of which are conserved in all life forms. The
structures of nine ribosomal
proteins have been solved to less than 3.0D resolution (i.e., S5, S6, 517, Ll,
L6, L9, L12, L14, L30),
revealing common motifs, such as b-a-b protein folds in addition to acidic and
basic RNA-binding
motifs positioned between b-strands. Most ribosomal proteins are believed to
contact rRNA directly
(reviewed in Liljas, A. and Garber, M. (1995) Curr. Opin. Struct. Biol. 5:721-
727; see also Woodson,
S.A. and Leontis, N.B. (1998) Curr. Opin. Struct. Biol. 8:294-300;
Ramakrishnan, V. and White, S.W.
(1998) Trends Biochem. Sci. 23:208-212).
Ribosomal proteins may undergo post-translational modifications or interact
with other
ribosome-associated proteins to regulate translation. For example, the highly
homologous 40S
ribosomal protein S6 kinases (S6K1 and S6K2) play a key role in the regulation
of cell growth by
controlling the biosynthesis of translational components which make up the
protein synthetic apparatus
(including the ribosomal proteins). In the case of S6K1, at least eight
phosphorylation sites are
believed to mediate kinase activation in a hierarchical fashion (Dufner and
Thomas (1999) Exp. Cell.
Res. 253:100-109). Some of the ribosomal proteins, including L1, also function
as translational
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repressors by binding to polycistronic mRNAs encoding ribosomal proteins
(reviewed in Liljas, supra
and Garber, supra).
Recent evidence suggests that a number of ribosomal proteins have secondary
functions
independent of their involvement in protein biosynthesis. These proteins
function as regulators of cell
proliferation and, in some instances, as inducers of cell death. For example,
the expression of human
ribosomal protein Ll3a has been shown to induce apoptosis by arresting cell
growth in the G2/M
phase of the cell cycle. Inhibition of expression of Ll3a induces apoptosis in
target cells, which
suggests that this protein is necessary, in the appropriate amount, for cell
survival. Similar results have
been obtained in yeast where inactivation-of yeast homologues of Ll3a, rp22
and rp23, results in
severe growth retardation and death. A closely related ribosomal protein, L7,
arrests cells in G1 and
also induces apoptosis. Thus, it appears that a subset of ribosomal proteins
may function as cell cycle
checkpoints and compose a new family of cell proliferation regulators.
Mapping of individual ribosomal proteins on the surface of intact ribosomes is
accomplished
using 3D immunocryoelectronmicroscopy, whereby antibodies raised against
specific ribosomal
proteins are visualized. Progress has been made toward the mapping of Ll, L7,
and L12 while the
structure of the intact ribosome has been solved to only 20-25D resolution and
inconsistencies exist
among different crude structures (Frank, J. (1997) Curr. Opin. Struct. Biol.
7:266-272).
Three distinct sites have been identified on the ribosome. The aminoacyl-tRNA
acceptor site
(A site) receives charged tRNAs (with the exception of the initiator-tRNA).
The peptidyl-tRNA site
(P site) binds the nascent polypeptide as the amino acid from the A site is
added to the elongating
chain. Deacylated tRNAs bind in the exit site (E site) prior to their release
from the ribosome. The
structure of the ribosome is reviewed in Stryer, L. (1995) Biochemistry, W.H.
Freeman and Company,
New York NY, pp. 888-9081; Lodish, H. et al. (1995) Molecular Cell BioloQV,
Scientific American
Books, New York NY, pp. 119-138; and Lewin, B (1997) Genes VI, Oxford
University Press, Inc.
New York, NY).
Various proteins are necessary for processing of transcribed RNAs in the
nucleus. Pre-
mRNA processing steps include capping at the 5' end with methylguanosine,
polyadenylating the 3'
end,. and splicing to remove introns. The primary RNA transript from DNA is a
faithful copy of the
gene containing both exon and intron sequences, and the latter sequences must
be cut out of the RNA
3o transcript to produce a mRNA that codes for a protein. This "splicing" of
the mRNA sequence takes
place in the nucleus with the aid of a large, multicomponent ribonucleoprotein
complex known as a
spliceosome. The s,pliceosomal complex is comprised of five small nuclear
ribonucleoprotein particles
(snRNPs) designated Ul, U2, U4, U5, and U6. Each snRNP contains a single
species of snRNA and
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about ten proteins. The RNA components of some snRNPs recognize and base-pair
with intron
consensus sequences. The protein components mediate spliceosome assembly and
the splicing
reaction. Autoantibodies to snRNP proteins are found in the blood of patients
with systemic lupus
erythematosus (Stryer, L. (1995) Biochemistry, W.H. Freeman and Company, New
York NY, p.
863).
Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that
have roles in
splicing, exporting of the mature RNAs to the cytoplasm, and mRNA translation
(Biamonti, G. et al.
(1998) Clip. Exp. Rheumatol. 16:317-326). Some examples of hn.RNPs include the
yeast proteins
Hrplp, involved in cleavage and polyadenylation at the 3' end of the RNA;
Cbp80p, involved in
l0 capping the 5' end of the RNA; and Npl3p, a homolog of mammalian hnRNP A1,
involved in export of
mRNA from the nucleus (Shen, E.C. et al. (1998) Genes Dev. 12:679-691). HnRNPs
have been
shown to be important targets of the autoimmune response in rheumatic diseases
(Biamonti, supra).
Many snRNP and hnRNP proteins are characterized by an RNA recognition motif
(RRM).
(Reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21:5803-5816.) The
RRM is about 80 amino
acids in length and forms four (3-strands and two a,-helices arranged in an a
/(3 sandwich. The RRM
contains a core RNP-1 octapeptide motif along with surrounding conserved
sequences. In addition to
snRNP proteins, examples of RNA-binding proteins which contain the above
motifs include
heteronuclear ribonucleoproteins which stabilize nascent RNA and factors which
regulate alternative
splicing. Alternative splicing factors include developmentally regulated
proteins, specific examples of
which have been identified in lower eukaryotes such as Drosophila melanogaster
and
Caenorhabditis elegans. These proteins play key roles in developmental
processes such as pattern
formation and sex determination, respectively. (See, for example, Hodgkin, J.
et al. (1994)
Development 120:3681-3689.)
The 3' ends of most eukaryote mRNAs are also posttranscriptionally modified by
polyadenylation. Polyadenylation proceeds through two enzymatically distinct
steps: (i) the
endonucleolytic cleavage of nascent mRNAs at cis-acting polyadenylation
signals in the
3 =untranslated (non-coding) region and (ii) the addition of a poly(A) tract
to the 5' mRNA fragment.
The presence of cis-acting RNA sequences is necessary for both steps. These
sequences include 5'-
AAUAAA-3' located 10-30 nucleotides upstream of the cleavage site and a less
well-conserved GU-
or U-rich sequence element located 10-30 nucleotides downstream of the
cleavage site. Cleavage
stimulation factor (CstF), cleavage factor I (CF I), and cleavage factor II
(CF II) are involved in the
cleavage reaction while cleavage and polyadenylation specificity factor (CPSF)
and poly(A)
polymerase (PAP) are necessary for both cleavage and polyadenylation. An
additional enzyme,
1s
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poly(A)-binding protein II (PAB II), promotes poly(A) tract elongation
(Ruegsegger, U. et al. (1996)
J. Biol. Chem. 271:6107-6113; and references within).
TRANSLATION
Correct translation of the genetic code depends upon each amino acid forming a
finkage with
the appropriate transfer RNA (tRNA). The aminoacyl-tRNA synthetases (aaRSs)
are essential
proteins found in all living organisms. The aaRSs are responsible for the
activation and correct
attachment of an amino acid with its cognate tRNA, as the first step in
protein biosynthesis.
Prokaryotic organisms have at least twenty different types of aaRSs, one for
each different amino
acid, while eukaryotes usually have two aaRSs, a cytosolic form and a
mitochondrial form, for each
l0 different amino acid. The 20 aaRS enzymes can be divided into two
structural classes. Class I
enzymes add amino acids to the 2' hydroxyl at the 3' end of tRNAs while Class
II enzymes add amino
acids to the 3' hydroxyl at the 3' end of tRNAs. Each class is characterized
by a distinctive topology
of the catalytic domain. Class I enzymes contain a catalytic domain based on
the nucleotide-binding
Rossman 'fold'. In particular, a consensus tetrapeptide motif is highly
conserved (Prosite Document
PDOC00161, Aminoacyl-transfer RNA synthetases class-I signature). Class I
enzymes are specific
for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine,
methionine, tyrosine, tryptophan,
and valine. Class II enzymes contain a central catalytic domain, which
consists of a seven-stranded
antiparallel B-sheet domain, as well as N- and C- terminal regulatory domains.
Class II enzymes are
separated into two groups based on the heterodimeric or homodimeric structure
of the enzyme; the
latter group is further subdivided by the structure of the N- and C-terminal
regulatory domains
(Hartlein, M. and Cusack, S. (1995) J. Mol. Evol. 40:519-530). Class II
enzymes are specific for
alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine,
proline, serine, and threonine.
Certain aaRSs also have editing functions. IleRS, for example, can misactivate
valine to form
Val-tRNAne, but this product is cleared by a hydrolytic activity that destroys
the mischarged product.
This editing activity is located within a second catalytic site found in the
connective polypeptide 1
region (CP1), a long insertion sequence within the Rossman fold domain of
Class I enzymes
(Schimmel, P. et al. (1998) FASEB J. 12:1599-1609). AaRSs also play a role in
tRNA processing. It
has been shown that mature tRNAs are charged with their respective amino acids
in the nucleus
before export to the cytoplasm, and charging may serve as a quality control
mechanism to insure the
tRNAs are functional (Martinis, S.A. et al. (1999) EMBO J. 18:4591-4596).
Under optimal conditions, polypeptide synthesis proceeds at a rate of
approximately 40 amino
acid residues per second. The rate of misincorporation during translation in
on the order of 10'' and is
primarily the result of aminoacyl-t-RNAs being charged with the incorrect
amino acid. Incorrectly
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charged tRNA are toxic to cells as they result in the incorporation of
incorrect amino acid residues
into an elongating polypeptide. The rate of translation is presumed to be a
compromise between the
optimal rate of elongation and the need for translational fidelity.
Mathematical calculations predict that
10~ is indeed the maximum acceptable error rate for protein synthesis in a
biological system (reviewed
in Stryer, supra; and Watson, J. et al. (1987) The Benjamin/C~mmings
Publishing Co., Inc. Menlo
Park, CA). A particularly error prone aminoacyl-tRNA charging event is the
charging of tRNAG~'
with Gln. A mechanism exits for the correction of this mischarging event which
likely has its origins in
evolution. Gln was among the last of the 20 naturally occurring amino acids
used in polypeptide
synthesis to appear in nature. Gram positive eubacteria, cyanobacteria,
Archeae, and eukaryotic
to organelles possess a noncanonical pathway for the synthesis of Gln-tRNAG~'
based on the
transformation of Glu-tRNAG~' (synthesized by Glu-tRNA synthetase, GIuRS)
using the enzyme Glu-
tRNAG~' amidotransferase (Glu-AdT). The reactions involved in the
transamidation pathway are as
follows (Curnow, A.W. et al. (1997) Nucleic Acids Symposium 36:2-4):
GluRS
tRNAG~" + Glu + ATP -~ Glu-tRNAG~' + AMP + PP;
Glu-AdT
Glu-tRNAG'" + Gln + ATP ~ Gln-tRNAG'" + Glu + ADP + P
A similar enzyme, Asp-tRNA~° amidotransferase, exists in Archaea, which
transforms Asp-
tRNA'~" to Asn-tRNA'~°. Formylase, the enzyme that transforms Met-
tRNA'M~' to fMet-tRNA'M~ in
eubacteria, is likely to be a related enzyme. A hydrolytic activity has also
been identified that destroys
mischarged Val-tRNAne (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609). One
likely scenario
for the evolution of Glu-AdT in primitive life forms is the absence of a
specific glutaminyl-tRNA
synthetase (GlnRS), requiring an alternative pathway for the synthesis of Gln-
tRNAG~". In fact,
deletion of the Glu-AdT operon in Gram positive bacteria is lethal (Curnow,
A.W. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:11819-11826). The existence of GIuRS activity in other
organisms has been
inferred by the high degree of conservation in translation machinery in
nature; however, GIuRS has not
been identified in all organisms, including Homo Sapiens. Such an enzyme would
be responsible for
ensuring translational fidelity and reducing the synthesis of defective
polypeptides.
In addition to their function in protein synthesis, specific aminoacyl tRNA
synthetases also
play roles in cellular fidelity, RNA splicing, RNA trafficking, apoptosis, and
transcriptional and
translational regulation. For example, human tyrosyl-tRNA synthetase can be
proteolytically cleaved
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into two fragments with distinct cytokine activities. The carboxy-terminal
domain exhibits monocyte
and leukocyte chemotaxis activity as well as stimulating production of
myeloperoxidase, tumor
necrosis factor-a, and tissue factor. The N-terminal domain binds to the
interleukin-8 type A receptor
and functions as an interleukin-8-like cytokine. Human tyrosyl-tRNA synthetase
is secreted from
apoptotic tumor cells and may accelerate apoptosis (Wakasugi, K.; and
Schimmel, P. (1999) Science
284:147-151). Mitochondrial Neurospora crassa TyrRS and S. cerevisiae LeuRS
are essential
factors for certain group I intron splicing activities, and human
mitochondrial LeuRS can substitute for
the yeast LeuRS in a yeast null strain. Certain bacterial aaRSs are involved
in regulating their own
transcription or translation (Martinis, supra). Several aaRSs are able to
synthesize diadenosine
l0 oligophosphates, a class of signalling molecules with roles in cell
proliferation, differentiation, and
apoptosis (Kisselev, L.L et al. (1998) FEBS Lett. 427:157-163; Vartanian, A.
et al. (1999) FEBS Lett.
456:175-180).
Autoantibodies against aminoacyl-tRNAs are generated by patients with
autoimmune diseases
such as rheumatic arthritis, dermatomyositis and polymyositis, and correlate
strongly with complicating
interstitial lung disease (1LD) (Freist, W. et al. (1999) Biol. Chem. 380:623-
646; Freist, W. et al.
(1996) Biol. Chem. Hoppe Seyler 377:343-356). These antibodies appear to be
generated in response
to viral infection, and coxsackie virus has been used to induce experimental
viral myositis in animals.
Comparison of aaRS structures between humans and pathogens has been useful in
the design
of novel antibiotics (Schimmel, supra). Genetically engineered aaRSs have been
utilized to allow site-
specific incorporation of unnatural amino acids into proteins in vivo (Liu,
D.R. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:10092-10097).
tRNA Modifications
The modified ribonucleoside, pseudouridine (yr), is present ubiquitously in
the anticodon regions
of transfer RNAs (tRNAs), large and small ribosomal RNAs (rRNAs), and small
nuclear RNAs
(snRNAs). y is the most common of the modified nucleosides (i.e., other than
G, A, U, and C) present
in tRNAs. Only a few yeast tRNAs that are not involved in protein synthesis do
not contain y1
(Cortese, R. et al. (1974) J. Biol. Chem. 249:1103-1108). The enzyme
responsible for the conversion
of uridine to yr, pseudouridine synthase (pseudouridylate synthase), was first
isolated from Salmonella
typhimurium (Arena, F. et al. (1978) Nucleic Acids Res. 5:4523-4536). The
enzyme has since been
isolated from a number of mammals, including steer and mice (Green, C.J. et
al. (1982) J. Biol. Chem.
257:3045-52; and Chen, J. and Patton, J.R. (1999) RNA 5:409-419). tRNA
pseudouridine synthases
have been the most extensively studied members of the family. They require a
thiol donor (e.g.,
cysteine) and a monovalent canon (e.g., ammonia or potassium) for optimal
activity. Additional
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cofactors or high energy molecules (e.g., ATP or GTP) are not required (Green,
supra). Other
eukaryotic pseudouridine synthases have been identified that appear to be
specific for rRNA
(reviewed in Smith, C.M. and Steitz, J.A. (1997) Cell 89:669-672) and a dual-
specificity enzyme has
been identified that uses both tRNA and rRNA substrates (Wrzesinski, J. et al.
(1995) RNA 1:
437-448). The absence of yr in the anticodon loop of tRNAs results in reduced
growth in both
bacteria (Singer, C.E. et al. (1972) Nature New Biol. 238:72-74) and yeast
(Lecointe, F. (1998) J.
Biol. Chem. 273:1316-1323), although the genetic defect is not lethal.
Another ribonucleoside modification that occurs primarily in eukaryotic cells
is the conversion
of guanosine to Nz,N2-dimethylguanosine (m2zG) at position 26 or 10 at the
base of the D-stem. of
cytosolic and mitochondrial tRNAs. This posttranscriptional modification is
believed to stabilize tRNA
structure by preventing the formation of alternative tRNA secondary and
tertiary structures. Yeast
tRNA~p is unusual in that it does not contain this modification. The
modification does not occur in
eubacteria, presumably because the structure of tRNAs in these cells and
organelles is sequence
constrained and does not require posttranscriptional modification to prevent
the formation of
alternative structures (Steinberg, S. and Cedergren, R. (1995) RNA 1:886-891,
and references within).
The enzyme responsible for the conversion of guanosine to mz2G is a 63 kDa S-
adenosylinethionine
(SAM)-dependent tRNA NZ,NZ-dimethyl-guanosine methyltransferase (also referred
to as the TRMI
gene product and herein referred to as TRM) (Edqvist, J. (1995) Biochimie
77:54-61). The enzyme
localizes to both the nucleus and the mitochondria (Li, J-M. et al. (1989) J.
Cell Biol. 109:1411-1419).
Based on studies with TRM from Xenopus laevis, there appears to be a
requirement for base pairing
at positions C11-G24 and G10-C25 immediately preceding the G26 to be modified,
with other
structural features of the tRNA also being required for the proper
presentation of the G26 substrate
(Edqvist. J. et al. (1992) Nucleic Acids Res. 20:6575-6581). Studies in yeast
suggest that cells
carrying a weak ochre tRNA suppressor (sup3-i) are unable to suppress
translation termination in the
absence of TRM activity, suggesting a role for TRM in modifying the frequency
of suppression in
eukaryotic cells (Niederberger, C. et al. (1999) FEBS Lett. 464:67-70), in
addition to the more general
function of ensuring the proper three-dimensional structures for tRNA.
Translation Initiation
Initiation of translation can be divided into three stages. The first stage
brings an initiator
transfer RNA (Met-tRNAf) together with the 40S ribosomal subunit to form the
43S preinitiation
complex. The second stage binds the 435 preinitiation complex to the mRNA,
followed by migration
of the complex to the correct AUG initiation codon. The third stage brings the
60S ribosomal subunit
to the 405 subunit to generate an 80S ribosome at the inititation codon.
Regulation of translation
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primarily involves the first and second stage in the initiation process (V.M.
Pain (1996) Eur. J.
Biochem. 236:747-771 ).
Several initiation factors, many of which contain multiple subunits, are
involved in bringing an
initiator tRNA and the 40S ribosomal subunit together. eIF2, a guanine
nucleotide binding protein,
recruits the initiator tRNA to the 40S ribosomal subunit. Only when eIF2 is
bound to GTP does it
associate with the initiator tRNA. eIF2B, a guanine nucleotide exchange
protein, is responsible for
converting eIF2 from the GDP-bound inactive form to the GTP-bound active form.
Two other
factors, eIFlA and eIF3 bind and stabilize the 40S subunit by interacting with
the 18S ribosomal RNA
and specific ribosomal structural proteins. elF3 is also involved in
association of the 40S ribosomal
subunit with mRNA. The Met-tRNAf, eIFlA, eIF3, and 40S ribosomal subunit
together make up the
43S preinitiation complex (Pain, supra).
eIF2 plays a central role in the maintenance of a rate-limiting step in mRNA
translation. In
this step, eIF2 binds GTP and Met-tRNAi and transfers Met-tRNAi to the 40S
ribosomal subunit. At
the end of the initiation process, GTP bound to eIF2 is hydrolyzed to GDP and
the eIF2.GDP complex
is released from the ribosome. The exchange of GDP bound to elF2 for GTP is a
prerequisite to
binding Met-tRNAi and is mediated by a second initiation factor, elF2B, a
guanine nucleotide-
exchange factor. Phosphorylation of eIF2 on its alpha- subunit converts eIF2
from a substrate of
eIF2B into a competitive inhibitor. Thus, phosphorylation of eIF2 alpha
effectively prevents formation
of the e1F'2.GTP.Met-tRNAi complex and inhibits global protein synthesis.
Phosphorylation of elF2
2o alpha occurs under a variety of conditions including viral infection,
apoptosis, nutrient deprivation,
heme-deprivation, and certain stresses. The 5 =untranslated region of
hepatitis C virus (HCV)
functions as an internal ribosome entry site (IRES) to initiate translation of
HCV proteins.
eIF2Bgamma and eIF2gamma are cellular factors involved in HCV IRES-mediated
translation
(Kimball, S.R. (1999) Int. J. Biochem. Cell Biol. 31:25-29; Webb, B.L. and
Proud, C.G. (1997) Int. J.
Biochem. Cell Biol. 29:1127-1131; Kruger M. et al. (2000) Proc. Natl. Acad.
Sci. U S A
97:8566-8571).
Additional factors are required for binding of the 43S preinitiation complex
to an mRNA
molecule, and the process is regulated at several levels. eIF4F is a complex
consisting of three
proteins: eIF4E, eIF4A, and eIF4G. eIF4E recognizes and binds to the mRNA 5'-
terminal m'GTP
3o cap, eIF4A is a bidirectional RNA-dependent helicase, and elF4G is a
scaffolding polypeptide. eIF4G
has three binding domains. The N-terminal third of eIF4G interacts with eIF4E,
the central third
interacts with eIF4A, and the C-terminal third interacts with eIF3 bound to
the 43S preinitiation
complex. Thus, eIF4G acts as a bridge between the 40S ribosomal subunit and
the mRNA (M.W.
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Hentze (1997) Science 275:500-501).
The ability of eIF4F to initiate binding of the 43S preinitiation complex is
regulated by
structural features of the mRNA. The mRNA molecule has an untranslated region
(UTR) between
the 5' cap and the AUG start codon. In some mRNAs this region forms secondary
structures that
impede binding of the 43S preinitiation complex. The helicase activity of
eIF4A is thought to function
in removing this secondary structure to facilitate binding of the 43S
preinitiation complex (Pain,
supra).
Translation Elongation
Elongation is the process whereby additional amino acids are joined to the
initiator methionine
to form the complete polypeptide chain. The elongation factors EFl a, EFl (3
'y, and EF2 are involved
in elongating the polypeptide chain following initiation. EFl a is a GTP-
binding protein. In EFl a's
GTP-bound form, it brings an aminoacyl-tRNA to the ribosome's A site. The
amino acid attached to
the newly arrived aminoacyl-tRNA forms a peptide bond with the initiatior
methionine. The GTP on
EFl a is hydrolyzed to GDP, and EF1 a -GDP dissociates from the ribosome. EF1
(3 'y binds EFl a -
GDP and induces the dissociation of GDP from EF1 a, allowing EFl a to bind GTP
and a new cycle
to begin.
As subsequent aminoacyl-tRNAs are brought to the ribosome, EF-G, another GTP-
binding
protein, catalyzes the translocation of tRNAs from the A site to the P site
and finally to the E site of
the ribosome. This allows the ribosome and the mRNA to remain attached during
translation.
The MCM domain is found in DNA-dependent ATPases required for the initiation
of
eukaryotic DNA replication. In eukaryotes there is a family of six proteins
that contain this domain,
MCM2 to MCM7 (Hu, B. et al. (1993) Nucleic Acids Res. 21:5289-5293).
Translation Termination
The release factor eRF carries out termination of translation. eRF recognizes
stop codons in
the mRNA, leading to the release of the polypeptide chain from the ribosome.
The apical ectodermal ridge (AER) is an essential structure for vertebrate
limb development.
Wnt3a is expressed during the induction of chick AER. Misexpression of Wnt3a
induces ectopic
expression of AER-specific genes in the limb ectoderm. The genes beta-catenin
and Lefl mimic the
effect of Wnt3a. Blocking the intrinsic Lefl activity disrupts AER formation.
Hence, Wnt3a
functions in AER formation through the beta-catenin/LEFl pathway. In contrast,
neither beta-catenin
nor Lefl affects the Wnt7a-regulated dorsoventral polarity of the limb. Thus,
two related Wnt genes
elicit distinct responses in the same tissues by using different intracellular
pathways (Kengaku, M. et
a1.(1998) Science 280:1274-1277).
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Treacher Collies Syndrome (TCS) is the most common of the human
mandibulofacial
dysostosis disorders. It shows autosomal dominant inheritance and occurs in 1
of 50,000 live births,
with approximately 60% arising from new mutations. TCS symptoms show wide
variability. The
disease is deduced to be a result of interference in the development of the
first and second branchial
arches. The TCS gene, TCOFl, is localized to chromosome Sq31-33.3. There are
ten identified
mutations in TCOFI consisting of nonsense mutations, insertions, deletions, or
splicing mutations that
apparently lead to premature termination of translation. Moreover, all are
unique to each human
family. TCOFI encodes a low complexity protein of 1,411 amino acids, with
repeated motifs that
mirror the organization of its exons. These motifs are shared with nucleolar
trafficking proteins in
other species and are highly phosphorylated by casein kinase. The full-length
TCOFl protein ,
sequence also contains nuclear and nucleolar localization signals and several
polymorphisms. This
data suggests that TCS results from defects in a nucleolar trafficking protein
that is critically required
during human craniofacial development (Wise, C.A. et al. (1997) Proc. Natl.
Acad. Sci. U.S.A.
94:3110-3115).
Breast Cancer
There are more than 180,000 new cases of breast cancer diagnosed each year,
and the
mortality rate for breast cancer approaches 10% of all deaths in females
between the ages of 45-54
(Gish, K. (1999) AWIS Magazine 28:7-10). However the survival rate based on
early diagnosis of
localized breast cancer is extremely high (97%), compared with the advanced
stage of the disease in
which the tumor has spread beyond the breast (22%). Current procedures for
clinical breast
examination are lacking in sensitivity and specificity, and efforts are
underway to develop
comprehensive gene expression profiles for breast cancer that may be used in
conjunction with
conventional screening methods to improve diagnosis and prognosis of this
disease (Perou, C.M. et al.
(2000) Nature 406:747-752).
Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a
woman to
breast cancer and may be passed on from parents to children (Gish, supra).
However, this type of
hereditary breast cancer accounts for only about S% to 9% of breast cancers,
while the vast majority
of breast cancer is due to non-inherited mutations that occur in breast
epithelial cells.
The relationship between expression of epidermal growth factor (EGF) and its
receptor,
EGFR, to human mammary carcinoma has been particularly well studied. (See
Khazaie, K. et al.
(1993) Cancer and Metastasis Rev. 12:2$5-274, and references cited therein for
a review of this
area.) Overexpression of EGFR, particularly coupled with down-regulation of
the estrogen receptor,
is a marker of poor prognosis in breast cancer patients. In addition, EGFR
expression in breast tumor
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metastases is frequently elevated relative to the primary tumor, suggesting
that EGFR is involved in
tumor progression and metastasis. This is supported by accumulating evidence
that EGF has effects
on cell functions related to metastatic potential, such as cell motility,
chemotaxis, secretion and
differentiation. Changes in expression of other members of the erbB receptor
family, of which EGFR
is one, have also been implicated in breast cancer. The abundance of erbB
receptors, such as HER-
2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their
functional importance in
the pathogenesis of the disease, and may therefore provide targets for therapy
of the disease (Bacus,
S. S. et al. (1994) Am. J. Clip. Pathol. 102:513-524). Other known markers of
breast cancer include
a human secreted frizzled protein mRNA that is downregulated in breast tumors;
the matrix G1a
protein which is overexpressed is human breast carcinoma cells; Drgl or RTP, a
gene whose
expression is diminished in colon, breast, and prostate tumors; maspin, a
tumor suppressor gene
downregulated in invasive breast carcinomas; and CaNl9, a member of the S100
protein family, all of
which are down regulated in mammary carcinoma cells relative to normal mammary
epithelial cells
(Zhou, Z. et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990)
Oncogene 5:1391-1395; Ulrix,
W. et al (1999) FEBS Lett 455:23-26; Sager, R. et al. (1996) Curr. Top.
Microbiol. Immunol. 213:51-
64; and Lee, S. W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508).
Cell lines derived from human mammary epithelial cells at various stages of
breast cancer
provide a useful model to study the process of malignant transformation and
tumor progression as it
has been shown that these cell lines retain many of the properties of their
parental tumors for lengthy
culture periods (Wistuba, LI. et al. (1998) Clip. Cancer Res. 4:2931-2938).
Such a model is
particularly useful for comparing phenotypic and molecular characteristics of
human mammary
epithelial cells at various stages of malignant transformation.
Preadipoc a Cells
The most important function of adipose tissue is its ability to store and
release fat during
periods of feeding and fasting. White adipose tissue is the major energy
reserve in periods of excess
energy use, and its primary purpose is mobilization during energy deprivation.
Understanding how the
various molecules regulate adiposity and energy balance in physiological and
pathophysiological
situations may lead to the development of novel therapeutics for human
obesity. Adipose tissue is also
one of the important target tissues for insulin. Adipogenesis and insulin
resistance in type II diabetes
are linked and present intriguing relations. Most patients with type II
diabetes are obese and obesity in
turn causes insulin resistance.
The majority of research in adipocyte biology to date has been done using
transformed mouse
preadipocyte cell fines. The culture condition, which stimulates mouse
preadipocyte differentiation is
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different from that for inducing human primary preadipocyte differentiation.
In addition, primary cells
are diploid and may therefore reflect the in vivo context better than
aneuploid cell lines.
Understanding the gene expression profile during adipogenesis in human will
lead to understanding the
fundamental mechanism of adiposity regulation. Furthermore, through comparing
the gene expression
profiles of adipogenesis between donor with normal weight and donor with
obesity, identification of
crucial genes, potential drug targets for obesity and type II diabetes, will
be possible.
Peroxisome Proliferator-activated Receptor Gamma A~onist
Thiazolidinediones (TZDs) act as agonists for the peroxisome-proliferator-
activated receptor
gamma (PPAR~y), a member of the nuclear hormone receptor superfamily. TZDs
reduce
hyperglycemia, hyperinsulinemia, and hypertension, in part by promoting
glucose metabolism and
inhibiting gluconeogenesis. Roles for PPARy and its agonists have been
demonstrated in a wide range
of pathological conditions including diabetes, obesity, hypertension,
atherosclerosis, polycystic ovarian
syndrome, and cancers such as breast, prostate, liposarcoma, and colon cancer.
The mechanism by which TZDs and other PPARY agonists enhance insulin
sensitivity is not
fully understood, but may involve the ability of PPARy to promote
adipogenesis. When ectopically
expressed in cultured preadipocytes, PPAR~y is a potent inducer of adipocyte
differentiation. TZDs, in
combination with insulin and other factors, can also enhance differentiation
of human preadipocytes in
culture (Adams et al. (1997) J. Clin. Invest. 100:3149-3153). The relative
potency of different TZDs
in promoting adipogenesis in vitro is proportional to both their insulin
sensitizing effects in vivo, and
their ability to bind and activate PPARy in vitro. Interestingly, adipocytes
derived from omental
adipose depots are refractory to the effects of TZDs. It has therefore been
suggested that the insulin
sensitizing effects of TZDs may result from their ability to promote
adipogenesis in subcutaneous
adipose depots (Adams et al., ibid). Further, dominant negative mutations in
the PPARy gene have
been identified in two non-obese subjects with severe insulin resistance,
hypertension, and overt non-
insulin dependent diabetes mellitus (N117DM) (Barroso et al. (1998) Nature
402:880-883).
N11.7DM is the most common form of diabetes mellitus, a chronic metabolic
disease that
affects 143 million people worldwide. NmDM is characterized by abnormal
glucose and lipid
metabolism that result from a combination of peripheral insulin resistance and
defective insulin
secretion. Nll7DM has a complex, progressive etiology and a high degree of
heritability. Numerous
complications of diabetes including heart disease, stroke, renal failure,
retinopathy, and peripheral
neuropathy contribute to the high rate of morbidity and mortality.
At the molecular level, PPARy functions as a ligand activated transcription
factor. In the
presence of ligand, PPARy forms a heterodimer with the retinoid X receptor
(RXR) which then
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activates transcription of target genes containing one or more copies of a
PPARy response element
(PPRE). Many genes important in lipid storage and metabolism contain PPREs and
have been
identified as PPARy targets, including PEPCK, aP2, LPL, ACS, and FAT-P
(Auwerx, J. (1999)
Diabetologia 42:1033-1049). Multiple ligands for PPARy have been identified.
These include a
variety of fatty acid metabolites; synthetic drugs belonging to the TZD class,
such as Pioglitazone and
Rosiglitazone (BRL49653); and certain non-glitazone tyrosine analogs such as
GI262570 and
GW1929. The prostaglandin derivative 15-dPGJ2 is a potent endogenous ligand
for PPARy.
Expression of PPARy is very high in adipose but barely detectable in skeletal
muscle, the
primary site for insulin stimulated glucose disposal in the body. PPARy is
also moderately expressed
in large intestine, kidney, liver, vascular smooth muscle, hematopoietic
cells, and macrophages. The
high expression of PPARy in adipose suggests that the insulin sensitizing
effects of TZDs may result
from alterations in the expression of one or more PPARy regulated genes in
adipose tissue.
Identification of PPAR~y target genes will contribute to better drug design
and the development of
novel therapeutic strategies for diabetes, obesity, and other conditions.
Systematic attempts to identify PPARY target genes have been made in several
rodent models
of obesity and diabetes (Suzuki et al. (2000) Jpn. J. Pharmacol. 84:113-123;
Way et al. (2001)
Endocrinology 142:1269-1277). However, a serious drawback of the rodent gene
expression studies is
that significant differences exist between human and rodent models of
adipogenesis, diabetes, and
obesity (Taylor (1999) Cell 97:9-12; Gregoire et al. (1998) Physiol. Reviews
78:783-809). Therefore,
an unbiased approach to identifying TZD regulated genes in primary cultures of
human tissues is
necessary to fully elucidate the molecular basis for diseases associated with
PPARy activity.
Lung Cancer
Lung cancer is the leading cause of cancer death for men and the second
leading cause of
cancer death for women in the U.S. The vast majority of lung cancer cases are
attributed to smoking
tobacco, and increased use of tobacco products in third world countries is
projected to lead to an
epidemic of lung cancer in these countries. Exposure of the bronchial
epithelium to tobacco smoke
appears to result in changes in tissue morphology, which are thought to be
precursors of cancer. Lung
cancers are divided into four histopathologically distinct groups. Three
groups (squamous cell
carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-
small cell lung cancers
(NSCLCs). The fourth group of cancers is referred to as small cell lung cancer
(SCLC). Collectively,
NSCLCs account for ~70% of cases while SCLCs account for --18% of cases. The
molecular and
cellular biology underlying the development and progression of lung cancer are
incompletely
understood.
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Deletions on chromosome 3 are common in this disease and are thought to
indicate the
presence of a tumor suppressor gene in this region. Activating mutations in K-
ras are commonly
found in lung cancer and are the basis of one of the mouse models for the
disease.
Colorectal Cancer
Colorectal cancer is the second leading cause of cancer deaths in the United
States, and is
thought to be a disease of aging since 90% of the total cases occur in
individuals over the age of 55.
A widely accepted hypothesis is that several mutations must accumulate over
time in an individual
who develops the disease. To understand the nature of gene alterations in
colorectal cancer, a
number of studies have focused on the inherited syndromes. The first, Familial
Adenomatous
l0 Polyposis (FAP), is caused by mutations in the Adenomatous Polyposis Coli
gene (APC), resulting in
truncated or inactive forms of the protein. This tumor suppressor gene has
been mapped to
chromosome Sq. The second known inherited syndrome is hereditary nonpolyposis
colorectal cancer
(HNPCC), which is caused by mutations in mismatch repair genes. Although
hereditary colon cancer
syndromes occur in a small percentage of the population, and most colorectal
cancers are considered
sporadic, knowledge from studies of the hereditary syndromes can be applied
broadly. For instance,
somatic mutations in APC occur in at least 80% of sporadic colon tumors. APC
mutations are
thought to be the initiating event in disease progression. Other mutations
occur subsequently.
Approximately SO% of colorectal cancers contain activating mutations in ras,
while 85% contain
inactivating mutations in p53. Changes in all of these genes lead to gene
expression changes in colon
cancer.
Ovarian Cancer
Ovarian cancer is the leading cause of death from a gynecologic cancer. The
majority of
ovarian can-cers are derived from epithelial cells, and 70% of patients with
epithelial ovarian cancers
present with late-stage disease. Identification of early-stage markers for
ovarian cancer would
significantly increase the survival rate. Some of the molecular events
implicated in ovarian cancer
include mutation of p53 and microsatellite instability.
Additional Diseases and Related Factors
Tangier disease (TD) is a genetic disorder characterized by near absence of
circulating HDL
and the accumulation of cholesterol esters in many tissues, including tonsils,
lymph nodes, liver, spleen,
thymus, and intestine. Low levels of HDL represent a clear predictor of
premature coronary artery
disease and homozygous TD correlates with a four- to six-fold increase in
cardiovascular disease
compared to controls. The major cardioprotective activity of HDL is ascribed
to its role in reverse
cholesterol transport, the flux of cholesterol from peripheral cells such as
tissue macrophages, through
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plasma lipoproteins to the liver. The HDL protein, apolipoprotein AI plays a
major role in this process,
interacting with the cell surface to remove excess cholesterol and
phospholipids. This pathway is
severely impaired in TD. The defect lies in a specific gene, the ABC1
transporter. This gene is a
member of the family of ATP-binding cassette transporters, which utilize ATP
hydrolysis to transport
a variety of substrates across membranes.
The effects upon liver metabolism and hormone clearance mechanisms are
important to
understand the pharmacodynamics of a drug. The human C3A cell line is a clonal
derivative of
HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver
tumor), which was selected
for strong contact inhibition of growth. The use of a clonal population
enhances the reproducibility of
the cells. C3A cells have many characteristics of primary human hepatocytes in
culture: i) expression
of insulin receptor and insulin-like growth factor 1I receptor; ii) secretion
of a high ratio of serum
albumin compared with a-fetoprotein iii) conversion of ammonia to urea and
glutamine; iv)
metabolism of aromatic amino acids; and v) proliferation in glucose-free and
insulin-free medium. The
C3A cell line is now well established as an in vitro model of the mature human
liver (Mickelson et al.
(1995) Hepatology 22:866-875; Nagendra et al. (1997) Am J Physiol 272:6408-
6416).
Dexamethasone (DEX) is a synthetic glucocorticoid used as an anti-inflammatory
or immuno-
suppressive agent. Due to its greater ability to reach the central nervous
system, DEX is usually the
treatment of choice to control cerebral edema. Glucocorticoids are naturally
occurring hormones that
prevent or suppress inflammation and immune responses when administered at
pharmacological doses.
At the molecular level, unbound glucocorticoids readily cross cell membranes
and bind with high
affinity to specific cytoplasmic receptors. Subsequent to binding,
transcription and protein synthesis
are affected. The result can include inhibition of leukocyte infiltration at
the site of inflammation,
interference in the function of mediators of inflammatory response, and
suppression of humoral
immune responses. The anti-inflammatory actions of corticosteroids are thought
to involve
phospholipase A 2 inhibitory proteins, collectively called lipocortins.
Lipocortins, in turn, control the
biosynthesis of potent mediators of inflammation such as prostaglandins and
leukotrienes by inhibiting
the release of the precursor molecule arachidonic acid.
Human aortic endothelial cells (HMVECdNeos) are primary cells derived from the
endothelium of the microvasculature of human skin. HMVECdNeos have been used
as an
3o experimental model for investigating in vitro the role of the endothelium
in human vascular biology.
Activation of the vascular endothelium is considered a central event in a wide
range of both
physiological and pathophysiological processes, such as vascular tone
regulation, coagulation and
thrombosis, atherosclerosis, and inflammation.
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Tumor necrosis factor alpha (TNF-a ) is a pleiotropic cytokine that plays a
central role in
mediation of the inflammatory response through activation of multiple signal
transduction pathways.
TNF-a is produced by activated lymphocytes, macrophages, and other white blood
cells, and is known
to activate endothelial cells. Monitoring the endothelial cell response to TNF-
a at the level of mRNA
expression can provide information necessary for better understanding of both
TNF-a signaling and
endothelial cell biology.
Dendritic cells (DCs), as antigen presenting cells, play a crucial role in the
initiation of the
immune response. DCs can be derived in vitro either from CD34+ bone marrow
precursors (IDCs)
or from peripheral blood monocytic cells (mDCs). In vivo, DCs reside in two
distinct compartments:
the peripheral tissues such as lung, skin, kidney, heart, and intestine; and
in secondary lymphoid organs
such as lymph node, spleen, and Peyer's patches. In the periphery, DCs are
efficient antigen
processing cells but are limited in their capacity to activate naive T cells.
Upon activation (injury,
inflammation, infection), DCs enter their final stage of maturation during
which they downregulate the
capacity to process new antigens, migrate out of the periphery into the
secondary lymphoid organs,
and acquire an extremely potent capacity to activate naive T cells. Factors
such as cross linking the
CD40 surface molecules or the presence of TNF-a can induce this final stage of
maturation.
CD40 is a type I integral membrane glycoprotein belonging to the TNF-receptor
family. It is
expressed on all mature B lymphocytes, dendritic cells, and some epithelial
cells. Antibodies specific
for CD40 molecules can induce proliferation of B cells when presented with IL-
4 or antibodies
specific for CD20 molecules. Also, stimulation of B cells with anti-CD40
antibodies and IL-4 can
induce the switch of immunoglobulin production to the IgE isotype.
Characterization of region-specific gene expression in the human brain
provides a context and
background for molecular neurobiology research in general. Information from
RNA expression in
these tissues may supply insight into the genetic basis of brain structure and
function, which may in
turn become useful in drug target discovery.
Array technology can provide a simple way to explore the expression of a
single polymorphic
gene or the expression profile of a large number of related or unrelated
genes. When the expression
of a single gene is examined, arrays are employed to detect the expression of
a specific gene or its
variants. When an expression profile is examined, arrays provide a platform
for examining which
genes are tissue specific, carrying out housekeeping functions, parts of a
signaling cascade, or
specifically related to a particular genetic predisposition, condition,
disease, or disorder. The potential
application of gene expression profiling is particularly relevant to improving
diagnosis, prognosis, and
treatment of disease. For example, both the levels and sequences expressed in
tissues from subjects
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with diabetes may be compared with the levels and sequences expressed in
normal tissue.
E~ression profiling
Microarrays are analytical tools used in bioanalysis. A microarray has a
plurality of molecules
spatially distributed over, and stably associated with, the surface of a solid
support. Microarrays of
polypeptides, polynucleotides, and/or antibodies have been developed and fmd
use in a variety of
applications, such as gene sequencing, monitoring gene expression, gene
mapping, bacterial
identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays find use is in gene expression
analysis. Array
technology can provide a simple way to explore the expression of a single
polymorphic gene or the
expression profile of a large number of related or unrelated genes. When the
expression of a single
gene is examined, arrays are employed to detect the expression of a specific
gene or its variants.
When an expression profile is examined, arrays provide a platform for
identifying genes that are tissue
specific, are affected by a substance being tested in a toxicology assay, are
part of a signaling
cascade, carry out housekeeping functions, or are specifically related to a
particular genetic
predisposition, condition, disease, or disorder.
There is a need in the art for new compositions, including nucleic acids and
proteins, for the
diagnosis, prevention, and treatment of cell proliferative, neurological,
developmental, and
autoimmune/inflammatory disorders, and infections.
2o SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides, nucleic
acid-associated
proteins, referred to collectively as 'NAAP' and individually as 'NAAP-1,'
'NAAP-2,' 'NAAP-3,'
'NAAP-4,' 'NAAP-5,' 'NAAP-6,' 'NAAP-7,' 'NAAP-8,' 'NAAP-9,' 'NAAP-10,' 'NAAP-
11,'
'NAAP-12,' 'NAAP-13,' 'NAAP-14,' 'NAAP-15,' 'NAAP-16,' 'NAAP-17,' 'NAAP-18,'
'NAAP-
19,' 'NAAP-20,' 'NAAP-21,' 'NAAP-22,' 'NAAP-23,' 'NAAP-24,' 'NAAP-25,' 'NAAP-
26,'
'NAAP-27,' 'NAAP-28,' 'NAAP-29,' 'NAAP-30,' 'NAAP-31,' 'NAAP-32,' 'NAAP-33,'
'NAAP-
34,' 'NAAP-35,' 'NAAP-36,' 'NAAP-37,' 'NAAP-38,' 'NAAP-39,' 'NAAP-40,' 'NAAP-
41,'
'NAAP-42,' 'NAAP-43,' 'NAAP-44,' 'NAAP-45,' 'NAAP-46,' 'NAAP-47,' 'NAAP-48>'
'NAAP-
49,' 'NAAP-50,' 'NAAP-51,' 'NAAP-52,' 'NAAP-53,' 'NAAP-54,' 'NAAP-55,' 'NAAP-
56,'
'NAAP-57,' and 'NAAP-58' and methods for using these proteins and their
encoding polynucleotides
for the detection, diagnosis, and treatment of diseases and medical
conditions. Embodiments also
provide methods for utilizing the purified nucleic acid-associated proteins
and/or their encoding
polynucleotides for facilitating the drug discovery process, including
determination of efficacy, dosage,
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toxicity, and pharmacology. Related embodiments provide methods for utilizing
the purified nucleic
acid-associated proteins and/or their encoding polynucleotides for
investigating the pathogenesis of
diseases and medical conditions.
An embodiment 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 >D NO:1-
58, b) a polypeptide comprising a naturally occurring amino acid sequence at
least 90% identical or at
least about 90% identical to an amino acid sequence selected from the group
consisting of SEQ )D
NO:1-58, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-58, and d) an immunogenic fragment of
a polypeptide
having an amino acid sequence selected from the group consisting of SEQ >D
NO:l-58. Another
embodiment provides an isolated polypeptide comprising an amino acid sequence
of SEQ )D NO:1-58.
Still another embodiment 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 )D NO:1-58, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical or at least about 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-58, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ D7
N0:1-58, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ >D NO:1-58. In another embodiment, the polynucleotide
encodes a polypeptide
selected from the group consisting of SEQ >D NO:1-58. In an alternative
embodiment, the
polynucleotide is selected from the group consisting of SEQ >D N0:59-116.
Still another embodiment 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 )D N0:1-58, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ >D NO:1-58, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ )D NO:1-58, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ >D NO:1-58.
Another embodiment provides a cell transformed with the recombinant
polynucleotide. Yet another
embodiment provides a transgenic organism comprising the recombinant
polynucleotide.
Another embodiment 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
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of SEQ )D NO:1-58, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ B7 NO:1-58, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ 117 NO:l-58, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-58.
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.
Yet another embodiment provides an isolated antibody which 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 )Z7 NO:1-58, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid
sequence selected from the group consisting of SEQ >D NO:1-58, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ >D NO:1-58,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ )D NO:1-58.
Still yet another embodiment provides an isolated polynucleotide selected from
the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:59-116, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical or at least about 90% identical to a
polynucleotide sequence selected
from the group consisting of SEQ )D N0:59-116, 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). In other embodiments, the polynucleotide can comprise at
least about 20, 30, 40,
60, 80, or 100 contiguous nucleotides.
Yet another embodiment provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide being selected from the group consisting of a) a
polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ )D N0:59-
116, b) a polynucleotide
comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ B7 N0:59-116, 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
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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. In a related embodiment, the method can include
detecting the amount of
the hybridization complex. In still other embodiments, the probe can comprise
at least about 20, 30,
40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide being selected from the group consisting of
a) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
>17 N0:59-116, b) a
l0 polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:59-116, 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. In a related embodiment, the method can include detecting
the amount of the
amplified target polynucleotide or fragment thereof.
Another embodiment 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 )D NO:1-58, b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical or at least about 90% identical to
an amino acid sequence
selected from the group consisting of SEQ )D NO:1-58, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-58,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-58, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition can comprise an amino acid sequence selected from
the group consisting
of SEQ )D NO:1-58. Other embodiments provide a method of treating a disease or
condition
associated with decreased or abnormal expression of functional NAAP,
comprising administering to a
patient in need of such treatment the composition.
Yet another embodiment 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:l-58, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
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amino acid sequence selected from the group consisting of SEQ >D NO:l-58, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ll~ NO:1-58, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID N0:1-58. The method comprises a)
exposing a sample
comprising the polypeptide to a compound, and b) detecting agonist activity in
the sample. Another
embodiment provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. Yet another embodiment provides a
method of treating a
disease or condition associated with decreased expression of functional NAAP,
comprising
administering to a patient in need of such treatment the composition.
Still yet another embodiment 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 )D NO:1-58, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical or
at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ
)D NO:1-58, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ >D NO:1-58, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ )17 NO:1-58. The
method comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
antagonist activity in
the sample. Another embodiment provides a composition comprising an antagonist
compound
identified by the method and a pharmaceutically acceptable excipient. Yet
another embodiment
provides a method of treating a disease or condition associated with
overexpression of functional
NAAP, comprising administering to a patient in need of such treatment the
composition.
Another embodiment 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 >D N0:1-58, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ >D NO:1-58, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
)D NO:l-58, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ >D N0:1-58. 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 ~ compound that
specifically binds to the
polypeptide.
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Yet another embodiment 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 >D NO:1-58, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ )D NO:1-58, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
)D N0:1-58, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ >D NO:1-58. 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 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.
Still yet another embodiment 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 )D N0:59-
116, the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
2o polynucleotide in the presence of varying amounts of the compound and in
the absence of the
compound.
Another embodiment 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
>D N0:59-116, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:59-116, 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
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)D N0:59-116, ii) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical or at least about 90% identical to a polynucleotide sequence
selected from the group
consisting of SEQ )D N0:59-116, 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 can comprise a fragment of a
polynucleotide 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.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for full length polynucleotide and
polypeptide
embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog, and the PROTEOME database identification numbers and annotations of
PROTEOME
database homologs, for polypeptide embodiments of the invention. The
probability scores for the
matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide embodiments, 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 embodiments, along with selected fragments of the
polynucleotides.
Table S shows representative cDNA libraries for polynucleotide embodiments.
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
polynucleotides and
polypeptides, along with applicable descriptions, references, and threshold
parameters.
3o DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is
understood that
embodiments of the invention are not limited to the particular machines,
instruments, materials, and
methods described, as these may vary. It is also to be understood that the
terminology used herein is
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for the purpose of describing particular embodiments only, and is not intended
to limit the scope of the
invention.
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 various embodiments of 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
"NAAP" refers to the amino acid sequences of substantially purified NAAP
obtained from
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
NAAP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of NAAP either by
directly interacting with
NAAP or by acting on components of the biological pathway in which NAAP
participates.
An "allelic variant" is an alternative form of the gene encoding NAAP. 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
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.
Each 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 NAAP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as NAAP or a
polypeptide with at least one functional characteristic of NAAP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
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the polynucleotide encoding NAAP, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide encoding
NAAP. 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
NAAP. Deliberate
amino acid substitutions may be made on the basis of one or more similarities
in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the
biological or immunological activity of NAAP 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" can refer to an oligopeptide,
a peptide, a
polypeptide, or a 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~ative amino acid sequence associated with the recited
protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid. Amplification
may be carried out using polymerase chain reaction (PCR) technologies or other
nucleic acid
amplification technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of NAAP. Antagonists may include proteins such as antibodies, anticalins,
nucleic acids,
carbohydrates, small molecules, or any other compound or composition which
modulates the activity of
NAAP either by directly interacting with NAAP or by acting on components of
the biological pathway
in which NAAP participates.
The term "antibody" refers to intact immunoglobulin 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 NAAP 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
Garners that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KL1T). The coupled peptide is then used to immunize
the animal.
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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
immunize 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.
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
ribonucleotide may be replaced by 2'-F or 2'-NHZ), 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 (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
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 "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
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 polynucleotide having 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 S-methyl cytosine, 2'-
deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense molecules may be produced by any method including
chemical synthesis
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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 NAAP, 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, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide" and a "composition
comprising a given
polypeptide" can refer to any composition containing the given polynucleotide
or polypeptide. The
composition may comprise a dry formulation or an aqueous solution.
Compositions comprising
polynucleotides encoding NAAP or fragments of NAAP 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.).
"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 GELV)EW fragment assembly system
(Accekys,
Burlington MA) 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.
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Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
to 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
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
immunological 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
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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 NAAP or a polynucleotide encoding NAAP
which can be
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 about 5 to about 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 SO%) 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 117 N0:59-116 can comprise a region of unique polynucleotide
sequence
that specifically identifies SEQ ID N0:59-116, for example, as distinct from
any other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:59-116
can be employed
in one or more embodiments of methods of the invention, for example, in
hybridization and
amplification technologies and in analogous methods that distinguish SEQ D7
N0:59-116 from related
polynucleotides. The precise length of a fragment of SEQ D7 N0:59-116 and the
region of SEQ ID
N0:59-116 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 ID N0:1-58 is encoded by a fragment of SEQ ll7 N0:59-116. A
fragment of SEQ ll~ NO:1-58 can comprise a region of unique amino acid
sequence that specifically
identifies SEQ ID NO:1-58. For example, a fragment of SEQ ID NO:1-58 can be
used as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ll~ NO:1-58.
The precise length of a fragment of SEQ ID N0:1-58 and the region of SEQ ID
NO:1-58 to which
the fragment corresponds can be determined based on the intended purpose for
the fragment using
one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide 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.
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"Homology" refers to sequence similarity or, alternatively, 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 identical 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 one
or more
computer algorithms or programs known in the art or described herein. For
example, percent identity
can 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.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
which can be used 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://www.ncbi.nlin.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
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
Open Gap: S and Extension Gap: 2 penalties
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Gap x drop-off. 50
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as deFmed 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
l0 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 amino 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 identical 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. The
phrases "percent similarity" and "% similarity," as applied to polypeptide
sequences, refer to the
percentage of residue matches, including identical residue matches and
conservative substitutions,
between at least two polypeptide sequences aligned using a standardized
algorithm. In contrast,
conservative substitutions are not included in the calculation of percent
identity between polypeptide
sequences.
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=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
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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
Open Gap: 11 and Extension Gap: I penalties
Gap x drop-off. SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ll7 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 S0, 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 abifity.
"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
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
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1% (w/v) SDS, and about 100 pg/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 carned 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. and D.W.
Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold
Spring Harbor Press,
Cold Spring Harbor NY, ch. 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 pg/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 acids 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 present in
solution and another nucleic acid 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
polynucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune 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.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of NAAP
which is
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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 fragment
of NAAP 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, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, antibody, or
other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of NAAP. For example,
modulation
l0 may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of NAAP.
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
15 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. For 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
20 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
25 may be pegylated to extend their fifespan in the cell.
"Post-translational modification" of an NAAP 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 NAAP.
30 "Probe" refers to nucleic acids encoding NAAP, their complements, or
fragments thereof,
which are used to detect identical, allelic or related nucleic acids. 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
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acids, usually DNA oligonucleotides, which may be annealed to a target
polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a
DNA polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic
acid, 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, for
example, Sambrook,
J. and D.W. Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed.,
vol. 1-3, Cold Spring
Harbor Press, Cold Spring Harbor NY), Ausubel, F.M. et al. (1999; Short
Protocols in Molecular
Biolo~y, 4'" ed., John Wiley & Sons, New York NY), and Innis, M. et al. (1990;
PCR Protocols, A
Guide to Methods and ApQlications, 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 Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
CA 02462660 2004-04-O1
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nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
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 nucleic acid 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,
l0 by the artificial manipulation of isolated segments of nucleic acids, e.g.,
by genetic engineering
techniques such as those described in Sambrook and Russell (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 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
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 molecule, is composed of the same
linear
sequence of nucleotides as the reference DNA molecule 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 NAAP,
nucleic acids encoding NAAP, or fragments thereof 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,
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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
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 an 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 about 60%
free, preferably at least about 75% free, and most preferably at least about
90% free from other
components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
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 surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" 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
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
limited to animals
and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art. The
nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell,
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by way of deliberate genetic manipulation, such as by microinjection or by
infection with a
recombinant virus. In another embodiment, the nucleic acid can be introduced
by infection with a
recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002)
Science 295:868-872). The
term genetic manipulation does not include classical cross-breeding, or in
vitro fertilization, but rather
is directed to the introduction of a recombinant DNA molecule. The transgenic
organisms
contemplated in accordance with the present invention include bacteria,
cyanobacteria, fungi, plants
and animals. The isolated DNA of the present invention can be introduced into
the host by methods
known in the art, for example infection, transfection, transformation or
transconjugation. Techniques
for transferring the DNA of the present invention into such organisms are
widely known and provided
in references such as Sambrook and Russell (supra).
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
2o polynucleotides due to alternate splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotides 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 or sequence similarity 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 85%,
at least 90%, at least
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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 or sequence similarity over a
certain defined length of one
of the polypeptides.
THE INVENTION
Various embodiments of the invention include new human nucleic acid-associated
proteins
(NAAP), the polynucleotides encoding NAAP, and the use of these compositions
for the diagnosis,
treatment, or prevention of cell proliferative, neurological, developmental,
and
autoimmune/inflammatory disorders, and infections.
Table 1 summarizes the nomenclature. for the full length polynucleotide and
polypeptide
embodiments of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to
a single Incyte project identification number (Incyte Project 117). Each
polypeptide sequence is
denoted by both a polypeptide sequence identification number (Polypeptide SEQ
ID NO:) and an
Incyte polypeptide sequence number (Incyte Polypeptide >17) as shown. Each
polynucleotide
sequence is denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ >l7
NO:) and an Incyte polynucleotide consensus sequence number (Incyte
Polynucleotide ID) as shown.
Column 6 shows the Incyte ID numbers of physical, full length clones
corresponding to the polypeptide
and polynucleotide sequences of the invention. The full length clones encode
polypeptides which have
at least 95% sequence identity to the polypeptide sequences shown in column 3.
Table 2 shows sequences with homology to polypeptide embodiments of the
invention as
identified by BLAST analysis against the GenBank protein (genpept) database
and the PROTEOME
database. Columns 1 and 2 show the polypeptide sequence identification number
(Polypeptide SEQ
ID NO:) and the corresponding Incyte 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 and the PROTEOME database identification
numbers
(PROTEOME ID NO:) of the nearest PROTEOME database homologs. Column 4 shows
the
probability scores for the matches between each polypeptide and its
homolog(s). Column 5 shows the
annotation of the GenBank and PROTEOME database homolog(s) along with relevant
citations where
applicable, all of which are expresslyincorporated 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 Incyte
polypeptide sequence number (Incyte Polypeptide )D) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
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phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Accekys, Burlington
MA). 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 nucleic acid-associated
proteins. For example,
SEQ ID N0:2 is 57% identical, from residue T192 to residue T586, to human DNA
binding protein
(GenBank ID g1020145) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 1.1e-149, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ D7 N0:2 is localized to
the nucleus, binds
DNA, and is a zinc finger protein containing a KRAB domain, as determined by
BLAST analysis
using the PROTEOME database. SEQ B~ N0:2 also contains a KRAB box domain and
14 zinc
forger, C2H2 type, domains as determined by searching for statistically
significant matches in the
hidden Markov model (HMM)-based PFAM database of conserved protein family
domains. (See
Table 3.) Data from BLIMPS, MOTIFS, and other BLAST analyses provide further
corroborative
evidence that SEQ )D N0:2 is a KRAB family zinc finger protein.
In an alternative example, SEQ B7 N0:16 is 93% identical, from residue M1 to
residue 8364,
to chicken transcription factor, LEF-1 (GenBank )D g3258665) as determined by
the Basic Local
2o Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score
is 9.8e-191, which
indicates the probability of obtaining the observed polypeptide sequence
alignment by chance. SEQ
ll~ N0:16 is localized to the nucleus, functions as a DNA-binding protein, and
is a transcriptional
activator, as determined by BLAST analysis using the PROTEOME database. SEQ )D
N0:16 also
contains a HMG (high mobility group) box domain as determined by searching for
statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLllVIPS, and other BLAST analyses
provide further
corroborative evidence that SEQ LD N0:16 is a LEF-1 transcription factor.
In an alternative example, SEQ >D N0:19 is 71% identical from residue H19 to
residue A113,
and 100% identical from residue Ml to residue Y48, to ribosomal protein L27a
(GenBank >D
g550017) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 1.4e-31, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ D7 N0:19 is a component of the
large 60S
ribosomal subunit, and is abnormally expressed in colorectal carcinomas, as
determined by BLAST
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analysis using the PROTEOME database. SEQ 1D N0:19 also contains a ribosomal
protein L15
domain as determined by searching for statistically significant matches in the
hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLIMPS, MOTIFS, and PROF1LESCAN analyses provide further corroborative
evidence that SEQ
>D N0:19 is a ribosomal protein.
In an alternative example, SEQ ID NO:51 is 98% identical, from residue M1 to
residue H477,
to a human transcription factor (GenBank ID 8516381) as determined by the
Basic Local Alignment
Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.5e-266,
which indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ 117 NO:51 also
to has homology to proteins that are localized to the neuronal cells, have DNA-
binding and transcriptional
regulation function, and are fork head proteins, as determined by BLAST
analysis using the
PROTEOME database. SEQ ID NO:S 1 also contains a fork head domain as
determined by searching
for statistically significant matches in the hidden Markov model (HNIlVI)-
based PFAM database of
conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ D7 NO:51
is a fork head
DNA-binding protein.
SEQ )D NO:1, SEQ >D N0:3-15, SEQ >D N0:17-18, SEQ )D N0:20-50, and SEQ ID
N0:52-58 were analyzed and annotated in a similar manner. The algorithms and
parameters for the
analysis of SEQ )D NO:1-58 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled
using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or any
combination of these two
types of sequences. Column 1 lists the polynucleotide sequence identification
number (Polynucleotide
SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number
(Incyte >D) for
each polynucleotide of the invention, and the length of each polynucleotide
sequence in basepairs.
Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA
and/or genomic
sequences used to assemble the full length polynucleotide embodiments, and of
fragments of the
polynucleotides which are useful, for example, in hybridization or
amplification technologies that
identify SEQ m N0:59-116 or that distinguish between SEQ ID N0:59-116 and
related
polynucleotides.
3o The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotides. In addition, the
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polynucleotide fragments described in column 2 may identify sequences derived
from the ENSEMBL
(The Sanger Centre, Cambridge, UK) database (i.e., those sequences including
the designation
"ENST"). Alternatively, the polynucleotide fragments described in column 2 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 polynucleotide fragments
described in column 2
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon
stitching" algorithm. For example, a polynucleotide sequence identified as
FL XXxXXX NI NZ_YYYYI' N3 N4 represents a "stitched" sequence in which XXxXXX
is the
identification number of the cluster of sequences to which the algorithm was
applied, and YYYYY 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
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
F'I,~~LXXXXX_gAAAAA_gBBBBB_1 N is 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 Ret~eq 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, Incyte cDNA coverage redundant with the sequence coverage shown
in
Table 4 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
polynucleotides 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 polynucleotides. 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 NAAP variants. Various embodiments of NAAP
variants
can have at least about 80%, at least about 90%, or at least about 95% amino
acid sequence identity
to the NAAP amino acid sequence, and can contain at least one functional or
structural characteristic
of NAAP.
Various embodiments also encompass polynucleotides which encode NAAP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ >D N0:59-116, which encodes NAAP. The
polynucleotide
sequences of SEQ ID N0:59-116, 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 variants of a polynucleotide encoding NAAP. In
particular,
such a variant polynucleotide will have at least about 70%, or alternatively
at least about 85%, or even
at least about 95 % polynucleotide sequence identity to a polynucleotide
encoding NAAP. A particular
aspect of the invention encompasses a variant of a polynucleotide comprising a
sequence selected
from the group consisting of SEQ ID N0:59-116 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 N0:59-116. Any one of
the polynucleotide
variants described above can encode a polypeptide which contains at least one
functional or structural
characteristic of NAAP.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide encoding NAAP. A splice variant may have portions which have
significant sequence
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identity to a polynucleotide encoding NAAP, but will generally have a greater
or lesser number of
polynucleotides due to additions or deletions of blocks of sequence arising
from alternate splicing of
exons during mRNA processing. A splice variant may have less than about 70%,
or alternatively less
than about 60%, or alternatively less than about 50% polynucleotide sequence
identity to a
polynucleotide encoding NAAP over its entire length; however, portions of the
splice variant will have
at least about 70%, or alternatively at least about 85%, or alternatively at
least about 95%, or
alternatively 100% polynucleotide sequence identity to portions of the
polynucleotide encoding NAAP.
For example, a polynucleotide comprising a sequence of SEQ ID NO:105 and a
polynucleotide
comprising a sequence of SEQ )D NO:l 10 are splice variants of each other. Any
one of the splice
variants described above can encode a polypeptide which contains at least one
functional or structural
characteristic of NAAP.
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 NAAP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
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 NAAP, and all such variations
are to be considered as
being specifically disclosed.
Although polynucleotides which encode NAAP and its variants are generally
capable of
hybridizing to polynucleotides encoding naturally occurring NAAP under
appropriately selected
conditions of stringency, it may be advantageous to produce polynucleotides
encoding NAAP 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 NAAP 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.
3o The invention also encompasses production of polynucleotides which encode
NAAP and
NAAP derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic polynucleotide 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
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introduce mutations into a polynucleotide encoding NAAP or any fragment
thereof.
Embodiments of the invention can also include polynucleotides that are capable
of hybridizing
to the claimed polynucleotides, and, in particular, to those having the
sequences shown in SEQ ID
N0:59-116 and fragments thereof, under various conditions of stringency (Wahl,
G.M. and S.L.
Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol. 152:507-S11).
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
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OIL, Taq polymerase
(Applied
l0 Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway
NJ), or combinations
of polymerases and proofreading exonucleases such as those found in the
ELONGASE amplification
system (Invitrogen, Carlsbad CA). 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 carried out using either the ABI 373 or 377 DNA sequencing
system (Applied
Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences),
or other
systems known in the art. The resulting sequences are analyzed using a variety
of algorithms which
are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995)
Molecular Biology and
Biotechnology, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding NAAP 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 (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 (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 (Lagerstrom, M. et al. (1991) PCR Methods
Applic. 1:111-119). In
this method, multiple restriction enzyme digestions and ligations 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
(Parker, J.D. et al.
(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested
primers, and
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PROMOTERF>NDER 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 30 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-
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, polynucleotides or fragments thereof
which encode
NAAP may be cloned in recombinant DNA molecules that direct expression of
NAAP, or fragments
or functional equivalents thereof, in appropriate host cells. Due to the
inherent degeneracy of the
genetic code, other polynucleotides which encode substantially the same or a
functionally equivalent
polypeptides may be produced and used to express NAAP.
The polynucleotides of the invention can be engineered using methods generally
known in the
art in order to alter NAAP-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
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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 NAAP, such as its biological or enzymatic
activity or its ability to bind to
other molecules or compounds. DNA shuffling is a process by which a library of
gene variants is
produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
selection or screening procedures that identify those gene variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selectionlscreening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular
evolution. For example, fragments of a single gene containing random point
mutations may be
recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
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, polynucleotides encoding NAAP may be synthesized, in
whole or in
part, using one or more chemical methods well known in the art (Caruthers,
M.H. et al. (1980)
Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232).
Alternatively, NAAP itself or a fragment thereof may be synthesized using
chemical methods known
in the art. For example, peptide synthesis can be performed using various
solution-phase or
solid-phase techniques (Creighton, T. (1984) Proteins, Structures and
Molecular Properties, WH
Freeman, New York NY, pp. SS-60; Roberge, J.Y. et al. (1995) Science 269:202-
204). Automated
synthesis may be achieved using the ABI 431A peptide synthesizer (Applied
Biosystems).
Additionally, the amino acid sequence of NAAP, 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 (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
(Creighton, supra, pp. 28-53).
In order to express a biologically active NAAP, the polynucleotides encoding
NAAP 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
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inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotides encoding
NAAP. Such elements may vary in their strength and specificity. Specific
initiation signals may also
be used to achieve more eif cient translation of polynucleotides encoding
NAAP. Such signals include
the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In
cases where a
polynucleotide sequence encoding NAAP 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 and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used (Scharf, D. et
al. ( 1994) Results Probl.
Cell Differ. 20:125-162).
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing polynucleotides encoding NAAP and appropriate
transcriptional and translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination (Sambrook and Russell, supra, ch. 1-4, and
8; Ausubel et al.,
supra, ch. 1, 3, and 15).
A variety of expression vector/host systems may be utilized to contain and
express
polynucleotides encoding NAAP. 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 (Sambrook and Russell,
supra; Ausubel et al.,
supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509;
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; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of
Science and
TechnoloQV (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T.
Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659; 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 polynucleotides to the
targeted organ, tissue, or
cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu,
M. et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6340-6344; Butler, R.M. et al. (1985) Nature 317:813-
815; McGregor, D.P.
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et al. (1994) Mol. Immunol. 31:219-226; 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 polynucleotides encoding NAAP. For example, routine
cloning, subcloning,
and propagation of polynucleotides encoding NAAP can be achieved using a
multifunctional E. coli
vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid
(Invitrogen).
Ligation of polynucleotides encoding NAAP 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
to sequencing, single strand rescue with helper phage, and creation of nested
deletions in the cloned
sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-
5509). When large
quantities of NAAP are needed, e.g. for the production of antibodies, vectors
which direct high level
expression of NAAP 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 NAAP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pa
storis. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign polynucleotide sequences into the host genome for stable
propagation (Ausubel et al.,
2o supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer,
C.A. et al. (1994)
Bio/Technology 12:181-184).
Plant systems may also be used for expression of NAAP. Transcription of
polynucleotides
encoding NAAP 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 may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue,
R. et al. (1984)
Science 224:838-843; 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
(The McGraw Hill Yearbook of Science and Technology (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, polynucleotides encoding
NAAP may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
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sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses NAAP in host cells (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 (Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355).
For long term production of recombinant proteins in mammalian systems, stable
expression of
NAAP in cell lines is preferred. For example, polynucleotides encoding NAAP
can be transformed
into cell lines using expression vectors which may contain viral origins of
replication and/or
endogenous expression elements and a selectable marker gene on the same or on
a separate vector.
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 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
(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, dhfr confers
resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively (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
hisD, which alter cellular
requirements for metabolites (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), (3-
glucuronidase and its substrate (3-glucuronide, or luciferase and its
substrate luciferin 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 (Rhodes, C.A. (1995)
Methods Mol. Biol. 55:121-131).
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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 NAAP is inserted within a marker gene sequence,
transformed cells containing
polynucleotides encoding NAAP can be identified by the absence of marker gene
function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding
NAAP 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 polynucleotide encoding NAAP and that
express NAAP
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 or 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.
Immunological methods for detecting and measuring the expression of NAAP 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 NAAP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art
(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 I-m_m__unoloQV, Greene
Pub. Associates and Wiley-
Interscience, New York NY; Pound, J.D. (1998) Immunochemical 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 NAAP
include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, polynucleotides encoding NAAP, 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 polymerase
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
Biosciences, 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
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well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with polynucleotides encoding NAAP 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 NAAP may be designed to contain signal sequences
which direct
secretion of NAAP 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 polynucleotides or to process the expressed protein in the desired
fashion. Such modifications
l0 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
polynucleotides
encoding NAAP may be ligated to a heterologous sequence resulting in
translation of a fusion protein
in any of the aforementioned host systems. For example, a chimeric NAAP
protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of NAAP 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-
transferase (GST), maltose
binding protein (MBP), thioredoxin (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
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 NAAP encoding sequence and the heterologous protein
sequence, so that NAAP
may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein
expression and purification are discussed in Ausubel et al. (supra, ch. 10 and
16). A variety of
commercially available kits may also be used to facilitate expression and
purification of fusion proteins.
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In another embodiment, synthesis of radiolabeled NAAP may be achieved in vitro
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 precursor, for
example, 35S-methionine.
NAAP, fragments of NAAP, or variants of NAAP may be used to screen for
compounds
that specifically bind to NAAP. One or more test compounds may be screened for
specific binding to
NAAP. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test
compounds can be screened
for specific binding to NAAP. Examples of test compounds can include
antibodies, anticalins,
oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
In related embodiments, variants of NAAP can be used to screen for binding of
test
compounds, such as antibodies, to NAAP, a variant of NAAP, or a combination of
NAAP and/or one
or more variants NAAP. In an embodiment, a variant of NAAP can be used to
screen for
compounds that bind to a variant of NAAP, but not to NAAP having the exact
sequence of a
sequence of SEQ ID NO:1-58. NAAP variants used to perform such screening can
have a range of
about 50% to about 99% sequence identity to NAAP, with various embodiments
having 60%, 70%,
75%, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to
NAAP can be
closely related to the natural ligand of NAAP, e.g., a ligand or fragment
thereof, a natural substrate, a
structural or functional mimetic, or a natural binding partner (Coligan, J.E.
et al. (1991) Current
Protocols in ImmunoloQV 1(2):Chapter S). In another embodiment, the compound
thus identified can
be a natural ligand of a receptor NAAP (Howard, A.D. et al. (2001) Trends
Pharmacol. Sci.22:132-
140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
In other embodiments, a compound identified in a screen for specific binding
to NAAP can be
closely related to the natural receptor to which NAAP binds, at least a
fragment of the receptor, or a
fragment of the receptor including all or a portion of the ligand binding site
or binding pocket. For
example, the compound may be a receptor for NAAP which is capable of
propagating a signal, or a
decoy receptor for NAAP which is not capable of propagating a signal
(Ashkenazi, A. and V.M. Divit
(1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends
Immunol. 22:328-336).
The compound can be rationally designed using known techniques. Examples of
such techniques
include those used to construct the compound etanercept (ENBREL; Amgen Inc.,
Thousand Oaks
CA), which is efficacious for treating rheumatoid arthritis in humans.
Etanercept is an engineered p75
tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human
IgG1 (Taylor, P.C. et al.
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(2001) Curr. Opin. Immunol. 13:611-616).
In one embodiment, two or more antibodies having similar or, alternatively,
different
specificities can be screened for specific binding to NAAP, fragments of NAAP,
or variants of .
NAAP. The binding specificity of the antibodies thus screened can thereby be
selected to identify
particular fragments or variants of NAAP. In one embodiment, an antibody can
be selected such that
its binding specificity allows for preferential identification of specific
fragments or variants of NAAP.
In another embodiment, an antibody can be selected such that its binding
specificity allows for
preferential diagnosis of a specific disease or condition having increased,
decreased, or otherwise
abnormal production of NAAP.
In an embodiment, anticalins can be screened for specific binding to NAAP,
fragments of
NAAP, or variants of NAAP. Anticalins are ligand-binding proteins that have
been constructed based
on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:8177-
8184; Skerra, A.
(2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can
include a beta-barrel
having eight antiparallel beta-strands, which supports four loops at its open
end. These loops form the
natural ligand-binding site of the lipocalins, a site which can be re-
engineered in vitro by amino acid
substitutions to impart novel binding specificities. The amino acid
substitutions can be made using
methods known in the art or described herein, and can include conservative
substitutions (e.g.,
substitutions that do not alter binding specificity) or substitutions that
modestly, moderately, or
significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to,
stimulate, or inhibit
NAAP involves producing appropriate cells which express NAAP, either as a
secreted protein or on
the cell membrane. Preferred cells can include cells from mammals, yeast,
Drosophila, or E. coli.
Cells expressing NAAP or cell membrane fractions which contain NAAP are then
contacted with a
test compound and binding, stimulation, or inhibition of activity of either
NAAP or the 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
NAAP, either in solution
or affixed to a solid support, and detecting the binding of NAAP 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.
An assay can be used to assess the ability of a compound to bind to its
natural ligand and/or to
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inhibit the binding of its natural ligand to its natural receptors. Examples
of such assays include radio-
labefing assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be
introduced into a polypeptide
compound (such as a receptor) to improve or alter its ability to bind to its
natural ligands (Matthews,
D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related
embodiment, one or more amino
acid substitutions can be introduced into a polypeptide compound (such as a
ligand) to improve or alter
its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells
(1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-
10988).
NAAP, fragments of NAAP, or variants of NAAP may be used to screen for
compounds
l0 that modulate the activity of NAAP. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for NAAP
activity, wherein NAAP is combined with at least one test compound, and the
activity of NAAP in the
presence of a test compound is compared with the activity of NAAP in the
absence of the test
compound. A change in the activity of NAAP in the presence of the test
compound is indicative of a
compound that modulates the activity of NAAP. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising NAAP under conditions suitable for
NAAP activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of
NAAP 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 NAAP 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 (March, J.D.
(1996) Clin. 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 transferred to
pseudopregnant dams, and
the resulting chimeric progeny are genotyped and bred to produce heterozygous
or homozygous
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strains. Transgenic animals thus generated may be tested with potential
therapeutic or toxic agents.
Polynucleotides encoding NAAP may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding NAAP 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 NAAP 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 overexpress NAAP, e.g., by secreting NAAP 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
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of NAAP and nucleic acid-associated proteins. In addition, examples of
tissues expressing
NAAP can be found in Table 6 and can also be found in Example XI. Therefore,
NAAP appears to
play a role in cell proliferative, neurological, developmental, and
autoimmune/inflammatory disorders,
and infections. In the treatment of disorders associated with increased NAAP
expression or activity,
it is desirable to decrease the expression or activity of NAAP. In the
treatment of disorders
associated with decreased NAAP expression or activity, it is desirable to
increase the expression or
activity of NAAP.
Therefore, in one embodiment, NAAP 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 NAAP. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myeloflbrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a
cancer of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder,
ganglia, gastrointestinal
tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,
penis, prostate, salivary glands,
skin, spleen, testis, thymus, thyroid, and uterus; a neurological disorder
such as epilepsy, ischemic
~1
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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 disorder of the central nervous system, cerebral
palsy, a neuroskeletal
disorder, an autonomic nervous system disorder, a cranial nerve disorder, a
spinal cord disease,
muscular dystrophy and other neuromuscular disorder, a peripheral nervous
system disorder,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathy, myasthenia
gravis, periodic paralysis, a mental disorder including mood, anxiety, and
schizophrenic disorder,
seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic
neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder; a
developmental disorder such as renal tubular acidosis, anemia, Cushing's
syndrome, achondroplastic
dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR syndrome
(Wiltns' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(A1DS), 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,
3o glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis,
hypereosinophilia, 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
72
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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; and an
infection, such as those
caused by a viral agent classified as adenovirus, arenavirus, bunyavirus,
calicivirus, coronavirus,
filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus,
papovavirus,
paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or
togavirus; an infection
caused by a bacterial agent classified as pneumococcus, staphylococcus,
streptococcus, bacillus,
corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella,
kingella, haemophilus,
legionella, bordetella, gram-negative enterobacterium including shigella,
salmonella, or campylobacter,
pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium,
actinomyces,
mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; an
infection caused by a fungal
agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus,
coccidioides, malasezzia,
histoplasma, or other mycosis-causing fungal agent; and an infection caused by
a parasite classified as
plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma,
toxoplasma,
pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue
nematode such as
trichinella, intestinal nematode such as ascaris, lymphatic filarial nematode,
trematode such as
schistosoma, and cestode such as tapeworm.
In another embodiment, a vector capable of expressing NAAP 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 NAAP including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
NAAP 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 NAAP including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of NAAP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of NAAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of NAAP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of NAAP.
Examples of such
disorders include, but are not limited to, those cell proliferative,
neurological, developmental, and
autoimmune/inflammatory disorders, and infections described above. In one
aspect, an antibody which
specifically binds NAAP 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 NAAP.
73
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In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding NAAP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of NAAP including, but not limited to, those
described above.
In other embodiments, any protein, agonist, antagonist, antibody,
complementary sequence, or
vector embodiments may be administered in combination with other appropriate
therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made
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 or prevention of the
various disorders described
above. Using this approach, one may be able to achieve therapeutic efficacy
with lower dosages of
to each agent, thus reducing the potential for adverse side effects.
An antagonist of NAAP may be produced using methods which are generally known
in the
art. In particular, purified NAAP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind NAAP.
Antibodies to NAAP 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. In an embodiment, neutralizing
antibodies (i.e., those
which inhibit dimer formation) can be used therapeutically. Single chain
antibodies (e.g., from camels
or llamas) may be potent enzyme inhibitors and may have application in the
design of peptide mimetics,
and in the development of immuno-adsorbents and biosensors (Muyldermans, S.
(2001) J. Biotechnol.
74:277-302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
dromedaries, llamas, humans, and others may be immunized by injection with
NAAP 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, oil emulsions, KLH,
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
NAAP 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 substantially identical to a portion of the amino acid sequence
of the natural protein.
Short stretches of NAAP amino acids may be fused with those of another
protein, such as KLH, and
74
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antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to NAAP 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 EBV-
hybridoma
technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Immunol. Methods
81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
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 (Morrison, S.L. et
al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608;
Takeda, 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 art, to produce NAAP-specific
single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic composition,
may be generated by chain
shuffling from random combinatorial immunoglobulin libraries (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 irnmunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature (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 NAAP may also be
generated.
For example, such fragments include, but are not limited to, F(ab')Z 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 may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity
(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 either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
NAAP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering NAAP epitopes is generally used, but a competitive
binding assay may also be
employed (Pound, supra).
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Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for NAAP. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of NAAP-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 NAAP epitopes, represents the average affinity, or avidity, of the
antibodies for NAAP. The
Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
NAAP epitope, represents a true measure of affinity. High-affinity antibody
preparations with K
ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in
which the NAAP-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka
ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of NAAP, preferably in active
form, from the
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 NAAP-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available
(Catty, supra; Coligan et al.,
supra).
In another embodiment of the invention, polynucleotides encoding NAAP, 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
NAAP. Such technology is well known in the art, and antisense oligonucleotides
or larger fragments
can be designed from various locations along the coding or control regions of
sequences encoding
NAAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, 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 (Slater, J.E. et
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al. (1998) J. Allergy Clip. Immunol. 102:469-475; Scanlon, K.J. et al. (1995)
9:1288-1296). Antisense
sequences can also be introduced intracellularly through the use of viral
vectors, such as retrovirus and
adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et
al., supra; Uckert, W.
and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery
mechanisms include
liposome-derived systems, artificial viral envelopes, and other systems known
in the art (Rossi, J.J.
(1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci.
87:1308-1315; Moms,
M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
In another embodiment of the invention, polynucleotides encoding NAAP may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene 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.,
2o against human retroviruses, 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
Trypanosoma cruzi). In
the case where a genetic deficiency in NAAP expression or regulation causes
disease, the expression
of NAAP from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
NAAP are treated by constructing mammalian expression vectors encoding NAAP
and introducing
these vectors by mechanical means into NAAP-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 transfection, (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.
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Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of NAAP include,
but are not
limited to, the PCDNA 3.1, EPTTAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
w
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
NAAP
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and 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
l0 (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-
REX plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding NAAP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
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.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to NAAP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding NAAP 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 vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) 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
~s
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al. (1998) J. Virol. 72:9873-9880). U.5. 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 an embodiment, an adenovirus-based gene therapy delivery system is used to
deliver
polynucleotides encoding NAAP to cells which have one or more genetic
abnormalities with respect to
the expression of NAAP. 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
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).
In another embodiment, a herpes-based, gene therapy delivery system is used to
deliver
polynucleotides encoding NAAP to target cells which have one or more genetic
abnormalities with
respect to the expression of NAAP. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing NAAP 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.5. 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 and use of recombinant HSV strains deleted
for ICP4, ICP27 and
ICP22. For HSV vectors, see also Goins, W.F. et al. (1999; J. Virol. 73:519-
532) and Xu, H. et al.
(1994; Dev. Biol. 163:152-161). The manipulation of cloned herpesvirus
sequences, the generation of
recombinant virus following the transfection of multiple plasmids containing
different segments of the
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large herpesvirus genomes, the growth and propagation of herpesvirus, and the
infection of cells with
herpesvirus are techniques well known to those of ordinary skill in the art.
In another embodiment, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding NAAP 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
NAAP into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
NAAP-coding RNAs and the synthesis of high levels of NAAP in vector transduced
cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of NAAP 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 (Gee, J.E. et al. (1994) in Huber, B.E. and
B.I. Carr, Molecular and
ImmunoloQic Approaches, Futura 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,
so
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engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of RNA molecules encoding NAAP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning 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 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
molecules encoding
NAAP. Such DNA sequences may be incorporated into a wide variety of 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 all 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, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
In other embodiments of the invention, the expression of one or more selected
polynucleotides
of the present invention can be altered, inhibited, decreased, or silenced
using RNA interference
(RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art.
RNAi is a post-
transcriptional mode of gene silencing in which double-stranded RNA (dsRNA)
introduced into a
targeted cell specifically suppresses the expression of the homologous gene
(i.e., the gene bearing the
sequence complementary to the dsRNA). This effectively knocks out or
substantially reduces the
expression of the targeted gene. PTGS can also be accomplished by use of DNA
or DNA fragments
as well. RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-
811) and Gura, T.
(2000; Nature 404:804-808). PTGS can also be initiated by introduction of a
complementary segment
of DNA into the selected tissue using gene delivery and/or viral vector
delivery methods described
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herein or known in the art.
RNAi can be induced in mammalian cells by the use of small interfering RNA
also known as
siRNA. SiRNA are shorter segments of dsRNA (typically about 21 to 23
nucleotides in length) that
result in vivo from cleavage of introduced dsRNA by the action of an
endogenous ribonuclease.
SiRNA appear to be the mediators of the RNAi effect in mammals. The most
effective siRNAs
appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs. The use of
siRNA for inducing
RNAi in mammalian cells is described by Elbashir, S.M. et al. (2001; Nature
411:494-498).
SiRNA can either be generated indirectly by introduction of dsRNA into the
targeted cell, or
directly by mammalian transfection methods and agents described herein or
known in the art (such as
liposome-mediated transfection, viral vector methods, or other polynucleotide
delivery/introductory
methods). Suitable SiRNAs can be selected by examining a transcript of the
target polynucleotide
(e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and
recording the
occurrence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as
potential siRNA target sites,
with sequences having a 21 nucleotide length being preferred. Regions to be
avoided for target
siRNA sites include the S' and 3' untranslated regions (UTRs) and regions near
the start codon (within
75 bases), as these may be richer in regulatory protein binding sites. UTR-
binding proteins and/or
translation initiation complexes may interfere with binding of the siRNP
endonuclease complex. The
selected target sites for siRNA can then be compared to the appropriate genome
database (e.g.,
human, etc.) using BLAST or other sequence comparison algorithms known in the
art. Target
sequences with significant homology to other coding sequences can be
eliminated from consideration.
The selected SiRNAs can be produced by chemical synthesis methods known in the
art or by in vitro
transcription using commercially available methods and kits such as the
SILENCER siRNA
construction kit (Ambion, Austin TX).
In alternative embodiments, long-term gene silencing and/or RNAi effects can
be induced in
selected tissue using expression vectors that continuously express siRNA. This
can be accomplished
using expression vectors that are engineered to express hairpin RNAs (shRNAs)
using methods
known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-
553; and Paddison, P.J.
et al. (2002) Genes Dev. 16:948-958). In these and related embodiments, shRNAs
can be delivered to
target cells using expression vectors known in the art. An example of a
suitable expression vector for
delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once
delivered to the
target tissue, shRNAs are processed in vivo into siRNA-like molecules capable
of carrying out gene-
specific silencing.
In various embodiments, the expression levels of genes targeted by RNAi or
PTGS methods
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can be determined by assays for mRNA and/or protein analysis. Expression
levels of the mRNA of a
targeted gene, can be determined by northern analysis methods using, for
example, the
NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real
time PCR
methods; and by other RNA/polynucleotide assays known in the art or described
herein. Expression
levels of the protein encoded by the targeted gene can be determined by
Western analysis using
standard techniques known in the art.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding NAAP. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
to 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 NAAP
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding NAAP may be therapeutically useful, and in the treatment of disorders
associated with
decreased NAAP expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding NAAP may be therapeutically useful.
In various embodiments, one or more 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 NAAP 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
NAAP 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 of the
polynucleotide encoding NAAP. 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
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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.
l0 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.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art (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 ReminQton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of NAAP,
antibodies to NAAP, and mimetics, agonists, antagonists, or inhibitors of
NAAP.
In various embodiments, the compositions described herein, such as
pharmaceutical
compositions, may be administered by any number of routes including, but not
limited to, oral,
intravenous, intramuscular, intra-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 delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
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proteins), recent developments in the field of pulmonary delivery 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 allows 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 NAAP or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, NAAP 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
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
NAAP or fragments thereof, antibodies of NAAP, and agonists, antagonists or
inhibitors of NAAP,
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 LDSO/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 /.cg 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 NAAP may be used for
the
diagnosis of disorders characterized by expression of NAAP, or in assays to
monitor patients being
treated with NAAP or agonists, antagonists, or inhibitors of NAAP. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
assays for NAAP include methods which utilize the antibody and a label to
detect NAAP 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 NAAP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
NAAP expression. Normal
or standard values for NAAP expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to NAAP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of NAAP
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, polynucleotides encoding NAAP may be
used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotides, 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 NAAP may be correlated
with disease. The
diagnostic assay may be used to determine absence, presence, and excess
expression of NAAP, and
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to monitor regulation of NAAP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotides,
including genomic sequences, encoding NAAP or closely related molecules may be
used to identify
nucleic acid sequences which encode NAAP. The specificity of the probe,
whether it is made from a
highly specific 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 NAAP, 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 NAAP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ )D
N0:59-116 or
from genomic sequences including promoters, enhancers, and introns of the NAAP
gene.
Means for producing specific hybridization probes for polynucleotides encoding
NAAP include
the cloning of polynucleotides encoding NAAP or NAAP derivatives into vectors
for the production of
mRNA probes. Such vectors are known in the art, are commercially available,
and may be used to
synthesize RNA probes in vitro 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 355, or by enzymatic
labels, such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling systems, and the
like.
Polynucleotides encoding NAAP may be used for the diagnosis of disorders
associated with
expression of NAAP. Examples of such disorders include, but are not limited
to, a cell profiferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a
cancer of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder,
ganglia, gastrointestinal
tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,
penis, prostate, salivary glands,
skin, spleen, testis, thymus, thyroid, and uterus; 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, 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
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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 disorder of the central nervous system, cerebral
palsy, a neuroskeletal
disorder, an autonomic nervous system disorder, a cranial nerve disorder, a
spinal cord disease,
muscular dystrophy and other neuromuscular disorder, a peripheral nervous
system disorder,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathy, myasthenia
gravis, periodic paralysis, a mental disorder including mood, anxiety, and
schizophrenic disorder,
seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic
neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder; a
developmental disorder such as renal tubular acidosis, anemia, Cushing's
syndrome, achondroplastic
dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR syndrome
(Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation),
Smith-Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; an
autoimmune/intlammatory 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
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, 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 helininthic infections, and trauma; and an
infection, such as those
caused by a viral agent classified as adenovirus, arenavirus, bunyavirus,
calicivirus, coronavirus,
filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus,
papovavirus,
paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or
togavirus; an infection
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caused by a bacterial agent classified as pneumococcus, staphylococcus,
streptococcus, bacillus,
corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella,
kingella, haemophilus,
legionella, bordetella, gram-negative enterobacterium including shigella,
salinonella, or campylobacter,
pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium,
actinomyces,
mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; an
infection caused by a fungal
agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus,
coccidioides, malasezzia,
histoplasma, or other mycosis-causing fungal agent; and an infection caused by
a parasite classified as
plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosome,
toxoplasma,
pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue
nematode such as
to trichinella, intestinal nematode such as ascaris, lymphatic filarial
nematode, trematode such as
schistosoma, and cestode such as tapeworm. Polynucleotides encoding NAAP 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 NAAP expression. Such qualitative or quantitative
methods are well known
in the art.
In a particular embodiment, polynucleotides encoding NAAP may be used in
assays that
detect the presence of associated disorders, particularly those mentioned
above. Polynucleotides
complementary to sequences encoding NAAP 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
polynucleotides encoding NAAP
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 regimen 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
NAAP, 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 NAAP, 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 for a disorder.
Deviation from standard
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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.
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
to 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 NAAP
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 NAAP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
NAAP, 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 polynucleotides
encoding NAAP
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 polynucleotides
encoding NAAP 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 tluorescently 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
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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).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
l0 fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) C~rr. Opin.
Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of NAAP include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244;
Duplaa, C. 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
polynucleotides 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
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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, NAAP, fragments of NAAP, or antibodies specific for
NAAP 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
l0 quantifying the number of expressed genes and their relative abundance
under given conditions and at
a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
Patent No. 5,840,484;
hereby 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
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). 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 for comparison of expression data after
treatment with different
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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.
In an embodiment, the toxicity of a test compound can be 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 embodiment relates to the use of the polypeptides disclosed herein 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 are separated by isoelectric
focusing in the first
dimension, and then according to molecular weight by sodium dodecyl sulfate
slab gel electrophoresis
in the second dimension (Steiner and Anderson, supra). The proteins are
visualized in the gel as
discrete and uniquely positioned spots, typically by staining the 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 interest. In some cases,
further sequence data
may be obtained for definitive protein identification.
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A proteomic profile may also be generated using antibodies specific for NAAP
to quantify the
levels of NAAP 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 may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol- or
amino-reactive fluorescent compound and detecting the amount of fluorescence
bound at each array
element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should 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 may be
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
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
(Brennan,
T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. USA
93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al. (1995)
PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci.
USA 94:2150-2155;
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Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of
microarrays are well known
and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A
Practical Approach, Oxford
University Press, London).
In another embodiment of the invention, nucleic acid sequences encoding NAAP
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 mufti-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 (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
355; Price, C.M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet.
7:149-154). Once
mapped, the nucleic acid sequences 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) (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 (Heinz-Ulrich, 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
NAAP on a physical
map and a specific disorder, or a-predisposition to a specific disorder, may
help define the 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 l 1q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation
(Gatti, R.A. et al. (1988) Nature 336:57?-580). The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
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among normal, carrier, or affected individuals.
In another embodiment of the invention, NAAP, 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 may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between NAAP 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 (Geysen, 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 NAAP, or fragments
thereof, and washed.
Bound NAAP is then detected by methods well known in the art. Purified NAAP
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 NAAP specifically compete with a test compound
for binding NAAP.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with NAAP.
In additional embodiments, the nucleotide sequences which encode NAAP 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
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. 60/348,442, U.S. Ser. No. 60/337,535, U.S. Ser. No.
60/335,544, U.S. Ser.
No. 60/344,650, and U.S. Ser. No. 60/334,762, are hereby expressly
incorporated by reference.
3o EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and
lysed in guanidinium
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isothiocyanate, while others were homogenized and lysed in phenol or in a
suitable mixture of
denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and
guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCI 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,
l0 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 (Invitrogen),
using the
recommended procedures or similar methods known in the art (Ausubel et al.,
supra, ch. 5). Reverse
transcription was initiated using oligo d(T) or random primers. Synthetic
oligonucleotide adapters were
ligated to double stranded cDNA, and the cDNA was digested with the
appropriate restriction enzyme
or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using
SEPHACRYL
S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham
Biosciences) 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 (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid
(Invitrogen), PBK-
CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid
(Stratagene),
pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY
(Incyte Genomics),
or derivatives thereof. Recombinant plasmids were transformed into competent
E. coli cells including
XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DHlOB, or ElectroMAX
DH10B
from Invitrogen.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: 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
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96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were 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
384-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 Biosciences 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 (Amersham
Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard
2o 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 (Ausubel et al.,
supra, ch. 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
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; PROTEOME databases with sequences from Homo
Sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans,
Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); hidden
Markov model (HMM)-based protein family databases such as PFAM, INCY, and
TIGRFAM (Haft,
D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain
databases such as
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SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;
Letunic, I. et al. (2002)
Nucleic Acids Res. 30:242-244). (IEVVIM 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
I~~IMER. 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 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, the PROTEOME databases, BLOCKS, PRINTS, DOMO,
PRODOM, Prosite, hidden Markov model (I-ftVIM)-based protein family databases
such as PFAM,
INCY, and TIGRFAM; and HIVIM-based protein domain databases such as SMART.
Full length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(MiraiBio, Alameda
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 )D
N0:59-116. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 2.
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IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative nucleic acid-associated proteins 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 (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94;
Burge, C. and S. Karlin
(1998) Curr. 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 nucleic acid-associated proteins, the encoded polypeptides
were analyzed by
querying against PFAM models for nucleic acid-associated proteins. Potential
nucleic acid-associated
proteins were also identified by homology to Incyte cDNA sequences that had
been annotated as
nucleic acid-associated proteins. 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 fmd 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" Seduences
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
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs 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" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example BI were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example 1V. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions 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.
VI. Chromosomal Mapping of NAAP Encoding Polynucleotides
The sequences which were used to assemble SEQ )D N0:59-116 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 m N0:59-116 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
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of all sequences of that cluster, including its particular SEQ 1D NO:, to that
map location.
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 were 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 (Sambrook and Russell,
supra, ch. 7; Ausubel et
al., supra, ch. 4).
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is much
faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer 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 (S 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
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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
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, polynucleotides encoding NAAP 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;
hemic 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 rerlect the tissue-
and disease-specific
expression of cDNA encoding NAAP. cDNA sequences and cDNA library/tissue
information are
found in the L1FESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of NAAP Encoding Polynucleotides
Full length polynucleotides are 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 S' 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 (MJ Research,
Inc.). The reaction
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mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme
(Invitrogen), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer pair
PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step
3: 60°C, 1 min; Step 4: 68°C, 2
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ 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 S: 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 p,1
PICOGREEN
l0 quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p.1 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 ~1 to 10 ~1 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
Biosciences). 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
Biosciences), 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 Biosciences) and Pfu DNA polymerase (Stratagene) 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 times; Step 6: 72°C, S 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
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and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides are verified using the above
procedure or are used
to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides designed for
such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in NAAP Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ )D N0:59-116 using the LIFESEQ database (Incyte Genomics).
Sequences from
the same gene were clustered together and assembled as described in Example
III, allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a nninimum Phred quality score of 15, and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original
chromatogram files
in the vicinity of the putative SNP. Clone error filters used statistically
generated algorithms to identify
errors introduced during laboratory processing, such as those caused by
reverse transcriptase,
polymerase, or somatic mutation. Clustering error filters used statistically
generated algorithms to
identify errors resulting from clustering of close homologs or pseudogenes, or
due to contamination by
non-human sequences. A final set of filters removed duplicates and SNPs found
in immunoglobulins
or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43 % Chinese, 31 % Japanese, 13 % Korean, 5 % Vietnamese, and 8% other
Asian. Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:59-116 are employed to screen
cDNAs,
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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 /.cCi of
['y 32P] adenosine triphosphate (Amersham Biosciences), 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 (Amersham Biosciences). 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,
to 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.
XI. 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 et al., 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, M., ed.
(1999) DNA Microarrays: A Practical Approach, Oxford University Press,
London). 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, UV, 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 any appropriate number of elements (Schena, M. et al. (1995)
Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
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
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
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biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized 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
l0 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/pl oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/p,l RNase inhibitor, 500 pM dATP, 500 pM dGTP, 500
p,M dTTP, 40 pM
dCTP, 40 pM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse
transcription
reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with
GEMBRIGHT kits
(Incyte Genomics). 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 O.SM sodium
hydroxide and incubated for
minutes at 85°C to the stop the reaction and degrade the RNA. Samples
are purified using two
successive CHROMA SPIN 30 gel filtration spin columns (Clontech, Palo Alto CA)
and after
20 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 p1 SX
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 pg.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
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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 p,1 of the array
element DNA, at an average
concentration of 100 ng/p,l, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about S 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 p,1 of sample mixture consisting of 0.2 p,g
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX 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
IS 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 SX SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (1X SSC, 0.1
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
2o Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
25 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,
30 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 simultaneously.
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, Inc., 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 obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte
Genomics). Array
elements that exhibit at least about a two-fold change in expression, a signal-
to-background ratio of at
least about 2.5, and an element spot size of at least about 40%, are
considered to be differentially
expressed.
Expression
SEQ >D N0:65 showed differential expression in cancer cell lines versus non-
cancerous cell
lines, as determined by microarray analysis. For example, the expression of
SEQ )D N0:65 was
decreased by at least two fold in breast tumor cell lines each isolated from
pleural effusion from
donors at different stages of tumor progression and malignant transformation
when grown in one of
two different chemically defined, serum-free media both supplemented with
growth factors and
growth hormones. Therefore, SEQ >D N0:65 is useful in diagnostic assays for
breast cancer.
Normal breast cell lines are obtained as follows. Primary mammary gland cells
are isolated
from a donor with fibrocystic breast disease. Tumorous breast cell fines are
obtained as follows.
Breast carcinoma cells are derived in vitro from cells emigrating from a
tumor. Alternately, breast
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tumor cells are isolated from invasive tumor of donors. Further, nonmalignant
or malignant primary
breast adenocarcinoma cells are obtained from the pleural effusion of donors.
Further, the expression of SEQ ll7 N0:65 was decreased at least two-fold in
treated human
adipocytes from obese and normal donors when compared to non-treated
adipocytes from the same
donors. The normal human primary subcutaneous preadipocytes were isolated from
adipose tissue of
a 28-year-old healthy female with a body mass index (BMI) of 23.59. The obese
human primary
subcutaneous preadipocytes were isolated from adipose tissue of a 40-year-old
healthy female with a
body mass index (BMI) of 32.47. The preadipocytes were cultured and induced to
differentiate into
adipocytes by culturing them in the differentiation medium containing the
active components, PPAR-'y
agonist and human insulin. Human preadipocytes were treated with human insulin
and PPAR-y
agonist for three days and subsequently were switched to medium containing
insulin for 24 hours, 48
hours, four days, 8 days or 15 days before the cells were collected for
analysis. Differentiated
adipocytes were compared to untreated preadipocytes maintained in culture in
the absence of inducing
agents. Between 80% and 90% of the preadipocytes finally differentiated
to.adipocytes as observed
under phase contrast microscope. Thus, SEQ >D N0:65 is useful for the
diagnosis, prognosis, or
treatment of diabetes mellitus and other disorders, such as obesity,
hypertension, atherosclerosis,
polycystic ovarian syndrome, and cancers including breast, prostate, and
colon.
For example, SEQ ll~ N0:72-74 showed differential expression in tumorous
tissue versus
non-tumorous tissues, as determined by microarray analysis. The expression of
cDNAs from lung
tumor tissue from several donors was compared with that of normal lung tissue
from the same donor,
respectively. Array elements that exhibited about at least a two-fold change
in expression and a signal
intensity over 250 units, a signal-to-background ratio of a least 2.5, and an
element spot size of at least
40% were identified as differentially expressed using the GEMTOOLS program
(Incyte Genomics).
The expression of SEQ )D N0:72 was increased at least two-fold in lung
squamous cell
carcinoma when matched with normal tissue from the same donor. The tumorous
lung tissue was
obtained from the lung of a 66-year-old male with lung squamous cell
carcinoma. Normal tissue was
obtained from grossly uninvolved lung tissue from the same donor. Therefore,
SEQ D.7 N0:72 is
useful in diagnostic assays for lung squamous cell carcinoma.
Alternately, the expression of SEQ >D N0:73 was decreased at least 2.7-fold in
lung
adenocarcinoma when matched with normal tissue from the same donor. The
tumorous lung tissue
was obtained from the right lung of a 60-year old donor with moderately
differentiated
adenocarcinoma. Normal tissue was obtained from grossly uninvolved tissue from
the right lung from
the same donor. Therefore, SEQ >D N0:73 is useful in diagnostic assays for
lung adenocarcinoma.
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Further, the expression of SEQ ID N0:74 was increased at least 2.7-fold in
lung
adenocarcinoma when matched with normal tissue from the same donor. The
tumorous lung tissue
was obtained from the lung of a 66-year old female with lung adenocarcinoma.
Normal tissue was
obtained from grossly uninvolved tissue from grossly uninvolved lung tissue
from the same donor. The
expression of SEQ >D N0:74 was increased at least 3.2-fold in lung squamous
cell carcinoma from
two donors when matched with normal tissue from the same donor. In one case,
the tumorous lung
tissue was obtained from the lung of a 66-year-old male with lung squamous
cell carcinoma. In the
other case, the tumorous lung tissue was obtained from the lung of a 73-year
old male with lung
squamous cell carcinoma. Normal tissue was obtained from grossly uninvolved
lung tissue from the
same donor, respectively. Therefore, SEQ 117 N0:74 is useful in diagnostic
assays for lung
adenocarcinoma and squamous cell carcinoma.
For example, SEQ )D N0:79 showed increased expression in colon tissue affected
by colon
cancer versus normal colon tissue as determined by microarray analysis. Gene
expression profiles
were obtained by comparing normal colon tissue from a 67 year-old donor with
moderately
differentiated adenocarcinoma (Dukes B, TNM classification) to cancer-affected
colon tissue from
the same donor. Samples were provided by the Huntsman Cancer Institute.
Therefore, SEQ >Z7
N0:79 is useful in diagnostic assays for disorders of cell proliferation
including colon cancer.
For example, SEQ ll~ N0:79 showed decreased expression in ovary tissue
affected by
ovarian cancer versus normal ovary tissue as determined by microarray
analysis. A normal ovary
from a 79 year-old female donor was compared to an ovarian tumor from the same
donor. Samples
were provided by the Huntsman Cancer Institute. Therefore, SEQ >D N0:79 is
useful in diagnostic
assays for disorders of cell proliferation including ovarian cancer.
For example, SEQ ID N0:79 showed decreased expression in C3A cells treated
with
dexamethasone, versus untreated C3A cells, as determined by microarray
analysis. The human C3A
cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated
from a 15-year-old male with
liver tumor), which was selected for strong contact inhibition of growth.
Early Confluent C3A cells
were treated with dexamethasone at l, 10, and 100 ~M for 1, 3, and 6 hours.
The treated cells were
compared to untreated early confluent C3A cells. Therefore, SEQ >D N0:79 is
useful in diagnostic
assays for, and monitoring treatment of, autoimmune/inflammatory disorders.
For example, SEQ ID N0:81 showed differential expression in fibroblasts
affected by
Tangiers Disease (TD) versus normal fibroblasts, when both were treated with
LDL cholesterol, as
determined by microarray analysis. Normal and TD-derived fibroblasts were
compared cultured in
the presence of cholesterol and compared with the same cell type cultured in
the absence of
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cholesterol. Human fibroblasts were obtained from skin explants from both
normal subjects and two
patients with homozygous TD. Cell lines were immortalized by transfection with
human
papillomavirus 16 genes E6 and E7 and a neomycin resistance selectable marker,
and TD was
confirmed in TD-derived cells by reduced apoA-I mediated tritiated cholesterol
efflux. Therefore,
SEQ ID N0:81 is useful in diagnostic assays for autoimmune/inflammatory
disorders including Tangier
Disease.
For example, SEQ ID N0:93 showed differential expression in mammary cells
affected by
breast carcinoma versus nonmalignant mammary epithelial cells as determined by
microarray analysis.
The gene expression profile of a nonmalignant mammary epithelial cell line was
compared to the gene
expression profiles of breast carcinoma lines at different stages of tumor
progression. Cell lines
compared included: a) MCF-10A, a breast mammary gland cell line isolated from
a 36-year-old
woman with fibrocystic breast disease; b) MCF7, a nonmalignant breast
adenocarcinoma cell line
isolated from the pleural effusion of a 69- year-old female; c) T-47D, a
breast carcinoma cell line
isolated from a pleural effusion obtained from a 54-year-old female with an
infiltrating ductal
carcinoma of the breast; d) Sk-BR-3, a breast adenocarcinoma cell line
isolated from a malignant
pleural effusion of a 43-year-old female; e) BT-20, a breast carcinoma cell
line derived in vitro from
tumor mass isolated from a 74-year-old female; f) MDA-mb-231, a breast tumor
cell line isolated from
the pleural effusion of a 51-year old female; and g) MDA-mb-4355, a spindle
shaped strain that
evolved from the parent line (435) isolated from the pleural effusion of a 31-
year-old female with
metastatic, ductal adenocarcinoma of the breast.
The cells were grown in the supplier's recommended medium to 70-80% confluence
prior to
RNA harvest. Expression was decreased by at least two-fold in 4 of the 6
breast carcinoma cell lines
as compared to the nonmalignant mammary epithelial cell line. Therefore, SEQ
)D N0:93 is useful in
diagnostic assays for and monitoring treatment of, cell proliferative
disorders including breast
carcinoma.
As another example, SEQ ID N0:94 showed decreased expression in tissue
affected by
adenocarcinoma versus normal tissue as determined by microarray analysis. A
sample of tissue right
lung tissue that showed moderately differentiated adenocarcinoma of was
compared to grossly
uninvolved lung tissue from the same donor (Huntsman Cancer Institute, Salt
Lake City, UT).
Therefore, SEQ >D N0:94 is useful in diagnostic assays for, and monitoring
treatment of, cell
proliferative disorders including adenocarcinoma.
As another example, SEQ ID N0:98 showed decreased expression in stimulated
dendritic
cells treated with CD40 antibodies versus stimulated dendritic cells not
treated with CD40 antibodies,
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as determined by microarray analysis. Human monocytic dendritic cells (mDCs)
were derived in vitro
from the adherent cellular fraction of the peripheral blood of 4 healthy
volunteer donors. The adherent
leukocytes, mostly monocytes, were incubated for 13 days in the presence of
recombinant interleukin-
4 (10 ng/ml) and granulocyte/macrophage colony stimulating factor (10 ng/ ml).
The differentiated
mDCs were collected after 13 days from the non-adherent cellular fraction and
activated in the
presence of soluble mouse anti-human CD40 antibodies for 2, 8, and 24 hours.
The anti-CD40 treated
mDCs were compared to untreated mDCs. Therefore, SEQ ID N0:98 is useful in
diagnostic assays
for, and monitoring treatment of, autoimmune/inflammatory disorders.
As another example, SEQ )D NO:100 showed decreased expression in cells treated
with
to tumor necrosis factor alpha (TNF-a ), which mediates the inflammatory
response through activation of
signal transduction pathways, versus untreated cells as determined by
microarray analysis. Human
aortic endothelial cells (HMVECdNeos) were grown to 85% confluence and then
treated for 1, 2, 4, 8,
and 24 hours with tumor necrosis factor alpha (TNF-a ). TNF-a -treated cells
were compared to
untreated HMVECdNeos collected at 85% confluence (0 hour). Therefore, SEQ >D
N0:100 is useful
IS in diagnostic assays for, and monitoring treatment of, cell proliferative
disorders.
In order to evaluate RNA expression, HMVECdNeo cells were grown to 85%
confluency
and then treated with TNF-a (10 ng/ml) for 2, 4, 8, and 24 hours. TNF-a-
treated cells were
compared to untreated HMVECdNeos collected at 85% confluency (0 hour). The
expression of SEQ
)D N0:108 was underexpressed by at least two-fold in TNF-a-treated versus
untreated cells at the
20 last three time points tested. Therefore, SEQ D7 N0:108 may be useful in
disease staging and
diagnostic assays for cell proliferative and inflammatbry disorders, including
those involving nucleic
acid-associated proteins.
Region-specific RNA expression in human brain tissue was evaluated using
specific dissected
brain regions from a non-demented human female brain. Brain regions were then
pooled and used as
25 the control. Specific brain regions were then compared to the mixed brain
control. The mixed brain
control was reconstituted from the purified mRNA isolated from the major
regions of the brain. The
expression of SEQ ID N0:109 was underexpressed by at least two-fold in the
dentate nuclear brain
tissue as compared to the mixed brain control tissue. Therefore, SEQ 117
N0:109 may be useful in
disease staging and diagnostic assays for cell proliferative and/or
neurological disorders, including
30 those involving nucleic acid-associated proteins.
XII. Complementary Polynucleotides
Sequences complementary to the NAAP-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring NAAP.
Although use of
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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 NAAP. 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 NAAP-encoding
transcript.
XIII. Expression of NAAP
Expression and purification of NAAP is achieved using bacterial or virus-based
expression
systems. For expression of NAAP 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
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors ate transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express NAAP upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of NAAP in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Autographica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding NAAP 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 (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).
In most expression systems, NAAP 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 Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from
NAAP 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 histidine residues, enables purification on metal-
chelate resins (QIAGEN).
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Methods for protein expression and purification are discussed in Ausubel et
al. (supra, ch. 10 and 16).
Purified NAAP obtained by these methods can be used directly in the assays
shown in Examples
XVII, XVIII, XIX, and XX, where applicable.
XIV. Functional Assays
NAAP function is assessed by expressing the sequences encoding NAAP 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 plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid
(Invitrogen), both of
which contain the cytomegalovirus promoter. S-10 ,ug 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 ~g of an additional plasmid
containing sequences
encoding a marker protein are co-transfected. Expression of a marker protein
provides a means to
distinguish transfected cells from 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 NAAP on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding NAAP 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 NAAP and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
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XV. Production of NAAP Specific Antibodies
NAAP 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 animals (e.g., rabbits, mice, etc.) and to produce antibodies using
standard protocols.
Alternatively, the NAAP 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 (Ausubel et al., 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
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-NAAP
activity by, for example, binding the peptide or NAAP to a substrate, blocking
with 1 % BSA, reacting
with rabbit antisera, washing, and reacting with radio-iodinated goat anti-
rabbit IgG.
XVI. Purification of Naturally Occurring NAAP Using Specific Antibodies
Naturally occurring or recombinant NAAP is substantially purified by
immunoaffinity
chromatography using antibodies specific for NAAP. An immunoaffvlity column is
constructed by
covalently coupling anti-NAAP antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin
is blocked and
washed according to the manufacturer's instructions.
Media containing NAAP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of NAAP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/NAAP 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 NAAP is collected.
XVII. Identification of Molecules Which Interact with NAAP
NAAP, or biologically active fragments thereof, are labeled with'zsI Bolton-
Hunter reagent
(Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539). Candidate
molecules previously
arrayed in the wells of a multi-well plate are incubated with the labeled
NAAP, washed, and any wells
with labeled NAAP complex are assayed. Data obtained using different
concentrations of NAAP are
used to calculate values for the number, affinity, and association of NAAP
with the candidate
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molecules.
Alternatively, molecules interacting with NAAP 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).
NAAP 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).
XVIII. Demonstration of NAAP Activity
NAAP activity is measured by its ability to stimulate transcription of a
reporter gene (Liu,
H.Y. et al. (1997) EMBO J. 16:5289-5298). The assay entails the use of a well
characterized
reporter gene construct, LexAop LacZ, that consists of LexA DNA
transcriptional control elements
(LexAop) fused to sequences encoding the E. coli LacZ enzyme. The methods for
constructing and
expressing fusion genes, introducing them into cells, and measuring LacZ
enzyme activity, are well
known to those skilled in the art. Sequences encoding NAAP are cloned into a
plasmid that directs
the synthesis of a fusion protein, LexA-NAAP, consisting of NAAP and a DNA
binding domain
derived from the LexA transcription factor. The resulting plasmid, encoding a
LexA-NAAP fusion
protein, is introduced into yeast cells along with a plasmid containing the
LexAop-LacZ reporter gene.
The amount of LacZ enzyme activity associated with LexA-NAAP transfected
cells, relative to
control cells, is proportional to the amount of transcription stimulated by
the NAAP.
Alternatively, NAAP activity is measured by its ability to bind zinc. A 5-10
~M sample
solution in 2.5 mM ammonium acetate solution at pH 7.4 is combined with 0.05 M
zinc sulfate solution
(Aldrich, Milwaukee WI) in the presence of 100 pM dithiothreitol with 10%
methanol added. The
sample and zinc sulfate solutions are allowed to incubate for 20 minutes. The
reaction solution is
passed through a VYDAC column (Grace Vydac, Hesperia, CA) with approximately
300 Angstrom
bore size and 5 ~M particle size to isolate zinc-sample complex from the
solution, and into a mass
spectrometer (PE Sciex, Ontario, Canada). Zinc bound to sample is quantified
using the functional
atomic mass of 63.5 Da observed by Whittal, R. M. et al. ((2000) Biochemistry
39:8406-8417).
In the alternative, a method to determine nucleic acid binding activity of
NAAP involves a
polyacrylamide gel mobility-shift assay. In preparation for this assay, NAAP
is expressed by
transforming a manunalian cell fine such as COS7, HeLa or CHO with a
eukaryotic expression vector
containing NAAP cDNA. The cells are incubated for 48-72 hours after
transformation under
conditions appropriate for the cell line to allow expression and accumulation
of NAAP. Extracts
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containing solubilized proteins can be prepared from cells expressing NAAP by
methods well known
in the art. Portions of the extract containing NAAP are added to [32P]-labeled
RNA or DNA.
Radioactive nucleic acid can be synthesized in vitro by techniques well known
in the art. The
mixtures are incubated at 25°C in the presence of RNase- and DNase-
inhibitors under buffered
conditions for S-10 minutes. After incubation, the samples are analyzed by
polyacrylamide gel
electrophoresis followed by autoradiography. The presence of a band on the
autoradiogram indicates
the formation of a complex between NAAP and the radioactive transcript. A band
of similar mobility
will not be present in samples prepared using control extracts prepared from
untransformed cells.
In the alternative, a method to determine methylase activity of NAAP measures
transfer of
l0 radiolabeled methyl groups between a donor substrate and an acceptor
substrate. Reaction mixtures
(50 p1 final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM
dithiothreitol, 3
polyvinylalcohol, 1.5 ~,Ci [methyl-3H]AdoMet (0.375 ~M AdoMet) (DuPont-NEN),
0.6 p.g NAAP, and
acceptor substrate (e.g., 0.4 pg [35S]RNA, or 6-mercaptopurine (6-MP) to 1 mM
final concentration).
Reaction mixtures are incubated at 30°C for 30 minutes, then
65°C for 5 minutes.
Analysis of [methyl-3H]RNA is as follows: (1) 50 ~.1 of 2 x loading buffer (20
mM Tris-HCI,
pH 7.6, 1 M LiCI, 1 mM EDTA, 1% sodium dodecyl sulphate (SDS)) and 50 p,1
oligo d(T)-cellulose
( 10 mg/ml in 1 x loading buffer) are added to the reaction mixture, and
incubated at ambient
temperature with shaking for 30 minutes. (2) Reaction mixtures are transferred
to a 96-well filtration
plate attached to a vacuum apparatus. (3) Each sample is washed sequentially
with three 2.4 ml
aliquots of 1 x oligo d(T) loading buffer containing 0.5% SDS, 0.1% SDS, or no
SDS. (4) RNA is
eluted with 300 p.1 of water into a 96-well collection plate, transferred to
scintillation vials containing
liquid scintillant, and radioactivity determined.
Analysis of [methyl-3H]6-MP is as follows: (1) 500 p1 0.5 M borate buffer, pH
10.0, and then
2.5 ml of 20% (v/v) isoamyl alcohol in toluene are added to the reaction
mixtures. (2) The samples
are mixed by vigorous vortexing for ten seconds. (3) After centrifugation at
700g for 10 minutes, 1.5
ml of the organic phase is transferred to scintillation vials containing 0.5
ml absolute ethanol and liquid
scintillant, and radioactivity determined. (4) Results are corrected for the
extraction of 6-MP into the
organic phase (approximately 41%).
In the alternative, type I topoisomerase activity of NAAP can be assayed based
on the
relaxation of a supercoiled DNA substrate. NAAP is incubated with its
substrate in a buffer lacking
Mg2+ and ATP, the reaction is terminated, and the products are loaded on an
agarose gel. Altered
topoisomers can be distinguished from supercoiled substrate
electrophoretically. This assay is specific
for type I topoisomerase activity because Mg2+ and ATP are necessary cofactors
for type II
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topoisomerases.
Type II topoisomerase activity of NAAP can be assayed based on the
decatenation of a
kinetoplast DNA (KDNA) substrate. NAAP is incubated with KDNA, the reaction is
terminated,
and the products are loaded on an agarose gel. Monomeric circular KDNA can be
distinguished from
catenated KDNA electrophoretically. Kits for measuring type I and type II
topoisomerase activities
are available commercially from Topogen (Columbus OH).
ATP-dependent RNA helicase unwinding activity of NAAP can be measured by the
method
described by Zhang and Grosse (1994; Biochemistry 33:3906-3912). The substrate
for RNA
unwinding consists of 32P-labeled RNA composed of two RNA strands of 194 and
130 nucleotides in
l0 length containing a duplex region of 17 base-pairs. The RNA substrate is
incubated together with
ATP, Mg2+, and varying amounts of NAAP in a Tris-HCl buffer, pH 7.5, at
37°C for 30 minutes. The
single-stranded RNA product is then separated from the double-stranded RNA
substrate by
electrophoresis through a 10% SDS-polyacrylamide gel, and quantitated by
autoradiography. The
amount of single-stranded RNA recovered is proportional to the amount of NAAP
in the preparation.
In the alternative, NAAP function is assessed by expressing the sequences
encoding NAAP
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 (Invitrogen
Corporation, Carlsbad CA), both of which contain the cytomegalovirus promoter.
5-10 ~g of
recombinant vector are transiently transfected into a human cell line,
preferably of endothelial or
hematopoietic origin, using either liposome formulations or electroporation. 1-
2 p,g of an additional
plasmid containing sequences encoding a marker protein are co-transfected.
Expression of a marker protein provides a means to distinguish transfected
cells from
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
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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 NAAP on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding NAAP and either CD64 or CD64-GFP.
CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Inc., Lake
l0 Success NY). mRNA can be purified from the cells using methods well known
by those of skill in the
art. Expression of mRNA encoding NAAP and other genes of interest can be
analyzed by northern
analysis or microarray techniques.
Pseudouridine synthase activity of NAAP is assayed using a tritium (3H)
release assay
modified from Nurse et al. ((1995) RNA 1:102-112), which measures the release
of 3H from the CS
position of the pyrimidine component of uridylate (U) when 3H-radiolabeled U
in RNA is isomerized to
pseudouridine (y1). A typical 500 /.d assay mixture contains 50 mM HEPES
buffer (pH 7.5), 100 mM
ammonium acetate, 5 mM dithiothreitol, 1 mM EDTA, 30 units RNase inhibitor,
and 0.1-4.2 pM
[5 3H]tRNA (approximately 1 pCi/nmol tRNA). The reaction is initiated by the
addition of <5 p1 of a
concentrated solution of NAAP (or sample containing NAAP) and incubated for 5
min at 37 °C.
Portions of the reaction mixture are removed at various times (up to 30 min)
following the addition of
NAAP and quenched by dilution into 1 ml 0.1 M HCl containing Norit-SA3 (12%
w/v). The
quenched reaction mixtures are centrifuged for 5 min at maximum speed in a
microcentrifuge, and the
supernatants are filtered through a plug of glass wool. The pellet is washed
twice by resuspension in 1
ml 0.1 M HCl, followed by centrifugation. The supernatants from the washes are
separately passed
through the glass wool plug and combined with the original filtrate. A portion
of the combined filtrate
is mixed with scintillation fluid (up to 10 ml) and counted using a
scintillation counter. The amount of
3H released from the RNA and present in the soluble filtrate is proportional
to the amount of
peudouridine synthase activity in the sample (Ramamurthy, V. (1999) J. Biol.
Chem.
274:22225-22230).
In the alternative, pseudouridine synthase activity of NAAP is assayed at 30
°C to 37 °C in a
mixture containing 100 mM Tris-HCl (pH 8.0), 100 mM ammonium acetate, 5 mM
MgClz, 2 mM
dithiothreitol, 0.1 mM EDTA, and 1-2 fmol of [3zP]-radiolabeled runoff
transcripts (generated in vitro
by an appropriate RNA polymerase, i.e., T7 or SP6) as substrates. NAAP is
added to initiate the .
120
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
reaction or omitted from the reaction in control samples. Following
incubation, the RNA is extracted
with phenol-chloroform, precipitated in ethanol, and hydrolyzed completely to
3-nucleotide
monophosphates using RNase TZ. The hydrolysates are analyzed by two-
dimensional thin layer
chromatography, and the amount of 32P radiolabel present in the yrMP and UMP
spots are evaluated
after exposing the thin layer chromatography plates to film or a
Phosphorlmager screen. Taking into
account the relative number of uridylate residues in the substrate RNA, the
relative amount yrMP and
UMP are determined and used to calculate the relative amount of y per tRNA
molecule (expressed in
mol tV /mol of tRNA or mol yr /mol of tRNA/minute), which corresponds to the
amount of
pseudouridine synthase activity in the NAAP sample (Lecointe, F. et al. (1998)
J. Biol. Chem.
273:1316-1323).
Nz,N2-dimethylguanosine transferase ((m22G)methyltransferase) activity of NAAP
is
measured in a 160 p1 reaction mixture containing 100 mM Tris-HCl (pH 7.5), 0.1
mM EDTA, 10 mM
MgCl2, 20 mM NHdCl, 1mM dithiothreitol, 6.2 p,M S-adenosyl-L-[methyl-
3H]methionine (30-70
Ci/mM), 8 pg m2zG-deficient tRNA or wild type tRNA from yeast, and
approximately 100 pg of
purified NAAP or a sample comprising NAAP. The reactions are incubated at 30
°C for 90 min and
chilled on ice. A portion of each reaction is diluted to 1 ml in water
containing 100 p,g BSA. 1 ml of 2
M HCl is added to each sample and the acid insoluble products are allowed to
precipitate on ice for 20
min before being collected by filtration through glass fiber filters. The
collected material is washed
several times with HCl and quantitated using a liquid scintillation counter.
The amount of 3H
incorporated into the mz2G-deficient, acid-insoluble tRNAs is proportional to
the amount of
N2,Nz-dimethylguanosine transferase activity in the NAAP sample. Reactions
comprising no
substrate tRNAs, or wild-type tRNAs that have already been modified, serve as
control reactions
which should not yield acid-insoluble 3H-labeled products.
Polyadenylation activity of NAAP is measured using an in vitro polyadenylation
reaction.
The reaction mixture is assembled on ice and comprises 10 p1 of 5 mM
dithiothreitol, 0.025% (v/v)
NONIDET P-40, 50 mM creative phosphate, 6.5% (w/v) polyvinyl alcohol, 0.5
unit/pl RNAGUARD
(Pharmacia), 0.025 pg/pl creative kinase, 1.25 mM cordycepin 5'-triphosphate,
and 3.75 mM MgClz, in
a total volume of 25 p1. 60 fmol of CstF, 50 fmol of CPSF, 240 fmol of PAP, 4
p,1 of crude or partially
purified CF II and various amounts of amounts CF I are then added to the
reaction mix. The volume
is adjusted to 23.5 p1 with a buffer containing 50 mM TrisHCl, pH 7.9, 10%
(v/v) glycerol, and 0.1 mM
Na-EDTA. The final ammonium sulfate concentration should be below 20 mM. The
reaction is
initiated (on ice) by the addition of 15 fmol of 32P-labeled pre-mRNA
template, along with 2.5 ~g of
unlabeled tRNA, in 1.5 p1 of water. Reactions are then incubated at 30
°C for 75-90 min and stopped
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WO 03/038052 PCT/US02/34846
by the addition of 75 p1 (approximately two-volumes) of proteinase K mix (0.2
M Tris-HCI, pH 7.9,
300 mM NaCI, 25 mM Na-EDTA, 2% (w/v) SDS), 1 p.1 of 10 mg/ml proteinase K,
0.25 p1 of 20 mg/ml
glycogen, and 23.75 p.1 of water). Following incubation, the RNA is
precipitated with ethanol and
analyzed on a 6% (w/v) polyacrylamide, 8.3 M urea sequencing gel. The dried
gel is developed by
autoradiography or using a phosphoimager. Cleavage activity is determined by
comparing the amount
of cleavage product to the amount of pre-mRNA template. The omission of any of
the polypeptide
components of the reaction and substitution of NAAP is useful for identifying
the specific biological
function of NAAP in pre-mRNA polyadenylation (Riiegsegger, U. et al. (1996) J.
Biol. Chem.
271:6107-6113; and references within).
tRNA synthetase activity is measured as the aminoacylation of a substrate tRNA
in the
presence of [14C]-labeled amino acid. NAAP is incubated with [14C]-labeled
amino acid and the
appropriate cognate tRNA (for example, [14C]alanine and tRNAa~a) in a buffered
solution. 14C-
labeled product is separated from free [14C]amino acid by chromatography, and
the incorporated'4C
is quantified by scintillation counter. The amount of 14C-labeled product
detected is proportional to the
activity of NAAP in this assay.
In the alternative, NAAP activity is measured by incubating a sample
containing NAAP in a
solution containing 1 mM ATP, 5 mM Hepes-KOH (pH 7.0), 2.5 mM KCI, 1.5 mM
magnesium
chloride, and 0.5 mM DTT along with misacylated ['4C]-Glu-tRNAGln (e.g., 1 pM)
and a similar
concentration of unlabeled L-glutamine. Following the quenching of the
reaction with 3 M sodium
acetate (pH 5.0), the mixture is extracted with an equal volume of water-
saturated phenol, and the
aqueous and organic phases are separated by centrifugation at 15,000 x g at
room temperature for 1
min. The aqueous phase is removed and precipitated with 3 volumes of ethanol
at -70°C for 15 min.
The precipitated aminoacyl-tRNAs are recovered by centrifugation at 15,000 x g
at 4°C forl5 min.
The pellet is resuspended in of 25 mM KOH, deacylated at 65°C for 10
min., neutralized with 0.1 M
HCl (to final pH 6-7), and dried under vacuum. The dried pellet is resuspended
in water and spotted
onto a cellulose TLC plate. The plate is developed in either
isopropanol/formic acid/water or
ammonia/water/chloroform/ methanol. The image is subjected to densitometric
analysis and the
relative amounts of Glu and Gln are calculated based on the Rf values and
relative intensities of the
spots. NAAP activity is calculated based on the amount of Gln resulting from
the transformation of
Glu while acylated as Glu-tRNAG~' (adapted from Curnow, A.W. et al. (1997)
Proc. Natl. Acad. Sci.
USA 94:11819-26).
XIX. Identification of NAAP Agonists and Antagonists
Agonists or antagonists of NAAP activation or inhibition may be tested using
the assays
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CA 02462660 2004-04-O1
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described in section XVIZI. Agonists cause an increase in NAAP activity and
antagonists cause a
decrease in NAAP activity.
XX. NAAP Secretion Assay
A high throughput assay may be used to identify polypeptides that are secreted
in eukaryotic
cells. In an example of such an assay, polypeptide expression libraries are
constructed by fusing 5'-
biased cDNAs to the 5 =end of a leaderless ~i-lactamase gene. (3-lactamase is
a convenient genetic
reporter as it provides a high signal-to-noise ratio against low endogenous
background activity and
retains activity upon fusion to other proteins. A dual promoter system allows
the expression of ~i-
lactamase fusion polypeptides in bacteria or eukaryotic cells, using the lac
or CMV promoter,
respectively.
Libraries are first transformed into bacteria, e.g., E. cofi, to identify
library members that
encode fusion polypeptides capable of being secreted in a prokaryotic system.
Mammalian signal
sequences direct the translocation of (3-lactamase fusion polypeptides into
the periplasm of bacteria
where it confers antibiotic resistance to carbenicillin. Carbenicillin-
selected bacteria are isolated on
solid media, individual clones are grown in liquid media, and the resulting
cultures are used to isolate
library member plasmid DNA.
Mammalian cells, e.g., 293 cells, are seeded into 96-well tissue culture
plates at a density of
about 40,000 cells/well in 100 ~1 phenol red-free DME supplemented with 10%
fetal bovine serum
(FBS) ( Life Technologies, Rockville, MD). The following day, purified plasmid
DNAs isolated from
carbenicillin-resistant bacteria are diluted with 15 ~l OPTI-MEM I medium
(Life Technologies) to a
volume of 25 p,1 for each well of cells to be transfected. In separate plates,
1 p.1 LF2000 Reagent (Life
Technologies) is diluted into 25 p,l/well OPTI-MEM I. The 25 p1 diluted LF2000
Reagent is then
combined with the 25 ~l diluted DNA, mixed briefly, and incubated for 20
minutes at room
temperature. The resulting DNA-LF2000 reagent complexes are then added
directly to each well of
293 cells. Cells are also transfected with appropriate control plasmids
expressing either wild-type (3-
lactamase, leaderless (3-lactamase, or, for example, CD4-fused leaderless ~i-
lactamase. 24 hrs
following transfection, about 90 p.1 of cell culture media are assayed at
37°C with 100 ~M Nitrocefin
(Calbiochem, San Diego CA) and 0.5 mM oleic acid (Sigma, St. Louis, MO) in 10
mM phosphate
buffer (pH 7.0). Nitrocefm is a substrate for (3-lactamase that undergoes a
noticeable color change
3o from yellow to red upon hydrolysis. (3-lactamase activity is monitored over
20 min in a microtiter plate
reader at 486 nm. Increased color absorption at 486 nm corresponds to
secretion of a (3-lactamase
fusion polypeptide in the transfected cell media, resulting from the presence
of a eukaryotic signal
sequence in the fusion polypeptide. Polynucleotide sequence analysis of the
corresponding library
123
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
member plasmid DNA is then used to identify the signal sequence-encoding cDNA.
(Described in
U.S. Patent application 09/803,317, filed March 9, 2001.)
Various modifications and variations of the described compositions, 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. It will be appreciated that the invention provides novel and
useful proteins, and their
encoding polynucleotides, which can be used in the drug discovery process, as
well as methods for
using these compositions for the detection, diagnosis, and treatment of
diseases and conditions.
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.
l0 Nor should the description of such embodiments be considered exhaustive or
limit the invention to the
precise forms disclosed. Furthermore, elements from one embodiment can be
readily recombined with
elements from one or more other embodiments. Such combinations can form a
number of
embodiments within the scope of the invention. It is intended that the scope
of the invention be
defined by the following claims and their equivalents.
124
CA 02462660 2004-04-O1
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CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
<110> INCYTE GENOMICS, INC.
BECHA, Shanya D.
BOROWSKY, Mark L.
BURFORD, Neil
CHAWLA, Narinder K.
ELLIOTT, Vicki S.
EMERLING, Brooke M.
FORSYTHE, Ian J.
GIETZEN, Kimberly J.
GORVAD, Ann E.
GRIFFIN, Jennifer A.
HAFALIA, April J.A.
ISON, Craig H.
LAL, Preeti G.
LEE, Ernestine A.
LEE, Sally
LEE, Soo Yeun
MARQUIS, Joseph P.
RAMKUMAR, Jayalaxmi
SPRAGUE, William W.
SWARNAKAR, Anita
TANG, Y. Tom
WARREN, Bridget A.
YANG, Junming
YUE, Henry
ZEBARJADIAN, Yeganeh
<120> NUCLEIC ACID-ASSOCIATED PROTEINS
<130> PF-1249 PCT
<140> To Be Assigned
<141> Herewith
<150> US 60/348,442
<151> 2001-10-29
<150> US 60/335,544
<151> 2001-11-O1
<150> US 60/337,535
<151> 2001-11-05
<150> US 60/344,650
<151> 2001-11-09
<150> US 60/334,762
<151> 2001-11-15
<160> 116
<170> PERL Program
<210> 1
<211> 1374
<212> PRT
<213> Homo sapiens
1/147
CA 02462660 2004-04-O1
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<220>
<221> misc_feature
<223> Incyte ID No: 7503848CD1
<400> 1
Met Ala Glu Ala Arg Lys Arg Arg Glu Leu Leu Pro Leu Ile Tyr
1 5 10 15
His His Leu Leu Arg Ala Gly Tyr Val Arg Ala Ala Arg Glu Val
20 25 30
Lys Glu Gln Ser Gly Gln Lys Cys Phe Leu Ala Gln Pro Val Thr
35 40 45
Leu Leu Asp Ile Tyr Thr His Trp Gln Gln Thr Ser Glu Leu Gly
50 55 60
Arg Lys Arg Lys Ala Glu Glu Asp Ala Ala Leu Gln Ala Lys Lys
65 70 75
Thr Arg Val Ser Asp Pro Ile Ser Thr Ser Glu Ser Ser Glu Glu
80 85 90
Glu Glu Glu Ala Glu Ala Glu Thr Ala Lys Ala Thr Pro Arg Leu
95 100 105
Ala Ser Thr Asn Ser Ser Val Leu Gly Ala Asp Leu Pro Ser Ser
110 115 120
Met Lys Glu Lys Ala Lys Ala Glu Thr Glu Lys Ala Gly Lys Thr
125 130 135
Gly Asn Ser Met Pro His Pro Ala Thr Gly Lys Thr Val Ala Asn
140 145 150
Leu Leu Ser Gly Lys Ser Pro Arg Lys Ser Ala Glu Pro Ser Ala
155 160 165
Asn Thr Thr Leu Val Ser Glu Thr Glu Glu Glu Gly Ser Val Pro
170 175 180
Ala Phe Gly Ala Ala Ala Lys Pro Gly Met Val Ser Ala Gly Gln
185 190 195
Ala Asp Ser Ser Ser Glu Asp Thr Ser Ser Ser Ser Asp Glu Thr
200 205 210
Asp Val Glu Val Lys Ala Ser Glu Lys Ile Leu Gln Val Arg Ala
215 220 225
Ala Ser Ala Pro Ala Lys Gly Thr Pro Gly Lys Gly Ala Thr Pro
230 235 240
Ala Pro Pro Gly Lys Ala Gly Ala Val Ala Ser Gln Thr Lys Ala
245 250 255
Gly Lys Pro Glu Glu Asp Ser Glu Ser Ser Ser Glu Glu Ser Ser
260 265 270
Asp Ser Glu Glu Glu Thr Pro Ala Ala Lys Ala Leu Leu Gln Ala
275 280 285
Lys Ala Ser Gly Lys Thr Ser Gln Val Gly Ala Ala Ser Ala Pro
290 295 300
Ala Lys Glu Ser Pro Arg Lys Gly Ala Ala Pro Ala Pro Pro Gly
305 310 315
Lys Thr Gly Pro Ala Val Ala Lys Ala Gln Ala Gly Lys Arg Glu
320 325 330
Glu Asp Ser Gln Ser Ser Ser Glu Glu Ser Asp Ser Glu Glu Glu
335 340 345
Ala Pro Ala Gln Ala Lys Pro Ser Gly Lys Ala Pro Gln Val Arg
350 355 360
Ala Ala Ser Ala Pro Ala Lys Glu Ser Pro Arg Lys Gly Ala Ala
365 370 375
Pro Ala Pro Pro Arg Lys Thr Gly Pro Ala Ala Ala Gln Val Gln
2/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
380 385 390
Val Gly Lys Gln Glu Glu Asp Ser Arg Ser Ser Ser Glu Glu Ser
395 400 405
Asp Ser Asp Arg Glu Ala Leu Ala Ala Met Asn Ala Ala Gln Val
410 415 420
Lys Pro Leu Gly Lys Ser Pro Gln Val Lys Pro Ala Ser Thr Met
425 430 435
Gly Met Gly Pro Leu Gly Lys Gly Ala Gly Pro Val Pro Pro Gly
440 445 450
Lys Val Gly Pro Ala Thr Pro Ser Ala Gln Val Gly Lys Trp Glu
455 460 465
Glu Asp Ser Glu Ser Ser Ser Glu Glu Ser Ser Asp Ser Ser Asp
470 475 480
Gly Glu Val Pro Thr Ala Val Ala Pro Ala Gln Glu Lys Ser Leu
485 490 495
Gly Asn Ile Leu Gln Ala Lys Pro Thr Ser Ser Pro Ala Lys Gly
500 505 510
Pro Pro Gln Lys Ala Gly Pro Val Ala Val Gln Val Lys Ala Glu
515 520 525
Lys Pro Met Asp Asn Ser Glu Ser Ser Glu Glu Ser Ser Asp Ser
530 535 540
Ala Asp Ser Glu Glu Ala Pro Ala Ala Met Thr Ala Ala Gln Ala
545 550 555
Lys Pro Ala Leu Lys Ile Pro Gln Thr Lys Ala Cys Pro Lys Lys
560 565 570
Thr Asn Thr Thr Ala Ser Ala Lys Val Ala Pro Val Arg Val Gly
575 580 585
Thr Gln Ala Pro Arg Lys Ala Gly Thr Ala Thr Ser Pro Ala Gly
590 595 600
Ser Ser Pro Ala Val Ala Gly Gly Thr Gln Arg Pro Ala Glu Asp
605 610 615
Ser Ser Ser Ser Glu Glu Ser Asp Ser Glu Glu Glu Lys Thr Gly
620 625 630
Leu Ala Val Thr Val Gly Gln Ala Lys Ser Val Gly Lys Gly Leu
635 640 645
Gln Val Lys Ala Ala Ser Val Pro Val Lys Gly Ser Leu Gly Gln
650 655 660
Gly Thr Ala Pro Val Leu Pro Gly Lys Thr Gly Pro Thr Val Thr
665 670 675
Gln Val Lys Ala Glu Lys Gln Glu Asp Ser Glu Ser Ser Glu Glu
680 685 690
Glu Ser Asp Ser Glu Glu Ala Ala Ala Ser Pro Ala Gln Val Lys
695 700 705
Thr Ser Val Lys Lys Thr Gln Ala Lys Ala Asn Pro Ala Ala Ala
710 715 720
Arg Ala Pro Ser Ala Lys Gly Thr Ile Ser Ala Pro Gly Lys Val
725 730 735
Val Thr Ala Ala Ala Gln Ala Lys Gln Arg Ser Pro Ser Lys Val
740 745 750
Lys Pro Pro Val Arg Asn Pro Gln Asn Ser Thr Val Leu Ala Arg
755 760 765
Gly Pro Ala Ser Val Pro Ser Val Gly Lys Ala Val Ala Thr Ala
770 775 780
Ala Gln Ala Gln Thr Gly Pro Glu Glu Asp Ser Gly Ser Ser Glu
785 790 795
Glu Glu Ser Asp Ser Glu Glu Glu Ala Glu Thr Leu Ala Gln Val
3/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
800 805 810
Lys Pro Ser Gly Lys Thr His Gln Ile Arg Ala Ala Leu Ala Pro
815 820 825
Ala Lys Glu Ser Pro Arg Lys Gly Ala Ala Pro Thr Pro Pro Gly
830 835 840
Lys Thr Gly Pro Ser Ala Ala Gln Ala Gly Lys Gln Asp Asp Ser
845 850 855
Gly Ser Ser Ser Glu Glu Ser Asp Ser Asp Gly Glu Ala Pro Ala
860 865 870
Ala Val Thr Ser Ala Gln Val Ile Lys Pro Pro Leu Ile Phe Val
875 880 885
Asp Pro Asn Arg Ser Pro Ala Gly Pro Ala Ala Thr Pro Ala Gln
890 895 900
Ala Gln Ala Ala Ser Thr Pro Arg Lys Ala Arg Ala Ser Glu Ser
905 910 915
Thr Ala Arg Ser Ser Ser Ser Glu Ser Glu Asp Glu Asp Val Ile
920 925 930
Pro Ala Thr Gln Cys Leu Thr Pro Gly Ile Arg Thr Asn Val Val
935 940 945
Thr Met Pro Thr Ala His Pro Arg Ile Ala Pro Lys Ala Ser Met
950 955 960
Ala Gly Ala Ser Ser Ser Lys Glu Ser Ser Arg Ile Ser Asp Gly
965 970 975
Lys Lys Gln Glu Gly Pro Ala Thr Gln Val Asp Ser Ala Val Gly
980 985 990
Thr Leu Pro Ala Thr Ser Pro Gln Ser Thr Ser Val Gln Ala Lys
995 1000 1005
Gly Thr Asn Lys Leu Arg Lys Pro Lys Leu Pro Glu Val Gln Gln
1010 1015 1020
Ala Thr Lys Ala Pro Glu Ser Ser Asp Asp Ser Glu Asp Ser Ser
1025 1030 1035
Asp Ser Ser Ser Gly Ser Glu Glu Asp Gly Glu Gly Pro Gln Gly
1040 1045 1050
Ala Lys Ser Ala His Thr Leu Val Gly Pro Thr Pro Ser Arg Thr
1055 1060 1065
Glu Thr Leu Val Glu Glu Thr Ala Ala Glu Ser Ser Glu Asp Asp
1070 1075 1080
Val Val Ala Pro Ser Gln Ser Leu Leu Ser Gly Tyr Met Thr Pro
1085 1090 1095
Gly Leu Thr Pro Ala Asn Ser Gln Ala Ser Lys Ala Thr Pro Lys
1100 1105 1110
Leu Asp Ser Ser Pro Ser Val Ser Ser Thr Leu Ala Ala Lys Asp
1115 1120 1125
Asp Pro Asp Gly Lys Gln Glu Ala Lys Pro Gln Gln Ala Ala Gly
1130 1135 1140
Met Leu Ser Pro Lys Thr Gly Gly Lys Glu Ala Ala Ser Gly Thr
1145 1150 1155
Thr Pro Gln Lys Ser Arg Lys Pro Lys Lys Gly Ala Gly Asn Pro
1160 1165 1170
Gln Ala Ser Thr Leu Ala Leu Gln Ser Asn Ile Thr Gln Cys Leu
1175 1180 1185
Leu Gly Gln Pro Trp Pro Leu Asn Glu Ala Gln Val Gln Ala Ser
1190 1195 1200
Val Val Lys Val Leu Thr Glu Leu Leu Glu Gln Glu Arg Lys Lys
1205 1210 1215
Val Val Asp Thr Thr Lys Glu Ser Ser Arg Lys Gly Trp Glu Ser
4/147
CA 02462660 2004-04-O1
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1220 1225 1230
Arg Lys Arg Lys Leu Ser Gly Asp Gln Pro Ala Ala Arg Thr Pro
1235 1240 1245
Arg Ser Lys Lys Lys Lys Lys Leu Gly Ala Gly Glu Gly Gly Glu
1250 1255 1260
Ala Ser Val Ser Pro Glu Lys Thr Ser Thr Thr Ser Lys Gly Lys
1265 1270 1275
Ala Lys Arg Asp Lys Ala Ser Gly Asp Val Lys Glu Lys Lys Gly
1280 1285 1290
Lys Gly Ser Leu Gly Ser Gln Gly Ala Lys Asp Glu Pro Glu Glu
1295 1300 1305
Glu Leu Gln Lys Gly Met Gly Thr Val Glu Gly Gly Asp Gln Ser
1310 1315 1320
Asn Pro Lys Ser Lys Lys Glu Lys Lys Lys Ser Asp Lys Arg Lys
1325 1330 ' 1335
Lys Asp Lys Glu Lys Lys Glu Lys Lys Lys Lys Ala Lys Lys Ala
1340 1345 1350
Ser Thr Lys Asp Ser Glu Ser Pro Ser Gln Lys Lys Lys Lys Lys
1355 1360 1365
Lys Lys Lys Thr Ala Glu Gln Thr Val
1370
<210> 2
<211> 588
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2608080CD1
<400> 2
Met Ala Ala Pro Ala Leu Ala Leu Val Ser Phe Glu Asn Val Val
1 5 10 15
Val Thr Phe Thr Gly Glu Glu Trp Gly His Leu Asp Leu Ala Gln
20 25 30
Arg Thr Leu Tyr Gln Glu Val Met Leu Glu Thr Cys Arg Leu Leu
35 40 45
Val Ser Leu Gly His Pro Val Pro Lys Pro Glu Leu Ile Tyr Leu
50 55 60
Leu Glu His Gly Gln Glu Leu Trp Thr Val Lys Arg Gly Leu Ser
65 70 75
Gln Ser Thr Cys Ala Gly Glu Lys Ala Lys Pro Lys Ile Thr Glu
80 85 90
Pro Thr Ala Ser Gln Leu Ala Phe Ser Glu Glu Ser Ser Phe Gln
95 100 105
Glu Leu Leu Ala Gln Arg Ser Ser Arg Asp Ser Arg Leu Gly Gln
110 115 120
Ala Arg Asp Glu Glu Lys Leu Ile Lys Ile Gln Glu Gly Asn Leu
125 130 135
Arg Pro Gly Thr Asn Pro His Lys Glu Ile Cys Pro Glu Lys Leu
140 145 150
Ser Tyr Lys His Asp Asp Leu Glu Pro Asp Asp Ser Leu Gly Leu
155 160 165
Arg Val Leu Gln Glu Arg Val Thr Pro Gln Asp Ala Leu His Glu
170 175 180
5/147
CA 02462660 2004-04-O1
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Cys Asp Ser Gln Gly Pro Gly Lys Asp Pro Met Thr Asp Ala Arg
185 190 195
Asn Asn Pro Tyr Thr Cys Thr Glu Cys Gly Lys Gly Phe Ser Lys
200 205 210
Lys Trp Ala Leu Val Arg His Gln Gln Ile His Ala Gly Val Lys
215 220 225
Pro Tyr Glu Cys Asn Glu Cys Gly Lys Ala Cys Arg Tyr Met Ala
230 235 240
Asp Val Ile Arg His Met Arg Leu His Thr Gly Glu Lys Pro Tyr
245 250 255
Lys Cys Ile Glu Cys Gly Lys Ala Phe Lys Arg Arg Phe His Leu
260 265 270
Thr Glu His Gln Arg Ile His Thr Gly Asp Lys Pro Tyr Glu Cys
275 280 285
Lys Glu Cys Gly Lys Ala Phe Thr His Arg Ser Ser Phe Ile Gln
290 295 300
His Asn Met Thr His Thr Arg Glu Lys Pro Phe Leu Cys Lys Glu
305 310 315
Cys Gly Lys Ala Phe Tyr Tyr Ser Ser Ser Phe Ala Gln His Met
320 325 330
Arg Ile His Thr Gly Lys Lys Leu Tyr Glu Cys Gly Glu Cys Gly
335 340 345
Lys Ala Phe Thr His Arg Ser Thr Phe Ile Gln His Asn Val Thr
350 355 360
His Thr Gly Glu Lys Pro Phe Leu Cys Lys Glu Cys Gly Lys Thr
365 370 375
Phe Cys Leu Asn Ser Ser Phe Thr Gln His Met Arg Ile His Thr
380 385 390
Gly Glu Lys Pro Tyr Glu Cys Gly Glu Cys Gly Lys Ala Phe Thr
395 400 405
His Arg Ser Thr Phe Ile Arg His Lys Arg Thr His Thr Gly Glu
410 415 420
Lys Pro Phe Glu Cys Lys Glu Cys Gly Lys Ala Phe Cys Asp Ser
425 430 435
Ser Ser Leu Ile Gln His Met Arg Ile His Thr Gly Glu Lys Pro
440 445 450
Tyr Glu Cys Ser Glu Cys Gly Lys Ala Phe Thr His His Ser Val
455 460 465
Phe Ile Arg His Asn Arg Thr His Ser Gly Gln Lys Pro Leu Glu
470 475 480
Cys Lys Glu Cys Ala Lys Ala Phe Tyr Tyr Ser Ser Ser Phe Thr
485 490 495
Arg His Met Arg Ile His Thr Gly Glu Lys Pro Tyr Val Cys Arg
500 505 510
Glu Cys Gly Lys Ala Phe Thr Gln Pro Ala Asn Phe Val Arg His
515 520 525
Asn Arg Ile His Thr Gly Glu Lys Pro Phe Glu Cys Lys Glu Cys
530 535 540
Glu Lys Ala Phe Cys Asp Asn Phe Ala Leu Thr Gln His Met Arg
545 550 555
Thr His Thr Gly Glu Lys Pro Phe Glu Cys Asn Glu Cys Gly Lys
560 565 570
Thr Phe Ser His Ser Ser Ser Phe Thr His His Arg Lys Ile His
575 580 585
Thr Arg Val
6/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
<210> 3
<211> 607
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7503402CD1
<400> 3
Met Leu Leu Ala Gln Ile Asn Arg Asp Ser Gln Gly Met Thr Glu
1 5 10 15
Phe Pro Gly Gly Gly Met Glu Ala Gln His Val Thr Leu Cys Leu
20 25 30
Thr Glu Ala Val Thr Val Ala Asp Ala Lys Leu Ile Asp Gly Gln
35 40 45
Val Ile Gln Leu Glu Asp Gly Ser Ala Ala Tyr Val Gln His Val
50 55 60
Pro Ile Pro Lys Ser Thr Gly Asp Ser Leu Arg Leu Glu Asp Gly
65 70 75
Gln Ala Val Gln Leu Glu Asp Gly Thr Thr Ala Phe Ile His His
80 85 90
Thr Ser Lys Asp Ser Tyr Asp Gln Ser Ala Leu Gln Ala Val Gln
95 100 105
Leu Glu Asp Gly Thr Thr Ala Tyr Ile His His Ala Val Gln Val
110 115 120
Pro Gln Ser Asp Thr Ile Leu Ala Ile Gln Ala Asp Gly Thr Val
125 130 135
Ala Gly Leu His Thr Gly Asp Ala Thr Ile Asp Pro Asp Thr Ile
140 145 150
Ser Ala Leu GIu Gln Tyr Ala Ala Lys Val Ser Ile Asp Gly Ser
155 160 165
Glu Ser Val Ala Gly Thr Gly Met Ile Gly Glu Asn Glu Gln Glu
170 175 180
Lys Lys Met Gln Ile Val Leu Gln Gly His Ala Thr Arg Val Thr
185 190 195
Ala Lys Ser Gln Gln Ser Gly Glu Lys AIa Phe Arg Cys Glu Tyr
200 205 210
Asp Gly Cys Gly Lys Leu Tyr Thr Thr Ala His His Leu Lys Val
215 220 225
His Glu Arg Ser His Thr Gly Asp Arg Pro Tyr Gln Cys Glu His
230 235 240
Ala Gly Cys Gly Lys Ala Phe Ala Thr Gly Tyr Gly Leu Lys Ser
245 250 255
His Val Arg Thr His Thr Gly Glu Lys Pro Tyr Arg Cys Ser Glu
260 265 270
Asp Asn Cys Thr Lys Ser Phe Lys Thr Ser Gly Asp Leu Gln Lys
275 280 285
His Ile Arg Thr His Thr Gly Glu Arg Pro Phe Lys Cys Pro Phe
290 295 300
Glu Gly Cys Gly Arg Ser Phe Thr Thr Ser Asn Ile Arg Lys Val
305 310 315
His Val Arg Thr His Thr Gly Glu Arg Pro Tyr Tyr Cys Thr Glu
320 325 330
Pro Gly Cys Gly Arg Ala Phe Ala Ser Ala Thr Asn Tyr Lys Asn
335 340 345
7/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
His Val Arg Ile His Thr Gly Glu Lys Pro Tyr Val Cys Thr Val
350 355 360
Pro Gly Cys Asp Lys Arg Phe Thr Glu Tyr Ser Ser Leu Tyr Lys
365 370 375
His His Val Val His Thr His Ser Lys Pro Tyr Asn Cys Asn His
380 385 390
Cys Gly Lys Thr Tyr Lys Gln Ile Ser Thr Leu Ala Met His Lys
395 400 405
Arg Thr Ala His Asn Asp Thr Glu Pro Ile Glu Glu Glu Gln Glu
410 415 420
Ala Phe Phe Glu Pro Pro Pro Gly Gln Gly Glu Asp Val Leu Lys
425 430 435
Gly Ser Gln Ile Thr Tyr Val Thr Gly Val Glu Gly Asp Asp Val
440 445 450
Val Ser Thr Gln Val Ala Thr Val Thr Gln Ser Gly Leu Ser Gln
455 460 465
Gln Val Thr Leu Ile Ser Gln Asp Gly Thr Gln His Val Asn Ile
470 475 480
Ser Gln Ala Asp Met Gln Ala Ile Gly Asn Thr Ile Thr Met Val
485 490 495
Thr Gln Asp Gly Thr Pro Ile Thr Val Pro Ala His Asp Ala Val
500 505 510
Ile Ser Ser Ala Gly Thr His Ser Val Ala Met Val Thr Ala Glu
515 520 525
Gly Thr Glu Gly Gln Gln Val Ala Ile Val Ala Gln Asp Leu Ala
530 535 540
Ala Phe His Thr Ala Ser Ser Glu Met Gly His Gln Gln His Ser
545 550 555
His His Leu Val Thr Thr Glu Thr Arg Pro Leu Thr Leu Val Ala
560 ~ 565 570
Thr Ser Asn Gly Thr Gln Ile Ala Val Gln Leu Gly Glu Gln Pro
575 580 585
Ser Leu Glu Glu Ala Ile Arg Ile Ala Ser Arg Ile Gln Gln Gly
590 595 600
Glu Thr Pro Gly Leu Asp Asp
605
<210> 4
<211> 422
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503517CD1
<400> 4
Met Glu Phe Gln Ala Val Val Met Ala Val Gly Gly Gly Ser Arg
1 5 10 15
Met Thr Asp Leu Thr Ser Ser Ile Pro Lys Pro Leu~Leu Pro Val
20 25 30
Gly Asn Lys Pro Leu Ile Trp Tyr Pro Leu Asn Leu Leu Glu Arg
35 40 45
Val Gly Phe Glu Glu Val Ile Val Val Thr Thr Arg Asp Val Gln
50 55 60
Lys Ala Leu Cys Ala Glu Phe Lys Met Lys Met Lys Pro Asp Ile
8/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
65 70 75
Val Cys Ile Pro Asp Asp Ala Asp Met Gly Thr Ala Asp Ser Leu
80 85 90
Arg Tyr Ile Tyr Pro Lys Leu Lys Thr Asp Val Leu Val Leu Ser
95 100 105
Cys Asp Leu Ile Thr Asp Val Ala Leu His Glu Val Val Asp Leu
110 115 120
Phe Arg Ala Tyr Asp Ala Ser Leu Ala Met Leu Met Arg Lys Gly
125 130 135
Gln Asp Ser Ile Glu Pro Val Pro Gly Gln Lys Gly Lys Lys Lys
140 145 150
Ala Val Glu Gln Arg Asp Phe Ile Gly Val Asp Ser Thr Gly Lys
155 160 165
Arg Leu Leu Phe Met Ala Asn Glu Ala Asp Leu Asp Glu Glu Leu
170 175 180
Val Ile Lys Gly Ser Ile Leu Gln Lys Ser Ile Thr Ser Ile Arg
185 190 195
Ser Glu Leu Ile Pro Tyr Leu Val Arg Lys Gln Phe Ser Ser Ala
200 205 210
Ser Ser Gln Gln Gly Gln Glu Glu Lys Glu Glu Asp Leu Lys Lys
215 220 225
Lys Glu Leu Lys Ser Leu Asp Ile Tyr Ser Phe Ile Lys Glu Ala
230 235 240
Asn Thr Leu Asn Leu Ala Pro Tyr Asp Ala Cys Trp Asn Ala Cys
245 250 255
Arg Gly Asp Arg Trp Glu Asp Leu Ser Arg Ser Gln Val Arg Cys
260 265 270
Tyr Val His Ile Met Lys Glu Gly Leu Cys Ser Arg Val Ser Thr
275 280 285
Leu Gly Leu Tyr Met Glu Ala Asn Arg Gln Val Pro Lys Leu Leu
290 295 300
Ser Ala Leu Cys Pro Glu Glu Pro Pro Val His Ser Ser Ala Gln
305 310 315
Ile Val Ser Lys His Leu Val Gly Val Asp Ser Leu Ile Gly Pro
320 325 330
Glu Thr Gln Ile Gly Glu Lys Ser Ser Ile Lys Arg Ser Val Ile
335 340 345
Gly Ser Ser Cys Leu Ile Lys Asp Arg Val Thr Ile Thr Asn Cys
350 355 360
Leu Leu Met Asn Ser Val Thr Val Glu Glu Gly Ser Asn Ile Gln
365 370 375
Gly Ser Val Ile Cys Asn Asn Ala Val Ile Glu Lys Gly Ala Asp
380 385 390
Ile Lys Asp Cys Leu Ile Gly Ser Gly Gln Arg Ile Glu Ala Lys
395 400 405
Ala Lys Arg Val Asn Glu Val Ile Val Gly Asn Asp Gln Leu Met
410 415 420
Glu Ile
<210> 5
<211> 142
<212> PRT
<213> Homo sapiens
<220>
9/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
<221> misc_feature
<223> Incyte ID No: 7500014CD1
<400> 5
Met Ser Glu Gly Glu Ser Gln Thr Val Leu Ser Ser Gly Ser Asp
1 5 10 15
Pro Lys Val Glu Ser Ser Ser Ser Ala Pro Gly Leu Thr Ser Pro
20 25 30
Val Val Pro Pro Ser Val Lys Thr Pro Thr Pro Glu Pro Ala Glu
35 40 45
Val Glu Thr Arg Lys Val Val Leu Met Gln Cys Asn Ile Glu Ser
50 55 60
Val Glu Glu Gly Val Lys His His Leu Thr Leu Leu Leu Lys Leu
65 70 75
Glu Asp Lys Leu Asn Arg His Leu Ser Cys Asp Leu Met Pro Asn
80 85 90
Glu Asn Ile Pro Glu Leu Ala Ala Glu Leu Val Gln Leu Gly Phe
95 100 105
Ile Ser Glu Ala Asp Gln Ser Arg Leu Thr Ser Leu Leu Glu Glu
110 115 120
Thr Leu Asn Lys Phe Asn Phe Ala Arg Asn Ser Thr Leu Asn Ser
125 130 135
Ala Ala Val Thr Val Ser Ser
140
<210> 6
<211> 433
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7501365CD1
<400> 6
Met Ala Arg Val Ala Trp Gly Leu Leu Trp Leu Leu Leu Gly Ser
1 5 10 15
Ala Gly Ala Gln Tyr Glu Lys Tyr Ser Phe Arg Gly Phe Pro Pro
20 25 30
Glu Asp Leu Met Pro Leu Ala Ala Ala Tyr Gly His Ala Leu Glu
35 40 45
Gln Tyr Glu Gly Glu Ser Trp Arg Glu Ser Ala Arg Tyr Leu Glu
50 55 60
Ala Ala Leu Arg Leu His Arg Leu Leu Arg Asp Ser Glu Ala Phe
65 70 75
Cys His Ala Asn Cys Ser Gly Pro Ala Pro Ala Ala Lys Pro Asp
80 85 90
Pro Asp Gly Gly Arg Ala Asp Glu Trp Ala Cys Glu Leu Arg Leu
95 100 105
Phe Gly Arg Val Leu Glu Arg Ala Ala Cys Leu Arg Arg Cys Lys
110 115 120
Arg Thr Leu Pro Ala Phe Gln Val Pro Tyr Pro Pro Arg Gln Leu
125 130 135
Leu Arg Asp Phe Gln Ser Arg Leu Pro Tyr Gln Tyr Leu His Tyr
140 145 150
Ala Leu Phe Lys Ala Asn Arg Leu Glu Lys Ala Val Ala Ala Ala
10/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
155 160 165
Tyr Thr Phe Leu Gln Arg Asn Pro Lys His Glu Leu Thr Ala Lys
170 175 180
Tyr Leu Asn Tyr Tyr Arg Gly Met Leu Asp Val Ala Asp Glu Ser
185 190 195
Leu Thr Asp Leu Glu Ala Gln Pro Tyr Glu Ala Val Phe Leu Arg
200 205 210
Ala Val Lys Leu Tyr Asn Ser Gly Asp Phe Arg Ser Ser Thr Glu
215 220 225
Asp Met Glu Arg Ala Leu Ser Glu Tyr Leu Ala Val Phe Ala Arg
230 235 240
Cys Leu Ala Gly Cys Glu Gly Ala His Glu Gln Val Asp Phe Lys
245 250 255
Asp Phe Tyr Pro Ala Ile Ala Asp Leu Phe Ala Glu Ser Leu Gln
260 265 270
Cys Lys Val Asp Cys Glu Ala Asn Leu Thr Pro Asn Val Gly Gly
275 280 285
Tyr Phe Val Asp Lys Phe Val Ala Thr Met Tyr His Tyr Leu Gln
290 295 300
Phe Ala Tyr Tyr Lys Leu Asn Asp Val Arg Gln Ala Ala Arg Ser
305 310 315
Ala Ala Ser Tyr Met Leu Phe Asp Pro Lys Asp Ser Val Met Gln
320 325 330
Gln Asn Leu Val Tyr Tyr Arg Phe His Arg Ala Arg Trp Gly Leu
335 340 345
Glu Glu Glu Asp Phe Gln Pro Arg Glu Glu Ala Met Leu Tyr His
350 355 360
Asn Gln Thr Ala Glu Leu Arg Glu Leu Leu Glu Phe Thr His Met
365 370 375
Tyr Leu Gln Ser Asp Asp Glu Ser Gln Ser Leu Asn Ser His Glu
380 385 390
Lys Gly Thr Pro His Thr Pro Gln Ala Trp Glu Ala Trp Cys Arg
395 400 405
Trp Pro His Pro His Gln Pro Gly Gln Gln Gln Glu Leu Phe Ile
410 415 420
Lys Asn Leu Arg Trp Ala Arg Cys Gly Gly Ser His Leu
425 430
<210> 7
<211> 1450
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503540CD1
<400> 7
Met Ser Leu Thr Ser Trp Phe Leu Val Ser Ser Gly Gly Thr Arg
1 5 10 15
His Arg Leu Pro Arg Glu Met Ile Phe Val Gly Arg Asp Asp Cys
20 25 30
Glu Leu Met Leu Gln Ser Arg Ser Val Asp Lys Gln His Ala Val
35 40 45
Ile Asn Tyr Asp Ala Ser Thr Asp Glu His Leu Val Lys Asp Leu
50 55 60
11/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
Gly Ser Leu Asn Gly Thr Phe Val Asn Asp Val Arg Ile Pro Glu
65 70 75
Gln Thr Tyr Ile Thr Leu Lys Leu Glu Asp Lys Leu Arg Phe Gly
80 85 90
Tyr Asp Thr Asn Leu Phe Thr Val Val Gln Gly Glu Met Arg Val
95 100 105
Pro Glu Glu Ala Leu Lys His Glu Lys Phe Thr Ile Gln Leu Gln
110 115 120
Leu Ser Gln Lys Ser Ser Glu Ser Glu Leu Ser Lys Ser Ala Ser
125 130 135
Ala Lys Ser Ile Asp Ser Lys Val Ala Asp Ala Ala Thr Glu Val
140 145 150
Gln His Lys Thr Thr Glu Ala Leu Lys Ser Glu Glu Lys Ala Met
155 160 165
Asp Ile Ser Ala Met Pro Arg Gly Thr Pro Leu Tyr Gly Gln Pro
170 175 180
Ser Trp Trp Gly Asp Asp Glu Val Asp Glu Lys Arg Ala Phe Lys
185 190 195
Thr Asn Gly Lys Pro Glu Glu Lys Asn His Glu Ala Gly Thr Ser
200 205 210
Gly Cys Ser Ile Asp Ala Lys Gln Val Glu Glu Gln Ser Ala Ala
215 220 225
Ala Asn Glu Glu Val Leu Phe Pro Phe Cys Arg Glu Pro Ser Tyr
230 235 240
Phe Glu Ile Pro Thr Lys GIu Phe Gln Gln Pro Ser Gln Ile Thr
245 250 255
Glu Ser Thr Ile His Glu Ile Pro Thr Lys Asp Thr Pro Ser Ser
260 265 270
His Ile Thr Gly Ala Gly His Ala Ser Phe Thr Ile Glu Phe Asp
275 280 285
Asp Ser Thr Pro Gly Lys Val Thr Ile Arg Asp His Val Thr Lys
290 295 300
Phe Thr Ser Asp Gln Arg His Lys Ser Lys Lys Ser Ser Pro Gly
305 310 315
Thr Gln Asp Leu Leu Gly Ile Gln Thr Gly Met Met Ala Pro Glu
320 325 330
Asn Lys Val Ala Asp Trp Leu Ala Gln Asn Asn Pro Pro Gln Met
335 340 345
Leu Trp Glu Arg Thr Glu Glu Asp Ser Lys Ser Ile Lys Ser Asp
350 355 360
Val Pro Val Tyr Leu Lys Arg Leu Lys Gly Asn Lys His Asp Asp
365 370 375
Gly Thr Gln Ser Asp Ser Glu Asn Ala Gly Ala His Arg Arg Cys
380 385 390
Ser Lys Arg Ala Thr Leu Glu Glu His Leu Arg Arg His His Ser
395 400 405
Glu His Lys Lys Leu Gln Lys Val Gln Ala Thr Glu Lys His Gln
410 415 420
Asp Gln Ala Val Val Phe GIy Val Asp Asp Asn Gln Asp Tyr Asn
425 430 435
Arg Pro Val Ile Asn Glu Lys His Lys Asp Leu Ile Lys Asp Trp
440 445 450
Ala Leu Ser Ser Ala Ala Ala Val Met Glu Glu Arg Lys Pro Leu
455 460 465
Thr Thr Ser Gly Phe His His Ser Glu Glu Gly Thr Ser Ser Ser
470 475 480
12/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
Gly Ser Lys Arg Trp Val Ser Gln Trp Ala Ser Leu Ala Ala Asn
485 490 495
His Thr Arg His Asp Gln Glu Glu Arg Ile Met Glu Phe Ser Ala
500 505 510
Pro Leu Pro Leu Glu Asn Glu Thr Glu Ile Ser Glu Ser Gly Met
515 520 525
Thr Val Arg Ser Thr Gly Ser Ala Thr Ser Leu Ala Ser Gln Gly
530 535 540
Glu Arg Arg Arg Arg Thr Leu Pro Gln Leu Pro Asn Glu Glu Lys
545 550 555
Ser Leu Glu Ser His Arg Ala Lys Val Val Thr Gln Arg Ser Glu
560 565 570
Ile Gly Glu Lys Gln Asp Thr Glu Leu Gln Glu Lys Glu Thr Pro
575 580 585
Thr Gln Val Tyr Gln Lys Asp Lys Gln Asp Ala Asp Arg Pro Leu
590 595 600
Ser Lys Met Asn Arg Ala Val Asn Gly Glu Thr Leu Lys Thr Gly
605 610 615
Gly Asp Asn Lys Thr Leu Leu His Leu Gly Ser Ser Ala Pro Gly
620 625 630
Lys Glu Lys Ser Glu Thr Asp Lys Glu Thr Ser Leu Val Lys Gln
635 640 645
Thr Leu Ala Lys Leu Gln Gln Gln Glu Gln Arg Glu Glu Ala Gln
650 655 660
Trp Thr Pro Thr Lys Leu Ser Ser Lys Asn Val Ser Gly Gln Thr
665 670 675
Asp Lys Cys Arg Glu Glu Thr Phe Lys Gln Glu Ser Gln Pro Pro
680 685 690
Glu Lys Asn Ser Gly His Ser Thr Ser Lys Gly Asp Arg Val Ala
695 700 705
Gln Ser Glu Ser Lys Arg Arg Lys Ala Glu Glu Ile Leu Lys Ser
710 715 720
Gln Thr Pro Lys Gly Gly Asp Lys Lys Glu Ser Ser Lys Ser Leu
725 730 735
Val Arg Gln Gly Ser Phe Thr Ile Glu Lys Pro Ser Pro Asn Ile
740 745 750
Pro Ile Glu Leu Ile Pro His Ile Asn Lys Gln Thr Ser Ser Thr
755 760 765
Pro Ser Ser Leu Ala Leu Thr Ser Ala Ser Arg Ile Arg Glu Arg
770 775 780
Ser Glu Ser Leu Asp Pro Asp Ser Ser Met Asp Thr Thr Leu Ile
785 790 795
Leu Lys Asp Thr Glu Ala Val Met Ala Phe Leu Glu Ala Lys Leu
800 805 810
Arg Glu Asp Asn Lys Thr Asp Glu Gly Pro Asp Thr Pro Ser Tyr
815 820 825
Asn Arg Asp Asn Ser Ile Ser Pro Glu Ser Asp Val Asp Thr Ala
830 835 840
Ser Thr Ile Ser Leu Val Thr Gly Glu Thr Glu Arg Lys Ser Thr
845 850 855
Gln Lys Arg Lys Ser Phe Thr Ser Leu Tyr Lys Asp Arg Cys Ser
860 865 870
Thr Gly Ser Pro Ser Lys Asp Val Thr Lys Ser Ser Ser Ser Gly
875 880 885
Ala Arg Glu Lys Met Glu Lys Lys Thr Lys Ser Arg Ser Thr Asp
890 895 900
13/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
Val Gly Ser Arg Ala Asp Gly Arg Lys Phe Val Gln Ser Ser Gly
905 910 915
Arg Ile Arg Gln Pro Ser Val Asp Leu Thr Asp Asp Asp Gln Thr
920 925 930
Ser Ser Val Pro His Ser Ala Ile Ser Asp Ile Met Ser Ser Asp
935 940 945
Gln Glu Thr Tyr Ser Cys Lys Pro His Gly Arg Thr Pro Leu Thr
950 955 960
Ser Ala Asp Glu His Val His Ser Lys Leu Glu Gly Ser Lys Val
965 970 975
Thr Lys Ser Lys Thr Ser Pro Val Val Ser Gly Ser Ser Ser Lys
980 985 990
Ser Thr Thr Leu Pro Arg Pro Arg Pro Thr Arg Thr Ser Leu Leu
995 1000 1005
Arg Arg Ala Arg Leu Gly Glu Ala Ser Asp Ser Glu Leu Ala Asp
1010 1015 1020
Ala Asp Lys Ala Ser Val Ala Ser Glu Val Ser Thr Thr Ser Ser
1025 1030 1035
Thr Ser Lys Pro Pro Thr Gly Arg Arg Asn Ile Ser Arg Ile Asp
1040 1045 1050
Leu Leu Ala Gln Pro Arg Arg Thr Arg Leu Gly Ser Leu Ser Ala
1055 1060 1065
Arg Ser Asp Ser Glu Ala Thr Ile Ser Arg Ser Ser Ala Ser Ser
1070 1075 1080
Arg Thr Ala Glu Ala Ile Ile Arg Ser Gly Ala Arg Leu Val Pro
1085 1090 1095
Ser Asp Lys Phe Ser Pro Arg Ile Arg Ala Asn Ser Ile Ser Arg
1100 1105 1110
Leu Ser Asp Ser Lys Val Lys Ser Met Thr Ser Ala His Gly Ser
1115 1120 1125
AIa Ser Ala Leu Lys Thr Thr Arg Leu Gln Ser Ala Gly Ser Ala
1130 1135 1140
Met Pro Thr Ser Ser Ser Phe Lys His Arg Ile Lys Glu Gln Glu
1145 1150 1155
Asp Tyr Ile Arg Asp Trp Thr Ala His Arg Glu Glu Ile Ala Arg
1160 1165 1170
Ile Ser Gln Asp Leu Ala Leu Ile Ala Arg Glu Ile Asn Asp Val
1175 1180 1185
Ala Gly Glu Ile Asp Ser Val Thr Ser Ser Gly Thr Ala Pro Ser
1190 1195 1200
Thr Thr Val Ser Thr Ala Ala Thr Thr Pro Gly Ser Ala Ile Asp
1205 1210 1215
Thr Arg Glu Glu Leu Val Asp Arg Val Phe Asp Glu Ser Leu Asn
1220 1225 1230
Phe Gln Lys Ile Pro Pro Leu Val His Ser Lys Thr Pro Glu Gly
1235 1240 1245
Asn Asn Gly Arg Ser Gly Asp Pro Arg Pro Gln Ala Ala Glu Pro
1250 1255 1260
Pro Asp His Leu Thr Ile Thr Arg Arg Arg Thr Trp Ser Arg Asp
1265 1270 1275
Glu Val Met Gly Asp Asn Leu Leu Leu Ser Ser Val Phe Gln Phe
1280 1285 1290
Ser Lys Lys Ile Arg Gln Ser Ile Asp Lys Thr Ala Gly Lys Ile
1295 1300 1305
Arg Ile Leu Phe Lys Asp Lys Asp Arg Asn Trp Asp Asp Ile Glu
1310 1315 1320
14/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
Ser Lys Leu Arg Ala Glu Ser Glu Val Pro Ile Val Lys Thr Ser
1325 1330 1335
Ser Met Glu Ile Ser Ser Ile Leu Gln Glu Leu Lys Arg Val Glu
1340 1345 1350
Lys Gln Leu Gln Ala Ile Asn Ala Met Ile Asp Pro Asp Gly Thr
1355 1360 1365
Leu Glu Ala Leu Asn Asn Met Gly Phe Pro Ser Ala Met Leu Pro
1370 1375 1380
Ser Pro Pro Lys Gln Lys Ser Ser Pro Val Asn Asn His His Ser
1385 1390 1395
Pro Gly Gln Thr Pro Thr Leu Gly Gln Pro Glu Ala Arg Ala Leu
1400 1405 1410
His Pro Ala Ala Val Ser Ala Ala Ala Glu Phe Glu Asn Ala Glu
1415 1420 1425
Ser Glu Ala Asp Phe Ser Ile His Phe Asn Arg Val Asn Pro Asp
1430 1435 1440
Gly Glu Glu Glu Asp Val Thr Val His Lys
1445 1450
<210> 8
<211> 647
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7504326CD1
<400> 8
Met Val Met Tyr Ala Arg Lys Gln Gln Arg Leu Ser Asp Gly Cys
1 5 10 15
His Asp Arg Arg Gly Asp Ser Gln Pro Tyr Gln Ala Leu Lys Tyr
20 25 30
Ser Ser Lys Ser His Pro Ser Ser Gly Asp His Arg His Glu Lys
35 40 45
Met Arg Asp Ala Gly Asp Pro Ser Pro Pro Asn Lys Met Leu Arg
50 55 60
Arg Ser Asp Ser Pro Glu Asn Lys Tyr Ser Asp Ser Thr Gly His
65 70 75
Ser Lys Ala Lys Asn Val His Thr His Arg Val Arg Glu Arg Asp
80 85 90
Gly Gly Thr Ser Tyr Ser Pro Gln Glu Asn Ser His Asn His Ser
95 100 105
Ala Leu His Ser Ser Asn Ser His Ser Ser Asn Pro Ser Asn Asn
110 115 120
Pro Ser Lys Thr Ser Asp Ala Pro Tyr Asp Ser Ala Asp Asp Trp
125 130 135
Ser Glu His Ile Ser Ser Ser Gly Lys Lys Tyr Tyr Tyr Asn Cys
140 145 150
Arg Thr Glu Val Ser Gln Trp Glu Lys Pro Lys Glu Trp Leu Glu
155 160 165
Arg Glu Gln Arg Gln Lys Glu Ala Asn Lys Met Ala Val Asn Ser
170 175 180
Phe Pro Lys Asp Arg Asp Tyr Arg Arg Glu Val Met Gln Ala Thr
185 190 ~ 195
Ala Thr Ser Gly Phe Ala Ser Gly Met Glu Asp Lys His Ser Ser
15/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
200 205 210
Asp Ala Ser Ser Leu Leu Pro Gln Asn Ile Leu Ser Gln Thr Ser
215 220 225
Arg His Asn Asp Arg Asp Tyr Arg Leu Pro Arg Ala Glu Thr His
230 235 240
Ser Ser Ser Thr Pro Val Gln His Pro Ile Lys Pro Val Val His
245 250 255
Pro Thr Ala Thr Pro Ser Thr Val Pro Ser Ser Pro Phe Thr Leu
260 265 270
Gln Ser Asp His Gln Pro Lys Lys Ser Phe Asp Ala Asn Gly Ala
275 280 285
Ser Thr Leu Ser Lys Leu Pro Thr Pro Thr Ser Ser Val Pro Ala
290 295 300
Gln Lys Thr Glu Arg Lys Glu Ser Thr Ser Gly Asp Lys Pro Val
305 310 315
Ser His Ser Cys Thr Thr Pro Ser Thr Ser Ser Ala Ser Gly Leu
320 325 330
Asn Pro Thr Ser Ala Pro Pro Thr Ser Ala Ser Ala Val Pro Val
335 340 345
Ser Pro Val Pro Gln Ser Pro Ile Pro Pro Leu Leu Gln Asp Pro
350 355 360
Asn Leu Leu Arg Gln Leu Leu Pro Ala Leu Gln Ala Thr Leu Gln
365 370 375
Leu Asn Asn Ser Asn Val Asp Ile Ser Lys Ile Asn Glu Val Leu
380 385 390
Thr Ala Ala Val Thr Gln Ala Ser Leu Gln Ser Ile Ile His Lys
395 400 405
Phe Leu Thr Ala Gly Pro Ser Ala Phe Asn Ile Thr Ser Leu Ile
410 415 420
Ser Gln Ala Ala Gln Leu Ser Thr Gln Ala Gln Pro Ser Asn Gln
425 430 435
Ser Pro Met Ser Leu Thr Ser Asp Ala Ser Ser Pro Arg Ser Tyr
440 445 450
Val Ser Pro Arg Ile Ser Thr Pro Gln Thr Asn Thr Val Pro Ile
455 ~ 460 465
Lys Pro Leu Ile Ser Thr Pro Pro Val Ser Ser Gln Pro Lys Val
470 475 480
Ser Thr Pro Val Val Lys Gln Gly Pro Val Ser Gln Ser Ala Thr
485 490 495
Gln Gln Pro Val Thr Ala Asp Lys Gln Gln Gly His Glu Pro Val
500 505 510
Ser Pro Arg Ser Leu Gln Arg Ser Ser Ser Gln Arg Ser Pro Ser
515 520 525
Pro Gly Pro Asn His Thr Ser Asn Ser Ser Asn Ala Ser Asn Ala
530 535 540
Thr Val Val Pro Gln Asn Ser Ser Ala Arg Ser Thr Cys Ser Leu
545 550 555
Thr Pro Ala Leu Ala Ala His Phe Ser Glu Asn Leu Ile Lys His
560 565 570
Val Gln Gly Trp Pro Ala Asp His Ala Glu Lys Gln Ala Ser Arg
575 580 585
Leu Arg Glu Glu Ala His Asn Met Gly Thr Ile His Met Ser Glu
590 595 600
Ile Cys Thr Glu Leu Lys Asn Leu Arg Ser Leu Val Arg Val Cys
605 610 615
Glu Ile Gln Ala Thr Leu Arg Glu Gln Arg Ile Leu Phe Leu Arg
16/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
620 625 630
Gln Gln Ile Lys Glu Leu Glu Lys Leu Lys Asn Gln Asn Ser Phe
635 640 645
Met Val
<210> 9
<211> 195
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7504388CD1
<400> 9
Met Ala Pro Pro Ala Ala Pro Gly Arg Asp Arg Val Gly Arg Glu
1 5 10 15
Asp Glu Asp Gly Trp Glu Thr Arg Gly Asp Arg Lys Val Gln Ala
20 25 30
Lys Leu Glu Asn Ala Glu Val Leu Glu Leu Thr Val Arg Arg Val
35 40 45
Gln Gly Val Leu Arg Gly Arg Ala Arg Glu Arg Glu Gln Leu Gln
50 55 60
Ala Glu Ala Ser Glu Arg Phe Ala Ala Gly Tyr Ile Gln Cys Met
65 70 75
His Glu Val His Thr Phe Val Ser Thr Cys Gln Ala Ile Asp Ala
80 85 90
Thr Val Ala Ala Glu Leu Leu Asn His Leu Leu Glu Ser Met Pro
95 100 105
Leu Arg Glu Gly Ser Ser Phe Gln Asp Leu Leu Gly Asp AIa Leu
110 115 120
Ala Gly Pro Pro Arg Ala Pro Gly Arg Ser Gly Trp Pro Ala Gly
125 130 135
Gly Ala Pro Gly Ser Pro Ile Pro Ser Pro Pro Gly Pro Gly Asp
140 145 150
Asp Leu Cys Ser Asp Leu Glu Glu Ala Pro Glu Ala Glu Leu Ser
155 160 165
Gln Ala Pro Ala Glu Gly Pro Asp Leu Val Pro Ala Ala Leu Gly
170 175 180
Ser Leu Thr Thr Ala Gln Ile Ala Arg Ser Val Trp Arg Pro Trp
185 190 195
<210> 10
<211> 781
<212> PRT
<213> Homo sapiens
<220>
<221> mist feature
<223> Incyte ID No: 2828380CD1
<400> 10
Met Ala Thr Gln Gly His Leu Thr Phe Lys Asp Val Ala Ile Glu
1 5 10 15
17/147
CA 02462660 2004-04-O1
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Phe Ser Gln Glu Glu Trp Lys Cys Leu Glu Pro Val Gln Lys Ala
20 25 30
Leu Tyr Lys Asp Val Met Leu Glu Asn Tyr Arg Asn Leu Val Phe
35 40 45
Leu Gly Ile Ser Pro Lys Cys Val Ile Lys Glu Leu Pro Pro Thr
50 55 60
Glu Asn Ser Asn Thr Gly Glu Arg Phe Gln Thr Val Ala Leu Glu
65 70 75
Arg His Gln Ser Tyr Asp Ile Glu Asn Leu Tyr Phe Arg Glu Ile
80 85 90
Gln Lys His Leu His Asp Leu Glu Phe Gln Trp Lys Asp Gly Glu
95 100 105
Thr Asn Asp Lys Glu Val Pro Val Pro His Glu Asn Asn Leu Thr
110 115 120
Gly Lys Arg Asp Gln His Ser Gln Gly Asp Val Glu Asn Asn His
125 130 135
Ile Glu Asn Gln Leu Thr Ser Asn Phe Glu Ser Arg Leu Ala Glu
140 145 150
Leu Gln Lys Val Gln Thr Glu Gly Arg Leu Tyr Glu Cys Asn Glu
155 160 165
Thr Glu Lys Thr Gly Asn Asn Gly Cys Leu Val Ser Pro His Ile
170 175 180
Arg Glu Lys Thr Tyr Val Cys Asn Glu Cys Gly Lys Ala Phe Lys
185 190 195
Ala Ser Ser Ser Leu Ile Asn His Gln Arg Ile His Thr Thr Glu
200 205 210
Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe His Arg Ala
215 220 225
Ser Leu Leu Thr Val His Lys Val Val His Thr Arg Gly Lys Ser
230 235 240
Tyr Gln Cys Asp Val Cys Gly Lys Ile Phe Arg Lys Asn Ser Tyr
245 250 255
Phe Val Arg His Gln Arg Ser His Thr Gly Gln Lys Pro Tyr Ile
260 265 270
Cys Asn Glu Cys Gly Lys Ser Phe Ser Lys Ser Ser His Leu Ala
275 280 285
Val His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Asn
290 295 300
Leu Cys Gly Lys Ser Phe Ser Gln Arg Val His Leu Arg Leu His
305 310 315
Gln Thr Val His Thr Gly Glu Arg Pro Phe Lys Cys Asn Glu Cys
320 325 330
Gly Lys Thr Phe Lys Arg Ser Ser Asn Leu Thr Val His Gln Val
335 340 345
Ile His Ala Gly Lys Lys Pro Tyr Lys Cys Asp Val Cys Gly Lys
350 355 360
Ala Phe Arg His Arg Ser Asn Leu Val Cys His Arg Arg Ile His
365 370 375
Ser Gly Glu Lys Gln Tyr Lys Cys Asn Glu Cys Gly Lys Val Phe
380 385 390
Ser Lys Arg Ser Ser Leu Ala Val His Arg Arg Ile His Thr Val
395 400 405
Glu Lys Pro Cys Lys Cys Asn Glu Cys Gly Lys Val Phe Ser Lys
410 415 420
Arg Ser Ser Leu Ala Val His Gln Arg Ile His Thr Gly Gln Lys
425 430 435
18/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
Thr Tyr Lys Cys Asn Lys Cys Gly Lys Val Tyr Ser Lys His Ser
440 445 450
His Leu Ala Val His Trp Arg Ile His Thr Gly Glu Lys Ala Tyr
455 460 465
Lys Cys Asn Glu Cys Gly Lys Val Phe Ser Ile His Ser Arg Leu
470 475 480
Ala Ala His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys
485 490 495
Asn Glu Cys Gly Lys Val Phe Ser Gln His Ser Arg Leu Ala Val
500 505 510
His Arg Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu
515 520 525
Cys Gly Lys Val Phe Ser Asp Arg Ser Ala Phe Ala Arg His Arg
530 535 540
Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly
545 550 555
Lys Val Phe Ser Gln Cys Ser Arg Leu Thr Val His Leu Arg Ile
560 565 570
His Ser Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Val
575 580 585
Tyr Ser Gln Tyr Ser His Leu Val Gly His Arg Arg Val His Thr
590 595 600
Gly Glu Lys Pro Tyr Lys Cys His Glu Cys Gly Lys Ala Phe Asn
605 610 615
Gln GIy Ser Thr Leu Asn Arg His GIn Arg Ile His Thr GIy Glu
620 625 630
Lys Pro Tyr Lys Cys Asn Gln Cys Gly Asn Ser Phe Ser Gln Arg
635 640 645
Val His Leu Arg Leu His Gln Thr Val His Thr Gly Asp Arg Pro
650 655 660
Tyr Lys Cys Asn Glu Cys Gly Lys Thr Phe Lys Arg Ser Ser Asn
665 670 675
Leu Thr Ala His Gln Ile Ile His Ala Gly Lys Lys Pro Tyr Lys
680 685 690
Cys Asp Glu Cys Gly Lys Val Phe Arg His Ser Ser His Leu Val
695 700 705
Ser His Gln Arg Ile His Thr Gly Glu Lys Arg Tyr Lys Cys Ile
710 715 720
Glu Cys Gly Lys Ala Phe Gly Arg Leu Phe Ser Leu Ser Lys His
725 730 735
Gln Arg Ile His Ser Gly Lys Lys Pro Tyr Lys Cys Asn Glu Cys
740 745 750
Gly Lys Ser Phe Ile Cys Arg Ser Gly Leu Thr Lys His Arg Ile
755 760 765
Arg His Thr Gly Glu Ser Leu Thr Thr Lys Leu Asn Val Thr Arg
770 775 780
Pro
<210> 11
<211> 595
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
19/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
<223> Incyte ID No: 6456919CD1
<400> 11
Met Asp Pro Val Ala Phe Lys Asp Val Ala Val Asn Phe Thr Gln
1 5 10 15
Glu Glu Trp Ala Leu Leu Asp Ile Ser Gln Arg Lys Leu Tyr Arg
20 25 30
Glu Val Met Leu Glu Thr Phe Arg Asn Leu Thr Ser Leu Gly Lys
35 40 45
Arg Trp Lys Asp Gln Asn Ile Glu Tyr Glu His Gln Asn Pro Arg
50 55 60
Arg Asn Phe Arg Ser Leu Ile Glu Glu Lys Val Asn Glu Ile Lys
65 70 75
Asp Asp Ser His Cys Gly Glu Thr Phe Thr Pro Val Pro Asp Asp
80 85 90
Arg Leu Asn Phe Gln Glu Lys Lys Ala Ser Pro Glu Val Lys Ser
95 100 105
Cys Glu Ser Phe Val Cys Gly Glu Val Gly Leu Gly Asn Ser Ser
110 115 120
Phe Asn Met Ser Ile Arg Gly Asp Ile Gly His Lys Ala Tyr Glu
125 130 135
Tyr Gln Glu Tyr Gly Pro Lys Pro Cys Lys Cys Gln Gln Pro Lys
140 145 150
Lys Ala Phe Arg Tyr Arg Pro Ser Phe Arg Thr Gln Glu Arg Asp
155 160 165
His Thr Gly Glu Lys Pro Asn Ala Cys Lys Val Cys Gly Lys Thr
170 175 180
Phe Ile Ser His Ser Ser Val Arg Arg His Met Val Met His Ser
185 190 195
Gly Asp Gly Pro Tyr Lys Cys Lys Phe Cys Gly Lys Ala Phe His
200 205 210
Cys Leu Arg Leu Tyr Leu Ile His Glu Arg Ile His Thr Gly Glu
215 220 225
Lys Pro Cys Glu Cys Lys Gln Cys Gly Lys Ser Phe Ser Tyr Ser
230 235 240
Ala Thr His Arg Ile His Lys Arg Thr His Thr Gly Glu Lys Pro
245 250 255
Tyr Glu Tyr Gln Glu Cys Gly Lys Ala Phe His Ser Pro Arg Ser
260 265 270
Tyr Arg Arg His Glu Arg Ile His Met Gly Glu Lys Ala Tyr Gln
275 280 285
Cys Lys Glu Cys Gly Lys Ala Phe Thr Cys Pro Arg Tyr Val Arg
290 295 300
Ile His Glu Arg Thr His Ser Arg Lys Asn Leu Tyr Glu Cys Lys
305 310 315
Gln Cys Gly Lys Ala Leu Ser Ser Leu Thr Ser Phe Gln Thr His
320 325 330
Val Arg Leu His Ser Gly Glu Arg Pro Tyr Glu Cys Lys Ile Cys
335 340 345
Gly Lys Asp Phe Cys Ser Val Asn Ser Phe Gln Arg His Glu Lys
350 355 360
Ile His Ser Gly Glu Lys Pro Tyr Lys Cys Lys Gln Cys Gly Lys
365 370 375
Ala Phe Pro His Ser Ser Ser Leu Arg Tyr His Glu Arg Thr His
380 385 390
Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe
20/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
395 400 405
Arg Ser Ala Ser His Leu Arg Val His Gly Arg Thr His Thr Gly
410 415 420
Glu Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Arg Tyr
425 430 435
Val Asn Asn Leu Gln Ser His Glu Arg Thr Gln Thr His Ile Arg
440 445 450
Ile His Ser Gly Glu Arg Arg Tyr Lys Cys Lys Ile Cys Gly Lys
455 460 465
Gly Phe Tyr Cys Pro Lys Ser Phe Gln Arg His Glu Lys Thr His
470 475 480
Thr Gly Glu Lys Leu Tyr Glu Cys Lys Gln Arg Ser Val Val Pro
485 490 495
Ser Val Val Pro Val Pro Phe Asp Ile Met Lys Gly Leu Thr Leu
500 505 510
Glu Arg Ser Pro Ile Asn Ala Ser Asn Val Gly Lys Pro Ser Glu
515 520 525
Leu Cys Gln Ser Phe Glu Cys Met Val Gly Leu Thr Leu Lys Arg
530 535 540
Asn Pro Met Ser Val Ser Asn Asp Gly Lys Pro Ser Asp Leu Pro
545 550 555
His Thr Phe Glu Tyr Val Val Gly His Thr Met Glu Arg Asn Pro
560 565 570
Met His Val Arg Asn Val Gly Asn Pro Ser Asp Leu Pro Arg Thr
575 580 585
Phe Glu Phe Met Lys Gly His Lys His Thr
590 595
<210> 12
<211> 226
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7502244CD1
<400> 12
Met Asp Gln Ala Arg Gly Leu Asp Asp Ala Ala Ala Arg Gly Gly
1 5 10 15
Gln Cys Pro Gly Leu Gly Pro Ala Pro Thr Pro Thr Pro Pro Gly
20 25 30
Arg Leu Gly Ala Pro Tyr Ser Glu Ala Trp Gly Tyr Phe His Leu
35 40 45
Ala Pro Gly Arg Pro Gly His Pro Ser Gly His Trp Ala Thr Cys
50 55 60
Arg Leu Cys Gly Glu Gln Val Gly Arg Gly Pro Gly Phe His Ala
65 70 75
Gly Thr Ser Ala Leu Trp Arg His Leu Arg Ser Ala His Arg Arg
80 85 90
Glu Leu Glu Ser Ser Gly Ala Gly Ser Ser Pro Pro Ala Ala Pro
95 100 105
Cys Pro Pro Pro Pro Val Pro Ala Ala Cys Pro Glu Gly Asp Trp
110 115 120
Ala Arg Leu Leu Glu Gln Met Gly Ala Leu Ala Val Arg Gly Ser
125 130 135
21/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
Leu Ala Gly Ala Gly Ala Gly Ser Gly Ala Glu Ala Ala Val Glu
140 145 150
Gln Gly Glu Arg Ala Leu Glu Arg Arg Arg Arg Ala Leu Gln Glu
155 160 165
Glu Glu Arg Ala Ala Ala Gln Ala Arg Arg Glu Leu Gln Ala Glu
170 175 180
Arg Glu Ala Leu Gln Ala Arg Leu Arg Asp Val Ser Arg Arg Glu
185 190 195
Gly Ala Leu Gly Trp Ala Pro Ala Ala Pro Pro Pro Leu Lys Asp
200 205 210
Asp Pro Glu Gly Asp Arg Asp Gly Cys Val Ile Thr Lys Val Leu
215 220 225
Leu
<210> 13
<211> 548
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7498718CD1
<400> 13
Met Phe Pro Val Phe Ser Gly Cys Phe Gln Glu Leu Gln Glu Lys
1 5 10 15
Asn Lys Ser Leu Glu Leu Val Ser Phe Glu Glu Val Ala Val His
20 25 30
Phe Thr Trp Glu Glu Trp Gln Asp Leu Asp Asp Ala Gln Arg Thr
35 40 45
Leu Tyr Arg Asp Val Met Leu Glu Thr Tyr Ser Ser Leu Val Ser
50 55 60
Leu Gly His Cys Ile Thr Lys Pro Glu Met Ile Phe Lys Leu Glu
65 70 75
Gln Gly Ala Glu Pro Trp Ile Val Glu Glu Thr Leu Asn Leu Arg
80 85 90
Leu Ser Ala Val Gln Ile Ile Asp Asp Leu Ile Glu Arg Ser His
95 100 105
Glu Ser His Asp Arg Phe Phe Trp Gln Ile Val Ile Thr Asn Ser
110 115 120
Asn Thr Ser Thr Gln Glu Arg Val Glu Leu Gly Lys Thr Phe Asn
125 130 135
Leu Asn Ser Asn His Val Leu Asn Leu Ile Ile Asn Asn Gly Asn
140 145 150
Ser Ser Gly Met Lys Pro Gly Gln Phe Asn Asp Cys Gln Asn Met
155 160 165
Leu Phe Pro Ile Lys Pro Gly Glu Thr Gln Ser Gly Glu Lys Pro
170 175 180
His Val Cys Asp Ile Thr Arg Arg Ser His Arg His His Glu His
185 190 195
Leu Thr Gln His His Lys Ile Gln Thr Leu Val Gln Thr Phe Gln
200 205 210
Cys Asn Glu Gln Gly Lys Thr Phe Asn Thr Glu Ala Met Phe Phe
215 220 225
Ile His Lys Arg Val His Ile Val Gln Thr Phe Gly Lys Tyr Asn
22/147
CA 02462660 2004-04-O1
WO 03/038052 PCT/US02/34846
230 235 240
Glu Tyr Glu Lys Ala Cys Asn Asn Ser Ala Val Ile Val Gln Gly
245 250 255
Ile Thr Gln Val Gly Gln Pro Thr Cys Cys Arg Lys Ser Asp Phe
260 265 270
Thr Lys His Gln Gln Thr His Thr Gly Glu Lys Pro Tyr Glu Cys
275 280 285
Val Glu Cys Glu Lys Pro Ser Ile Ser Lys Ser Asp Leu Met Leu
290 295 300
Gln Cys Lys Met Pro Thr Glu Glu Lys Pro Tyr Ala Cys Asn Trp
305 310 315
Cys Glu Lys Leu Phe Ser Tyr Lys Ser Ser Leu Ile Ile His Gln
320 325 330
Arg Ile His Thr Gly Glu Lys Pro Tyr Gly Cys Asn Glu Cys Gly
335 340 345
Lys Thr Phe Arg Cys Lys Ser Phe Leu Thr Leu His Glu Arg Thr
350 355 360
His Thr Gly Asp Lys Pro Tyr Lys Cys Ile Glu Cys Gly Lys Thr
365 370 375
Phe His Cys Lys Ser Leu Leu Thr Leu His His Arg Thr His Ser
380 385 390
Gly Glu Lys Pro Tyr Gln Cys Ser Glu Cys Gly Lys Thr Phe Ser
395 400 405
Gln Lys Ser Tyr Leu Thr Ile His His Arg Thr His Thr Gly Glu
410 415 420
Lys Pro Tyr Ala Cys Asp His Cys Glu Glu Ala Phe Ser His Lys
425 430 435
Ser Arg Leu Thr Val His Gln Arg Thr His Thr Gly Glu Lys Pro
440 445 450
Tyr Glu Cys Asn Glu Cys Gly Lys Pro Phe Ile Asn Lys Ser Asn
455 460 465
Leu Arg Leu His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Glu
470 475 480
Cys Asn Glu Cys Gly Lys Thr Phe His Arg Lys Ser Phe Leu Thr
485 490 495
Ile His Gln Trp Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Asn
500 505 510
Glu Cys Gly Lys Thr Phe Arg Cys Lys Ser Phe Leu Thr Val His
515 520 525
Gln Arg Thr His Ala Gly Glu Lys Pro Tyr Ala Cys Asn Glu Cys
530 535 540
Gly Lys Thr Tyr Ser His Lys Ser
545
<210> 14
<211> 264
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6259308CD1
<400> 14
Met Pro Asp Ser Ala Pro Ala Met Ala Asp Lys Met Asp Met Ser
1 5 10 15
23/147
CA 02462660 2004-04-O1
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Leu Asp Asp Ile Ile Lys Leu Asn Arg Ser Gln Arg Gly Gly Arg
20 25 30
Gly Gly Gly Arg Gly Arg Gly Arg Ala Gly Ser Gln Gly Gly Arg
35 40 45
Gly Gly Gly Ala Gln Ala Ala Ala Arg Val Asn Arg Gly Gly Gly
50 55 60
Pro Ile Arg Asn Arg Pro Ala Ile Ala Arg Gly Ala Ala Gly Gly
65 70 75
Gly Gly Arg Asn Arg Pro Ala Pro Tyr Ser Arg Pro Lys Gln Leu
80 85 90
Pro Asp Lys Trp Gln His Asp Leu Phe Asp Ser Gly Phe Gly Gly
95 100 105
Gly Ala Gly Val Glu Thr Gly Gly Lys Leu Leu Val Ser Asn Leu
110 115 120
Asp Phe Gly Val Ser Asp Ala Asp Ile Gln Glu Leu Phe Ala Glu
125 130 135
Phe Gly Thr Leu Lys Lys Ala Ala Val His Tyr Asp Arg Ser Gly
140 145 150
Arg Ser Leu Gly Thr Ala Asp Val His Phe Glu Arg Lys Ala Asp
155 160 165
Ala Leu Lys Ala Met Lys Gln Tyr Asn Gly Val Pro Leu Asp Gly
170 175 180
Arg Pro Met Asn Ile Gln Leu Val Thr Ser Gln Ile Asp Ala Gln
185 190 195
Arg Arg Pro Ala Gln Ser Val Asn Arg Gly Gly Met Thr Arg Asn
200 205 210
Arg Gly Ala Gly Gly Phe Gly Gly Gly Gly Gly Thr Arg Arg Gly
215 220 225
Thr Arg Gly Gly Ala Arg Gly Arg Gly Arg Gly Ala Gly Arg Asn
230 235 240
Ser Lys GIn Gln Leu Ser Ala Glu Glu Leu Asp Ala Gln Leu Asp
245 250 255
Ala Tyr Asn Ala Arg Met Asp Thr Ser
260
<210> 15
<211> 611
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7504104CD1
<400> 15
Met His His Gln Gln Arg Met Ala Ala Leu Gly Thr Asp Lys Glu
1 5 10 15
Leu Ser Asp Leu Leu Asp Phe Ser Ala Met Phe Ser Pro Pro Val
20 25 30
Ser Ser Gly Lys Asn Gly Pro Thr Ser Leu Ala Ser Gly His Phe
35 40 45
Thr Gly Ser Asn Val Glu Asp Arg Ser Ser Ser Gly Ser Trp Gly
50 55 60
Asn Gly Gly His Pro Ser Pro Ser Arg Asn Tyr Gly Asp Gly Thr
65 70 75
Pro Tyr Asp His Met Thr Ser Arg Asp Leu Gly Ser His Asp Asn
24/147
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 300
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 300
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
