Disease associated protein kinases

The invention provides human disease associated protein kinases and polynucleotides (collectively designated DAPK) which identify and encode them. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention further provides methods for diagnosing and treating disorders associated with expression of human disease associated protein kinases.

FIELD OF THE INVENTION 
This invention relates to nucleic acid and amino acid sequences of human 
protein kinases which are important in disease and to the use of these 
sequences in the diagnosis, prevention, and treatment of diseases 
associated with cell proliferation. 
BACKGROUND OF THE INVENTION 
Kinases regulate many different cell proliferation, differentiation, and 
signaling processes by adding phosphate groups to proteins. Uncontrolled 
signaling has been implicated in a variety of disease conditions including 
inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein 
phosphorylation is the main strategy for controlling activities of 
eukaryotic cells. It is estimated that more than 1000 of the 10,000 
proteins active in a typical mammalian cell are phosphorylated. The high 
energy phosphate which drives activation is generally transferred from 
adenosine triphosphate molecules (ATP) to a particular protein by protein 
kinases and removed from that protein by protein phosphatases. 
Phosphorylation occurs in response to extracellular signals (hormones, 
neurotransmitters, growth and differentiation factors, etc), cell cycle 
checkpoints, and environmental or nutritional stresses and is roughly 
analogous to turning on a molecular switch. When the switch goes on, the 
appropriate protein kinase activates a metabolic enzyme, regulatory 
protein, receptor, cytoskeletal protein, ion channel or pump, or 
transcription factor. 
The kinases comprise the largest known protein group, a superfamily of 
enzymes with widely varied functions and specificities. They are usually 
named after their substrate, their regulatory molecules, or some aspect of 
a mutant phenotype. With regard to substrates, the protein kinases may be 
roughly divided into two groups; those that phosphorylate tyrosine 
residues (protein tyrosine kinases, PTK) and those that phosphorylate 
serine or threonine residues (serine/threonine kinases, STK). A few 
protein kinases have dual specificity and phosphorylate threonine and 
tyrosine residues. Almost all kinases contain a similar 250-300 amino acid 
catalytic domain. The N-terminal domain, which contains subdomains I-IV, 
generally folds into a two-lobed structure which binds and orients the ATP 
(or GTP) donor molecule. The larger C terminal lobe, which contains 
subdomains VI A-XI, binds the protein substrate and carries out the 
transfer of the gamma phosphate from ATP to the hydroxyl group of a 
serine, threonine, or tyrosine residue. Subdomain V spans the two lobes. 
The kinases may be categorized into families by the different amino acid 
sequences (generally between 5 and 100 residues) located on either side 
of, or inserted into loops of, the kinase domain. These added amino acid 
sequences allow the regulation of each kinase as it recognizes and 
interacts with its target protein. The primary structure of the kinase 
domains is conserved and can be further subdivided into 11 subdomains. 
Each of the 11 subdomains contain specific residues and motifs or patterns 
of amino acids that are characteristic of that subdomain and are highly 
conserved (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Books, 
Vol I:7-20 Academic Press, San Diego, Calif.). 
The second messenger dependent protein kinases primarily mediate the 
effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, 
inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, 
cyclic-ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin. 
The cyclic-AMP dependent protein kinases (PKA) are important members of 
the STK family. Cyclic-AMP is an intracellular mediator of hormone action 
in all procaryotic and animal cells that have been studied. Such 
hormone-induced cellular responses include thyroid hormone secretion, 
cortisol secretion, progesterone secretion, glycogen breakdown, bone 
resorption, and regulation of heart rate and force of heart muscle 
contraction. PKA is found in all animal cells and is thought to account 
for the effects of cyclic-AMP in most of these cells. Altered PKA 
expression is implicated in a variety of disorders and diseases including 
cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular 
disease (Isselbacher, K. J. et al. (1994) Harrison's Principles of 
Internal Medicine, McGraw-Hill, New York, N.Y., pp. 416-431, 1887). 
Calcium-calmodulin (CaM) dependent protein kinases are also members of STK 
family. Calmodulin is a calcium receptor that mediates many calcium 
regulated processes by binding to target proteins in response to the 
binding of calcium. The principle target protein in these processes is CaM 
dependent protein kinases. CaM-kinases are involved in regulation of 
smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase 
kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM 
kinase I phosphorylates a variety of substrates including the 
neurotransmitter related proteins synapsin I and II, the gene 
transcription regulator, CREB, and the cystic fibrosis conductance 
regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal 
14:3679-86). CaM II kinase also phosphorylates synapsin at different 
sites, and controls the synthesis of catecholamines in the brain through 
phosphorylation and activation of tyrosine hydroxylase. Many of the CaM 
kinases are activated by phosphorylation in addition to binding to CaM. 
The kinase may autophosphorylate itself, or be phosphorylated by another 
kinase as part of a "kinase cascade". 
Another ligand-activated protein kinase is 5'-AMP-activated protein kinase 
(AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81). Mammaliam AMPK is 
a regulator of fatty acid and sterol synthesis through phosphorylation of 
the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase 
and mediates responses of these pathways to cellular stresses such as heat 
shock and depletion of glucose and ATP. AMPK is a heterotrimeric complex 
comprised of a catalytic alpha subunit and two non-catalytic beta and 
gamma subunits that are believed to regulate the activity of the alpha 
subunit. Subunits of AMPK have a much wider distribution in non-lipogenic 
tissues such as brain, heart, spleen, and lung than expected. This 
distribution suggests that its role may extend beyond regulation of lipid 
metabolism alone. 
The mitogen-activated protein kinases (MAP) are also members of the STK 
family. MAP kinases also regulate intracellular signaling pathways. They 
mediate signal transduction from the cell surface to the nucleus via 
phosphorylation cascades. Several subgroups have been identified, and each 
manifests different substrate specificities and responds to distinct 
extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature 
365:781-783). MAP kinase signaling pathways are present in mammalian cells 
as well as in yeast. The extracellular stimuli which activate mammalian 
pathways include epidermal growth factor (EGF), ultraviolet light, 
hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and 
pro-inflammatory cytokines such as tumor necrosis factor (TNF) and 
interleukin-1 (IL-1). 
PRK (proliferation-related kinase) is a serum/cytokine inducible STK that 
is involved in regulation of the cell cycle and cell proliferation in 
human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem. 
271:19402-8). PRK is related to the polo (derived from Drosophila polo 
gene) family of STKs implicated in cell division. PRK is downregulated in 
lung tumor tissue and may be a proto-oncogene whose deregulated expression 
in normal tissue leads to oncogenic transformation. Altered MAP kinase 
expression is implicated in a variety of disease conditions including 
cancer, inflammation, immune disorders, and disorders affecting growth and 
development. 
The cyclin-dependent protein kinases (CDKs) are another group of STKs that 
control the progression of cells through the cell cycle. Cyclins are small 
regulatory proteins that act by binding to and activating CDKs which then 
trigger various phases of the cell cycle by phosphorylating and activating 
selected proteins involved in the mitotic process. CDKs are unique in that 
they require multiple inputs to become activated. In addition to the 
binding of cyclin, CDK activation requires the phosphorylation of a 
specific threonine residue and the dephosphorylation of a specific 
tyrosine residue. 
Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosine 
residues on their target proteins and may be divided into transmembrane, 
receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane 
protein-tyrosine kinases are receptors for most growth factors. Binding of 
growth factor to the receptor activates the transfer of a phosphate group 
from ATP to selected tyrosine side chains of the receptor and other 
specific proteins. Growth factors (GF) associated with receptor PTKs 
include; epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, 
insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and 
macrophage colony stimulating factor. 
Non-receptor PTKs lack transmembrane regions and, instead, form complexes 
with the intracellular regions of cell surface receptors. Such receptors 
that function through non-receptor PTKs include those for cytokines, 
hormones (growth hormone and prolactin) and antigen-specific receptors on 
T and B lymphocytes. 
Many of these PTKs were first identified as the products of mutant 
oncogenes in cancer cells where their activation was no longer subject to 
normal cellular controls. In fact, about one third of the known oncogenes 
encode PTKs, and it is well known that cellular transformation 
(oncogenesis) is often accompanied by increased tyrosine phosphorylation 
activity (Carbonneau H and Tonks NK (1992) Annu Rev Cell Biol 8:463-93). 
Regulation of PTK activity may therefore be an important strategy in 
controlling some types of cancer. 
An additional family of protein kinases previously thought to exist only in 
procaryotes is the histidine protein kinase family (HPK). HPKs bear little 
homology with mammalian STKs or PTKs but have distinctive sequence motifs 
of their own (Davie, J. R. et al. (1995) J. Biol. Chem. 270:19861-67). A 
histidine residue in the N-terminal half of the molecule (region I) is an 
autophosphorylation site. Three additional motifs located in the 
C-terminal half of the molecule include an invariant asparagine residue in 
region II and two glycine-rich loops characteristic of nucleotide binding 
domains in regions III and IV. Recently a branched chain alpha-ketoacid 
dehydrogenase kinase has been found with characteristics of HPK in rat 
(Davie et al., supra). 
The discovery of new human disease associated protein kinases which are 
important in disease development, and the polynucleotides encoding them, 
satisfies a need in the art by providing new compositions which are useful 
in the diagnosis, prevention and treatment of diseases associated with 
cell proliferation, particularly and immune responses and cancers. 
SUMMARY OF THE INVENTION 
The invention features substantially purified polypeptides, human disease 
associated protein kinases, collectively referred to as DAPK and 
individually referred to as DAPK-1, DAPK-2, DAPK-3, DAPK-4, DAPK-5, 
DAPK-6, and DAPK-7, having the amino acid sequences selected from the 
group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, 
SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively. 
The invention further provides isolated and substantially purified 
polynucleotide sequences encoding DAPK. In a particular aspect, the 
polynucleotide is at least one of the nucleotide sequences selected from 
the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID 
NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. 
In addition, the invention provides a polynucleotide sequence, or fragment 
thereof, which hybridizes under stringent conditions to any of the 
polynucleotide sequences of SEQ ID NOs:8-14. In another aspect the 
invention provides compositions comprising isolated and purified 
polynucleotide sequences of SEQ ID NOs:8-14 or fragments thereof. 
The invention further provides a polynucleotide sequence comprising the 
complement or fragments thereof of any one of the polynucleotide sequences 
encoding DAPK. In another aspect the invention provides compositions 
comprising isolated and purified polynucleotide sequences comprising the 
complements of SEQ ID NOs:8-14, or fragments thereof. 
The present invention further provides an expression vector containing at 
least a fragment of any one of the polynucleotide sequences of SEQ ID 
NOs:8-14. In yet another aspect, the expression vector containing the 
polynucleotide sequence is contained within a host cell. 
The invention also provides a method for producing a polypeptide or a 
fragment thereof, the method comprising the steps of: a) culturing the 
host cell containing an expression vector containing at least a fragment 
of the polynucleotide sequence encoding an DAPK under conditions suitable 
for the expression of the polypeptide; and b) recovering the polypeptide 
from the host cell culture. 
The invention also provides a pharmaceutical composition comprising a 
substantially purified DAPK in conjunction with a suitable pharmaceutical 
carrier. 
The invention also provides a purified antagonist of DAPK. In one aspect 
the invention provides a purified antibody which binds to an DAPK. 
Still further, the invention provides a purified agonist of DAPK. 
The invention also provides a method for treating or preventing a cancer 
comprising administering to a subject in need of such treatment an 
effective amount of a pharmaceutical composition containing DAPK. 
The invention also provides a method for treating or preventing a cancer 
comprising administering to a subject in need of such treatment an 
effective amount of a pharmaceutical composition containing DAPK. 
The invention also provides a method for treating or preventing an immune 
response associated with the increased expression or activity of DAPK 
comprising administering to a subject in need of such treatment an 
effective amount of an antagonist of DAPK. 
The invention also provides a method for stimulating cell proliferation 
comprising administering to a cell an effective amount of DAPK. 
The invention also provides a method for detecting a polynucleotide which 
encodes a disease associated protein kinase in a biological sample 
comprising the steps of: a) hybridizing a polynucleotide sequence 
complementary to a polynucleotide encoding DAPK to nucleic acid material 
of a biological sample, thereby forming a hybridization complex; and b) 
detecting the hybridization complex, wherein the presence of the complex 
correlates with the presence of a polynucleotide encoding the disease 
associated protein kinase in the biological sample. 
The invention also provides a microarray which contains at least a fragment 
of at least one of the polynucleotide sequences encoding DAPK. In a 
particular aspect, the microarray contains at least a fragment of at least 
one of the sequences selected from the group consisting of SEQ ID NO:8, 
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and 
SEQ ID NO:14. 
The invention also provides a method for the simultaneous detection of the 
levels of expression of polynucleotides which encode disease associated 
protein kinases in a biological sample comprising the steps of: a) 
hybridizing said microarray to labeled complementary nucleotides of a 
biological sample, comprising at least a fragment of at least one of the 
polynucleotides encoding DAPK, thereby forming hybridization complexes; 
and b) quantifying expression, wherein the signal produced by the 
hybridization complexes correlates with expression of particular 
polynucleotides encoding disease associated protein kinases in the 
biological sample. In a preferred embodiment, prior to hybridization, the 
nucleic acid material of the biological sample is amplified and labeled by 
the polymerase chain reaction.

DESCRIPTION OF THE INVENTION 
Before the present proteins, nucleotide sequences, and methods are 
described, it is understood that this invention is not limited to the 
particular methodology, protocols, cell lines, vectors, and reagents 
described, as these may vary. It is also to be understood that the 
terminology used herein is for the purpose of describing particular 
embodiments only, and is not intended to limit the scope of the present 
invention which will be limited only by the appended claims. 
It must be noted that as used herein and in the appended claims, the 
singular forms "a", "an", and "the" include plural reference unless the 
context clearly dictates otherwise. Thus, for example, reference to "a 
host cell" includes a plurality of such host cells, reference to the 
"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 commonly understood by one of ordinary skill in the 
art to which this invention belongs. Although any methods and materials 
similar or equivalent to those described herein can be used in the 
practice or testing of the present invention, the preferred methods, 
devices, and materials are now described. All publications mentioned 
herein are incorporated herein by reference for the purpose of describing 
and disclosing the cell lines, vectors, arrays and methodologies which are 
reported in the publications which might be used in connection with the 
invention. Nothing herein is to be construed as an admission that the 
invention is not entitled to antedate such disclosure by virtue of prior 
invention. 
DEFINITIONS 
DAPK, as used herein, refers to the amino acid sequences of substantially 
purified DAPK obtained from any species, particularly mammalian, including 
bovine, ovine, porcine, murine, equine, and preferably human, from any 
source whether natural, synthetic, semi-synthetic, or recombinant. 
The term "agonist", as used herein, refers to a molecule which, when bound 
to DAPK, increases or prolongs the duration of the effect of DAPK. 
Agonists may include proteins, nucleic acids, carbohydrates, or any other 
molecules which bind to and modulate the effect of DAPK. 
An "allele" or "allelic sequence", as used herein, is an alternative form 
of the gene encoding DAPK. Alleles may result from at least one mutation 
in the nucleic acid sequence and may result in altered mRNAs or 
polypeptides whose structure or function may or may not be altered. Any 
given natural or recombinant gene may have none, one, or many allelic 
forms. Common mutational changes which give rise to alleles 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 DAPK as used herein include those 
with deletions, insertions, or substitutions of different nucleotides 
resulting in a polynucleotide that encodes the same or a functionally 
equivalent DAPK. Included within this definition are polymorphisms which 
may or may not be readily detectable using a particular oligonucleotide 
probe of the polynucleotide encoding DAPK, and improper or unexpected 
hybridization to alleles, with a locus other than the normal chromosomal 
locus for the polynucleotide sequence encoding DAPK. The encoded protein 
may also be "altered" and contain deletions, insertions, or substitutions 
of amino acid residues which produce a silent change and result in a 
functionally equivalent DAPK. Deliberate amino acid substitutions may be 
made on the basis of similarity in polarity, charge, solubility, 
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the 
residues as long as the biological or immunological activity of DAPK is 
retained. For example, negatively charged amino acids may include aspartic 
acid and glutamic acid; positively charged amino acids may include lysine 
and arginine; and amino acids with uncharged polar head groups having 
similar hydrophilicity values may include leucine, isoleucine, and valine, 
glycine and alanine, asparagine and glutamine, serine and threonine, and 
phenylalanine and tyrosine. 
"Amino acid sequence" as used herein refers to an oligopeptide, peptide, 
polypeptide, or protein sequence, and fragment thereof, and to naturally 
occurring or synthetic molecules. Fragments of DAPK are preferably about 5 
to about 15 amino acids in length and retain the biological activity or 
the immunological activity of DAPK. Where "amino acid sequence" is recited 
herein to refer to an amino acid sequence of a naturally occurring protein 
molecule, amino acid sequence, and like terms, are not meant to limit the 
amino acid sequence to the complete, native amino acid sequence associated 
with the recited protein molecule. 
"Amplification" as used herein refers to the production of additional 
copies of a nucleic acid sequence and is generally carried out using 
polymerase chain reaction (PCR) technologies well known in the art 
(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory 
Manual, Cold Spring Harbor Press, Plainview, N.Y.). 
The term "antagonist" as used herein, refers to a molecule which, when 
bound to DAPK, decreases the amount or the duration of the effect of the 
biological or immunological activity of DAPK. Antagonists may include 
proteins, nucleic acids, carbohydrates, or any other molecules which 
decrease the effect of DAPK. 
As used herein, the term "antibody" refers to intact molecules as well as 
fragments thereof, such as Fab, F(ab').sub.2, and Fv, which are capable of 
binding the epitopic determinant. Antibodies that bind DAPK polypeptides 
can be prepared using intact polypeptides or fragments containing small 
peptides of interest as the immunizing antigen. The polypeptide or 
oligopeptide used to immunize an animal can be derived from the 
translation of RNA or synthesized chemically and can be conjugated to a 
carrier protein, if desired. Commonly used carriers that are chemically 
coupled to peptides include bovine serum albumin and thyroglobulin, 
keyhole limpet hemocyanin. The coupled peptide is then used to immunize 
the animal (e.g., a mouse, a rat, or a rabbit). 
The term "antigenic determinant", as used herein, refers to that fragment 
of a molecule (i.e., an epitope) that makes contact with a particular 
antibody. When a protein or 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 a given region or three-dimensional 
structure on the protein; these regions or structures are referred to as 
antigenic determinants. 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 "antisense", as used herein, refers to any composition containing 
nucleotide sequences which are complementary to a specific DNA or RNA 
sequence. The term "antisense strand" is used in reference to a nucleic 
acid strand that is complementary to the "sense" strand. Antisense 
molecules include peptide nucleic acids and may be produced by any method 
including synthesis or transcription. Once introduced into a cell, the 
complementary nucleotides combine with natural sequences produced by the 
cell to form duplexes and block either transcription or translation. The 
designation "negative" is sometimes used in reference to the antisense 
strand, and "positive" is sometimes used in reference to the sense strand. 
The term "biologically active", as used herein, refers to a protein having 
structural, regulatory, or biochemical functions of a naturally occurring 
molecule. Likewise, "immunologically active" refers to the capability of 
the natural, recombinant, or synthetic DAPK, or any oligopeptide thereof, 
to induce a specific immune response in appropriate animals or cells and 
to bind with specific antibodies. 
The terms "complementary" or "complementarity", as used herein, refer to 
the natural binding of polynucleotides under permissive salt and 
temperature conditions by base-pairing. For example, the sequence "A-G-T" 
binds to the complementary sequence "T-C-A". Complementarity between two 
single-stranded molecules may be "partial", in which only some of the 
nucleic acids bind, or it may be complete when total complementarity 
exists between the single stranded molecules. The degree of 
complementarity between nucleic acid strands has significant effects on 
the efficiency and strength of hybridization between nucleic acid strands. 
This is of particular importance in amplification reactions, which depend 
upon binding between nucleic acids strands and in the design and use of 
PNA molecules. 
A "composition comprising a given polynucleotide sequence" as used herein 
refers broadly to any composition containing the given polynucleotide 
sequence. The composition may comprise a dry formulation or an aqueous 
solution. Compositions comprising polynucleotide sequences encoding DAPK 
(SEQ ID NOs:8-14) or fragments thereof 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., SDS) and other components 
(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.). 
"Consensus", as used herein, refers to a nucleic acid sequence which has 
been resequenced to resolve uncalled bases, has been extended using XL-PCR 
kit (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3' direction and 
resequenced, or has been assembled from the overlapping sequences of more 
than one Incyte Clone using a computer program for fragment assembly 
(e.g., GELVIEW fragment assembly system, GCG, Madison, Wis.). Some 
sequences have been both extended and assembled to produce the consensus 
sequence. 
The term "correlates with expression of a polynucleotide", as used herein, 
indicates that the detection of the presence of a ribonucleic acid that is 
similar to a polynucleotide encoding an DAPK by northern analysis is 
indicative of the presence of mRNA encoding DAPK in a sample and thereby 
correlates with expression of the transcript from the polynucleotide 
encoding the protein. 
The term "DAPK" refers to any one or all of the human polypeptides, DAPK-1, 
DAPK-2, DAPK-3, DAPK-4, DAPK-5, DAPK-6, DAPK-7, and DAPK-8. 
A "deletion", as used herein, refers to a change in the amino acid or 
nucleotide sequence and results in the absence of one or more amino acid 
residues or nucleotides. 
The term "derivative", as used herein, refers to the chemical modification 
of a nucleic acid encoding or complementary to DAPK or the encoded DAPK. 
Such modifications include, for example, replacement of hydrogen by an 
alkyl, acyl, or amino group. A nucleic acid derivative encodes a 
polypeptide which retains the biological or immunological function of the 
natural molecule. A derivative polypeptide is one which is modified by 
glycosylation, pegylation, or any similar process which retains the 
biological or immunological function of the polypeptide from which it was 
derived. 
The term "homology", as used herein, refers to a degree of complementarity. 
There may be partial homology or complete homology (i.e., identity). A 
partially complementary sequence that at least partially inhibits an 
identical sequence from hybridizing to a target nucleic acid is referred 
to using the functional term "substantially homologous." The inhibition of 
hybridization of the completely complementary sequence to the target 
sequence may be examined using a hybridization assay (Southern or northern 
blot, solution hybridization and the like) under conditions of low 
stringency. A substantially homologous sequence or hybridization probe 
will compete for and inhibit the binding of a completely homologous 
sequence to the target sequence under conditions of low stringency. This 
is not to say that conditions of low stringency are such that non-specific 
binding is permitted; low stringency conditions require that the binding 
of two sequences to one another be a specific (i.e., selective) 
interaction. The absence of non-specific binding may be tested by the use 
of a second target sequence which lacks even a partial degree of 
complementarity (e.g., less than about 30% identity). In the absence of 
non-specific binding, the probe will not hybridize to the second 
non-complementary target sequence. 
Human artificial chromosomes (HACs) are linear microchromosomes which may 
contain DNA sequences of 10K to 10M in size and contain all of the 
elements required for stable mitotic chromosome segregation and 
maintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355). 
The term "humanized antibody", as used herein, refers to antibody molecules 
in which amino acids have been replaced in the non-antigen binding regions 
in order to more closely resemble a human antibody, while still retaining 
the original binding ability. 
The term "hybridization", as used herein, refers to any process by which a 
strand of nucleic acid binds with a complementary strand through base 
pairing. 
The term "hybridization complex", as used herein, refers to a complex 
formed between two nucleic acid sequences by virtue of the formation of 
hydrogen bonds between complementary G and C bases and between 
complementary A and T bases; these hydrogen bonds may be further 
stabilized by base stacking interactions. The two complementary nucleic 
acid sequences hydrogen bond in an antiparallel configuration. A 
hybridization complex may be formed in solution (e.g., C.sub.0 t or 
R.sub.0 t analysis) or between one nucleic acid sequence present in 
solution and another nucleic acid sequence immobilized on a solid support 
(e.g., paper, membranes, filters, chips, pins or glass slides, or any 
other appropriate substrate to which cells or their nucleic acids have 
been fixed). 
An "insertion" or "addition", as used herein, refers to a change in an 
amino acid or nucleotide sequence resulting in the addition of one or more 
amino acid residues or nucleotides, respectively, as compared to the 
naturally occurring molecule. 
"Microarray" refers to an array (or arrangement) of distinct 
oligonucleotides synthesized on a substrate, such as paper, nylon or other 
type of membrane, filter, gel, polymer, chip, glass slide, or any other 
suitable support. 
The term "modulate", as used herein, refers to a change in the activity of 
DAPK. For example, modulation may cause an increase or a decrease in 
protein activity, binding characteristics, or any other biological, 
functional or immunological properties of DAPK. 
"Nucleic acid sequence" as used herein refers to an oligonucleotide, 
nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNA of 
genomic or synthetic origin which may be single- or double-stranded, and 
represent the sense or antisense strand. "Fragments" are those nucleic 
acid sequences which are greater than 60 nucleotides than in length, and 
most preferably includes fragments that are at least 100 nucleotides or at 
least 1000 nucleotides, and at least 10,000 nucleotides in length. 
The term "oligonucleotide" refers to a nucleic acid sequence of at least 
about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 
nucleotides, and more preferably about 20 to 25 nucleotides, which can be 
used in PCR amplification or hybridization assays. As used herein, 
oligonucleotide is substantially equivalent to the terms 
"amplimers","primers", "oligomers", and "probes", as commonly defined in 
the art. 
"Peptide nucleic acid", PNA as used herein, refers to an antisense molecule 
or anti-gene agent which comprises an oligonucleotide of at least five 
nucleotides in length linked to a peptide backbone of amino acid residues 
which ends in lysine. The terminal lysine confers solubility to the 
composition. PNAs may be pegylated to extend their lifespan in the cell 
where they preferentially bind complementary single stranded DNA and RNA 
and stop transcript elongation (Nielsen, P. E. et al. (1993) Anticancer 
Drug Des. 8:53-63). 
The term "portion", as used herein, with regard to a protein (as in "a 
portion of a given protein") refers to fragments of that protein. The 
fragments may range in size from five amino acid residues to the entire 
amino acid sequence minus one amino acid. Thus, a protein "comprising at 
least a portion of the amino acid sequence of an DAPK encompasses the 
full-length DAPK and fragments thereof. 
The term "sample", as used herein, is used in its broadest sense. A 
biological sample suspected of containing nucleic acid encoding DAPK, or 
fragments thereof, or DAPK itself may comprise a bodily fluid, extract 
from a cell, chromosome, organelle, or membrane isolated from a cell, a 
cell, genomic DNA, RNA, or cDNA (in solution or bound to a solid support, 
a tissue, a tissue print, and the like. 
The terms "specific binding" or "specifically binding", as used herein, 
refers to that interaction between a protein or peptide and an agonist, an 
antibody and an antagonist. The interaction is dependent upon the presence 
of a particular structure (i.e., the antigenic determinant or epitope) of 
the protein recognized by the binding molecule. For example, if an 
antibody is specific for epitope "A", the presence of a protein containing 
epitope A (or free, unlabeled A) in a reaction containing labeled "A" and 
the antibody will reduce the amount of labeled A bound to the antibody. 
The terms "stringent conditions" or "stringency", as used herein, refer to 
the conditions for hybridization as defined by the nucleic acid, salt, and 
temperature. These conditions are well known in the art and may be altered 
in order to identify or detect identical or related polynucleotide 
sequences. Numerous equivalent conditions comprising either low or high 
stringency depend on factors such as the length and nature of the sequence 
(DNA, RNA, base composition), nature of the target (DNA, RNA, base 
composition), milieu (in solution or immobilized on a solid substrate), 
concentration of salts and other components (e.g., formamide, dextran 
sulfate and/or polyethylene glycol), and temperature of the reactions 
(within a range from about 5.degree. C. below the melting temperature of 
the probe to about 20.degree. C. to 25.degree. C. below the melting 
temperature). One or more factors be may be varied to generate conditions 
of either low or high stringency different from, but equivalent to, the 
above listed conditions. 
The term "substantially purified", as used herein, refers to nucleic or 
amino acid sequences that are removed from their natural environment, 
isolated or separated, and are at least 60% free, preferably 75% free, and 
most preferably 90% free from other components with which they are 
naturally associated. 
A "substitution", as used herein, refers to the replacement of one or more 
amino acids or nucleotides by different amino acids or nucleotides, 
respectively. 
"Transformation", as defined herein, describes a process by which exogenous 
DNA enters and changes a recipient cell. It may occur under natural or 
artificial conditions using various methods well known in the art. 
Transformation may rely on any known method for the insertion of foreign 
nucleic acid sequences into a prokaryotic or eukaryotic host cell. The 
method is selected based on the type of host cell being transformed and 
may include, but is not limited to, viral infection, electroporation, heat 
shock, lipofection, and particle bombardment. Such "transformed" cells 
include stably transformed cells in which the inserted DNA is capable of 
replication either as an autonomously replicating plasmid or as part of 
the host chromosome. They also include cells which transiently express the 
inserted DNA or RNA for limited periods of time. 
A "variant" of DAPK, as used herein, refers to an amino acid sequence that 
is altered by one or more amino acids. The variant may have "conservative" 
changes, wherein a substituted amino acid has similar structural or 
chemical properties, e.g., replacement of leucine with isoleucine. More 
rarely, a variant may have "nonconservative" changes, e.g., replacement of 
a glycine with a tryptophan. Analogous minor variations may also include 
amino acid deletions or insertions, or both. Guidance in determining which 
amino acid residues may be substituted, inserted, or deleted without 
abolishing biological or immunological activity may be found using 
computer programs well known in the art, for example, DNASTAR software. 
THE INVENTION 
The invention is based on the discovery of human disease associated protein 
kinases (DAPK) and the polynucleotides encoding DAPK, and the use of these 
compositions for the diagnosis, prevention, or treatment of diseases 
associated with cell proliferation. Table 1 shows the protein and 
nucleotide sequence identification numbers, Incyte Clone number, cDNA 
library, NCBI homolog and NCBI sequence identifier for each of the human 
disease associated protein kinases disclosed herein. 
TABLE 1 
______________________________________ 
NCBI 
Polypeptide 
Polynucleotide 
Inctye Clone 
Incyte Library 
Homolog 
______________________________________ 
Seq 1 Seq 8 39043 HUVENOB01 
Human GI 
1488263 
Seq 2 Seq 9 40194 YNOT01 
Human GI 
1827450 
Seq 3 Seq 10 402339 TMLR3DT01 
Rat GI 
303804 
Seq 4 Seq 11 705365 SYNORAT04 
Human GI 
854170 
Seq 5 Seq 12 827431 PROSNOT06 
Human GI 
790790 
Seq 6 Seq 13 1340712 COLNTUT03 
Rat GI 
924921 
Seq 7 Seq 14 1452972 PENITUT01 
Human GI 
1335856 
______________________________________ 
DAPK-1 (SEQ ID NO:1) was first identified in Incyte Clone 39043 from the 
HUVENOB01 cDNA library using a computer search for amino acid sequence 
alignments. A consensus sequence, SEQ ID NO:8, was derived from the 
extended and overlapping nucleic acid sequences: Incyte Clones 
39043/HUVENOB01, 86618/LIVRNOT01, 241996/HIPONOT01, 486079/HNT2RAT01, 
1255087/LUNGFET03, 1294238/PGANNOT03, and 2375745/ISLTNOT01. 
Therefore, in one embodiment, the invention encompasses a polypeptide 
comprising the amino acid sequence of SEQ ID NO:1. DAPK-1 is 685 amino 
acids in length and has a potential ATP-binding sequence at G.sub.89 
KGGFAKC. As shown in FIG. 1, DAPK-1 has sequence homology with 
cytokine-inducible, human proliferation-related kinase, PRK (GI 1488263). 
In particular, DAPK-1 and PRK share 53% homology. DAPK-1 and PRK share the 
ATP binding region described above and, in addition, share a presumed 
regulatory sequence at K.sub.506 WVDYS common to members of the polo 
family of protein kinases. DAPK-1 is associated with cDNA libraries which 
are immortalized or cancerous and show inflammatory or immune responses. 
DAPK-2 (SEQ ID NO:2) was first identified in Incyte Clone 40194 from the 
YNOT01 cDNA library using a computer search for amino acid sequence 
alignments. A consensus sequence, SEQ ID NO:9, was derived from the 
extended and overlapping nucleic acid sequences: Incyte Clones 
40194/YNOT01, 278198/TESTNOT03, and 1683885/PROSNOT15. 
Therefore, in one embodiment, the invention encompasses a polypeptide 
comprising the amino acid sequence of SEQ ID NO:2. DAPK-2 is 448 amino 
acids in length and has a potential ATP-binding sequence at G.sub.36 
SGGFGLI and an STK specific signature sequence at Y.sub.162 VHGDVKAANLLL. 
As shown in FIG. 2, DAPK-2 has sequence homology with the human vaccina 
virus related kinase, VRK1 (GI1827450). In particular, DAPK-2 and VRK1 
share 65% homology. DAPK-2 and VRK1 share the glycine-rich ATP-binding 
sequence and the STK signature sequence described above. DAPK-2 is 
associated with cDNA libraries which are immortalized or cancerous and 
show inflammatory or immune responses. 
DAPK-3 (SEQ ID NO:3) was first identified in Incyte Clone 402339 from the 
TMLR3DT01 cDNA library using a computer search for amino acid sequence 
alignments. A consensus sequence, SEQ ID NO:10, was derived from the 
extended and overlapping nucleic acid sequences: Incyte Clones 
402339/TMLR3DT01, 495759/HNT2NOT01, and 1931950/COLNNOT16. 
Therefore, in one embodiment, the invention encompasses a polypeptide 
comprising the amino acid sequence of SEQ ID NO:3. DAPK-3 is 400 amino 
acids in length and contains various sequence motifs characteristic of the 
catalytic domain of protein kinases. An ATP-binding sequence is found at 
G.sub.79 AGNGGVV of subdomain I, and K.sub.101 and E.sub.118 are invariant 
residues found in subdomains II and II, respectively. The "catalytic loop" 
of subdomain VIB is found in the sequence H.sub.112 RDVKPSN, and the 
triplet codons D.sub.212 FG and A.sub.275 PE are characteristic of 
subdomains VII and VIII, respectively. As shown in FIG. 3, DAPK-3 has 
sequence homology with the rat MAP kinase kinase, MEK2 (GI 303804). In 
particular, DAPK-3 and MEK3 share 94% homology. DAPK-3 is associated with 
cDNA libraries which are immortalized or cancerous and show inflammatory 
or immune responses. 
DAPK-4 (SEQ ID NO:4) was first identified in Incyte Clone 705365 from the 
SYNORAT04 cDNA library using a computer search for amino acid sequence 
alignments. A consensus sequence, SEQ ID NO:11, was derived from the 
extended and overlapping nucleic acid sequences: Incyte Clones 
705365/SYNORAT04, 2529903/GBLANOT02, and 2729238/OVARTUT05. 
Therefore, in one embodiment, the invention encompasses a polypeptide 
comprising the amino acid sequence of SEQ ID NO:4. DAPK-4 is 464 amino 
acids in length and contains various sequence motifs characteristic the 
catalytic domain of protein kinases. An ATP-binding sequence is found at 
G.sub.97 RGAFGEV and the catalytic loop is found at H.sub.211 RDIKPDN. 
DAPK-4 also contains a nuclear localization signal at K.sub.266 
RKAETWKKNR. As shown in FIG. 4, DAPK-4 has sequence homology with human 
nuclear protein kinase, Ndr (GI 854170). In particular, DAPK-4 and Ndr 
share 87% homology. DAPK-4 is associated with cDNA libraries which are 
immortalized or cancerous and show inflammatory or immune responses. 
DAPK-5 (SEQ ID NO:5) was first identified in Incyte Clone 827431 from the 
PROSNOT06 cDNA library using a computer search for amino acid sequence 
alignments. A consensus sequence, SEQ ID NO:12, was derived from the 
extended and overlapping nucleic acid sequences: Incyte Clones 755081, 
758002 and 760552/BRAITUT02, 827431/PROSNOT06, 1286067/COLNNOT16, and 
1503272/BRAITUT07. 
Therefore, in one embodiment, the invention encompasses a polypeptide 
comprising the amino acid sequence of SEQ ID NO:5. DAPK-5 is 343 amino 
acids in length and contains various sequence motifs characteristic of the 
catalytic domain of protein kinases. An ATP-binding sequence is found at 
G.sub.22 SGAFSEV and the catalytic loop is found at H.sub.134 RDLKPEN. The 
triplet codons D.sub.157 FG and A.sub.180 PE characteristic of subdomains 
VII and VIII, respectively, are also found. As shown in FIG. 5, DAPK-5 has 
sequence homology with the human CaM-kinase, CaMKI (GI 790790). In 
particular, DAPK-5 and CaMKI share 64% homology. In addition to the 
typical protein kinase motifs mentioned above, DAPK-5 and CaMKI share 
T.sub.171 which is a phosphorylation site for CaMKI kinase and an 
auto-inhibitory and CaM-binding domain found between I.sub.280 and 
L.sub.313 of DAPK-5. DAPK-5 is associated with cDNA libraries which are 
immortalized or cancerous. 
DAPK-6 (SEQ ID NO:6) was first identified in Incyte Clone 1340712 from the 
COLNTUT03 cDNA library using a computer search for amino acid sequence 
alignments. A consensus sequence, SEQ ID NO:13, was derived from the 
extended and overlapping nucleic acid sequences: Incyte Clones 
1340712/COLNTUT03, 1350483/LATRTUT02 and 2631495/COLNTUT15. 
Therefore, in one embodiment, the invention encompasses a polypeptide 
comprising the amino acid sequence of SEQ ID NO:6. DAPK-6 is 412 amino 
acids in length and has characteristics of a histidine protein kinase 
(HPK). H.sub.211 in DAPK-6 corresponds to a potential autophosphorylation 
site in subdomain I of HPK, and N.sub.279 is also an invariant residue of 
subdomain II. The sequences D.sub.315 RGGG and G.sub.365 FGFG are 
characteristic of subdomain III and IV of HPK. As shown in FIG. 6, DAPK-6 
has sequence homology with a rat branched-chain alpha-ketoacid 
dehydrogenase kinase, BCKDH kinase (GI 924921). In particular, DAPK-6 and 
BCKDH kinase share 98% homology. BCKDH kinase shares the characteristic 
sequences of HPKs described above, but differs by the presence of a 
distinctive N-terminal leader sequence in DAPK-6 that may target DAPK-6 to 
a different subcellular site. DAPK-6 is associated with cDNA libraries 
which are immortalized or cancerous and show inflammatory or immune 
responses. 
DAPK-7 (SEQ ID NO:7) was first identified in Incyte Clone 1452972 from the 
PENITUT01 cDNA library using a computer search for amino acid sequence 
alignments. A consensus sequence, SEQ ID NO:14, was derived from the 
extended and overlapping nucleic acid sequences: Incyte Clones 
307571/HEARNOT01, 842220/PROSTUT05, 1364737/SCORNON02, 1452972 and 
1454802/PENITUT01, and 1479332/CORPNOT02. 
Therefore, in one embodiment, the invention encompasses a polypeptide 
comprising the amino acid sequence of SEQ ID NO:7. DAPK-7 is 328 amino 
acids in length and has potential cAMP-dependent protein kinase 
phosphorylation sites at S.sub.72 and S.sub.217. As shown in FIG. 7, 
DAPK-7 has sequence homology with human fetal liver AMPK gamma-subunit (GI 
1335856). In particular, DAPK-7 and AMPK gamma share 73% homology. Several 
sequences that are conserved among AMPK gamma isoforms are shared by 
DAPK-7 and AMPK gamma. These include L.sub.77 TITDFINLHRYYKS, S.sub.217 
ALPVVDE, V.sub.228 VDIYSKFDVI, and A.sub.286 EVHRRLVVV. Sequence 
differences between DAPK-7 and other AMPK gamma isoforms, particularly the 
distinctive N-terminal portion of DAPK-7, L.sub.2 EKLEFEDEAVEDSESG, may 
signify different tissue expression and/or regulatory roles for DAPK-7. 
DAPK-7 is associated with cDNA libraries which are immortalized or 
cancerous and show inflammatory or immune responses. 
The invention also encompasses DAPK variants which retain the biological or 
functional activity of DAPK. A preferred DAPK variant is one having at 
least 80%, and more preferably 90%, amino acid sequence identity to the 
DAPK amino acid sequence. A most preferred DAPK variant is one having at 
least 95% amino acid sequence identity to an DAPK disclosed herein (SEQ ID 
NOs:1-7). 
The invention also encompasses polynucleotides which encode DAPK. 
Accordingly, any nucleic acid sequence which encodes the amino acid 
sequence of DAPK can be used to produce recombinant molecules which 
express DAPK. In a particular embodiment, the invention encompasses a 
polynucleotide consisting of a nucleic acid sequence selected from the 
group consisting of SEQ ID NOs:8-14. 
It will be appreciated by those skilled in the art that as a result of the 
degeneracy of the genetic code, a multitude of nucleotide sequences 
encoding DAPK, some bearing minimal homology to the nucleotide sequences 
of any known and naturally occurring gene, may be produced. Thus, the 
invention contemplates each and every possible variation of nucleotide 
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 nucleotide sequence of naturally 
occurring DAPK, and all such variations are to be considered as being 
specifically disclosed. 
Although nucleotide sequences which encode DAPK and its variants are 
preferably capable of hybridizing to the nucleotide sequence of the 
naturally occurring DAPK under appropriately selected conditions of 
stringency, it may be advantageous to produce nucleotide sequences 
encoding DAPK or its derivatives possessing a substantially different 
codon usage. 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 DAPK and its derivatives without altering the 
encoded amino acid sequences include the production of RNA transcripts 
having more desirable properties, such as a greater half-life, than 
transcripts produced from the naturally occurring sequence. 
The invention also encompasses production of DNA sequences, or fragments 
thereof, which encode DAPK and its derivatives, entirely by synthetic 
chemistry. After production, the synthetic sequence may be inserted into 
any of the many available expression vectors and cell systems using 
reagents that are well known in the art. Moreover, synthetic chemistry may 
be used to introduce mutations into a sequence encoding DAPK or any 
fragment thereof. 
Also encompassed by the invention are polynucleotide sequences that are 
capable of hybridizing to the claimed nucleotide sequences, and in 
particular, those shown in SEQ ID NOs:8-14, under various conditions of 
stringency as taught in Wahl, G. M. and S. L. Berger (1987; Methods 
Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol. 
152:507-511). 
Methods for DNA sequencing which are well known and generally available 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 DNA polymerase (US Biochemical Corp, 
Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 
polymerase (Amersham, Chicago, Ill.), or combinations of polymerases and 
proofreading exonucleases such as those found in the ELONGASE 
amplification system marketed by (GIBCO/BRL, Gaithersburg, Md.). 
Preferably, the process is automated with machines such as the Hamilton 
MICROLAB 2200 (Hamilton, Reno, Nev.), Peltier thermal cycler (PTC200; MJ 
Research, Watertown, Mass.) and the ABI CATALYST and 373 and 377 DNA 
SEQUENCERS (Perkin Elmer). 
The nucleic acid sequences encoding DAPK may be extended utilizing a 
partial nucleotide sequence and employing various 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 primers to retrieve unknown sequence adjacent to a known 
locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). In particular, 
genomic DNA is first amplified in the presence of primer to a linker 
sequence and a primer specific to the known region. The amplified 
sequences are then subjected to a second round of PCR with the same linker 
primer and another specific primer internal to the first one. Products of 
each round of PCR are transcribed with an appropriate RNA polymerase and 
sequenced using reverse transcriptase. 
Inverse PCR may also be used to amplify or extend sequences using divergent 
primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids 
Res. 16:8186). The primers may be designed using commercially available 
software such as OLIGO 4.06 primer analysis software (National Biosciences 
Inc., Plymouth, Minn.), or another appropriate program, to be 22-30 
nucleotides in length, to have a GC content of 50% or more, and to anneal 
to the target sequence at temperatures about 68.degree.-72.degree. C. The 
method uses several restriction enzymes to generate a suitable fragment in 
the known region of a gene. The fragment is then circularized by 
intramolecular ligation and used as a PCR template. 
Another method which may be used is capture PCR which involves PCR 
amplification of DNA fragments adjacent to a known sequence 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 also be used to place an engineered double-stranded 
sequence into an unknown fragment of the DNA molecule before performing 
PCR. 
Another method which may be used to retrieve unknown sequences is that of 
Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060). 
Additionally, one may use PCR, nested primers, and PROMOTERFINDER 
libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). This process 
avoids the need to screen libraries and is useful in finding intron/exon 
junctions. 
When screening for full-length cDNAs, it is preferable to use libraries 
that have been size-selected to include larger cDNAs. Also, random-primed 
libraries are preferable, in that they will contain more sequences which 
contain the 5' regions of genes. Use of a randomly primed library may be 
especially 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 fluorescent dyes 
(one for each nucleotide) which are laser activated, and detection of the 
emitted wavelengths by a charge coupled devise camera. Output/light 
intensity may be converted to electrical signal using appropriate software 
(e.g. GENOTYPER and SEQUENCE NAVIGATOR software, Perkin Elmer) 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 the sequencing of small pieces of DNA which 
might be present in limited amounts in a particular sample. 
In another embodiment of the invention, polynucleotide sequences or 
fragments thereof which encode DAPK may be used in recombinant DNA 
molecules to direct expression of DAPK, fragments or functional 
equivalents thereof, in appropriate host cells. Due to the inherent 
degeneracy of the genetic code, other DNA sequences which encode 
substantially the same or a functionally equivalent amino acid sequence 
may be produced, and these sequences may be used to clone and express 
DAPK. 
As will be understood by those of skill in the art, it may be advantageous 
to produce DAPK-encoding nucleotide sequences possessing non-naturally 
occurring codons. For example, codons preferred by a particular 
prokaryotic or eukaryotic host can be selected to increase the rate of 
protein expression or to produce an RNA transcript having desirable 
properties, such as a half-life which is longer than that of a transcript 
generated from the naturally occurring sequence. 
The nucleotide sequences of the present invention can be engineered using 
methods generally known in the art in order to alter DAPK encoding 
sequences for a variety of reasons, including but not limited to, 
alterations which modify 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, site-directed mutagenesis may be used 
to insert new restriction sites, alter glycosylation patterns, change 
codon preference, produce splice variants, introduce mutations, and so 
forth. 
In another embodiment of the invention, natural, modified, or recombinant 
nucleic acid sequences encoding DAPK may be ligated to a heterologous 
sequence to encode a fusion protein. For example, to screen peptide 
libraries for inhibitors of DAPK activity, it may be useful to encode a 
chimeric DAPK protein that can be recognized by a commercially available 
antibody. A fusion protein may also be engineered to contain a cleavage 
site located between the DAPK encoding sequence and the heterologous 
protein sequence, so that DAPK may be cleaved and purified away from the 
heterologous moiety. 
In another embodiment, sequences encoding DAPK may be synthesized, in whole 
or in part, using chemical methods well known in the art (see Caruthers, 
M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:215-223; Horn, T. et al. 
(1980) Nucl. Acids Res. Symp. Ser. 7:225-232). Alternatively, the protein 
itself may be produced using chemical methods to synthesize the amino acid 
sequence of DAPK, or a fragment thereof. For example, peptide synthesis 
can be performed using various solid-phase techniques (Roberge, J. Y. et 
al. (1995) Science 269:202-204) and automated synthesis may be achieved, 
for example, using the ABI 431A peptide synthesizer (Perkin Elmer). 
The newly synthesized peptide may be substantially purified by preparative 
high performance liquid chromatography (e.g., Creighton, T. (1983) 
Proteins, Structures and Molecular Principles, WH Freeman and Co., New 
York, N.Y.). The composition of the synthetic peptides may be confirmed by 
amino acid analysis or sequencing (e.g., the Edman degradation procedure; 
Creighton, supra). Additionally, the amino acid sequence of DAPK, or any 
part thereof, may be altered during direct synthesis and/or combined using 
chemical methods with sequences from other proteins, or any part thereof, 
to produce a variant polypeptide. 
In order to express a biologically active DAPK, the nucleotide sequences 
encoding DAPK or functional equivalents, may be inserted into appropriate 
expression vector, i.e., a vector which contains the necessary elements 
for the transcription and translation of the inserted coding sequence. 
Methods which are well known to those skilled in the art may be used to 
construct expression vectors containing sequences encoding DAPK and 
appropriate transcriptional and translational control elements. These 
methods include in vitro recombinant DNA techniques, synthetic techniques, 
and in vivo genetic recombination. Such techniques are described in 
Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold 
Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) 
Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. 
A variety of expression vector/host systems may be utilized to contain and 
express sequences encoding DAPK. 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 virus expression vectors (e.g., baculovirus); plant cell systems 
transformed with virus expression vectors (e.g., cauliflower mosaic virus, 
CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors 
(e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is 
not limited by the host cell employed. 
The "control elements" or "regulatory sequences" are those non-translated 
regions of the vector--enhancers, promoters, 5' and 3' untranslated 
regions--which interact with host cellular proteins to carry out 
transcription and translation. Such elements may vary in their strength 
and specificity. Depending on the vector system and host utilized, any 
number of suitable transcription and translation elements, including 
constitutive and inducible promoters, may be used. For example, when 
cloning in bacterial systems, inducible promoters such as the hybrid lacZ 
promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or 
PSPORT1 plasmid (Gibco BRL) and the like may be used. The baculovirus 
polyhedrin promoter may be used in insect cells. Promoters or enhancers 
derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and 
storage protein genes) or from plant viruses (e.g., viral promoters or 
leader sequences) may be cloned into the vector. In mammalian cell 
systems, promoters from mammalian genes or from mammalian viruses are 
preferable. If it is necessary to generate a cell line that contains 
multiple copies of the sequence encoding DAPK, vectors based on SV40 or 
EBV may be used with an appropriate selectable marker. 
In bacterial systems, a number of expression vectors may be selected 
depending upon the use intended for DAPK. For example, when large 
quantities of DAPK are needed for the induction of antibodies, vectors 
which direct high level expression of fusion proteins that are readily 
purified may be used. Such vectors include, but are not limited to, the 
multifunctional E. coli cloning and expression vectors such as the 
BLUESCRIPT phagemid (Stratagene), in which the sequence encoding DAPK may 
be ligated into the vector in frame with sequences for the amino-terminal 
Met and the subsequent 7 residues of .beta.-galactosidase so that a hybrid 
protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) 
J. Biol. Chem. 264:5503-5509); and the like. PGEX vectors (Promega, 
Madison, Wis.) may also be used to express foreign polypeptides as fusion 
proteins with glutathione S-transferase (GST). In general, such fusion 
proteins are soluble and can easily be purified from lysed cells by 
adsorption to glutathione-agarose beads followed by elution in the 
presence of free glutathione. Proteins made in such systems may be 
designed to include heparin, thrombin, or factor XA protease cleavage 
sites so that the cloned polypeptide of interest can be released from the 
GST moiety at will. 
In the yeast, Saccharomyces cerevisiae, a number of vectors containing 
constitutive or inducible promoters such as alpha factor, alcohol oxidase, 
and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et 
al. (1987) Methods Enzymol. 153:516-544. 
In cases where plant expression vectors are used, the expression of 
sequences encoding DAPK may be driven by any of a number of promoters. For 
example, viral promoters such as the 35S and 19S promoters of CaMV may be 
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; Broglie, R. et al. (1984) 
Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell 
Differ. 17:85-105). These constructs can be introduced into plant cells by 
direct DNA transformation or pathogen-mediated transfection. Such 
techniques are described in a number of generally available reviews (see, 
for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science 
and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196. 
An insect system may also be used to express DAPK. For example, in one such 
system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used 
as a vector to express foreign genes in Spodoptera frugiperda cells or in 
Trichoplusia larvae. The sequences encoding DAPK may be cloned into a 
non-essential region of the virus, such as the polyhedrin gene, and placed 
under control of the polyhedrin promoter. Successful insertion of DAPK 
will render the polyhedrin gene inactive and produce recombinant virus 
lacking coat protein. The recombinant viruses may then be used to infect, 
for example, S. frugiperda cells or Trichoplusia larvae in which DAPK may 
be expressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 
91:3224-3227). 
In mammalian host cells, a number of viral-based expression systems may be 
utilized. In cases where an adenovirus is used as an expression vector, 
sequences encoding DAPK may be ligated into an adenovirus 
transcription/translation complex consisting of the late promoter and 
tripartite leader sequence. Insertion in a non-essential E1 or E3 region 
of the viral genome may be used to obtain a viable virus which is capable 
of expressing DAPK in infected host cells (Logan, J. and Shenk, T. (1984) 
Proc. Natl. Acad. Sci. 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. 
Human artificial chromosomes (HACs) may also be employed to deliver larger 
fragments of DNA than can be contained and expressed in a plasmid. HACs of 
6 to 10M are constructed and delivered via conventional delivery methods 
(liposomes, polycationic amino polymers, or vesicles) for therapeutic 
purposes. 
Specific initiation signals may also be used to achieve more efficient 
translation of sequences encoding DAPK. Such signals include the ATG 
initiation codon and adjacent sequences. In cases where sequences encoding 
DAPK, its initiation codon, and upstream 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 the ATG initiation codon should be 
provided. Furthermore, the initiation codon should be in the correct 
reading frame to ensure translation of the entire insert. 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 which are appropriate for the particular 
cell system which is used, such as those described in the literature 
(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162). 
In addition, a host cell strain may be chosen for its ability to modulate 
the expression of the inserted sequences or to process the expressed 
protein in the desired fashion. Such modifications of the polypeptide 
include, but are not limited to, acetylation, carboxylation, 
glycosylation, phosphorylation, lipidation, and acylation. 
Post-translational processing which cleaves a "prepro" form of the protein 
may also be used to facilitate correct insertion, folding and/or function. 
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; Bethesda, Md.) and may be chosen to ensure the 
correct modification and processing of the foreign protein. 
For long-term, high-yield production of recombinant proteins, stable 
expression is preferred. For example, cell lines which stably express DAPK 
may be transformed 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 1-2 days in 
an enriched media before they are switched to selective media. The purpose 
of the selectable marker is to confer resistance to selection, and its 
presence allows growth and recovery of cells which successfully express 
the introduced sequences. Resistant clones of stably transformed cells may 
be proliferated 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 (Wigler, M. et al. (1977) Cell 11:223-32) and adenine 
phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes 
which can be employed in tk.sup.- or aprt.sup.- cells, respectively. Also, 
antimetabolite, antibiotic or herbicide resistance can be used as the 
basis for selection; for example, dhfr which confers resistance to 
methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); 
npt, which confers resistance to the aminoglycosides neomycin and G-418 
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, 
which confer resistance to chlorsulfuron and phosphinotricin 
acetyltransferase, respectively (Murry, supra). Additional selectable 
genes have been described, for example, trpB, which allows cells to 
utilize indole in place of tryptophan, or hisD, which allows cells to 
utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan 
(1988) Proc. Natl. Acad. Sci. 85:8047-51). Recently, the use of visible 
markers has gained popularity with such markers as anthocyanins, .beta. 
glucuronidase and its substrate GUS, and luciferase and its substrate 
luciferin, being widely 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. et al. (1995) 
Methods Mol. Biol. 55:121-131). 
Although the presence/absence of marker gene expression suggests that the 
gene of interest is also present, its presence and expression may need to 
be confirmed. For example, if the sequence encoding DAPK is inserted 
within a marker gene sequence, transformed cells containing sequences 
encoding DAPK can be identified by the absence of marker gene function. 
Alternatively, a marker gene can be placed in tandem with a sequence 
encoding DAPK 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. 
Alternatively, host cells which contain the nucleic acid sequence encoding 
DAPK and express DAPK 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 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. The presence of polynucleotide sequences encoding DAPK can be 
detected by DNA-DNA or DNA-RNA hybridization or amplification using probes 
or fragments or fragments of polynucleotides encoding DAPK. Nucleic acid 
amplification based assays involve the use of oligonucleotides or 
oligomers based on the sequences encoding DAPK to detect transformants 
containing DNA or RNA encoding DAPK. 
A variety of protocols for detecting and measuring the expression of DAPK, 
using either polyclonal or monoclonal antibodies specific for the protein 
are known in the art. Examples include enzyme-linked immunosorbent assay 
(ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting 
(FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal 
antibodies reactive to two non-interfering epitopes on DAPK is preferred, 
but a competitive binding assay may be employed. These and other assays 
are described, among other places, in Hampton, R. et al. (1990; 
Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and 
Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216). 
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 DAPK include 
oligolabeling, nick translation, end-labeling or PCR amplification using a 
labeled nucleotide. Alternatively, the sequences encoding DAPK, 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 (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison 
Wis.); and U.S. Biochemical Corp., Cleveland, Ohio). Suitable reporter 
molecules or labels, which may be used for ease of detection, include 
radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic 
agents as well as substrates, cofactors, inhibitors, magnetic particles, 
and the like. 
Host cells transformed with nucleotide sequences encoding DAPK 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 contained 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 DAPK may be 
designed to contain signal sequences which direct secretion of DAPK 
through a prokaryotic or eukaryotic cell membrane. Other constructions may 
be used to join sequences encoding DAPK to nucleotide sequence encoding a 
polypeptide domain which will facilitate purification of soluble proteins. 
Such purification facilitating domains include, but are not limited to, 
metal chelating peptides such as histidine-tryptophan modules that allow 
purification on immobilized metals, protein A domains that allow 
purification on immobilized immunoglobulin, and the domain utilized in the 
FLAG extension/affinity purification system (Immunex Corp., Seattle, 
Wash.). The inclusion of cleavable linker sequences such as those specific 
for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the 
purification domain and DAPK may be used to facilitate purification. One 
such expression vector provides for expression of a fusion protein 
containing DAPK and a nucleic acid encoding 6 histidine residues preceding 
a thioredoxin or an enterokinase cleavage site. The histidine residues 
facilitate purification on IMIAC (immobilized metal ion affinity 
chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 
3: 263-281) while the enterokinase cleavage site provides a means for 
purifying DAPK from the fusion protein. A discussion of vectors which 
contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell 
Biol. 12:441-453). 
In addition to recombinant production, fragments of DAPK may be produced by 
direct peptide synthesis using solid-phase techniques (Merrifield J. 
(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed 
using manual techniques or by automation. Automated synthesis may be 
achieved, for example, using Applied Biosystems 431A peptide synthesizer 
(Perkin Elmer). Various fragments of DAPK may be chemically synthesized 
separately and combined using chemical methods to produce the full length 
molecule. 
THERAPEUTICS 
Chemical and structural homology exits among the human protein kinases of 
the invention. The expression of DAPK is closely associated with cell 
proliferation. Therefore, in cancers or immune disorders where DAPK is 
being expressed, or is promoting cell proliferation; it is desirable to 
decrease the expression of DAPK. In cancers where expression of DAPK is 
decreased, it is desirable to provide the protein or increase the 
expression of DAPK. 
In one embodiment, DAPK or a fragment or derivative thereof may be 
administered to a subject to prevent or treat cancer which is associated 
with decreased expression of DAPK. Such cancers include, but are not 
limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, 
sarcoma, and teratocarcinoma and cancers of the adrenal gland, bladder, 
bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, 
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, 
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, 
testis, thymus, thyroid, and uterus. 
In another embodiment, an agonist which is specific for DAPK may be 
administered to a subject to prevent or treat cancer including, but not 
limited to, those cancers listed above. In another further embodiment, a 
vector capable of expressing DAPK, or a fragment or a derivative thereof, 
may be administered to a subject to prevent or treat cancer including, but 
not limited to, those cancers listed above. 
In a further embodiment, antagonists which decrease the expression and 
activity of DAPK may be administered to a subject to prevent or treat 
cancer which is associated with increased expression of DAPK. Such cancers 
include, but are not limited to, adenocarcinoma, leukemia, lymphoma, 
melanoma, myeloma, sarcoma, and teratocarcinoma and cancers of the adrenal 
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, 
ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, 
ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, 
spleen, testis, thymus, thyroid, and uterus. In one aspect, antibodies 
which specifically bind DAPK may be used directly as an antagonist or 
indirectly as a targeting or delivery mechanism for bringing a 
pharmaceutical agent to cells or tissue which express DAPK. 
In another embodiment, a vector expressing the complement of the 
polynucleotide encoding DAPK may be administered to a subject to treat or 
prevent cancer including, but not limited to, those cancers listed above. 
In one embodiment, an antagonist of DAPK may be administered to a subject 
to prevent or treat an immune response. Such responses may be associated 
with AIDS, Addison's disease, adult respiratory distress syndrome, 
allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis, 
Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, 
diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, 
gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus 
erythematosus, multiple sclerosis, myasthenia gravis, myocardial or 
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, 
polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and 
autoimmune thyroiditis; complications of cancer, hemodialysis, 
extracorporeal circulation; viral, bacterial, fungal, parasitic, 
protozoal, and helminthic infections and trauma. In one aspect, antibodies 
which specifically bind DAPK may be used directly as an antagonist or 
indirectly as a targeting or delivery mechanism for bringing a 
pharmaceutical agent to cells or tissue which express DAPK. 
In another embodiment, a vector expressing the complement of the 
polynucleotide encoding DAPK may be administered to a subject to treat or 
prevent an immune response including, but not limited to, those listed 
above. 
In a further embodiment, DAPK or a fragment or derivative thereof may be 
added to cells to stimulate cell proliferation. In particular, DAPK may be 
added to a cell in culture or cells in vivo using delivery mechanisms such 
as liposomes, viral based vectors, or electroinjection for the purpose of 
promoting cell proliferation and tissue or organ regeneration. 
Specifically, DAPK may be added to a cell, cell line, tissue or organ 
culture in vitro or ex vivo to stimulate cell proliferation for use in 
heterologous or autologous transplantation. In some cases, the cell will 
have been preselected for its ability to fight an infection or a cancer or 
to correct a genetic defect in a disease such as sickle cell anemia, 
.beta. thalassemia, cystic fibrosis, or Huntington's chorea. 
In another embodiment, an agonist which is specific for DAPK may be 
administered to a cell to stimulate cell proliferation, as described 
above. 
In another embodiment, a vector capable of expressing DAPK, or a fragment 
or a derivative thereof, may be administered to a cell to stimulate cell 
proliferation, as described above. 
In other embodiments, any of the therapeutic proteins, antagonists, 
antibodies, agonists, complementary sequences or vectors of the invention 
may be administered in combination with other appropriate therapeutic 
agents. Selection of the appropriate agents for use in combination therapy 
may be made 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 each agent, thus reducing the 
potential for adverse side effects. 
Antagonists or inhibitors of DAPK may be produced using methods which are 
generally known in the art. In particular, purified DAPK may be used to 
produce antibodies or to screen libraries of pharmaceutical agents to 
identify those which specifically bind DAPK. 
Antibodies to DAPK may be generated using methods that are well known in 
the art. Such antibodies may include, but are not limited to, polyclonal, 
monoclonal, chimeric, single chain, Fab fragments, and fragments produced 
by a Fab expression library. Neutralizing antibodies, (i.e., those which 
inhibit dimer formation) are especially preferred for therapeutic use. 
For the production of antibodies, various hosts including goats, rabbits, 
rats, mice, humans, and others, may be immunized by injection with DAPK or 
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, keyhole limpet hemocyanin, 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 DAPK have an amino acid sequence consisting of at 
least five amino acids and more preferably at least 10 amino acids. It is 
also preferable that they are identical to a portion of the amino acid 
sequence of the natural protein, and they may contain the entire amino 
acid sequence of a small, naturally occurring molecule. Short stretches of 
DAPK amino acids may be fused with those of another protein such as 
keyhole limpet hemocyanin and antibody produced against the chimeric 
molecule. 
Monoclonal antibodies to DAPK 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. 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", 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. 
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 DAPK-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. 
88:11120-3). 
Antibodies may also be produced by inducing in vivo production in the 
lymphocyte population or by screening immunoglobulin libraries or panels 
of highly specific binding reagents as disclosed in the literature 
(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, 
G. et al. (1991) Nature 349:293-299). Antibody fragments which contain 
specific binding sites for DAPK may also be generated. For example, such 
fragments include, but are not limited to, the F(ab')2 fragments which can 
be produced by pepsin digestion of the antibody molecule and the Fab 
fragments which can be 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 
254:1275-1281). 
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 DAPK and its specific antibody. A two-site, monoclonal-based 
immunoassay utilizing monoclonal antibodies reactive to two 
non-interfering DAPK epitopes is preferred, but a competitive binding 
assay may also be employed (Maddox, supra). 
In another embodiment of the invention, the polynucleotides encoding DAPK, 
or any fragment or complement thereof, may be used for therapeutic 
purposes. In one aspect, the complement of the polynucleotide encoding 
DAPK may be used in situations in which it would be desirable to block the 
transcription of the mRNA. In particular, cells may be transformed with 
sequences complementary to polynucleotides encoding DAPK. Thus, 
complementary molecules or fragments may be used to modulate DAPK 
activity, or to achieve regulation of gene function. Such technology is 
now well known in the art, and sense or antisense oligonucleotides or 
larger fragments, can be designed from various locations along the coding 
or control regions of sequences encoding DAPK. 
Expression vectors derived from retro viruses, adenovirus, herpes or 
vaccinia viruses, or from various bacterial plasmids may be used for 
delivery of nucleotide sequences to the targeted organ, tissue or cell 
population. Methods which are well known to those skilled in the art can 
be used to construct vectors which will express nucleic acid sequence 
which is complementary to the polynucleotides of the gene encoding DAPK. 
These techniques are described both in Sambrook et al. (supra) and in 
Ausubel et al. (supra). 
Genes encoding DAPK can be turned off by transforming a cell or tissue with 
expression vectors which express high levels of a polynucleotide or 
fragment thereof which encodes DAPK. Such constructs may be used to 
introduce untranslatable sense or antisense sequences into a cell. Even in 
the absence of integration into the DNA, such vectors may continue to 
transcribe RNA molecules until they are disabled by endogenous nucleases. 
Transient expression may last for a month or more with a non-replicating 
vector and even longer if appropriate replication elements are part of the 
vector system. 
As mentioned above, modifications of gene expression can be obtained by 
designing complementary sequences or antisense molecules (DNA, RNA, or 
PNA) to the control, 5' or regulatory regions of the gene encoding DAPK 
(signal sequence, promoters, enhancers, and introns). Oligonucleotides 
derived from the transcription initiation site, e.g., between positions 
-10 and +10 from the start site, are preferred. 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 
Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). The 
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. Examples which may be 
used include engineered hammerhead motif ribozyme molecules that can 
specifically and efficiently catalyze endonucleolytic cleavage of 
sequences encoding DAPK. 
Specific ribozyme cleavage sites within any potential RNA target are 
initially identified by scanning the target molecule for ribozyme cleavage 
sites which include the following sequences: GUA, GUU, and GUC. Once 
identified, short RNA sequences of between 15 and 20 ribonucleotides 
corresponding to the region of the target gene containing the cleavage 
site may be evaluated for secondary structural features which may render 
the oligonucleotide inoperable. The suitability of candidate targets may 
also be evaluated by testing accessibility to hybridization with 
complementary oligonucleotides using ribonuclease protection assays. 
Complementary ribonucleic acid molecules and ribozymes of the invention may 
be prepared by any method known in the art for the synthesis of nucleic 
acid molecules. These include techniques for chemically synthesizing 
oligonucleotides such as solid phase phosphoramidite chemical synthesis. 
Alternatively, RNA molecules may be generated by in vitro and in vivo 
transcription of DNA sequences encoding DAPK. 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 
S' 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. 
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 polycationic 
amino polymers (Goldman, C. K. et al. (1997) Nature Biotechnology 
15:462-66; incorporated herein by reference) may be achieved using methods 
which are well known in the art. 
Any of the therapeutic methods described above may be applied to any 
subject in need of such therapy, including, for example, mammals such as 
dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. 
An additional embodiment of the invention relates to the administration of 
a pharmaceutical composition, in conjunction with a pharmaceutically 
acceptable carrier, for any of the therapeutic effects discussed above. 
Such pharmaceutical compositions may consist of DAPK, antibodies to DAPK, 
mimetics, agonists, antagonists, or inhibitors of DAPK. The compositions 
may be administered alone or in combination with at least one other agent, 
such as stabilizing compound, which may be administered in any sterile, 
biocompatible pharmaceutical carrier, including, but not limited to, 
saline, buffered saline, dextrose, and water. The compositions may be 
administered to a patient alone, or in combination with other agents, 
drugs or hormones. 
The pharmaceutical compositions utilized in this invention may be 
administered by any number of routes including, but not limited to, oral, 
intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, 
intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, 
enteral, topical, sublingual, or rectal means. 
In addition to the active ingredients, these pharmaceutical compositions 
may contain suitable pharmaceutically-acceptable carriers comprising 
excipients and auxiliaries which facilitate processing of the active 
compounds into preparations which can be used pharmaceutically. Further 
details on techniques for formulation and administration may be found in 
the latest edition of Remington's Pharmaceutical Sciences (Maack 
Publishing Co., Easton, Pa.). 
Pharmaceutical compositions for oral administration can be formulated using 
pharmaceutically acceptable carriers well known in the art in dosages 
suitable for oral administration. Such carriers enable the pharmaceutical 
compositions to be formulated as tablets, pills, dragees, capsules, 
liquids, gels, syrups, slurries, suspensions, and the like, for ingestion 
by the patient. 
Pharmaceutical preparations for oral use can be obtained through 
combination of active compounds with solid excipient, optionally grinding 
a resulting mixture, and processing the mixture of granules, after adding 
suitable auxiliaries, if desired, to obtain tablets or dragee cores. 
Suitable excipients are carbohydrate or protein fillers, such as sugars, 
including lactose, sucrose, mannitol, or sorbitol; starch from corn, 
wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, 
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums 
including arabic and tragacanth; and proteins such as gelatin and 
collagen. If desired, disintegrating or solubilizing agents may be added, 
such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a 
salt thereof, such as sodium alginate. 
Dragee cores may be used in conjunction with suitable coatings, such as 
concentrated sugar solutions, which may also contain gum arabic, talc, 
polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium 
dioxide, lacquer solutions, and suitable organic solvents or solvent 
mixtures. Dyestuffs or pigments may be added to the tablets or dragee 
coatings for product identification or to characterize the quantity of 
active compound, i.e., dosage. 
Pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a coating, such as glycerol or sorbitol. Push-fit capsules can contain 
active ingredients mixed with a filler or binders, such as lactose or 
starches, lubricants, such as talc or magnesium stearate, and, optionally, 
stabilizers. In soft capsules, the active compounds may be dissolved or 
suspended in suitable liquids, such as fatty oils, liquid, or liquid 
polyethylene glycol with or without stabilizers. 
Pharmaceutical formulations suitable for parenteral administration may be 
formulated in aqueous solutions, preferably in physiologically compatible 
buffers such as Hanks' solution, Ringer's solution, or physiologically 
buffered saline. Aqueous injection suspensions may contain substances 
which increase the viscosity of the suspension, such as sodium 
carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions 
of the active compounds may be prepared as appropriate oily injection 
suspensions. Suitable lipophilic solvents or vehicles include fatty oils 
such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate 
or triglycerides, or liposomes. Non-lipid polycationic amino polymers may 
also be used for delivery. Optionally, the suspension may also contain 
suitable stabilizers or agents which increase the solubility of the 
compounds to allow for the preparation of highly concentrated solutions. 
For topical or nasal administration, penetrants appropriate to the 
particular barrier to be permeated are used in the formulation. Such 
penetrants are generally known in the art. The pharmaceutical compositions 
of the present invention may be manufactured in a manner that is known in 
the art, e.g., by means of conventional mixing, dissolving, granulating, 
dragee-making, levigating, emulsifying, encapsulating, entrapping, or 
lyophilizing processes. 
The pharmaceutical composition may be provided as a salt and can be formed 
with many acids, including but not limited to, hydrochloric, sulfuric, 
acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more 
soluble in aqueous or other protonic solvents than are the corresponding 
free base forms. In other cases, the preferred preparation may be a 
lyophilized powder which may contain any or all of the following: 1-50 mM 
histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 
5.5, that is combined with buffer prior to use. 
After pharmaceutical compositions have been prepared, they can be placed in 
an appropriate container and labeled for treatment of an indicated 
condition. For administration of DAPK, such labeling would include amount, 
frequency, and method of administration. 
Pharmaceutical 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. 
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, usually mice, rabbits, dogs, or pigs. The 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 DAPK or fragments thereof, antibodies of DAPK, 
agonists, antagonists or inhibitors of DAPK, which ameliorates the 
symptoms or condition. Therapeutic efficacy and toxicity may be determined 
by standard pharmaceutical procedures in cell cultures or experimental 
animals, e.g., ED50 (the dose therapeutically effective in 50% of the 
population) and LD50 (the dose lethal to 50% of the population). The dose 
ratio between therapeutic and toxic effects is the therapeutic index, and 
it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions 
which exhibit large therapeutic indices are preferred. The data obtained 
from cell culture assays and animal studies is used in formulating a range 
of dosage for human use. The dosage contained in such compositions is 
preferably within a range of circulating concentrations that include the 
ED50 with little or no toxicity. The dosage varies within this range 
depending upon the dosage form employed, 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 that requires treatment. Dosage and 
administration are adjusted to provide sufficient levels of the active 
moiety or to maintain the desired effect. Factors which may be taken into 
account include the severity of the disease state, general health of the 
subject, age, weight, and gender of the subject, diet, time and frequency 
of administration, drug combination(s), reaction sensitivities, and 
tolerance/response to therapy. Long-acting pharmaceutical compositions may 
be administered every 3 to 4 days, every week, or once every two weeks 
depending on half-life and clearance rate of the particular formulation. 
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a 
total dose of about 1 g, 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 DAPK may be used 
for the diagnosis of conditions or diseases characterized by expression of 
DAPK, or in assays to monitor patients being treated with DAPK, agonists, 
antagonists or inhibitors. The antibodies useful for diagnostic purposes 
may be prepared in the same manner as those described above for 
therapeutics. Diagnostic assays for DAPK include methods which utilize the 
antibody and a label to detect DAPK in human body fluids or extracts of 
cells or tissues. The antibodies may be used with or without modification, 
and may be labeled by joining them, either covalently or non-covalently, 
with a reporter molecule. A wide variety of reporter molecules which are 
known in the art may be used, several of which are described above. 
A variety of protocols including ELISA, RIA, and FACS for measuring DAPK 
are known in the art and provide a basis for diagnosing altered or 
abnormal levels of DAPK expression. Normal or standard values for DAPK 
expression are established by combining body fluids or cell extracts taken 
from normal mammalian subjects, preferably human, with antibody to DAPK 
under conditions suitable for complex formation The amount of standard 
complex formation may be quantified by various methods, but preferably by 
photometric, means. Quantities of DAPK expressed in control and disease, 
samples from biopsied tissues are compared with the standard values. 
Deviation between standard and subject values establishes the parameters 
for diagnosing disease. 
In another embodiment of the invention, the polynucleotides encoding DAPK 
may be used for diagnostic purposes. The polynucleotides which may be used 
include oligonucleotide sequences, complementary RNA and DNA molecules, 
and PNAs. The polynucleotides may be used to detect and quantitate gene 
expression in biopsied tissues in which expression of DAPK may be 
correlated with disease. The diagnostic assay may be used to distinguish 
between absence, presence, and excess expression of DAPK, and to monitor 
regulation of DAPK levels during therapeutic intervention. 
In one aspect, hybridization with PCR probes which are capable of detecting 
polynucleotide sequences, including genomic sequences, encoding DAPK or 
closely related molecules, may be used to identify nucleic acid sequences 
which encode DAPK. The specificity of the probe, whether it is made from a 
highly specific region, e.g., 10 unique nucleotides in the 5' regulatory 
region, or a less specific region, e.g., especially in the 3' coding 
region, and the stringency of the hybridization or amplification (maximal, 
high, intermediate, or low) will determine whether the probe identifies 
only naturally occurring sequences encoding DAPK, alleles, or related 
sequences. 
Probes may also be used for the detection of related sequences, and should 
preferably contain at least 50% of the nucleotides from any of the DAPK 
encoding sequences. The hybridization probes of the subject invention may 
be DNA or RNA and derived from the nucleotide sequence of SEQ ID No:8-14 
or from genomic sequence including promoter, enhancer elements, and 
introns of the naturally occurring DAPK. 
Means for producing specific hybridization probes for DNAs encoding DAPK 
include the cloning of nucleic acid sequences encoding DAPK or DAPK 
derivatives into vectors for the production of mRNA probes. Such vectors 
are known in the art, 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, 
radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline 
phosphatase coupled to the probe via avidin/biotin coupling systems, and 
the like. 
Polynucleotide sequences encoding DAPK may be used for the diagnosis of 
conditions, disorders, or diseases which are associated with either 
increased or decreased expression of DAPK. Examples of such conditions or 
diseases include adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, 
sarcoma, teratocarcinoma, and cancers of the adrenal gland, bladder, bone, 
brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, 
heart, kidney, liver, lung, bone marrow, muscle, ovary, pancreas, 
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, 
thymus, thyroid, and uterus; and immune disorders such as AIDS, Addison's 
disease, adult respiratory distress syndrome, allergies, anemia, asthma, 
atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerative 
colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, 
atrophic gastritis, glomerulonephritis, gout, Graves' disease, 
hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple 
sclerosis, myasthenia gravis, myocardial or pericardial inflammation, 
osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid 
arthritis, scleroderma, Sjogren's syndrome, and thyroiditis. The 
polynucleotide sequences encoding DAPK may be used in Southern or northern 
analysis, dot blot, or other membrane-based technologies; in PCR 
technologies; or in dipstick, pin, ELISA assays or microarrays utilizing 
fluids or tissues from patient biopsies to detect altered DAPK expression. 
Such qualitative or quantitative methods are well known in the art. 
In a particular aspect, the nucleotide sequences encoding DAPK may be 
useful in assays that detect activation or induction of various cancers, 
particularly those mentioned above. The nucleotide sequences encoding DAPK 
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 quantitated and compared with a standard value. 
If the amount of signal in the biopsied or extracted sample is 
significantly altered from that of a comparable control sample, the 
nucleotide sequences have hybridized with nucleotide sequences in the 
sample, and the presence of altered levels of nucleotide sequences 
encoding DAPK in the sample indicates the presence of the associated 
disease. Such assays may also be used to evaluate the efficacy of a 
particular therapeutic treatment regimen in animal studies, in clinical 
trials, or in monitoring the treatment of an individual patient. 
In order to provide a basis for the diagnosis of disease associated with 
expression of DAPK, 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, which encodes DAPK, under conditions 
suitable for hybridization or amplification. Standard hybridization may be 
quantified by comparing the values obtained from normal subjects with 
those from an experiment where a known amount of a substantially purified 
polynucleotide is used. Standard values obtained from normal samples may 
be compared with values obtained from samples from patients who are 
symptomatic for disease. Deviation between standard and subject values is 
used to establish the presence of disease. 
Once disease is established and a treatment protocol is initiated, 
hybridization assays may be repeated on a regular basis to evaluate 
whether the level of expression in the patient begins to approximate that 
which is observed in the normal patient. 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 a relatively high amount of 
transcript in biopsied tissue from an individual may indicate a 
predisposition for the development of the disease, or may provide a means 
for detecting the disease prior to the appearance of actual clinical 
symptoms. A more definitive diagnosis of this type may allow health 
professionals to employ preventative measures or aggressive treatment 
earlier thereby preventing the development or further progression of the 
cancer. 
Additional diagnostic uses for oligonucleotides designed from the sequences 
encoding DAPK may involve the use of PCR. Such oligomers may be chemically 
synthesized, generated enzymatically, or produced in vitro. Oligomers will 
preferably consist of two nucleotide sequences, one with sense orientation 
(5'-&gt;3') and another with antisense (3'&lt;-5'), employed under optimized 
conditions for identification of a specific gene or condition. The same 
two oligomers, nested sets of oligomers, or even a degenerate pool of 
oligomers may be employed under less stringent conditions for detection 
and/or quantitation of closely related DNA or RNA sequences. 
Methods which may also be used to quantitate the expression of DAPK include 
radiolabeling or biotinylating nucleotides, coamplification of a control 
nucleic acid, and standard curves onto which the experimental results are 
interpolated (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 an ELISA format where the oligomer of interest is presented in various 
dilutions and a spectrophotometric or colorimetric response gives rapid 
quantitation. 
In further embodiments, oligonucleotides derived from any of the 
polynucleotide sequences described herein may be used as targets in a 
microarray. The microarray can be used to monitor the expression level of 
large numbers of genes simultaneously (to produce a transcript image), and 
to identify genetic variants, mutations and polymorphisms. This 
information may be used to determine gene function, understanding the 
genetic basis of disease, diagnosing disease, and in developing and in 
monitoring the activities of therapeutic agents. 
In one embodiment, the microarray is prepared and used according to the 
methods described in PCT application WO95/11995 (Chee et al.), Lockhart, 
D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. 
(1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are 
incorporated herein in their entirety by reference. 
The microarray is preferably composed of a large number of unique, 
single-stranded nucleic acid sequences, usually either synthetic antisense 
oligonucleotides or fragments of cDNAs, fixed to a solid support. The 
oligonucleotides are preferably about 6-60 nucleotides in length, more 
preferably 15-30 nucleotides in length, and most preferably about 20 
nucleotides in length. For a certain type of microarray, it may be 
preferable to use oligonucleotides which are only 7-10 nucleotides in 
length. The microarray may contain oligonucleotides which cover the known 
5', or 3', sequence, or contain sequential oligonucleotides which cover 
the full length sequence; or unique oligonucleotides selected from 
particular areas along the length of the sequence. Polynucleotides used in 
the microarray may be oligonucleotides that are specific to a gene or 
genes of interest in which at least a fragment of the sequence is known or 
that are specific to one or more unidentified cDNAs which are common to a 
particular cell type, developmental or disease state. In certain 
situations it may be appropriate to use pairs of oligonucleotides on a 
microarray. The "pairs" will be identical, except for one nucleotide which 
preferably is located in the center of the sequence. The second 
oligonucleotide in the pair (mismatched by one) serves as a control. The 
number of oligonucleotide pairs may range from 2 to one million. 
In order to produce oligonucleotides to a known sequence for a microarray, 
the gene of interest is examined using a computer algorithm which starts 
at the 5' or more preferably at the 3' end of the nucleotide sequence. The 
algorithm identifies oligomers of defined length that are unique to the 
gene, have a GC content within a range suitable for hybridization, and 
lack predicted secondary structure that may interfere with hybridization. 
The oligomers are synthesized at designated areas on a substrate using a 
light-directed chemical process. The substrate may be paper, nylon or 
other type of membrane, filter, chip, glass slide or any other suitable 
solid support. 
In another aspect, the oligonucleotides may be synthesized on the surface 
of the substrate by using a chemical coupling procedure and an ink jet 
application apparatus, as described in PCT application WO95/251116 
(Baldeschweiler et al.) which is incorporated herein in its entirety by 
reference. In another aspect, a "gridded" array analogous to a dot (or 
slot) blot may be used to arrange and link cDNA fragments or 
oligonucleotides to the surface of a substrate using a vacuum system, 
thermal, UV, mechanical or chemical bonding procedures. An array may be 
produced by hand or using available devices (slot blot or dot blot 
apparatus), materials and machines (including robotic instruments) and may 
contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other 
multiple from 2 to one million which lends itself to the efficient use of 
commercially available instrumentation. 
In order to conduct sample analysis using the microarrays, the RNA or DNA 
from a biological sample is made into hybridization probes. The mRNA is 
isolated, and cDNA is produced and used as a template to make antisense 
RNA (aRNA). The aRNA is amplified in the presence of fluorescent 
nucleotides, and labeled probes are incubated with the microarray so that 
the probe sequences hybridize to complementary oligonucleotides of the 
microarray. Incubation conditions are adjusted so that hybridization 
occurs with precise complementary matches or with various degrees of less 
complementarity. After removal of nonhybridized probes, a scanner is used 
to determine the levels and patterns of fluorescence. The scanned images 
are examined to determine degree of complementarity and the relative 
abundance of each oligonucleotide sequence on the microarray. The 
biological samples may be obtained from any bodily fluids (such as blood, 
urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or 
other tissue preparations. A detection system may be used to measure the 
absence, presence, and amount of hybridization for all of the distinct 
sequences simultaneously. This data may be used for large scale 
correlation studies or functional analysis of the sequences, mutations, 
variants, or polymorphisms among samples (Heller, R. A. et al., (1997) 
Proc. Natl. Acad. Sci. 94:2150-55). 
In another embodiment of the invention, the nucleic acid sequences which 
encode DAPK may also be used to generate hybridization probes which are 
useful for mapping the naturally occurring genomic sequence. The sequences 
may be mapped to a particular chromosome, to a specific region of a 
chromosome or to artificial chromosome constructions, such as human 
artificial chromosomes (HACs), yeast artificial chromosomes (YACs), 
bacterial artificial chromosomes (BACs), bacterial P1 constructions or 
single chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood 
Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154. 
Fluorescent in situ hybridization (FISH as described in Verma et al. (1988) 
Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, 
N.Y.) may be correlated with other physical chromosome mapping techniques 
and genetic map data. Examples of genetic map data can be found in various 
scientific journals or at Online Mendelian Inheritance in Man (OMIM). 
Correlation between the location of the gene encoding DAPK on a physical 
chromosomal map and a specific disease, or predisposition to a specific 
disease, may help delimit the region of DNA associated with that genetic 
disease. The nucleotide sequences of the subject invention may be used to 
detect differences in gene sequences between normal, carrier, or affected 
individuals. 
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 number or arm of a particular human 
chromosome is not known. New sequences can be assigned to chromosomal 
arms, or parts thereof, by physical mapping. This provides valuable 
information to investigators searching for disease genes using positional 
cloning or other gene discovery techniques. Once the disease or syndrome 
has been crudely localized by genetic linkage to a particular genomic 
region, for example, AT to 11q22-23 (Gatti, R. A. et al. (1988) Nature 
336:577-580), any sequences mapping to that area may represent associated 
or regulatory genes for further investigation. The nucleotide sequence of 
the subject invention may also be used to detect differences in the 
chromosomal location due to translocation, inversion, etc. among normal, 
carrier, or affected individuals. 
In another embodiment of the invention, DAPK, 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 DAPK and the agent being tested, 
may be measured. 
Another technique for drug screening which may be used provides for high 
throughput screening of compounds having suitable binding affinity to the 
protein of interest as described in published PCT application WO84/03564. 
In this method, as applied to DAPK large numbers of different small test 
compounds are synthesized on a solid substrate, such as plastic pins or 
some other surface. The test compounds are reacted with DAPK, or fragments 
thereof, and washed. Bound DAPK is then detected by methods well known in 
the art. Purified DAPK 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 DAPK specifically compete 
with a test compound for binding DAPK. In this manner, the antibodies can 
be used to detect the presence of any peptide which shares one or more 
antigenic determinants with DAPK. 
In additional embodiments, the nucleotide sequences which encode DAPK 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. 
The examples below are provided to illustrate the subject invention and are 
not included for the purpose of limiting the invention. 
EXAMPLES 
For purposes of example, the preparation and sequencing of the TMLR3DT01 
cDNA library, from which Incyte Clone 402339 was isolated, is described. 
Preparation and sequencing of cDNAs in libraries in the LIFESEQ.TM. 
database have varied over time, and the gradual changes involved use of 
particular kits, plasmids, and machinery available at the particular time 
the library was made and analyzed. 
I TMLR3DT01 cDNA Library Construction 
The TMLR3DT01 cDNA library was constructed from normal peripheral blood 
T-lymphocytes obtained from two unrelated Caucasian males aged 25 and 29 
years. This library represents a mixture of allogeneically stimulated 
human T cell populations obtained from FICOLL/HYPAQUE gradient purified 
buffy coats. The cells from the two different donors (not typed for HLA 
alleles) were incubated at a density of 1.times.10.sup.6 /ml, cultured for 
96 hours in DME containing 10% human serum, washed in PBS, scraped and 
lyzed immediately in buffer containing guanidinium isothiocyanate. The 
lysate was extracted twice with a mixture of phenol and chloroform, pH 8.0 
and centrifuged over a CsCl cushion using an Beckman SW28 rotor in a 
L8-70M ultracentrifuge (Beckman Instruments). The RNA was precipitated 
using 0.3M sodium acetate and 2.5 volumes of ethanol, resuspended in water 
and DNase treated for 15 min at 37.degree. C. The total RNA was isolated 
using the OLIGOTEX kit (QIAGEN Inc, Chatsworth Calif.). B lymphocytes were 
not removed, and some contaminating macrophages may also have been 
present. 
Stratagene (La Jolla Calif.) used the total RNA to construct a custom cDNA 
library. First strand cDNA synthesis was accomplished using an oligo d(T) 
primer/linker which also contained an XhoI restriction site. Second strand 
synthesis was performed using a combination of DNA polymerase I, E. coli 
ligase and RNase H, followed by the addition of an EcoRI adaptor to the 
blunt ended cDNA. The EcoRI adapted, double-stranded cDNA was then 
digested with XhoI restriction enzyme and fractionated on Sephacryl S400 
to obtain sequences which exceeded 800 bp in size. The size-selected cDNAs 
were inserted into the LAMBDAZAP vector system (Stratagene); and the 
vector which contains the BLUESCRIPT phagemid (Stratagene) was transformed 
into cells of E. coli, strain XL1-BLUEMRF (Stratagene). 
The phagemid forms of individual cDNA clones were obtained by the in vivo 
excision process. Enzymes from both BLUESCRIPT phagemid and a 
co-transformed f1 helper phage nicked the DNA, initiated new DNA 
synthesis, and created the smaller, single-stranded, circular phagemid 
molecules which contained the cDNA insert. The phagemid DNA was released, 
purified, and used to reinfect fresh host cells (SOLR, Stratagene). 
Presence of the phagemid which carries the gene for .beta.-lactamase 
allowed transformed bacteria to grow on medium containing ampicillin. 
II Isolation and Sequencing of cDNA Clones 
Plasmid DNA was released from the cells and purified using the MINIPREP kit 
(Catalogue #77468; Advanced Genetic Technologies Corporation, Gaithersburg 
Md.). This kit consists of a 96 well block with reagents for 960 
purifications. The recommended protocol was employed except for the 
following changes: 1) the 96 wells were each filled with only 1 ml of 
sterile Terrific Broth (Catalog #22711, Gibco/BRL, Gaithersburg Md.) with 
carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteria were 
cultured for 24 hours after the wells were inoculated and then lysed with 
60 .mu.l of lysis buffer; 3) a centrifugation step employing the Beckman 
GS-6R @2900 rpm for 5 min was performed before the contents of the block 
were added to the primary filter plate; and 4) the optional step of adding 
isopropanol to Tris buffer was not routinely performed. After the last 
step in the protocol, samples were transferred to a Beckman 96-well block 
for storage. 
The cDNAs were sequenced by the method of Sanger F and AR Coulson (1975; J 
Mol Biol 94:441f), using a Hamilton MICROLAB 2200 (Hamilton, Reno Nev.) in 
combination with four Peltier thermal cyclers (PTC200 from MJ Research, 
Watertown Mass.) and Applied Biosystems 377 or 373 DNA sequencing systems 
(Perkin Elmer) and reading frame was determined. 
III Homology Searching of cDNA Clones and Their Deduced Proteins 
The nucleotide sequences and/or amino acid sequences of the Sequence 
Listing were used to query sequences in the GenBank, SwissProt, BLOCKS, 
and Pima II databases. These databases, which contain previously 
identified and annotated sequences, were searched for regions of homology 
using BLAST, which stands for Basic Local Alignment Search Tool (Altschul, 
S. F. (1993) J. Mol. Evol 36:290-300; Altschul, et al. (1990) J. Mol. 
Biol. 215:403-410). 
BLAST produced alignments of both nucleotide and amino acid sequences to 
determine sequence similarity. Because of the local nature of the 
alignments, BLAST was especially useful in determining exact matches or in 
identifying homologs which may be of prokaryotic (bacterial) or eukaryotic 
(animal, fungal, or plant) origin. Other algorithms such as the one 
described in Smith, T. et al. (1992, Protein Engineering 5:35-51), 
incorporated herein by reference, could have been used when dealing with 
primary sequence patterns and secondary structure gap penalties. The 
sequences disclosed in this application have lengths of at least 49 
nucleotides, and no more than 12% uncalled bases (where N is recorded 
rather than A, C, G, or T). 
The BLAST approach searched for matches between a query sequence and a 
database sequence. BLAST evaluated the statistical significance of any 
matches found, and reported only those matches that satisfy the 
user-selected threshold of significance. In this application, threshold 
was set at 10.sup.-25 for nucleotides and 10.sup.-14 for peptides. 
Incyte nucleotide sequences were searched against the GenBank databases for 
primate (pri), rodent (rod), and other mammalian sequences (mam); and 
deduced amino acid sequences from the same clones were then searched 
against GenBank functional protein databases, mammalian (mamp), vertebrate 
(vrtp), and eukaryote (eukp) for homology. The relevant database for a 
particular match were reported as GIxxx.+-.p (where xxx is pri, rod, etc., 
and if present, p=peptide). 
IV Northern Analysis 
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 et al., supra). 
Analogous computer techniques use BLAST to search for identical or related 
molecules in nucleotide databases such as GenBank or the LIFESEQ.TM. 
database (Incyte Pharmaceuticals). 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 homologous. 
The basis of the search is the product score which is defined as: 
##EQU1## 
The product score takes into account both the degree of similarity between 
two sequences and the length of the sequence match. For example, with a 
product score of 40, the match will be exact within a 1-2% error; and at 
70, the match will be exact. Homologous molecules are usually identified 
by selecting those which show product scores between 15 and 40, although 
lower scores may identify related molecules. 
The results of northern analysis are reported as a list of libraries in 
which the transcript encoding DAPK occurs. Abundance and percent abundance 
are also reported. Abundance directly reflects the number of times a 
particular transcript is represented in a cDNA library, and percent 
abundance is abundance divided by the total number of sequences examined 
in the cDNA library. 
V Extension of DAPK Encoding Polynucleotides 
The nucleic acid sequence of an Incyte Clone disclosed in the Sequence 
Listing was used to design oligonucleotide primers for extending a partial 
nucleotide sequence to full length. One primer was synthesized to initiate 
extension in the antisense direction, and the other was synthesized to 
extend sequence in the sense direction. Primers were used to facilitate 
the extension of the known sequence "outward" generating amplicons 
containing new, unknown nucleotide sequence for the region of interest. 
The initial primers were designed from the cDNA using OLIGO 4.06 primer 
analysis software (National Biosciences), or another appropriate program, 
to be about 22 to about 30 nucleotides in length, to have a GC content of 
50% or more, and to anneal to the target sequence at temperatures of about 
68.degree. to about 72.degree. C. Any stretch of nucleotides which would 
result in hairpin structures and primer-primer dimerizations was avoided. 
Selected human cDNA libraries (Gibco/BRL) were used to extend the sequence 
If more than one extension is necessary or desired, additional sets of 
primers are designed to further extend the known region. 
High fidelity amplification was obtained by following the instructions for 
the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and 
reaction mix. Beginning with 40 pmol of each primer and the recommended 
concentrations of all other components of the kit, PCR was performed using 
the Peltier thermal cycler (PTC200; M. J. Research, Watertown, Mass.) and 
the following parameters: 
______________________________________ 
Step 1 94.degree. C. for 1 min (initial denaturation) 
Step 2 65.degree. C. for 1 min 
Step 3 68.degree. C. for 6 min 
Step 4 94.degree. C. for 15 sec 
Step 5 65.degree. C. for 1 min 
Step 6 68.degree. C. for 7 min 
Step 7 Repeat step 4-6 for 15 additional cycles 
Step 8 94.degree. C. for 15 sec 
Step 9 65.degree. C. for 1 min 
Step 10 68.degree. C. for 7:15 min 
Step 11 Repeat step 8-10 for 12 cycles 
Step 12 72.degree. C. for 8 min 
Step 13 4.degree. C. (and holding) 
______________________________________ 
A 5-10 .mu.l aliquot of the reaction mixture was analyzed by 
electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel 
to determine which reactions were successful in extending the sequence. 
Bands thought to contain the largest products were excised from the gel, 
purified using QIAQUICK DNA gel purification (QIAGEN Inc., Chatsworth, 
Calif.), and trimmed of overhangs using Klenow enzyme to facilitate 
religation and cloning. 
After ethanol precipitation, the products were redissolved in 13 .mu.l of 
ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1 .mu.l T4 
polynucleotide kinase were added, and the mixture was incubated at room 
temperature for 2-3 hours or overnight at 16.degree. C. Competent E. coli 
cells (in 40 .mu.l of appropriate media) were transformed with 3 .mu.l of 
ligation mixture and cultured in 80 .mu.l of SOC medium (Sambrook et al., 
supra). After incubation for one hour at 37.degree. C., the E. coli 
mixture was plated on Luria Bertani (LB)-agar (Sambrook et al., supra) 
containing 2.times. Carb. The following day, several colonies were 
randomly picked from each plate and cultured in 150 .mu.l of liquid 
LB/2.times. Carb medium placed in an individual well of an appropriate, 
commercially-available, sterile 96-well microtiter plate. The following 
day, 5 .mu.l of each overnight culture was transferred into a non-sterile 
96-well plate and after dilution 1:10 with water, 5 .mu.l of each sample 
was transferred into a PCR array. 
For PCR amplification, 18 .mu.l of concentrated PCR reaction mix 
(3.3.times.) containing 4 units of rTth DNA polymerase, a vector primer, 
and one or both of the gene specific primers used for the extension 
reaction were added to each well. Amplification was performed using the 
following conditions: 
______________________________________ 
Step 1 94.degree. C. for 60 sec 
Step 2 94.degree. C. for 20 sec 
Step 3 55.degree. C. for 30 sec 
Step 4 72.degree. C. for 90 sec 
Step 5 Repeat steps 2-4 for an additional 29 cycles 
Step 6 72.degree. C. for 180 sec 
Step 7 4.degree. C. (and holding) 
______________________________________ 
Aliquots of the PCR reactions were run on agarose gels together with 
molecular weight markers. The sizes of the PCR products were compared to 
the original partial cDNAs, and appropriate clones were selected, ligated 
into plasmid, and sequenced. 
In like manner, the nucleotide sequence of SEQ ID NO:8-14 are used to 
obtain 5' regulatory sequences using the procedure above, oligonucleotides 
designed for 5' extension, and an appropriate genomic library. 
VI Labeling and Use of Individual Hybridization Probes 
Hybridization probes derived from SEQ ID NOs:8-14 are employed to screen 
cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, 
consisting of about 20 base-pairs, is specifically described, essentially 
the same procedure is used with larger nucleotide fragments. 
Oligonucleotides are designed using state-of-the-art software such as 
OLIGO 4.06 primer analysis software (National Biosciences), labeled by 
combining 50 pmol of each oligomer and 250 .mu.Ci of .gamma.-.sup.32 P! 
adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPont 
NEN.RTM., Boston, Mass.). The labeled oligonucleotides are substantially 
purified with SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn). A 
aliquot containing 10.sup.7 counts per minute of the labeled probe is used 
in a typical membrane-based hybridization analysis of human genomic DNA 
digested with one of the following endonucleases (Ase I, Bgl II, Eco RI, 
Pst I, Xba 1, or Pvu II; DuPont NEN.RTM.). 
The DNA from each digest is fractionated on a 0.7 percent agarose gel and 
transferred to nylon membranes (NYTRAN PLUS membrane, Schleicher & 
Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 
40.degree. C. To remove nonspecific signals, blots are sequentially washed 
at room temperature under increasingly stringent conditions up to 
0.1.times. saline sodium citrate and 0.5% sodium dodecyl sulfate. After 
XOMATAR autoradiography film (Kodak, Rochester, N.Y.) is exposed to the 
blots or the blots are placed in a PHOSPHOIMAGER cassette (Molecular 
Dynamics, Sunnyvale, Calif.) for several hours, hybridization patterns are 
compared visually. 
VII Microarrays 
To produce oligonucleotides for a microarray, SEQ ID No:8-14 were examined 
using a computer algorithm which starts at the 3' end of the nucleotide 
sequence. The algorithm identified oligomers of defined length that are 
unique to the gene, have a GC content within a range suitable for 
hybridization, and lack predicted secondary structure that would interfere 
with hybridization. The algorithm identified approximately 20 
sequence-specific oligonucleotides of 20 nucleotides in length (20-mers). 
A matched set of oligonucleotides was created in which one nucleotide in 
the center of each sequence was altered. This process was repeated for 
each gene in the microarray, and double sets of twenty 20 mers were 
synthesized and arranged on the surface of the silicon chip using a 
light-directed chemical process (Chee, M. et al., PCT/WO95/11995, 
incorporated herein by reference). 
In the alternative, a chemical coupling procedure and an ink jet device 
were used to synthesize oligomers on the surface of a substrate 
(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein by 
reference). In another alternative, a "gridded" array analogous to a dot 
(or slot) blot was used to arrange and link cDNA fragments or 
oligonucleotides to the surface of a substrate using a vacuum system, 
thermal, UV, mechanical or chemical bonding procedures. A typical array 
may be produced by hand or using available materials and machines and 
contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 
dots. After hybridization, the microarray was washed to remove 
nonhybridized probes, and a scanner was used to determine the levels and 
patterns of fluorescence. The scanned images were examined to determine 
degree of complementarity and the relative abundance/expression level of 
each oligonucleotide sequence in the microarray. 
VIII Complementary Polynucleotides 
Sequence complementary to the sequence encoding DAPK, or any part thereof, 
is used to detect, decrease or inhibit expression of naturally occurring 
DAPK. Although use of oligonucleotides comprising from about 15 to about 
30 base-pairs is described, essentially the same procedure is used with 
smaller or larger sequence fragments. Appropriate oligonucleotides are 
designed using OLIGO 4.06 primer analysis software and the coding sequence 
of DAPK, SEQ ID NOs:8-14. 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 transcript encoding DAPK. 
IX Expression of DAPK 
Expression of DAPK is accomplished by subcloning the cDNAs into appropriate 
vectors and transforming the vectors into host cells. In this case, the 
cloning vector is also used to express DAPK in E. coli. Upstream of the 
cloning site, this vector contains a promoter for .beta.-galactosidase, 
followed by sequence containing the amino-terminal Met, and the subsequent 
seven residues of .beta.-galactosidase. Immediately following these eight 
residues is a bacteriophage promoter useful for transcription and a linker 
containing a number of unique restriction sites. 
Induction of an isolated, transformed bacterial strain with IPTG using 
standard methods produces a fusion protein which consists of the first 
eight residues of .beta.-galactosidase, about 5 to 15 residues of linker, 
and the full length protein. The signal residues direct the secretion of 
DAPK into the bacterial growth media which can be used directly in the 
following assay for activity. 
X Demonstration of DAPK Activity 
DAPK activity may be measured by phosphorylation of a protein substrate 
using gamma-labeled .sup.32 P-ATP and quantitation of the incorporated 
radioactivity using a gamma radioisotope counter. DAPK is incubated with 
the protein substrate, .sup.32 P-ATP, and a kinase buffer. The .sup.32 p 
incorporated into the substrate is then separated from free .sup.32 P-ATP 
by electrophoresis and the incorporated .sup.32 P is counted. The amount 
of .sup.32 P recovered is proportional to the activity of DAPK in the 
assay. A determination of the specific amino acid residues phosphorylated 
is made by phosphoamino acid analysis of the hydrolyzed protein. 
XI Production of DAPK Specific Antibodies 
DAPK that is substantially purified using PAGE electrophoresis (Sambrook, 
supra), or other purification techniques, is used to immunize rabbits and 
to produce antibodies using standard protocols. The amino acid sequence 
deduced from SEQ ID NOs:8-14 is analyzed using DNASTAR software (DNASTAR 
Inc) 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. Selection of appropriate epitopes, such as 
those near the C-terminus or in hydrophilic regions, is described by 
Ausubel et al. (supra), and others. 
Typically, the oligopeptides are 15 residues in length, synthesized using 
an Applied Biosystems peptide synthesizer model 431 A using 
fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma, St. 
Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester 
(MBS; Ausubel et al., supra). Rabbits are immunized with the 
oligopeptide-KLH complex in complete Freund's adjuvant. The resulting 
antisera are tested for antipeptide activity, for example, by binding the 
peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, 
washing, and reacting with radio iodinated, goat anti-rabbit IgG. 
XII Purification of Naturally Occurring DAPK Using Specific Antibodies 
Naturally occurring or recombinant DAPK is substantially purified by 
immunoaffinity chromatography using antibodies specific for DAPK. An 
immunoaffinity column is constructed by covalently coupling DAPK antibody 
to an activated chromatographic resin, such as CNBr-activated SEPHAROSE 
(Pharmacia & Upjohn). After the coupling, the resin is blocked and washed 
according to the manufacturer's instructions. 
Media containing DAPK is passed over the immunoaffinity column, and the 
column is washed under conditions that allow the preferential absorbance 
of DAPK (e.g., high ionic strength buffers in the presence of detergent). 
The column is eluted under conditions that disrupt antibody/protein 
binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, 
such as urea or thiocyanate ion), and DAPK is collected. 
XIII Identification of Molecules which Interact with DAPK 
DAPK or biologically active fragments thereof are labeled with .sup.125 I 
Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529). 
Candidate molecules previously arrayed in the wells of a multi-well plate 
are incubated with the labeled DAPK, washed and any wells with labeled 
DAPK complex are assayed. Data obtained using different concentrations of 
DAPK are used to calculate values for the number, affinity, and 
association of DAPK with the candidate molecules. 
All publications and patents mentioned in the above specification are 
herein incorporated by reference. Various modifications and variations of 
the described method and system of the invention will be apparent to those 
skilled in the art without departing from the scope and spirit of the 
invention. Although the invention has been described in connection with 
specific preferred embodiments, it should be understood that the invention 
as claimed should not be unduly limited to such specific embodiments. 
Indeed, various modifications of the described modes for carrying out the 
invention which are obvious to those skilled in molecular biology or 
related fields are intended to be within the scope of the following 
claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 21 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 685 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: HUVENOB01 
(B) CLONE: 39043 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetGluLeuLeuArgThrIleThrTyrGlnProAlaAlaSerThrLys 
151015 
MetCysGluGlnAlaLeuGlyLysGlyCysGlyAlaAspSerLysLys 
202530 
LysArgProProGlnProProGluGluSerGlnProProGlnSerGln 
354045 
AlaGlnValProProAlaAlaProHisHisHisHisHisHisSerHis 
505560 
SerGlyProGluIleSerArgIleIleValAspProThrThrGlyLys 
65707580 
ArgTyrCysArgGlyLysValLeuGlyLysGlyGlyPheAlaLysCys 
859095 
TyrGluMetThrAspLeuThrAsnAsnLysValTyrAlaAlaLysIle 
100105110 
IleProHisSerArgValAlaLysProHisGlnArgGluLysIleAsp 
115120125 
LysGluIleGluLeuHisArgIleLeuHisHisLysHisValValGln 
130135140 
PheTyrHisTyrPheGluAspLysGluAsnIleTyrIleLeuLeuGlu 
145150155160 
TyrCysSerArgArgSerMetAlaHisIleLeuLysAlaArgLysVal 
165170175 
LeuThrGluProGluValArgTyrTyrLeuArgGlnIleValSerGly 
180185190 
LeuLysTyrLeuHisGluGlnGluIleLeuHisArgAspLeuLysLeu 
195200205 
GlyAsnPhePheIleAsnGluAlaMetGluLeuLysValGlyAspPhe 
210215220 
GlyLeuAlaAlaArgLeuGluProLeuGluHisArgArgArgThrIle 
225230235240 
CysGlyThrProAsnTyrLeuSerProGluValLeuAsnLysGlnGly 
245250255 
HisGlyCysGluSerAspIleTrpAlaLeuGlyCysValMetTyrThr 
260265270 
MetLeuLeuGlyArgProProPheGluThrThrAsnLeuLysGluThr 
275280285 
TyrArgCysIleArgGluAlaArgTyrThrMetProSerSerLeuLeu 
290295300 
AlaProAlaLysHisLeuIleAlaSerMetLeuSerLysAsnProGlu 
305310315320 
AspArgProSerLeuAspAspIleIleArgHisAspPhePheLeuGln 
325330335 
GlyPheThrProAspArgLeuSerSerSerCysCysHisThrValPro 
340345350 
AspPheHisLeuSerSerProAlaLysAsnPhePheLysLysAlaAla 
355360365 
AlaAlaLeuPheGlyGlyLysLysAspLysAlaArgTyrIleAspThr 
370375380 
HisAsnArgValSerLysGluAspGluAspIleTyrLysLeuArgHis 
385390395400 
AspLeuLysLysThrSerIleThrGlnGlnProSerLysHisArgThr 
405410415 
AspGluGluLeuGlnProProThrThrThrValAlaArgSerGlyThr 
420425430 
ProAlaValGluAsnLysGlnGlnIleGlyAspAlaIleArgMetIle 
435440445 
ValArgGlyThrLeuGlySerCysSerSerSerSerGluCysLeuGlu 
450455460 
AspSerThrMetGlySerValAlaAspThrValAlaArgValLeuArg 
465470475480 
GlyCysLeuGluAsnMetProGluAlaAspCysIleProLysGluGln 
485490495 
LeuSerThrSerPheGlnTrpValThrLysTrpValAspTyrSerAsn 
500505510 
LysTyrGlyPheGlyTyrGlnLeuSerAspHisThrValGlyValLeu 
515520525 
PheAsnAsnGlyAlaHisMetSerLeuLeuProAspLysLysThrAla 
530535540 
HisTyrTyrAlaGluLeuGlyGlnCysSerValPheProAlaThrAsp 
545550555560 
AlaProGluGlnPheIleSerGlnValThrValLeuLysTyrPheSer 
565570575 
HisTyrMetGluGluAsnLeuMetAspGlyGlyAspLeuProSerVal 
580585590 
ThrAspIleArgArgProArgLeuTyrLeuLeuGlnTrpLeuLysSer 
595600605 
AspLysAlaLeuMetMetLeuPheAsnAspGlyThrPheGlnValAsn 
610615620 
PheTyrHisAspHisThrLysIleIleIleCysSerGlnAsnGluGlu 
625630635640 
TyrLeuLeuThrTyrIleAsnGluAspArgIleSerThrThrPheArg 
645650655 
LeuThrThrLeuLeuMetSerGlyCysSerSerGluLeuLysAsnArg 
660665670 
MetGluTyrAlaLeuAsnMetLeuLeuGlnArgCysAsn 
675680685 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 448 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: YNOT01 
(B) CLONE: 40194 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetProProLysArgAsnGluLysTyrLysLeuProIleProPhePro 
151015 
GluGlyLysValLeuAspAspMetGluGlyAsnGlnTrpValLeuGly 
202530 
LysLysIleGlySerGlyGlyPheGlyLeuIleTyrLeuAlaPhePro 
354045 
ThrAsnLysProGluLysAspAlaArgHisValValLysValGluTyr 
505560 
GlnGluAsnGlyProLeuPheSerGluLeuLysPheTyrGlnArgVal 
65707580 
AlaLysLysAspCysIleLysLysTrpIleGluArgLysGlnLeuAsp 
859095 
TyrLeuGlyIleProLeuPheTyrGlySerGlyLeuThrGluPheLys 
100105110 
GlyArgSerTyrArgPheMetValMetGluArgLeuGlyIleAspLeu 
115120125 
GlnLysIleSerGlyGlnAsnGlyThrPheLysLysSerThrValLeu 
130135140 
GlnLeuGlyIleArgMetLeuAspValLeuGluTyrIleHisGluAsn 
145150155160 
GluTyrValHisGlyAspValLysAlaAlaAsnLeuLeuLeuGlyTyr 
165170175 
LysAsnProAspGlnValTyrLeuAlaAspTyrGlyLeuSerTyrArg 
180185190 
TyrCysProAsnGlyAsnHisLysGlnTyrGlnGluAsnProArgLys 
195200205 
GlyHisAsnGlyThrIleGluPheThrSerLeuAspAlaHisLysGly 
210215220 
ValGlyGluIleAlaGlnPheLeuValCysAlaHisSerLeuAlaTyr 
225230235240 
AspGluLysProAsnTyrGlnAlaLeuLysLysIleLeuAsnProHis 
245250255 
GlyIleProLeuGlyProLeuAspPheSerThrLysGlyGlnSerIle 
260265270 
AsnValHisThrProAsnSerGlnLysValAspSerGlnLysAlaAla 
275280285 
ThrLysGlnValAsnLysAlaHisAsnArgLeuIleGluLysLysVal 
290295300 
HisSerGluArgSerAlaGluSerCysAlaThrTrpLysValGlnLys 
305310315320 
GluGluLysLeuIleGlyLeuMetAsnAsnGluAlaAlaGlnGluSer 
325330335 
ThrArgArgArgGlnLysTyrGlnGluSerGlnGluProLeuAsnGlu 
340345350 
ValAsnSerPheProGlnLysIleSerTyrThrGlnPheProAsnSer 
355360365 
PheTyrGluProHisGlnAspPheThrSerProAspIlePheLysLys 
370375380 
SerArgSerProSerTrpTyrLysTyrThrSerThrValSerThrGly 
385390395400 
IleThrAspLeuGluSerSerThrGlyLeuTrpProThrIleSerGln 
405410415 
PheThrLeuSerGluGluThrAsnAlaAspValTyrTyrTyrArgIle 
420425430 
IleIleProValLeuLeuMetLeuValPheLeuAlaLeuPhePheLeu 
435440445 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 400 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: TMLR3DT01 
(B) CLONE: 402339 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetLeuAlaArgArgLysProValLeuProAlaLeuThrIleAsnPro 
151015 
ThrIleAlaGluGlyProSerProThrSerGluGlyAlaSerGluAla 
202530 
AsnLeuValAspLeuGlnLysLysLeuGluGluLeuGluLeuAspGlu 
354045 
GlnGlnLysLysArgLeuGluAlaPheLeuThrGlnLysAlaLysVal 
505560 
GlyGluLeuLysAspAspAspPheGluArgIleSerGluLeuGlyAla 
65707580 
GlyAsnGlyGlyValValThrLysValGlnHisArgProSerGlyLeu 
859095 
IleMetAlaArgLysLeuIleHisLeuGluIleLysProAlaIleArg 
100105110 
AsnGlnIleIleArgGluLeuGlnValLeuHisGluCysAsnSerPro 
115120125 
TyrIleValGlyPheTyrGlyAlaPheTyrSerAspGlyGluIleSer 
130135140 
IleCysMetGluHisMetAspGlyGlySerLeuAspHisLeuLeuLys 
145150155160 
GluAlaLysArgIleProGluGluIleLeuGlyLysValSerIleAla 
165170175 
ValLeuArgGlyLeuAlaTyrLeuArgGluLysHisGlnIleMetHis 
180185190 
ArgAspValLysProSerAsnIleLeuValAsnSerArgGlyGluIle 
195200205 
LysLeuCysAspPheGlyValSerGlyGlnLeuIleAspSerMetAla 
210215220 
AsnSerPheValGlyThrArgSerTyrMetAlaProGluArgLeuGln 
225230235240 
GlyThrHisTyrSerValGlnSerAspIleTrpSerMetGlyLeuSer 
245250255 
LeuValGluLeuAlaValGlyArgTyrProIleProProProAspAla 
260265270 
LysGluLeuGluAlaIlePheGlyArgProValValAspGlyGluGlu 
275280285 
GlyGluProHisSerIleSerProArgProArgProProGlyArgPro 
290295300 
ValSerGlyHisGlyMetAspSerArgProAlaMetAlaIlePheGlu 
305310315320 
LeuLeuAspTyrIleValAsnGluProProProLysLeuProAsnGly 
325330335 
ValPheThrProAspPheGlnGluPheValAsnLysCysLeuIleLys 
340345350 
AsnProAlaGluArgAlaAspLeuLysMetLeuThrAsnHisThrPhe 
355360365 
IleLysArgSerGluValGluGluValAspPheAlaGlyTrpLeuCys 
370375380 
LysThrLeuArgLeuAsnGlnProGlyThrProThrArgThrAlaVal 
385390395400 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 464 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: SYNORAT04 
(B) CLONE: 705365 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetAlaMetThrAlaGlyThrThrThrThrPheProMetSerAsnHis 
151015 
ThrArgGluArgValThrValAlaLysLeuThrLeuGluAsnPheTyr 
202530 
SerAsnLeuIleLeuGlnHisGluGluArgGluThrArgGlnLysLys 
354045 
LeuGluValAlaMetGluGluGluGlyLeuAlaAspGluGluLysLys 
505560 
LeuArgArgSerGlnHisAlaArgLysGluThrGluPheLeuArgLeu 
65707580 
LysArgThrArgLeuGlyLeuAspAspPheGluSerLeuLysValIle 
859095 
GlyArgGlyAlaPheGlyGluValArgLeuValHisLysLysAspThr 
100105110 
GlyHisIleTyrAlaMetLysIleLeuArgLysSerAspMetLeuGlu 
115120125 
LysGluGlnValAlaHisIleArgAlaGluArgAspIleLeuValGlu 
130135140 
AlaAspGlyAlaTrpValValLysMetPheTyrSerPheGlnAspLys 
145150155160 
ArgAsnLeuTyrLeuIleMetGluPheLeuProGlyGlyAspMetMet 
165170175 
ThrLeuLeuMetLysLysAspThrLeuThrGluGluGluThrGlnPhe 
180185190 
TyrIleSerGluThrValLeuAlaIleAspAlaIleHisGlnLeuGly 
195200205 
PheIleHisArgAspIleLysProAspAsnLeuLeuLeuAspAlaLys 
210215220 
GlyHisValLysLeuSerAspPheGlySerCysThrGlyLeuLysLys 
225230235240 
AlaHisArgThrGluPheTyrArgAsnLeuThrHisAsnProProSer 
245250255 
AspPheSerPheGlnAsnMetAsnSerLysArgLysAlaGluThrTrp 
260265270 
LysLysAsnArgArgGlnLeuAlaTyrSerThrValGlyThrProAsp 
275280285 
TyrIleAlaProGluValPheMetGlnThrGlyTyrAsnLysLeuCys 
290295300 
AspTrpTrpSerLeuGlyValIleMetTyrGluMetLeuIleGlyTyr 
305310315320 
ProProPheCysSerGluThrProGlnGluThrTyrArgLysValMet 
325330335 
AsnTrpLysGluThrLeuValPheProProGluValProIleSerGlu 
340345350 
LysAlaLysAspLeuIleLeuArgPheCysIleAspSerGluAsnArg 
355360365 
IleGlyAsnSerGlyValGluGluIleLysGlyHisProPhePheGlu 
370375380 
GlyValAspTrpGluHisIleArgGluArgProAlaAlaIleProIle 
385390395400 
GluIleLysSerIleAspAspThrSerAsnPheAspAspPheProGlu 
405410415 
SerAspIleLeuGlnProValProAsnThrThrGluProAspTyrLys 
420425430 
SerLysAspTrpValPheLeuAsnTyrThrTyrLysArgPheGluGly 
435440445 
LeuThrGlnArgGlySerIleProThrTyrMetLysAlaGlyLysLeu 
450455460 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 343 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PROSNOT06 
(B) CLONE: 827431 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
MetLeuLeuLeuLysLysHisThrGluAspIleSerSerValTyrGlu 
151015 
IleArgGluArgLeuGlySerGlyAlaPheSerGluValValLeuAla 
202530 
GlnGluArgGlySerAlaHisLeuValAlaLeuLysCysIleProLys 
354045 
LysAlaLeuArgGlyLysGluAlaLeuValGluAsnGluIleAlaVal 
505560 
LeuArgArgIleSerHisProAsnIleValAlaLeuGluAspValHis 
65707580 
GluSerProSerHisLeuTyrLeuAlaMetGluLeuValThrGlyGly 
859095 
GluLeuPheAspArgIleMetGluArgGlySerTyrThrGluLysAsp 
100105110 
AlaSerHisLeuValGlyGlnValLeuGlyAlaValSerTyrLeuHis 
115120125 
SerLeuGlyIleValHisArgAspLeuLysProGluAsnLeuLeuTyr 
130135140 
AlaThrProPheGluAspSerLysIleMetValSerAspPheGlyLeu 
145150155160 
SerLysIleGlnAlaGlyAsnMetLeuGlyThrAlaCysGlyThrPro 
165170175 
GlyTyrValAlaProGluLeuLeuGluGlnLysProTyrGlyLysAla 
180185190 
ValAspValTrpAlaLeuGlyValIleSerTyrIleLeuLeuCysGly 
195200205 
TyrProProPheTyrAspGluSerAspProGluLeuPheSerGlnIle 
210215220 
LeuArgAlaSerTyrGluPheAspXaaProPheTrpAspAspIleSer 
225230235240 
GluSerGlyLysAspPheIleArgHisLeuLeuGluArgAspLeuGln 
245250255 
LysArgPheThrCysGlnGlnAlaLeuArgAspLeuTrpIlePheTrp 
260265270 
AspThrGlyPheGlyArgAspIleLeuGlyPheValSerGluGlnIle 
275280285 
ArgLysAsnPheAlaTrpThrHisTrpLysArgAlaPheAsnAlaThr 
290295300 
LeuPheLeuArgHisIleArgLysLeuGlyGlnIleProGluGlyGlu 
305310315320 
GlyAlaSerGluGlnGlyMetXaaArgHisSerHisXaaGlyLeuArg 
325330335 
AlaGlyGlnProProLysTrp 
340 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 412 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: COLNTUT03 
(B) CLONE: 1340712 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetIleLeuAlaSerValLeuArgSerGlyProGlyGlyGlyLeuPro 
151015 
LeuArgProLeuLeuGlyProAlaLeuAlaLeuArgAlaArgSerThr 
202530 
SerAlaThrAspThrHisHisValGluMetAlaArgGluArgSerLys 
354045 
ThrValThrSerPheTyrAsnGlnSerAlaIleAspAlaAlaAlaGlu 
505560 
LysProSerValArgLeuThrProThrMetMetLeuTyrAlaGlyArg 
65707580 
SerGlnAspGlySerHisLeuLeuLysSerAlaArgTyrLeuGlnGln 
859095 
GluLeuProValArgIleAlaHisArgIleLysGlyPheArgCysLeu 
100105110 
ProPheIleIleGlyCysAsnProThrIleLeuHisValHisGluLeu 
115120125 
TyrIleArgAlaPheGlnLysLeuThrAspPheProProIleLysAsp 
130135140 
GlnAlaAspGluAlaGlnTyrCysGlnLeuValArgGlnLeuLeuAsp 
145150155160 
AspHisLysAspValValThrLeuLeuAlaGluGlyLeuArgGluSer 
165170175 
ArgLysHisIleGluAspGluLysLeuValArgTyrPheLeuAspLys 
180185190 
ThrLeuThrSerArgLeuGlyIleArgMetLeuAlaThrHisHisLeu 
195200205 
AlaLeuHisGluAspLysProAspPheValGlyIleIleCysThrArg 
210215220 
LeuSerProLysLysIleIleGluLysTrpValAspPheAlaArgArg 
225230235240 
LeuCysGluHisLysTyrGlyAsnAlaProArgValArgIleAsnGly 
245250255 
HisValAlaAlaArgPheProPheIleProMetProLeuAspTyrIle 
260265270 
LeuProGluLeuLeuLysAsnAlaMetArgAlaThrMetGluSerHis 
275280285 
LeuAspThrProTyrAsnValProAspValValIleThrIleAlaAsn 
290295300 
AsnAspValAspLeuIleIleArgIleSerAspArgGlyGlyGlyIle 
305310315320 
AlaHisLysAspLeuAspArgValMetAspTyrHisPheThrThrAla 
325330335 
GluAlaSerThrGlnAspProArgIleSerProLeuPheGlyHisLeu 
340345350 
AspMetHisSerGlyAlaGlnSerGlyProMetHisGlyPheGlyPhe 
355360365 
GlyLeuProThrSerArgAlaTyrAlaGluTyrLeuGlyGlySerLeu 
370375380 
GlnLeuGlnSerLeuGlnGlyIleGlyThrAspValTyrLeuArgLeu 
385390395400 
ArgHisIleAspGlyArgGluGluSerPheArgIle 
405410 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 328 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PENITUT01 
(B) CLONE: 1452972 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
MetLeuGluLysLeuGluPheGluAspGluAlaValGluAspSerGlu 
151015 
SerGlyValTyrMetArgPheMetArgSerHisLysCysTyrAspIle 
202530 
ValProThrSerSerLysLeuValValPheAspThrThrLeuGlnVal 
354045 
LysLysAlaPhePheAlaLeuValAlaAsnGlyValArgAlaAlaPro 
505560 
LeuTrpGluSerLysLysGlnSerPheValGlyMetLeuThrIleThr 
65707580 
AspPheIleAsnIleLeuHisArgTyrTyrLysSerProMetValGln 
859095 
IleTyrGluLeuGluGluHisLysIleGluThrTrpArgGluLeuTyr 
100105110 
LeuGlnGluThrPheLysProLeuValAsnIleSerProAspAlaSer 
115120125 
LeuPheAspAlaValTyrSerLeuIleLysAsnLysIleHisArgLeu 
130135140 
ProValIleAspProIleSerGlyAsnAlaLeuTyrIleLeuThrHis 
145150155160 
LysArgIleLeuLysPheLeuGlnLeuPheMetSerAspMetProLys 
165170175 
ProAlaPheMetLysGlnAsnLeuAspGluLeuGlyIleGlyThrTyr 
180185190 
HisAsnIleAlaPheIleHisProAspThrProIleIleLysAlaLeu 
195200205 
AsnIlePheValGluArgArgIleSerAlaLeuProValValAspGlu 
210215220 
SerGlyLysValValAspIleTyrSerLysPheAspValIleAsnLeu 
225230235240 
AlaAlaGluLysThrTyrAsnAsnLeuAspIleThrValThrGlnAla 
245250255 
LeuGlnHisArgSerGlnTyrPheGluGlyValValLysCysAsnLys 
260265270 
LeuGluIleLeuGluThrIleValAspArgIleValArgAlaGluVal 
275280285 
HisArgLeuValValValAsnGluAlaAspSerIleValGlyIleIle 
290295300 
SerLeuSerAspIleLeuGlnAlaLeuIleLeuThrProAlaGlyAla 
305310315320 
LysGlnLysGluThrGluThrGlu 
325 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2770 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: HUVENOB01 
(B) CLONE: 39043 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TAGTCGGCACCAGAGGCAAGGGTGCGAGGACCACGGCCGGCTCGGACGTGTGACCGCGCC60 
TAGGGGGTGGCAGCGGGCAGTGCGGGGCGGCAAGGCGACCATGGAGCTTTTGCGGACTAT120 
CACCTACCAGCCAGCCGCCAGCACCAAAATGTGCGAGCAGGCGCTGGGCAAGGGTTGCGG180 
AGCGGACTCGAAGAAGAAGCGGCCGCCGCAGCCCCCCGAGGAATCGCAGCCACCTCAGTC240 
CCAGGCGCAAGTGCCCCCGGCGGCCCCTCACCACCATCACCACCATTCGCACTCGGGGCC300 
GGAGATCTCGCGGATTATCGTCGACCCCACGACTGGGAAGCGCTACTGCCGGGGCAAAGT360 
GCTGGGAAAGGGTGGCTTTGCAAAATGTTACGAGATGACAGATTTGACAAATAACAAAGT420 
CTACGCCGCAAAAATTATTCCTCACAGCAGAGTAGCTAAACCTCATCAAAGGGAAAAGAT480 
TGACAAAGAAATAGAGCTTCACAGAATTCTTCATCATAAGCATGTAGTGCAGTTTTACCA540 
CTACTTCGAGGACAAAGAAAACATTTACATTCTCTTGGAATACTGCAGTAGAAGGTCAAT600 
GGCTCATATTTTGAAAGCAAGAAAGGTGTTGACAGAGCCAGAAGTTCGATACTACCTCAG660 
GCAGATTGTGTCTGGACTGAAATACCTTCATGAACAAGAAATCTTGCACAGAGATCTCAA720 
ACTAGGGAACTTTTTTATTAATGAAGCCATGGAACTAAAAGTTGGGGACTTCGGTCTGGC780 
AGCCAGGCTAGAACCCTTGGAACACAGAAGGAGAACGATATGTGGTACCCCAAATTATCT840 
CTCTCCTGAAGTCCTCAACAAACAAGGACATGGCTGTGAATCAGACATTTGGGCCCTGGG900 
CTGTGTAATGTATACAATGTTACTAGGGAGGCCCCCATTTGAAACTACAAATCTCAAAGA960 
AACTTATAGGTGCATAAGGGAAGCAAGGTATACAATGCCGTCCTCATTGCTGGCTCCTGC1020 
CAAGCACTTAATTGCTAGTATGTTGTCCAAAAACCCAGAGGATCGTCCCAGTTTGGATGA1080 
CATCATTCGACATGACTTTTTTTTGCAGGGCTTCACTCCGGACAGACTGTCTTCTAGCTG1140 
TTGTCATACAGTTCCAGATTTCCACTTATCAAGCCCAGCTAAGAATTTCTTTAAGAAAGC1200 
AGCTGCTGCTCTTTTTGGTGGCAAAAAAGACAAAGCAAGATATATTGACACACATAATAG1260 
AGTGTCTAAAGAAGATGAAGACATCTACAAGCTTAGGCATGATTTGAAAAAGACTTCAAT1320 
AACTCAGCAACCCAGCAAACACAGGACAGATGAGGAGCTCCAGCCACCTACCACCACAGT1380 
TGCCAGGTCTGGAACACCCGCAGTAGAAAACAAGCAGCAGATTGGGGATGCTATTCGGAT1440 
GATAGTCAGAGGGACTCTTGGCAGCTGTAGCAGCAGCAGTGAATGCCTTGAAGACAGTAC1500 
CATGGGAAGTGTTGCAGACACAGTGGCAAGGGTTCTTCGGGGATGTCTGGAAAACATGCC1560 
GGAAGCTGATTGCATTCCCAAAGAGCAGCTGAGCACATCATTTCAGTGGGTCACCAAATG1620 
GGTTGATTACTCTAACAAATATGGCTTTGGGTACCAGCTCTCAGACCACACCGTCGGTGT1680 
CCTTTTCAACAATGGTGCTCACATGAGCCTCCTTCCAGACAAAAAAACAGCTCACTATTA1740 
CGCAGAGCTTGGCCAATGCTCAGTTTTCCCAGCAACAGATGCTCCTGAGCAATTTATTAG1800 
TCAAGTGACGGTGCTGAAATACTTTTCTCATTACATGGAGGAGAACCTCATGGATGGTGG1860 
AGATCTGCCTAGTGTTACTGATATTCGAAGACCTCGGCTCTACCTCCTTCAGTGGCTAAA1920 
ATCTGATAAGGCCCTAATGATGCTCTTTAATGATGGCACCTTTCAGGTGAATTTCTACCA1980 
TGATCATACAAAAATCATCATCTGTAGCCAAAATGAAGAATACCTTCTCACCTACATCAA2040 
TGAGGATAGGATATCTACAACTTTCAGGCTGACAACTCTGCTGATGTCTGGCTGTTCATC2100 
AGAATTAAAAAATCGAATGGAATATGCCCTGAACATGCTCTTACAAAGATGTAACTGAAA2160 
GACTTTTCGAATGGACCCTATGGGACTCCTCTTTTCCACTGTGAGATCTACAGGGAAGCC2220 
AAAAGAATGATCTAGAGTATGTTGAAGAAGATGGACATGTGGTGGTACGAAAACAATTCC2280 
CCTGTGGCCTGCTGGACTGGGTGGAACCAGAACAGGCTAAGGCATACAGTTCTTGACTTT2340 
GGACAATCCAAGAGTGAACCAGAATGCAGTTTTCCTTGAGATACCTGTTTTAAAAGGTTT2400 
TTCAGACAATTTTGCAGAAAGGTGCATTGATTCTTAAATTCTCTCTGTTGAGAGCATTTC2460 
AGCCAGAGGACTTTGGAACTGTGAATATACTTCCTGAAGGGGAGGGAGAAGGGAGGAAGC2520 
TCCCATGTTGTTTAAAGGCTGTAATTGGAGCAGCTTTTGGCTGCGTAACTGTGAACTATG2580 
GCCATATATAATTTTTTTTCATTAATTTTTGAAGATACTTGTGGCTGGAAAAGTGCATTC2640 
CTTGTTAATAAACTTTTTATTTATTACAGCCCAAAGAGCAGTATTTATTATCAAAATGTC2700 
TTTTTTTTTATGTTGACCATTTTAAACCGTTGGCAATAAAGAGTATGAAAACGCAGAAAA2760 
AAAAAAAAAA2770 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1593 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: YNOT01 
(B) CLONE: 40194 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CTAGGCGTCCCCTTCTACTCACGTTTGCCAAAAGCGGGTCCGACGTGTTAGCGGAAAAAA60 
GTGATGCCACCAAAAAGAAATGAAAAATACAAACTTCCTATTCCATTTCCAGAAGGCAAG120 
GTTCTGGATGATATGGAAGGCAATCAGTGGGTACTGGGCAAGAAGATTGGCTCTGGAGGA180 
TTTGGATTGATATATTTAGCTTTCCCCACAAATAAACCAGAGAAAGATGCAAGACATGTA240 
GTAAAAGTGGAATATCAAGAAAATGGCCCGTTATTTTCAGAACTTAAATTTTATCAGAGA300 
GTTGCAAAAAAAGACTGTATCAAAAAGTGGATAGAACGCAAACAACTTGATTATTTAGGA360 
ATTCCTCTGTTTTATGGATCTGGTCTGACTGAATTCAAGGGAAGAAGTTACAGATTTATG420 
GTAATGGAAAGACTAGGAATAGATTTACAGAAGATCTCAGGCCAGAATGGTACCTTTAAA480 
AAGTCAACTGTCCTGCAATTAGGTATCCGAATGTTGGATGTACTGGAATATATACATGAA540 
AATGAATATGTTCATGGTGATGTAAAAGCAGCAAATCTACTTTTGGGTTACAAAAATCCA600 
GACCAGGTTTATCTTGCAGATTATGGACTTTCCTACAGATATTGTCCCAATGGGAACCAC660 
AAACAGTATCAGGAAAATCCTAGAAAAGGCCATAATGGGACAATAGAGTTTACCAGCTTG720 
GATGCCCACAAGGGAGTAGGTGAAATAGCCCAATTTTTGGTATGTGCTCATAGTTTAGCA780 
TATGATGAAAAGCCAAACTATCAAGCCCTCAAGAAAATTTTGAACCCTCATGGAATACCT840 
TTAGGACCACTGGACTTTTCCACAAAAGGACAGAGTATAAATGTCCATACTCCAAACAGT900 
CAAAAAGTTGATTCACAAAAGGCTGCAACAAAGCAAGTCAACAAGGCACACAATAGGTTA960 
ATCGAAAAAAAAGTCCACAGTGAGAGAAGCGCTGAGTCCTGTGCAACATGGAAAGTGCAG1020 
AAAGAGGAGAAACTGATTGGATTGATGAACAATGAAGCAGCTCAGGAAAGCACAAGGAGA1080 
AGACAGAAATATCAAGAGTCTCAAGAACCTTTGAATGAAGTAAACAGTTTCCCACAAAAA1140 
ATCAGCTATACACAATTCCCAAACTCATTTTATGAGCCTCATCAAGATTTTACCAGTCCA1200 
GATATATTCAAGAAGTCAAGATCTCCATCTTGGTATAAATACACTTCCACAGTCAGCACG1260 
GGGATCACAGACTTAGAAAGTTCAACTGGACTTTGGCCTACAATTTCCCAGTTTACTCTT1320 
AGTGAAGAGACAAACGCAGATGTTTATTATTATCGCATCATCATACCTGTCCTTTTGATG1380 
TTAGTATTTCTTGCTTTATTTTTTCTCTGAAGATGATACCAAAATTCCTTTTGATAATTT1440 
TTTAAGTTTCCAGCTCTTCACCGAAATGTTGTATTCTTATTTCAGTGTTTCCTTCCAGAC1500 
ATTTTTAAGGTAATTGGCTTTAAAAAGAGAACATATTTTAACAAAGTTTGTGGACACTCT1560 
AAAAAATAAAATTGCTTTGTACTAGAAAAAAAA1593 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1504 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: TMLR3DT01 
(B) CLONE: 402339 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CCGGCCCGCGGAGCCCCGATGCTGGCCCGGAGGAAGCCGGTGCTGCCGGCGCTCACCATC60 
AACCCTACCATCGCCGAGGGCCCATCCCCTACCAGCGAGGGCGCCTCCGAGGCAAACCTG120 
GTGGACCTGCAGAAGAAGCTGGAGGAGCTGGAACTTGACGAGCAGCAGAAGAAGCGGCTG180 
GAAGCCTTTCTCACCCAGAAAGCCAAGGTCGGCGAACTCAAAGACGATGACTTCGAAAGG240 
ATCTCAGAGCTGGGCGCGGGCAACGGCGGGGTGGTCACCAAAGTCCAGCACAGACCCTCG300 
GGCCTCATCATGGCCAGGAAGCTGATCCACCTTGAGATCAAGCCGGCCATCCGGAACCAG360 
ATCATCCGCGAGCTGCAGGTCCTGCACGAATGCAACTCGCCGTACATCGTGGGCTTCTAC420 
GGGGCCTTCTACAGTGACGGGGAGATCAGCATTTGCATGGAACACATGGACGGCGGCTCC480 
CTGGACCATCTGCTGAAAGAGGCCAAGAGGATTCCCGAGGAGATCCTGGGGAAAGTCAGC540 
ATCGCGGTTCTCCGGGGCTTGGCGTACCTCCGAGAGAAGCACCAGATCATGCACCGAGAT600 
GTGAAGCCCTCCAACATCCTCGTGAACTCTAGAGGGGAGATCAAGCTGTGTGACTTCGGG660 
GTGAGCGGCCAGCTCATCGACTCCATGGCCAACTCCTTCGTGGGCACGCGCTCCTACATG720 
GCTCCGGAGCGGTTGCAGGGCACACATTACTCGGTGCAGTCGGACATCTGGAGCATGGGC780 
CTGTCCCTGGTGGAGCTGGCCGTCGGAAGGTACCCCATCCCCCCGCCCGACGCCAAAGAG840 
CTGGAGGCCATCTTTGGCCGGCCCGTGGTCGACGGGGAAGAAGGAGAGCCTCACAGCATC900 
TCGCCTCGGCCGAGGCCCCCCGGGCGCCCCGTCAGCGGTCACGGGATGGATAGCCGGCCT960 
GCCATGGCCATCTTTGAACTCCTGGACTATATTGTGAACGAGCCACCTCCTAAGCTGCCC1020 
AACGGTGTGTTCACCCCCGACTTCCAGGAGTTTGTCAATAAATGCCTCATCAAGAACCCA1080 
GCGGAGCGGGCGGACCTGAAGATGCTCACAAACCACACCTTCATCAAGCGGTCCGAGGTG1140 
GAAGAAGTGGATTTTGCCGGCTGGTTGTGTAAAACCCTGCGGCTGAACCAGCCCGGCACA1200 
CCCACGCGCACCGCCGTGTGACAGTGGCCGGGCTCCCTGCGTCCCGCTGGTGACCTGCCC1260 
ACCGTCCCTGTCCATGCCCCGCCCTTCCAGCTGAGGACAGGCTGGCGCCTCCACCCACCC1320 
TCCTGCCTCACCCCTGCGGAGAGCACCGTGGCGGGGCGACAGCGCATGCAGGAACGGGGG1380 
TCTCCTCTCCTGCCCGTCCTGGCCGGGGTGCCTCTGGGGACGGGCGACGCTGCTGTGTGT1440 
GGTCTCAGAGGCTCTGCTTCCTTAGGTTACAAAACAAAACAGGGAGAGAAAAAGCAAAAA1500 
AAAA1504 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1935 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: SYNORAT04 
(B) CLONE: 705365 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GCGGAGGCTGAGCCGGCCGCGGGCGCGACCGGAGGCAGTTTCCGTTACTATGGCAATGAC60 
GGCAGGGACTACAACAACCTTTCCTATGAGCAACCATACCCGGGAAAGAGTGACTGTAGC120 
CAAGCTCACATTGGAGAATTTTTATAGCAACCTAATTTTACAGCATGAAGAGAGAGAAAC180 
CAGGCAGAAGAAATTAGAAGTGGCCATGGAAGAAGAAGGATTAGCAGATGAAGAGAAAAA240 
GTTACGTCGATCACAACACGCTCGCAAAGAAACAGAGTTCTTACGGCTCAAAAGGACCAG300 
ACTTGGCTTGGATGACTTTGAGTCTCTGAAAGTTATAGGAAGAGGAGCTTTTGGAGAGGT360 
GCGGTTGGTCCACAAAAAAGATACAGGCCATATCTATGCAATGAAGATATTGAGAAAGTC420 
TGATATGCTTGAAAAAGAGCAGGTGGCCCATATCCGAGCAGAAAGAGATATTTTGGTAGA480 
AGCAGATGGTGCCTGGGTGGTGAAGATGTTTTACAGTTTTCAGGATAAGAGGAATCTTTA540 
TCTAATCATGGAATTTCTCCCTGGAGGTGACATGATGACATTGCTAATGAAGAAAGACAC600 
CTTGACAGAAGAGGAAACACAGTTCTACATTTCAGAGACTGTTCTGGCAATAGATGCGAT660 
CCACCAGTTGGGTTTCATCCATCGGGATATTAAGCCAGACAACCTTTTATTGGATGCCAA720 
GGGTCATGTAAAATTATCTGATTTTGGTTCATGTACGGGATTAAAGAAAGCTCACAGGAC780 
TGAATTTTATAGAAATCTCACACACAACCCACCAAGTGACTTCTCATTTCAGAACATGAA840 
CTCAAAGAGGAAAGCAGAAACTTGGAAGAAGAACAGGAGACAACTGGCATATTCCACAGT900 
TGGGACACCAGATTACATTGCTCCAGAAGTATTCATGCAGACTGGTTACAACAAATTGTG960 
TGACTGGTGGTCTTTGGGAGTGATTATGTATGAAATGCTAATAGGATATCCACCTTTCTG1020 
CTCTGAAACACCTCAAGAAACATACAGAAAAGTGATGAACTGGAAAGAAACTCTGGTATT1080 
TCCTCCAGAGGTACCTATATCTGAGAAAGCCAAGGACTTAATTCTCAGATTTTGTATTGA1140 
TTCTGAAAACAGAATTGGAAATAGTGGAGTAGAAGAAATAAAAGGTCATCCCTTTTTTGA1200 
AGGTGTCGACTGGGAGCACATAAGGGAAAGGCCAGCAGCAATCCCTATAGAAATCAAAAG1260 
CATTGATGATACTTCAAATTTTGATGACTTCCCTGAATCTGATATTTTACAACCAGTGCC1320 
AAATACCACAGAACCGGACTACAAATCCAAAGACTGGGTTTTTCTCAATTATACCTATAA1380 
AAGGTTTGAAGGGTTGACTCAACGTGGCTCTATCCCCACCTACATGAAAGCTGGGAAGTT1440 
ATGAATGAAGATAACATTCACCCATAACCAAGAGAACTCAGGTAGCTGCATCACCAGGCT1500 
TGCTTGGCGTAGATAACAATACACTGAAATACTCCTGAAGATGGTGGTGCTTATTGACTA1560 
CAAGAGGAAATTCTACAGGATTAGGATTTCTAAGACTACTATAGGAATTGGTTGGCAGTG1620 
CCAGCTGGCTCTTTTTTTTAATATTTTATTATTTTTGTTAACTTTATTATATGAAGGTAC1680 
TGGAATAAAAGGAACAGACATCCCTTTCTAACTGCACTGCCTACATGCGTATTAAGGTCC1740 
ATTCTGCCTGTGTGTGCTGTGGCTTTGAACTGTAACACCTCTAATCAATTCAGGAGAAAC1800 
ACATATCATTTAAAGCAACATAGGCTAACCTGTANGTAACACTGCAGTATTGATGTTTTA1860 
CTGCAAATCTTATGGGTCTAGATAATCAGTAAAAGCCATCTTCCATAGTTGGTGTTAGAA1920 
CATTGCCCTATTGGT1935 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1282 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PROSNOT06 
(B) CLONE: 827431 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GAAGTTTCTCACTAGGGTCTTCTCTGGCCCAGCCTTTGACTGAAGCTGGTCTGGAGACAG60 
GGGCATTAGAGAAGTGACTCATAGATGGCCTAAAGAAGCGGGGCCACTCAAGGACCCAGG120 
ACAGAGGGAAGAGGGCCAACCCAGCTGGACCACAGGCAAACCCCATTGCCTTTGAGAGAA180 
AGAAGAGGACCCGGTGAAACATGCTGCTGCTGAAGAAACACACGGAGGACATCAGCAGCG240 
TCTACGAGATCCGCGAGAGGCTCGGCTCGGGTGCCTTCTCCGAGGTGGTGCTGGCCCAGG300 
AGCGGGGCTCCGCACACCTCGTGGCCCTCAAGTGCATCCCCAAGAAGGCCCTCCGGGGCA360 
AGGAGGCCCTGGTGGAGAACGAGATCGCAGTGCTCCGTAGGATCAGTCACCCCAACATCG420 
TCGCTCTGGAGGATGTCCACGAGAGCCCTTCCCACCTCTACCTGGCCATGGAACTGGTGA480 
CGGGTGGCGAGCTGTTTGACCGCATCATGGAGCGCGGCTCCTACACAGAGAAGGATGCCA540 
GCCATCTGGTGGGTCAGGTCCTTGGCGCCGTCTCCTACCTGCACAGCCTGGGGATCGTGC600 
ACCGGGACCTCAAGCCCGAAAACCTCCTGTATGCCACGCCCTTTGAGGACTCGAAGATCA660 
TGGTCTCTGACTTTGGACTCTCCAAAATCCAGGCTGGGAACATGCTAGGCACCGCCTGTG720 
GGACCCCTGGATATGTGGCCCCAGAGCTCTTGGAGCAGAAACCCTACGGGAAGGCCGTAG780 
ATGTGTGGGCCCTGGGCGTCATCTCCTACATCCTGCTGTGTGGGTACCCCCCCTTCTACG840 
ACGAGAGCGACCCTGAGCTCTTCAGCCAGATCCTGAGGGCCAGCTATGAGTTTGACTNTC900 
CTTTCTGGGATGACATCTCAGAATCAGGCAAAGACTTTATTCGGCACCTTCTGGAGCGAG960 
ACCTTCAGAAGAGGTTCACCTGCCAACAGGCCTTGCGGGACCTTTGGATCTTTTGGGACA1020 
CAGGCTTTGGCAGGGACATCTTAGGGTTTGTCAGTGAGCAGATCCGGAAGAACTTTGCTT1080 
GGACACACTGGAAGCGAGCCTTCAATGCCACCTTGTTCCTGCGCCACATCCGGAAGCTGG1140 
GGCAGATCCCAGAGGGCGAGGGGGCCTCTGAGCAGGGCATGGSCCGNCACAGCCACTNAG1200 
GCCTTCGTGCTGGCCAGCCCCCCAAGTGGTGATGCCCAGGNAGATGCCGAGGCCAAGTGG1260 
ANTGANCCCCAGATTTNCTTNC1282 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1866 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: COLNTUT03 
(B) CLONE: 1340712 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CGGCGGAGGGCGCAGGCGGCTGGGCGCCTGGCGAGTGGACTGTTCGAGCCCTTCCGCTGG60 
GACCCGGGCCCTGGCTCCGGCCCCGCGATGGGAGCTGCTCTCCGCGGGCTGAGCCTGTCA120 
GCATCCTCGACGCACCCTGGTCCCTGAAGTCGGAGAAGAGCCCCTACCCACCCACACCCC180 
CTTGCCCCATTTTGGGTCGCCTGGGTCCTCAGTCCTAGCGGATCCTCAGTCCTAGCGGCC240 
ACCGGGTCTGAAAGGAGCAAGACGATGATCCTGGCGTCGGTGCTGAGGAGCGGTCCCGGG300 
GGCGGGCTTCCGCTCCGGCCCCTCCTGGGACCCGCACTCGCGCTCCGGGCCCGCTCGACG360 
TCGGCCACCGACACACACCACGTGGAGATGGCTCGGGAGCGCTCCAAGACCGTCACCTCC420 
TTTTACAACCAGTCGGCCATCGACGCGGCAGCGGAGAAGCCCTCAGTCCGCCTAACGCCC480 
ACCATGATGCTCTACGCTGGCCGCTCTCAGGACGGCAGCCACCTTCTGAAAAGTGCTCGG540 
TACCTGCAGCAAGAACTTCCAGTGAGGATTGCTCACCGCATCAAGGGCTTCCGCTGCCTT600 
CCTTTCATCATTGGCTGCAACCCCACCATACTGCACGTGCATGAGCTATATATCCGTGCC660 
TTCCAGAAGCTGACAGACTTCCCTCCGATCAAGGACCAGGCGGACGAGGCCCAGTACTGC720 
CAGCTGGTGCGACAGCTGCTGGATGACCACAAGGATGTGGTGACCCTCTTGGCAGAGGGC780 
CTACGTGAGAGCCGGAAGCACATAGAGGATGAAAAGCTCGTCCGCTACTTCTTGGACAAG840 
ACGCTGACTTCGAGGCTTGGAATCCGCATGTTGGCCACGCATCACCTGGCGCTGCATGAG900 
GACAAGCCTGACTTTGTCGGCATCATCTGTACTCGTCTCTCACCAAAGAAGATTATTGAG960 
AAGTGGGTGGACTTTGCCAGACGCCTGTGTGAGCACAAGTATGGCAATGCGCCCCGTGTC1020 
CGCATCAATGGCCATGTGGCTGCCCGGTTCCCCTTCATCCCTATGCCACTGGACTACATC1080 
CTGCCGGAGCTGCTCAAGAATGCCATGAGAGCCACAATGGAGAGCCACCTAGACACTCCC1140 
TACAATGTCCCAGATGTGGTCATCACCATCGCCAACAATGATGTCGATCTGATCATCAGG1200 
ATCTCAGACCGTGGTGGAGGAATCGCTCACAAAGATCTGGACCGGGTCATGGACTACCAC1260 
TTCACTACTGCTGAGGCCAGCACACAGGACCCCCGGATCAGCCCCCTCTTTGGCCATCTG1320 
GACATGCATAGTGGCGCCCAGTCAGGACCCATGCACGGCTTTGGCTTCGGGTTGCCCACG1380 
TCACGGGCCTACGCGGAGTACCTCGGTGGGTCTCTGCAGCTGCAGTCCCTGCAGGGCATT1440 
GGCACGGACGTCTACCTGCGGCTCCGCCACATCGATGGCCGGGAGGAAAGCTTCCGGATC1500 
TGACCCCACAGCCTTTGGCCTGCTCACCCGACCAGCCTGGGCCGCATTCCCTGCAGGACC1560 
TCCCGGGTCAGGCAGGGCGGCCCCCTGCTCCACACACTGCTGCATCTTGGGTCTCAGGGA1620 
CCCAGACAGATGGACTTACATGGAGCTGGGCACTGCCCCTGCCTCAACAGGGTCCATTGC1680 
TCTCTCGCCTCAGAACTTGGAGCAGGGAAGTGGGCACCTGAGGCCTCAGCACAGTGTCGT1740 
CATTCTCTTCTGGGGGACCCCACTCTGAGCTGTTATTAAAGTTCACATTTTGGAATGGCC1800 
AGAAAAGAAGGAAGGTGGATGGTGGTGAGGAGGGGTGGGGAGAGGTGAGGTGGTTGTGGT1860 
TTGTGT1866 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1435 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PENITUT01 
(B) CLONE: 1452972 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GGCCCCAGCGCTCGGCCGGCCGCGAGCCCGCCGGCCGGGGACGAGCGTCGCAGCTCATGC60 
TGATCGCTGTCCTCCTCCTCCCCCTCAGGCGGCGCTGGCGGCGGCCCTGGGACCCGCGGA120 
AGCCGGCATGCTGGAGAAGCTGGAGTTCGAGGACGAAGCAGTAGAAGACTCAGAAAGTGG180 
TGTTTACATGCGATTCATGAGGTCACACAAGTGTTATGACATCGTTCCAACCAGTTCAAA240 
GCTTGTTGTCTTTGATACTACATTACAAGTTAAAAAGGCCTTCTTTGCTTTGGTAGCCAA300 
CGGTGTCCGAGCAGCGCCACTGTGGGAGAGTAAAAAACAAAGTTTTGTAGGAATGCTAAC360 
AATTACAGATTTCATAAATATACTACATAGATACTATAAATCACCTATGGTACAGATTTA420 
TGAATTAGAGGAACATAAAATTGAAACATGGAGGGAGCTTTATTTACAAGAAACATTTAA480 
GCCTTTAGTGAATATATCTCCAGATGCAAGCCTCTTCGATGCTGTATACTCCTTGATCAA540 
AAATAAAATCCACAGATTGCCCGTTATTGACCCTATCAGTGGGAATGCACTTTATATACT600 
TACCCACAAAAGAATCCTCAAGTTCCTCCAGCTTTTTATGTCTGATATGCCAAAGCCTGC660 
CTTCATGAAGCAGAACCTGGATGAGCTTGGAATAGGAACGTACCACAACATTGCCTTCAT720 
ACATCCAGACACTCCCATCATCAAAGCCTTGAACATATTTGTGGAAAGACGAATATCAGC780 
TCTGCCTGTTGTGGATGAGTCAGGAAAAGTTGTAGATATTTATTCCAAATTTGATGTAAT840 
TAATCTTGCTGCTGAGAAAACATACAATAACCTAGATATCACGGTGACCCAGGCCCTTCA900 
GCACCGTTCACAGTATTTTGAAGGTGTTGTGAAGTGCAATAAGCTGGAAATACTGGAGAC960 
CATCGTGGACAGAATAGTAAGAGCTGAGGTCCATCGGCTGGTGGTGGTAAATGAAGCAGA1020 
TAGTATTGTGGGTATTATTTCCCTGTCGGACATTCTGCAAGCCCTGATCCTCACACCAGC1080 
AGGTGCCAAACAAAAGGAGACAGAAACGGAGTGACCGCCGTGAATGTAGACGCCCTAGGA1140 
GGAGAACTTGAACAAAGTCTCTGGGTCACGTTTTGCCTCATGAACACTGGCTGCAAGTGG1200 
TTAAGAATGTATATCAGGGTTTAACAATAGGTATTTCTTCCAGTGATGTTGAAATTAAGC1260 
TTAAAAAAGAAAGATTTTATGTGCTTGAAGATTCAGGCTTGCATTAAAAGACTGTTTTCA1320 
GACCTTTGTCTGAAGGATTTTAAATGCTGTATGTCATTAAAGTGCACTGTGTCCTGAAGT1380 
TTTCATTATTTTTCATTTCAAAGAATTCACTGGTATGGAACAGGTGATGTGGCAT1435 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 607 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 1827450 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
MetLeuAlaGlyLeuProThrSerAspProGlyArgLeuIleThrAsp 
151015 
ProArgSerGlyArgThrTyrLeuLysGlyArgLeuLeuGlyLysGly 
202530 
GlyPheAlaArgCysTyrGluAlaThrAspThrGluThrGlySerAla 
354045 
TyrAlaValLysValIleProGlnSerArgValAlaLysProHisGln 
505560 
ArgGluLysIleLeuAsnGluIleGluLeuHisArgAspLeuGlnHis 
65707580 
ArgHisIleValArgPheSerHisHisPheGluAspAlaAspAsnIle 
859095 
TyrIlePheLeuGluLeuCysSerArgLysSerLeuAlaHisIleTrp 
100105110 
LysAlaArgHisThrLeuLeuGluProGluValArgTyrTyrLeuArg 
115120125 
GlnIleLeuSerGlyLeuLysTyrLeuHisGlnArgGlyIleLeuHis 
130135140 
ArgAspLeuLysLeuGlyAsnPhePheIleThrGluAsnMetGluLeu 
145150155160 
LysValGlyAspPheGlyLeuAlaAlaArgLeuGluProProGluGln 
165170175 
ArgLysLysThrIleCysGlyThrProAsnTyrValAlaProGluVal 
180185190 
LeuLeuArgGlnGlyHisGlyProGluAlaAspValTrpSerLeuGly 
195200205 
CysValMetTyrThrLeuLeuCysGlySerProProPheGluThrAla 
210215220 
AspLeuLysGluThrTyrArgCysIleLysGlnValHisTyrThrLeu 
225230235240 
ProAlaSerLeuSerLeuProAlaArgGlnLeuLeuAlaAlaIleLeu 
245250255 
ArgAlaSerProArgAspArgProSerIleAspGlnIleLeuArgHis 
260265270 
AspPhePheThrLysGlyTyrThrProAspArgLeuProIleSerSer 
275280285 
CysValThrValProAspLeuThrProProAsnProAlaArgSerLeu 
290295300 
PheAlaLysValThrLysSerLeuPheGlyArgLysLysLysSerLys 
305310315320 
AsnHisAlaGlnGluArgAspGluValSerGlyLeuValSerGlyLeu 
325330335 
MetArgThrSerValGlyHisGlnAspAlaArgProGluAlaProAla 
340345350 
AlaSerGlyProAlaProValSerLeuValGluThrAlaProGluAsp 
355360365 
SerSerProArgGlyThrLeuAlaSerSerGlyAspGlyPheGluGlu 
370375380 
GlyLeuThrValAlaThrValValGluSerAlaLeuCysAlaLeuArg 
385390395400 
AsnCysIleAlaPheMetProProAlaGluGlnAsnProAlaProLeu 
405410415 
AlaGlnProGluProLeuValTrpValSerLysTrpValAspTyrSer 
420425430 
AsnLysPheGlyPheGlyTyrGlnLeuSerSerArgArgValAlaVal 
435440445 
LeuPheAsnAspGlyThrHisMetAlaLeuSerAlaAsnArgLysThr 
450455460 
ValHisTyrAsnProThrSerThrLysHisPheSerPheSerValGly 
465470475480 
AlaValProArgAlaLeuGlnProGlnLeuGlyIleLeuArgTyrPhe 
485490495 
AlaSerTyrMetGluGlnHisLeuMetLysGlyGlyAspLeuProSer 
500505510 
ValGluGluValGluValProAlaProProLeuLeuLeuGlnTrpVal 
515520525 
LysThrAspGlnAlaLeuLeuMetLeuPheSerAspGlyThrValGln 
530535540 
ValAsnPheTyrGlyAspHisThrLysLeuIleLeuSerGlyTrpGlu 
545550555560 
ProLeuLeuValThrPheValAlaArgAsnArgSerAlaCysThrTyr 
565570575 
LeuAlaSerHisLeuArgGlnLeuGlyCysSerProAspLeuArgGln 
580585590 
ArgLeuArgTyrAlaLeuArgLeuLeuArgAspArgSerProAla 
595600605 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 396 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 1827450 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
MetProArgValLysAlaAlaGlnAlaGlyArgGlnSerSerAlaLys 
151015 
ArgHisLeuAlaGluGlnPheAlaValGlyGluIleIleThrAspMet 
202530 
AlaLysLysGluTrpLysValGlyLeuProIleGlyGlnGlyGlyPhe 
354045 
GlyCysIleTyrLeuAlaAspMetAsnSerSerGluSerValGlySer 
505560 
AspAlaProCysValValLysValGluProSerAspAsnGlyProLeu 
65707580 
PheThrGluLeuLysPheTyrGlnArgAlaAlaLysProGluGlnIle 
859095 
GlnLysTrpIleArgThrArgLysLeuLysTyrLeuGlyValProLys 
100105110 
TyrTrpGlySerGlyLeuHisAspLysAsnGlyLysSerTyrArgPhe 
115120125 
MetIleMetAspArgPheGlySerAspLeuGlnLysIleTyrGluAla 
130135140 
AsnAlaLysArgPheSerArgLysThrValLeuGlnLeuSerLeuArg 
145150155160 
IleLeuAspIleLeuGluTyrIleHisGluHisGluTyrValHisGly 
165170175 
AspIleLysAlaSerAsnLeuLeuLeuAsnTyrLysAsnProAspGln 
180185190 
ValTyrLeuValAspTyrGlyLeuAlaTyrArgTyrCysProGluGly 
195200205 
ValHisLysGluTyrLysGluAspProLysArgCysHisAspGlyThr 
210215220 
IleGluPheThrSerIleAspAlaHisAsnGlyValAlaProSerArg 
225230235240 
ArgGlyAspLeuGluIleLeuGlyTyrCysMetIleGlnTrpLeuThr 
245250255 
GlyHisLeuProTrpGluAspAsnLeuLysAspProLysTyrValArg 
260265270 
AspSerLysIleArgTyrArgGluAsnIleAlaSerLeuMetAspLys 
275280285 
CysPheProGluLysAsnLysProGlyGluIleAlaLysTyrMetGlu 
290295300 
ThrValLysLeuLeuAspTyrThrGluLysProLeuTyrGluAsnLeu 
305310315320 
ArgAspIleLeuLeuGlnGlyLeuLysAlaIleGlySerLysAspAsp 
325330335 
GlyLysLeuAspLeuSerValValGluAsnGlyGlyLeuLysAlaLys 
340345350 
ThrIleThrLysLysArgLysLysGluIleGluGluSerLysGluPro 
355360365 
GlyValGluAspThrGluTrpSerAsnThrGlnThrGluGluAlaIle 
370375380 
GlnThrArgSerArgThrArgLysArgValGlnLys 
385390395 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 400 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 854170 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
MetLeuAlaArgArgLysProValLeuProAlaLeuThrIleAsnPro 
151015 
ThrIleAlaGluGlyProSerProThrSerGluGlyAlaSerGluAla 
202530 
HisLeuValAspLeuGlnLysLysLeuGluGluLeuAspLeuAspGlu 
354045 
GlnGlnArgLysArgLeuGluAlaPheLeuThrGlnLysAlaLysVal 
505560 
GlyGluLeuLysAspAspAspPheGluArgIleSerGluLeuGlyAla 
65707580 
GlyAsnGlyGlyValValThrLysAlaArgHisArgProSerGlyLeu 
859095 
IleMetAlaArgLysLeuIleHisLeuGluIleLysProAlaValArg 
100105110 
AsnGlnIleIleArgGluLeuGlnValLeuHisGluCysAsnSerPro 
115120125 
TyrIleValGlyPheTyrGlyAlaPheTyrSerAspGlyGluIleSer 
130135140 
IleCysMetGluHisMetAspGlyGlySerLeuAspGlnValLeuLys 
145150155160 
GluAlaLysArgIleProGluAspIleLeuGlyLysValSerIleAla 
165170175 
ValLeuArgGlyLeuAlaTyrLeuArgGluLysHisGlnIleMetHis 
180185190 
ArgAspValLysProSerAsnIleLeuValAsnSerArgGlyGluIle 
195200205 
LysLeuCysAspPheGlyValSerGlyGlnLeuIleAspSerMetAla 
210215220 
AsnSerPheValGlyThrArgSerTyrMetSerProGluArgLeuGln 
225230235240 
GlyThrHisTyrSerValGlnSerAspIleTrpSerMetGlyLeuSer 
245250255 
LeuValGluLeuAlaIleGlyArgTyrProIleProProProAspAla 
260265270 
LysGluLeuGluAlaSerPheGlyArgProValValAspGlyAlaAsp 
275280285 
GlyGluProHisSerValSerProArgProArgProProGlyArgPro 
290295300 
IleSerGlyHisGlyMetAspSerArgProAlaMetAlaIlePheGlu 
305310315320 
LeuLeuAspTyrIleValAsnGluProProProLysLeuProSerGly 
325330335 
ValPheSerSerAspPheGlnGluPheValAsnLysCysLeuIleLys 
340345350 
AsnProAlaGluArgAlaAspLeuLysLeuLeuThrAsnHisAlaPhe 
355360365 
IleLysArgSerGluGlyGluAspValAspPheAlaGlyTrpLeuCys 
370375380 
ArgThrLeuArgLeuLysGlnProSerThrProThrArgThrAlaVal 
385390395400 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 465 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 8541070 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
MetAlaMetThrGlySerThrProCysSerSerMetSerAsnHisThr 
151015 
LysGluArgValThrMetThrLysValThrLeuGluAsnPheTyrSer 
202530 
AsnLeuIleAlaGlnHisGluGluArgGluMetArgGlnLysLysLeu 
354045 
GluLysValMetGluGluGluGlyLeuLysAspGluGluLysArgLeu 
505560 
ArgArgSerAlaHisAlaArgLysGluThrGluPheLeuArgLeuLys 
65707580 
ArgThrArgLeuGlyLeuGluAspPheGluSerLeuLysValIleGly 
859095 
ArgGlyAlaPheGlyGluValArgLeuValGlnLysLysAspThrGly 
100105110 
HisValTyrAlaMetLysIleLeuArgLysAlaAspMetLeuGluLys 
115120125 
GluGlnValGlyHisIleArgAlaGluArgAspIleLeuValGluAla 
130135140 
AspSerLeuTrpValValLysMetPheTyrSerPheGlnAspLysLeu 
145150155160 
AsnLeuTyrLeuIleMetGluPheLeuProGlyGlyAspMetMetThr 
165170175 
LeuLeuMetLysLysAspThrLeuThrGluGluGluThrGlnPheTyr 
180185190 
IleAlaGluThrValLeuAlaIleAspSerIleHisGlnLeuGlyPhe 
195200205 
IleHisArgAspIleLysProAspAsnLeuLeuLeuAspSerLysGly 
210215220 
HisValLysLeuSerAspPheGlyLeuCysThrGlyLeuLysLysAla 
225230235240 
HisArgThrGluPheTyrArgAsnLeuAsnHisSerLeuProSerAsp 
245250255 
PheThrPheGlnAsnMetAsnSerLysArgLysAlaGluThrTrpLys 
260265270 
ArgAsnArgArgGlnLeuAlaPheSerThrValGlyThrProAspTyr 
275280285 
IleAlaProGluValPheMetGlnThrGlyTyrAsnLysLeuCysAsp 
290295300 
TrpTrpSerLeuGlyValIleMetTyrGluMetLeuIleGlyTyrPro 
305310315320 
ProPheCysSerGluThrProGlnGluThrTyrLysLysValMetAsn 
325330335 
TrpLysGluThrLeuThrPheProProGluValProIleSerGluLys 
340345350 
AlaLysAspLeuIleLeuArgPheCysCysGluTrpGluHisArgIle 
355360365 
GlyAlaProGlyValGluGluIleLysSerAsnSerPhePheGluGly 
370375380 
ValAspTrpGluHisIleArgGluArgProAlaAlaIleSerIleGlu 
385390395400 
IleLysSerIleAspAspThrSerAsnPheAspGluPheProGluSer 
405410415 
AspIleLeuLysProThrValAlaThrSerAsnHisProGluThrAsp 
420425430 
TyrLysAsnLysAspTrpValPheIleAsnTyrThrTyrLysArgPhe 
435440445 
GluGlyLeuThrAlaArgGlyAlaIleProSerTyrMetLysAlaAla 
450455460 
Lys 
465 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 370 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 790790 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
MetLeuGlyAlaValGluGlyProArgTrpLysGlnAlaGluAspIle 
151015 
ArgAspIleTyrAspPheArgAspValLeuGlyThrGlyAlaPheSer 
202530 
GluValIleLeuAlaGluAspLysArgThrGlnLysLeuValAlaIle 
354045 
LysCysIleAlaLysGluAlaLeuGluGlyLysGluGlySerMetGlu 
505560 
AsnGluIleAlaValLeuHisLysIleLysHisProAsnIleValAla 
65707580 
LeuAspAspIleTyrGluSerGlyGlyHisLeuTyrLeuIleMetGln 
859095 
LeuValSerGlyGlyGluLeuPheAspArgIleValGluLysGlyPhe 
100105110 
TyrThrGluArgAspAlaSerArgLeuIlePheGlnValLeuAspAla 
115120125 
ValLysTyrLeuHisAspLeuGlyIleValHisArgAspLeuLysPro 
130135140 
GluAsnLeuLeuTyrTyrSerLeuAspGluAspSerLysIleMetIle 
145150155160 
SerAspPheGlyLeuSerLysMetGluAspProGlySerValLeuSer 
165170175 
ThrAlaCysGlyThrProGlyTyrValAlaProGluValLeuAlaGln 
180185190 
LysProTyrSerLysAlaValAspCysTrpSerIleGlyValIleAla 
195200205 
TyrIleLeuLeuCysGlyTyrProProPheTyrAspGluAsnAspAla 
210215220 
LysLeuPheGluGlnIleLeuLysAlaGluTyrGluPheAspSerPro 
225230235240 
TyrTrpAspAspIleSerAspSerAlaLysAspPheIleArgHisLeu 
245250255 
MetGluLysAspProGluLysArgPheThrCysGluGlnAlaLeuGln 
260265270 
HisProTrpIleAlaGlyAspThrAlaLeuAspLysAsnIleHisGln 
275280285 
SerValSerGluGlnIleLysLysAsnPheAlaLysSerLysTrpLys 
290295300 
GlnAlaPheAsnAlaThrAlaValValArgHisMetArgLysLeuGln 
305310315320 
LeuGlyThrSerGlnGluGlyGlnGlyGlnThrAlaSerHisGlyGlu 
325330335 
LeuLeuThrProValAlaGlyGlyProAlaAlaGlyCysCysCysArg 
340345350 
AspCysCysValGluProGlyThrGluLeuSerProThrLeuProHis 
355360365 
GlnLeu 
370 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 382 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 924921 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
SerThrSerAlaThrAspThrHisHisValGluLeuAlaArgGluArg 
151015 
SerLysThrValThrSerPheTyrAsnGlnSerAlaIleAspValVal 
202530 
AlaGluLysProSerValArgLeuThrProThrMetMetLeuTyrSer 
354045 
GlyArgSerGlnAspGlySerHisLeuLeuLysSerGlyArgTyrLeu 
505560 
GlnGlnGluLeuProValArgIleAlaHisArgIleLysGlyPheArg 
65707580 
SerLeuProPheIleIleGlyCysAsnProThrIleLeuHisValHis 
859095 
GluLeuTyrIleArgAlaPheGlnLysLeuThrAspPheProProIle 
100105110 
LysAspGlnAlaAspGluAlaGlnTyrCysGlnLeuValArgGlnLeu 
115120125 
LeuAspAspHisLysAspValValThrLeuLeuAlaGluGlyLeuArg 
130135140 
GluSerArgLysHisIleGluAspGluLysLeuValArgTyrPheLeu 
145150155160 
AspLysThrLeuThrSerArgLeuGlyIleArgMetLeuAlaThrHis 
165170175 
HisLeuAlaLeuHisGluAspLysProAspPheValGlyIleIleCys 
180185190 
ThrArgLeuSerProLysLysIleIleGluLysTrpValAspPheAla 
195200205 
ArgArgLeuCysGluHisLysTyrGlyAsnAlaProArgValArgIle 
210215220 
AsnGlyHisValAlaAlaArgPheProPheIleProMetProLeuAsp 
225230235240 
TyrIleLeuProGluLeuLeuLysAsnAlaMetArgAlaThrMetGlu 
245250255 
SerHisLeuAspThrProTyrAsnValProAspValValIleThrIle 
260265270 
AlaAsnAsnAspValAspLeuIleIleArgIleSerAspArgGlyGly 
275280285 
GlyIleAlaHisLysAspLeuAspArgValMetAspTyrHisPheThr 
290295300 
ThrAlaGluAlaSerThrGlnAspProArgIleSerProLeuPheGly 
305310315320 
HisLeuAspMetHisSerGlyGlyGlnSerGlyProMetHisGlyPhe 
325330335 
GlyPheGlyLeuProThrSerArgAlaTyrAlaGluTyrLeuGlyGly 
340345350 
SerLeuGlnLeuGlnSerLeuGlnGlyIleGlyThrAspValTyrLeu 
355360365 
ArgLeuArgHisIleAspGlyArgGluGluSerPheArgIle 
370375380 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 331 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 1335856 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
MetGluThrValIleSerSerAspSerSerProAlaValGluAsnGlu 
151015 
HisProGlnGluThrProGluSerAsnAsnSerValTyrThrSerPhe 
202530 
MetLysSerHisArgCysTyrAspLeuIleProThrSerSerLysLeu 
354045 
ValValPheAspThrSerLeuGlnValLysLysAlaPhePheAlaLeu 
505560 
ValThrAsnGlyValArgAlaAlaProLeuTrpAspSerLysLysGln 
65707580 
SerPheValGlyMetLeuThrIleThrAspPheIleAsnIleLeuHis 
859095 
ArgTyrTyrLysSerAlaLeuValGlnIleTyrGluLeuGluGluHis 
100105110 
LysIleGluThrTrpArgGluValTyrLeuGlnAspSerPheLysPro 
115120125 
LeuValCysIleSerProAsnAlaSerLeuPheAspAlaValSerSer 
130135140 
LeuIleArgAsnLysIleHisArgLeuProValIleAspProGluSer 
145150155160 
GlyAsnThrLeuTyrIleLeuThrHisLysArgIleLeuLysPheLeu 
165170175 
LysLeuPheIleThrGluPheProLysProGluPheMetSerLysSer 
180185190 
LeuGluGluLeuGlnIleGlyThrTyrAlaAsnIleAlaMetValArg 
195200205 
ThrThrThrProValTyrValAlaLeuGlyIlePheValGlnHisArg 
210215220 
ValSerAlaLeuProValValAspGluLysGlyArgValValAspIle 
225230235240 
TyrSerLysPheAspValIleAsnLeuAlaAlaGluLysThrTyrAsn 
245250255 
AsnLeuAspValSerValThrLysAlaLeuGlnHisArgSerHisTyr 
260265270 
PheGluGlyValLeuLysCysTyrLeuHisGluThrLeuGluThrIle 
275280285 
IleAsnArgLeuValGluAlaGluValHisArgLeuValValValAsp 
290295300 
GluAsnAspValValLysGlyIleValSerLeuSerAspIleLeuGln 
305310315320 
AlaLeuValLeuThrGlyGlyGluLysLysPro 
325330 
__________________________________________________________________________