AU2008201683A1 - Diagnosis and treatment of diseases associated with altered expression of HIPK1 - Google Patents

Description

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AUSTRALIA

F B RICE CO Patent and Trade Mark Attorneys Patents Act 1990 UNIVERSITY OF AARHUS COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Diagnosis and treatment of diseases associated with altered expression ofHIPK1 The following statement is a full description of this invention including the best method of performing it known to us:- 00 SMETHODS FOR DIAGNOSIS AND TREATMENT OF DISEASES ASSOCIATED WITH ALTERED EXPRESSION OF HIPKI This is a divisional of AU 2002328896, the entire contents of which are incorporated 5 herein by reference.

FIELD OF THE INVENTION 00 ID The present invention relates to methods for use in diagnosis and treatment of diseases, Sincluding lymphoma and leukemia, associated with altered gene expression of HIPK1.

00 SBACKGROUND OF THE INVENTION c Lymphomas are a collection of cancers involving the lymphatic system and are generally categorized as Hodgkin's disease and Non-Hodgkin lymphoma. Hodgkin's lymphomas are of B lymphocyte origin. Non-Hodgkin lymphomas are a collection of over 30 different types of cancers including T and B lymphomas. Leukemia is a disease of the blood forming tissues and includes B and T cell lymphocytic leukemias. It is characterized by an abnormal and persistent increase in the number of leukocytes and the amount of bone marrow, with enlargement of the spleen and lymph nodes.

Oncogenes are genes that can cause cancer. Carcinogenesis can occur by a wide variety of mechanisms, including infection of cells by viruses containing oncogenes, activation of protooncogenes in the host genome, and mutations of protooncogenes and tumor suppressor genes.

There are a number of viruses known to be involved in human cancer as well as in animal cancer. Of particular interest here are viruses that do not contain oncogenes themselves; these are slow- transforming retroviruses. They induce tumors by integrating into the host genome and affecting neighbouring protooncogenes in a variety of ways, including promoter insertion,

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00 and/or truncaUon of a protooncogene or tumor suppressor gene. The analysis of sequences at or near the Insertion sites has led to the identification of a number of new protooncogenes.

S With respect to lymphoma and leukemia. murine leukemia retrovirus (MuLV). such as SL3-3 or Akv, is a potent inducer of tumors when inoculated into susceptible newborn mice, or when carried in the \O germline. A number of sequences have been identified as relevant in the induction of lymphoma and leukemia by analyzing the insertion sites; see Sorensen et al,, J. of Virology 74:2161 (2000); Hansen et al., Genome Res. 10(2):237-43 (2000); Sorensen et al.. J. Virology 70:4063 (1996); Sorensen etal., S J. Virology 67:7118 (1993); Joosten et al.. Virology 268:308 (2000): and LI et el., Nature Genetics O 23:348 (1999); all of which are expressly incorporated by reference herein.

OO As demonstrated herein, a HIPK1 gene is also implicated In lymphomas and leukemias. HIPK1 is a member of a novel family of nuclear protein kinases that act as transcriptional co-repressors for NK class of homeoproteins (Kim YH et al.. J. Biol. Chem. 1698, 273:25875-25879). Homeoproteins are transcription factors that regulate homeobox genes, which are Involved in various developmental processes, such as pattern formation and organogenesis (McGinnis, W, and Krumlauf, Cell 1992, 68:283-302).

Homeoproteins may play a role in human disease. Aberrant expression of the NKX2-5 homeodomain transcription factor has been found to be Involved in a congenitel heart disease. (Schott. et al., Science 1998. 281:108-111).

Accordingly, it is an object of the invention to provide methods for detection and screening of drug candidates for diseases involving HIPK1, particularly with respect to lymphomas.

SUMMARY OF THE INVENTION In accordance with the objects outlined above, the present invention provides methods for screening for compositions which modulate diseases including lymphomas. Also provided herein are methods of inhibiting proliferation of a cell, preferably a lymphoma cell. Methods of treatment of diseases including lymphomas. and their diagnosis, are also provided herein.

In one aspect, a method of screening drug candidates comprises providing a cell that expresses a HIPK1 gene or fragments thereof. The method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of a HIPK1 gene.

00 0 C In one embodiment, the method of screening drug candidates includes comparing the level of expression in hie absence of the drug candidate to the level of expresslon In the presence of the drug candidate.

Also provided herein Is a method of screening for a bloactive agent capable of b(nding to a protein encoded by a HIPK1 gene, the method comprising combining a HIPK1 protein and a candidate bioactive agent. end determining the binding of the candidate agent to a HIPK1 protein.

0 S Further provided herein is a method for screening for a bioactive agent capable of modulating the

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0 activity of a protein encoded bya HIPK1 gene. In one embodiment, the method comprises combining 0 a HIPK1 protein and a candidate bioactlve agent, and determining the effect of the candidate agent on C< the bloactivity of a HIPK1 protein.

Also provided is a method of evaluating the effect of a candidate lymphoma drug comprising administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. This method may further comprise comparing the expression profile of the patient to an expression profile of a heathy Individual.

In a further espect, a method for inhibiting the activity of a protein encoded by a HIPK1 gene Is provided. In one embodiment the method comprises administering to a patient an inhibitor of a HIPK1 protein.

A method of neutralizing the effect of HIPK1 protein is also provided, Preferably, the method comprises contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.

Moreover. provided herein is a biochip comprilng a nucleic acid segment which encodes HIPK1 protein.

Also provided herein is a method for diagnosilg or determining the propensity to disease, including lymphomas. by sequencing at least one HIPK1 gene of an Individual. In yet another aspect of the invention, a method Is provided for determining HIPK1 gene copy number In an individual.

Other aspects of the Invention will become apparent to the skilled artisan by the following description of the invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a sequence associated with lymphoma. The use of oncogenic 00 0 retroviruses, whose sequences insert into the genome of the host organism resulting In lymphoma, allows the identification of host sequences Involved in lymphoma. These sequences may then be used In a number of different ways. including diagnosis, prognoals, ecreening for modulators (including both agonlets and antagonists), antibody generation (for immunotherapy and imaging), etc.

Accordingly, the present invention provides HIPK1 nucleic acid and protein sequences that are associated with lymphoma. HIPK1 nucleic acid and protein sequences as outlined herein also are cf known as SGRS29 nucleic acid and protein sequences. Association In this context means that the 00 Snucleotide or protein sequences are either differentially expressed or altered in lymphome as compared to normal lymphold tissue. As outlined below, HIPK1 sequences may be up-regulated (I.e.

C expressed at a higher level) In lymphoma, or down-regulated expressed at a lower level), in 00 lymphoma. HIPK1 sequences also include sequences which have been altered truncated O sequences or sequences with a point mutation) and show either the same expression profile or an altered profile. In a preferred embodiment, the HIPK1sequences are from humans; however, as will be appreciated by those in the art, HIPK1 sequences from other organisms may be useful in animal models of disease and drug evaluation: thus. other HIPK1 sequences are provided, from vertebrates, Including mammals, including rodents (rats, mice, hamsters, guinea pigs etc.), primates, farm animals (including sheep, goals, pigs, cows. horses, etc). HIPK1 sequences from other organisms may be obtained using the techniques outlined below.

Sequences of the invention can include both nucleic acid and amino acid sequences. In a preferred embodiment, the HIPK1 sequences are recombinant nucleic acids. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed In vitro. In general, by the manipulation of nucleic acid by polymerases and endonucleases, in a form not normally found in nature. Thus an isolated nucleic acid, In a linear form, or an expression vector formed In vitro by ligating DNA molecules that are not normally Joined, are both considered recombinant for the purposes of this invention, It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly. I.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly.

although subsequently replicated non-reombinantly, are still considered recombinent for the purposes of the invention.

Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e, through the expression of a recombinant nucleic acid as depicted above. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host. and thus may be substantially pure. For example, an isolated protein 0 Is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about more preforably at least about 5% by weight of the total protein in a given sample. A substantially pure protein comprises at least about 75% by weight of the total protein. with at least about 80% being preferred, and at least about 80% being particularly 0 preferred. The definition includes the production of a HIPK1 protein from one organism in a different organism or host cell. Altematively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that C the protein is made at increased concentration levels, Altematively, the protein may be In a form not 00 \0 normally found In nature, as In the addition of an epitope tag or amino adcd substitulions, insertions and deletions, as discussed below.

00 In a preferred embodiment, the sequences of the invention are nucleic acids. As will be appreciated by those in the art and is more fully outlined below. HIPK1 sequences are useful in a variety of applications, including diagnostic applications, which will detect naturally occurring nucleic acids, as well as screening applications: for example, biochips comprising nucleic acid probes to a HIPK1 sequences can be generated. In the broadest sense, then, by "nucleic acid or "ollgonucleotide" or grammatical equivalents herein means at least two nucleotides covalenUy linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although In some cases, au outlined below (for example in antisense applications or when a candidate agent Is a nucleic acid), nucleic acid analogs may be used that have alternate backbones, comprising, for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger.

J. Org. Chem. 35:3800 (1970): Sprinzl et al., Eur. J. Biochem, 81:579 (1977); Letslnger et al., Nucl.

Acids Res. 14:3487 (1986): Sawai et al. Chem. Lett 805 (1984). Letslnger et al., J. Am. Chem. Soc.

110:4470 (1988); and Pauwels et al.. Chemice Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991): and U.S. Patent No. 6.644.048), phosphorodithioate (Briu et al.. J.

Am. Chem. Soc. 111:2321 (1989), 0-methylphophoroamidite linkages (see Eckstein, Oligonucleotldes and Analogues; A Practical Approach. Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (19.92): Meier et al.. Chem. Int Ed. Engl.

31:1008 (1992): Nielsen. Nature. 365:566 (1993); Carlsson et al., Nature 380:207 (1996). all of which are incorporated by reference). Other analog nucleic acds Include those with positive backbones (Denpcy et al.. Proc. Natl. Acad. Sci. USA 92:6097 (1995): non-ionic backbones Patent Nos.

5.386,023. 5,637,684, 5.602.240, 5.216.141 and 4,469.863; Kledrowshi et al.. Angew. Chem. Intl. Ed.

English 30'423 (1991); Letalnger et al., J. Am. Chem. Soc. 110:4470 (1986); Letsinger et al., Nucleoside Nucleotide 13:1697 (1994): Chapters 2 and 3, ASC Symposium Series 580.

"Carbohydrate Modifications In Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic Medicinal Chem. Lett. 4:395 (1994): Jeffs et al., J. Blomolecular NMR 34:17 (1994): Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones. Including those described in U.S.

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SPatent Nos. 5.235.033 and 5,034.506. and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antieense Research". Ed, Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within one defniltlon of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are described in Rawls, C E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example to increase the stability and half-life of such molecules in physiological C environments or as probes on a biochip.

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As will be appreciated by those In the art. all of these nucleic acid analogs may find use in the present NK Invention, In addition, mixtures of naturally occurring nucleic acids and analogs can be made; 00 alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includes peptide nucleic acid analogs.

These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes In the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4'C drop in Tm for an intemal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9'C. Similarly. due to their non-ionic nature, hybridization of the bases attached to these backbones Is relatively Insensitive to salt concentration. In addition, PNAs are not degraded by cellular enzymes, and thus can be more stable.

The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand ("Watson") also defines the sequence of the other strand ("Crick"); thus the sequences described herein also includes the complement of the sequence. The nucleic acid may be DNA. both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil. adenine. thymlne, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. As used herein, the term "nucleoslde" Includes nucleotides and nucleoside and nucleotide analogs. and modified nucleosides such as amino modified nucleosides. In addition, 'nucleoslde" Includes non-naturally occurring analog structures. Thus for example the Individual units of a peptide nucleic acid, each contalning a base, are referred to herein as a nucleoside.

A HIPK1 sequence can be initially identified by substantial nucleic acid end/or amino acid sequence 00 S homology to the HIPK1 sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and iL generally determined as outlined below, using either homology programs or hybridization conditions.

The HIPKI sequences of the Invention were identified as follows: basically. infection of mice with murine leukemia viruses (MuLV; including SL3-3. Akv and mutants thereof) resulted in lymphoma.

The HIPK1 sequences outlined herein comprise the insertion sites for the virus. In general, the retrpvirus can cause lymphoma in three basic ways: first of all, by Inserting upstream of a normally O silent host gene and activating it promoter insertion): secondly, by truncating a host gene that leads to oncogenesis; or by enhancing the transcription of a neighboring gene. For example, N retrovirus enhancers, including SL3-3, are known to act on genes up to approximately 200 kilobases 0 0 of the insertion site.

In a preferred embodiment, HIPKI sequences are those that are up-regulated in lymphoma: that is, the expression of these genes is higher in lymphoma as compared to normal lymphoid tissue of the same differentiation stage. 'Up-regulation" as used herein means at least about 50%, more preferably at least about 100%. more preferably at least about 150%. more preferably, at least about 200%, with from 300 to at least 1000% being especially preferred.

In a preferred embodiment HIPK1 sequences are those that are down-regulated in lymphoma; that is, the expression of these genes is lower In lymphoma as compared to normal lymphoid tissue of the same differentiation stage. "Down-regulation" as used herein means at least about 50%. more preferably at least about 100%, more preferably at least about 150%, more preferably, at least about 200%, with from 300 to at least 1000% being especially preferred.

In a preferred embodiment. HIPK1 sequences are those that are altered but show either the same expression profile or an altered profile as compared to normal lymphoid tissue of the same differentiation stage. "Altered HIPK1 sequences as used herein refers to sequences which are truncated, contain insertions or contain point mutations.

In its native form, HIPK1 is an intracellular protein that is localized in the nucleus. In general, intracellular proteins may be found in the cytoplasm and/or in the nucleus, Intracellular proteins are involved in all aspects of cellular function and replication (including, for example, signaling pathways); aberrant expression of such proteins results in unregulated or disregulated cellular processes. For example, many Intracellular proteins have enzymatic activity such as protein kinase activity.

phosphatldyl inositol-conjugated lipid klnase activity, protein phosphatase activity, phosphatldyl 00 inoeitol-conjugated lipid phosphatase activity, protease activity, nucleotide cyclase activity, polymerasa 0 activity and the like. Intracellular proteins also serve as docking proteins that are involved in organizing complexes of proteins, or targeting proteins to various subcellular localizations, and are O- Involved in maintaining the structural Integrity of organelles.

Intracellular proteins found in the nucleus Include DNA-birding transcription regulatory factors, or transcription factors. These proteins typically bind to specific nucleic acid sequences located in the regulatory regions of target genes and modulate the transcription of these target genes. Without being 00 bound by theory. DNA-blnding transcription factors can act, directly or indirectly, on a number of factors associated with the transcriptional apparatus including RNA polymerases and basal S transcription factors. DNA binding transcription factors can also act at a number of stages during 00 assembly of the transcriptional apparatus, initiation of transcription, and transcript elongation.

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An Increasingly appreciated concept in characterizing intracallular proteins is the presence in the proteins of one or more motifs for which defined functions have been attributed. In addition to the highly conserved sequences found in the enzymatic domain of proteins, highly conserved sequences have been identified in proteins that are involved In protein-protein. interaction. For example. Srchomology-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence dependent manner.

PTB domains, which are distinct from SH2 domains, also bind tyrosine phosphorylated targets. SH3 domains bind to proline-rich targets. In addition, PH domains, telratrlcopeptide repeats and WD domains to name only a few, have been shown to mediate protein-protein interactions. Some of these may also be involved in binding to phospholipids or other second messengers. As will be appreciated by one of ordinary skill in the art, these motifs can be identfied on the basis of primary sequence: thus, an analysis of the sequence of proteins may provide insight into both the enzymatic potential of the molecule and/or molecules with which the protein may associate.

Common protein motifs have also been identified among transcription factors and have been used to divide these factors into families. These motifs include the basic helix-loop-helix, basic leuclne zipper.

zinc finger and homeodomeln motifs, HIPK1 is known to contain several conserved domains. Including a homeoprotein interaction domain, a protein kinase domain, a PEST domain, and a YH domain enriched In tyrosine and histidine residues (Kim et al., J. Biol. Chem. 273:25875 (1998). In the mouse HIPK1 amino add sequence depicted In Table 2 as SEQ ID NO. 3, the homeoprotein interaction domain is from about amino acid 190 to about amino acid 518, the protein kinase domain is from about amino acid 581 to about amino add 848, the PEST domain is from about amino acid 890 to about amino acid 974. and the YH domain is from about amino acid 1057 to about amino acid 1210.

00 It is recognized that through recombinant techniques, HIPK1 sequences can be made lo be transmembrane proteins. Trarnmembrane proteins are molecules that span the phospholipid bilayer of a cell. They generally include approximately 20 consecutive hydrophobic amino acids that may be L followed by charged amino acids. They may have an intracellular domain, an extracellular domain, or both. The intracellular domains of such proteins may have a number of functions including those IND already described for Intracellular proteins. For example, the Intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins. Frequently the Intracellular domain of transmembrane proteins serves both roles. For example certain receptor tyroslne kinases have 00 both protein kinase activity and SH2 domains. In addition, autophosphorylation of tyrosines on the receptor molecule Itself, creates binding sites for additional SH2 domain containing proteins, 00 It will also be appreciated by those in the art that a transmembrane protein can be mads soluble by removing transmembrane sequences, for example through recombinant methods. Furthermore, C transmembrmne proteins that have been made soluble can be made to be secreted through recombinant means by adding an appropriate signal sequence.

It is further recognized that HIPK1 proteins can be made to be secreted proteins through techniques well recognized in the art the secretion of which can be either constitutive or regulated. These proteins have a signal peptide or signal sequence that targets the molecule to the secretory pathway.

In another preferred embodiment, the HIPK1 proteins are nuclear proteins, preferably transcripton factors. Transcription factors are involved in numerous physiological events and act by regulating gene expression at the transcriptional level. Transcription factors often serve as nodal points of regulation controlling multiple genes. They ar capable of effecting a multifarious change in gene expression and can integrate many convergent signals to effect such a change. Transcription factors are often regarded as "master regulators" of a particular cellular state or event. Accordingly, transcription factors have often been found to faithfully mark a particular cell state, a quality which makes them attractive for use as diagnostic markers. In addition, because of their important role as coordinators of pattems of gene expression associated with particular cell states, transcription factors are attractive therapeutic targets. Intervention at the level of transcripflonal regulation allows one to effectively target multiple genes associated with a dysfunction which fall under the regulation of a "master regulator' or transcription factor.

A HIPK1 sequence is initially identified by substantial nucleic acid and/or amino acid sequence homology to the HIPK1 sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below. using either homology programs or hybridization conditions.

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OO As used heroin, a nucleic acid is a "HIPK1 nucleic acid" If the overall homology of the nucleic acid S sequence to one of the nucleic acids of Tables 1, 2. and 3 is preferably greater than about 75%, more preferably greater than about 80%. even more preferably greater than about 85% and most preferably O greater than 90%. In some embodiments the homology will be as high as about 93 to 95 or 98%. In a preferred embodiment, the sequences which are used to determine sequence identity or similarity are S selected from those of the nucleic acids of SEQ ID NOS: 1. 2. 4. In another embodiment, the sequences are naturally occurring allelic variants of the sequences of the nucleic acids of SEQ ID NOS: 1. 2, 4. In another embodiment, the sequences are sequence variants as further described 00 herein.

Homology In this context means sequence similarity or Identity, with identity being preferred. A 00 preferred comparison for homology purposes is to compare the sequence containing sequencing errors to the correct sequence. This homology will be determined using standard techniques known in the art, including, but not limited to, the local homology algorithm of Smith Waterman, Adv. Appl.

Math. 2:482 (1981). by the homology alignment algorithm of Needleman Wunsch, J. Mol. Biol, 48:443 (1970). by the search for similarity method of Pearson Lipman. PNAS USA 85:2444 (1988).

by computerized implementations of these algorithms (GAP. BESTFIT. FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group. 575 Science Drive, Madison, W), the Best Fit sequence program described by Devereux et al., NUCl. Acid Res. 12:387-395 (1984).

preferably using the default settings, or by Inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simpllfication of the progressive alignment method of Feng Doolittle, J. Mol. Evol. 36:351-360 (1987): the method is similar to that described by Higglns Sharp CABIOS 5:151-163 (1989). Useful PILEUP parameters including a default gap weight of 3.00. a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al,, J. Mol. Biol.

215. 403-410, (1990) and Karlin et al., PNAS USA 90:6873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altechul et al., Methods in Enzymology. 266: 460-480 (1996); http://blasLwustl]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span overlap fraction 0.125, word threshold 11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence end composition of the particular database against which the sequence of interest is being searched: however, the values may be adjusted to increase sensitivity.

00 A amino acid sequence identity value is determined by the number of matching identical residues

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divided by the total number of rei;dues of the "longer" sequence in the aligned region. The "longer" sequence Is the one having the most actual residues in the aligned region (gaps Introduced by WU- Blast-2 to maximize the alignment score are ignored).

S Thus, "percent nucleic acid sequence identity" is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotlde residues of the nucleic acids of the SEQ ID NOS. 1, 2, 4. A preferred method utilizes the BLASTN module of WU-BLAST-2 sat to the default 00 parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

0 The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for 00 sequences which contain either more or fewer nucleotides than those of the nucleic acids of the SEQ NOS. 1. 2. 4. it is understood that the percentage of homology will be determined based on the Cr number of homologous nucleosides in relation to the total number of nucleosides. Thus, for example, homology of sequences shorter than those of the sequences identified herein and as discussed below, will be determined using the number ofnucleosides in the shorter sequence.

In one embodiment, the nucleic acid homology is determined through hybridization studies. Thus. for example. nucleic acids which hybridize under high stringency to the nucleic acids identified in the figures, or their complements, are considered HIPK1 sequences. High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology. ed. Ausube., et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen. Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays' (1993). Generally, stringent conditions are selected to be about 5-10'C lower than the thermal melting point (Tm) for the specific sequence at a defined Ionic strength pH. The Tm Is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 60% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm. 50% of the probes are occupied at equilibrium). Stringent conditions will be those In which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH to 8.3 and the temperature Is at least about 30'C for short probes 10 to 50 nucleotldes) and at leasl about 60'C for long probes greater than 50 nucleotides). Stringent conditions may also be achieved wilh the addition of destabilizing agents such as formamide.

-11 00 In another embodiment, less stringent hybridization conditions are used: for example, moderate or low stringency conditions may be used. as are known in the art see Maniatis and Ausubel, supra, and Cl Tijssen, supra, In addition, the HIPK1 nucleic acid sequences of the Invention include fragments of larger genes.. ie.

\0 they are nucleic acid segments. "Genes" in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions. Accordingly, as win be appreciated by those in the art using the sequences provided herein, additional sequences of HIPK1 genes can be obtained, OO using techniques well known in the art for cloning either longer sequences or the full length "C sequences: see Maniatis et al.. and Ausubel, et al., supra, hereby expressly incorporated by S reference. In general, this is done using PCR, for example, kinetic PCR.

00 Once a HIPK1 nucleic acid is Identified, it can be cloned and, if necessary, its constituent parts C recombined to form the entire HIPK1 nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment the recombinant HIPK1 nucleic acid can be further used as a probe to identify and isolate other HIPK1 nucleic acids, for example additional coding regions. It can also be used as a "precursor nucleic acid to make modified or variant HIPK1 nucleic acids and proteins.

The HIPK1 nucleic acids of the present invention are used In several ways. In a first embodiment, nucleic acid probes to a HIPK1 nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, for example for gene therapy and/or antisense applications. Alternatively. HIPK1 nucleic acids that Include coding regions of HIPK1 proteins can be put into expression vectors for the expression of HIPK1 proteins, again either for screening purposes or for administration to a patient.

In a preferred embodiment nucleic acid probes to HIPK1 nucleic acids (both the nucleic acid sequences outlined In the figures and/or the complements thereof) are made. The nucleic add probes attached to the blochlp are designed to be substantially complementary to HIPK1 nucleic acids, i.e. the target sequence (eIther the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will Interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, If the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence Is not a complementary target sequence. Thus, by "substantially complementary" herein is -12- 0 meant that the probes are sufficiently complementary to the target sequences to hybridize under 0 normal reaction conditions, particularly high stringency conditions, as outlined herein.

k A nucleic acid probe is generally single stranded but can be partially single and partially double stranded. The strandedness of the probe Is dictated by the structure, composition, and properties of Sthe target sequence. In general,. the nucleic acid probes range from about 8 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred. That is, generally whole genes are not used. In some embodiments, much Slonger nucleic acids can be used, up to hundreds of bases.

00 In a preferred embodiment, more than one probe per sequence 1o used, with either overlapping probes C or probes to different sections of the target being used. That Is. two, three, four or more probes, with 00 three being preferred, are used to build in a redundancy for a particular targeL The probes can be overlapping have some sequence in common), or separate.

As will be appreciated by those in the art, nucleic acids can be attached or immobilized to a solid support in a wide variety of ways. By "immobilized" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can be covalent or non-covalent, By "non-covalent binding" and grammatical equivalents herein Is meant one or more of either electrostatic, hydrophilic. and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as. streptavidln to the support and the noncovalent binding of the biotinylated probe to the streptevidin. By 'covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sIgma bonds, pi bonds end coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules.

Immobilization may also involve a combinatlon of covalent and non-covalent interactions.

In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip. or can be directly synthesized on the biochip.

The biochip comprises a suitable solid substrate. By "substrate' or "solid support" or other grammatical equivalents herein is meant any material that can be modified to contain dlsorete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. Ac will be appreciated by those in the art, the number of -13- 00 possible substrates are very large, and include, but are not limited to. glass and modified or 0 functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other C~ materials, polypropylene, polyethylene. polybutylene, polyurethanes, TefonJ. etc.), polysaccharides, k nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, etc. In general, Ihe substrates allow optical detection and do not appreciably fluoresce.

In a preferred embodiment the surface of the blochlp and the probe may be derivatized with chemical c functional groups for subsequent attachment of the two. Thus, for example, the blochip Is derlvatlzed 00 0C with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo C groups and thlol groups, with amino groups being particularly preferred. Using these functional r1 groups, the probes can be atetched using functional groups on the probes. For example, nucleic 00 S acids containing amino groups can be attached to surfaces comprising amino groups, for example C using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference). In addition, in some cases, additional linkers, such as alkyl groups (including substituted and heteroalkyl groups) may be used.

In this embodiment, the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5' or 3' terminus may be attached to the solid support or attachment may be via an internal nucleoside.

In an additional embodiment, the immobilization to the solid support may be very strong, yet noncovalent For example, biotinylated oligonucleotldes can be made, which bind to surfaces covalently coated with streptavidin. resulting in attachment Alternatively, the oligonucleotides may be synthesized on the surface, as is known in the art For example, photoactivation techniques utilizing photopolymerization compounds and techniques are used. In a preferred embodiment, the nucleic acids can be synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505: U.S. Patent Nos. 5.700,637 and 5,445,934; and references cited within, all of which are expressly incorporated by reference; these methods of attachment form the basis of the Affimetrix GeneChip" technology.

In addition to the solid-phase technology represented by biochip arrays, gene expression can also be quantified using liquid-phase arrays. One such system is kinetic polymerase chain reaction (PCR) Kinetic PCR allows for the simultaneous amplification and quantification of specific nucleic acid sequences. The specificity Is derived from synthetic oligonucleotide primers designed to preferentially -14- OO adhere to single-stranded nucleic acid sequences bracketing the target site. This pair of 0 oligonucleotide primers form specific, non-covalently bound complexes on each strand of the target C sequence. These complexes facilitate in vitro transcription of double-stranded DNA in opposite k orientations: Temperature cycling of the reaction mixture creates a continuous cycle of primer binding, transcription, and re-melting of the nucleic acid to individual strands. The result is an exponential increase of the target dsDNA product This product can be quantified in real time either through the use of an intercalating dye or a sequence specific probe. SYBR® Greene I, is an example of an intercalating dye, that preferentially binds to deDNA resulting in a concomitant increase in the fluorescent signal. Sequence specific probes, such as used with TaqMan* technology, consist of a

OO

.C fluorochrome and a quenching molecule covalently bound to opposite ends of an oligonucleotide. The probe is designed to selectively bind the target DNA sequence between the two primers..When the DNA strands are synthesized during the PCR reaction, the fluorochrome is cleaved from the probe by 00 0 the exonuclease activity of the polymerase resulting In signal dequenching. The probe signaling i method can be more specific than the intercalating dye method. but In each case, signal strength is proportional to the dsDNA product produced. Each type of quantification method can be used In multiwell liquid phase arrays with each well representing primers and/or probes specific to nucleic add sequences of interest. When used with messenger RNA preparations of tissues or cell lines, and an array of probe/prlmer reactions can simultaneously quantify the expression of multiple gene products of interest. See Germer, et al., Genome Res. 10:258-266 (2000); Held, C. at al.. Genome Res.

6. 986-994 (1996).

In a preferred embodiment, HIPK1 nucleic acids encoding HIPK1 proteins are used to make a variety of expression vectors to express HIPK1 proteins which can then be used in screening assays, as described below. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate Into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding a HIPK1 protein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, Include a promoter, optionally an operator sequence. and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylatdon signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it Is expressed as a preprotein that participates in the secretion of the polypeptide: a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence: or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to 00 facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leaoer, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction Ssites. If such sites do not exist, synthetic ollgonucleotlde adaptors or linkers are used in accordance with conventional practice. The transcriptional and translational regulatory nucleic acid will generally Sbe appropriate to the host cell used to express HIPK1 protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bscillus are preferably used to express a HIPK1 protein in Bacillus. Numerous types ofapproprrate expression vectors, and suitable regulatory 00 sequences are known in the art for a variety of host cells.

\0 0 In general, the transcriptional and translational regulatory sequences may include, but are not limited 00 to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences.

translational start end stop sequences, and enhancer or activator sequences. In a preferred CN embodiment the regulatory sequences include a promoter and transcriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art. end are useful in the present invention.

In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genore, and preferably two homologous sequences which flank the expression construct The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are woll known in the art and will vary with the host cell used.

The HIPK1 proteins of the present invention are produced by culturing a host cell transformed with an expression veclor containing nucleic acid encoding a HIPK1 protein, under the appropriate conditions to Induce or cause expression of HIPK1 protein. The conditions appropriate for HIPK1 protein expression will vary with the choice of the expression vector and the host cell, and will be easily -16.

00 ascertained by one skilled in the art through routine experimentation. For example, the use of O constitutive promoters in the expression vector will require optimizing the growth and proliferation of C the host cell, while the use of an inducible promoter requires the appropriate growth conditions for Sinduction. In addition, In some embodiments, the timing of the harvest is important. For example, the baculoviral systems used In Insect cell expression are lytic viruses, and thus harvest time selectior 'C can be crucial for product yield.

Appropriate host cells Include yeast, bacteria, archaebacteria, fungi, and insect, plant and animal cells, 00 including mammalian cells. Of particular interest are Drosophila melanogaster cells. Saccharomyces )O cerevisiae and other yeasts, E. coli, Bacillus subtflis, Sf9 cells, C129 cells, 293 cells, Neurospora.

SBHK. CHO, COS, HeLa cells, THP1 cell line (a macrophage cell line) and human cells and cell lines.

(NO

00 0 In a preferred embodiment, HIPK1 protein is expressed in mammalian cells. Mammalian expression K systems are also known in the art, and include retrovlral systems. A preferred expression vector system is a retroviral vector system such as Is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumorvlrus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter.

and the CMV promoter. Typically, transcription termination and polyadenylatlon sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. Examples of transcription terminator and polyadenylatlon signals include those derived form The methods of Introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art. and will vary with the host cell used. Techniques include dextran-mediated transfection. calcium phosphale precipitation, polybrene mediated transfection, protoplast fusion.

eleclroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

In a preferred embodiment. HIPK1 proteins are expressed in bacterial systems. Bacteral expression systems are well known in the art. Promoters from bacteriophage may also be used and are known In the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence. an efficient ribosome binding site is desirable. The expression vector may also include a signal peptide sequence -17that provides for secretion of HIPK1 protein in bacteria. The protein is either secreted into the growth 00 0 media (gram-positive bacteria) or Into the periplasmic space, located between the inner and outer c membrane of the cell (gram-negative bacteria). The bacterial expression vector may also Include a selectable marker gene to allow for the selection of bacterial strains that have been transformed.

Suitable selection genes include genes which render the baoteria resistant to drugs such as ampicillin, chloramphenicol, erythromycln, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidlne. tryptophan and leucine blosynthetic pathways. These components are assembled into expression vectors. Expresslon vectors for bacteria C are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and 00 0 Streptococcus lividens, among others. The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, CK and others.

00 In one embodiment. HIPK1 proteins are produced in insect cells. Expression vectors for the transformation of insect cells; and in particular, beculovirus-based expression vectors, are well known in the art.

In a preferred embodiment, HIPK1 protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candlda alb/cans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytlca.

HIPK1 protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, for the creation of monoclonal antibodies. If the desired epitope is small, a HIPK1 protein may be fused to a carrier protein to form an immunogen. Alternatively, a HIPK1 protein may be made as a fusion protein to increase expression, or for other reasons. For example, when a HIPK1 protein is a HIPK1 peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes, In one embodiment, the HIPK1 nucleic acids, proteins and antibodies of the invention are labeled. By "labeled" herein is meant that a compound has at least one element isotope or chemical compound attached to enable the detectlon of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy Isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes The labels may be incorporated into a HIPK1 nucleic acids, proteins and antibodies at any position. For example, the label should be capable of producing, either directly or indirectly, a detectable signal. The detectable moiety may be a radioisotope. such as P. or a fluorescent or chemiluminescent compound, such as -19- 00 luorescein isothiocyanate. rhodamlne, or luclferin, or an enzyme, such as alkaline phosphetase, beta- 0 galactosldase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962): David at al., Biochemistry. 13:1014 (1974): Pain et al.. J. Immunol. Meth.. 40:219 (1981); and SNygren, J. Histochem. and Cytochen., 30:407 (1982).

\N

Accordingly, the present invention also provides HIPK1 protein sequences. A HIPK1 protein of the present invention may be identified in several ways. "Protein" in this sense includes proteins, 00 polypeptides. and peptides. As will be appreciated by those in the art, the nucleic acid sequences of Sthe invention can be used to generate protein sequences. There are a variety of ways to do this, 0 including cloning the entire gene and verifying its frame and amino acid sequence, or by oomparing it 00 to known sequences to search for homology to provide a frame, assuming a HIPK1 protein has homology to some protein in the database being used. Generally, the nucleic acid sequences are C' input into a program that will search all three frames for homology. This Is done in a preferred embodiment using the following NCBI Advanced BLAST parameters. The program Is blastx or blastn.

The database is nr. The input data is as "Sequence in FASTA format". The organism list is "none".

The "expect" Is 10; the filter is default The "descriptions' Is 500. the "alignments" is 600, and the "alignment view" is pairwise. The 'Query Genetic Codes" is standard The matrix Is BLO5UM62; gap existence cost is 11, per residue gap cost is 1; and the lambda ratio is .85 default This results in the generation of a putative protein sequence.

Also included within one embodiment of HIPKI proteins are amino acid variants 6f the naturally occurring sequences, as determined herein. Preferably, the variants are preferably greater than about homologous to the wild-type sequence. more preferably greater than about 80%. even more preferably greater than about 85% and most preferably greater than 90%. In some embodiments the homology will be as high as about 93 to 95 or 98%. As for nucleic acids, homology in this context means sequence similarity or Identity, with identity being preferred. This homology will be determined using standard techniques known in the art as are outlined above for the nucleic acid homologies.

HIPK1 proteins of the present invention may be shorter or longer than the wild type amino acid sequences. Thus, in a preferred embodiment, included within the definition of HIPK1 proteins are portions or fragments of the wild type sequences herein. In addition, as outlined above, the HIPK1 nucleic acids of the invention may be used to obtain additional coding regions, and thus additional protein sequence, using techniques known in the art In a preferred embodiment, the HIPK1 proteins are derivative or variant HIPK1 proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative HIPK1 peptide will -19- 00 contain at least one amino aoid substitution, deletion or insertion, with arino acid substitutions being particularly preferred. The amino acid substitution, insertion or deletion may occur at any residue within a HIPK1 peptlde.

Also included in an embodiment of HIPK1 proteins of the present Invention are amino ecid sequence \0 variants. These variants fall Into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding a HIPK1 protein, using cassette or PCR mutagenesis or other techniques well known In the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell \0 culture as outlined above. However, variant HIPK1 protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nalure of the variation, a feature that sets them apart 0 from naturally occurring allellc or interspecies variation ofa HIPK1 protein amino acid sequence. The C= variants typically exhibit the seme qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as wil be more fully outlined below.

While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per so need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed HIPK1 variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites In DNA having a known sequence are well known. for example, M13 primer mutagenesis and LAR mutagenesis. Screening of the mutants is done using assays of HIPK1 protein activities.

Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although In some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the elteratlon of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of a HIPK1 protein are desired, substitutions are generally made In accordance with the following chart 00 Chart I O Original Residue Exemplary Substitutions C Ala Ser Arg Lys Asn Gin. His Asp Glu 0 Cys Ser Gin Asn Glu Asp Gly Pro His Asn, Gin 00 lie Leu, Val Leu lie, Val Lys Arg, Gin, Glu S Met Leu, lie C Phe Met, Leu, Tyr 00 Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ila, Leu Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone In the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the aide chain. The substitutions which in general are expected to produce the greatest changes In the polypeptide's properties are those In which a hydrophilic residue, e.g. seryl or threonyl is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl. valyl or alanyl; a cysteine or proline is substituted for (or by) any other residue; a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or espartyl: or a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain. e.g. glycine.

The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the 3 characteristics of the HIPK1 proteins as needed. Alternatively, the variant may be designed such that the biological activity of a HIPK1 protein is altered. For example, glycosylation lstes may be altered or removed, dominant negative mutations created, etc.

Covalent modifications of HIPK1 polypeptides are included within the scope of this invention, for example for use in screening. One type of covalent modification includes reacting targeted amino acid 3 residues of a HIPK1 polypeptide with an organic derivatizing agent that is capable of reacting with -21- 00 selected side chains or the N-or C-terminal residues of an HI-PKl polypeptlde. Derivatization with bifunctional agents is useful, for instance, for crosslinking -IiPKI to a water-insoluble support Matrix or ri surface for use in'the method for pur ifying anti-HIPK1. antibodies or screening assays, as is more flly described below. Commonly used crosslinking agents include, 1.1-bis(dlazoacety)...2 phenyisthane, glutaraidehyde,. N-hydroxysuccinimide esters. for example, esters with 4-azidosalicylic INO acid, homobifunctional imidloesters, including disuccinimidyl esters such as 3.3'dithiobis(succinimidylproplonate). bifunctionai maleimideS such as bis-N-meleimido. .8-octane and agents such as methyl-3-[(P-ezidophenylI)dithiolpro pioim'd ate.

00 Other modifications include deemidation of giutaminyl and asparaginyl residues to the corresponding S glutamyl and aspartyi residues, respectively, hydroxylation of proline and lysine, phosphorylation of 00 hydroxyl groups of seryl, throonyi or tyrosyl residues. methylatiori of the a-amino groups of lysine, arginine, and hiatidine side chains Creig hton, Proteins: Structure and Molecular Properties, W.H.

Freeman Co., San Francisco, pp. 79-86 (1983)), acetyistion of the N-terminai amine, and amidation of any C-terminai carboxyl group.

Another type of covalent modification of a HIPK1 poiypeptide included within the scope of this invention comprises altering the native giycosylation pattern, of the polypeptide. "Altertng the native 9 lycosylation pattern" is intended for purposes herein to mean deleting one or more csrbohydrate moieties found in native sequence HIPKI polypeptide. and/or adding one or more glycosylation sites that are not present in the native sequence HIPK1 polypeptide.

Addition of glycosyiation sites to HIPKI polypeptides may be accomplished by altering the amnino acid sequence thereof. The alteration may be made, for example, by the addition of. or substitution by. one or more serine or threonine residues to the native sequence HIMK polypeptide (for O-linked glycosylation sites). A HIPIK1 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding a HIPKI polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

Another means of Increasing the number of carbohydrate moieties on a HIP) i potypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described In the a rt in WO 67/05330 published 11 September 1987, and in Apiin and Wriston, LA Crit. Rev.

Biochem., pp. 259-306 (198 1).

Removal of carbohydrate moieties present on a HIPKI polypeptide may be accompiished chemically or enzymaticaly or by mutational substtution of .codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, -22 00 for instance, by Haklmuddln, et.el., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.

Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidase. as descrlbad by Thotakura et al., Meth, SEnzymol.. 138:350 (1987).

O Another type of covalent modification of HIPK1 comprises linking a HIPK1 polypepbde to one of a variety of nonproteinaceous polymers, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes. in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689: 4,301,144: 00 4.670.417; 4,791,192 or 4,179.337.

\O

0 HIPK1 polypeptides of the present invention may also be modified in a way to form chimeric molecules 00 comprising a HIPK1 polypeptide fused to another, heterologous polypeptide or amino acid sequence.

S Inr one embodiment such a chimeric molecule comprises a fusion of a HIPK1 polypeptide with a tag CN polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope Lag is generally placed at the amino-or carboxyl-terminus of a HIPK1 polypeptide. although Internal fusions may also be tolerated in some instances. The presence of such epitope-tagged forms of a HIPK1 polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epltope tag enables a HIPK1 polypeptide to be readily purified by affinity purification using an antitag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment the chimeric molecule may comprise a fusion of a HIPK1 polypeptide with an immunoglobulin or a particular region of an immunoglobulln. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well known In the art. Examples include poly.histidine (poly-hl$) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell; Biol., 8:2159-2165 (1988)]: the e-mye tag and the 8F9. 3C7.

6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and.Cellular Biology. 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204.1210 (1988)]; the KT3 epitope peptide [Martin et al.. Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem.. 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Nail. Acad. Sci. USA. 87:6393-6397 (1990)].

Also included with the definition of HIPK1 protein In one embodiment are other HIPK1 proteins of the HIPK family, and HIPK1 proteins from other organisms, which are cloned and expressed as outlined below. Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related HIPK1 proteins from humans or other organisms. As will be appreciated by those -23- 00 in the art, particularly useful probe and/or PCR primer sequences include the unique areas of a HIPK1 0 nucleic acid sequence. As is generally knpwn in the art, preferred PCR primers are from about 15 to K about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain k inosine as needed. The conditions for the PCR reaction are well known in the art.

In addition, as is outlined herein, HIPK1 proteins can be made that are longer than those encoded by the nucleic acids of the figures, for example, by the elucidation of additional sequences, the addition of epitope or purification tags. the addition of other fusion sequences, etc.

00 HIPK1 proteins may also be identified as being encoded by HIPK1 nucleic acids. Thus, HIPK1 Q proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein.

00 In a preferred embodiment, the invention provides HIPK1 antibodies, In a preferred embodiment, when a HIPK1 protein is to be used to generate antibodies, for example for immunotherapy, a HIPK1 protein should share at least one epitope or determinant with the full length protein. By "epitope* or "determinant" herein is meant a portion of a protein which will generate and/or bind an antibody or Tcall receptor in the context of MHC. Thus, in most instances, antibodies made to a smaller HIPK1 protein will be able to bind to the full length protein. In a preferred embodiment, the epitope Is unique: that Is, antibodies generated to a unique epitope show little or no cross-reactivity.

In one embodiment, the term "antibody" includes antibody fragments, as are known in the art, Including Fab, Fab, single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.

Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more Injections of an immunizing agent and. if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may Include a protein encoded by a nucleic acid of the figures or fragment thereof or a fusion protein thereof. It may be useful to conjugate the Immunizing agent to a protein known to be Immunogenic in the mammal being immunized. Examples of such immunogenic proteins Include but are not limited to keyhole limpet hemocyanln, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophospnoryl Lipid A, synthetic trehalose dlcorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

-24- 00 The antibodies may. alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared Susing hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).

In a hybridoma method, a mouse, hamster, or other appropriate host animal. Is typically immunized sa with an immunizing agent to elicit lymphocytes that.produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in 'sC vitro. The immunizing agent will typically include a polypeptide encoded by a nucleic acid of Tables 1, 2, and 3 or fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph nodA cells Sare used if non-human mammalian sources are desired. The lymphocytes are then fused with an 0 immortalized cell line using a suitable fusing agent such as polyethylene glycol, to form a hybridoma 0 cell IGoding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp. 59-103).

Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, O bovine and human origin. Usually. rat or mouse myeloma cell lines are employed. The hybridoma c- cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthlne, aminopterin, and thymldine ("HAT medium"), which substances prevent the growth of HGPRT-deflclent cells.

In one embodiment, the antibodies are bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a protein encoded by a nucleic acid of the Tables 1, 2, and 3. or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific.

Ir a preferred embodiment, the antibodies to HIPK1 are capable of reducing or eliminating the biological function of HIPK1, as is described below. That is. the addition of antl-HIPK1 antibodies (either polyclonal or preferably monoclonal) to HIPK1 (or cells containing HIPK1) may reduce or eliminate a HIPK1 activity. Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred end about a 95-100% decrease being especially preferred.

In e preferred embodiment the antibodies to the HIPK1 proteins are humanized antibodies.

Humanized forms of non-human murine) antibodies are chimeric molecules of Immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv. Fab, Fab', or other entigen binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) In which 00 residues form a complementary determining region (CDR) of the recipient are replaced by residues S from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired CN specificity, affinity and capacity. In some Instances, Fv framework residues of the human S immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also S comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of et least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework r residues (FR) regions are those of a human immunoglobuiln consensus sequence. The humanized 00 .C antibody optimally also will comprise at least a portion of an Immunoglobulln constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986): Riechmann et al..

Nature, 332:323-329 (1988): and Presta, Curr. Op. Struct Biol.,.2:593-596 (1992)].

00 Methods for humanizing non-human antibodies are well known In the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from e source which is non-human.

These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al.. Nature. 321:522-525 (1986): Riechmann at al.. Nature.

332:323-327 (1988): Verhoeyen et al., Science, 239:1534-1536 (1988)]. by substituting rodent CORs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies Patent No. 4.816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et at are also available for the preparation of human monoclonal antibodies [Cole et al.. Monoclonal Antibodies and Cancer Therapy, Alan R.

Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)). Similarly, human antibodies 0 can be made by introducing human immunoglobulin loci into transgenic animals, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, Including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5.545,807; 5,545,806; 5.569,625: 5,625,126; 5.633,425; .5.661,016, and in the following scientific publications: Marks et al., BiofTechnology 10, 779-783 26 00 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994): Fishwild et al.. Nature Biotechnology 14, 845-51 (1996): Neuberger. Nature Biotechnology 14. 826 (1996); Lonberg and Huszar, Intem. Rev. Immunol. 13 65-93 (1995).

SBy immunotherapy Is meant treatment of lymphoma with an antibody raised against a HIPK1 protein.

As used herein. Immunotherapy can be passive or active. Passive immunotherapy as defined herein is the passive transfer of antibody to a recipient (patient). Active immunization is the Induction of antibody and/or T-cell responses in a recipient (patient). Induction of an immune response is the 00 result of providing the recipient with an antigen to which antibodies are raised. As appreciated by one IN of ordinary skill in the art, the antigen may be provided by injecting a polypeptlde against which 0 antibodies are desired to be raised into a recipient, or contacting the recipient with a nucleic acid 00 capable of expressing the antigen and under conditions for expression of the antigen.

0 C' In another preferred embodiment, the antibody is conjugated to a therapeutic moiety. In one aspect the therapeutic moiety is a small molecule that modulates the activity of a HIPK1 protein. In another aspect the therapeutic moiety modulates the activity of molecules associated with or in close proximity to a HIPK1 protein. The therapeutic moiety may inhibit enzymatic activity such as protease or protein kinase activity associated with lymphoma.

In a preferred embodiment, the therapeutic moiety may also be a cytotoxic agent. In this method, targeting the cytotoxic agent to tumor tissue or cells, results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with lymphoma. Cytotoxic agents are numerous and varied and include, but are not limited to. cytotoxic drugs or toxins or active fragments of such toxins.

Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricln A chain, abrin A chain, curcin, croUtn, phenomycin. enomycin and the like. Cytotoxic agents also Include radiochemicale made by conjugating radioisotopes to antibodies raised against HIPK1 proteins, or binding of a radlonuclide to a chelating agent that has been covalently attached to the antibody.

Targeting the therapeutic moiety to transmembrane HIPK1 proteins not only serves to increase the local concentration of therapeutic moiety in the lymphoma. but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety.

In a preferred embodiment, a HIPK1 protein against which the antibodies are raised is an intracellular protein. In this case. the antibody may be conjugated to a protein which facilitates entry into the cell.

In one case, the antibody enters the cell by endocytosis. In another embodiment, a nucleic acid encoding the antibody is administered to the individual or cell. Moreover, wherein a HIPK1 protein can be targeted within a cell, the nucleus, an antibody thereto contains a signal for that target localization. a nuclear localization signal.

-27 00 The HIPK1 antibodies of the invention specifically bind to HIPK1 proteins. By "specifically bind" herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10- 10 with a preferred range being 10" 10 M 1 In a preferred embodiment a HIPK1 protein is purified or isolated after expression. HIPK1 proteins Smay be Isolated or purifed in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including Ion exchange, hydrophobic.

00 affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, a HIPK1 protein may be purified using a standard antl-G,a antibody column. Ultraflltration and diafiltration C techniques, In conjunction with protein concentration, are also useful. For general guidance in suitable 00 purification techniques, see Scopes. Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of a HIPK1 protein. In some instances no C] purification will be necessary.

Once expressed and purified If necessary, the HIPK1 protelns and nucleic acids are useful in a number of applications.

In one aspect, the expression levels of genes are determined for different cellular states In the lymphoma phenotype; that is, the expression levels of genes in normal tissue and in lymphoma tissue (and in some cases, for varying severities of lynphoma that relate to prognosis, as outlined below) are evaluated to provide expression profiles. An expression profile of a particular cell state or point of development Is essentially a "fingerprint' of the state; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that IS unique to the state of the cell. By comparing expression profiles of cells In different states, information regarding which genes are important (including both up- and downregulation of genes) in each of these states is obtained. Then. diagnosis may be done or confirmed: does tissue from a particular patient have the gene expression profile of normal or lymphoma tissue.

"Differential expression," or grammatical equivalents as used herein, refers to both qualitative as well as quantitative differences in the genee' temporal and/or cellular expression patterns within and among the cells. Thus, a differentially expressed gene can qualitatively have its expression altered.

including an activation or inactivation, in, for example, normal versus lymphoma tissue. That is. genes may be turned on or turned off in a particular state, relative to another state. As Is apparent to the skilled artisan, any comparison of two or more states can be made. Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but Is not detectable in both. Alternatively, the determination -28- 00 is quantitative in that expression is-increased or decreased; that is, the expression of the gene is either O upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased CK amount of transcript The degree to which.expression differs need only be large enough to quantify via standard characterization techniques as outlined below, such as by use of Affymetrx GeneChip'" expression arrays. Lockhart Nature Biotechnology. 14:1675-1680 (1996). hereby expressly \0 Incorporated by reference. Other techniques include, but are not limited to. quantitative reverse transcriptase PCR, Northern analysis and RNase protection. As outlined above, preferably the change in expression upregulatlon or downregulation) is at least about 60%, more preferably at least about 100%. more preferably at least about 150%. more preferably, at least about 200%, with from .C 300 to at least 1000% being especlally preferred.

As will be appreciated by those in the art. this may be done by evaluation at either the gene transcript.

00 or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product Itself (protein) can be monitored, for example through the use of antibodies to a HIPK1 protein and standard immunoassays (ELISAs, etc.) or other techniques, including mass spectroscopy assays, 20 gel electrophoresis assays. etc. Thus, the proteins corresponding to HIPKI genes, i.e. those Identified as being important in a lymphoma phenotype, can be evaluated in a lymphoma diagnostic test.

In a preferred embodiment, gene expression monitoring Is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well. Similarly, these assays may be done on an individual basis as well.

In this embodiment, the HIPK1 nucleic acid probes may be attached to biochips as outlined herein for the detection and quantification nf HIPK1 sequences in a particular cell. The assays are done as is known in the art. As will be appreciated by those in the art. any number of different HIPK1 sequences may be used as probes, with single sequence assays being used in some cases, and a plurality of the sequences described herein being used in other embodiments. In addition, while solid-phase assay s are described, any number of solution based assays may be done as well.

In a preferred embodiment, both solid and solution based assays may be used to detect HIPK1 sequences that are up-regulated or down-regulated In lymphoma as compared to normal tymphoid 0 tissue, In instances where a HIPK1 sequence has been altered but shows the same expression profile or an altered expression profile, the protein will be detected as outlined herein -29- 00 In a preferred embodiment nucleic acids encoding a HIPK1 protein are detected. Although DNA or SRNA encoding a HIPK1 protein may be detected, of particular interest are methods wherein the mRNA N encoding HIPK1 protein is detected. The presence of mRNA in a sample is an indication that a HIPK1 S gene has been transcribed to form the mRNA, and suggests that the protein is expressed. Probes to detect the mRNA can be any nucleotlde/deoxynucleotide probe that is complementary to and base pairs with the mRNA and includes but is not limited to oligonucleotides, cDNA or RNA. Probes also should contain a detectable label, as defined herein. In one method the mRNA is detected after immobilizing the nucleic add to be examined on a solid support such as nylon membranes and Cr hybridizing the probe with the sample. Following washing to remove the non-specifically bound probe, 00 0C the label is detected. In another method detection of the mRNA Is performed in situ. In this method C permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for C sufficient time to allow the probe to hybridize with the target mRNA. Following washing to remove the 00 0 non-epecifically bound probe, the label is detected. For example a digoxygenin labeled riboprobe Ci (RNA probe) that is complementary to the mRNA encoding HIPK1 protein is detected by binding the digoxygenin with an anti-dlgoxygenin secondary antibody and developed with nitro blue tetrazollum and 5-bromo-4-chloro-3-indoyl phosphate.

In a preferred embodiment, the HIPK1 proteins, antibodies, nucleic acids, modified HIPK1 proteins and cells containing HIPK1 sequences are used in diagnostic assays. This can be done on an individual gene or corresponding polypeptide level, or as sets of assays.

As described and defined herein, HIPK1 proteins find use as markers of lymphoma, Detection of these proteins in putative lymphomic tissue or patients allows for a determination or diagnosis of lymphoma. Numerous methods known to those of ordinary skill in the art find use in detecting lymphoma, In one embodiment, antibodies are used to detect HIPK1 proteins. A preferred method separates proteins from a sample or patient by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be any other type of gel including isoelectric focusing gels and the like).

Following separation of proteins, a HIPK1 protein Is detected by immunoblOtting with antibodies raised against a HIPK1 protein, Methods of immunoblotting are well known to those of ordinary skill in the art.

In another preferred method, antibodies to a HIPK1 protein find use in in situ imaging techniques. In this method cells are contacted with from one to many antibodies to a HIPK1 protein(s), Following washing to remove non-specific antibody binding, the presence of Iho antibody or antibodies is detected. In one embodiment the antibody is detected by incubating with a secondary antibody that contains a detectable label. In another method the primary antibody to a HIPK1 protein(s) contains a detectable label. In another preferred embodiment each one of multiple primary antibodies contains a 00 distinct and detectable label. This method finds particular use in simultaneous screening for a plurality of HIPK1 proteins. As will be appreciated by one of ordinary skill in the art, numerous other histological imaging techniques are useful In the invention.

In a preferred embodiment the label Is detected in a fluommeter which has the ability to detect and distinguish emissions of different wavelengths. In addition, a fluorescence activated cell sorter (FACS) can be used In the method.

00 In a preferred embodiment, in situ hybridization of labeled HIPK1 nucleic acid probes to tissue arrays 0 is done. For example, arrays of tissue samples, including leukemlellymphoma tissue and/or normal S tissue, are made. In situ hybridization as is known in the art can then be done.

00 It is understood that when comparing the expression fingerprints between an individual and a C standard, the skilled artisan can make a diagnosis as well as a prognosis. It Is further understood that the genes which indicate the diagnosis may differ from those which Indicate the prognosis.

In a preferred embodiment, the HIPK1 proteins, antibodies, nucleic acids, modified HtPK1 proteins and cells containing HIPK1 sequences are used in prognosis assays. As above, gene expression profiles can be generated that correlate to lymphoma severity. in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of genes being preferred. As.above, the HIPK1 probes are attached to biochips for the detection end quantification of HIPK1 sequences in a tissue or patient The assays proceed as outlined for diagnosis.

In a preferred ambodiment, any of the HIPK1 sequences as described herein are used In drug screening essays, The HIPK1 proteins, antibodies, nucleic acids, modified HIPK1 proteins and cells containing HIPK1 sequences are used In drug screening assays or by evaluating the effect of drug candidates on a "gene expression profile" or expression profile of polypeptides. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, Zlokarnik, et al., Science 279. 84-8 (1998). Heid. et al., Genome Res., 6:988-994 (1896).

In a preferred embodiment, the HIPK' proteins, antibodies, nucleic acids, modified HIPK1 proteins and cells containing the native or modified HIPK1 proteins are used In screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the lymphoma phenotype. As above, this can be done by screening for modulators of gene expression or for modulators of protein activity. Similarly, this may be done on an individual gene or protein level or by evaluating the effect of drug candidates on a "gene expression profile'.. In a preferred embodiment.

S31

I

00 the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, see Zlokamik.

supra.

Having identified the HIPK1 genes herein, a variety of essays to evaluate the effects of agents on IN gene expression may be executed. In a preferred embodiment, assays may be run on an individual gene or protein level. That is, having identified a particular gene as aberrantly regulated in lymphoma.

candidate bioactive agents may be screened to modulate the gene's response. 'Modulation" thus 00 includes both an increase and a decrease In gene expression or activity. The preferred amount of S modulation will depend on the original change of the gene expression in normal versus tumor tissue, with changes of at least 10%, preferably 50%. more preferably 100-300%. and in some embodiments 00 300-1000% or greater. Thus, if a gene exhibits a 4 fold increase in tumor compared to normal tissue.

0 a decrease of about four fold Is desired: a 10 fold decrease in tumor compared to normal tissue gives CK a 10 fold increase In expression for a candidate agent is desired, etc. Alternatively, where a HIPK1 sequence has been altered but shows the same expression profile or an altered expression profile, the protein will be detected as outlined herein.

As will be appreciated by those in the art, this may be done by evaluation at either the gene or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, the level of the gene product itself can be monitored, for example through the use of antibodies Lo a HIPK1 protein and standard immunoassays.

Alternatively, binding and bioactlvity assays with the protein may be done as outlined below.

In a preferred embodiment, gene expression monitoring is done and a number of genes, I.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well.

In this embodiment, the HIPK1 nucleic acid probes are attached to blochips as outlined herein for the detection and quantification of HIPK1 sequences in a particular cell, The assays are further described below.

Generally, in a preferred embodiment, a candidate bioactive agent is added to the cells prior to analysis. Moreover. screens are provided to Identify a candidate bioactive agent which modulates lymphoma, modulates HIPK1 proteins, binds to HIPK1 protein, or interferes between the binding of HIPK1 protein and an antibody.

-32- 00 The term "candidate bioactive agent" or'drug candidata" or grammatical equivalents as used herein describes any molecule, protein, oligopeptide, small organic or inorganic molecule, polysaccharide, polynucleotide, etc.. to be tested for bloactive agents that are capable of directly or Indirectly altering either the lymphoma phenotype, binding to and/or modulating the bioactivity of an HIPK1 protein, or the expression of a MIPK1 sequence, including both nucleic acid sequences and IN) protein sequences. In a particularly preferred embodiment, the candidate agent suppresses a lymphoma/leukemla associated (LA) phenotype, for example to a normal tissue fingerprint. Similarly, the candidate agent preferably suppresses a severe LA phenotype. Generally a plurality of assay 00 mixtures are run in parallel with different agent concentrations to obtain a differential response to the IND various concentrations. Typically, one of these concentrations serves as a negative control. at zero concentration or below the level of detection.

00 S In one aspect a candidete agent will neutralize the effect of a HIPK1 protein. By "neutralize" is meant C1 that activity of a protein Is either inhibited or counter acted against so as to have substantially no effect on a cell.

Candidate agents encompass numerous chemical classes, though typically they are organic or inorganic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Preferred small molecules are less than 2000. or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural Interaction with proteins, particularly hydrogen bonding. and typically include at least an amine, carbonyl. hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.

The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids. steroids, purines, pyrimidlnes, derivatives; structural analogs or combinations thereof. Particularly preferred are peptldes.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules. including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced, Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation. alkylation, esterification. amidification to produce structural analogs.

-33 00 In a preferred embodiment, the candidate bioactlve agents are proteins, By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptidee, oligopeptides and i peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or p synthetic peplidomimetic structures. Thus "amino acid", or "peptide residue". as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and S noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also Includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the or the configuration. In the preferred embodiment the amino acids are in the or L-configuration.

00 If non-naturally occurring side chains are used, non-amino acid substituents may be used. for example S to prevent or retard in vivo degradations.

00 In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or O fragments of naturally occurring proteins. Thus, for example. cellular extracts containing proteins, or Cl random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of procaryotic and eucaryotic proteins may be made for screening in the methods of the invention.

Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred, In a preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized' or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bloactive proteinaceous agents.

In one embodiment, the library Is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library it biased. That is, some positions within the sequence are either held constant or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cystelnes, for cross-linking. prollnes for SH-3 domains, serlnes, threonines. tyrosines or histidines for phosphorylatlon sites, etc.. or to purines, etc.

-34- 00 In a preferred embodiment, the candidate bloactive agents are nucleic acids, as defined above.

O

S As described above generally for proteins, nucleic acid candidate bioaotive agents may be naturally S occurring nucleic acids, random nucleic acids, or *biased" random nucleic acids. For example, digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.

In a preferred embodiment, the candidate bloactive agents are organic chemical moieties, a wide van'ety of which are available in the literature.

00 C In assays for altering the expression profile of one or more HIPK1 genes, after the candidate agent has been added and the cells allowed to incubate for some period of time, the sample containing the 1 target sequences to be analyzed is added to the blochip. If required, the target sequence is prepared 00 using known techniques. For example. the sample may be treated to lyse the cells, using known lysis buffers, electroporation. etc., with purification and/or amplification such as PCR occurring as needed.

as will be appreciated by those in the art. For example, an in vitro transcription with labels covalently attached to the nucleosides is done, Generally, the nucleic acids are labeled with a label as defined herein, with biotin-FITC or PE, cy3 and cy5 being particularly preferred.

In a preferred embodiment, the target sequence is labeled with, for example, a fluorescent chemlluminescent, chemical, or radioactive signal, to provide a means of detecting the target sequenoe's specific binding to a probe. The label also can be an enzyme, such as, alkaline phosphatose or horseradish peroxidase. which wnen provided with an appropriate substrate produces a product that can be detected. Alternatively, the label can be a labeled compound or small molecule.

such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or blotin which specifically binds to streptavidin.

For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. As known in the art. unbound labeled streptavidin is removed prior to analysis.

As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise "sandwich assays', which include the use of multiple probes, as Is generally outlined in U.S.

Patent Nos. 5,681,702. 5.597,909, 5,545.730. 5,594,117. 5,591,584. 5.571.670. 5,580,731, 5,571.670, 5.591,584, 5,624,802. 5,635,352, 5,594,118, 5.359,100, 5,124,246 and 6,681,697, all of which are hereby incorporated by reference. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes. under conditions that allow the formation of a hybridization complex.

0 0 A variety of hybridization conditions may be used in te present invention, including high, moderate S and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of O- target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature. formamide concentration, salt concentration, chaotropic salt concentration pH. organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as is generally outlined in U.S.

00 Patent No. 5,681,697. Thus it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.

00 The reactions outlined herein may be accomplished in a variety of ways. as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially. in any order, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents may be Included in the assays.. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assey, such as protease inhibitors, nuclease inhibitors, anti-microblal agents, etc., may be used, depending on the sample preparation methods and purity of the target. In addition, either solid phase or solution based kinetic PCR) assays may be used.

Once the assay is run, the data is analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile, In a preferred embodiment, as for the diagnosis and prognosis applications, having identified the differentially expressed gene(s) or mutated gene(s) important in any one state, screens can be run to alter the expression of the genes individually. That is, screening for modulation of regulation of expression of a single gene can be done. Thus, for example, particularly in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.

In addition screens can be done for novel genes that are induced in response to a candidate agent After identifying a candidate agent based upon its ability to suppress a HIPK1 expression pattern leading to a normal expression pattern, or modulate a single HIPK1 gone expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to identify genes that are specifically modulated In response to the agent. Comparing expression profiles between normal tissue and agent treated LA tissue reveals genes that are not expressed In -36.

00 normal tissue or LA tissue, but are expressed in agent treated tissue. These agent specific sequences S can be identified and used by any of the methods described herein for HIPK1 genes or proteins. In CN particular these sequences and the proteins they encode find use In marking or identifying agent treated cells. In addition, antibodies can be raised against the agent induced proteins end used to target novel therapeutics to the treated LA tissue sample.

Thus. in one embodiment, a candidate agent is administered to a population of LA cells, that thus has an associated HIPK1 expression profile. By "administration' or'contacting" herein is meant that the 00 candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, O whether by uptake and Intracellular action, or by action at the cell surface. In some embodiments, nucleic acid encoding a proteinaceous candidate agent a peptide) may be put into a viral construct such as a ratroviral construct and added to the cell, such that expression of the peptide

O

0 agent is accomplished; see PCT US97/01019, hereby expressly Incorporated by reference.

Once the candidate agent has been administered to the cells, the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein, Thus, for example. LA tissue may be screened for agents that reduce or suppress the LA phenotype.

A change in at least one gene of the expression profile indicates that the agent has an effect on HIPK1 activity. By defining such a signature for the LA phenotype, screens for new drugs that alter the phenotype can be devised. With this approach, the drug target need not be known and need not be represented In the original expression screening platform, nor does the level of transcript for the target protein need to change.

In a preferred embodiment, as outlined ebove. screens may be done on individual genes and gene products (proteins); That is, having identified a particular differentially expressed gene as important In a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done. A HIPK1 product may be a fragment, or alternatively, be the full length protein to the fragment encoded by the nucleic acids of the figures. Preferably, a HIPK1 is a fragment. In another embodiment, the sequences are sequence variants as further described herein.

Preferably, a HIPK1 is a fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment. Preferably, the fragment includes a non-trarsmembrane region, In e preferred embodiment, the fragment has an N-termnal. Cys to aid in solubility. In one embodiment, the c-terminua of the fragment is kept as a free acid and the n-terrr.inus is a free amine to aid in coupling, to cysteine.

-37- 00 In one embodiment, the HIPK1 proteins are conjugated to an immunogenic agent as discussed herein.

In one embodiment a HIPK1 protein is conjugated to BSA.

In a preferred embodiment, screens for agents that alter the biological function of the expression product of a HIPK1 gene are done. Again, having identified the Impuotance of a gene in a particular state, screening for agents that bind and/or modulate the biological activity of the gene product can be run as is more fully outlined below, 00 In a preferred embodiment screens are designed to first find candidate agents that can bind to HIPK1 proteins, and then these agents may be used in essays that evaluate the ability of the candidate agent N to modulate a HIPK1 activity and the lymphoma phenotype. Thus. as will be appreciated by those in 0 the art, there are a number of different assays which may be run; binding assays.and activity essays.

0 In a preferred embodiment binding assays are done. In general, purified or isolated gene product is used; that is, the gene products of one or more HIPK1 nucleic acids are made. In general, this Is done as is known In the art. For example, antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present Alternatively, cells comprising the HIPK1 proteins can be used in the assays.

Thus, In a preferred embodiment the methods comprise combining HIPK1 protein and a candidate bioactlve agent, and determining the binding of the candidate agent to a HIPK1 protein. Preferred embodiments utilize the human or mouse HIPK1 protein, although other mammalian proteins may also be used, for example for the development of animal models of human disease. In some embodiments, as outlined herein, variant or derivative HIPK1 proteins may be used.

Generally, In a preferred embodiment of the methods herein, a HIPK1 protein or the candidate agent Is non-diffusably bound to an insoluble support having Isolated sample receiving areas a microtfter plate, an array, etc.). The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include mIcrotiter plates, arrays, membranes and beads. These are typically made of glass, plastic polystyrene), polysaccharides, nylon or nitrocellulose, Teflon"', etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies (which -38do not sterically block either the ligand binding site or activation sequence when the protein is bound Sto the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the C protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound ;4 material is removed by washing. The sample receiving areas may.then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other molety, In a preferred embodiment, a HIPK1 protein is bound to the support, and a candidate bloactive agent is added to the assay. Alternatively, the candidate agent is bound to the support and a HIPK1 protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified In 00 screens of chemical libraries, peptlde analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretlc mobility shift assays, 00 immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.

The determination of the binding of the candidate bioactive agent to a HIPK1 protein may be done In a number of ways. In a preferred embodiment, the candidate bioactive agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of a HIPK1 protein to a solid support, adding a labeled candidate agent (for example a fluorescent label). washing off excess reagent, and determining whether the label Is present on the solid support Various blocking and washing steps may be utilized as is known in the art.

By "labeled* herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radloIsotope, fluorescers, enzyme. antibodies, particles such as magnetic particles, chemiluminescers. or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavldin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal In some embodiments, only one of the components is labeled, For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using or with fluorophores.

Alternatively, more than one component may be labeled with different labels; using for the proteins, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bloactive agent is determined through the use of competitive binding assays. In this embodiment the competitor is a binding moiety known to bind to the targel molecule HIPK1 protein), such as an antibody, peptlde, binding partner, ligand, etc.

-39- 00 Under certain circumstances, there may be competitive binding as between the bloactive agent and the binding moiety, with the binding moiety displacing the bloactive agent.

C- In one embodiment, the candidate bioactive agent is labeled. Either the candidate bioactive agent, or the competitor, or. both. is added first to the protein for a time sufficient to allow binding, if present Incubatlons may be performed at eny temperature which facilitates optimal activity, typically between 4 and 40'C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess-reagent 00 is generally removed or washed awey. The second component is then added, and the presence or absence of the labeled component Is followed, to indicate binding.

0 00 In a preferred embodiment. the competitor is added first, followed by the candidate bioactlve agent.

Displacement of the competitor is an Indication that the candidate bloactive agent is binding to a 1 HIPK1 protein and thus is capable of binding to, and potentially modulating, the activity of a HIPK1 protein. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive agent is added first with incubation and washing, followed by the competitor. The absence of binding by the competitor may Indicate that the bloactive agent is bound to a HIPK1 protein with a higher affinity. Thus. if the candidate bioactive agent Is labeled, the presence of the label on the support, coupled with a lack of competitor binding.

may indicate that the candidate agent is capable of binding to a HIPK1 protein.

In a preferred embodiment, the methods comprise differentlel screening to identity bloactive agents thatare capable of modulatng the activity of a HIPK1 proteins. In this embodiment the methods comprise combining HIPK1 protein and a competitor in a first sample. A second sample comprises a candidate bioactive agent. HIPK1 protein and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to a HIPI1 protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to a HIPK1 protein.

Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native HIPK1 protein, but cannot bind to modified HIPK1 proteins. The structure of a HIPK1 protein may be modeled, and used in rational drug design to synthesize agents that interact with that site. Drug candidates that affect HIPK1 blosctlvity are also identified by screening drugs for 00 the abilly to either enhance or reduce the activity of the protein.

S Positive controls and negative controls may be used in the assays. Preferably all control and test S samples are performed in at least triplicate to obtain statistically significant results. Incubation of all O samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted (f in a scintillation counter to determine the amount of bound compound.

00

NO

A variety of other reagents may be Included in the screening assays. These include reagents like CN salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal 00 protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the essay, such as protease inhibitors, nuolease Inhibitors.

anti-microbial agents, etc., may be used. The mixture of components may be added In any order that provides for the requisite binding.

Screening for agents that modulate the activity of HIPK1 proteins may also be done. In a preferred embodiment methods for screening for a bioactive agent capable of modulating the activity of HIPK1 proteins comprise the steps of adding a candidate bioactive agent to a sample of HIPK1 proteins, as above, and determining an alteration in the biological activity of HIPK1 proteins. "Modulating the activity of a HIPK1 protein' includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment the candidate agent should both bind to HIPK1 proteins (although this may rot be necessary). and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of HIPK1 proteins.

Thus, in this embodiment, the methods comprise combining HIPK1 sample and a candidate bioactive agent, and evaluating the effect on HIPK1 activity. By "HIPK1 activity" or grammatical equivalents herein is meant one of a HIPK1 protein's biological activities, including, but not limited to, its role in lymphoma, including cell division, preferably in lymphoid tissue, cell proliferation, tumor growth and transformation of cells. In one embodiment, HIPK1 activity includes activation of or by a protein encoded by a nucleic acid of the tables. An inhibitor of HIPK1 activity is the inhibition of any one or more HIPK1 activities.

-41- In a preferred embodiment, the activity of a HIPK1 protein is increased: in another preferred S embodiment, the activity of a HIPK1 protein is decreased. Thus, bioactive agents that are antagonists are preferred In some embodiments, and bioacive agents that are agonists may be preferred in other S embodiments.

S In a preferred embodiment, the Invention provides methods for screening for bloactlve agents capable of modulating the activity of HIPK1 proteiri. The methoda comprise adding a candidate bioactive agent, as defined above, to a cell comprising HIPK1 proteins. Preferred cell types include almost any C cell. The cells contain a recombinant nucleic acid that encodes HIPK1 protein. In a preferred 00 embodiment, a library of candidate agents are tested on a plurality of cells.

S In one aspect. the assays are evaluated In the presence or absence or previous or subsequent 00 exposure of physiological signals, for example hormones, antibodies, peptides. antigens, cytokines.

S growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation.

carcinogenics, or other cells cell.cell contacts). In another example, the determinations are determined at different stages of the cell cycle process.

In this way, bioactive agents are Identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of a HIPK1 protein.

In one embodiment, a method of inhibiting lymphoma cancer cell division is provided. The method comprises administration of a lymphoma cancer inhibitor. In a preferred embodiment, the method compriass administration of a HIPK1 Inhibitor.

In another embodiment, a method of inhibiting tumor growth is provided. The method comprises administration of a lymphoma cancer inhibitor. In a preferred embodiment, the method.comprises administration of a HIPK1 inhibitor.

In a further embodiment methods of treating cells or individuals with cancer are provided. The method comprises administration of a lymphoma cancer inhibitor. In a preferred embodiment, tho method comprises administration of a HIPK1 inhibitor, In one embodiment, a lymphoma cancer inhibitor is an antibody as discussed above. In another embodiment, the lymphoma cancer inhibitor is an antisense molecule. Antisense molecules as used herein include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or ONA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for lymphoma cancer molecules. Antisense or sense oligonucleotides, according to the present invention, -42 comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 0 nucleotldes. The ability to derive an antisense or a sense oligonucleotide. based upon a cDNA Ssequence encoding a given protein is described In. for example, Stein and Cohen. Cancer Res.

i 48:2659, (1988) and var der Krol et al.. BioTechnlques 6:958. (1988).

SAntsense molecules may be introduced into a cell containing the target nucleolide sequence by formation of a conjugate with a ligand binding molecule, as described In WO 91/04753. Suitable ligand binding molecules include, but are not limited to. cell surface receptors, growth factors. other CM cytokines. or other ligands that bind to cell surface receptors. Preferably, conjugation of the Ilgand 00 binding molecule does not substantially interfere with the ability of the lgand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or Cl its conjugated version into the cell. Alternatively, a sense or an antisense oligonucleotide may be 00 o introduced into a cell containing the target nucleic acid sequence by formation of an ollgonucleotide- 0 lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of trootment.

The compounds heving the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described. The agents may be administered In a variety of ways, orally, parenterally subcutaneously. intraperltoneally, intravascularly. etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100% wgt/vol. The agents may be administered alone or in combination with other treatments, radiation.

The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers end/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.

Without being bound by theory, it appears that the various HIPK1 sequences are important in lymphoma. Accordingly, disorders based on mutant or variant HIPK1 genes may be determined. In one embodiment the invention provides methods for Identifying cells containing verlant HIPK1 genes comprising determining all or part of te sequence of at least one endogenous HIPK1 genes in a cell.

As will be appreciated by those In the art, this may be done using any number of sequencing -43techniques. In a preferred embodiment, the invention provides methods of identifying a HIPK1 O genotype of an individual comprising determining all or pert of the sequence of at least one HIPK1 S gene of the individual. This is generally done in at least one tissue of the individual, and may Include the evaluation of a number of tissues or different samples of the same tissue. The method may inlclude comparing the sequence of the sequenced HIPK1 gene to a known HIPK1 gene, a wildtype gene. As will be appreciated by those In the art. alterations In the sequence of some oncogenes can be an Indication of either the presence of.the disease, or propensity to develop the disease, or prognosis evaluations.

00 The sequence of all or part of a HIPK1 gene can then be compared to the sequence of a known HIPK1 gene to determine if any differences exist This can be done using any number of known CN homology programs, such as Bestfit etc. In a preferred embodiment, the presence of a difference in the sequence between a HIPK1 gene of the patient and the known HIPK1 gene Is indicative of a disease state or a propensity for a disease state, as outlined herein.

In a preferred embodiment, the HIPK1 genes are used as probes to determine the number of copies of a HIPK1 gene in the genome. For example, some cancers exhibit chromosomal deletions or Insertions, resulting in en alteration in the copy number of a gene.

In another preferred embodiment HIPKI genes are used as probes to determine the chromosomal location of the HIPK1 genes. Information such as chromosomal location finds use In providing a diagnosis or prognosis In particular when chromosomal abnormalities such as translocations, and the like are identified in HIPK1 gene loci.

Thus, In one embodiment, methods of modulating HIPK1 in cells.or organisms are provided. In one embodiment, the methods comprise administering to a cell an anti-HIPK1 antbody that reduces or eliminates the biological activity of an endogenous HIPK1 protein. Altematively, the methode comprise administering to a cell or organism a recombinant nucleic acid encoding HIPK1 protein. As will be appreciated by those in the art, this may be accomplished in any number of ways. In a preferred embodiment. for example when a HIPK1 sequence is down-regulated in lymphoma, the activity of a HIPK1 gene is increased by increasing the amount of HIPK1 in the cell. for example by overexpresslng the endogenous HIPK1 or by administering a gene encoding a HIPK1 sequence, using known gene-therapy techniques, for example. In a preferred embodiment, the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCTiUS93/03868, hereby incorporated by reference in its entirety. Alternatively, for example when a HIPK1 sequence Is up-regulated in -44.

OO lymphoma, the activity of the endogenous HIPK1 gene is decreased, for example by the administration 0 of a HIPK1 antisense nucleic acid.

i In one embodiment, the HIPK1 proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to HIPK1 proteins, which are useful as described herein. Similarly, the I\ HIPK1 proteins can be coupled, using standard technology, to affinity chromatography columns.

These columns may then be used to purify HIPK1 antibodies. In a preferred embodiment the antibodies are generated to epitopes unique to HIPK1 protein; that is, the antJbodies show little or no creoa-reactivity to other proteins. These antibodies find use in a. number of applications. For example.

00 S the HIPK1 antibodies may be coupled to standard affinity chromatography columns and used to purify HIPK1 proteins. The antibodies may also be used as blocking polypeptides, as outlined above, since CN they will specifically bind to a HIPK1 protein.

00 O In one embodiment, a therapeutically effective dose of HIPK1 or modulator thereof is administered to a patient. By "therapeutically effective dose" herein Is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As Is known in the art, adjustments for HIPK1 degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary. and will be ascertainable with routine experimentation by those skilled in the art.

A "patient" for the purposes of the present Invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient Is human.

The administration of the HIPK1 proteins and modulators of the present invention can be done In a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermalty, intraperitoneally, intramuscularly. intrapulmonary, vaginally, rectally. or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the HIPK1 proteins and modulators may be directly applied as a solution or spray.

The pharmaceutical compositions of the present invenlion comprise HIPK1 protein in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are In a water soluble form, such as being present as pharmaceutically acceplable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition sa31' refers to 00 those salts that retain the biological effectiveness of the free bases and that are not biologically or Sotherwise undesirable, formed with inorganic acids such ac hydrochloric acid, hydrobromic acid.

C' sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic ecid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonlcacld. succinic acid. fumaric acid.

S tartaric acid; citric acid, benzoic acid. cinnamic acid, mandelic acid, methanesulfonic acid, 0C ethanesulfonic acid. p-toluenesulfonic acid. salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium.

ammonium, calcium, magnesium, iron. zinc. copper, manganese, aluminum salts and the like.

OC Particularly preferred are the ammonium. potassium, sodium, calcium, and magnesium salts. Salts \0 derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines 0 and basic ion exchange resins, such as isopropylamlne, trimethylamine, diethylamlne, riethylamine, tripropylamlne, and ethanolamine.

The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin: buffers; fillers such as microcrystalllne cellulose, lactose, corn and other starches: binding agents: sweeteners and other flavoring agents: coloring agents; and polyethylene glycol, Additives are well known in the art, and are used in a variety of formulations.

In a preferred embodiment HIPK1 proteins and modulators are administered as therapeutic agents.

and can be formulated as outlined above. Similarly. HIPK1 genes (Including both the full-length sequence, partial sequences., or regulatory sequences of the HIPK1 coding regions) can be administered in gene therapy applications, as is known in the art. These HIPK1 genes can Include antisense applications, either as gene therapy for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.

In a preferred embodiment, HIPK1 genes are administered as DNA vaccines, either single genes or combinations of HIPK1 genes. Naked DNA vaccines are generally known in the art. Brower. Nature Biotechnology, 16:1304-1305 (1998).

In one embodiment, HIPK1 genes of the present invention are used as DNA vaccines. Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a HIPKI gene or portion of a HIPK1 gene under the control of a promoter for expression in a LA patient. A HIPK1 gene used for DNA vaccines can encode full-length HIPK1 proteins, but more preferably encodes portions of a HIPKi proteins including peptides derived from a HIPK1 protein. In e preferred embodiment a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a HIPK1 gene. Similarly, it is possible to immunize a patient with a plurality of HIPKI genes or Portions thereof as defined herein. Without being bound by theory, expression of the 00 polypeptide encoded by the DNA vaccine. cytotoxic T-cels, helper T-ceiis and antibodies are induced C 'which recognize and destroy or eliminate cells expre sing HIMK proteins.

in a-preferred embodiment the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine. Such adjuvant molecules include cytokJnes that increase the imtnunogenic response to a HIPKI polypeptide encoded by the DNA vaccine. Additional or aitemative adjuvants are known to those of ordinary skill in the art and find use in the invention.

00 In another *preferred embodiment HiPKI genes find Use In generating animal models of Lymphoma.

As is appreciated by one of ordinary skill in the art, when a HIPK1 gene identified is repressed or diminished In tissue, gene therapy technology wherein antlsense RNA directed to a HIPK1I gene will 00 also diminish or repress expression of the gene. An animal generated as such serves as an animal model of lymphomna that rinds use in screening bioactive drug candidates. Similarly, gene knockout rl technology, for example as a result of homologous recomnbination with an appropriate gene targeting vector, will result in the absence of HIPK1I protein, When desired. tissue-specific expression of knockout of HIPKI proteln may be necessary.

It is also possible thet HIPi( protein is overexpressed in lymphoma. As such, trarisgenic snImals can be generated ftht overexpress HIMK protein. Depending on the desired expression level, promoters of various strengths can be employed to express the transgene. Also, the number of copies of the integrated transgene can be determined and compared for a determInation of the expression level of the transgene. Anilmals; generated by such methods find use as animal models of HIPK1 and are additionally useful in screening for bioactive molecules to treat lymphoma.

A HIPK1 nucleic acid sequence of the invention is depicted in Table I as SEQ ID NO. 1. The nucleic acid sequence shown is from mouse.

TAB3LE 1 TAG SEQ. ID -SEQUENCE NO. SOOl13 1 CTCCG GGAGC A CNTGACGGGTGTOpG0GACCGGTHTCCCAGTCNTCTCCGCA ACGCTCTCCNAGGTGCTACCGGGTTGGTGGGGCQOOTCfl'ACGTTA-

GAGCNTACATTAACCCCPCATTATTCCCAA

CCCAMTCACATCCCAOCGATTGGGCAGCOCAGGGAGACATTOACTACCTGGGGGATCA

CTCTCAGGGTTTAGATCTC-AGTTTTTACTTA.AATTGTTTG.CTGCCAkTGTCGATTTC ______AOC;CcAGCNAoGGOzGATTrAoATGCCTCCCTGTCCTTIJOA A contig assembled from the mouse EST database by the National Center for Biotechnology Information (NCBI) having homology with all or parts of a lHlPt1 nucleic acid sequence of the invention Is depicted in -47.

Table 2 as SEQ ID NO. 2. SEQ ID NO. 3 represents the amino 91cid sequence of a'protein encoded by 00 SEQ ID NO. 2; CI TABLE 2

MOUSE

SAGRES REF SEQ SEQUENC4E TAGO 9 IOi SOOO013 F3 2 CCGCCACCAACGCCGrAAACCACCTCGAGACTGCTGOCGAGAGGACTGGGAAACC GGTCCCCACAcACTGTC4CACGCTGGCTCCCCACGGAG;GCCCACCCACACCCOCGGCCCGGG GCAAGATGCACTGATCTCAGCCCTCCCGCTCCrCCGCACTTCCOCCTCAGTATGOCCTCACA 00 OCTGCAGGTOTMCGCCCCCATAGTGTCGTCGAGTGCC1CTGCAGITGCAAAGAAMCTGA WTAGAGCCCTCTGGCTGGAT1TTCAGGACAGAGCAGCMoCACAAATACTATACCCACA OCAAAACCCTCOCAGCTACACAAGGGCAAGCCAC3CTCCTCTCACC.AGQZTAGCAAATr~T.AATC *1rCCTGCTACGAAGGGCCTCCrCTCCAGCTCCrGCCGTGGAGATA-mGGTAACAG 00 ~CTGCTGATAGCTCAGGCAGCQCCGCTACAGCAACCTTCCAAGCAGCCAGACCCTOGACTCAC AGGAGCAACG I I I 1 1 1 GAGCCATATCAA4ATGGArTGMGAGAAMGAGTGAGGAA

GTGGAGAGCAACGGTAGCGTOCAGATCATAGMGCAACACCCCCCTCTCATGCTGCAGMACAG

AACCGTGGTOOGTGCTGCTGCCACGACCACCACTOTACCACCAAGAQTAGCAGTrCOAOTG

GAGAAGGGGATTACCAGCTGGTCCAGCATGAGATCCTTTGCTCTATGACCACAGCTATGAA

GTCCTGGAGTTrCCTAGGCCGGGGOACATTTMGACAGOTGGCAAAGTGCTQG(AAGCGGAGCA CCAAGGAAATrOTQGCCATTMGATC-TGAAGAACCACCCCTCCTATGCCAACAA GGACAGA TTGAAGTGAGCATCC-rrTCCCGCCTAAGCAGTOAAAATGCrGATGAG;TATAACTTrTGTCCQTT CTTATGAOTGTTCAGCACMAGMTCATACCT(cCC,'TGTGMrToAATGrGGAGCAQMCTT CGTAC(GArfCTAAAGCACMCAGTTAOCCCACTGCCACTCMGTACATAGACCATCTTG CAGCAGGTGGCCACAGCCCTGAkTGAOCTc3AAGAGTCTTGOTCTGATTATGCTOACCrAA ACCTrGAMACATMTGCTAGTCGATCCAGTcdcCA~kCCCTACCGAGTGMAGGTATTGACTT TGGTTrCTGCTAGTCATGITrrCAAAGCCGTGTGTCAkcCTACCTGCAATCACGCTACACAG AGCTCCTGAATTATCTTGGArAtcA-rCTGTGAAOCTArrGACATGTGG7CACTGGGCTGT GTAATAGCTGAGCTGrrCCTGGGATGGCCTCT-rATCCrGOTGCICAGAATACOATCAGATr CGCTATAMTCACAAACACA.AGGCCTGCCAO3CTGAGTATCTTCTCAGTGCCOOMACAAAAMCA ACCAGGTrrlMTCAAGACCTAATOGGTACCCACTGTOAGGCTAAGACACCTG3 AAGAACATOATGGAMCTGGATAAAOTCAAAAOAAGCTCOGAAGTACATrTYACT GTrAATACATGGCTCAGGTAAATATGTCTACAGACTAAGGGGACAGATATGrAG CAGAGAAAOCAGATCGGAGAGAGTATATTOATCTrCTAAAGAAAATGCTGACGArrGATG CAGATAAGAGAATrCACOCCTCTGAAGACTCTTAACCACCAAMTGTGACGATOAGTCACC TCCTGGACMrCCTCACAGCAGCCACGTTAAGTCCTG(rTCCAGACATOGAGATCTGCA AGCGGAGGGflCACATGTATGACACAOTGATCAATCAGATCCClCACTACACATG TCGCTCCAAMTACMGCACAAATCTAACCATGAGCTrCAGCACCAGCTCAACAr-AGTGC ACAATCAGGCCAGTGT'TCTAGCTTCCAOCTCrACTGCAGCAGCAGCTACCCTTTCTCTGO CATTCAGATGTCTCCTGCTcACACCMTCGG3CTGTACCCATCGTCGGCAGcCC CAGrrcCTGGAGTTCCCAGCAGOOTGTrTTcrAcAACCTGGAACCACC-CAGAtCtGCA CTCAGACAGATCCATTCCAGCAAACATTATAOTATGCCCACCTGC1rMCAGACTGGAC TACMGCAACACAAGCATCTGGA-rCCCTGTGAGGATGGATAAGCTTGCCAATTG 'rACCCCAGGCGCCGCTocAGCCCTOCAGATCCAOTCAGGAG-ACTCACACAGGOAA GCTGTACACCACTAATGGTAGCAACTCTCCACCCTCAGTAGCCACCATACGCCCkGT ________ATGCGGTGCCCrrrACCCTGAGCGCGCAGCAGGCCGCCGGCGCTGGTTGAACAOACG 00

IND

00

MOUSE

SAGRES REF SEQ SEQUENCE TAQ# A D

CTGCTGTACTGCAAGCCTGGCCTGCAGGACCCAACAIATTCTCCTGCCTTCAGCCTGGC

AGCACTGCCCGGGrGTAGCTCTGCACAACTCTGTCCAGCCTGCTGcAGTGA1TrcCACAO CCATGGGGAGCAGCCAAC-AOCTAGCTOACTGO3AOGMTGCCCACTCTCATGGCAACCAGT ACAGCACTATTAGCAGCACCCATCMGTCTGACCACCATGTGACC~rGGCCACTGCTC

AGCCTCTGAATGTTGGTGTTGCCCATOTTGTCAGACAACAACAGTCTAGTTCCCTCCCTT

CAAGAAGM TAAGCAOTCTCCAGMTCATCCAAATCCTrCTCTGGAAGTCCTGCCTT CTCAAGTATCTCTGGTGOATAGTCCTTCGTACCACATcrrcrTATAA1TTcC TAGTTCCTGTCCAAGACCAGCATCAGCCAATCATCATTCCAGATACCCCCAGCccTccTG

TGAGTGTCATCACTATCCGTAGTOACACTGATGMAGAAGAGGACAACAAATACMAGCCCA

ATAGCTCGAGCCTGAAOOCGAOO0TCTAATGTCATCAGTTATGTCACTGTcAATGArrcTc CAGACTCTGACTrCCTCCCTrGAOCAOCCCACATCCCACAGACACiCTOAGTGCTCTGCOO GCAACAGTGOOACCCrrCTGGAGOGACCTOGC.AOACCTGCAGCAGATGGcATrGGCACCC GTACTATCArrGTGCCTCCT~TTAAA~ACACTGGCGACTGCACGTAGCAACACAGG CCTCAGGTCTCCrrAGCAGTAAGACCAAGCCAGTGGCCTCAGTGAGTGGGCAGTCATCTG GATGCTGTATCACrCCCACGGGGtACCOGOCTCAGCGAGGGGGAGCCAGCGCdGTGCAG;C CACTCACCrAGCCAGAACCAGrCAGTCATCGTCAOCTTCAACCTCGC-AGGAAGAAGCA GOAACCCTGCTCCCCGCAGACAGCAGGrCATflTGGCCCCOCTCTCCCMGCCCCCTACG CCTTCCAGCATGGCAGCCCACTGCACTCGACGGGGCACCCACACTTGGCCC4CAGCCCCTG CTCACCTOCCAAGCCAGCCTCACCTGTATACGTACGCTGCCCCCACTTCTGCTGCrGCAT TOOOCTCCACCAGTTCCArrGCTCATCToTTctCCCCCAOOGVtCCTCAAGGCATGCTG CAGC1TATACCACACACCCTAGCACTCTGGTGCATr-AOOTTrCCTGTCAGTGTCGGGCCCA

GCCTCCTCACTTCTGCCAGTGTGGCCCCTGCTCAOTACCAACACCAGMTGCCACTCAGT

CCTACATCGGOTCTTCCGAGGCTCAACAMACACTOGATACCCGCTGAGTCCTACCA

AGATCAOTCAG3TATTcTrAcrrCGTAGT1GATOAGCACGAGGAOCTCCO'TGGC TOCCTG CTAAGTAGCCCTGAGTTCTTAATGGGCTCTGGAGAGCACCTCr-ATATCTCCTCTTGAAA GrrCCTAOCCAGCAGCGCGTrrCTGCOGGGGCCCACTGAAGCAGMOGCrTCCCTGGGAA CAOCTCTCGGTGTTGACTGcAT TGTrGCAGTCTCCC/AAOTCTGCCCTGTr1TTTTAA11c TTTATTCTTG;TGACAGCAnTGGACGTrGGAAGAGCTCAGAAOCCCA~Tr.'CGCAGT TACCAAGGAAGAAGATCGTCTGMGTTACCCTCTG7rCATACAMGGTCTC1TGACT TGGTrrCTATAAATGrMTAAAATGMGTAAOCTCTCn7ACGAGGGGAATGCTGA CrrGA.AATCCTGTAGCAGATGAGAMGAGTCATTAC I I I i IGCTAAMACTAAA ACACAAGACTCC'fGTCTTMArTGAAGCAOCTAOCAAGGOTGTGCTATGGCGT ATGGAAACAGMATOAT TCAT TCATtTCGTGCTGTccT-rACTGGGCAGTTGTTAOAOT

TTAGTACAACGAGTCACTGAAACCTCYOCAOCTOCTOCYOAGCTGCTCGCAGAGCAGCA

CTGAACAGGCAGCCAGCGCTGCTGGGAAGOAAOOTGAGOGTGAGGACTGTGCCGACCAGG

ArATT~CTAATGAAGACCATGAGrrCAAGTCCTCCTCCTCTCTC7TAG1TTAAC17APA T7CTCCTTrATAGAAAMGCCAGTGAGGTGGTAAGTGTATGGTGGTGGTT1GCATACAATAG TATGCAATCTCTCTCTAGAATGAGATACTOOCACTGATAAACATTGCC7MkGATTTCT

ATGCAATATACACGTCTGTGTCCTCATCTCTCCCTCTGTTCATGTGACT

TATTTGAGGGGAAACTAAGAACTAACCAGATAGTGTGTATAGCTTTATAM~

TAAAGTAGCTITCCTTGTATGCCAACAGCAAATrGAATGCTCTCTTACTAGACTTATGT AATAAGTGCATGTAGGArGCAGAATATAGM-ATACTGAAMAAoAAT AT-1-AGAAGT=GTAATGGTGGTGTMAATATTTGCATAATTAAATATGTAr-ATAT

TGATTAGAAGMATATAACAATT~CCTCTAACCCAAAATGTATTGTAATCAAATGT

.49 00

IND

00

MOUSE

SAGRES REF SEQ SEQUENCE TAG# A IONl GTAGTTACACTTGATTGTGTAT7AGTGTGTACTGATCCTCCAGTGTACCCCGG AGTG~AGCrCrGAMA~AA~.A~,TcrT~3AmA GTGAACTATGCAATCTATTAAGCATATACrGTACTCTGAGAAGCAOGCATT GCCTGCAGAGAGGAGACCTTGGGATTGT7rGCACAGGTGTGTCTGGTGAGAGTGTTC AGTGTGTGTCTITCCTrCCTCCTCTCCTCTCTCCCCTATTGTAGTGCCTATATGATA AT OTAGTGGTT.ATAGAGTT1ACAGTGAGCTTGCMfAGGATGACCAGCAAGCCCCAGTG ACCCCAAGCTGTrCGTGGGATrAACAGAGCAGGGAGTAGCTGTGTG1TWATGC OrrCGTGTrCTCAGTO=CCTACCGACAGTGACAAGTCAAAGCCGCAGct.rCICTCCTTA ACTGCCACCTCTGTCCCGTCCATTT7GGATCTCAGCTrAGTCTCACAGAoAGCATOCC CTAACGTGGCTCTCTCAC7TOTCCTGCTACTOCTTCTGTGAGAGTCAGGAAGrCAGG CGAGAAGGACGCCAGTGCTAATATGCATArr'GAAGGTTTGTGCArACTAGGGT GGGATrCCT=rCTCTCCTCCATGTGATATGATAGTCCMTCTGCATAGCTGTCGTTTCC TG3TAMCTCT~111TG GMrGTT IITTIITPAAGCATGTAA CAGATGTG1TATACCAAMGAGCCTGTrGTATTGCTTAATATGTCCCATACTACGAGAAG GGTTrGAGACTACTGGTGACAAGAGCTCACAGAAAGGmrCrAATrAGTGACGAA TAGAAGAGAACT~GArWA~CTAG-T~TGAA CATGrTrCTGGGGCCCTGCACACTGTAAArTGTCTATATCAACCCCTCCATOG ArrG(aTcAAG-TwGGTAcTAGGGG-GGGGAATrcTTGCCCATGAGGGA1TrGTGG GGAGAAGGTTrAACCCTAAGCTACAGAGTGGTCCACCQAA~rAATATATCAGAGTGGT AArrCTAGGAflGTTGTTAGTGTGTCAGGAGG'GCAGGATGGAGATGGGAGATT TCATGGAACCcG-rCAGGAAAGCTCTG.&ACCAGCGTGGA.ArACCGAGGGGC Tu ICAACGAA CTTOCAGTTrCTTCATCArOGOO)A(c3AAC3AGTTTCCAOOQaCAOOOQCAOQTAaTCAOTA GCCTGCCGOCAACQTQQTOTOTQTTGTCrTTTMATCATATATTAAQCTGTGCGTT CAOCAQTCTTGGTTrGAGATACCACGCATCATTGTUAGTrTCACTAGTGTrATAC CCTTTATGTCATTCTGTGTGTGATCTTTGTGTTTC CTTTOCCCCAAGCATTCTdGGTfl TOTTTATrATCAMTGCAAA~r'rACrrTTT.

OGOACAAOAMlATTOcTrrrAAGAATOAGATGCAGGOAAAAAACAAACCAACrCTGT CCCCACTCCTCACCrCCCTAATCCAATAAGCAGrATGAAGATGGGAGTCTrAAATTTA TGGOA&GAGGATGCCTAGGAGTTGCATCG11ACCTGAGACATCTGGCTAGCAGI G I~

ACMACAGACTTGAGGTGTCACTCTGCAACTGACATTCAGATMCCTAATAAC.

CCATCTGTGTCTGOTGAATGTCOTATOCGCCAOACATACOTT7ACATTCATTrCTGGCrCTGG GGCTTAACATrGACTGCTTOCCCTGATOOCATGGAG3GAGAACCCTACGAACATAGCGCTG ACTAOOTCAOCA1-rCCTGACCTTGtAACAGCTAAGGCrrTAACCrCTCTTAGAACG TGCAMrCCAGTTTCTCCCT-CCCAGrGTGAGAGAGGAACTGGAAGGGTGCATAGGCACA CACCAGGACACTrAGTCACTCCAAGTCcccAGTGCAACTAGGGGTGG17ACCCTGTT AACCCCAGGJAAGAAGACCCCA1TCAAACA.GTrCCGGCCATTGAC-AGCCTGC1T1IT IGTG GTTrGCTCATCCOTCATCATCCGCTAGAGGGGCTTAGCCAGGCCAGCACAOTACTGGCTOT CC TATrCTGCATTAGTATGCAGGAATTTACTAGrrGAGATOG1 GTMTAGGATAGGAG ATGAAATTGCC'TT1'CGTGACAGGAATGGCCAGCCTGCTTGTGTTrrrMAATGA TGGATGGTGCAGCATOTTCAAGIMCCATGGGTTGrrGCTAAAA1TATATMTQ TGrGGTTTCAATTCAMTTCAOCrrGAAAAATAAMTCACTATATGTAGCAGTACATTATA TGTACATTATATTMrTTTAGTATrTTGCTTTGAATCCTrGATATTOCMTOGMTTC CTAAMATrAAATGTA1TrGATATGCTAAAAAA 00

IND

00

MOUSE

SAGRES REF SE0 StQtJEPICE TAG# it 3 FNLPAYDOGWAPAVEHIVTMADSSOSAATATPQSSQfLHRSNVSU..EPYQKCoIJ(KS2V ESNGSVOIIEEHPPLMLQNRTWGAAATTTVrrK3SSSQEGOYQLVCHILCSMTNSYEVLEP:L OMOTFGQVAKCWKRSTKEivAJI(ILKNlHPSYARQGOI EVSILSRLSSENADEYNIFVRSYECFaI4KN HTrCLVFEMLEONLYIDFt.KQNKFSPL.PLKYIRPILQQVATALMKU(SLGLIHAOLKPENIMLVDPVRQ PYRvKVIPOSACHSIVCSYLORYRPEIILGLPFCOtD'VSLGCVAELLGWLYPGAS EYORIQQLA-LSGXTPNONLYLVJTrEEEOKKAKIN LODMAQVNMSTOLEGTOMLAEKADRRE-YIDLLKKMLTIDAOKRITPU(TLNH'QFVtMASHULflFPHS

SHVKSCFONMEICKRRVHMYDTVSQIKSPFTHVAPNTSTNLTMSFSNQLNTVHNQASVLASSST

AAAATLSLANSDVSLLNYOSALYPSSAPVPGVAQQGVSLOPG17QICTQTOPFOQTFiVCPPAFQ TGLQATT)SGFPVRMDNAVPIVPQAPMOPLQIQSGVLTOGSCTPLMVAT.4POVAT1TPQYAV

PTLSCAAGRPALVEOTAVLOAWPGGTOQILLPSAWQOLPGVALHNSVQFAAVIPEAMGSSQQ*

LAOWRNANSHGNQYSTIMQQPSLLTNMVTLATAOPLNVGVAk4WRQQQSSSLPSKKNKOSAPVS

SKSSLEVLPSQVYSLVGSSPLRTTSSYNSLVPVOIDONQPIIIPDTPSPPVSVITIRSOMEEENKYK

PNSSSLKARS NvLSYVTVNDSPDSDSSLSSPHPTDTLSALRGNS OTLLEGPGRPAADGIGTRTIIVP PLKTQLGOC1VATQASGLLSSKTKPVASvSGQSSGCCITPTGYRAOP.GGASAVQPLNLSNOOS SSASTSOERSSNPAPRROCQAFVAPLSQAP'rAFQHGSPLHSTGHLAPAPAHLPS QPHLYrYAA PYTSpALSTSSAHLFSPQGSSRHAAAYTTHIPSTLVHQVPVSVPSLLTSASVAPAQY)4OFAT QSYIG3SSRGSTIYTYPLSPTKSQYSYL Also suitable for use in th~e present invention is the sequence provided in Genbank Accession No. AF077658.

A contig assembled from the human EST database by the NCBI having homology with an or parts of a HIPKI nucleic acid sequence of the Invention Is depicted in Table 3 as SEQ ID NO. 4. SEQ 10 NO. 5 depicts the amino acid sequence orea open-readinq frame of SEQ 10 NO. 4 which encodes the C-terminal portion of human HIPK1 protetn.

TABLE 3

H-UMAN

SAQRES REF SEQ SEQUENCE TAGII 0 ION 5000013 F30 4 CACACOGCAGTATGCGTGCCcTACTCTGAGCTGCGCAGCCGGCCO~tCcGCGCTGGT ACTGGCAArAGTTCCCTGGGGTAGCTCTACACAACTCT(3CCAOCCCACAGCAAfGA.T TCAOGCCATGGGGAGTGGACAGAOCTA(GACTOTGGAATGCCACCTICArGG CAcAATCAGCACTATvATGCAGCAGCCArCcTOCTGATAACCATGTGAAn-Qoc CACTGCTCAoCCTCTAArTGGTGTTGCCCATTrTCAGA~CAACAATCCAGTTC CCrCC1TrCAGAAMTAAoCAOTcAGCTCCAGT~CTC-CCAGrCCTCTAGATGr TCTGCCTTCCCAGTCTArCTTGGGGAGCAGrCCCCTCcOCCAcATCrc.TrA TTCAGTCOATACCACTC~rAOTCTCA 61 00

IND

00

HUMAN

SAGRFES REF SEOUENCE TAG#S 0 IO#P CCCTCCTGTGAGTGTCATCACTATCCGAAGTGACACrGATGAOQMAGAGGACAACAA'TA CMAGCCCAGTAGCTCTGGACT3AAGCCAAGGTCTMTGCATCATTATGTCACTGTCAA TGATTCTCCAGACTC-rGACTCrrC1TGAGCAGCCCTTATrCCACTOATACCCTGAGTGC TCTCCGAGGCAATAGTGGATCCGTTTTGGAGGGGCCTGGCAGAGTTrGTGOCAGATGGCAC TOQCA CCCGCACTATCATTGTGCCTCCACTGAAAACTCAGCTrGGTGACTGCACTGTAGC MACCCAGGCCTCAGGTCTCC'TGAGCMTMGACTMGCCAGTCGCTrCAGTAGTOQGCA GTCATCTGGATGCTGTATCACCCCCACAOG1ATCGAGCTCAACGCGGGGGGACCAGTGC AGCACAACCACTCMATCrrAGCCAQACCAGCAGTCATCGGCGGCTCCAACCTrACGGA GAGAAGCAGCAACCCAGCCCCCCOCAGGCAGCAGGCGMGrTGGCCCCTCTCTCCAAGC CCCCTACACCTltCAGCATGOCACCCGCACACTCGACAGGGCACCCACACC1GCCCC GOCCCCTGCTCACCTGCCAAGCCAQGCTCATCTGTATACGTATGCTGCCCCGACTTrCTOC TGCTGCACTrGGGCTCAACCAGCTCCArTGCTCATCTm7CTCCCCACAGGGTCCTCAAG .GCATGCTCCAGCCTATACCACTCACCCTAO.CACMn-TGCACCAGCGTCCCTGTCAGTrGt TGGGCCCAGCCTCCTCACTTCTOCCAGCGYGGCCCCTGCTCAGTACCAACACrCAOTrTGC CACCCMTCCTACATrGGGTCTTCCCGOGCT'CACAATTACACTGGATACCCGCTGAG TCCTACCAAGATCAGCCAGTAM7CCTACtTATAG1TGGTGAGrCATGAGGGAGGAOGAATO ATGGCTACCTrTCCTGCCCrCCGrTCT-AATATrGCGGCTATQOGAGAGATCcTCCTrA CCCTCTTOAAomCTTAGCCAGCAACTTGTCTCAGGGGCCCACTGAAGCAGAAGGTT TTCTCGGGGGAACCTGTCTCAGTGTTOACTOCArGTTCTATCTCCCAAAGMTGC CCTATTTTPATTCATTATrGTGACAOTM I I I GTACTGGAAGAG1TTAGATG

CCCATCTTCTGCAGTTACCAAGGAAGAGAOATTQTTCTGAAGTTACCCTCTGAAATAT

MGCTCTCTQCTCGATTCTATAATGC-TrMAAAACAAGTGAMGCCCCTCMTAT 1TCAT17TGTTATrGTGATTGCTGGTCAGGAMMTG3CTGATAGMGGAGTTGAAATC TGATOACAAAAAAGAAATrACTrMGGTTTATAAACTCAGACTTGCCTATT A1TMAAA.AGCGGcTAcAcMATCTCCCTTTTATTGACATTAACTACAGAGrT T'CGTTrATTAATTCrAOGA~~r~MCAAT GTTrACGTATTACTCTGGTTACTATTGAGATnCTCTCAATTGCTCCTGTGTfl'G7ATAA AGTACGTGTI7AAAAGGCAGCrCACCAMITCTGGTACTAAGYGAGAAATCCATATC TGCGTGAAAACAcCAAGTATTrC1TTMATOAACACCATAATTCTrrrTAAArrAT TT-AAAGTCTTCTCTCTCTGATCAGcTTrArrrTT TATCGAAAAAGCATAA GGTGGTTATTATTACATGGTGGTGGTGGTTTAT-rATATGCAAAATCTCTTCTATtATG AOATACTGGCATTOATGAGC7TrGCCTAAAGATTAG;TATCMAflTCAGTAATACACCTC TG1-1T-GCTCATCTCTCCCTTCTGTMATGTGAMTGTrrGGGGAGMA~GCTAAAAAAA CCTGAAACCAOATAACGMCAMTCTTGTTATAGCTATACTCAAAGTAGcCCTr TGTATGCCAOCAOCAATGATGCTCTCrYrAGACTTATATATAATGCATGTAG GAATTGCAAAAATArrAAAAAT1-TAIrAcTGAATI-AAAAATATI-I-AGAAGTMTT TAATGGTGGTGT-MTAATATMrACATAATWAATATGTACATATTGATTAOAAAATAT AACMGCATTTrCCTGCTAACCCAA.AATGTTrATrTGTAATCMATGTGTAGTGATTAC AC1TGATGTGTACTTAGTTTATGTGATCCCCATOTATCCCGGAGATOOATGA TGTCTCCArrGTATrrAAACCAAAATGAACTGATACTTOTTGGATGTATGTGACTAT TGCAATATATAGAGCATATACTTAGTGCTGATAGCGGGGCArTCCTOAGGv AGAGGAGACCC~rGGAArGnTOCACAGGTGTGTCTrgqrGAGGAG1TrrT~CAGTVGT GTCTCTrOCrrCCCTCCCTCCTTCCCTATTGTAGTGCCTTATATGATAATGTAG' _______GGTrAATAGAGTTTACAGTOAGCTrGCC-rA(GATGGACCAGCMAGCCCCCGTGGAoccT S2 7-- 00

IND

00

HUMAN

SAGRES REF SEQ

SEQUENCE

TAG# it IDO AATGrACGAACGAAGTATOTTTGGATCTG- CTCATCCCTGCCACAT$dAATMAAACAGCAGCTrCTCC1-rACCACCCC TCACrTTCTrOATTGCGGTTAAAGATTCCTT GCCTCTCACMTGCGTGCTACCTGCCGTGAGA1TCAOGAAGCAC;TAGAGGA GTCMAGCCMTATTAAATATGCATTCTT7ACTATGTGCATCAC1-T-AGATAT rrrT'C' CTTOATCrCO~AArGCTCTGAA TATT- rG-GAAAr-TTACGT;GTATCAAACTrOA

TGTACTTCCTCAGGAMTGGTCGTGGCAGA

TCCGAG rACGTAOATTGGAOACAGCGTAT ATGGCGGTT~r-TAACI TTOCTGvCCTAGT

TGAAGTCCTTTACTTCAGCTCCTGCATGGATCTGGTAATAGAGGTACTGGG

GAGGAATCGCAAAOTTGGAAAGVACTAATCG

T1TGTCCATCCGAATTGAAATGATATATTTAGATATAT.IT-.AGACTG,-1CTGTG

TAGATAGAGATGTTCAGGAGGTGCAGGATAGATGOAATTCATGGCCTGGT

CACACCGACO'TACCO(GGTTAATT7G~lCC TTrGTAAOQAGTAAGGGcTTrccAGATGGGGCAOTATCCCTACAOCCTACCAG.ACAT G-TTC1ArAATATTTTATGGMA3-CAAr GTCACATAGCCAAGCAGTTGATMCTGTCACTATGTATAAGTT-mCGGTC AACATGTOTGATCrGTGTCTCcTTGCCAAGCACATTCTOATTC-rGQGAAC ACGTTG'7TAGAAArMrCTGCT-fCGAGGAA GAGr7TTTAAAGCAOACAAAACATCA7COA CCATCAAGAAACCrAAAG~.CAkCCAAAAoAA

TACAACTTTGTCTATACTCTGATTTGTTAAC

AGG~MATTATT

ACGGACGTCACTAATC

CCTGTGTCTGCTGTGTGTATOTATOTGCTCACTGTGQ.TTAACTQrTCCCGGGG TTAGCCCTGTGGCCCTAr-AGGAAGGAGCCTG~oA~AGTGGOCTGGC CTG~-CCrACGCTAACC~AGC-rATCCCCGAr GGCATrCCAACTCrTMCGOTGAGAGAOAACGGAC-AGQCA

GT,.AGC,

CACTCTGGGCTCATAGGGACACTTOTCACTCCAGAGrAATAGTCCCAGGAGGTG

ATTAMATCCOTAAACACCAGAAGATCTTAA

AGnrCTGGCCATrCTGAOCCTGCTMGTArTTGCTCATCCATTGTCCTCCCTAGAGGG GCAGrGCGCTACAOAGCCGATTT~rGTAcr~ TAOAAArATG'GGTGGrrGAAGATAATCTT AGGCGATGCGGCGTCTrO r 'MCTGATAG ATGOACTTTCTGT OrCAAr*TTAGOO~TA

TTA~'AAAATTATCTTACGAATTTTC~~AGA

TGrGACGMATCTAATGATGATCA~rTAAG AMAAOTGT~GGGGMAAT~rrC~CCC~rG rrTCCrcT'AACACTTAAAAAAMTA~kTCGG TAGGATCGTTCrAGATTTMA~A~rAAAGA

TAAATAAMAATATTGG&GOCA'GGAACTG

ATMOTTAT-rGCG'MAAATGTrT~rCGAM TPQYAvPSCAAGRPALVEQTAAVAWPGGTQQILLPSWQLPGVALHNSVOPAMIPWJMG 53 00 SAGRES REF SEQ TAG* D

SGQQLAOWRN*ASHGNQYSTIMQ

APVSSKSSLDVLPSQVYSLVGSSPI

NKYKPSSSGLKPRSNVISYVTVNOD

00 TIIVPPLKTOLGDCrVATOASGLLSP 00

QQSSAAPTGQE-RSSNPAPRRQQA

APTSAAALOSTSSIAHLFSPOGSS

TOSYIGSSRG2STIY-rGYPLS-PTKISC 00 SAll references cjtad herein are incorporated by reference.

HUMAN

SEQUENCE

OPSL.LTNHVTLATAQPLNVGVAHIWRQQQSSSLPSKKNKQS

LRTSSNSLVPVQDHQPIIIPTPSPPVSVMTRSDTPEEeD.

iPDSDSSLSSPYSTLSALRtONSGSVAGPGRWVADGTGTR ~KTKPVASVSGQSSCCTPTYV(AQROGGTGAQPLNLSQ1 VAPLSOAPYFHSPLSTGPH4APAPAHLPSOAHLYTY RHAAAYrrl.PSTLVHQVPVSVGPSLLTSASVAPAQYQ.HQFA

IYSYL

.54.

Claims (11)

1. A method of screening drug candidates comprising: a) providing a cell that expresses a HIPK1 gene selected from the group consisting of SEQ ID NOS. 1, 2, and 4. or fragment thereof: b) adding a drug candidate to said cell: and c) determining the effect of said drug candidate on the expression of said HIPK1 gene. 00 OO S 2. A method according to claim 1 wherein said determining comprises comparing the level of expresalon in the N absence of said drug candidate to the level of expression In the presence of said drug candidate. 00 0 3. A method of screening for a bioactlve agent capable of binding to HIPK1 protein, wherein said HIPK1 protein is encoded by a nucleic acid selected from the group consisting of SEQ ID NOS. 1. 2. and 4, said method comprising: a) combining said HIPK1 protein and a candidate bioactlve agent; and b) determining the binding of said candidate agent to said HIPK1 protein.

4. A method for screening for a bioactive agent capable of modulating the activity of HIPK1 protein, wherein said HIPK1 protein is encoded by a nucleic acid selected from the group consisting of SEQ ID NOS. 1, 2, and 4. said method comprising: a) combining said HIPK1 protein and a candidate bioactive agent and b) determining the effect of said candidate agent on the bioact)vity of said HIPK1 protein. A method of evaluating the effect of a candidate lymphoma drug comprising: a) administering said drug to a patient; b) removing a cell sample from said patient; and c) determining alterations In the expression or activation of a gene selected from the group consisting SEQ ID NQS. 1. 2, and 4

6. A method of diagnosing lymphoma comprising: a) determining the expression of a HIPK1 gene selected from the group consisting of SEQ ID NOS. 1. 2. and 4. or a polypeptide encoded thereby In a first tissue type of a first individual: and b) comparing said expression of said 9ene(s) from a second normal tissue type from said first Individual or a second unaffected individual: wherein a difference in said expression indicates that the first individual has lymphoma. 00 S7. A method for inhibiting the activity of a HIPK1 protein, wherein said HIPK1 protein is encoded by a nucleic Sacid selected from the group consisting of.SEQ ID NOS. 1. 2. and 4. said method comprising binding.an i inhibitor to said HIPK1 protein.

8. A method of treating lymphoma comprising administering to a patient an Inhibitor of HIPK1 protein, wherein said HIPK1 protein is encoded by a nucleic acid selected from the group consisting of SEQ ID NOS. 1, 2, and 4. 00

9. A method of neutralizing the effect of a HIPK1 protein, wherein said HIPK1 protein is encoded by a nucleic acid selected from the group consisting or SEQ ID NOS. 1. 2. and 4, comprising contacting an agent specific Nl for said HIPK1 protein with said HIPK1 protein in an amount sufficient to effect neutralization. 00 A polypeptide which specifically binds to a HIPK1 protein encoded by a nucleic acid selected from the group consisting of SEQ ID NOS. 1, 2. and 4.

11. A polypeptide according to claim 10 comprising an antibody which specifically binds to HIPK1 protein encoded by a nucleic acid selected from the group consisting of SEQ ID NOS. 1, 2, and 4.

12. A blochip comprising one or more nucleic acid segments selected from the group consisting or SEQ ID NOS. 1, 2, end 4.

13. A method of diagnosing lymphomas or e propensity to lymphomas by sequencing at least one HIPK1 gene of an individual.

14. A method of determining HIPK .gene copy number comprising adding a HIPK1 gene probe to a sample of genomic DNA from an individual under conditions suitable for hybrldlzation. -56- 00 SEQUENCE LISTING 0> PEDERSEN, FINN S SOCRENSEN, ANNETTE. B HERNAINDEZ, JAVIER M METHODS FOR DIAGNOSIS AND TREATMENT OF DISEASES ASSOCIATED WITH ALJTERED I'DC )RESSION OF tIIPK1 A-70O19/M~S/DCF ifO> 00 POC Patentln version J 0>1 00.1> 331 2> DNA 3> mus nmusculus 0> misc feature at positions 16, 18, 26, 41, 50, 61.70, 66, 96, 124. 129, 299, 306, or 329 can be any base. 1 :cgtnggg agccancnto gac-ggngtgt ggqgaccggt ntcccagicn tctccgCaaa ;gtctccn aggtggttta accggngttt ggtggnggtc gggtttctta cagttagatg 120 *anctC'anc tagtgt.gaca tcaccccaaa ccagtgtgat ttttccccca acate-ccaat 180 catcccag c gattgggca gcgcagggag acattgacta cctggqqgat gactctgagg 240 ttageatt ctcagttttt acttaaattg tttgctgcca tgtcgatttc agggcagcna 300 gggnattt agatgcctcc ctgetcctt ng a 331 <210> 2 (211> 7594 <212> DNA <213> Mus umusculus <400> 2 ccgccaccaa acgccggtta aaccacctcg gagactgctg tgcggagagg actgggaaac cggtccccac acactgtcca cgctggctcc ccacggaggc ccacccacac ccgcggcccg 120 gggcaagatg cagtgatctc agccctcccg ctcctccgca cttccgcctc agtatggcct 180 cacagctoca ggjtgttttcg cccccatcag tgtcgtcgag tgccttctgc agtgcaaaga 740 aactgaaaat agagccctcr ggctaggatq tttcaggaCa gagcagcaac qacaaatact 300 00 icccacag iatttcaa ttgtggt :aga ccct __iagagaaa :ctctcat iccaa gag 00 IND :gctc tat ci tagcaaa 00 S:acccctc c-1 aaaatgc Icctgcct .ttaaccc itgaagct ;tcgatc ;tttccaa Itccttgg jagctgtt lLttCaa Iggttttt laacatga gcttagatga cagagaaagc cagataagag tcctggactt agcggagggt tcgctccaaa acaatcaggc caaaacCCEC tcttcctgct aacagctgct gactcacagg gagtgaggaa gctgcagaac tagcagttcc gaccaacagc gtgctggaag ctatgcCaga tgatgagtat tgtgtttgag actgccactc gaagagtctt agttcgccaa agccgtgtgt attaccattc cctgggatgg aacacaag9c teacagagat attggaaact catggctcag agatcggaga aatcacgcct tccteacagc tcacatgtat tacaagcaca cagtgttcta ccagctacac tacgaccagg gatagctcag agcaacgttz gtggagagca agaaccgtgg agtggagaag tatgaagtcc cggagcacca caaggacaga aactttgtcc atgttggagc aagtacataa ggtctgattc cCctaccgag tcaacctacc tgtgaagcta Cctctttatc ctgcCagctq cctaatttgg ggaataaa.gt. gtaaatatgt gagtatattg ctgaagactc agccacgtta gacacagtga aatctaacca gcttzccaqct aagggcaagc gcctccttct qcagcgccgc ctttgcttga acg giagc gt tgggtgctgc gggattacca tggagttcct aggaaattgt ttgaagtgag gttcttatga agaacttgta gaccaatctt atgctgacct tgaaggtcat tgcaatcacg ttgacatgtg ctggtgcttc agtatcttct ggtacccact caaaagaagc ctacagactt atcttctaaa ttaaccaCCa agtcctgttt gtcagatcaa tgagcttcag ctactgcagc cagctcctct cccagctcct tacagcaacc gccatatcsa gcagatcata tgccacgacc gct ggt cca g aggccggggg ggccettaag catcctttec gtqttttcag cgattttcta gcagcaggtg taaacctga tgactttggt ctactacaa gtcactgggc agaatacgat cagtgccgga gtggaggctt tcggaagtac agaggggaca gaaaatgctg atttgtgacg ccagaacatg gagtcccttc caaccagctc ageagctacc caccaggtag gccgtggagc ttccaaagca aaatatggat gaegaacacc accactgtga catgagatcc acatttggac atcttgaaga cgcctaagca cacaagstc aagcagaaca gccacagccc aacataatgc tctgctagtc gctcctgaaa tgtgtaatag cagattcgct acaaaaaca aagacacctg atttttaact gatatgt tag acgattgatg atgagtcacc gagatctgca actacacatg aacacagtgc ctttctctgg 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1.200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 ccaattcaga tgictcgctg ctaaaclzacc aatcggcttt gtocCcatcg tcggcagcoc 00 0gttcctgg C1cagacaga *,caagcaac 11C ccccaggc .tgtacacc 00 \Qgctgtact 0 cagctgcc N0 00atggggag Sagcactat cctctgaa aagaagaa caagttta gttcctgt agtgtcat agctcgag gactctga aacaatgg actatcat tcaggtct- gatgctgtat Cactcaacct gcaaccetqc ccttccagca ctcacctgcc tgggctccac caqcttata~c agttgcccag tccattccag aacaaagcat gcctgctgct actaatggta ctttaccctg qcaagcctgg cggggtagct cagccaacag tatgcagcag tgttggtgtt taagcaatct ttctctggtt ccaagaccag cactatccgt cctgaaaacg ctcCtccctg gacccttctg tgtgcctcct ccttaocagt cactcccacg tagccagaac tocccgcaga tggcagccca aagccagcct caqttccatt cacacaccct cagggcgttt~ caaacattta tctggattcc cagccgctgc gcaactct cc agctgcgcag cctggaggaa ctgcacaa ct ctagctgact CCatctttgc gcccatgttg gctccagttt gggagtagtc catcagccaa agtgacactg aggtctaatg agcagcccac gagggacctg ttgaaaacac aaaaccaagc gggtaccggg cagcagt cat cagcaggcat ctgcactcga cacctgtata gCtcatctgt agcactctgg ccttacaacc taatatgccc ctgtgaggat aqatccagtc accctcaagt caggccggcc cccaacaaat ctgtccagcc aggaggaatgc tgaccaacca tcagacaaca cat ccaaatc ctcttcgtac tcatcattcc at gaagaaga tcatcagtta atcccacaga geagacctgc agcttggcga cagtggcctc Ct cagcgagg cgtcagcttc ttgtggcccc cggggcaccc cgtacgctgc tctcccccca tgcatcaggt tggaaccacc a CCt gct ttt ggataatgct aggjagtactc agccaccatc ggcgctggt tctcctgcct tgctgcagtg ccactctcat tgtgaccttg acagtctagt ctctctggaa cacatcttct agataeccccc ggacaacaaa tgtcactgtc cactctgagt agcagatggc Ctgcactgta actgagtggg gggagccagc aacctcgcag gctctcccaa acacttggcc CCccacttct gggttcctca tcctgtcmgt Csgatctgca cagactagac gtgccaattg acacagggaa acgccgcagt gaacagactg tCagcctgqc 'attccagagg ggcaaccagt gccactgctc tccctccctt cQtcctgcctt tataattccc agecclzcctg tacaagccca natgattctc gctctgcggg attggcaccc gcaacacagg cagtcacctg gcggtocagc gaaagaagca gccccctacg ccagcccctg gctgctgcat aggcatgctg gtcgggccca 2100 2160 2220 2280 2340 2400 2460

2520. 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 32360 3420 3480 3540 3600 3660 3'720 gcctcctcac ttctgccagt gtggcccctg ctcagtacca acaccagttt gccactcagt 0 ctacatlcgg gtcttcccga gatcagtca gtattcttac $:).taagtagcc ctgagttctt rttcctagcc agcagcgcgt .agc.tctcgg tgttgactgc :ttattcttg tgacsgcatt 00 :accaaggaa gaaagatcgt 00 :ttgaaatcc tgtagcagat x cacaagact tccttgtctt itggaaacao aatgatttca :ttagtacaa cgagtcactg :cgaacaggc agc-agccjct Bttcattcta aatgaagaec ttctcettat agaaaagcca tatacaaaat ctctctctag atgaatttca ataatacacg tatttgaggg gAaaactaaa taa.agtagct tcctttgtat aataagtgca~ tgtaggaatt attttagaag ttttgtahtg tgattagaag aaatataaca gtagtgatta cacttgaatt agatggatta tgtctccatt gtgaactaat tgcaattcta gcctgcagag aggagacctt agtgtgtgtc ttttccttcc atgtagtggt tzatagagri accccaagct gttcgctggc ggctcaacaa ttgtagttga aatgggctct tctgcggggc attgttgcag tttgga-cqtt tctgaagtta aaaatgaagt gagaaAgagt ttattttgaa ttttcatgtc aaacctgtgc gctggg&ggg ato~agttcaa gtgaggtggt aatgagatac tctIgtgtttt gaaactaaaa gcca acagca gcagaaaata gtggtgtttt atttttcctc gtgtatttag gtatttaaac ittagagcata gggattgttt tcctctcctc tttacactgg tgagcacgag ggagagcacc ceactgaagc tctcccaagt ggaagagctc ccctctgtca aaagctcttc cattactttt agcagcttag gtgctgtcct aactgctgct aaggtgaggg. gtcctcctcC aagtgtatgg tggcactgat cctcatctct ccagaiaagt at tgaatgc ttttaaaagt aatattttgc taacccaaaa tgtgtatctg caaaatgaac *ttactgtagt *tgcaceqigtg *tctcccctta ~tacc cgctg a ;agggctccg t :ccattatct agaaggcttt1 :tgccctgtt agaagcccat tacatttggt tttacgaggg tgtttgctta caagggtgtg tactgggcag gagctgctcg t ga gg act qt tctctctagt tggtgjgtttg aaacattgcc cccttctgtt' tqtgtatagc tctcttacta ttattactga ataattaaat tgttatttgt atcctccagt tgatacttgt gctgagagag tgtctqqtga ttgtagtgcc atgaccagca igtcctacca :ggctgcctg :ctct tgaaa :Ccctgggaa :ttttaattc ctt c tgca gt ctctttgact gaaatgctga aaaaactaaa cttatggcgt ttgttagagt cagagcagca qcccaccagg ttaacttaaa catacaatag taagatttct tcatgtgact ttttatactt agacttstgt atttaaaaat atgtacatat aatcaaatgt gttaccccgg tggaatgtat caggggcatt ggagttqttc tf~aratqata agccccagtg gtgtaaatgc 3780 3840 3900 3960 4020 4080 4140 4200 4260 4.320 4380 4440 4500 4560 4620 '16S0 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 tacagtgagc. ttgccttagg atttaacaga gcaggttgag 00 cgtvttc ~-:gccacct C.k a agtgg-ac gaagagt I.attcctt ;taaactt 00 ;atgtgtt 00 :gaaaaag S:gt *tgttc .tgggtca agaaggtt ttctagga atggaacc tggagttt ctgccgC qcagtctg tttatgtc ct at ttaa gacaagat ccccactcct tggqaaaaga actrtacaga ccatctgtgt ggcttaa cat act a ggtca c tgcatttcci tcagtctccc ctgtcccgtt tctctcactg gacgccagtg ttctctCctC tgcttggttt tataccaaag aactactggt aaagcaaaac .tt ggg gccc t agttgaaggt aaccctaagc ttgqttctat cg ttcaggaa ctt cat catg aacgtggtgt ttggttgaga attctgtgtg atacagttct ttattgtttt caccteccta *ggatgcctag ctttgaggtt *ctgctgaatg *tgactgcttg icat-tgcctga kgtttctccct taccgacagt ccattttgga tgccttgcta Ctaaatatgc catgtgatat tttttttttt aqcctqttgt gacaagaagc Ctct tga at c gcacactgta actaggggtg tacagagtgg gtaggtqqtg agctctgaac gggaggaaga gtgttgtctt taaccacgca tgatc-tttgt agtttctagg tataggaatg atccaacaag gagtttacat atcactctgc tgtatgcgcc ccctgotggc ccttgqaaca tcccaggtga gacaagtcaa tCttCagctC cctggcttct atatttgaag gatagtcctt tgtttgttgt attgcttaat tcacagaaag tgaacaattc aaattgtcct gggacattCt tccacctgaa tcaggaggtg caggtggaac gtttccaggg ttctttaatc tcattgtgta gtttcctttc caaacatttt agatgcaggg cagttattga cgttacctga aaactgaca. agacatagtt atggaggaga gcttaaggct gagaggaact agccgcagct. agttct.caca gtgaqagttc gtttgtgcat tctcgcatagc atgtcccata gtttcttaat ctgaggtttc ag tat tcaa C tgcccatgag ttaaattata caggatggag accgaggggc cagggcaggt attatattaa gtttgtcact ccccaagcat ttttaacctt aaaaaacaaa agatgggagt gacatctggc ttcagatttt ttacattcat 4gccctacgaa ttaaaccttC ggaagggttg ttcctcctta gaagcattcc aggaagcagg tacttagggt tgtcgtttcc aagcatgtaa ctacgagaag tagtgacgaa tttgggacaa ccctccatgg ggatttgtgg tcagagtggt at gggag at t tgtcaacgaa agtcagttta gctgtgcgtt agtgttatac tctgggtttt ttctctataa ccaaccctgt cttaaactta tagcagtgtg cctagataac tctgqcCtgg catagcgctg tcttagaacg cataggcaca 5520 5580 5640 5700 5760 5820 5680 5940 6000 6060 6120 61e0 6240 6300 6360* 6420 6480 6040 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 caccaggaca cttagtcact ccagagtcc cagttgcaac taggagqtgg ttacCCtgtt 00 IND 00 aaccccagga gttgctcatc cctattctgc atgaaattgc tggatggtgc tgtggtttca tgtacattat ctaatttatt agaagaaccc cgtccaccatc attagtatgc cttccggtga a gcat gt ttc attcaattca atgteatgtt aaatgcattt catttcaaac cgctagaggg aglaazttac caggaatgc caagtttcea gcttgaaaaa agt at tt tt g gatatgctaa agttccggcc gcttagccag tagttgagat caagcctgct tggttgtttg taatttcact ctttgaatcc aea attgagagcc gccagcacag ggtttgtttt ttgtgttttt ttgctaaaat atatgtagca ttgatattgc tgcttttgtg tactggctgt aggataggag ttttaaatga ttatataaty gtacattata aatggoattc 7200 7260 7320 7380 7440 '7500 7560 7594 <210> 3 <211> 1210 <212> PRT <2 13> Mus musculus <400> 3 Met. Ala 1 Ser Gin Leu Gin Val Phe Ser 5 Pro Ser Val Sex Ser Ser is Ala Phe Cys Vai Ser Giy Leu Pro Ala Ala Lys Lys Leu Lys Ile Giu Pro Ser Gly Trp Asp Set Lys Thr Gin Ser Ser Asn Asp 40 Lys Tyr Tyr Thr His Thr Gin Giy Gin Aia Ser Ser Setr Gin Vai Aia Asn Asn Leu Pro Ala Tyr.*Asp Gin Gly Leu Leu Pro Aia Pro Val Giu His IlefVal1Val) Thr A-la Ala Ser Sex Giy Ser Ala Ala Thr Aia Thr Ser Lev Lau 115 Gin Ser Ser Gin Leu Thr His Arg Ser Asn VJa. 110 Lys Ser Giu Giu Pro Tyr Gin Lys 120 Cys Gly Lev Lys Arg 125 Giu Val Giu Ser Asn Giy Ser Vai Gn Ile Ile Giu Giu His Pro Pro 00 Le u 145 Met Lou Gin Asn Arg 150 Thr Val Val Gly Ala Ala'Ala. Thr Thr Thr Val Thr Thr Ser Ser Ser Ser Gly Glu Gly ASP Tyr Gin 175 Leu Val Gin Leu Giiu Phe 195 Lys Arg Ser 210 Glu Ile Leu Cys Ser 185 Met Thr Asn Ser Tyr Glu Val 190 Lys Cys.Trp Leu Gly Arg GIly Thr Lys Glu Ile 215 Phe GJly Gin Val Val A-La Ile Lys Lau Lys Asn His Ser Tyr Ala Arg -Gin 230 Gly Gin Ile Giu Ssr Ile Leu Ser Arg 240 Leu Ser Ser Glu Asn 245 Ala Asp GlU Tyr As n 250 Phe Val Arg Ser Tyr Glu 255 Cys Phe Gin Gin Asn Lau 2-75 Lys Asn His Thr L6U Val Phe Giu Met Lou Glu 270 Pro Leu Pro Tyr Asp Phe Leu Gin Asn Lys Phe Ser 285 Leu Lys 290 Tyr Ile Axrg Pro Ile 295 LOU Gin Gin Val Ala 300 Thr Ala Leu met LgU Lys Ser Leu Leu Ile His Ala Asp 315 Lau Lys Pro Giu Ile Met Leu Val Pro Vai Ara Gin Tyr Axg Val Lys Vai Ile 335 Asp ?he Gly Ala Ser His Val Ser Lys Ala Val] Cys 345 Ser Thr Tyr 350 La .u Gin Ser Arg Tyr Tyr Arg Ala Pro Giu 'Ile Ile Leu Gly Leu Pro 335 360 365 00 Phe Cys 370 Giu Ala Ile Asp met 375 Trp Ser Leu Gly Val. Ile Ala Glu Phe Lou Gly 'rrp Leu Tyr Pro Ile Arg Tyr 1ie Ser 405 Gin Thr Gin Gly Gly Ala Ser Glu 395 Leu Pro Ala GJlu 410 Phe Asn.Arg Asp Tyr Asp Tyr Leu 415 Ser Ala Gly Gly Tyr Pro 435 LYS Thr Thr Axrg Phe 425 Pro Asri Leu 430 Giu Leu Giu Leu Trp, Axg Leu Lys Thr Pro Glu Glu 440 Thr Gly 450 Ile Lys Ser Lys 'Ala Arg Lys Tyr Ile Phe Asn Cys ~160 Leu Glu Gly Thr Leu Asp 480 Asp 465 Asp Met Ala Gin A~sn met Ser Thr Asp 475 Hot Leu Ala Giu Ala Asp Arg Arg Tyr Ile Asp Leu Leu Lys 495 Lys Met Leu Leu Asn His 515 Ile Asp Ala Asp Arg Ile Thr Pro Leu Lys Thr 510 Phe Pro His Gin ?h~e Val Thr Met 520 Ser His Lau Leu Asp 525 Ser Ser 530 His Val Lys Ser Phe Gin Asri Met Ile Cys Lys Arg Arg 545 Val His Met*Tyr Thr Val. Ser Gin Ile* 555 Lys Ser Pro Phe Thr His Val Ala Pro 565 Aan Thr 580 A-sn Thr Ser Thr Leu Thr Met Ser Phe Ser 575 Asn Gin Leu val His Asn Ala Ser Val Leu Ala Ser Ser 590 Ser Thx Ala 595 Ala Ala Ala Thr Leu 600 Ser Leu A.Ila Asn Asp Val Ser Lou Leu 610 Asn Tyr Gin Ser Ala 615 Leu Tyr Pro Sex' Ala Ala Pro Val. Giy Thr. Thr Gin Pro 625 Gly Val Ala G3.n Giy Val Ser tLeu Gin Pro Thr Phe Ile Ile Cys Thr Gin Asp Pro Phe Gin Val Cys Pre 655 Ser Gly Ph. 670 Ala Pro Ala Pro Ala. Phe Pro Vai Arg 675 Gin 660 Thr Gly Leu Gin Aia 665 Thr Thr Lys His Met Asp Asn Ala Pro Ile Val Pro Gin 685 Ala Gin 690 Pr~o Leu Gin Ilie Ser Gly Val Leu Thr Gin Gly Ser 700 Val Ala Thr Ile Cys Thr 720 Thx 705 Pro Leu Met Val Thr Leu His Pro Pro Gin Tyr Ala Pro Phe Thr Leu Ser 730 Cys Ala Ala Giy Arg Pro 735 Ala Leu Val Thr Gin Gin 155 Giu 740 Gin Thr Ala Ala Leu Gin Ala Trp Pro Giy Gly 750 Pro Gly Val Ile Leu Leu Pro Ser 760 Al1a Trp Gin Gin Ala Lcu 770 His Asn Ser Val Pro Ala Ala Val Ile Pro Glu Ala Met 760 Ala His Ser His Glv Gly 785 Ser Ser Gin Gin Ala Asp Trp Arg As n '795 Asn Gin Tyr Ser Thr 805 Ile Met Gin Gin Pro 810 Ser Leui Leu Thr Asn His 8is Val Thr Leu Val Arg Gin 835 Thr Ala Gin Pro Leu 825 Asn Val Gly Val Ala His Val 830 Asm Lys Gin Gin Gin Ser Ser Ser 840 Leu Pro Ser Lys Ser A1a 850 Pro Val Ser Ser Lys Ser.Ser Leu 855 Ser Ser Pro Leu Giu Leu Pro Se-r Gin Thr Ser Ser Tyr Val 865 Tyr Ser Leu Val Gly 870 Arg Thr 875 Gin Pro Ile Asn Ser Leu Val Val. Gin Asp Gin Ile Ile Pro 895 Ser Asp Thr 910 Ser Leu Lys Asp Thr Pro Asp Giu Giu 915 Pro Pro Val Ser Ile Thr Ile Axrg Giu Asp Asn Lys Lys Pro Asn Ser Aia Axg 930 Ser Asn Val Ile Tyr Val Thr Vai Asn Asp Ser Pro Asp 940 Asp Thr Leu Ser Ala Ser Asp Ser Ser Leu 945 Leu Arg Gly Asn Ser 965 Ser 950 Ser Pro His Pro Thr 955 Giy Thr Leu Leu Giu 970 Giy Pro Gly Arg Pro Ala 975 A-14, Asp Gly Gin Leu Gly 995 Gly Thr Arg Thr Ile Val Pro Pro Lei; Lys Thr 990 Azp Cys Thr Val Aia Thr Gin. Ala Ser Gly teu Leu Ser 1000 100.5 Ser Lya Thr Lys Pro Vai Ala Ser Val Ser Gly Gin Ser Ser Gly 1010 1015. 1020 Cys Cys Ile Thr Pro Thr. Gly Tyr Arg Ala Gin Arg Gly Gly Ala 1025 1030 1035 Ser Ala Val Gin Pro Leu Asn Leu Ser Gin Asn Gin Gin Ser Ser 00 1040 C1Ser Ala 1055 IN Ara Gin 1070 Phe Gin 00 1065 SAla Pro C1 1100 00 Tyr Ala C1 1115 1045 1050 Ser Thr Ser Gln* Gin Ala Phe Val His Gly Ser Pro Ala Pro Ala His Ala Pro Thr Ser His Lau Phe Ser Thr Thr His Pro Gly Pro Ser Leu Gin His Gin Phe Ser Thr Ile Tyr 1iQ Ala 1130 Ala Tryr 1145 Ser Val 1160 Gin Tyr- 1175 Arg Gly 1190 Glu 1060 Al a 1075 Lmu 1090 Leu 1 105 Al a 1120 Pro 113S Ser 1150 1165 Ala 1180 Thr 1195 Leu 1210 Arg Ser Ser Asn Pro Leu Ser Gin His Soy Thr Gly Pro Ser Gin Pro Ala Ala Lev Gly G.1n Gly Ser Ser Thr Lou Val His Thr Ser Ala Ser Thr Gin Ser Tyr Gly Tyr Pro Leu Pro 1065 Ala 1080 His 1095 His 1110 Ser 1125 Arg 1140 Gin 1155 Val1 1170 Ile 1les Ser 1200 Ala Pro *Arq Pro Tyr Ala Pro His Leu Leu Tyr Thr Thr Ser Ser His Ala Ala Val Pro Val Ala Pro Ala Gly Ser Sex Pro Thr Lys Ile'Ser Gl.n Tyr Sar Tyr 1205 <210> 4 <211> 5161 <212> DNA <213> Homno sapiens cocaccgcag tatgcggtge cctttactct gagctgcgca gccggccggc cggcgctggt tgaacagact 9ccgctgtac tggcgtggcc tggagggact cagcaaattc tcctgccttc 00 aacttgjgcaa Stccagaggcc caaccagtac <cactgctcag __cctcccttcg tccqccttcc 00 taattccttg 00 Stgattctcca C1tctccaaggc tggcacccgc ~aaccaggcc gtcatcizgga agcacaacca gagaagcagc c ccctcacc ggcCCctgct tgctgcactg gcatgctgca tgggcccagc cacccaatcc tcctaocaag atggctacct ccctcttgaa tttctctgg cctatttt cccatcttct tttgtctctc cagttgcctg atgggagt 9 agcactatca cctctgaatg aagaaqaata caagtctatt gtccctgtcc agtgtcatca agctctgga c gactctgact aatagtggat actatcattg tcaggtzctcc tgctgtatca ctcaatctta aacccagccc ttccagcatg cacctgccaa ggctcaacca gcctatacca CtCCtCactt tacattgggt atcagccagt tctcctggcc .attttrt a gc ggaacc tgtc aactCatrt gcagttacca tgacttgatt gggtagjctet gacagcagct tgcagcagcc ttggtgttgc agCagtcagc Ctctggttgg aagatcagca ctatccgaag tgaagccaag cttctttgag ccgttttgga tgcctccact tgagcaataa CCCCCacagg 9ccagaacca cccgcaggca gcagcccgct gccaggctca gctccattgc Ctcaccctag Ctgccagcgt CttccCgagg attcctactt ctgcgttett cagcaacttg tCaatgttga ttttgtgaca agqaagagag tctataaatg *acacaactct agctgactgg atccttgctg ccatgttgtc tccagtctct gagcagr ccc tcagcccatc tgacactgat gtctaatgrtc cagcccttat ggggcctggc gaaaactcag gactaagcca gtatcgagct gcagtcatcg gcaggcgttt acactcgaca tctgtatacg cacttrggtg ggcccctgct ctcaacaatt atagttgg aacattgggc ttctgcaggg ctgcattgtt otaatttqgg attgttc tga cttttaaaaa gtccagccc& aggaatgccc actaaccatg agacaacaac CCcaagtcct ctCcgcacco atcattccag gaggaagagg atcagtt.Lg tCcactgata agagttgtgg cttggtgact gtcgcttcag caacgcgcgg gcggctccaa gtggccctc gggcac-ccac tatgctgccc tccccaca~gg caccaggrcce cagtaccaac tacactggat agcatgaggg tatggagaga gcccactgaa gtagtcttcc tacttggaag Qgttaccctc caagtga age CagCaatgat acrctcatgg tgarratggc aatccagttc ctctagatgt catcttctta a tact cccag acaaCaaata tcactgtcaa ccctgagtgc cagatggcac gcactgtagc tgagtgggca ggaccagtgc cctcacagga tctcccaagc accttgccce cgacttctgc gttcetcaag- Ctgtcagtgt accagtttgc acccgctgag Aggaggaatc tcctccttt~a gcagaaggtt Caaagtttgc agttcagatg tgaaaaaiat ccctctttat 180 240 300 360 420. 480 540 600 660 720 780 940 900 960 1020 1080 1140 1200 1260 1320 13S0 1440 1500 J. 560 1620 1680 1740 1800 1860 00 00D 00 ttcattttgt tgatgacaaa attttaaaag tteagttttg gttacgtatt agtagtgttt tgcgtgaaaa tttttaaaag ggtggttatt agatactggc tgttttgctc cctgaaacca tgtatgccag gaattgcaaa taatggtggt aacaagcaat acttgaattg Sgt ct cc at tgcaattata agaggagacc gtctct tcct ggttaitaga aagttgttca ctcagtttcc tc tacccctt gctctctcac gtcaagccaa ttttttttcc gttattgtga aaaagaaaaa cggcttacac ttttastgtc actctgtgtt aaaaggcagc caccaagtat tctttctctc attacatggt attgatgagc atctct c ccc gataagaaca cagcaaattg a aa tat ttt a gttttaa tat ttttcctgct tgtacttage gtatttaaac ttagagcata cttggaattg tccctttctt gtttacagtq ccgggattta ctgccaacat t ccat t ttgg tgtgcqg.tgc tattaaatat ttttcccatg t'tgctggtca ttactttttg aatctccctt atattatact a ttat tgaga tcaccatttg tctttttaaa tctgattcog gqtggtggtt tttgcCCaaa tctgttttat tttettgtgt aatgctctct aaaatttatt t t tac ataa t aa~ccaaaat gtgtatgtga caaaatgaac ttactgtagt .ttttgcacag cctccttccc agcttgcctt tcagaacagg tgaaaaataa attctcggct taccttgctt gcattctttt tggcagtcct ggaaaaatgc tttgtttata ttqtttattg taatgggcaa ttctctcaat ctggtaactt tgaagcacca cttaaatttt ttattatatg gattagtatg gtgatttgtt atagctttta tattaagact actgaattta taaatatgta gttatttgta tcctccagtg tgatacttgt gctgazatgag gtgtgtctgg ttattgtagt aggatggacc attagtagct aaacagcagc gagttctcac ct g tqggaat aaagtatgtg tcctgcacat tgatagaagg aactcagact gacatttaaa tt gttatttt tgctcctgtg aatgtgaqag tgaattcttt tttatcgaua caaaatctct aatttteag~t tggggagaaa tacttcaaag tatataataa aaaatatttt catattgatt atcaaatgtg ttatcccgga tggaatgtat caggggcatt tgagqsgttt gccttatatg agcaagccc gtattgtgca ttttctcct agaageattt tcaggaagca caatCacttt agttgacatt agttgaaatc tgcctatttt cttacagagt tgcaaaactg tttgttataa aatceatatc tttaaattat aagccattaa gtctattatg aatacacctc gctaaaaaaa' tagcttcctt gtgcatgtag agaagttttg agaaaaatat tagtgattac gatggattg4 gtgaactaat gCctgcaagg ttcagtgtgt ataatgtagt cgtggaccct atgcattgtt taccaccacc tccccatgtg ggtgagagqa tagaatgaat cctagtaaaa 1920 1960 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3160 3240 330o 3360 3420 3480 3540 00 tatttgcttg tgcttaccat tcacagaaag attgaggctt tgaaattgtc gatggggaca tgtgttccat cagatagaga cagccagctc t t gta ag gag gttgtgttt gtcaagatag aacatgtgtg acaggtctag gatttgttgt cccagtccaa taccaagagc agagattttt ttgaaaaaaa gtccccatac gtttcttagc ttgaggtttc Ct rg vtactc t ttcctgccca ccgaattgaa t gg tgt caa g tgtacacaggt taagggcttc ctttattttt Ccaagcagtt atctttgtgt tttctaaagg ttttgtaaga taagcagata ttgtattgtt cagtcttagt ctgaatgtgt tggccCtga c qtgacctgac acttcttcct tcatagggac cagtgctcag attctgagcc actgccctta ttcactagtt gtggcccgag acgtctacat *catgttaaca *tatgaggaga *tggtgaaqaa ttttttaaca Cagctcctqc taaaggatt aatgatacat gagqtgcagg tgaacaccga caagatggga taaaatcatt tgtataattt ctcctttttg acaaattttt aatgagatgc ccact taaga accttagtca Ctacaaactg atgtatgtgc aggaagggag ctcaaaccag ttccgggtga Acttggtcac ctqaaat acc tgcttttgtg gccaggcaag gaga ~ggttr c Ctgct tcct ggttgtttgt *gatqtgttta taccaaagag cctgttgtat agttttgtgg tgccgetggt gacaaggaac .tatagagaag gaaccaaaqc Ctgttgagtc gcttgtatag tcttggggcc cttcaagctg atggatctgq gtcaagcaga aggtactgg~g ggggaaacqaa gattaatcct aaaatacagg ttgagAtata attttaggac tgqttctgtg atggagatgg gagatttcat ggagcctggt ggagctgtca aagtatttgg agtttcttca caggtagtcc gtacagccta ccaggaacat atattgagtt gtgttttcag cactatattg ctgtcactag tgtcatacag ttttctggtc ccaagcacat tctgattttc ttgttggaac tgttccttgt Cttttttctg taagggacea a ggaaageea accaaatccc attcctgcac tagjgagtcta aactccacag aaaaggataa cttgcctagc agtgtggc tttaaaaact gcatttccga ttttccagca taaaaatcca tcactgtggc tttagattct gtccctgggg gaagcctggt gaatttagtg agcagctggc cttaaggctt taaqtcctct ctcagaactt gagaagaagc ggagaagggt tc-agtgtagc tccagagttt ttautagccc ccaggagqtg aaccccagga ataagaactc. catttcaaac attgctcatc cattgtcctc cactagaggg cacagtaatg tgtgttttgt tcagcattat gttttaggat aggaaatgaa attgCCtCtc tgctaaactt tatataotgt gtggtttcaa 3600 3660 3720 3780 38.40 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560

4620- 4680 4740 4600 486.0 4920 4980 5040 5100 5160 5220 5280 Cctgtgtctg ttagccctgt ctgggtcaca ggcatttcca cactctgggc at at tat ttt Bgttctggcc gctaagcttg tatgcaaaaa agtgacagga at ggtgcagc 00 ~t a' S t4 00 tcagcttga gttagtatt tttgatatg tgtgcgctt atgtttgta agatatztta tctttggat acataatat aaaataatct tctgetttga ctagttattg tcttttacaa atgcagatgt aaatttaata tcaatgtttg ggtaaaatgt cactacatgt atCcttgata tgtgcgattt ceagcctcta aettagggag ctaactatgg tctttggttt agagcaattg agcagta cat ttgcaatgga aaactttlt gaaacagata tatgtaaaat gaaaagggtc tacaaagtag caatgcatca tatatgtaca a~tOCtactt tgctttctcc gtttctgaga aatcatttta cattgtgtaa cttgtatttt ataaaatggg ttatatgtaa tattaaatgt Ctttttttgg attactgagc Scaaaagaaa Aacatagttt cagtattttc taaattttct Gly Arg is 5340 5400 5460 5520 558a0 5640 5100 5760 5761 <210> <211> 490 <212> PRT <213> Hom~o sapiens (400 Thr Pro Gin Tyr Ala 1 5 Val Pro Phe Thr Leu 10 Ser Cys Ala Ala Pro Ala Leu Thr Gin Gin Val Glu Gin Thr Ala A.1a 25 Val. Leu Ala Trp Pro Gly Val lie Leu Leu Pro Ser Thr Trp Gin Gin Ala Leu s0 His Asn Ser Val Pro Thr Ala Met Ile Pro Glu Ala Mt'e Gly Ser Gly Gin Gin Akla Asp Trp Arg Ala His Ser His hsn Gin Tyr Ser Ile Met Gin Gin Ser Leu Leu Thr Asn His Val Thr Leu Thr Ala Gin Pro Asn Val Gly Val Ala Hi~s Val 110 Asn Lys Cin Val Arg Gin Gin Gin Ser Ser Ser Leu Pro Ser Lys 00 sor Ala 130 Pro Val Ser Ser 3er Sex Leu Asp Leu Pro Ser Gin Tyr Ser Leu Val Gly 150 Ser Ser Pro Leu Thr Thr Ser Ser Tyr 160 ASn Ser Leu Val Val Gin Asp Gin GIn Pro Ile Asp Thr Pro Asp GiU Glu 195 Pro Pro Val Ser Ile Thr Ile Arg Ile Ile Pro 1,75 Ser Asp Thr 190. Gly Leu Lys GIlu Asp Asn Lys Lys Pro Ser Ser Ser 205 Pro Arg 210 Ser Asn Val Ile Tyr Val Thr Val Asn Asp Ser Pro Asp 220 Asp Thr Leu Ser Ala Ser 225 Asp Ser Ser Leu Ser 230 Ser Pro Tyr Sex Leu Arg Gly Asn Se r 245 Gly Ser Val Leu Gly Pro Gly.Arg Vai Val 255 Ala Asp Gly Gin Lou Gly 275 Thr 260 Gly Thr Arg Thr ile Val. Pro Pro Leu Lys Thr 210 Lau Leu Ser Asp Cys Thz Vai Thr Gin Ala Ser Asn Lys 290 Thr L~ys Pro Val Al a 295 Sar Val Ser Giy Gin 300 Ser Ser Cly Cys Ile Thr Pro Thr Gi y 310 Tyr Arg Ala GIn Arg 315 GlY Gly Thr Sar Ala Cin Pro Lev Leu Ser Gin Asn Gin Ser Ser Ala Ala Pro 335 Thr Ser GIn Glu 340 Arg Ser Ser Asn Ala Pro Arg Arg Gin Gin Aia 350 00 Phe Val A~la 355 Pro Lou His 370 Pro Lau Ser Gln Ser Thr-Gly MH,* 375 Al a 360 Pro Tyr Thr Phe Gin.His Gly Ser 365 Ala Pro Ala His Pro His Leu Ala Leu 385 Pro Ser Gin Ala His 390 LoU Tyr Thr Tyr Ala Pro Thr Ser Ala 400 Pro Gin 415* Ala Ala Leu Gly Ser 405 Thr Sar Sex Ile His Lau Phe Ser Gly Ser Ser Va. His Gin 435 His Ala Ala Ala Tyr 425 Thr Thr His Pro Ser Thr Leu 430 Thr Ser Ala Val Pro Val Ser Vai2 440 Gly Pro Ser Leu Lau 445 Ser Val. 4 SO Al& Pro'Ale GIn Ser Ser Arg Gly 470 Gin His Gin Phe Thr Gln Ser Tyr GI y Ser Thr Ile Tyr Th r 475 Gly Tyr Pro Leu Pro Thr Lys Ile Ser 4 8 Gln Tyr Ser Tyr