TANKYRASE H, COMPOSITIONS INVOLVED IN THE CELL CYCLE AND METHODS OF USE

Abstract: The present invention is directed to novel polypeptides, nucleic acids and related molecules which have an effect on or are related to the cell cycle. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided by the present invention are methods for identifying novel compositions which mediate cell cycle bioactivity, and the use of such compositions in diagnosis and treatment of disease.

Description: TANKYRASE H, COMPOSITIONS INVOLVED IN THE CELL CYCLE AND METHODS OF USE FIELD OF THE INVENTION The present invention is directed to compositions involved in cell cycle regulation and methods of use More particularly, the present invention is directed to genes encoding proteins and proteins involved in cell cycle regulation, particularly those having homoiogy to tankyrase Methods of use include use in assays screening for modulators of the cell cycle and use as therapeutics BACKGROUND OF THE INVENTION Cells cycle through various stages of growth, starting with the M phase, where mitosis and cytoplasmic division (cytokinesis) occurs The M phase is followed by the G1 phase, in which the cells resume a high rate of biosynthesis and growth The S phase begins with DNA synthesis, and ends when the DNA content of the nucleus has doubled The cell then enters G2 phase, which ends when mitosis starts, signaled by the appearance of condensed chromosomes Terminally differentiated cells are arrested in the G1 phase, and no longer undergo cell division The hallmark of a malignant cell is uncontrolled proliferation This phenotype is acquired through the accumulation of gene mutations, the majority of which promote passage through the cell cycle Cancer cells ignore growth regulatory signals and remain committed to cell division Classic oncogenes, such as ras, lead to inappropriate transition from G1 to S phase of the cell cycle, mimicking proliferative extracellular signals Cell cycle checkpoint controls ensure faithful replication and segregation of the genome The loss of cell cycle checkpoint control results in genomic instability, greatly accelerating the accumulation of mutations wh.ch drive malignant transformation Thus, modulating cell cycle checkpoint pathways and other such pathways with therapeutic agents could exploit the differences between normal and tumor cells, both improving the selectivity of radio- and chemotherapy, and leading to novel cancer treatments As another example, it would be useful to control entry into apoptosis It is also sometimes desirable to enhance proliferation of cells in a controlled manner For example, proliferation of cells is useful in wound healing and where growth of tissue is desirable Thus, identifying modulators which promote, enhance or deter the inhibition of proliferation is desirable Continuous cell proliferation, as in cancer, requires the replication of DNA including chromosome ends known as telomeres Without being bound by theory, it appears that telomeres decrease in size with successive cell divisions, that the number of divisions a cell is capable of negatively correlates with telomere length, and that a cell cannot divide further once a cπtical telomere length has been reached Further, the normal process of telomere shortening with successive cell divisions appears to be circumvented in cancer, suggesting the maintenance of telomore length may be cπtical to normal and oncogenic growth Without being bound by theory, it appears that the synthesis of telomeres involves unique DNA replication mechanisms These mechanisms act to extend telomeres prior to cell division, and are critical to the determination of telomere length in daughter cells Several molecules involved in telomere synthesis have been identified, including the proteins telomerase, TRF-1 and tankyrase These and other molecules involved in telomere synthesis provide unique targets for intervention strategies designed to modulate cell proliferation Recognized in this application is the hypothesis that these two aspects of cell proliferation control, namely check point modulation and telomere maintenance, intersect in some aspect The present application sets forth tankyrase h nucleic acids and proteins which, without being bound by theory, appear to bndge the gap that currently exists between these two points of control Despite the desirability of identifying cell cycle components and modulators, there is a deficit in the field of such compounds Accordingly, it would be advantageous to provide compositions and methods useful in screening for modulators of the cell cycle It would also be advantageous to provide novel compositions which are involved in the cell cycle SUMMARY OF THE INVENTION The present invention provides cell cycle proteins and nucleic acids which encode such proteins Also provided are methods for screening for a bioactive agent capable of modulating the cell cycle The method compπses combining a cell cycle protein and a candidate bioactive agent and a ceil or a population of cells, and determining the effect on the cell in the presence and absence of the candidate agent Therapeutics for regulating or modulating the cell cycle are also provided In one aspect, a recombinant nucleic acid encoding a cell cycle protein comprising a nucleic acid that hybridizes under high stringency conditions to a sequence as set forth in Figure 1 or Figure 2, or their complements, is provided In a preferred embodiment, the recombinant nucleic acid encodes a protein which binds to p21 In another aspect, a recombinant nucleic acid encoding a cell cycle protein compnsmg a nucleic acid having at least 85% sequence identity to a sequence as set forth in Figure 1 or Figure 2 is provided In one embodiment, a recombinant nucleic acid is provided which encodes a polypeptide having the sequence as shown in Figure 3 or Figure 4 In yet another aspect, a recombinant nucleic acid is provided herein which has a nucleic acid sequence as set forth in Figure 1 or Figure 2 In a further aspect, expression vectors are provided herein In one embodiment, the vector comprises any one of the recombinant nucleic acids described herein, operably linked to regulatory sequences recognized by a host cell transformed with the nucleic acid Moreover, host cells comprising any one of the nucleic acids or vectors descπbed herein are provided Also provided herein is a process for producing a cell cycle protein comprising cultuπng any one of the host cells described herein under conditions suitable for expression of a cell cycle protein In one embodiment, the cell cycle protein is recovered In a further aspect herein, a recombinant cell cycle protein encoded by any one of the nucleic acids described herein is provided In yet another aspect, a recombinant polypeptide comprising an ammo acid sequence having at least 85% sequence identity with the sequence set forth in Figure 3 or Figure 4 is provided herein In one embodiment, the recombinant polypeptide provided herein bind to p21 In another embodiment, the recombinant polypeptide provided herein has a sequence as set forth in Figure 3 or Figure 4 Also provided herein is an isolated polypeptide which specifically binds to a cell cycle protein In one aspect, the polypeptide is an antibody In a preferred embodiment, the antibody is a monoclonal antibody In another preferred embodiment, the antibody reduces or eliminates the biological function of said ceil cycle protein In a further aspect, the present invention provides a method for screening for a bioactive agent capable of binding to a cell cycle protein tankyrase H In a preferred embodiment, said method comprises combining a cell cycle protein tankyrase H and a candidate bioactive agent, and determining the binding of said candidate agent to said cell cycle protein tankyrase H In one embodiment, a method for screening for agents capable of interfering with the binding of a cell cycle protein tankyrase H and p21 is provided In a preferred embodiment, a screening method as described herein comprises combining a cell cycle protein tankyrase H, a candidate bioactive agent and a p21 protein, and determining the binding of said cell cycle protein and said p21 protein In one case, said ceil cycle protein and said p21 protein are combined first In yet another embodiment a method for screening for a bioactive agent capable of modulating the activity of a cell cycle protein tankyrase H is provided In one aspect, the method compnses the steps of adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a cell cycle protein tankyrase H and determining the effect of the candidate bioactive agent on said cell In another embodiment, a library of candidate bioactive agents are added to a plurality of cells comprising a recombinant nucleic acid encoding a cell cycle protein Also provided herein is a method of diagnosing cancer In one embodiment, the method comprises determining the level of expression of tankyrase H from a test sample of an individual and compaπng the level with a control which indicates there is no cancer, wherein an increase in the level of expression of tankyrase H in the test sample over the control sample indicates that the individual has cancer In another embodiment, the method comprises determining the activity of tankyrase H, preferably poly ADP-πbose polymerase (PARP) activity, in a test sample from an individual and comparing the level with a control An alteration (eg an increase) in the activity of tankyrase H in the test sample over the control sample indicates that the individual has cancer Further provided herein are methods for the treatment of individuals affected by dysfunction of tankyrase activity, tankyrase H activity, telomerase activity, or cancer, using a pharmaceutical composition comprising modulators of tankyrase H activity, including antisense oligonucleotides and bioactive agents capable of binding to and/or modulating the activity of tankyrase H BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleic acid sequence of SEQ ID NO 1 , corresponding to clone TH-1 and encoding tankyrase H isoform 1 (TaHo-1), wherein the stop codon is bold and underlined Figure 2 shows the nucleic acid sequence of SEQ ID NO 2, corresponding to clone K23 and encoding tankyrase H isoform 2 (TaHo-2), wherein the stop codon is bold and underlined Figure 3 shows the ammo acid sequence of SEQ ID NO 3, corresponding to tankyrase H isoform 1 (TaHo-1) and predicted from the nucleic acid sequence set forth by SEQ ID NO 1 Figure 4 shows the am o acid sequence of SEQ ID NO 4, corresponding to tankyrase H isoform 2 (TaHo-2) and predicted from the nucleic acid sequence set forth by SEQ ID NO 2 DETAILED DESCRIPTION OF THE INVENTION The present invention provides cell cycle proteins and nucleic acids which encode such proteins Also provided are methods for screening for a bioactive agent capable of modulating the cell cycle The method compπses combining a cell cycle protein and a candidate bioactive agent and a cell or a population of cells, and determining the effect on the cell in the presence and absence of the candidate agent Other screening assays including binding assays are also provided herein as descnbed below Further provided are activity assays, including PARP activity assays, for screening of bioactive agents Therapeutics for regulating or modulating the cell cycle are also provided and described herein Diagnostics, as further described below, are also provided herein A cell cycle protein of the present invention may be identified in several ways "Protein" in this sense includes proteins, polypeptides, and peptides A cell cycle protein may be initially identified by its association with a protein known to be involved in the cell cycle Wherein the cell cycle proteins and nucleic acids are novel, compositions and methods of use are provided herein In the case that the cell cycle proteins and nucleic acids were known but not known to be involved in cell cycle activity as described herein, methods of use, i e functional screens and therapeutic uses, are provided In one embodiment provided herein, a cell cycle protein as defined herein has one or more of the following characteristics binding to p21 (also called CIP), homoiogy to tankyrase and homoiogy to poly adenosine diphosphate-πbose polymerase (PARP), and PARP activity The homoiogy to tankyrase and PARP is found using the following database and parameters Altschul, et al , Nucleic Acid Res , 25 3389-3402 (1997), non-redundant GenBank+EMBL+DDBJ+PDB sequences with a lambda of 1 37, k of 0 711 , H of 0, gapped lambda of 1 37, k of 0 711 , H of 4 94e-324, matrix of blastn matrix 1 -3, gap penalties existence 5, extension 2 Preferably, as further discussed below, the cell cycle protein provided herein shares at least 50% homoiogy (identity or similarity) with tankyrase or the catalytic domain of PARP, and at least 80% identity, preferably at least 85%, with the sequence in Figure 3 or Figure 4, or portions thereof In one embodiment, the cell cycle protein is termed tankyrase H, sometimes referred to as "TaHo" herein The ammo acid sequence is shown in Figure 4 and Figure 4, and the nucleic acid sequence is shown in Figure 1 and Figure 2 The ammo acid sequence of tankyrase H bears homoiogy to tankyrase, but preferably, less than 80% Tankyrase is an enzyme which binds to TRF1 and which has been indicated as having a role in maintaining telomere length. Smith, et al., Science, 282(5393): 1484-7 (1998). More particularly, tankyrase has homoiogy to ankyrins and binds to the telomeric protein TRF1, a negative regulator of telomere length maintenance. Ankyrins have been reported to have homoiogy to tissue-differentiation and cell cycle control proteins. Lux, et al., Nature, 344(6261 ):36-42 (1990). Telomeres shorten progressively with every cell division, ultimately causing cessation of cell division thereby inducing a cell death pathway. This process, telomeres, and the role of telomerase are further described in, e.g., Bryan and Cech, Curr Opin Cell Biol., 11(3):318-24 (1999); Hiyama, et al, Virchows Arch, 434(6):438-7 (1999); Krejc, Genomics, 58(2):202-6 (1999); Holt and Shay, J Cell Physiol., 180(1):10-8 (1999); and Tan, J Theor Biol., 198(2):259-68 (1999). Tankyrase H shares full length homoiogy with a catalytic domain of PARP which is also identified with tankyrase (tankyrase shares homoiogy to the catalytic domain of PARP). The protein p21, to which cell cycle proteins described herein preferably bind, has been reported on as being a cell cycle protein. P21 encodes a universal inhibitor of cyclin-dependent kinases. See, e.g., Skomedal, et ai., Gynecol. Oncol., 73(2):223-8 (1999); Skomedal, et al., J Pathol., 187(5):556-562 (1999); Shimizu, et al., Cancer, 85(3):669-77 (1999); Li, et al., Oncogene, 9(8):2261-8 (1994). In one embodiment, cell cycle nucleic acids or cell cycle proteins are initially identified by substantial nucleic acid and/or amino acid sequence identity or similarity to the sequence(s) provided herein. In a preferred embodiment, cell cycle nucleic acids or cell cycle proteins have sequence identity or similarity to the sequences provided herein as described below and one or more of the cell cycle protein bioactivities as further described below. Such sequence identity or similarity can be based upon the overall nucleic acid or amino acid sequence. A cell cycle protein, tankyrase H, is shown in and described in Figure 3 and Figure 4. In a preferred embodiment, a protein is a "cell cycle protein" as defined herein if the overall sequence identity of the amino acid sequence of Figure 3 or Figure 4 is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%. In some embodiments the sequence identity will be as high as about 93 to 95 or 98%. In another preferred embodiment, a cell cycle protein has an overall sequence similarity with the amino acid sequence of Figure 3 or Figure 4 of greater than about 80%, more preferably greater than about 85%, even more preferably greater than about 90% and most preferably greater than 93%. In some embodiments the sequence identity will be as high as about 95 to 98 or 99%. As is known in the art, a number of different programs can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv Appl Math 2 482 (1981), by the sequence identity alignment algoπthm of Needleman & Wunsch, J Mol Biool 48 443 (1970), by the search for similaπty method of Pearson & Lipman, PNAS USA 85 2444 (1988), by computerized implementations of these algoπthms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wl), the Best Fit sequence program described bv Devereux et al , Nucl Acid Res 12 387-395 (1984), preferably using the default settings, or by inspection Preferably, percent identity is calculated by FastDB based upon the following parameters mismatch penalty of 1 , gap penalty of 1, gap size penalty of 0 33, and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R Liss, Inc An 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 simplification of the progressive alignment method of Feng & Doolittle, J Mol Evol 35 351-360 (1987), the method is similar to that described by Higgms & Sharp CABIOS 5 151-153 (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, descπbed in Altschul et al , J Mol Biol 215, 403-410, (1990) and Karlm et al , PNAS USA 90 5873-5787 (1993) A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al Methods in Enzvmoloqy. 266 460-480 (1996), http //blast wustl/edu/blast/ README html] 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 =1 , overlap fraction = 0 125, word threshold (T) = 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 and composition of the particular database against which the sequence of interest is being searched, however, the values may be adjusted to increase sensitivity An additional useful algorithm is gapped BLAST as reported by Altschul et al Nucleic Acids Res 25 3389-3402 Gapped BLAST uses BLOSUM-62 substitution scores threshold T parameter set to 9, the two-hit method to trigger ungapped extensions, charges gap lengths of k a cost of 10+/ , X u set to 16 and X g set to 40 for database search stage and to 67 for the output stage of the algorithms Gapped alignments are triggered by a score corresponding to ~22 bits A % ammo acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues 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) In a similar manner, "percent (%) nucleic acid sequence identity" is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the given nucleic acid sequence A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0 125, respectively The alignment may include the introduction of gaps in the sequences to be aligned In addition, for sequences which contain either more or fewer ammo acids than the protein encoded by the sequences in the Figures, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical ammo acids in relation to the total number of ammo acids Thus, for example, sequence identity of sequences shorter than that shown in the Figure, as discussed below, will be determined using the number of am o acids in the shorter sequence, in one embodiment In percent identity calculations relative weight is not assigned to vanous manifestations of sequence vaπation, such as, insertions, deletions, substitutions, etc In one embodiment, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0", which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations Percent sequence identity can be calculated, for example * by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100 The "longer" sequence is the one having the most actual residues in the aligned region As will be appreciated by those skilled in the art, the sequences of the present invention may contain sequencing errors That is, there may be incorrect nucleosides, frameshifts, unknown nucleosides, or other types of sequencing errors in any of the sequences, however, the correct sequences will fall within the homoiogy and stringency definitions herein Cell cycle proteins of the present invention may be shorter or longer than the ammo acid sequence encoded by the nucleic acid shown in the Figures Thus, in a preferred embodiment, included within the definition of cell cycle proteins are portions or fragments of the ammo acid sequence encoded by the nucleic acid sequence provided herein In one embodiment herein, fragments of cell cycle proteins are considered cell cycle proteins if a) they share at least one antigenic epitope, b) have at least the indicated sequence identity, c)preferably have cell cycle biological activity as further defined herein, d) and have PARP activity as further defined herein In some cases, where the sequence is used diagnostically, that is, when the presence or absence of cell cycle protein nucleic acid is determined, only the indicated sequence identity is required The nucleic acids of the present invention may also be shorter or longer than the sequence in the Figures The nucleic acid fragments include any portion of the nucleic acids provided herein which have a sequence not exactly previously identified, fragments having sequences with the indicated sequence identity to that portion not previously idertified are provided in an embodiment herein In addition, as is more fully outlined below, cell cycle proteins can be made that are longer than those depicted in the Figure, for example, by the addition of epitope or purification tags, the addition of other fusion sequences, or the elucidation of additional coding and non-coding sequences As described below, the fusion of a cell cycle peptide to a fluorescent peptide, such as Green Fluorescent Peptide (GFP), is particularly preferred Cell cycle proteins may also be identified as encoded by cell cycle nucleic acids which hybπdize to the sequence depicted in the Figures, or the complement thereof, as outlined herein Hybridization conditions are further described below In a preferred embodiment, a cell cycle protein has PARP activity which may be assayed in vitro The PARP activity may be auto-PARP activity, causing modification of the cell cycle protein itself Alternatively, the PARP activity may be trans-PARP activity, with other molecules serving as substrates for the cell cycle protein PARP activity may be assayed through the determination of ADP-πbosyl groups on substrates of cell cycle proteins In a preferred embodiment, the determination of ADP-πbosyl groups is achieved using an anti-polyADP πbose monoclonal antibody In another preferred embodiment, the determination of ADP-πbosyl groups is achieved by using labeled ADP-πbosyl groups These labels as defined below are preferably radioisotopes or secondary labels such as biotm In a preferred embodiment, the in vitro PARP assay is performed in microtiter wells using biotinylated NAD as a source of biotin-labeled poly-ADP πbose In this preferred embodiment, a cell lysate comprising a cell cycle protem-GFP fusion protein serves as the source of a cell cycle protein, which protein is adhered to the well surface by means of an affixed anti-GFP antibody Further in this preferred embodiment, biotm labeled-ADP-πbosyl groups are detected on cell cycle protein substrate, which may include the cell cycle protein itself, using streptavidin linked to an enzyme, such as HRP, which enzyme is capable of generating a detectable signal upon cleavage of an appropriate substrate In a preferred embodiment, when a cell cycle protein is to be used to generate antibodies, a cell cycle protein must 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. Thus, in most instances, antibodies made to a smaller cell cycle 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 The term "antibody" includes antibody fragments, as are known in the art, including Fab Fab 2 , single chain antibodies (Fv for example), chimeπc antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies In a preferred embodiment, the antibodies to a cell cycle protein are capable of reducing or eliminating the biological function of the cell cycle proteins descπbed herein, as is descπbed below. That is, the addition of anti-cell cycle protein antibodies (either polyclonal or preferably monoclonal) to cell cycle proteins (or cells containing cell cycle proteins) may reduce or eliminate the cell cycle activity Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred The cell cycle antibodies of the invention specifically bind to cell cycle proteins In a preferred embodiment, the antibodies specifically bind to cell cycle 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 " 6 M ~ \ with a preferred range being 10 "7 - 10 9 M '1 Antibodies are further descπbed below In the case of the nucleic acid, the overall sequence identity of the nucleic acid sequence is commensurate with ammo acid sequence identity but takes into account the degeneracy in the genetic code and codon bias of different organisms Accordingly, the nucleic acid sequence identity may be either lower or higher than that of the protein sequence Thus the sequence identity of the nucleic acid sequence as compared to the nucleic acid sequence of the Figures is preferably greater than 75%, more preferably greater than about 80%, particularly greater than about 85% and most preferably greater than 90% In some embodiments the sequence identity will be as high as about 93 to 95 or 98% In a preferred embodiment, a cell cycle nucleic acid encodes a cell cycle protein As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the cell cycle proteins of the present invention Thus, having identified a particular am o acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the ammo acid sequence of the cell cycle protein In one embodiment, the nucleic acid is determined through hybridization studies Thus, for example, nucleic acids which hybridize under high stringency to the nucleic acid sequence shown in the Figures, or its complement is considered a cell cycle nucleic acid 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 Ausubel, et al , both of which are hereby incorporated by reference Stπngent 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 pπnαples of hybπdization and the strategy of nucleic acid assays" (1993), which is hereby incorporated in its entirety by reference Generally, stnngent conditions are selected to be about 5-10'C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength pH The T m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybπdize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium) Stπngent conditions will be those in which the salt concentration is less than about 1 0 sodium ion, typically about 0 01 to 1 0 M sodium ion concentration (or other salts) at pH 7 0 to 8 3 and the temperature is at least about 30"C for short probes (e g 10 to 50 nucleotides) and at least about 60"C for long probes (e g greater than 50 nucleotides) Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide In another embodiment, less stringent hybridization conditions are used, for example, moderate or low stπngency conditions may be used, as are known in the art, see Maniatis and Ausubel, supra, and Tijssen, supra The cell cycle proteins and nucleic acids of the present invention are preferably recombinant As used herein and further defined below, "nucleic acid" may refer to either DNA or RNA, or molecules which contain both deoxy- and πbonucleotides The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids Such nucleic acids may also contain modifications in the πbose-phosphate backbone to increase stability and half life of such molecules in physiological environments The nucleic acid may be double stranded, single stranded, 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 depicted in the Figures also include the complement of the sequence By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature Thus an isolated cell cycle 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 remtroduced 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 recombmantly, although subsequently replicated non-recombinantly, are still considered recombinant 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 characteπstics 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 is unaccompanied by at least some of the matenal with which it is normally associated in its natural state, preferably constituting at least about 0 5%, more preferably 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 90% being particularly preferred The definition includes the production of a cell cycle protein from one organism in a different organism or host cell Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a mducible promoter or high expression promoter, such that the protein is made at increased concentration levels Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or ammo acid substitutions, insertions and deletions, as discussed below In one embodiment, the present invention provides cell cycle protein vaπants 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 ceil cycle 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 ceil culture as outlined above However, variant cell cycle protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques Am o acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or mterspecies variation of the cell cycle protein ammo acid sequence The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below While the site or region for introducing an ammo acid sequence vaπation is predetermined, the mutation per se 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 cell cycle 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 pπmer mutagenesis and PCR mutagenesis Screening of the mutants is done using assays of cell cycle protein activities Am o acid substitutions are typically of single residues, insertions usually will be on the order of from about 1 to 20 ammo 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 am o acids to minimize the alteration of the molecule However, larger changes may be tolerated in certain circumstances When small alterations in the characteristics of the cell cycle protein are desired, substitutions are generally made in accordance with the following chart Chart I Original Residue Exemplary Substitutions Ala Ser Arg Lys Asp Glu Cys Ser Gin Asn Glu Asp Gly Pro lie Leu, Val Leu lie, Val Lys Arg, Gin, Glu Met Leu, He Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val He, 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 side chain The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e g seryl or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, isoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteine or proline is substituted for (or by) any other residue, (c) 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 aspartyl, or (d) a residue having a bulky side chain, e g phenylalamne, 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 characteristics of the cell cycle proteins as needed Alternatively, the vaπant may be designed such that the biological activity of the cell cycle protein is altered For example, glycosylation sites may be altered or removed Covalent modifications of cell cycle polypeptides are included within the scope of this invention One type of covalent modification includes reacting targeted am o acid residues of a cell cycle polypeptide with an organic deπvatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a cell cycle polypeptide Denvatization with bifunctional agents is useful, for instance, for crosslinking cell cycle to a water-insoluble support matrix or surface for use in the method for purifying anti-cell cycle antibodies or screening assays, as is more fully described below Commonly used crosslinking agents include, e g , 1 ,1-bιs(dιazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccmimide esters, for example, esters with 4-azιdo- salicyhc acid, homobifunctional imidoesters, including disuccmimidyl esters such as 3,3'-dιthιobιs- (sucαnimidylpropionate), bifunctional maleimides such as bιs-N-maleιmιdo-1,8-octane and agents such as methyl-3-[(p-azιdophenyl)dιthιo]propιoιmιdate Other modifications include deamidation of glutaminyl and asparagmyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the "-am o groups of lysine, argmine, and histid e side chains [T E Creighton, Proteins Structure and Molecular Properties, W H Freeman & Co , San Francisco, pp 79-86 (1983)], acetylation of the N-termmal amine, and amidation of any C-termmal carboxyl group Another type of covalent modification of the cell cycle polypeptide included within the scope of this invention compπses altenng the native glycosylation pattern of the polypeptide "Alteπng the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence cell cycle polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence cell cycle polypeptide Addition of glycosylation sites to cell cycle polypeptides may be accomplished by alteπng the ammo acid sequence thereof The alteration may be made, for example, by the addition of, or substituton by, one or more seπne or threonme residues to the native sequence cell cycle polypeptide (for O- linked glycosylation sites) The cell cycle ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the cell cycle polypeptide at preselected bases such that codons are generated that will translate into the desired ammo acids Another means of increasing the number of carbohydrate moieties on the cell cycle polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are descπbed in the art, e g , in WO 87/05330 published 11 September 1987, and in Aplm and Wπston, CRC Cπt Rev Biochem . pp 259-306 (1981) Removal of carbohydrate moieties present on the cell cycle polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for ammo acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , 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-glycosidases as described by Thotakura et al , Meth Enzvmol , 138 350 (1987) Another type of covalent modification of cell cycle comprises linking the cell cycle polypeptide to one of a variety of nonproteinaceous polymers, e g , 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, 4,670,417, 4,791 ,192 or 4,179,337 Cell cycle polypeptides of the present invention may also be modified in a way to form chimeπc molecules comprising a cell cycle polypeptide fused to another, heterologous polypeptide or ammo acid sequence In one embodiment, such a chimeπc molecule comprises a fusion of a cell cycle polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the amino-or carboxyl-terminus of the cell cycle polypeptide The presence of such epitope-tagged forms of a cell cycle polypeptide can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the cell cycle polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matπx that binds to the epitope tag In an alternative embodiment, the chimeπc molecule may compnse a fusion of a cell cycle polypeptide with an immunoglobulm or a particular region of an immunoglobulm For a bivalent form of the chimeπc molecule, such a fusion could be to the Fc region of an IgG molecule as discussed further below Various tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidine (poly-his) or poly-histidine-glycme (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol , 8.2159-2165 (1988)], the c-myc 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 Engmeeπng. 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 Natl Acad Sci USA. 87 6393-6397 (1990)] In an embodiment herein, cell cycle proteins of the cell cycle family and cell cycle proteins from other organisms are cloned and expressed as outlined below Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related cell cycle proteins from humans or other organisms As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the cell cycle nucleic acid sequence As is generally known in the art, preferred PCR pπmers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain mosine as needed The conditions for the PCR reaction are well known in the art It is therefore also understood that provided along with the sequences in the sequences listed herein are portions of those sequences, wherein unique portions of 15 nucleotides or more are particularly preferred The skilled artisan can routinely synthesize or cut a nucleotide sequence to the desired length 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 cell cycle nucleic acid can be further- used as a probe to identify and isolate other cell cycle nucleic acids It can also be used as a "precursor" nucleic acid to make modified or variant ceil cycle nucleic acids and proteins Using the nucleic acids of the present invention which encode a cell cycle protein, a variety of expression vectors are made The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome Generally, these expression vectors include transcπptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the cell cycle 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 nbosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation 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 preprotem 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 nbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation As another example, operably linked refers to DNA sequences linked so as to be contiguous, and, in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic o gonucleotide adaptors or linkers are used in accordance with conventional practice The transcnptional and translational regulatory nucleic acid will generally be appropπate to the host cell used to express the cell cycle protein, for example, transcnptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the cell cycle protein in Bacillus Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells In general, the transcnptional and translational regulatory sequences may include, but are not limited to, promoter sequences, πbosomal binding sites, transcnptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences In a preferred embodiment, the regulatory sequences include a promoter and transcnptional start and stop sequences Promoter sequences encode either constitutive or mducible 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, and 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 genome, 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 appropπate 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 well known in the art and will vary with the host cell used 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 Cell cycle proteins of the present invention are produced by cultuπng a host cell transformed with an expression vector containing nucleic acid encoding a cell cycle protein, under the appropπate conditions to induce or cause expression of the cell cycle protein The conditions appropriate for cell cycle protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine expeπmentation For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an mducible promoter requires the appropriate growth conditions for induction 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 selection can be crucial for product yield Appropriate host cells include yeast, bacteria, archebactena, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E coll, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, A549 cells, fibrobiasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines In a preferred embodiment, the cell cycle proteins are expressed in mammalian cells Mammalian expression systems are also known in the art, and include retroviral systems A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for cell cycle protein into mRNA A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site A mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation 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 tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter Typically, transcπption termination and polyadenylation 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 The 3' terminus of the mature mRNA is formed by site- specific post-translational cleavage and polyadenylation Examples of transcription terminator and polyadenlytion signals include those deπved form SV40 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 phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotιde(s) in liposomes, and direct microinjection of the DNA into nuclei In a preferred embodiment, cell cycle proteins are expressed in bacteπal systems Bacterial expression systems are well known in the art A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of cell cycle protein into mRNA A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence This transcription initiation region typically includes an RNA polymerase binding site and a transcπption initiation site Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan Promoters from bacteπophage may also be used and are known in the art In addition, synthetic promoters and hybrid promoters are also useful, for example, the fac 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 nbosome binding site is desirable In E coli, the nbosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon The expression vector may also include a signal peptide sequence that provides for secretion of the cell cycle protein in bacteπa The signal sequence typically encodes a signal peptide compnsed of hydrophobic ammo acids which direct the secretion of the protein from the cell, as is well known in the art The protein is either secreted into the growth media (gram-positive bacteπa) or into the peπplasmic space, located between the inner and outer membrane of the cell (gram- πegative bacteπa) The bacteπal 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 bacteπa resistant to drugs such as ampicillm, chloramphenicol, erythromycin, kanamycm, neomycm and tetracycline Selectable markers also include biosynthetic genes, such as those in the histidme, tryptophan and leucme biosynthetic pathways These components are assembled into expression vectors Expression vectors for bacteπa are well known in the art, and include vectors for Bacillus subtilis, E coli, Streptococcus cremons, and Streptococcus hvidans, among others The bacteπal expression vectors are transformed into bactenal host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others In one embodiment, cell cycle proteins are produced in insect cells Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art In a preferred embodiment, cell cycle protein is produced in yeast cells Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K lactis, Pichia guillenmondii and P pastoπs, Schizosaccharomyces pombe, and Yarrowia hpolytica Preferred promoter sequences for expression in yeast include the mducible GAL1.10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokmase, phosphofructokmase, 3- phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene Yeast selectable markers include ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to tunicamyαn, the neomycm phosphotransferase gene, which confers resistance to G418, and the CUP1 gene, which allows yeast to grow in the presence of copper ions The cell cycle 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, the cell cycle protein may be fused to a carrier protein to form an immunogen Alternatively, the cell cycle protein may be made as a fusion protein to increase expression, or for other reasons For example, when the cell cycle protein is a cell cycle peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes Similarly, cell cycle proteins of the invention can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc In one embodiment, the cell cycle 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 detection 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, c) colored (particularly fluorescent) dyes, and d) secondary labels such as biotm The labels may be incorporated into the compound at any position In a preferred embodiment, the cell cycle protein is purified or isolated after expression Cell cycle proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample Standard punfication methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing For example, the cell cycle protein may be purified using a standard anti-cell cycle antibody column Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful For general guidance in suitable purification techniques, see Scopes, R , Protein Purification, Spπnger-Verlag, NY (1982) The degree of purification necessary will vary depending on the use of the cell cycle protein In some instances no purification will be necessary A preferred method of protein purification is 2-dιmensιonal (2-D) gel electrophoresis Following purification using 2-D gel electrophoresis or other methods, the cell cycle protein may be identified in a number of ways, including but not limited to mass spectroscopy and peptide sequence analysis Following identification, nucleic acid encoding the cell cycle protein may be isolated from a cDNA or genomic DNA library with the use of standard methods Once expressed and purified if necessary, the cell cycle proteins and nucleic acids are useful in a number of applications The nucleotide sequences (or their complement) encoding cell cycle proteins have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA Cell cycle protein nucleic acid will also be useful for the preparation of cell cycle proteins by the recombinant techniques described herein The full-length native sequence cell cycle protein gene, or portions thereof, may be used as hybπdization probes for a cDNA library to isolate other genes (for instance, those encoding naturally-occurring variants of cell cycle protein or cell cycle protein from other species) which have a desired sequence identity to the cell cycle protein coding sequence Optionally, the length of the probes will be about 20 to about 50 bases The hybridization probes may be derived from the nucleotide sequences herein or from genomic sequences including promoters, enhancer elements and introns of native sequences as provided herein By way of example, a screening method will comprise isolating the coding region of the cell cycle protein gene using the known DNA sequence to synthesize a selected probe of about 40 bases Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32 P or ^S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotm coupling systems Labeled probes having a sequence complementary to that of the cell cycle protein gene of the present invention can be used to screen braπes of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybπdizes The isolation of mRNA comprises isolating total cellular RNA by disrupting a cell and performing differential centπfugation Once the total RNA is isolated, mRNA is isolated by making use of the adenme nucleotide residues known to those skilled in the art as a poly (A) tail found on virtually every eukaryotic mRNA molecule at the 3'end thereof Oligonucleotides composed of only deoxythymidine [olgo(dT)] are linked to cellulose and the olιgo(dT)-cellulose packed into small columns When a preparation of total cellular RNA is passed through such a column, the mRNA molecules bind to the olιgo(dT) by the poly (A) tails while the rest of the RNA flows through the column The bound mRNAs are then eluted from the column and collected Nucleotide sequences encoding a cell cycle protein can also be used to construct hybridization probes for mapping the gene which encodes that cell cycle protein and for the genetic analysis of individuals with genetic disorders The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries Hybridization probes may be used to screen for alterations in gene number or gene location in individuals with genetic disorders Nucleic acids which encode cell cycle protein or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents A transgenic animal (e g , a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e g , an embryonic stage A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops In one embodiment, cDNA encoding a cell cycle protein can be used to clone genomic DNA encoding a cell cycle protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express the desired DNA Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos 4,736,866 and 4,870,009 Typically, particular cells would be targeted for the cell cycle protein transgene incorporation with tissue-specific enhancers Transgenic animals that include a copy of a transgene encoding a cell cycle protein introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of the desired nucleic acid Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition Alternatively, non-human homologues of the cell cycle protein can be used to construct a cell cycle protein "knock out" animal which has a defective or altered gene encoding a cell cycle protein as a result of homologous recombination between the endogenous gene encoding a cell cycle protein and altered genomic DNA encoding a cell cycle protein introduced into an embryonic cell of the animal For example, cDNA encoding a cell cycle protein can be used to clone genomic DNA encoding a cell cycle protein in accordance with established techniques A portion of the genomic DNA encoding a cell cycle protein can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e g , Thomas and Capecchi, Cell, 51 503 (1987) for a description of homologous recombination vectors] The vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombmed with the endogenous DNA are selected [see e g , Li et al , CeH, 69 915 (1992)] The selected cells are then injected into a blastocyst of an animal (e g , a mouse or rat) to form aggregation chimeras [see e g , Bradley, in Teratocarcmomas and Embryonic Stem Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 113-152] A chimeπc embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal Progeny harboring the homologously recombmed DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombmed DNA Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the cell cycle protein A cDNA encoding a cell cycle protein, or a variant, may be introduced into a specific locus, which locus need not comprise a cell cycle gene In a preferred embodiment, the locus is the HPRT gene locus In one aspect, the expression of the cDNA may be regulated by endogenous DNA sequence In another aspect, the cDNA may be regulated by exogenous DNA sequence In one aspect, this exogenous DNA sequence may comprise a conditional promoter In a preferred embodiment, transgenic animals comprising a loss of cell cycle protein function exhibit decreased cell proliferation, a decrease in the potential for proliferation, a decrease in the rate of progression through a stage of the cell cycle, a reduction in the number of cells, or an alteration in apoptosis Potential for proliferation in this regard refers to the potential of a cell to respond to an additional cue, intrinsic or extrinsic, which response is characteπzed by a change in proliferation In another preferred embodiment, transgenic animals comprising a gam of cell cycle protein function exhibit an increase in the rate of cell proliferation, an increase in the potential for proliferation, an increase in the rate of progression through a stage of the cell cycle, an increase in the number of cells, or an alteration in apoptosis It is understood that the models described herein can be vaπed For example, "knock-in" models can be formed, or the models can be cell-based rather than animal models Nucleic acid encoding the cell cycle polypeptides, antagonists or agonists may also be used in gene therapy In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low mtracellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik et al . Proc Natl Acad Sci USA 83. 4143-4146 [1986]) The oligonucleotides can be modified to enhance their uptake, e g by substituting their negatively charged phosphodiester groups by uncharged groups In a preferred embodiment, the introduction of cell cycle protein nucleic acid results in the potentiation of cell proliferation, increased cell proliferation, an increase in the rate of passage through a phase of the cell cycle, an increase in the number of cells, or an alteration in apoptosis In another embodiment, the introduction of cell cycle protein antisense nucleic acid results in a reduction of cell proliferation, a decrease in the potential for proliferation, a decrease in the rate of passage through a phase of the cell cycle, a decrease in the number of cells, or an alteration in apoptosis In a preferred embodiment, the introduction of nucleic acid encoding a dominant negative cell cycle protein compπsmg a cell cycle protein lacking the PARP domain or PARP activity results in decreased proliferation, a decrease in the potential to proliferate, deceleration through the G2/M phase, arrest at the G2/M phase of the cell cycle, or an alteration in apoptosis There are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, micromjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protem-liposome mediated transfection (Dzau et al . Trends in Biotechnology 11. 205-210 [1993]) In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a ceil surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e g capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo mternalization in cycling, proteins that target mtracellular localization and enhance mtracellular half-life The technique of receptor-mediated endocytosis is described, for example, by Wu ef a/ , J Biol Chem 262. 4429-4432 (1987), and Wagner et al , Proc Natl Acad Sci USA 87, 3410-3414 (1990) For review of gene marking and gene therapy protocols see Anderson et al , Science 256, 808-813 (1992) In a preferred embodiment, the cell cycle proteins, nucleic acids, variants, modified proteins, cells and/or transgenics containing the said nucleic acids or proteins are used in screening assays Identification of the cell cycle protein provided herein permits the design of drug screening assays for compounds that bind the cell cycle protein, that interfere with the binding of the cell cycle protein to another molecule, that affect cell cycle protein activity as described herein, or which modulate the cell cycle The assays described herein, preferred embodiments utilize the human cell cycle protein, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc ), farm animals (cows, sheep, pigs, horses, etc ) and pπmates These latter embodiments may be preferred in the development of animal models of human disease In some embodiments, as outlined herein, vaπant or derivative cell cycle proteins may be used, including deletion cell cycle proteins as outlined above In a preferred embodiment, the methods comprise combining a cell cyle protein and a candidate bioactive agent, and determining the binding of the candidate agent to the cell cycle protein In other embodiments, further discussed below, binding interference or bioactivity is determined The term "candidate bioactive agent" or "exogeneous compound" as used herein describes any molecule, e g , protein, small organic molecule, carbohydrates (including polysacchaπdes), polynucleotide, lipids, etc Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the vanous concentrations Typically, one of these concentrations serves as a negative control, / e , at zero concentration or below the level of detection In addition, positive controls, i e the use of agents known to alter cell cycling, may be used For example, p21 is a molecule known to arrest cells in the G1 cell phase, by binding G1 cyclm-CDK complexes Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons 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, sacchaπdes, fatty acids, steroids, puπnes, pyπmidines, derivatives, structural analogs or combinations thereof Particularly preferred are peptides 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, estenfication, amidification to produce structural analogs In a preferred embodiment, a library of different candidate bioactive agents are used Preferably, the library should provide a sufficiently structurally diverse population of randomized agents to effect a probabilistically sufficient range of diversity to allow binding to a particular target Accordingly, an interaction library should be large enough so that at least one of its members will have a structure that gives it affinity for the target Although it is difficult to gauge the required absolute size of an interaction library, nature provides a hint with the immune response a diversity of 10 7 -10 8 different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organism Published in vitro selection techniques have also shown that a library size of 10 7 to 10 8 is sufficient to find structures with affinity for the target A library of all combinations of a peptide 7 to 20 ammo acids in length, such as generally proposed herein, has the potential to code for 20 7 (10 9 ) to 20 20 Thus, with libraries of 10 7 to 10* different molecules the present methods allow a "working" subset of a theoretically complete interaction library for 7 ammo acids, and a subset of shapes for the 20 20 library Thus, in a preferred embodiment, at least 10 6 , preferably at least 10 7 , more preferably at least 10 β and most preferably at least 10 9 different sequences are simultaneously analyzed in the subject methods Preferred methods maximize library size and diversity In a preferred embodiment, the candidate bioactive agents are proteins By "protein" herein is meant at least two covalently attached ammo acids, which includes proteins, polypeptides, oligopeptides and peptides The protein may be made up of naturally occurπng ammo acids and peptide bonds, or synthetic peptidomimetic structures Thus "ammo acid", or "peptide residue", as used herein means both naturally occurring and synthetic ammo acids For example, homo- phenylalanme, citrulline and noreleucme are considered ammo acids for the purposes of the invention "Ammo acid" also includes imino acid residues such as proline and hydroxyprolme The side chains may be in either the (R) or the (S) configuration In the preferred embodiment, the ammo acids are in the (S) or L-configuration If non-naturally occurring side chains are used, non- ammo acid substituents may be used, for example to prevent or retard in vivo degradations Chemical blocking groups or other chemical substituents may also be added In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins Thus, for example, cellular extracts containing proteins, or random or directed digests of protemaceous cellular extracts, may be used In this way libraries of procaryotic and eukaryotic proteins may be made for screening in the systems described herein 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 30 ammo acids, with from about 5 to about 20 ammo acids being preferred, and from about 7 to about 15 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 ammo acids, respectively Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or ammo 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 bioactive protemaceous agents In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position In a preferred embodiment, the library is 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 ammo acid residues are randomized within a defined class, for example, of hydrophobic ammo acids, hydrophilic residues, steπcally biased (either small or large) residues, towards the creation of cysteines, for cross-linking, pralines for SH- 3 domains, seπnes, threon es, tyrosmes or histidines for phosphorylation sites, etc , or to puπnes, etc In a preferred embodiment, the candidate bioactive agents are nucleic acids By "nucleic acid" or "o gonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage, et al , Tetrahedron, 49(10) 1925 (1993) and references therein, Letsmger, J Orq Chem , 35 3800 (1970), Spπnzl, et al , Eur J Biochem . 81 579 (1977), Letsmger, et al , Nucl Acids Res . 14 3487 (1986), Sawai, et a/ , Chem Lett . 805 (1984), Letsmger, et al , J Am Chem Soc , 110 4470 (1988), and Pauwels, et al , Chemica Scπpta. 26 141 (1986)), phosphorothioate (Mag, et al , Nucleic Acids Res , 19 1437 (1991), and U S Patent No 5,644,048), phosphorodithioate (Bπu, et al , J Am Chem Soc . 111 2321 (1989)), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J Am Chem Soc . 114 1895 (1992), Meier, ef al , Chem Int Ed Enol . 31 1008 (1992), Nielsen, Nature. 365 566 (1993), Carlsson, ef al , Nature. 380 207 (1996), all of which are incorporated by reference)) Other analog nucleic acids include those with positive backbones (Denpcy, ef al , Proc Natl Acad Sci USA, 92 6097 (1995)), nonionic backbones (U S Patent Nos 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863, Kiedrowshi, ef al , Angew Chem Intl Ed English, 30 423 (1991), Letsmger, et al , J Am Chem Soc . 110 4470 (1988), Letsmger, et al., Nucleoside & Nucleotide, 13 1597 (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. Biomolecular NMR. 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) and non-πbose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins, ef al., Chem Soc. Rev.. (1995) pp. 169- 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 to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurnng nucleic acids and analogs may be made The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyπbo- and πbo-nucleotides, and any combination of bases, including uracil, adenme, thymme, cytosme, guanme, inosme, xathanme hypoxathanme, isocytosme, isoguanme, etc. As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of procaryotic or eukaryotic genomes may be used as is outlined above for proteins In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature In a preferred embodiment, the candidate bioactive agents are linked to a fusion partner By "fusion partner" or "functional group" herein is meant a sequence that is associated with the candidate bioactive agent, that confers upon all members of the library in that class a common function or ability Fusion partners can be heterologous (i e not native to the host cell), or synthetic (not native to any cell) Suitable fusion partners include, but are not limited to: a) presentation structures, which provide the candidate bioactive agents in a conformationally restπcted or stable form, b) targeting sequences, which allow the localization of the candidate bioactive agent into a subcellular or extracellular compartment, c) rescue sequences which allow the purification or isolation of either the candidate bioactive agents or the nucleic acids encoding them; d) stability sequences, which confer stability or protection from degradation to the candidate bioactive agent or the nucleic acid encoding it, for example resistance to proteolytic degradation, e) dimenzation sequences, to allow for peptide dimeπzation, or f) any combination of a), b), c), d), and e), as well as linker sequences as needed In one embodiment of the methods described herein, portions of cell cycle proteins are utilized; in a preferred embodiment, portions having cell cycle activity are used Cell cycle activity is descπbed further below and includes binding activity to p21 or cell cycle protein modulators as further described below, and PARP activity In addition, the assays descπbed herein may utilize either isolated cell cycle proteins or cells comprising the cell cycle proteins Generally, in a preferred embodiment of the methods herein, for example for binding assays, the cell cycle protein or the candidate agent is non-diffusibly bound to an insoluble support having isolated sample receiving areas (e g a microtiter 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 (e g , polystyrene), polysacchandes, 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 In some cases magnetic beads and the like are included 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 do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc In some embodiments, p21 can be used Following binding of the protein or agent, excess unbound 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 moiety Also included in this invention are screening assays wherein solid supports are not used, examples of such are descπbed below in a preferred embodiment, the cell cycle protein is bound to the support, and a candidate bioactive agent is added to the assay Alternatively, the candidate agent is bound to the support and the cell cycle protein is added Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide 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, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc ) and the like The determination of the binding of the candidate bioactive agent to the cell cycle protein may be done in a number of ways In a preferred embodiment, the candidate bioactive agent is labelled, and binding determined directly For example, this may be done by attaching all or a portion of the cell cycle protein to a solid support, adding a labelled 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 radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemilummescers, or specific binding molecules, etc Specific binding molecules include pairs, such as biotm and streptavidm, 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 protemaceous candidate agents) may be labeled at tyrosme positions using 125 l, or with fluorophores Alternatively, more than one component may be labeled with different labels, using 125 l for the proteins, for example, and a fluorophor for the candidate agents In a preferred embodiment the binding of the candidate bioactive agent is determined through the use of competitive binding assays In this embodiment, the competitor is a binding moiety known to bind to the target molecule (i e cell cycle protein), such as an antibody, peptide, binding partner, ligand, etc In a preferred embodiment, the competitor is p21 Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent This assay can be used to determine candidate agents which interfere with binding between cell cycle proteins and p21 "Interference of binding" as used herein means that native binding of the ceil cycle protein differs in the presence of the candidate agent The binding can be eliminated or can be with a reduced affinity Therefore, in one embodiment, interference is caused by, for example, a conformation change, rather than direct competition for the native binding site 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 Incubations may be performed at any 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 is generally removed or washed away The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding In a preferred embodiment, the competitor is added first, followed by the candidate bioactive agent Displacement of the competitor is an indication that the candidate bioactive agent is binding to the cell cycle protein and thus is capable of binding to, and potentially modulating, the activity of the cell cycle 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 bioactive agent is bound to the cell cycle 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 the cell cycle protein In a preferred embodiment, the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the cell cycle proteins Such assays can be done with the cell cycle protein or cells comprising said cell cycle protein In one embodiment, the methods comprise combining an cell cycle protein and a competitor in a first sample A second sample compπses a candidate bioactive agent, an cell cycle 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 the cell cycle 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 the cell cycle protein Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native cell cycle protein, but cannot bind to modified cell cycle proteins The structure of the cell cycle protein may be modeled, and used in rational drug design to synthesize agents that interact with that site Drug candidates that affect cell cycle bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein Positive controls and negative controls may be used in the assays Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results Incubation of all 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 in a scintillation counter to determine the amount of bound compound A variety of other reagents may be included in the screening assays These include reagents like salts, neutral proteins, e g albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease 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 cell cycle may also be done In a preferred embodiment, methods for screening for a bioactive agent capable of modulating the cell cycle compnse the steps of adding a candidate bioactive agent to a sample comprising a cell cycle protein (or cells compπsmg a cell cycle protein) and determining an alteration in the biological activity of the cell cycle protein "Modulating the activity of a cell cycle 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 may bind to cell cycle protein (although this may not be necessary), and should 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 cell cycle protein Thus, in this embodiment, the methods comprise combining a sample comprising a cell cycle protein and a candidate bioactive agent, and evaluating the effect of the agent on cell cycle protein activity By "cell cycle protein activity" or grammatical equivalents herein is meant at least one of the cell cycle protein's biological activities, including, but not limited to, its ability to affect the cell cycle, bind to p21 , and PARP activity In another embodiment, tankyrase H binds to a TRF (telomeπc repeat binding factor) In yet another embodiment, the cell cycle protein provided herein regulates telomere length, aging and/or apoptosis In a preferred embodiment, the activity of the cell cycle protein is decreased, in another preferred embodiment, the activity of the cell cycle protein is increased Thus, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments In a preferred embodiment, the invention provides methods for screening for bioactive agents capable of modulating the activity of the cell cycle The methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising a cell cycle protein Preferred cell types include almost any cell The cell comprises a recombinant nucleic acid that encodes a cell cycle protein In a preferred embodiment, a library of candidate agents are tested on a plurality of cells that comprise a recombinant nucleic acid that encodes a cell cycle protein Detection of cell cycle regulation may be done as will be appreciated by those in the art In one embodiment, indicators of the cell cycle are used There are a number of parameters that may be evaluated or assayed to allow the detection of alterations in cell cycle regulation, including, but not limited to, cell viability assays, assays to determine whether cells are arrested at a particular cell cycle stage ("cell proliferation assays"), and assays to determine at which cell stage the cells have arrested ("cell phase assays") By assaying or measuring one or more of these parameters, it is possible to detect not only alterations in cell cycle regulation, but alterations of different steps of the cell cycle regulation pathway This may be done to evaluate native cells, for example to quantify the aggressiveness of a tumor cell type, or to evaluate the effect of candidate drug agents that are being tested for their effect on cell cycle regulation In this manner, rapid, accurate screening of candidate agents may be performed to identify agents that modulate cell cycle regulation Thus, the present compositions and methods are useful to elucidate bioactive agents that can cause a cell or a population of cells to either move out of one growth phase and into another, or arrest in a growth phase In some embodiments, the cells are arrested in a particular growth phase, and it is desirable to either get them out of that phase or into a new phase Alternatively, it may be desirable to force a cell to arrest in a phase, for example G1 , rather than continue to move through the cell cycle Similarly, it may be desirable in some circumstances to accelerate a non- arrested but slowly moving population of cells into either the next phase or just through the cell cycle, or to delay the onset of the next phase For example, it may be possible to alter the activities of certain enzymes, for example kmases, phosphatases, proteases or ubiquitination enzymes, that contribute to initiating cell phase changes In a preferred embodiment, the methods outlined herein are done on cells that are not arrested in the G1 phase, that is, they are rapidly or uncontrollably growing and replicating, such as tumor cells In this manner, candidate agents are evaluated to find agents that can alter the cell cycle regulation, i e cause the cells to arrest at cell cycle checkpoints, such as in G1 (although arresting in other phases such as S, G2 or M are also desirable) Alternatively, candidate agents are evaluated to find agents that can cause proliferation of a population of cells, i e that allow cells that are generally arrested in G1 to start proliferating again, for example, peripheral blood cells, terminally differentiated cells, stem cells in culture, etc Accordingly, the invention provides methods for screening for alterations in cell cycle regulation of a population of cells By "alteration' or "modulation" (used herein interchangeably), is generally meant one of two things In a preferred embodiment, the alteration results in a change in the cell cycle of a cell, i e a proliferating cell arrests in any one of the phases, or an arrested cell moves out of its arrested phase and starts the cell cycle, as compared to another cell or in the same cell under different conditions Alternatively, the progress of a cell through any particular phase may be altered, that is, there may be an acceleration or delay in the length of time it takes for the cells to move thorough a particular growth phase For example, the cell may be normally undergo a G1 phase of several hours, the addition of an agent may prolong the G1 phase In a preferred embodiment, the introduction of cell cycle protein or cell cycle protein nucleic acid into a cell results in acceleration through the G2/M phase of the cell cycle In another preferred embodiment, the reduction of cell cycle protein, preferably with the use of antisense oligonucleotide or bioactive agent affecting cell cycle regulation as described herein, results in deceleration through the G2/M phase or arrest at the G2/M phase of the cell cycle In a preferred embodiment, the introduction of a dominant negative cell cycle protein comprising a cell cycle protein lacking the PARP domain or PARP activity results in decreased proliferation, a decrease in the potential to proliferate, deceleration through the G2/M phase, arrest at the G2/M phase of the cell cycle, or an alteration in apoptosis The measurements of cell cycle can be determined wherein all of the conditions are the same for each measurement, or under various conditions, with or without bioactive agents, or at different stages of the cell cycle process For example, a measurement of cell cycle regulation can be determined in a cell or cell population wherein a candidate bioactive agent is present and wherein the candidate bioactive agent is absent In another example, the measurements of cell cycle regulation are determined wherein the condition or environment of the cell or populations of cells differ from one another For example, the cells may be evaluated in the presence or absence cr previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokmes, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i e cell-cell contacts) In another example, the measurements of cell cycle regulation are determined at different stages of the cell cycle process In yet another example, the measurements of cell cycle regulation are taken wherein the conditions are the same, and the alterations are between one cell or cell population and another cell or cell population By a "population of cells" or "library of cells" herein is meant at least two cells, with at least about 10 3 being preferred, at least about 10 6 being particularly preferred, and at least about 10 8 to 10 9 being especially preferred The population or sample can contain a mixture of different cell types from either pπmary or secondary cultures although samples containing only a single cell type are preferred, for example, the sample can be from a cell line, particularly tumor cell lines, as outlined below The cells may be in any cell phase, either synchronously or not, including M, G1, S, and G2. In a preferred embodiment, cells that are replicating or proliferating are used; this may allow the use of retroviral vectors for the introduction of candidate bioactive agents. Alternatively, non- rep cating cells may be used, and other vectors (such as adenovirus and lentivirus vectors) can be used. In addition, although not required, the cells are compatible with dyes and antibodies. Preferred cell types for use in the invention include, but are not limited to, mammalian cells, including animal (rodents, including mice, rats, hamsters and gerbils), pπmates, and human cells, particularly including tumor cells of all types, including breast, skin, lung, cervix, colonrectal, leukemia, brain, etc. In a preferred embodiment, the methods comprise assaying one or more of several different cell parameters, including, but not limited to, cell viability, cell proliferation, and cell phase Other parameters include assaying telomere length. In a preferred embodiment, cell viability is assayed, to ensure that a lack of cellular change is due to expeπmental conditions (i e. the introduction of a candidate bioactive agent) not cell death. There are a variety of suitable cell viability assays which can be used, including, but not limited to, light scattering, viability dye staining, and exclusion dye staining. In a preferred embodiment, a light scattering assay is used as the viability assay, as is well known in the art For example, when viewed in the FACS, cells have particular characteristics as measured by their forward and 90 degree (side) light scatter properties These scatter properties represent the size, shape and granule content of the cells These properties account for two parameters to be measured as a readout for the viability. Briefly, the DNA of dying or dead cells generally condenses, which alters the 90 * scatter; similarly, membrane blebbmg can alter the forward scatter. Alterations in the intensity of light scattering, or the cell-refractive index indicate alterations in viability Thus, in general, for light scattering assays, a live cell population of a particular ceil type is evaluated to determine it's forward and side scattering properties This sets a standard for scattering that can subsequently be used In a preferred embodiment, the viability assay utilizes a viability dye There are a number of known viability dyes that stain dead or dying cells, but do not stain growing cells For example, annexm V is a member of a protein family which displays specific binding to phospholipid (phosphotidylsenne) in a divalent ion dependent manner This protein has been widely used for the measurement of apoptosis (programmed cell death) as cell surface exposure of phosphatidyiseπne is a hallmark early signal of this process Suitable viability dyes include, but are not limited to, annexin, ethidium homodιmer-1, DEAD Red, propidium iodide, SYTOX Green, etc , and others known in the art, see the Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Haugland, Sixth Edition, hereby incorporated by reference, see Apoptosis Assay on page 285 in particular, and Chapter 16 Protocols for viability dye staining for cell viability are known, see Molecular Probes catalog, supra In this embodiment, the viability dye such as annexin is labeled, either directly or indirectly, and combined with a cell population Annexin is commercially available, i e , from PharM gen, San Diego, California, or Caltag Laboratories, Millbrae, California Preferably, the viability dye is provided in a solution wherein the dye is in a concentration of about 100 ng/ml to about 500 ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and most preferably, from about 1 μg/ml to about 5 μg/ml In a preferred embodiment, the viability dye is directly labeled, for example, annexin may be labeled with a fluorochrome such as fluorecein isothiocyanate (FITC), Alexa dyes, TRITC, AMCA, APC, tπ-color, Cy-5, and others known in the art or commercially available In an alternate preferred embodiment, the viability dye is labeled with a first label, such as a hapten such as biotm, and a secondary fluorescent label is used, such as fluorescent streptavidin Other first and second labeling pairs can be used as will be appreciated by those in the art Once added, the viability dye is allowed to incubate with the cells for a period of time, and washed, if necessary The cells are then sorted as outlined below to remove the non-viable cells In a preferred embodiment, exclusion dye staining is used as the viability assay Exclusion dyes are those which are excluded from living cells, i e they are not taken up passively (they do not permeate the cell membrane of a live cell) However, due to the permeability of dead or dying cells, they are taken up by dead cells Generally, but not always, the exclusion dyes bind to DNA, for example via intercalation Preferably, the exclusion dye does not fluoresce, or fluoresces poorly, in the absence of DNA, this eliminates the need for a wash step Alternatively, exclusion dyes that require the use of a secondary label may also be used Preferred exclusion dyes include, but are not limited to, ethidium bromide, ethidium homodιmer-1, propidium iodine, SYTOX green nucleic acid stain, Calcem AM, BCECF AM, fluorescein diacetate, TOTO® and TO-PRO™ (from Molecular Probes, supra, see chapter 16) and others known in the art Protocols for exclusion dye staining for cell viability are known, see the Molecular Probes catalog, supra In general, the exclusion dye is added to the cells at a concentration of from about 100 ng/ml to about 500 ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and most preferably, from about 0 1 μg/ml to about 5 μg/ml, with about 0 5 μg/ml being particularly preferred The cells and the exclusion dye are incubated for some period of time, washed, if necessary, and then the cells sorted as outlined below, to remove non-viable ceils from the population In addition, there are other cell viability assays which may be run, including for example enzymatic assays, which can measure extracellular enzymatic activity of either live cells (i e secreted proteases, etc ), or dead cells (i e the presence of mtracellular enzymes in the media, for example, mtracellular proteases, mitochondnal enzymes, etc ) See the Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Haugland, Sixth Edition, hereby incorporated by reference, see chapter 16 in particular In a preferred embodiment, at least one cell viability assay is run, with at least two different cell viability assays being preferred, when the fluors are compatible When only 1 viability assay is run, a preferred embodiment utilizes light scattering assays (both forward and side scattering) When two viability assays are run, preferred embodiments utilize light scattering and dye exclusion, with light scattering and viability dye staining also possible, and all three being done in some cases as well Viability assays thus allow the separation of viable cells from non-viable or dying cells In addition to a cell viability assay, a preferred embodiment utilizes a cell proliferation assay By "proliferation assay" herein is meant an assay that allows the determination that a cell population is either proliferating, i e replicating, or not replicating In a preferred embodiment, the proliferation assay is a dye inclusion assay A dye inclusion assay relies on dilution effects to distinguish between cell phases Briefly, a dye (generally a fluorescent dye as outlined below) is introduced to cells and taken up by the cells Once taken up, the dye is trapped in the cell, and does not diffuse out As the cell population divides, the dye is proportionally diluted That is, after the introduction of the inclusion dye, the cells are allowed to incubate for some period of time, cells that lose fluorescence over time are dividing, and the cells that remain fluorescent are arrested in a non-growth phase Generally, the introduction of the inclusion dye may be done in one of two ways Either the dye cannot passively enter the cells (e g it is charged), and the ceils must be treated to take up the dye, for example through the use of a electric pulse Alternatively, the dye can passively enter the cells, but once taken up, it is modified such that it cannot diffuse out of the cells For example, enzymatic modification of the inclusion dye may render it charged, and thus unable to diffuse out of the cells For example, the Molecular Probes CellTracker™ dyes are fluorescent chloromethyl derivatives that freely diffuse into cells, and then glutathione S-transferase-mediated reaction produces membrane impermeant dyes Suitable inclusion dyes include, but are not limited to, the Molecular Probes line of CellTracker™ dyes , including, but not limited to CellTracker™ Blue, CellTracker™ Yellow-Green, CellTracker™ Green, CellTracker™ Orange, PKH26 (Sigma), and others known in the art; see the Molecular Probes Handbook, supra; chapter 15 in particular. In general, inclusion dyes are provided to the cells at a concentration ranging from about 100 ng/ml to about 5 μg/ml, with from about 500 ng/ml to about 1 μg/ml being preferred A wash step may or may not be used. In a preferred embodiment, a candidate bioactive agent is combined with the cells as described herein. The cells and the inclusion dye are incubated for some peπod of time, to allow cell division and thus dye dilution. The length of time will depend on the cell cycle time for the particular cells; in general, at least about 2 cell divisions are preferred, with at least about 3 being particularly preferred and at least about 4 being especially preferred The cells are then sorted as outlined below, to create populations of cells that are replicating and those that are not As will be appreciated by those in the art, in some cases, for example when screening for anti- proliferation agents, the bright (i e fluorescent) cells are collected, in other embodiments, for example for screening for proliferation agents, the low fluorescence cells are collected. Alterations are determined by measuring the fluorescence at either different time points or in different cell populations, and comparing the determinations to one another or to standards. In a preferred embodiment, the proliferation assay is an antimetabolite assay. In general, antimetabolite assays find the most use when agents that cause cellular arrest in G1 or G2 resting phase is desired In an antimetabolite proliferation assay, the use of a toxic antimetabolite that will kill dividing cells will result in survival of only those cells that are not dividing Suitable antimetabolites include, but are not limited to, standard chemotherapeutic agents such as methotrexate, cisplatm, taxol, hydroxyurea, nucleotide analogs such as AraC, etc In addition, antimetabolite assays may include the use of genes that cause cell death upon expression The concentration at which the antimetabolite is added will depend on the toxicity of the particular antimetabolite, and will be determined as is known in the art The antimetabolite is added and the cells are generally incubated for some period of time, again, the exact period of time will depend on the characteristics and identity of the antimetabolite as well as the cell cycle time of the particular cell population. Generally, a time sufficient for at least one cell division to occur In a preferred embodiment, at least one proliferation assay is run, with more than one being preferred Thus, a proliferation assay results in a population of proliferating cells and a population of arrested cells In a preferred embodiment, either after or simultaneously with one or more of the proliferation assays outlined above, at least one cell phase assay is done A "cell phase" assay determines at which cell phase the cells are arrested, M, G1, S, or G2. In a preferred embodiment, the cell phase assay is a DNA binding dye assay. Briefly, a DNA binding dye is introduced to the cells, and taken up passively. Once inside the cell, the DNA binding dye binds to DNA, generally by intercalation, although in some cases, the dyes can be either major or minor groove binding compounds The amount of dye is thus directly correlated to the amount of DNA in the cell, which vanes by cell phase, G2 and M phase cells have twice the DNA content of G1 phase cells, and S phase cells have an intermediate amount, depending on at what point in S phase the cells are. Suitable DNA binding dyes are perrπeant, and include, but are not limited to, Hoechst 33342 and 33258, acndine orange, 7-AAD, LDS 751, DAPI, and SYTO 16, Molecular Probes Handbook, supra; chapters 8 and 16 in particular In general, the DNA binding dyes are added in concentrations ranging from about 1 μg/ml to about 5 μg/ml. The dyes are added to the cells and allowed to incubate for some period of time; the length of time will depend in part on the dye chosen In one embodiment, measurements are taken immediately after addition of the dye. The cells are then sorted as outlined below, to create populations of cells that contain different amounts of dye, and thus different amounts of DNA; in this way, cells that are replicating are separated from those that are not. As will be appreciated by those in the art, in some cases, for example when screening for anti-proliferation agents, cells with the least fluorescence (and thus a single copy of the genome) can be separated from those that are replicating and thus contain more than a single genome of DNA Alterations are determined by measuring the fluorescence at either different time points or in different cell populations, and comparing the determinations to one another or to standards In a preferred embodiment, the cell phase assay is a cyclin destruction assay In this embodiment, pnor to screening (and generally prior to the introduction of a candidate bioactive agent, as outlined below), a fusion nucleic acid is introduced to the cells The fusion nucleic acid comprises nucleic acid encoding a cyclin destruction box and a nucleic acid encoding a detectable molecule "Cyclin destruction boxes" are known in the art and are sequences that cause destruction via the ubiquitmation pathway of proteins containing the boxes during particular cell phases That is, for example, G1 cyclms may be stable during G1 phase but degraded during S phase due to the presence of a G1 cyclin destruction box Thus, by linking a cyclin destruction box to a detectable molecule, for example green fluorescent protein, the presence or absence of the detectable molecule can serve to identify the cell phase of the cell population In a preferred embodiment, multiple boxes are used, preferably each with a different fluor, such that detection of the cell phase can occur A number of cyclin destruction boxes are known in the art, for example, cyclin A has a destruction box comprising the sequence RTVLGVIGD, the destruction box of cyclin B1 comprises the sequence RTALGDIGN See Glotzer et al , Nature 349 132-138 (1991 ) Other destruction boxes are known as well YMTVSIIDRFMQDSCVPKKMLQLVGVT (rat cyclin B), KFRLLQETMYMTVSIIDRFMQNSCVPKK (mouse cyclin B), RAILIDWLIQVQMKFRLLQETMYMTVS (mouse cyclin B1), DRFLQAQLVCRKKLQWGITALLLASK (mouse cyclin B2), and MSVLRGKLQLVGTAAMLL (mouse cyclin A2) The nucleic acid encoding the cyclin destruction box is operably linked to nucleic acid encoding a detectable molecule The fusion proteins are constructed by methods known in the art For example, the nucleic acids encoding the destruction box is ligated to a nucleic acid encoding a detectable molecule By "detectable molecule" herein is meant a molecule that allows a cell or compound comprising the detectable molecule to be distinguished from one that does not contain it, i e , an epitope, sometimes called an antigen TAG, a specific enzyme, or a fluorescent molecule Preferred fluorescent molecules include but are not limited to green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and enzymes including luciferase and β-galactosidase When antigen TAGs are used, preferred embodiments utilize cell surface antigens The epitope is preferably any detectable peptide which is not generally found on the cytoplasmic membrane, although in some instances, if the epitope is one normally found on the cells, increases may be detected, although this is generally not preferred Similarly, enzymatic detectable molecules may also be used, for example, an enzyme that generates a novel or chromogenic product Accordingly, the results of sorting after cell phase assays generally result in at least two populations of cells that are in different cell phases The proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the cell cycle proteins can be identified Genetic systems have been descπbed to detect protein-protein interactions The first work was done in yeast systems, namely the "yeast two-hybrid" system The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene Subsequent work was done in mammalian cells See Fields et al , Nature 340 245 (1989), Vasavada et al , PNAS USA 88 10686 (1991), Fearon et al , PNAS USA 89 7958 (1992), Dang et al , Mol Cell Biol 11 954 (1991), Chien et al , PNAS USA 88 9578 (1991), and U S Patent Nos 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463 a preferred system is descπbed in Serial Nos 09/050,863, filed March 30, 1998 and 09/359,081 filed July 22, 1999, entitled "Mammalian Protein Interaction Cloning System" For use in conjunction with these systems, a particularly useful shuttle vector is described in Serial No 09/133,944, filed August 14, 1998, entitled "Shuttle Vectors" In general, two nucleic acids are transformed into a cell, where one is a "bait" such as the gene encoding a cell cycle protein or a portion thereof, and the other encodes a test candidate Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed Expression of the indicator indicates when a test candidate binds to the cell cycle protein and can be identified as an cell cycle protein Using the same system and the identified cell cycle proteins the reverse can be performed Namely, the cell cycle proteins provided herein can be used to identify new baits, or agents which interact with cell cycle proteins Additionally, the two-hybrid system can be used wherein a test candidate is added in addition to the bait and the cell cycle protein encoding nucleic acids to determine agents which interfere with the bait, such as p21, and the cell cycle protein In one embodiment, a mammalian two-hybrid system is preferred Mammalian systems provide post-translational modifications of proteins which may contπbute significantly to their ability to interact In addition, a mammalian two-hybπd system can be used in a wide vaπety of mammalian cell types to mimic the regulation, induction, processing, etc of specific proteins within a particular cell type For example, proteins involved in a disease state (i e , cancer, apoptosis related disorders) could be tested in the relevant disease cells Similarly, for testing of random proteins, assaying them under the relevant cellular conditions will give the highest positive results Furthermore, the mammalian cells can be tested under a variety of expeπmental conditions that may affect mtracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytok es, radiation, chemotherapeutics, cellular and chemical stimuli, etc , that may contribute to conditions which can effect protein-protein interactions, particularly those involved in cancer Assays involving binding such as the two-hybrid system may take into account non-specific binding proteins (NSB) Expression in various cell types, and assays for cell cycle activity are described above The activity assays, such as having an effect on telomere length and aging can be performed to confirm the activity of cell cycle proteins which have already been identified by their sequence identity/similarity or binding to p21 as well as to further confirm the activity of lead compounds identified as modulators of the cell cycle, particularly, telomere length as it relates to aging Telomeres shorten progressively with every cell division, ultimately causing cessation of cell division thereby inducing a cell death pathway Thus, the cell cycle proteins are involved in the cell death pathway, or apoptosis Further, without being bound by theory, telomere synthesis is required for subsequent cell division In a preferred embodiment, a cell cycle protein regulates cell proliferation through the regulation of telomere synthesis In a prefered embodiment, this regulation involves PARP activity Thus a cell cycle protein may affect the cell cycle in at least two ways, including the modulation of telomere length and interaction witn p21. The components provided herein for the assays provided herein may also be combined to form kits The kits can be based on the use of the protein and/or the nucleic acid encoding the cell cycle proteins In one embodiment, other components are provided in the kit Such components include one or more of packaging, instructions, antibodies, and labels. Additional assays such as those used in diagnostics are further described below. In this way, bioactive agents are identified Compounds with pharmacological activity are able to enhance or interfere with the activity of the cell cycle protein The compounds having the desired pharmacological activity may be administered in a physiologically acceptable earner to a host, as further described below The present discovery relating to the role of cell cycle proteins in the cell cycle thus provides methods for inducing or preventing cell proliferation in cells. In a preferred embodiment, the cell cycle proteins, and particularly cell cycle protein fragments, are useful in the study or treatment of conditions which are mediated by the cell cycle proteins, i e to diagnose, treat or prevent cell cycle associated disorders Thus, "cell cycle associated disorders" or "disease state" include conditions involving both insufficient or excessive cell proliferation, preferably cancer. In another embodiment, states such as the state of "normal" aging which are not necessarily disorders can be modulated by the agents identified herein Thus, in one embodiment, methods of cell cycle regulation in cells or organisms are provided. In one embodiment, the methods comprise administering to a cell or individual in need thereof, a cell cycle protein in a therapeutic amount Alternatively, an anti-cell cycle antibody that reduces or eliminates the biological activity of the endogeneous cell cycle protein is administered In another embodiment, a bioactive agent as identified by the methods provided herein is administered Alternatively, the methods comprise administering to a cell or individual a recombinant nucleic acid encoding an cell cycle protein As will be appreciated by those in the art, this may be accomplished in any number of ways In a preferred embodiment, proliferation, the potential for proliferation, or the rate of passage through a stage of the cell cycle is increased by increasing the amount of cell cycle protein in the cell, for example by overexpressmg the endogeneous cell cycle or by administering a gene encoding a cell cycle protein, using known gene-therapy techniques, for example In a preferred embodiment, the gene therapy techniques include the incorporation of the exogeneous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety In a preferred embodiment, increasing cell cycle protein activity increase cell proliferation, the potential for proliferation, or the rate of passage through a stage of the cell cycle In another embodiment, decreasing cell cycle protein activity decreases cell proliferation, the potential for proliferation, or the rate of passage through a stage of the cell cycle Without being bound by theory, it appears that cell cycle protein is an important protein in the cell cycle. Accordingly, disorders based on mutant or variant cell cycle genes may be determined. In one embodiment, the invention provides methods for identifying cells containing variant cell cycle genes comprising determining all or part of the sequence of at least one endogeneous cell cycle genes in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the cell cycle genotype of an individual compπsmg determining all or part of the sequence of at least one cell cycle 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 include comparing the sequence of the sequenced cell cycle gene to a known cell cycle gene, i e. a wild-type gene The sequence of all or part of the cell cycle gene can then be compared to the sequence of a known cell cycle gene to determine if any differences exist This can be done using any number of known sequence identity programs, such as Bestfit, etc. In a preferred embodiment, the presence of a difference in the sequence between the cell cycle gene of the patient and the known cell cycle gene is indicative of a disease state or a propensity for a disease state In one embodiment, the invention provides methods for diagnosing a cell cycle related condition in an individual The methods comprise measuring of cell cycle activity in a tissue from the individual or patient, which may include a measurement of the amount or specific activity of a cell cycle protein This activity is compared to cell cycle activity from either a unaffected second individual or from an unaffected tissue from the first individual When these activities are different, the first individual may be at risk for a cell cycle associated disorder In this way, for example, monitoring of various disease conditions may be done, by monitoring the levels of the protein or the expression of mRNA therefor Similarly, expression levels may correlate to the prognosis In one aspect, the activity of the cell cycle protein is determined to diagnose a cell cycle related condition In a preferred embodiment, the activity is PARP activity In one aspect, the expression levels of cell cycle protein genes are determined in different patient samples or cells for which either diagnosis or prognosis information is desired Gene expression monitoring is done on genes encoding cell cycle proteins In one aspect, the expression levels of cell cycle protein genes are determined for different cellular states, such as normal cells and cells undergoing apoptosis or transformation By comparing cell cycle protein gene expression levels in cells in different states, information including both up- and down-regulation of cell cycle protein genes is obtained, which can be used in a number of ways For example, the evaluation of a particular treatment regime may be evaluated does a chemotherapeutic drug act to improve the long-term prognosis in a particular patient Similarly, diagnosis may be done or confirmed by compaπng patient samples Furthermore, these gene expression levels allow screening of drug candidates with an eye to mimicking or altering a particular expression level This may be done by making biochips comprising sets of important cell cycle protein genes, such as those of the present invention, which can then be used in these screens These methods can also be done on the protein basis; that is, protein expression levels of the cell cycle proteins can be evaluated for diagnostic purposes or to screen candidate agents In addition, the cell cycle protein nucleic acid sequences can be administered for gene therapy purposes, including the administration of antisense nucleic acids, or the cell cycle proteins administered as therapeutic drugs Ccell cycle protein sequences bound to biochips include both nucleic acid and ammo acid sequences as defined above In a preferred embodiment, nucleic acid probes to cell cycle protein nucleic acids (both the nucleic acid sequences having the sequences outlined in the Figures and/or the complements thereof) are made The nucleic acid probes attached to the biochip are designed to be substantially complementary to the cell cycle protein 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 hybπdization conditions, the sequence is not a complementary target sequence Thus, by "substantially complementary" herein is meant that the probes are sufficiently complementary to the target sequences to hybndize under normal reaction conditions, particularly high stringency conditions, as outlined herein 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 the target sequence In general, the nucleic acid probes ιange 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 In some embodiments, much longer nucleic acids can be used, up to hundreds of bases (e g , whole genes) 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, streptavidin to the support and the non-covalent binding of the biotmylated probe to the streptavidin. 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 and 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 combination 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 compπses a suitable solid substrate. By "substrate" or "solid support" or other grammatical equivalents herein is meant any mateπal that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method As will be appreciated by those in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysacchandes, nylon or nitrocellulose, resins, silica or silica-based mateπals including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc In general, the substrates allow optical detection and do not appreciably show fluorescence. In a preferred embodiment, the surface of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two Thus, for example, the biochip is derivatized with a chemical functional group including, but not limited to, ammo groups, carboxy groups, oxo groups and thiol groups, with ammo groups being particularly preferred Using these functional groups, the probes can be attached using functional groups on the probes For example, nucleic acids containing ammo groups can be attached to surfaces comprising ammo groups, for example 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, oligonucleotides, corresponding to the nucleic acid probe, 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 non- covalent For example, biotmylated oligonucleotides 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 photopolymeπzation 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 Affimetπx GeneChip™ technology "Differential expression," or grammatical equivalents as used herein, refers to both qualitative as well as quantitative differences in the genes' 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 mactivation, in, for example, normal versus apoptotic cell 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 is quantitative in that expression is increased or decreased, that is, the expression of the gene is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased 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 Affymetπx GeneChip™ expression arrays, Lockhart, Nature Biotechnology 14 1675-1680 (1996), hereby expressly incorporated by reference Other techniques include, but are not limited to, quantitative reverse transcπptase PCR, Northern analysis and RNase protection As will be appreciated by those in the art, this may be done by evaluation at either the gene transcript, 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 the cell cycle protein and standard immunoassays (ELISAs, etc ) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc In another method detection of the mRNA is performed in situ In this method permeabihzed cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybndize with the target mRNA Following washing to remove the non- specifically bound probe, the label is detected For example a digoxygenm labeled nboprobe (RNA probe) that is complementary to the mRNA encoding an cell cycle protein is detected by binding the digoxygenm with an anti-digoxygenin secondary antibody and developed with nitro blue tetrazo um and 5-bromo-4-chloro-3-ιndoyl phosphate In another preferred method, expression of cell cycle proteins is performed using in situ imaging techniques employing antibodies to cell cycle proteins In this method cells are contacted with from one to many antibodies to the cell cycle proteιn(s) Following washing to remove non-specific antibody binding, the presence of the 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 the cell cycle proteιn(s) contains a detectable label In another preferred embodiment each one of multiple primary antibodies contains a distinct and detectable label This method finds particular use in simultaneous screening for a plurahlty of cell cycle proteins The label may be detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths In addition, a fluorescence activated cell sorter (FACS) can be used in this method As will be appreciated by one of ordinary skill in the art, numerous other histological imaging techniques are useful in the invention and the antibodies can be used in ELISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE technology, In one embodiment, the cell cycle proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to ceil cycle proteins, which are useful as described herein Similarly, the cell cycle proteins can be coupled, using standard technology, to affinity chromatography columns These columns may then be used to purify cell cycle antibodies In a preferred embodiment, the antibodies are generated to epitopes unique to the cell cycle protein, that is, the antibodies show little or no cross-reactivity to other proteins These antibodies find use in a number of applications For example, the cell cycle antibodies may be coupled to standard affinity chromatography columns and used to purify cell cycle proteins as further described below The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the cell cycle protein The anti-cell cycle protein antibodies may comprise polyclonal antibodies 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 intrapeπtoneal injections The immunizing agent may include the cell cycle protein 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 hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid a, synthetic trehalose dicorynomycolate) The immunization protocol may be selected by one skilled in the art without undue experimentation The anti-cell cycle protein antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybπdoma methods, such as those descπbed by Kohler and Milstein, Nature. 256 495 (1975) In a hybπdoma method, a mouse, hamster, or other appropπate host animal, is typically immunized 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 vitro The immunizing agent will typically include the cell cycle polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human oπgin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybπdoma cell [Godmg, Monoclonal Antibodies Principles and Practice, Academic Press, (1986) pp 59-103] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed The hybπdoma 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 hypoxanthme guanine phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybndomas typically will include hypoxanthme, aminopteπn, and thymidme ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are muπne myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J Immunol , 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications. Marcel Dekker, Inc , New York, (1987) pp 51-63] The culture medium in which the hybπdoma ceils are cultured can then be assayed for the presence of monoclonal antibodies directed against cell cycle protein Preferably, the binding specificity of monoclonal antibodies produced by the hybπdoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked immunosorbent assay (ELISA) Such techniques and assays are known in the art The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem , 107 220 (1980) After the desired hybπdoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Godmg, supral Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybndoma cells may be grown in vivo as ascites in a mammal The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulm purification procedures such as, for example, protein a-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of muπne antibodies) The hybndoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulm protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous muπne sequences [U S Patent No 4,816,567, Morrison et al , supral or by covalently joining to the immunoglobulm coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeπc bivalent antibody The antibodies may be monovalent antibodies Methods for prepamg monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulm light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking Alternatively, the relevant cysteine residues are substituted with another ammo acid residue or are deleted so as to prevent crosslinking In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. The anti-cell cycle protein antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , muπne) antibodies are chimeπc immunoglobulins, immunoglobulm chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulm Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulm are replaced by corresponding non-human residues Humanized antibodies may also 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 at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulm and all or substantially all of the FR regions are those of a human immunoglobulm consensus sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulm constant region (Fc), typically that of a human immunoglobulm [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)] Methods for humanizing non-human antibodies are well known in the art Generally, a humanized antibody has one or more ammo acid residues introduced into it from a source which is non- human These non-human ammo 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 et al , Nature. 332 323-327 (1988), Verhoeyen et al , Science. 239 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeπc antibodies (U S 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 al. 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 Boerner et al., J. Immunol., 147(1): 86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulm loci into transgenic animals, e.g., 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; 5,569,825; 5,625,126; 5,633,425; 5,661 ,016, and in the following scientific publications: Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg ef 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. Intern. Rev. Immunol. 13 65-93 (1995). 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 the cell cycle protein, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy- chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J.. 10:3655- 3659 (1991 ). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, compπsmg at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulm light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzvmology. 121 210 (1986). Heteroconjugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360, WO 92/200373, EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxms may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyπmιdate and those disclosed, for example, in U.S. Patent No. 4,676,980. The anti-cell cycle protein antibodies of the invention have various utilities. For example, anti-cell cycle protein antibodies may be used in diagnostic assays for an cell cycle protein, e.g., detecting its expression in specific cells, tissues, or serum Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunopreαpitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies a Manual of Technigues. CRC Press, Inc (1987) pp. 147-158] The antibodies used in the diagnostic assays can be labeled with a detectable moiety The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal For example, the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 3S S, or 1 5 l, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or lucifeπn, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al , Nature. 144 945 (1962), David et al., Biochemistry. 13:1014 (1974); Pain et al , J Immunol Meth . 40 219 (1981), and Nygren, J Histochem and Cvtochem . 30 407 (1982) Anti-Cell cycle protein antibodies also are useful for the affinity purification of cell cycle protein from recombinant cell culture or natural sources. In this process, the antibodies against cell cycle protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art The immobilized antibody then is contacted with a sample containing the cell cycle protein to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the cell cycle protein, which is bound to the immobilized antibody Finally, the support is washed with another suitable solvent that will release the cell cycle protein from the antibody The anti-cell cycle protein antibodies may also be used in treatment In one embodiment, the genes encoding the antibodies are provided, such that the antibodies bind to and modulate the cell cycle protein within the cell In one embodiment, a therapeutically effective dose of an cell cycle protein, agonist or antagonist 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 cell cycle degradation, and systemic versus localized delivery, 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 expeπmentation 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 veteπnary applications In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human The administration of the cell cycle protein, agonist or antagonist of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, tranasally, transdermally, intrapeπtoneally, intramuscularly, intrapulmonary, vag ally, rectally, or intraocularly In some instances, for example, in the treatment of wounds and inflammation, the composition may be directly applied as a solution or spray 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 wt % The pharmaceutical compositions of the present invention comprise an cell cycle protein, agonist or antagonist (including antibodies and bioactive agents as described herein) 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 acceptable salts, which is meant to include both acid and base addition salts "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuπc acid, nitnc acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glyco c acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaπc acid, tartanc acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, 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 Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurπng substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, tπmethylamine, diethylamine, triethylamine, tπpropylamine, and ethanolamme The pharmaceutical compositions may also include one or more of the following earner proteins such as serum albumin, buffers, fillers such as microcrystalline 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 Combinations of the compositions may be administered Moreover, the compositions may be administered in combination with other therapeutics, including growth factors or chemotherapeutics and/or radiation Targeting agents (i e ligands for receptors on cancer cells) may also be combined with the compositions provided herein In one embodiment provided herein, the antibodies are used for immunotherapy, thus, methods of immunotherapy are provided By "immunotherapy" is meant treatment of cell cycle protein related disorders with an antibody raised against cell cycle proteins 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 can be the consequence of providing the recipient with an cell cycle protein antigen to which antibodies are raised As appreciated by one of ordinary skill in the art, the cell cycle protein antigen may be provided by injecting an cell cycle polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with an cell cycle protein nucleic acid, capable of expressing the cell cycle protein antigen, under conditions for expression of the cell cycle protein antigen In a preferred embodiment, a therapeutic compound is conjugated to an antibody, preferably an cell cycle protein antibody The therapeutic compound may be a cytotoxic agent In this method, targeting the cytotoxic agent to apoptotic cells or tumor tissue or cells, results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with apoptosis, cancer cell cycle protein related disorders 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 diptheπa A chain, exotoxm A chain, πcin A chain, abπn A chain, curcin, crotin, phenomycm, enomycm and the like Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against cell cycle proteins, or binding of a radionuclide to a chelatmg agent that has been covalently attached to the antibody In a preferred embodiment, cell cycle protein genes are administered as DNA vaccines, either single genes or combinations of cell cycle protein genes Naked DNA vaccines are generally known in the art, see Brower, Nature Biotechnology 16 1304-1305 (1998) Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing an cell cycle protein gene or portion of an cell cycle protein gene under the control of a promoter for expression in a patient The cell cycle protein gene used for DNA vaccines can encode full-length cell cycle proteins, but more preferably encodes portions of the cell cycle proteins including peptides deπved from the cell cycle protein In a preferred embodiment a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences deπved from a cell cycle protein gene Similarly, it is possible to immunize a patient with a plurality of cell cycle protein genes or portions thereof, as defined herein Without being bound by theory, following expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced which recognize and destroy or eliminate cells expressing cell cycle proteins In a preferred embodiment, the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine Such adjuvant molecules include cytokmes that increase the immunogenic response to the cell cycle protein encoded by the DNA vaccine Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention The above examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes All references and accession numbers cited herein are expressly incorporated by reference in their entirety

Claims: CLAIMS We claim 1 A method for screening for a bioactive agent capable of binding to a cell cycle protein tankyrase H, said method comprising combining a cell cycle protein tankyrase H and a candidate bioactive agent, and determining the binding of said candidate agent to said cell cycle protein tankyrase H 2 A method for screening for agents capable of interfering with the binding of a cell cycle protein tankyrase H and p21 comprising- a) combining a cell cycle protein tankyrase H, a candidate bioactive agent and a p21 protein, and b) determining the binding of said cell cycle protein and said p21 protein. 3 A method according to claim 2 wherein said cell cycle protein and said p21 protein are combined first 4 A method for screening for an bioactive agent capable of modulating the activity of an cell cycle protein tankyrase H, said method comprising the steps of a) adding a candidate bioactive agent to a cell compπsmg a recombinant nucleic acid encoding a cell cycle protein tankyrase H, b) determining the effect of the candidate bioactive agent on said cell 5 A method according to claim 4 wherein a library of candidate bioactive agents are added to a plurality of cells comprising a recombinant nucleic acid encoding a cell cycle protein 6 A method of diagnosing cancer, said method comprising determining the activity of tankyrase H from a test sample of an individual and comparing said level with a control which indicates there is no cancer, wherein an increase in the activity of tankyrase H in the test sample over the control sample indicates that the individual has cancer 7 A method for screening for a bioactive agent capable of modulating the activity of a cell cycle protein tankyrase H, said method comprising the steps of a) adding a candidate bioactive agent to a reaction mixture, said mixture comprising i) a source of poly ADP-πbose, II) a recombinant cell cycle protein tankyrase H, in) a substrate of cell cycle protein tankyrase H, and b) determining the effect of the candidate bioactive agent on the PARP activity of said cell cycle protein tankyrase by determining the poly ADP-nbose content of the substrate 8 The method of claim 25 wherein the source of poly ADP πbose is biotmylated NAD, and the determination of poly ADP πbose content involves streptavidm-based-detection of biotm 9 A method for treating an individual with a cell cycle related disorder, said method comprising administering to said individual an inhibitor of TaHo 10 A recombinant nucleic acid encoding a cell cycle protein compπsing a nucleic acid that hybridizes under high stringency conditions to a sequence complementary to that set forth by SEQ ID N0 1 or SEQ ID NO 2 11 The recombinant nucleic acid of claim 10 wherein said protein binds to p21 12 A recombinant nucleic acid encoding a cell cycle protein compπsing a nucleic acid having at least 85% sequence identity to a sequence as set forth by SEQ ID NO 1 or SEQ ID NO 2 13 A recombinant nucleic acid according to claim 10 having the sequence set forth by SEQ ID NO 1 or SEQ ID NO 2 13 A recombinant nucleic acid encoding a polypeptide sequence as set forth by SEQ ID NO 3 or SEQ ID NO 4 14 An expression vector comprising the recombinant nucleic acid according to any one of claims 10-13 operably linked to regulatory sequences recognized by a host cell transformed with the nucleic acid 15 A host cell comprising the recombinant nucleic acid according to any of claims 10-15 16 A host cell comprising the vector of claim 14 17 A process for producing a cell cycle protein comprising cultuπng the host cell of claim 15 or 16 under conditions suitable for expression of a cell cycle protein 18 A process according to claim 17 further comprising recovering said cell cycle protein 19 A recombinant cell cycle protein encoded by the nucleic acid of any of claims 10-15 20 A recombinant polypeptide comprising an am o acid sequence having at least 85% sequence identity with the sequence set forth by SEQ ID NO 3 or SEQ ID NO 4 21. A recombinant polypeptide according to claim 20 wherein said polypeptide binds to p21. 22. A recombinant polypeptide according to claim 20 having an ammo acid sequence as set forth by SEQ ID NO:3 or SEQ ID NO:4. 23. An isolated polypeptide which specifically binds to a cell cycle protein according to claim 20. 24. A polypeptide according to claim 23 that is an antibody. 25. A polypeptide according to claim 24 wherein said antibody is a monoclonal antibody.

Inventors: CHAN EVA, HUANG BETTY, LUO YING, XU XIANG, CHAN, EVA, HUANG, BETTY, LUO, YING, XU, XIANG
Applicants: RIGEL PHARMACEUTICALS INC, RIGEL PHARMACEUTICALS, INC.
Application year: 2000
Main classification: C12N9
IPC codes: A61K38, A61K45, A61P35, A61P43, C07K16, C12N1, C12N5, C12N9, C12N15, C12P21, C12Q1, G01N33