WO1998058064A1 - Kinesin-related protein - Google Patents

Abstract

The invention provides a human kinesin-related protein (KINRELP) and polynucleotides which identify and encode KINRELP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of KINRELP.

Description

KINESIN-RELATED PROTEIN

TECHNICAL FIELD

This invention relates to nucleic acid and amino acid sequences of a new human kinesm- related protein and to the use of these sequences in the diagnosis, prevention, and treatment of inflammation and disorders associated with cell proliferation and apoptosis

BACKGROUND OF THE INVENTION

Transportation and communication within cells are essential for cell proliferation, regulation, and function Membrane-bound vesicles and proteins are frequenth transported along well defined routes in the cytosol and delivered to particular cellular compartments The transport occurs in both directions, namely, anterograde and retrograde Several ATP-dependent proteins including kinesins. kinesin-related proteins ( RPs), and dvneins have been identified as the motors that drive the transport Among these, kinesins function in vesicle transport, and KRPs have been implicated in spindle assembly and chromosome segregation during meiosis and mitosis Kinesins and KRPs belong to a superfamily of motor proteins with over 12 different family members These proteins generally contain two heavy chains and several intermediate or light chains The kinesin heavy chain (KHC) consists of an a helical coiled-coil stalk domain, a motor domain which generates force for movement on microtubules by ATP h\drolysιs, and a fan-like, globular domain which interacts with intermediate or light chains and possibly with vesicle cargos Depending on its position in the KHC sequence the motor domain drives anterograde or retrograde transport the amino-termmal motor domain is associated with generating anterograde motion, and the central or carboxyl terminal motor domain, retrograde motion

A series of genes encoding proteins related to KHC have been identified in various tissues and organisms KIF2 is a mouse kinesin-related, neuron-specific protein which consists of an ammo-terminal globular domain, a central motor domain, and a carboxyl-terminal α-helical stalk domain (Aizawa, H et al ( 1992) J Cell Biol 1 19 1287- 1296) Five rat testis-specific KRPs are shown to be important in meiosis and morphogenesis (Sperry, A O and Zhao. L -P ( 1996) Mol Biol Cell 7 289-305) In a addition, a human kinesin-related protein that associates with the centromere region of mitotic chromosomes (Wordeman, L and Mitchison, T J ( 1995) 128. 95- 105), and a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly (Walczak, C E et al (1996) Cell 84 37-47) have been described

The discovery of a new human kinesin-related protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of inflammation and disorders associated with cell proliferation and apoptosis

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, human kinesin-related protein (KINRELP), having the amino acid sequence shown in SEQ LD NO 1 , or fragments thereof The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO 1 or fragments thereof and a composition comprising said polynucleotide sequence The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO 1 , or fragments of said polynucleotide sequence The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO 1 , or fragments or variants of said polynucleotide sequence

The invention also provides an isolated and purified sequence comprising SEQ ID NO 2 or variants thereof In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO 2 In another aspect the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO 2 or fragments or variants thereof The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO 2 The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences 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 comprising the am o acid sequence of SEQ ID NO 1 or a fragment thereof, the method comprising the steps of a) culturmg the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding KINRELP 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 KINRELP having the amino acid sequence of SEQ ED NO 1 in conjunction with a suitable pharmaceutical carrier

The invention also provides a purified antagonist which decreases the effect of the polypeptide of SEQ ID NO 1 In one aspect, the invention provides a purified antibody which binds to a polypeptide comprising at least a fragment of the amino acid sequence of SEQ ID NO: l

Still further, the invention provides a purified agonist which modulates the activity of the polypeptide of SEQ ID NO 1

The invention also provides a method for stimulating cell proliferation comprising administering to a cell an effectiv e amount of purified KINRELP

The invention also provides a method for treating a disorder associated with increased apoptosis comprising administering to a subject in need of such treatment an effective amount of purified KINRELP

The invention also prov ides a method for treating a disorder associated with cancer comprising administering to a subject in need of such treatment an effective amount of an antagonist which decreases the ef fect of KINRELP

The invention also prov ides a method for treating inflammation comprising administering to a subject in need of such treatment an effective amount of an antagonist which decreases the effect of KINRELP The invention also piovides a method for detecting a polynucleotide which encodes

KINRELP in a biological sample comprising the steps of a) hybridizing a polynucleotide sequence complementary to the polynucleotide sequence encoding KINRELP (SEQ ID NO 1 ) 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 KINRELP in the biological sample In a preferred embodiment, prior to hybridization, the nucleic acid material of the biological sample is amplified by the polymerase chain reaction

BRIEF DESCRIPTION OF THE FIGURES Figures 1A, IB, IC, ID, IE, IF, 1G, and IH show the amino acid sequence (SEQ ID NO 1) and nucleic acid sequence (SEQ ID NO 2) of KINRELP The alignment was produced using MacDNASIS PRO™ software (Hitachi Software Engineering Co. Ltd San Bruno, CA)

Figures 2A and 2B show the amino acid sequence alignments between KINRELP (SEQ ID NO 1) and a mouse kinesin-related protein, KIF2 (GI 220468, SEQ ID NO 3), produced using the multisequence alignment program of DNASTAR™ software (DNASTAR Inc. Madison WI)

Figures 3A and 3B show the hydrophobicity plots for KINRELP, SEQ ID NO 1 and KIF2 (SEQ LD NO 3), respectively The positive X axis reflects amino acid position, and the negative Y axis, hydrophobicity (MacDNASIS PRO software)

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 as 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 incoφorated herein by reference for the purpose of describing and disclosing the cell lines, vectors, 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

KINRELP, as used herein, refers to the amino acid sequences of substantially purified KINRELP obtained from any species, particularly mammalian, including bovine, ovine, porcine, muπne, 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 KINRELP, increases or prolongs the duration of the effect of KINRELP Agonists may include proteins nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of KINRELP

An "allele" or "allelic sequence", as used herein, is an alternative form of the gene encoding KINRELP 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 KINRELP 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 KINRELP Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding KINRELP, and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus tor the polynucleotide sequence encoding KINRELP 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 KINRELP Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity hvdrophilicity, and/or the amphipathic nature of the residues as long as the biological or immunological activ lty of KINRELP 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 hydrophihcity 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 o gopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Fragments of KINRELP are preferably about 5 to about 15 amino acids in length and retain the biological activity or the immunological activity of KINRELP 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, NY). The term "antagonist" as used herein, refers to a molecule which, when bound to

KINRELP, decreases the amount or the duration of the effect of the biological or immunological activity of KINRELP. Antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules which and decrease the effect of KINRELP.

As used herein, the term "antibody" refers to intact molecules as well as fragments thereof, such as Fa, F(ab')_:, and Fv, which are capable of binding the epitopic determinant. Antibodies that bind KINRELP polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or ohgopeptide 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 KINRELP, 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 KINRELP (SEQ ID NO: l ) or fragments thereof (e.g., SEQ ID NO:2 and 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™ (Perkin Elmer, Norwalk, CT) 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, WI). 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 ribonucleic acid that is similar to SEQ ID NO:2 by northern analysis is indicative of the presence of mRNA encoding KINRELP in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.

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 KINRELP or the encoded KINRELP 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 teim 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 , C₀t or R₀t 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 of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane filter chip, glass slide, or any other suitable solid support

The term modulate", as used herein, refers to a change in the activity of KINRELP For example, modulation may cause an increase or a decrease in piotein activity binding characteristics, or any other biological, functional or immunological properties of KINRELP

"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 oi 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 amphmers '/'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 fespan 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 SEQ ID NO 1" encompasses the full-length KINRELP and fragments thereof The term "sample", as used herein, is used in its broadest sense A biological sample suspected of containing nucleic acid encoding KINRELP, or fragments thereof, or KINRELP 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(ιn 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 natuie 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°C below the melting temperature of the probe to about 20°C to 25°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 know n 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, pofection, 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

A "variant" of KINRELP 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 v ariant 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 know n in the art for example, DNASTAR software

THE INVENTION

The invention is based on the discovery of a new human kinesin-related protein (hereinafter referred to as KINRELP ) the polynucleotides encoding KINRELP, and the use of these compositions for the diagnosis prevention, or treatment of inflammation and disorders associated with cell proliferation and apoptosis Nucleic acids encoding the KINRELP of the present invention were first identified in

Incyte Clone 93542 from a hypothalamus tissue tissue cDNA library (HYPONOB01) using a computer search for amino acid sequence alignments A consensus sequence, SEQ ID NO 2, was derived from the following overlapping and/or extended nucleic acid sequences Incyte Clones 309565 (TMLR2DT01 ), 969305 (BRSTNOT05), 382869 (HYPONOB01), and 155679 (THP1PLB02)

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO 1 , as shown in Figures 1A-H KINRELP is 222 amino acids in length KINRELP has an ATP-binding P-loop encompassing residues G286-T293 and a kinesin motor domain signature encompassing residues G424-E235 KINRELP also has four potential N- glycosylation sites encompassing residues N9-V12, N106-V 109, N244-F247, and N455-L458, two potential cAMP- and cGMP-dependent protein kinase phosphorylation sites encompassing residues R127-S 130 and K208-T21 1 , eleven potential casein kinase II phosphorylation sites encompassing residues S4-E7, T27-E30, S 108-D 1 1 1, T187-D 190, T273-E280, T338-E341,

S427-D430, S441-D444, T447-E450, T582-E585, and T634-E637, two potential tyrosine kinase phosphorylation sites encompassing residues K233-Y241 and K329-Y236. and fourteen potential protein kinase C phosphorylation sites encompassing residues S58-K60, S73-K75, S88-R90, S 126-R128, T246-R248, T266-R268, S290-K292, S301-K303, S344-K346 S392-R394, S405- R407, T516-R518, S608-R610 and S656-R658 As shown in Figures 2 A and 2B. KINRELP has chemical and structural homology with a mouse kinesin-related protein, KIF2 (GI 220468, SEQ ID NO 3) In particular KINRELP shares 97% identity with KIF2 As illustrated by Figures 3A and 3B, KINRELP and KIF2 have rather similar hydrophobicity plots Northern analysis shows the expression of this sequence in various libraries, at least 42% of which are immortalized or cancerous, at least 21 % of which involve immune response and at least 10% of which involve infant/fetal tissues or organs

The invention also encompasses KINRELP variants A preferred KINRELP variant is one having at least 80% and more preferably 90% amino acid sequence identity to the KINRELP amino acid sequence (SEQ ID NO 1 ) and which retain the biological, immunological or other functional characteristics of the activity of KINRELP A most preferred KINRELP variant is one having at least 95% amino acid sequence identity to SEQ ID NO 1

The invention also encompasses polynucleotides which encode KINRELP Accordingly, any nucleic acid sequence which encodes the amino acid sequence of KINRELP can be used to produce recombinant molecules which express KINRELP In a particular embodiment, the invention encompasses the polynucleotide comprising the nucleic acid sequence of SEQ ID NO 2 as shown in Figures 1A-H

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 KINRELP, 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 KINRELP, and all such variations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode KINRELP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring KINRELP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding KINRELP 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 KINRELP 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 KINRELP 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, s nthetic chemistry may be used to introduce mutations into a sequence encoding KINRELP 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 NO:2, 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. 152:507-51 1 )

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® (US Biochemical Corp, Cleveland. OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham. Chicago, EL), 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 Micro Lab 2200 (Hamilton. Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer). The nucleic acid sequences encoding KINRELP 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 transcπptase

Inverse PCR may also be used to amplify or extend sequences using dι\ ergent 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, MN), 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°-72° 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 hgation 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 1 1 1- 1 19) In this method multiple restriction enzyme digestions and hgations may also be used to place an engineered double-stranded sequence into an unknown fragment ot 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, C A) 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™, 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 KINRELP may be used in recombinant DNA molecules to direct expression of KINRELP, 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 KINRELP

As will be understood bv those ot skill in the art, it may be advantageous to produce KENRELP-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred bv a particular prokaryotic or eukarvotic 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 ot the present invention can be engineered using methods generally known in the art in oider to alter KINRELP 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 KINRELP may be gated to a heterologous sequence to encode a fusion protein For example, to screen peptide libraries for inhibitors of KINRELP activity, it may be useful to encode a chimeπc KINRELP 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 KINRELP encoding sequence and the heterologous protein sequence, so that KINRELP may be cleaved and purified away from the heterologous moiety

In another embodiment, sequences encoding KINRELP 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 215-223, Horn, T et al ( 1980) Nucl Acids Res Symp Ser 225-232) Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of KINRELP, 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 431 A 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, NY) 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 KINRELP, 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 KINRELP, the nucleotide sequences encoding KINRELP 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 KINRELP and appropriate transcπptional and translational control elements These methods include m 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, NY, and Ausubel, F M et al ( 1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY

A variety of expression vector/host systems may be utilized to contain and express sequences encoding KINRELP These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteπophage, 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, CA) or pSport l™ plasmid (Gibco BRL) and the like may be used. The baculovirus polyhedπn 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 KINRELP. vectors based on SV40 or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be ^elected depending upon the use intended for KINRELP. For example, when large quantities ot KINRELP 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 Bluescript® (Stratagene ). in which the sequence encoding KINRELP may be hgated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced; pEN vectors (Van Heeke. G. and S.M. Schuster ( 1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors (Promega, Madison, WI) 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 KINRELP 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. 6:307-31 1 ) Alternatively, plant promoters such as the small subumt of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. ( 1984) EMBO J. 3.1671- 1680; Broghe, 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, NY; pp 191- 196

An insect system may also be used to express KINRELP Foi example, in one such system, Auto rapha cahfornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Tπchoplusia larvae The sequences encoding KINRELP may be cloned into a non-essential region of the virus, such as the polyhedπn gene, and placed under control of the polyhedπn promoter Successful insertion of KINRELP will render the polyhedπn gene inactive and produce recombinant virus lacking coat protein The recombinant viruses may then be used to infect, for example, S frugiperda cells or Tπchoplusia larvae in which KINRELP 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 KINRELP may be gated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing KINRELP 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 ( posomes, polycationic ammo polymers, or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficient translation of sequences encoding KINRELP Such signals include the ATG initiation codon and adjacent sequences In cases where sequences encoding KINRELP, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcπptional 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 mav 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 acetvlation, carboxylation, glycosylation. phosphorylation lipidation, and acylation Post-translational processing which cleaves a "prepro^" form ot 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 KINRELP 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 1 1.223-32) and adenine phosphoπbosyltransferase (Lowy, I et al (1980) Cell 22 817-23) genes which can be employed in tk or aprt 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 phosphinotπcin 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, β glucuromdase and its substrate GUS, and luciferase and its substrate lucifenn. 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 KINRELP is inserted within a marker gene sequence, transformed cells containing sequences encoding KINRELP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding KINRELP 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 KINRELP and express KINRELP 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 KINRELP can be detected by

DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding KINRELP. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding KINRELP to detect transformants containing DNA or RNA encoding KINRELP. A variety of protocols for detecting and measuring the expression of KINRELP, 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 KINRELP 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, MN) and Maddox, D E et al (1983, J Exp Med 158 121 1- 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 KINRELP include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide Alternatively, the sequences encoding KINRELP. or any fragments thereof may be cloned into a vector for the production ot an mRNA probe Such vectors are known in the art. are commercially available, and may be used to synthesize RNA probes m vitro by addition ot an appropriate RNA polymerase such as T7 3 or SP6 and labeled nucleotides These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI), Promega (Madison WI), and U S Biochemical Corp , Cleveland, OH) Suitable reporter molecules oi labels, which may be used for ease of detection, include radionuchdes. enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors. inhibitors, magnetic particles, and the like

Host cells transformed with nucleotide sequences encoding KINRELP 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 bv those of skill in the art, expression vectors containing polynucleotides which encode KINRELP mav be designed to contain signal sequences which direct secretion of KINRELP through a prokaryotic or eukaryotic cell membrane Other constructions may be used to join sequences encoding KINRELP 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 FLAGS extension/affinity purification system (Immunex Corp , Seattle, WA) The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and KINRELP may be used to facilitate purification One such expression vector provides for expression of a fusion protein containing KINRELP and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMAC (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 KINRELP 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 KINRELP 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 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of KINRELP may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

THERAPEUTICS

Chemical and structural homology exists between KINRELP and a kinesin-related protein, KIF2 (GI 220468; SEQ ID NO:3). Northern analysis shows that the expression of

KINRELP is associated with cancer, inflammation and immune response, and fetal/infant development.

During fetal development, decreased expression of KINRELP may cause an increase in apoptosis with no adverse effects to the subject. However, in other situations and in adults, decreased expression of KINRELP may cause an increase in apoptosis which is detrimental to the subject. Therefore, in one embodiment, KINRELP or a fragment or derivative thereof may be administered to a subject to prevent or treat a disorder associated with an increase in apoptosis.

Such disorders include, but are not limited to, AIDS and other infectious or genetic immunodeficiencies, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, and cerebellar degeneration, myelodysplastic syndromes such as aplastic anemia, ischemic injuries such as myocardial infarction, stroke, and reperfusion injury, toxin-induced diseases such as alcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseases such as cachexia, viral infections such as those caused by hepatitis B and C, and osteoporosis. In another embodiment, an agonist which is specific for KINRELP may be used to prevent or treat a disorder associated with increased apoptosis including, but not limited to, those listed above

In still another embodiment, a vector capable of expressing KINRELP, or a fragment or a derivative thereof, may be used to prevent or treat a disorder associated with increased apoptosis including, but not limited to, those listed above

In a further embodiment. KINRELP or a fragment or derivative thereof may be added to cells to stimulate cell proliferation In particular, KINRELP may be added to a cell or cells in vivo using delivery mechanisms such as liposomes, viral based vectors, or electroinjection for the purpose of promoting regeneration or cell differentiation of the cell or cells In addition, KINRELP 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 selected for its ability to fight an infection or a cancer or to correct a genetic defect in a disease such as sickle cell anemia β thalassemia, cystic fibrosis, or Huntington's chorea

In another further embodiment an agonist which is specific for KINRELP may be administered to a cell to stimulate cell proliferation, as described above

In another further embodiment a vector capable of expressing KINRELP, or a fragment or a derivative thereof, may be administered to a cell or cells in

o using delivery mechanisms, or to a cell to stimulate cell proliferation as described above

Increased expression ot KINRELP appears to be associated with increased cell proliferation Therefore in one embodiment, an antagonist of KINRELP, or a fragment or a derivative thereof, may be administered to a subject to prevent or treat cancer Such disorders include various types of cancer including but not limited to, adenocarcinoma leukemia, lymphoma. melanoma, myeloma sarcoma, and teratocarcinoma and particularly, cancers of the adrenal gland, bladder bone bone marrow brain breast cervix gall bladder ganglia, gastrointestinal tract, heart kidnev liver, lung, muscle ovary, pancreas parathyroid, penis prostate, salivary glands skin, spleen testis, thymus, thyroid, and uterus In one aspect, an antibody specific for KINRELP 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 KINRELP

In still another embodiment a vector expressing the complementary sequence or antisense of the polynucleotide encoding KINRELP, or a fragment or a derivative thereof, may be administered to a subject to prevent or treat a disorder associated with cell proliferation including, but not limited to, the types of cancer listed above

In a further embodiment, an antagonist of KINRELP or a fragment or a derivative thereof, may be administered to a subject to prevent or treat inflammation of any type and, in particular, that which results from a particular disorder or conditions Such disorders and conditions associated with inflammation include, but are not limited to,Addιson 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, glomerulonephπtis, gout, Graves' disease, hypereosinophiha, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis myocardial or peπcardial ιnflammatιon,osteoarthπtιs, 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, an antibody specific for KINRELP 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 KINRELP

In another further embodiment a vector expressing the complementary sequence or antisense of the polynucleotide encoding KINRELP, or a fragment or a derivative thereof, may be administered to a subject to prevent or treat inflammation of any type including, but not limited to, those listed 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 KINRELP may be produced using methods which are generally known in the art In particular, purified KINRELP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind KINRELP

Antibodies to KINRELP may be generated using methods that are well known in the art Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeπc, single chain, Fab fragments, and fragments produced by a Fab expression library Neutralizing antibodies, (l 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 KINRELP or any fragment or o gopeptide thereof which has lmmunogenic 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 Corynebacteπum parvum are especially preferable

It is preferred that the ohgopeptides, peptides, or fragments used to induce antibodies to KINRELP 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 KINRELP amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeπc molecule

Monoclonal antibodies to KINRELP 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 hybπdoma technique, the human B-cell hybπdoma technique, and the EBV-hybπdoma 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 ot "chimeπc 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 l ( 1984) Proc Natl Acad. Sci 81 :6851-6855. Neuberger. M S et al. ( 1984) Nature 3 12 604-608. 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 KINRELP-specific single chain antibodies. Antibodies with related specificity, but of distinct ldiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton D.R. ( 1991) Proc. Natl. Acad. Sci 88- 1 1 120-3)

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 KINRELP 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 KINRELP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering KINRELP epitopes is preferred, but a competitive binding assay may also be employed (Maddox. supra).

In another embodiment of the invention, the polynucleotides encoding KINRELP. or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding KINRELP 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 KINRELP. Thus, complementary molecules or fragments may be used to modulate KINRELP 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 KINRELP.

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 KINRELP. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).

Genes encoding KINRELP 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 KINRELP. 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 KINRELP (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. NY) The complementary sequence or antisense molecule may also be designed to block translation ot mRNA by preventing the transcript from binding to πbosomes

Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA The mechanism ot πbozvme action involves sequence-specific hybridization of the πbozyme molecule to complementary target RNA, followed by endonucleolytic cleavage Examples which may be used include engineered hammerhead motif πbozyme molecules that can specifically and efficiently cataly/e endonucleolytic cleavage of sequences encoding KINRELP Specific πbozyme cleav age sites within any potential RNA target are initially identified by scanning the target molecule f or πbozyme cleavage sites which include the following sequences GUA. GUU. and GUC Once identified, short RNA sequences ot between 15 and 20 πbonucleotides 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 πbonuclease protection assays

Complementary πbonucleic 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 KINRELP Such DNA sequences may be incoφorated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6 Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues

RNA molecules may be modified to increase intracellular stability and half-life Possible modifications include, but are not limited to, the addition of flanking sequences at the 5^' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule This concept is inherent in the production of

PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine. queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine. cytidine, guanine, thymine, and undine 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 m 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 hposome injections or polycatiomc amino polymers (Goldman, C K et al ( 1997) Nature Biotechnology 15 462-66, incoφorated 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 ot a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above Such pharmaceutical compositions may consist of KINRELP. antibodies to KINRELP, mimetics. agonists, antagonists or inhibitors of KINRELP 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, lntramedullary, mtrathecal, lntraventπcular, transdermal, subcutaneous, intraperitoneal, intranasal. enteral, topical, subhngual, 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 arable and tragacanth. and proteins such as gelatin and collagen If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrohdone. agar, algimc 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 arable, talc, polyvinvlpyrrohdone. carbopol gel, polyethylene glycol. and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures Dyestuffs oi pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound l 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's 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 KINRELP, 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 puφose. 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

KINRELP or fragments thereof, antibodies of KINRELP, agonists, antagonists or inhibitors of KINRELP, 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 KINRELP may be used for the diagnosis of conditions or diseases characterized by expression of KINRELP, or in assays to monitor patients being treated with KINRELP, agonists, antagonists or inhibitors. The antibodies useful for diagnostic puφoses may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for KINRELP include methods which utilize the antibody and a label to detect KINRELP 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 KINRELP are known in the art and provide a basis for diagnosing altered or abnormal levels of KINRELP expression Normal or standard values for KINRELP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to KINRELP 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 KINRELP expressed in subject, control and disease, samples from biopsied tissues are compared with the standard values Deviation between standard and subject values establishes the parameters for diagnosing disease

In another embodiment of the invention, the polynucleotides encoding KINRELP may be used for diagnostic puφoses 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 KINRELP may be correlated with disease The diagnostic assay may be used to distinguish between absence, presence and excess expression of KINRELP and to monitor regulation of KINRELP levels during therapeutic intervention

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences including genomic sequences encoding KINRELP or closely related molecules, may be used to identify nucleic acid sequences which encode KINRELP The specificity ot 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 ot the hybridization or amplification (maximal, high intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding KINRELP, 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 KINRELP encoding sequences The hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of SEQ ED NO 2 or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring KINRELP

Means for producing specific hybridization probes for DNAs encoding KINRELP include the cloning ot nucleic acid sequences encoding KINRELP or KINRELP 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, radionuchdes 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 KINRELP may be used for the diagnosis of conditions, disorders, or diseases which are associated with expression of KINRELP Examples of such disorders include various types of cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, and particularly, 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, disorders associated with inflammation such as Addison s disease, adult respiratory distress syndrome, allergies, anemia asthma, atherosclerosis, bronchitis, cholecystitus, Crohn s disease ulcerative colitis, atopic dermatitis, dermatomyositis. diabetes mel tus. emphysema, atrophic gastritis glomerulonephπtis. gout, Graves' disease, hypereosinophi a, irritable bowel syndrome lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or peπcardial inflammation osteoarthπtis. osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma Sjogren's syndrome, and autoimmune thyroiditis, complications of cancer, hemodialysis extracoφoreal circulation, viral, bacterial, fungal, parasitic protozoal, and helminthic infections and trauma, and disorders associated an increase in apoptosis such as AIDS and other infectious or genetic immunodeficiencies, neurodegenerative diseases such as Alzheimer^'s disease. Parkinson's disease, amyotrophic lateral sclerosis, retimtis pigmentosa and cerebellar degeneration, myelodysplastic syndromes such as aplastic anemia, lschemic injuries such as myocardial infarction, stroke, and reperfusion injury, toxin-induced diseases such as alcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseases such as cachexia, viral infections such as those caused by hepatitis B and C, and osteoporosis The polynucleotide sequences encoding

KINRELP 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 KINRELP expression Such qualitative or quantitative methods are well known in the art In a particular aspect, the nucleotide sequences encoding KINRELP may be useful in assays that detect activation or induction of various cancers, particularly those mentioned above The nucleotide sequences encoding KINRELP 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 KINRELP 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 KINRELP, 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 KINRELP 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 ot 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 KINRELP 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'->3') and another with antisense (3'<-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 KINRELP include radiolabeling or biotinylating nucleotides, coamphfication of a control nucleic acid, and standard curves onto which the experimental results are inteφolated (Melby, P C et al ( 1993) J Immunol Methods, 159 235-244, Duplaa C et al ( 1993) Anal Biochem 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 spectrophotometπc or coloπmetπc response gives rapid quantitation

In further embodiments oligonucleotides derived from any of the polynucleotide sequences described herein mav be used as probes in microarrays The microarrays can be used to monitor the expression lev el ot large numbers of genes simultaneously (to produce a transcript image), and to identify genetic v ariants mutations and polymoφhisms This information will be useful in determining gene function understanding the genetic basis of disease, diagnosing disease, and in developing and monitoring the activity of therapeutic agents

In one embodiment the microarray is prepared and used according to the methods described in PCT application W095/1 1995 (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 incoφorated herein in their entirety by reference

The microarray is preterablv composed of a large number of unique, single-stranded nucleic acid sequences, usuallv either synthetic antisense oligonucleotides or fragments of cDNAs fixed to a solid support Microarrays 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 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 ate 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 oligomers 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 W095/251 1 16 (Baldeschweiler et al ) which is incoφorated herein in its entirety by reference In another aspect, a "gπdded ' 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 devises (slot blot or dot blot apparatus) materials and machines (including robotic instruments) and contain grids of 8 dots 24 dots 96 dots, 384 dots, 1536 dots or 6144 dots, or any other multiple which lends itself to the efficient use of commercially available instrumentation In ordei 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 ot less complementarity After removal of nonhybπdized 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 on the sequences, mutations, variants, or polymoφhisms among samples In another embodiment of the invention, the nucleic acid sequences which encode

KINRELP may also be used to generate hybridization probes which aie 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 PI 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, NY) 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 KINRELP 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 ot another mammalian species such as mouse, may reveal associated markers even if the number or arm ot 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 1 lq22-23 (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, KINRELP, its catalytic or lmmunogenic fragments or ohgopeptides 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 KINRELP 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 KINRELP 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 KINRELP, or fragments thereof, and washed Bound KINRELP is then detected by methods well known in the art Purified KINRELP 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 KINRELP specifically compete with a test compound for binding KINRELP In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with KINRELP

In additional embodiments the nucleotide sequences which encode KINRELP 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 puφose of limiting the invention

EXAMPLES I HYPONOB01 cDNA Library Construction

The hypothalamic cDNA library, HYPONOB01 was constructed using RNA isolated from the hypothalamus tissue ot 51 male and female Caucasian donors 16 to 75 years old (CLON 6579-2 Clontech, Palo Alto, CA) cDNA synthesis was initiated using a combination of ohgo(dT) and random priming Double-stranded cDNA was blunted, hgated to EcoRI adaptors, digested with Xhol, size-selected, and cloned into the Xhol and EcoRI sites of the Lambda Uni- ZAP™ vector system (Stratagene, La Jolla, CA)

The cDNA library can be screened with either DNA probes or antibody probes and the pBluescπpt® phagemid (Stratagene) can be rapidly excised in vivo The custom-constructed library phage particles were transfected into E coll host strain XL 1 -Blue® (Stratagene) Alternative unidirectional vectors include but are not limited to pcDNAI (Invitrogen, San Diego, CA) and pSHlox- 1 (Novagen, Madison, WI)

II Isolation and Sequencing of cDNA Clones The phagemid forms of individual cDNA clones were obtained by the in vivo excision process, in which the host bacterial strain was coinfected with both the lambda library phage and an f 1 helper phage Polypeptides derived from both the library-containing phage and the helper phage nicked the lambda DNA, initiated new DNA synthesis from defined sequences on the lambda target DNA and created a smaller, single stranded circular phagemid DNA molecule that included all DNA sequences of the pBluescπpt® plasmid and the cDNA insert The phagemid DNA was secreted from the cells, purified, and used to re-infect fresh host cells, where the double stranded phagemid DNA was produced Because the phagemid carries the gene for β-lactamase, the newly-transformed bacteria are selected on medium containing ampicillm Phagemid DNA was purified using the Magic Mimpieps™ DNA Purification System

(Promega catalogue #A7100 Promega Madison, WI) The DNA was eluted from the purification resin already prepared tor DNA sequencing and other analytical manipulations Phagemid DNA was also purified using the QIAwell-8 Plasmid, QIAwell PLUS, and QIAwell ULTRA DNA Purification System (QIAGEN, Chatsworth. CA) The DNA was eluted from the purification resin already piepared tor DNA sequencing and other analytical manipulations

The cDNA inserts from random isolates were sequenced in part Conventional enzymatic methods employ DNA polymerase Klenow fragment, Sequenase™ or Taq polymerase to extend DNA chains from an oligonucleotide primer annealed to the DNA template of interest Methods have been developed tor the use of both single- and double stranded templates The chain termination reaction products are usually electrophoresed on urea acrylamide gels and are detected either by autoradiography ( tor radionuchde-labelled precursors) or by fluorescence (for fluorescent-labelled precursors) Recent improvements in mechanized reaction preparation, sequencing and analysis using the fluorescent detection method have permitted expansion in the number of sequences that can be determined per day (such as the Applied Biosystems 373 DNA sequencer and Catalyst 800)

III Homology Searching of cDNA Clones and Their Deduced Proteins

Using the nucleotide sequences derived from the cDNA clones as query sequences (the sequences of the Sequence Listing), databases containing previously identified sequences are searched for areas of homology (similarity) Such databases include Genbank and EMBL Two homology search algorithms were used Homology algorithms help identify identical as well as non-identical sequences

The first algorithm was originally developed by D J Lipman and W R Pearson, "Rapid and Sensitive Protein Similarity Searches , ( 1985) Science 227 1435 In this algorithm, the homologous regions are searched in a two step manner In the first step, the highest homologous regions are determined by calculating a matching score using a homology score table The parameter Xtup is used in this step to establish the minimum window size to be shifted for comparing two sequences Ktup also sets the number of bases that must match to extract the highest homologous region among the sequences In this step, no insertions or deletions are applied and the homology is displayed as an initial (LNIT) value

In the second step, the homologous regions are aligned to obtain the highest matching score by inserting a gap in order to add a probable deleted portion The matching score obtained in the first step is recalculated using the homology score Table and the insertion score Table to an optimized (OPT) value in the final output

DNA homologies between two sequences can be examined graphically using the Harr method of constructing dot matrix homology plots (Needleman, S B and Wunsch C O ( 1970) J Mol Biol 48 443) This method produces a two-dimensional plot which can be useful in determining regions of homology versus regions of repetition

The second algorithm was developed by Applied Biosystems Inc and has been incoφorated into the Inherit 670 Sequence Analysis System In this algorithm Pattern Specification Language (developed by TRW Inc ) is used to determine regions of homology There are three parameters that determine how the sequence comparisons are run window size, window offset, and error tolerance Using a combination of these three parameters the DNA database is searched for sequences containing regions of homology and the appropriate sequences are scored with an initial value Subsequently, these homologous regions are examined using dot matrix homology plots to determine regions of homology versus regions of repetition Smith- Waterman alignments were used to display the results of the homology search Following the search for homologous regions, the sequences from the cDNA clones were classified as to whether they are exact matches (regions of exact homology) homologous human matches (regions of high similarity, but not exact matches), homologous non-human matches (regions of high similarity present in species other than human), or nonmatches (no significant regions of homology to previously identified nucleotide sequences) Searches of the deduced polypeptides and peptides are done in a manner analogous to that done with the cDNA sequences The sequence of the polypeptide is used as a query sequence and compared to the previously identified sequences contained in a database such as Swiss/Prot or the NBRF Protein database to find homologous polypeptides These polypeptides are initially scored for homology using a homology score Table (Orcutt, B C and Dayhoff, M O Scoring Matrices, PIR Report MAT - 0285 (February 1985)) resulting in an INIT score The homologous regions are aligned to obtain the highest matching scores by inserting a gap which adds a probable deleted portion The matching score is recalculated using the homology score Table and the insertion score Table resulting in an optimized (OPT) score Even in the absence of knowledge of the proper reading frame of an isolated sequence, the above-described polypeptide homology search may be performed by searching all 3 reading frames

Peptide and protein sequence homologies can also be ascertained using the Inherit 670 Sequence Analysis System in an analogous way to that used in DNA sequence homologies Pattern Specification Language and parameter windows are used to search polypeptide databases for sequences containing regions of homology which are scored with an initial value Subsequent examination with a dot-matrix homology plot determines regions of homology versus regions of repetition

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 using BLAST (Altschul S F 1993 and 1990, supra) are used to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQ™ database (Incyte Pharmaceuticals) This analysis is much faster than multiple, membrane-based hybridizations In addition, the sensitivity ot 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 % sequence identity x % maximum BLAST score

100 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 KINRELP 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 KINRELP Encoding Polynucleotides

The nucleic acid sequence of the Incyte Clone 93542 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 amphcons containing new, unknown nucleotide sequence for the region of interest The initial primers were designed from the cDNA using OLIGO 4 06 (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°to about 72° C Any stretch of nucleotides which would result in haiφin structures and primer-primer dimeπzations 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, MA) and the following parameters

Step 1 94° C for 1 min (initial denaturation) Step 2 65° C for 1 min

Step 3 68 ° C for 6 m

Step 4 94° C for 15 sec

Step 5 65 ° C for 1 mm

Step 6 68° C for 7 mm Step 7 Repeat step 4-6 for 15 additional cycles

Step 8 94° C for 15 sec

Step 9 65° C for 1 mm

Step 10 68° C for 7 15 mm

Step 1 1 Repeat step 8-10 for 12 cycles Step 12 72° C for 8 mm

Step 13 4° C (and holding)

A 5-10 μl 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™ (QIAGEN Inc , Chatsworth, CA), and trimmed of overhangs using Klenow enzyme to facilitate rehgation and cloning After ethanol precipitation, the products were redissolved in 13 μl of hgation buffer, lμl

T4-DNA ligase (15 units) and lμl T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2-3 hours or overnight at 16° C Competent E_ coh cells (in 40 μl of appropriate media) were transformed with 3 μl of hgation mixture and cultured in 80 μl of SOC medium (Sambrook et al . supra) After incubation for one hour at 37° C, the E_ coh mixture was plated on Luna Bertani (LB)-agar (Sambrook et al , supra) containing 2x Carb The following day, several colonies were randomly picked from each plate and cultured in 150 μl of liquid LB/2x Carb medium placed in an individual well of an appropriate, commercially- available, sterile 96-well microtiter plate The following day, 5 μl of each overnight culture was transferred into a non-sterile 96 well plate and after dilution 1 10 with water 5 μl of each sample was transferred into a PCR array

For PCR amplification 18 μl of concentrated PCR reaction mix (3 3x) 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° C for 60 sec

Step 2 94° C for 20 sec

Step 3 55 ° C for 30 sec

Step 4 72° C for 90 sec

Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6 72° C for 180 sec

Step 7 4° 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, hgated into plasmid, and sequenced

In like manner, the nucleotide sequence of SEQ ID NO 2 is 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 ED NO:2 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 (National Biosciences), labeled by combining 50 pmol of each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN^®, Boston, MA). The labeled oligonucleotides are substantially purified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn). A aliquot containing 10⁷ 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 Rl, Pst I, Xba 1, or Pvu II; DuPont NEN^®).

The DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film (Kodak, Rochester, NY) is exposed to the blots in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale, CA) for several hours, hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, the nucleotide sequence described herein is examined using a computer algorithm which starts 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 would interfere with hybridization. The algorithm identifies 20 sequence-specific oligonucleotides of 20 nucleotides in length (20-mers). A matched set of oligonucleotides is created in which one nucleotide in the center of each sequence is altered. This process is repeated for each gene in the microarray, and double sets of twenty 20 mers are synthesized and arranged on the surface of the silicon chip using a light-directed chemical process (Chee, M. et al., PCT/W095/1 1995, incoφorated herein by reference).

In the alternative, a chemical coupling procedure and an ink jet device are used to synthesize oligomers on the surface of a substrate (Baldeschweiler, J.D. et al., PCT/W095/251 16, incoφorated herein by reference). In another alternative, a "gridded" array analogous to a dot (or slot) blot is 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 materials and machines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots After hybridization, the microarray is washed to remove nonhybπdized probes, and 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 micro-array

VIII Complementary Polynucleotides

Sequence complementary to the KINRELP-encoding sequence, or any part thereof, is used to decrease or inhibit expression of naturally occurring KINRELP 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 software and the coding sequence of KINRELP. SEQ ID NO 1 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 πbosomal binding to the KINRELP- encoding transcript

IX Expression of KINRELP

Expression of KINRELP 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 KINRELP in E coh Upstream of the cloning site, this vector contains a promoter for β-galactosidase, followed by sequence containing the amino-terminal Met, and the subsequent seven residues of β-galactosidase. Immediately following these eight residues is a bacteπophage promoter useful for transcription and a linker containing a number of unique restriction sites

Induction of an isolated, transformed bacterial strain with EPTG using standard methods produces a fusion protein which consists of the first eight residues of β-galactosidase, about 5 to 15 residues of linker, and the full length protein The signal residues direct the secretion of

KINRELP into the bacterial growth media which can be used directly in the following assay for activity X Demonstration of KINRELP Activity

KINRELP can be expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with an eukaryotic expression vector encoding KINRELP. Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression of KINRELP. Then, phase microscopy is used to compare the mitotic index of transformed versus control cells. An increase in the mitotic index indicates KINRELP activity.

XI Production of KINRELP Specific Antibodies

KINRELP 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 NO:2 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 KINRELP Using Specific Antibodies

Naturally occurring or recombinant KINRELP is substantially purified by immunoaffinity chromatography using antibodies specific for KINRELP. An immunoaffinity column is constructed by covalently coupling KINRELP 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 KINRELP is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of KINRELP (e.g., high ionic strength buffers in the presence of detergent) The column is eluted under conditions that disrupt antibody/KINRELP binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion), and KINRELP is collected

XIII Identification of Molecules Which Interact with KINRELP

KINRELP or biologically active fragments thereof are labeled with ¹²⁵I 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 KINRELP, washed and any wells with labeled KINRELP complex are assayed Data obtained using different concentrations of KINRELP are used to calculate values for the number, affinity, and association of KINRELP with the candidate molecules

All publications and patents mentioned in the above specification are herein incoφorated 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

(i) APPLICANT: INCYTE PHARMACEUTICALS, INC. (ii) TITLE OF THE INVENTION: KINESIN-RELATED PROTEIN

(iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Incyte Pharmaceuticals, Inc.

(B) STREET: 3174 Porter Drive

(C) CITY: Palo Alto

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 94304

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (3) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) PCT APPLICATION NUMBER: To Be Assigned

(B) FILING DATE: Filed Herewith

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/878,865

(B) FILING DATE: 19 June 1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Billings, Lucy J.

(B) REGISTRATION NUMBER: 36,749

(C) REFERENCE/DOCKET NUMBER: PF-0324 PCT

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 650-855-0555 (3) TELEFAX: 650-845-4166

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 679 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: HYPONOB01

(B) CLONE: 93542 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

Met Val Thr Ser Leu Asn Glu Asp Asn Glu Ser Val Thr Val Glu Trp

1 5 10 15 lie Glu Asn Gly Asp Thr Lys Gly Lys Glu Thr Asp Leu Glu Ser lie

20 25 30

Phe Ser Leu Asn Pro Asp Leu Val Pro Asp Glu Glu lie Glu Pro Ser

35 40 45

Pro Glu Thr Pro Pro Pro Pro Ala Ser Ser Ala Lys Val Asn Lys lie

50 55 60

Val Lys Asn Arg Arg Thr Val Ala Ser lie Lys Asn Asp Pro Pro Ser 65 70 75 80

Arg Asp Asn Arg Val Val Gly Ser Ala Arg Ala Arg Pro Ser Gin Phe

85 90 95

Pro Glu Gin Ser Ser Ser Ala Gin Gin Asn Gly Ser Val Ser Asp lie

100 105 110

Ser Pro Val Gin Ala Ala Lys Lys Glu Phe Gly Pro Pro Ser Arg Arg

115 120 125

Lys Ser Asn Cys Val Lys Glu Val Glu Lys Leu Gin Glu Lys Arg Glu

130 135 140

Lys Arg Arg Leu Gin Gin Gin Glu Leu Arg Glu Lys Arg Ala Gin Asp 145 150 155 160

Val Asp Ala Thr Asn Pro Asn Tyr Glu lie Met Cys Met lie Arg Asp

165 170 175

Phe Arg Gly Ser Leu Asp Tyr Arg Pro Leu Thr Thr Ala Asp Pro lie

180 185 190

Asp Glu His Arg lie Cys Val Cys Val Arg Lys Arg Pro Leu Asn Lys

195 200 205

Lys Glu Thr G n Met Lys Asp Leu Asp Val lie Thr lie Pro Ser Lys

210 215 220

Asp Val Val Met Val His Glu Pro Lys Gin Lys Val Asp Leu Thr Arg 225 230 235 240

Tyr Leu Glu Asn Gin Thr Phe Arg Phe Asp Tyr Ala Phe Asp Asp Ser

245 250 255

Ala Pro Asn Glu Met Val Tyr Arg Phe Thr Ala Arg Pro Leu Val Glu

260 265 270

Thr lie Phe Glu Arg Gly Met Ala Thr Cys Phe Ala Tyr Gly Gin Thr

275 280 285

Gly Ser Gly Lys Thr His Thr Met Gly Gly Asp Phe Ser Gly Lys Asn

290 295 300

Gin Asp Cys Ser Lys Gly lie Tyr Ala Leu Ala Ala Arg Asp Val Phe 305 310 315 320

Leu Met Leu Lys Lys Pro Asn Tyr Lys Lys Leu Glu Leu Gin Val Tyr

325 330 335

Ala Thr Phe Phe Glu lie Tyr Ser Gly Lys Val Phe Asp Leu Leu Asn

340 345 350

Arg Lys Thr Lys Leu Arg Val Leu Glu Asp Gly Lys Gin Gin Val Gin

355 360 365

Val Val Gly Leu Gin Glu Arg Glu Val Lys Cys Val Glu Asp Val Leu

370 375 380

Lys Leu lie Asp lie Gly Asn Ser Cys Arg Thr Ser Gly Gin Thr Ser 385 ^" 390 395 400

Ala Asn Ala His Ser Ser Arg Ser His Ala Val Phe Gin lie lie Leu

405 410 415

Arg Arg Lys Gly Lys Leu His Gly Lys Phe Ser Leu lie Asp Leu Ala

420 ' 425 430

Gly Asn Glu Arg Gly Ala Asp Thr Ser Ser Ala Asp Arg Gin Thr Arg

435 440 445

Leu Glu Gly Ala Glu lie Asn Lys Ser Leu Leu Ala Leu Lys Glu Cys

450 455 460 lie Arg Ala Leu Gly Arg Asn Lys Pro His Thr Pro Phe Arg Ala Ser 465 470 475 480

Lys Leu Thr Gin Val Leu Arg Asp Ser Phe lie Gly Glu Asn Ser Arg

485 490 495

Thr Cys Met lie Ala Thr lie Ser Pro Gly Met Ala Ser Cys Glu Asn

500 505 510

Thr Leu Asn Thr Leu Arg Tyr Ala Asn Arg Val Lys Glu Leu Thr Val 515 520 525

Asp Pro Thr Ala Ala Gly Asp Val Arg Pro lie Met His His Pro Pro

530 535 540

Asn Gin lie Asp Asp Leu Glu Thr Gin Trp Gly Val Gly Ser Ser Pro 545 550 555 560

Gin Arg Asp Asp Leu Lys Leu Leu Cys Glu Gin Asn Glu Glu Glu Val

565 570 575

Ser Pro Gin Leu Phe Thr Phe His Glu Ala Val Ser Gin Met Val Glu

580 585 590

Met Glu Glu Gin Val Val Glu Asp His Arg Ala Val Phe Gin Glu Ser

595 600 605 lie Arg Trp Leu Glu Asp Glu Lys Ala Leu Leu Glu Met Thr Glu Glu

610 615 620

Val Asp Tyr Asp Val Asp Ser Tyr Ala Thr Gin Leu Glu Ala lie Leu 625 630 635 640

Glu Gin Lys lie Asp lie Leu Thr Glu Leu Arg Asp Lys Val Lys Ser

645 650 655

Phe Arg Ala Ala Leu Gin Glu Glu Glu Gin Ala Ser Lys Gin lie Asn

660 665 670

Pro Lys Arg Pro Arg Ala Leu 675

(2) INFORMATION FOR SEQ ID NO : 2 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2956 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: 93542

(B) CLONE: HYPONOB01

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

GCAACGGCCA ACTTCGGCAA GATCCAGATC GGGATTTACG TGGAGATCAA GCGCAGTTAG 60

GCCGAATACA TCAAGCAATG GTAACATCTT TAAATGAAGA TAATGAAAGT GTAACTGTTG 120

AATGGATAGA AAATGGAGAT ACAAAAGGCA AAGAGACTGA CCTGGAGAGC ATCTTTTCAC 180

TTAACCCTGA CCTTGTTCCT GATGAAGAAA TTGAACCCAG TCCAGAAACA CCTCCACCTC 240

CAGCATCCTC AGCCAAAGTA AACAAAATTG TAAAGAATCG ACGGACTGTA GCTTCTATTA 300

AGAATGACCC TCCTTCAAGA GATAATAGAG TGGTTGGTTC AGCACGTGCA CGGCCCAGTC 360

AATTTCCTGA ACAGTCTTCC TCTGCACAAC AGAATGGTAG TGTTTCAGAT ATATCTCCAG 420

TTCAAGCTGC AAAAAAGGAA TTTGGACCCC CTTCACGTAG AAAATCTAAT TGTGTGAAAG 480

AAGTAGAAAA ACTGCAAGAA AAACGAGAGA AAAGGAGATT GCAACAGCAA GAACTTAGAG 540

AAAAAAGAGC CCAGGACGTT GATGCTACAA ACCCAAATTA TGAAATTATG TGTATGATCA 600

GAGACTTTAG AGGAAGTTTG GATTATAGAC CATTAACAAC AGCAGATCCT ATTGATGAAC 660

ATAGGATATG TGTGTGTGTA AGAAAACGAC CACTCAATAA AAAAGAAACT CAAATGAAAG 720

ATCTTGATGT AATCACAATT CCTAGTAAAG ATGTTGTGAT GGTACATGAA CCAAAACAAA 780

AAGTAGATTT AACAAGGTAC CTAGAAAACC AAACATTTCG TTTTGATTAT GCCTTTGATG 840

ACTCAGCTCC TAATGAAATG GTTTACAGGT TTACTGCTAG ACCACTAGTG GAAACTATAT 900

TTGAAAGGGG AATGGCTACA TGCTTTGCTT ATGGGCAGAC TGGAAGTGGA AAAACTCATA 960

CTATGGGTGG TGACTTTTCA GGAAAGAACC AAGATTGTTC TAAAGGAATT TATGCATTAG 1020

CAGCTCGAGA TGTCTTTTTA ATGCTAAAGA AGCCAAACTA TAAGAAGCTA GAACTTCAAG 1080

TATATGCAAC CTTCTTTGAA ATTTATAGTG GAAAGGTGTT TGACTTGCTA AACAGGAAAA 1140

CAAAATTAAG AGTTCTAGAA GATGGAAAAC AGCAGGTTCA AGTGGTGGGA TTACAGGAAC 1200

GGGAGGTCAA ATGTGTTGAA GATGTACTGA AACTCATTGA CAT GGCAAC AGTTGCAGAA 1260

CATCCGGTCA AACATCTGCA AATGCACATT CATCTCGGAG CCATGCAGTG TTTCAGATTA 1320

TTCTTAGAAG GAAAGGAAAA CTACATGGCA AATTTTCTCT CATTGATTTG GCTGGAAATG 1380

AAAGAGGAGC TGATACTTCC AGTGCGGACA GGCAAACTAG GCTTGAAGGT GCTGAAATTA 1440

ATAAAAGCCT TTTAGCACTC AAGGAGTGCA TCAGAGCCTT AGGTAGAAAT AAACCTCATA 1500

CTCCTTTCCG TGCAAGTAAA CTCACTCAGG TGTTAAGAGA TTCTTTCATA GGTGAAAACT 1560

CTCGTACCTG CATGATTGCC ACAATCTCTC CAGGAATGGC ATCCTGTGAA AATACTCTTA 1620

ATACATTAAG ATATGCAAAT AGGGTCAAAG AATTGACTGT AGATCCAACT GCTGCTGGTG 1680

ATGTTCGTCC AATAATGCAC CATCCACCAA ACCAGATTGA TGACTTAGAG ACACAGTGGG 1740

GTGTGGGGAG TTCCCCTCAG AGAGATGATC TAAAACTTCT TTGTGAACAA AATGAAGAAG 1800

AAGTCTCTCC ACAGTTGTTT ACTTTCCACG AAGCTGTTTC ACAAATGGTA GAAATGGAAG 1860 AACAAGTTGT AGAAGATCAC AGGGCAGTGT TCCAGGAATC TATTCGGTGG TTAGAAGATG 1920

AAAAGGCCCT CTTAGAGATG ACTGAAGAAG TAGATTATGA TGTCGATTCA TATGCTACAC 1980

AACTTGAAGC TATTCTTGAG CAAAAAATAG ACATTTTAAC TGAACTGCGG GATAAAGTGA 2040

AATCTTTCCG TGCAGCTCTA CAAGAGGAGG AACAAGCCAG CAAGCAAATC AACCCGAAGA 2100

GACCCCGTGC CCTTTAAACC GGCATTTGCT GCTAAAGGAT ACCCAGAACC CTCACTACTG 2160

TAACATACAA CGGTTCAGCT GTAAGGGCCA TTTGAAAGTT TGGAATTTTA AGTGTCTGTG 2220

GAAAATGTTT TGTCCTTCAC CTGAATTACA TTTCAATTTT GTGAAACACT CTTTTGTCTA 2280

CAAAATGCTT CTAGTCCAGG AGGCACAACC AAGAACTGGG ATTAATGAAG CATTTTGTTT 2340

CATTTACACA AATAGTGATT TACTTTTGGA GATCCTTGTC AGTTTTATTT TCTATTTGAT 2400

GAAGTAAGAC TGTGGACTCA ATCCAGAGCC AGATAGTAGG GGGAAGCCAC AGCATTTCCT 2460

TTTAACTCAG TTCAATTTTT GTAGTGAGAC TGAGCAGTTT TAAATCCTTT GCGTGCATGC 2520

ATACCTCATC AGTGATTGTA CATACCTTGC CCACTCCTAG AGACAGCTGT GCTCACCTTT 2580

TCCTGCTTTG TGCCTTGATT AAGGCTACTG ACCCTAAATT TCTGAAGCAC AGCCAAGAAA 2640

AATTACATTC CTTGTCATTG TAAATTACCT TTGTGTGTAC ATTTTTACTG TATTTGAGAC 2700

ATTTTTTGTG TGTGACTAGT TAATTTTGCA GGATGTGCCA TATCATTGAA CGGAACTAAA 2760

GTCTGTGACA GTGGATATAG CTGCTGGACC ATTCCATCTT ATATGTAAAG AAATCTGGAA 2820

TTATTATTTT AAAACCATAT AACATGTGAT TATAATTTTT CTTAGCATTT TCTTTGTAAA 2880

GAACTACAAT ATAAACTAGT TGGTGTATAA TAAAAAGTAA TGAAATTCTG AGAAGAGTTT 2940

TATCTTAGGA AAATAC 2956

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 716 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE.

(A) LIBRARY: GenBank

(B) CLONE: 220468

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

Met Val Thr Ser Leu Asn Glu Asp Asn Glu Ser Val Thr Val Glu Trp

1 5 10 15 lie Glu Asn Gly Asp Thr Lys Gly Lys Glu lie Asp Leu Glu Ser lie

20 25 30

Phe Ser Leu Asn Pro Asp Leu Val Pro Asp Glu Asp lie Glu Pro Ser

35 40 45

Pro Glu Leu Pro Pro Pro Ser Ser Ser Ser Lys Val Asn Lys lie Val

50 55 60

Lys Asn Arg Arg Thr Val Ala Ala Val Lys Asn Asp Pro Pro Pro Arg 65 70 75 80

Asp Asn Arg Val Val Gly Ser Ala Arg Ala Arg Pro Ser Gin Leu Pro

85 90 95

Glu Gin Ser Ser Ser Ala Gin Gin Asn Gly Ser Val Ser Asp lie Ser

100 105 110

Pro Val Gin Ala Ala Lys Lys Glu Phe Gly Pro Pro Ser Arg Arg Lys

115 120 125

Ser Asn Cys Val Lys Glu Val Glu Lys Leu Gin Glu Lys Arg Glu Lys

130 135 140

Arg Arg Leu Gin Gin Gin Glu Leu Arg Glu Lys Arg Ala Gin Asp Val 145 150 155 160

Asp Ala Thr Asn Pro Asn Tyr Glu lie Met Cys Met lie Arg Asp Phe

165 170 175

Arg Gly Ser Leu Asp Tyr Arg Pro Leu Thr Thr Ala Asp Pro lie Asp

180 185 190

Glu His Arg lie Cys Val Cys Val Arg Lys Arg Pro Leu Asn Lys Lys

195 200 205

Glu Thr Gin Met Lys Asp Leu Asp Val lie Thr lie Pro Ser Lys Asp

210 215 220

Val Val Met Val His Glu Pro Lys Gin Lys Val Asp Leu Thr Arg Tyr 225 230 235 240

Leu Glu Asn Gin Thr Phe Arg Phe Asp Tyr Ala Phe Asp Asp Ser Ala

245 250 255

Pro Asn Glu Met Val Tyr Arg Phe Thr Ala Arg Pro Leu Val Glu Thr 260 265 270 lie Phe Glu Arg Gly Met Ala Thr Cys Phe Ala Tyr Gly Gin Thr Gly

275 280 285

Ser Gly Lys Thr His Thr Met Gly Gly Asp Phe Ser Gly Lys Asn Gin

290 295 300

Asp Cys Ser Lys Gly lie Tyr Ala Leu Ala Ala Arg Asp Val Phe Leu 305 310 315 320

Met Leu Lys Lys Pro Asn Tyr Lys Lys Leu Glu Leu Gin Val Tyr Ala

325 330 335

Thr Phe Phe Glu lie Tyr Ser Gly Lys Val Phe Asp Leu Leu Asn Arg

340 345 350

Lys Thr Lys Leu Arg Val Leu Glu Asp Gly Lys Gin Gin Val Gin Val

355 360 365

Val Gly Leu Gin Glu Arg Glu Val Lys Cys Val Glu Asp Val Leu Lys

370 375 380

Leu He Asp He Gly Asn Ser Cys Arg Thr Ser Gly Gin Thr Ser Ala 385 390 395 400

Asn Ala His Ser Ser Arg Ser His Ala Val Phe Gin He He Leu Arg

405 410 415

Arg Lys Gly Lys Leu His Gly Lys Phe Ser Leu He Asp Leu Ala Gly

420 425 430

Asn Glu Arg Gly Ala Asp Thr Ser Ser Ala Asp Arg Gin Thr Arg Le__

435 440 445

Glu Gly Ala Glu He Asn Lys Ser Leu Leu Arg Leu Lys Glu Cys He

450 455 460

Arg Ala Lea Gly Arg Asn Lys Pro His Thr Pro Phe Arg Ala Ser Lys 465 470 475 480

Leu Thr Gin Val Leu Arg Asp Ser Phe He Gly Glu Asn Ser Arg Thr

485 490 495

Cys Met He Ala Thr He Ser Pro Gly Met Ala Ser Cys Glu Asn Thr

500 505 510

Leu Asn Thr Leu Arg Tyr Ala Asn Arg Val Lys Glu Phe Gly He Ser

515 520 525

Pro Ser Asp He Pro Phe Ser Gin Gly Gly Gly Ser Arg Pro Asp Leu

530 535 540

Ser Pro Ser Tyr Asp Tyr Asp Asp Phe Ser Pro Ser He Thr Arg Val 545 550 555 560

Lys Glu Leu Thr Val Asn Pro Ala Ala Ala Gly Asp Val His Pro He

565 570 575

Met His His Pro Pro Ser Gin He Asp Asp Leu Glu Thr Gin Trp Gly

580 585 590

Val Gly Ser Ser Pro Gin Arg Asp Asp Leu Lys Leu Leu Cys Glu Glr.

595 600 605

Asn Glu Giα Glu Val Ser Pro Gin Leu Phe Thr Phe His Glu Ala Val

610 615 620

Ser Gin Met Val Glu Met Glu Glu Gin Val Val Glu Asp His Arg Ala 625 630 635 640

Val Phe Gin Glu Ser He Arg Trp He Glu Asp Glu Lys Ala Leu Leu

645 650 655

Glu Met Thr Glu Glu Val Asp Tyr Asp Val Asp Ser Tyr Ala Thr Gin

660 665 670

Leu Glu Ala He Leu Glu Gin Lys He Asp He Leu Thr Glu Leu Arg

675 680 685

Asp Lys Val Lys Ser Phe Arg Ala Ala Leu Gin Glu Glu Glu Gin Ala

690 695 700

Ser Lys Gin He Asn Pro Lys Arg Pro Arg Ala Leu 705 710 715

Claims

What is claimed is:

1. A substantially purified human kinesin-related protein comprising the amino acid sequence of SEQ ID NO: 1 or fragments thereof.

2. An isolated and purified polynucleotide sequence encoding the human kinesin- related protein of claim 1.

3. A polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of claim 2.

4 A composition comprising the polynucleotide sequence of claim 2

5 An isolated and purified polynucleotide sequence comprising SEQ ID NO.2 or variants thereof

6. A composition comprising the polynucleotide sequence of claim 5

7 A polynucleotide sequence which is complementary to the polynucleotide sequence of claim 2 or variants thereof

8 A composition comprising the polynucleotide sequence of claim 7

9. An expression vector containing at least a fragment of the polynucleotide sequence of claim 2.

10. A host cell containing the vector of claim 9

1 1. A method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, or a fragment thereof, the method comprising the steps of a) culturmg the host cell of claim 10 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture. 12 A pharmaceutical composition comprising a substantially purified human kinesin- related protein having the amino acid sequence of SEQ ID NO 1 in conjunction with a suitable pharmaceutical carrier

13 A purified antibody which specifically binds to the polypeptide of claim 1

14 A purified agonist which modulates the activity of the polypeptide of claim 1

15 A purified antagonist which decreases the effect of the polypeptide of claim 1

16 A method for stimulating cell proliferation comprising administering to a cell an effective amount of the pharmaceutical composition of claim 12

17 A method for treating a disorder associated with an increase in apoptosis comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 12

18 A method for treating cancer comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 15

19 A method for treating inflammation comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 15

20 A method for detecting a polynucleotide which encodes a human kinesin-related protein in a biological sample comprising the steps of a) hybridizing the polynucleotide of claim 7 to nucleic acid material of a biological sample, thereby forming a hybridization complex, and b) detecting said hybridization complex, wherein the presence of said complex correlates with the presence of a polynucleotide encoding the human kinesin- related protein in said biological sample