Source: http://www.google.com/patents/US5443962?dq=4182933
Timestamp: 2015-01-27 23:30:47
Document Index: 164085585

Matched Legal Cases: ['ART3', 'ART3', 'ART3', 'ART3', 'ART3', 'ART3', 'ART3', 'ART3', 'ART3']

Patent US5443962 - Methods of identifying inhibitors of cdc25 phosphatase - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe present invention makes available assays and reagents for identifying anti-proliferative agents, such as mitotic and meiotic inhibitors, especially inhibitors of cdc25 phosphatase. The present assay provides a simple and rapid screening test which relies on scoring for positive cellular proliferation...http://www.google.com/patents/US5443962?utm_source=gb-gplus-sharePatent US5443962 - Methods of identifying inhibitors of cdc25 phosphataseAdvanced Patent SearchPublication numberUS5443962 APublication typeGrantApplication numberUS 08/073,383Publication dateAug 22, 1995Filing dateJun 4, 1993Priority dateJun 4, 1993Fee statusLapsedAlso published asCA2163524A1, CA2163524C, DE69430573D1, DE69430573T2, EP0708653A1, EP0708653A4, EP0708653B1, US6251585, WO1994028914A1Publication number073383, 08073383, US 5443962 A, US 5443962A, US-A-5443962, US5443962 A, US5443962AInventorsGiulio Draetta, Guillaume Cottarel, Veronique DamagnezOriginal AssigneeMitotix, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (3), Non-Patent Citations (48), Referenced by (43), Classifications (19), Legal Events (6) External Links: USPTO, USPTO Assignment, EspacenetMethods of identifying inhibitors of cdc25 phosphataseUS 5443962 AAbstract The present invention makes available assays and reagents for identifying anti-proliferative agents, such as mitotic and meiotic inhibitors, especially inhibitors of cdc25 phosphatase. The present assay provides a simple and rapid screening test which relies on scoring for positive cellular proliferation as indicative of anti-mitotic or anti-meiotic activity, and comprises contacting a candidate agent with a cell which has an impaired cell-cycle checkpoint and measuring the level of proliferation in the presence and absence of the agent. The checkpoint impairment is such that it either causes premature progression of the cell through at least a portion of a cell-cycle or inhibition of normal progression of the cell through at least a portion of a cell-cycle, but can be off-set by the action of an agent which inhibits at least one regulatory protein of the cell-cycle in a manner which counter-balances the effect of the impairment.
What is claimed: 1. An assay for identifying an inhibitor of a cdc25 phosphatase, comprisingi. providing a cell expressing a recombinant cdc25 phosphatase, and having an impaired checkpoint which can cause premature entry of the cell into mitosis resulting in inhibition of proliferation of the cell, the premature entry into mitosis being mediated at least in part by the cdc25 phosphatase; ii. contacting the cell with a candidate agent; iii. measuring a level of proliferation of the cell in the presence of the candidate agent; and iv. comparing the level of proliferation of the cell in the presence of the candidate agent to a level of proliferation of the cell in the absence of the candidate agent, wherein an increase in the level of proliferation in the presence of the candidate agent is indicative of inhibition of the cdc25 phosphatase by the candidate agent. 2. The assay of claim 1, wherein the cell-cycle checkpoint impairment results in entry of the cell into mitosis before completion of replication or repair of genomic DNA of the cell.
3. The assay of claim 1, wherein the cell-cycle checkpoint impairment comprises a reduction of inhibitory phosphorylation of a cydin-dependent kinuse (cdk).
4. The assay of claim 3, wherein the cell-cycle checkpoint impairment comprises an impaired wee1 protein kinase activity, an impaired mik 1 protein kinase activity, or an over-expression of a nim1 gene product.
5. The assay of claim 1, wherein the cell-cycle checkpoint impairment is induced by treatment of the cell with a hyper-mitotic agent.
6. The assay of claim 5, wherein the hyper-mitotic agent is selected from a group consisting of caffeine, 2-aminopurine, 6-dimethylaminopurine, and okadaic acid.
7. The assay of claim 1, wherein the cell-cycle checkpoint is conditionally impairable and the level of proliferation of the cell in the presence and the absence of the candidate agent is measured under conditions wherein the cell-cycle checkpoint is impaired.
8. The assay of claim 1, wherein the cell is a yeast cell.
9. The assay of claim 8, wherein the yeast cell is a species of the genus Schizosaccharomyces.
10. The assay of claim 1, wherein the cdc25 phosphatase is a human cdc25.
11. The assay of claim 1, wherein the cdc25 phosphatase is a cdc25 of a human pathogen.
12. The assay of claim 11, wherein the cdc25 phosphatase is derived from a human pathogen which causes a mycotic infection.
13. The assay of claim 12, wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.
14. The assay of claim 12, wherein the human pathogen is selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus.
15. The assay of claim 11, wherein the human pathogen is Pneumocystis carinii.
16. An assay for identifying an inhibitor of a cdc25 phosphatase, comprisingi. providing a Schizosaccharomyces cella. expressing a recombinant cdc25 phosphatase, the Schizosaccharomyces cell lacking a functional endogenous cdc25 phosphatase activity, and b. having a conditionally impairable wee1 protein kinase which can cause inhibition of proliferation of the Schizosaccharomyces cell by facilitating premature entry of the Schizosaccharomyces cell into mitosis under conditions wherein the wee1 kinase is impaired, the premature entry into mitosis being mediated at least in part by the cdc25 phosphatase and a reduced level of inhibitory phosphorylation of a cdc2 protein kinase by the wee1 protein kinase; ii. contacting the Schizosaccharomyces cell with a test compound under the conditions wherein the wee1 kinase is impaired; iii. measuring a level of proliferation of the Schizosaccharomyces cell in the presence of the test compound; and iv. comparing the level of proliferation of the Schizosaccharomyces cell in the presence of the test compound to a level of proliferation of the Schizosaccharomyces cell in the absence of the test compound,wherein an increase in the level of proliferation in the presence of the test compound is indicative of inhibition of the cdc25 phosphatase by the test compound. 17. The assay of claim 16, wherein the Schizosaccharomyces cell is a Schizosaccharomyces pombe cell.
18. The assay of claim 16, wherein the Schizosaccharomyces cell is a conditional wee phenotype.
19. The assay of claim 18, wherein the Schizosaccharomyces cell is a wee1-50 mutant.
20. The assay of claim 16, wherein the impairment of the wee1 protein kinase activity is caused by the overexpression of a nim1 activator in the Schizosaccharomyces cell.
21. The assay of claim 20, wherein the Schizosaccharomyces cell is an OP-nim1 mutant.
22. The assay of claim 16, wherein the cdc25 phosphatase activity is a human cdc25.
23. The assay of claim 22, wherein the human cdc25 is selected from a group consisting of cdc25A, cdc25B and cdc25C.
24. The assay of claim 16, wherein the cdc25 phosphatase activity is a human pathogen cdc25.
25. The assay of claim 24, wherein the human pathogen is a fungus implicated in a mycotic infection selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis, nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, and sporotrichosis.
26. A Schizosaccharomyces cell comprisingi). an expressible recombinant gene encoding an exogenous cdc25 phosphatase; and ii). a conditionally impairable wee1 protein kinase which can cause inhibition of cell proliferation by facilitating premature entry of the cell into mitosis under conditions wherein the wee1 protein kinase is impaired, the premature entry into mitosis being mediated at least in part by the exogenous cdc25 phosphatase and a reduced level of inhibitory phosphorylation of a cdc2 protein kinase by the impaired wee1 protein kinase. 27. The Schizosaccharomyces cell of claim 26, wherein the exogenous cdc25 phosphatase comprises a human cdc25 phosphatase.
28. The Schizosaccharomyces cell of claim 26, wherein the human cdc25 phosphatase is selected from a group consisting of cdc25A, cdc25B, and cdc25C.
29. The Schizosaccharomyces cell of claim 26, wherein the recombinant cdc25 phosphatase is a human pathogen cdc25.
30. The Schizosaccharomyces cell of claim 29, wherein the human pathogen cdc25 is a cdc25 phosphatase of a fungus that causes a mycotic infection.
31. The Schizosaccharomyces cell of claim 26, wherein the wee1 protein kinase is temperature sensitive and is impaired at a temperature above a permissive temperature.
32. The Schizosaccharomyces cell of claim 31, wherein the Schizosaccharomyces cell is a wee1-50 mutant.
33. The Schizosaccharomyces cell of claim 26, further comprising an overexpressed nim1 gene product which impairs the wee1 protein kinase.
34. The Schizosaccharomyces cell of claim 33, wherein the Schizosaccharomyces cell is an OP-nim1 mutant.
35. An assay for identifying an inhibitor of a cdc25 phosphatase, comprisingi. providing a cell having an impaired checkpoint induced by treatment of the cell with a hyper-mitotic agent and which can cause premature entry of the cell into mitosis resulting in inhibition of proliferation of the cell, the premature entry into mitosis being mediated at least in part by a cdc25 phosphatase; ii. contacting the cell with a candidate agent; iii. measuring a level of proliferation of the cell in the presence of the candidate agent; and iv. comparing the level of proliferation of the cell in the presence of the candidate agent to a level of proliferation of the cell in the absence of the candidate agent,wherein an increase in the level of proliferation in the presence of the candidate agent is indicative of inhibition of the cdc25 phosphatase by the candidate agent. 36. The assay of claim 35, wherein the cell-cycle checkpoint impairment results in entry of the cell into mitosis before completion of replication or repair of genomic DNA of the cell.
37. The assay of claim 35, wherein the hyper-mitotic agent is selected from a group consisting of caffeine, 2-aminopurine, 6-dimethylaminopurine, and okadaic acid.
38. The assay of claim 37, wherein the cell-cycle checkpoint is conditionally impairable and the level of proliferation of the cell in the presence and the absence of the candidate agent is measured under conditions wherein the cell-cycle checkpoint is impaired.
39. The assay of claim 35, wherein the cell is a yeast cell.
40. An assay for identifying an inhibitor of a cdc25 phosphatase, comprisingi. providing a cell having an impaired checkpoint which can cause premature entry of the cell into mitosis resulting in inhibition of proliferation of the cell, the premature entry into mitosis being mediated at least in part by a human cdc25 phosphatase; ii. contacting the cell with a candidate agent; iii. measuring a level of proliferation of the cell in the presence of the candidate agent; and iv. comparing the level of proliferation of the cell in the presence of the candidate agent to a level of proliferation of the cell in the absence of the candidate agent,wherein an increase in the level of proliferation in the presence of the candidate agent is indicative of inhibition of the cdc25 phosphatase by the candidate agent. 41. An assay for identifying an inhibitor of a cdc25 phosphatase, comprisingi. providing a cell having an impaired checkpoint which can cause premature entry of the cell into mitosis resulting in inhibition of proliferation of the cell, the premature entry into mitosis being mediated at least in part by a cdc25 phosphatase of a human pathogen; ii. contacting the cell with a candidate agent; iii. measuring a level of proliferation of the cell in the presence of the candidate agent; and iv. comparing the level of proliferation of the cell in the presence of the candidate agent to a level of proliferation of the cell in the absence of the candidate agent,wherein an increase in the level of proliferation in the presence of the candidate agent is indicative of inhibition of the cdc25 phosphatase by the candidate agent. 42. The assay of claim 25, wherein the human pathogen is selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus.
BACKGROUND OF THE INVENTION Entry of cells into mitosis characteristically involves coordinated and simultaneous events, which include, for example, cytoskeletal rearrangements, disassembly of the nuclear envelope and of the nucleoli, and condensation of chromatin into chromosomes. Cell-cycle events are thought to be regulated by a series of interdependent biochemical steps, with the initiation of late events requiring the successful completion of those proceeding them. In eukaryotic cells mitosis does not normally take place until the G1, S and G2 phases of the cell-cycle are completed. For instance, at least two stages in the cell cycle are regulated in response to DNA damage, the G1/S and the G2/M transitions. These transitions serve as checkpoints to which cells delay cell-cycle progress to allow repair of damage before entering either S phase, when damage would be perpetuated, or M phase, when breaks would result in loss of genomic material. Both the G1/S and G2/M checkpoints are known to be under genetic control as there are mutants that abolish arrest or delay which ordinarily occur in wild-type cells in response to DNA damage.
The progression of a proliferating eukaryotic cell through the cell-cycle checkpoints is controlled by an array of regulatory proteins that guarantee that mitosis occurs at the appropriate time. These regulatory proteins can provide exquisitely sensitive feedback-controlled circuits that can, for example, prevent exit of the cell from S phase when a fraction of a percent of genomic DNA remains unreplicated (Dasso et al. (1990) Cell 61:811-823 ) and can block advance into anaphase in mitosis until all chromosomes are aligned on the metaphase plate (Rieder et al. (1990) J. Cell Biol. 110:81-95). In particular, the execution of various stages of the cell-cycle is generally believed to be under the control of a large number of mutually antagonistic kinases and phosphatases. For example, genetic, biochemical and morphological evidence implicate the cdc2 kinase as the enzyme responsible for triggering mitosis in eukaryotic cells (for reviews, see Hunt (1989) Curr. Opin. Cell Biol. 1:268-274; Lewin (1990) Cell 61:743-752; and Nurse (1990) Nature 344:503-508). The similarities between the checkpoints in mammalian cells and yeast have suggested similar roles for cdc protein kinases across species. In support of this hypothesis, a human cdc2 gene has been found that is able to substitute for the activity of an S. Pombe cdc2 gene in both its G 1/S and G2/M roles (Lee et al (1987) Nature 327:31). Likewise, the fact that the cdc2 homolog of S. Cerevisae (cdc28) can be replaced by the human cdc2 also emphasizes the extent to which the basic cell-cycle machinery has been conserved in evolution.
As mitosis progresses, the cdc2 kinase appears to trigger a cascade of downstream mitotic phenomena such as metaphase alignment of chromosomes, segregation of sister chromatids in anaphase, and cleavage furrow formation. Many target proteins involved in mitotic entry of the proliferating cell are directly phosphorylated by the cdc2 kinase. For instance, the cdc2 protein kinase acts by phosphorylating a wide variety of mitotic substrates such as nuclear lamins, histones, and microtubule-associated proteins (Moreno et al. (1990) Cell 61:549-551; and Nigg (1991) Semin. Cell Biol. 2:261-270). The cytoskeleton of cultured cells entering mitosis is rearranged dramatically. Caldesmon, an actin-associated protein, has also been shown to be a cdc2 kinase substrate (Yamashiro et al. (1991) Nature 349:169-172), and its phosphorylation may be involved in induction of M-phase-specific dissolution of actin cables. The interphase microtubule network disassembles, and is replaced by a mitosis-specific astral array emanating from centrosomes. This rearrangement has been correlated with the presence of mitosis-specific cdc2 kinase activity in cell extracts (Verde et al (1990) Nature 343:233- 238). Changes in nuclear structure during mitotic entry are also correlated with cdc2 kinase activity. Chromatin condensation into chromosomes is accompanied by cdc2 kinase-induced phosphorylation of histone H1 (Langan et al. (1989) Molec. Cell. Biol. 9:3860-3868), nuclear envelope dissolution is accompanied by cdc2-specific phosphorylation of lamin B (Peter et al. (1990) Cell 61:591-602) nucleolar disappearance is coordinated with the cdc2-dependent phosphorylation of nucleolin and NO38.
The cdc2 kinase is subject to multiple levels of control. One well-characterized mechanism regulating the activity of cdc2 involves the phosphorylation of tyrosine, threonine, and serine residues; the phosphorylation level of which varies during the cell-cycle (Draetta et al. (1988) Nature 336:738-744; Dunphy et al. (1989) Cell 58:181-191; Morla et al. (1989) Cell 58:193-203; Gould et al. (1989) Nature 342:39-45; and Solomon et al. (1990) Cell 63:1013-1024). The phosphorylation of cdc2 on Tyr-15 and Thr-14, two residues located in the putative ATP binding site of the kinase, negatively regulates kinase activity. This inhibitory phosphorylation of cdc2 is mediated at least impart by the wee1 and mik1 tyrosine kinases (Russel et al. (1987) Cell 49:559-567; Lundgren et al. (1991) Cell 64:1111-1122; Featherstone et al. (1991) Nature 349:808-811; and Parker et al. (1992) PNAS 89:2917-2921 ). These kinases act as mitotic inhibitors, over-expression of which causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of wee1 causes a modest advancement of mitosis, whereas loss of both wee1 and mik1 function causes grossly premature mitosis, uncoupled from all checkpoints that normally restrain cell division (Lundgren et al. (1991) Cell 64:1111-1122).
As the cell is about to reach the end of G2, dephosphorylation of the cdc2-inactivating Thr-14 and Tyr-15 residues occurs leading to activation of the cdc2 complex as a kinase. A stimulatory phosphatase, known as cdc25, is responsible for Tyr-15 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al. (1991 ) Cell 67:189-196; Lee et al. (1992) Mol Biol Cell 3:73-84; Millar et al. (1991) EMBO J 10:4301-4309; and Russell et al. (1986) Cell 45:145-153). Recent evidence indicates that both the cdc25 phosphatase and the cdc2-specific tyrosine kinases are detectably active during interphase, suggesting that there is an ongoing competition between these two activities prior to mitosis (Kumagai et al. (1992) Cell 70:139-151; Smythe et al. (1992) Cell 68:787-797; and Solomon et al. (1990) Cell 63:1013-1024. This situation implies that the initial decision to enter mitosis involves a modulation of the equilibrium of the phosphorylation state of cdc2 which is likely controlled by variation of the rate of tyrosine dephosphorylation of cdc2 and/or a decrease in the rate of its tyrosine phosphorylation. A variety of genetic and biochemical data appear to favor a decrease in cdc2-specific tyrosine kinase activity near the initiation of mitosis which can serve as a triggering step to tip the balance in favor of cdc2 dephosphorylation (Smythe et al. (1992) Cell 68:787-797; Matsumoto et al. (1991) Cell 66:347-360; Kumagai et al. (1992) Cell 70:139-151; Rowley et al. (1992) Nature 356:353-355; and Enoch et al. (1992) Genes Dev. 6:2035-2046). Moreover, recent data suggest that the activated cdc2 kinase is responsible for phosphorylating and activating cdc25. This event would provide a self-amplifying loop and trigger a rapid increase in the activity of the cdc25 protein, ensuring that the tyrosine dephosphorylation of cdc2 proceeds rapidly to completion (Hoffmann et al. (1993) EMBO J. 12:53).
The impaired checkpoint can be generated, for example, by molecular biological, genetic, and/or biochemical means. The checkpoint to be impaired can comprise a regulatory protein or proteins which control progression through the cell-cycle, such as those which control the G2/M transition or the G1/S transition. By way of example, the impaired checkpoint can comprise regulatory proteins which control the phosphorylation/dephosphorylation of a cdc protein kinase, such as the gene products of wee1, mik1, or nim1.
DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic representation of the construction of the "5'-half ura4-adh promoter- cdc25A-3'-half ura4" nucleic acid fragment of Example 1 for transforming ura4+S. pombe cells.
FIG. 2 is a schematic representation of the construction of the "5'-half ura4-adh promoter- cdc25B-3'-half ura4" nucleic acid fragment of Example 2 for transforming ura4+S. pombe cells.
FIG. 4 is a schematic representation of the construction of the "5'-half ura4-adh promoter- cdc25C-3'-half ura4" nucleic acid fragment of Example 3 for transforming ura4+S. pombe cells.
FIG. 5A and 5B are photographs of yeast colonies formed by S. pombe cells transformed with pART3 plasmid, grown at 25� C. and 37� C. respectively.
Genetic studies in eukaryotic systems, including mammalian and fungi, have identified several genes that are important for the proper timing of mitosis. For instance, in the fission yeast S. pombe, genes encoding regulators of cell division have been extensively characterized (for review see MacNeil et al. (1989) Curr. Genet. 16:1). As set out above, initiation of mitosis in fission yeast correlates with activation of the cdc2 protein kinase. cdc2 is a component of M phase promoting factor (MPF) purified from frogs and starfish, and homologs of cdc2 have been identified in a wide range of eukaryotes, suggesting that cdc2 plays a central role in mitotic control in all eukaryotic cells (Norbury et al. (1989) Biochem. Biophys. Acta 989:85). For purposes of the present disclosure, the term "cdc2" or "cdc protein kinase" is used synonymously with the recently adopted "cell division kinase" (cdk) nomenclature. Furthermore as used herein, the term cdc2 is understood to denote members of the cell division kinase family. Representative examples of cdc protein kinases include cdc 2-SP, cdc28 (S. Cerevisiae), cdk2-XL, cdc2-HS and cdk2-HS, where "HS" designates homosapiens, SP designates S. pombe, and "XL" designates Xenopus Laevis. As set out above, the switch that controls the transition between the inactive cdc2/cyclin B complex (phosphorylated on Tyr-15 and Thr-14) present during S-G2-prophase and the active form of the cdc2/cyclin B kinase (dephosphorylated on Tyr-15 and Thr-14) present at metaphase is believed to correspond to a change in the relative activities of the opposing kinases and phosphatase(s) that act on the sites. Given that many regulatory pathways appear to converge on cdc protein kinases, as well as their activating role at both G1/S and G2/M transitions, the hyper-mitotic cell of the present assay can be employed to develop inhibitors specific for particular cdc protein kinases.
Loss-of-function strains, such as wee1-50, mik1::ura, or stf1-1 (Rowley et al. (1992) Nature 356:353), are well known. In addition, each of the wee1, mik1, and nim1 genes have been cloned (see for example Coleman et al. (1993) Cell 72:919; and Feilotter et al. (1991) Genetics 127:309), such that disruption of wee1 and/or mik1 expression or over-expression of nim1 can be carried out to create the hyper-mitotic cell of the present assay. In a similar fashion, over-expression of wee1 and/or mik1 or disruption of nim1 expression can be utilized to generate the hypo-mitotic cell of the present assay. Furthermore, each of these negative mitotic regulators can also be a potential target for an anti-mitotic agent scored for using the hypo-mitotic cell of the present assay.
Acting antagonistically to the wee1/mik1 kinases, genetic and biochemical studies have indicated that the cdc25 protein is a central player in the process of cdc2-specific dephosphorylation and crucial to the activation of the cdc2 kinase activity. In the absence of cdc25, cdc2 accumulates in a tyrosine phosphorylated state and can cause inhibition of mitosis. The phosphatase activity of cdc25 performs as a mitotic activator and is therefore a suitable target for inhibition by an anti-mitotic agent in the present assay. It is strongly believed that this aspect of the mitotic control network is generally conserved among eukaryotes, though the particular mode of regulation of cdc25 activity may vary somewhat from species to species. Homologs of the fission yeast cdc25 have been identified in the budding yeast S. cerevisiae (Millar et al. (1991) CSH Symp. Quant. Biol. 56:577), humans (Galaktinov et al. (1990) Cell 67:1181; and Sadhu et al. (1989) PNAS 87:5139), mouse (Kakizuka et al. (1992) Genes Dev. 6:578), Drosophila (Edgar et al. (1989) Cell 57:177; and Glover (1991) Trends Genet. 7:125), and Xenopus (Kumagai et al., (1992) Cell 70:139; and Jessus et al. (1992) Cell 68:323). Human cdc25 is encoded by a multi-gene family now consisting of at least three members, namely cdc25A, cdc25B and cdc25C. As described below, all three homologs are able to rescue temperature-sensitive mutations of the S. Pombe cdc25. Early evidence suggests that these different homologs may have different functions. For instance, microinjection of anti-cdc25-C antibodies into mammalian cells prevents them from dividing. They appear to arrest in interphase with a flattened morphology, consistent with a role for cdc25C in the entry into mitosis. On the contrary, microinjection of antibodies to cdc25A results in a rounded-up mitotic-like state, suggesting that the different homologs may have distinct functions and represent an additional level of complexity to the control of M-phase onset by cdc25 in higher eukaryotes. Comparison of the human cdc 25's with each other and with cdc25 homologs from other species has been carried out. Comparison of cdc25A with cdc25C demonstrates a 48% identity in the 273 C-terminal region between the two proteins; and comparison between cdc25B and cdc25C reveals a 43% identify. The Drosophila cdc25 homolog "string" shares 34.5% identity to cdc25A in a 362 amino acid region and 43.9% in an 269 amino acid region with cdc25B. S. Pombe cdc25 is also related to the human cdc25's, but to a lesser extent. Interestingly, the overall similarity between different human cdc25 proteins does not greatly exceed that between humans and such evolutionary distinct species as Drosophila. Biochemical experiments have demonstrated that bacterially produced cdc25 protein from Drosophila and human activates the histone H1 kinase activity of cdc2 in Xenopus or starfish extracts (Kumagai et al. (1991 ) Cell 64:903; and Strausfield et al. (1991) Nature 351:242).
If the cdc25 phosphatase activity is the desired target for development of an anti-mitotic agent, it may be advantageous to choose the hyper-mitotic cell of the present assay so as to more particularly select for anti-mitotic agents which act directly or indirectly on cdc25. As set out above, it will generally be expected that in order to score for an anti-mitotic agent in an assay relying on a hyper-mitotic cell, the inhibited mitotic activator (e.g. cdc25) must be sufficiently connected to the abherent checkpoint so as to rescue the cell before it concludes in mitotic catastrophe. Furthermore, the hyper-mitotic cell of the present assay can be generated by manipulation of the cell in which a cdc25 homolog is endogenously expressed, as for example, by generating a wee1 mutation (a "wee" phenotype), or by exposure of the cell to 2-aminopurine or caffeine after a γ-radiation induced G2 arrest. Alternatively, the cdc25 gene from one species or cell type can be cloned and subsequently expressed in a cell to which it is not endogenous but in which it is known to rescue lack-of-function mutations of the endogenous cdc25 activity. For example, the exogenous cdc25, such as a human cdc25, could be expressed in an hyper-mitotic Schizosaccharomyces cell, such as an S. pombe cell like the temperature-sensitive wee1-50 mutant. It may be possible to take advantage of the structural and functional differences between the human cdc25 phosphatases to provide anti-mitotic agents which selectively inhibit particular human cell types. In a similar manner, it may be feasible to develop cdc25 phosphatase inhibitors with the present assay which act specifically on pathogens, such as fungus involved in mycotic infections, without substantially inhibiting the human homologs.
Other checkpoints which could be impaired to generate the hyper-mitotic and hypo-mitotic systems have been identified by examination of mitotic events in cells treated in a manner which disrupts DNA synthesis or DNA repair. Radiation-induced arrest is one example of a checkpoint mechanism which has been used to identify both negative and positive regulators of mitosis. In this instance, mitosis is delayed until the integrity of the genome is checked and, as far as possible, restored. Checkpoint controls also function to delay mitosis until DNA synthesis is complete. The observation of cell-cycle arrest points indicate that the regulation of progression into mitosis in response to both DNA damage and the DNA synthesis requires components of the mitotic control. For example, analysis of radiation-sensitive mutations in budding yeast have identified a number of defective regulatory proteins which can prevent the arrest of the cell-cycle in response to DNA damage and are therefore potential candidates for impairment to generate the hyper-mitotic or hypo-mitotic cell of the present assay. By way of illustration, a number of genes involved in this mitotic feedback control have been identified, and include the rad9, rad17, rad24, mec1, mec 2 and mec3 genes (Weinert et al. (1988) Science 241:317). All six genes have been shown to be negative regulators of cell-cycle progression and act in response to damaged DNA. Two genes, mec 1 and mec2, are also involved in arresting the cell-cycle in response to unreplicated DNA.
The response to DNA damage has also been investigated in the fission yeast S. pombe. Mutations in a number of genes have been identified which allow cells with damaged or unreplicated DNA to enter mitosis. For example, the HUS 12 and HUS 16 genes have been implicated as negative regulators of mitosis which respond to unreplicated DNA, while RAD21 is a negative regulator sensitive to damaged DNA. The HUS14, HUS17, HUS22, HUS26, RAD 1, RAD3, RAD9 and RAD 17 genes of S. Pombe each appear to be negative regulators of mitosis which are able to respond to either unreplicated or damaged DNA. (Rowley et al. (1992) EMBO 11:1343; and Enoch et al (1991) CSH Symp. Quant. Biol. 56:409)
Recently, mutations in the S. cerevisiae genes BUB and MAD have been isolated which fail to arrest in mitosis with microtubule-destabilizing drugs. (Hayt et al. (1991 ) Cell 66:507; and Li et al. (1991) Cell 66:519). The S. cerevisiae cell can also be affected by a number of environmental cues. One such effector is the a-mating factor which induces G1 arrest. Mutants in the FUS3 or FAR1 genes fail to arrest in G1 in response to α-factor. While mutations in either gene are phenotypically similar, they affect different regulatory pathways. For example, the FUS3 gene has been cloned and exhibits strong sequence similarity to the serine/threonine family of protein kinases (Goebl et al. (1991 ) Curr. Opin. Cell Biol. 3:242).
In the fungus Asperqillus nidulans, the bimE gene is believed to code for a negative regulator of mitosis that normally functions to prevent mitosis by controlling expression of a putative mitotic inducer, nimA. The absence of bimE function is believed to override cell-cycle control systems normally operative to prevent chromosome condensation and spindle formation from occurring during interphase. Temperature sensitive mutants of the bimE gene, such as the bimE7 mutant, allow cells with unreplicated DNA to prematurely enter mitosis (Osmani et al. (1988) Cell 52:241) and can be lethal phenotypes useful as hyper-mitotic cells of the present assay.
Many of the regulatory proteins involved in the progression of a cell through meiosis have also been identified. Because of the commonalty of certain mitotic and meiotic pathways, several mitotic regulatory proteins or their homologs, such as cdc protein kinases, cyclins, and cdc25 homologs, also serve to regulate meiosis. For example, cell division cycle mutants defective in certain mitotic cell-cycle events have been tested for sporulation at semi-restrictive temperatures (Gralbert et al. (1991) Curr Genet 20:199). The mitotic defective routants cdc10-129, cdc20-M10, cdc21-M6B, cdc23-M36 and cdc24-M38 formed four-spored asci but with low efficiency. Mutants defective in the mitotic initiation genes cdc2, cdc25 and cdc13 were blocked at meiosis II, though none of the wee1-50, ddh. nim1+ and win1+ alleles had any affect on sporulation, suggesting that their interactions with cdc25 and cdc2 are specific to mitosis in yeast. Other regulatory genes and gene products which can be manipulated to form the hyper- or hypo-meiotic cells of the present invention include rec102, spo13, cut1, cut2, IME1, MAT, RME1, cdc35, BCY1, TPK1, TPK2, TPK3, spd1, spd3, spd4, spo50, spo51, and spo53. As above, the hyper- or hypo-meiotic cells can be generated genetically or chemically using cells to which the intended target of the anti-meiotic agent is endogenous, or alternatively, using cells in which the intended target is exogenously expressed.
In addition, certain meiotic regulatory proteins are able to rescue loss-of-function mutations in the mitotic cell-cycle. For example, the Drosophila meiotic. cdc25 homolog, "twine", is able to rescue mitosis in temperature-sensitive cdc25 mutants of fission yeast. Thus, anti-meiotic agents can be identified using hyper- or hypo-meiotic cells, and in some instances, hyper- or hypo-mitotic cells.
It is also deemed to be within the scope of this invention that the hyper- and hypo-proliferative cells of the present assay, whether for identifying anti-mitotic or anti-meiotic agents, can be generated so as to comprise heterologous cell-cycle proteins (i.e. cross-species expression). As exemplified above in the instance of cdc25, cell-cycle proteins from one species can be expressed in the cells of another and have been shown to be able to rescue loss-of-function mutations in the host cell. In addition to those cell-cycle proteins which are ideally to be the target of inhibition by the candidate agent, cell-cycle proteins which interact with the intended inhibitor target can also be expressed across species. For example, in an hyper-proliferative yeast cell in which a human cdc25 (e.g. exogenously expressed) is the intended target for development of an anti-mitotic agent, a human cdc protein kinase and human cyclin can also be expressed in the yeast cell. Likewise, when a hypo-proliferative yeast expressing human wee1 is used, a human cdc protein kinase and human cyclin with which the human cdc25 would interact can be used to replace the corresponding yeast cell-cycle proteins. To illustrate, a triple cln deletion mutant of S. Cerevisae which is also conditionally deficient in cdc28 (the budding yeast equivalent of cdc2) can be rescued by the co-expression of a human cyclin and human cdc2 proteins, demonstrating that yeast cell-cycle machinery can be at least in part replaced with corresponding human regulatory proteins. Roberts et al. (1993) PCT Publication Number WO 93/06123. In this manner, the reagent cells of the present assay can be generated to more closely approximate the natural interactions which a particular cell-cycle protein might experience.
Manipulation of these regulatory pathways with certain drugs, termed here "hyper-mitotic agents", can induce mitotic aberrations and result in generation of the hyper-mitotic cell of the present assay. For instance, caffeine, the protein kinase inhibitors 2-aminopurine and 6-dimethylaminopurine, and the protein phosphatase inhibitor okadaic acid can cause cells that are arrested in S phase by DNA synthesis inhibitors to inappropriately enter mitosis (Schlegel et al. (1986) Science 232:1264; Schlegel et al. (1987) PNAS 84:9025; and Schlegel et al. (1990) Cell Growth Differ. 1:171). Further, 2-aminopurine is believed to be able to override a number of cell-cycle checkpoints from G 1, S phase, G2, or mitosis. (Andreassen et al. (1992) PNAS 89:2272; Andreassen et al. (1991) J. Cell Sci. 100:299, and Steinmann et al. (1991) PNAS 88:6843). For example, 2-aminopurine permits cells to overcome a G2/M block induced by γ-irradiation. Additionally, cells continuously exposed to 2-aminopurine alone are able to exit S phase without completion of replication, and exit mitosis without metaphase, anaphase, or telophase events.
To aid in the facilitation of mitotic catastrophe in the hyper-mitotic cell it may be desirable to expose the cell to an agent (i.e. a chemical or environmental stimulus) which ordinarily induces cell-cycle arrest at that checkpoint. Inappropriate exit from the chemically- or environmentally-induced arrested state due to the impairment of the negative regulatory checkpoint can ultimately be lethal to the cell. Such arresting agents can include exposure to DNA damaging radiation or DNA damaging agents; inhibition of DNA synthesis and repair using DNA polymerase inhibitors such as hydroxyurea or aphidicolin; topoisomerase inhibitors such as 4'-dimethly-epipodophyllotoxin (VM-26); or agents which interfere with microtubule-assembly, such as Nocadazole and taxol. By way of example, BHK and HeLa cells which receive 250 rads of γ radiation have been shown to undergo G2 arrest that was reversed without further treatment within 4-5 hours. However, in the presence of either caffeine, 2-aminopurine, or 6-dimethyl-aminopurine, this mitotic delay was suppressed in both the hamster and human cells, and allowed the cells undergo mitosis before DNA repair had been completed (Steinmann et al. (1991) PNAS 88:6843). Additionally, in certain cells, nutritional status of the cell, as well as mating factors, can cause arrest of the normal cell during mitosis.
The present assay can be used to develop inhibitors of fungal infections. The most common fungal infections are superficial and are presently treated with one of several topical drugs or with the oral drugs ketoconazole or griseofulvin. The systemic mycoses constitute quite a different therapeutic problem. These infections are often very difficult to treat and long-term, parenteral therapy with potentially toxic drugs may be required. The systemic mycoses are sometimes considered in two groups according to the infecting organism. The "opportunistic infections" refer to those mycoses--candidiasis, aspergillosis, cryptococcosis, and phycomycosis--that commonly occur in debilitated and immunosuppressed patients. These infections are a particular problem in patients with leukemias and lymphomas, in people who are receiving immunosuppressive therapy, and in patients with such predisposing factors as diabetes mellitus or AIDS. Other systemic mycoses--for example, blastomycosis, histoplasmosis, coccidiodomycosis, and sporotrichosis--tend to have a relatively low incidence that may vary considerably according to geographical area.
To develop an assay for anti-mitotic or anti-meiotic agents having potential therapeutic value in the treatment of a certain mycotic infection, a yeast implicated in the infection can be used to generate the appropriate reagent cell of the present assay. For example, the hyper-mitotic or hypo-mitotic cell can be generated biochemically as described above, or engineered, as for example, by screening for radiation-sensitive mutants having impaired checkpoints. Additionally, a putative mitotic regulator of the mycotic yeast, such as a cdc25 homolog, can be cloned and expressed in a heterologous cell which may be easier to manipulate or facilitate easier measurement .of proliferation, such as member of the Schizosaccharomyces genus like S. pombe.
By way of illustration, the present assays can be used to screen for anti-mitotic and anti-meiotic agents able to inhibit at least one fungus implicated in such mycosis as candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis, nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, or sporotrichosis. For example, if the mycotic infection to which treatment is desired is candidiasis, the present assay can comprise either a hyper-mitotic or hypo-mitotic cells generated directly from, or with genes cloned from, yeast selected from the group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, and Candida rugosa. Likewise, the present assay can be used to identify anti-mitotic and anti-meiotic agents which may have therapeutic value in the treatment of aspergillosis by making use of yeast such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus. Where the mycotic infection is mucormycosis, the yeast can be selected from a group consisting of Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus. Another pathogen which can be utilized in the present assay is Pneumocystis carinii.
Agents to be tested for their ability to act as anti-mitotic and/or anti-meiotic agents in the present assay can be those produced by bacteria, yeast or other organisms, or those produced chemically. The assay can be carried out in any vessel suitable for the growth of the cell, such as microtitre plates or petri dishes. As potent inhibitors mitosis and/or meiosis can fully inhibit proliferation of a cell, it may be useful to perform the assay at various concentrations of the candidate agent. For example, serial dilutions of the candidate agents can be added to the hyper-mitotic cell such that at at least one concentration tested the anti-mitotic agent inhibits the mitotic activator to an extent necessary to adequately'slow the progression of the cell through the cell-cycle but not to the extent necessary to inhibit entry into mitosis all together. In a like manner, where the assay comprises a hypo-mitotic cell, serial dilutions of a candidate agent can be added to the cells such that, at at least one concentration, an anti-mitotic agent inhibits a negative mitotic regulator to an extent necessary to adequately enhance progression of the cell through the cell-cycle, but not to an extent which would cause mitotic catastrophe.
To test compounds that might specifically inhibit the human cdc25A, cdc25B or cdc25C gene products, the genes were introduced into the genome of an S. pombe strain which was engineered to be conditionally hyper-mitotic. Three linear DNA fragments were constructed, each carrying one of the three human cdc25A, B or C genes under the control of an S. pombe promoter, and flanked by nucleic acid sequences which allow integration of the DNA into the S. pombe genome. The cdc25-containing DNA fragments are then used to transform an appropriate S. pombe strain. For example, in one embodiment, the expression of the human cdc25 gene is driven by the strong adh promoter and the flanking sequences of the fragment contain the ura4 gene to allow integration of the fragment at the ura4 locus by homologous recombination (Grimm et al. (1988) Molec. gen. Genet 81-86). The S. pombe strain is a wee1 temperature-sensitive mutant which becomes hyper-mitotic at temperatures above 36 � C., and carries a wild-type ura4 gene in which the cdc25 DNA fragment can be integrated.
EXAMPLE 1 The human cdc25A gene has been previously cloned (see Galaktinov et al. (1991) Cell 67:1181). The sequence of the cdc25A gene containing the open reading frame is shown in Seq. ID No. 1, and is predicted to encode a protein of 523 amino acids (Seq. ID No. 2). A 2.0 kb Ncol-KpnI fragment encoding amino acids 1-523 of human cdc25A was subcloned into a NcoI-KpnI-(partially) digested pARTN expression vector, resulting in the pARTN-cdc25A construct harboring human cdc25A cDNA in sense orientation to the constitutive adh promoter. The S. Pombe autonomously replicating pARTN vector is derived from pART3 (McLeod et al. (1987) EMBO 6:729) by ligation of a NcoI linker (New England Biolabs) into the SmaI site.
A 2.3 kb DNA fragment corresponding to the adh promoter and amino acids 1-523 of the human cdc25A gene, was isolated by digesting the pARTN-cdc25A plasmid with HindIII and Asp718. While HindIII is sufficient to isolate the adh promoter/human cdc25A gene fragment from the plasmid, we also used Asp718 to cut the close migrating 2.2 kb HindIII-HindIll S. cerevisiae LEU2 gene in two smaller fragments which makes isolation of the cdc25A fragment easier.
The HindIII/HindIII fragment was then blunt ended with Klenow enzyme and dNTPs (see Molecular Cloning: A Laboratory Manual 2ed, eds. Sambrook et al., CSH Laboratory Press: 1989) and ligated into a pKS-/ura4 plasmid previously digested with StuI and dephosphorylated with alkaline phosphatase. Massive amounts of the recombinant plasmid were prepared, and a 4.1 kb DNA fragment corresponding to "5'-half ura4-adh promoter-cdc 25A-3'-half ura4" (see FIG. 1 ) was isolated.
EXAMPLE 2 The human cdc25B gene has been previously cloned (see Galaktinov et al. (1991 ) Cell 67:1181 ). The sequence of the cdc25B gene containing the open reading frame is shown in Seq. ID. No. 3, and is predicted to encode a protein of 566 amino acids (Seq. ID No. 4). A 2.4 kb SmaI fragment from the p4x1.2 plasmid (Galaktinov et al., supra) encoding amino acids 32-566 was subcloned into a SmaI-digested pART3 vector, resulting in the pARTN-cdc25B vector containing the human cdc25B cDNA. While the site of initiation of translation is not clear (there is no exogenous ATG 5' to the SmaI cloning site in the cdc25B open reading frame) we speculate that the first ATG corresponds to the Met-59 of the human cdc25B open reading frame, or alternatively, an ATG at an NdeI site of pART3. In any event, the pARTN-cdc25B plasmid has been shown to be capable of transforming S. pombe cells and able to rescue temperature-sensitive mutations of the yeast cdc25 gene (Galaktinov et at., supra).
As above, a 2.7 kb DNA fragment, corresponding to the adh promoter and amino acids 32-566 of the human cdc25B gene, was isolated by digesting pARTN-cdc25B with HindIII and Asp718. The HindIII/HindIII cdc25B fragment was blunt ended with Klenow enzyme and dNTPs, and ligated into a pKS-/ura4 vector previously digested with StuI and dephosphorylated with alkaline phosphatase. A 4.4 kb DNA fragment corresponding to "5'-half ura4-adh promoter-cdc-25B-3'-half ura4" (see FIG. 2) was isolated.
A 2.5 kbp fragment corresponding to the adh promoter and amino acids 1-473 of the human cdc25C gene was isolated by digesting pART3-cdc25C with HindIII and Asp718. The HindIII/HindIII cdc25C fragment was blunt ended with Klenow enzyme and dNTPs, and ligated into a pKS-/ura4 plasmid previously digested with StuI and dephosphorylated with alkaline phosphatase. A 4.3 kbp DNA fragment corresponding to "5'-half ura4-adh promoter-cdc25C-3'-half ura4" (see FIG. 4) was isolated.
EXAMPLE 4 Each of the cdc25 plasmid constructs pARTN-cdc25A, pARTN-cdc25B, and pART3-cdc25C, as well as the original pART3 plasmid, were used to transform the S. Pombe strain Sp553 (h+N, cdc25-22, wee1-50, leu1-32) using well known procedures. Briefly, cells were grown in YE medium at 25� C. until they were in exponential phase (�-107 cells/ml). The cells were then spun down from the media at 3000 rpm for 5 minutes, and resuspended in LiCl/TE at a concentration of �108 cells/ml (LiCl/TE=10 mM Tris, 1 mM EDTA, 50 mM LiCl, Ph 8). The resuspended cells were incubated at room temperature for 10 minutes, then spun again at 3000 rpm for 5 minutes, resuspended in LiCl/TE to a concentration of �5�108 cells/ml, and shaken for 30 minutes at 25� C.
To an aliquot of 150 μl of cells, 500 ng of plasmid DNA and 3501 μL of PEG/TE (10 mM Tris, 1 mM EDTA, 50% PEG 4000, Ph 8) was added. The cell/plasmid mixture was then incubated for 30 minutes at 25� C., heat shocked at 42� C. for 20 minutes, then spun at 15,000 rpm for 10 seconds after the addition of 0.5 mL of Edinburgh Minimal Medium (EMM). The cells were resuspended in 0.6 mL EMM, and 0.2 mL aliquots were plated.
FIGS. 5A and 5B illustrate the ability of the pART3 transformed yeast to grow at 25� C. and 37� C. respectively. As set out above, at the non-permissive temperature of 37� C., both the endogenous wee1 and cdc25 activities are impaired such that they mutually off-set each other's effects, and the cells are still able to proliferate (pART3 lacks any cdc25 gene).
FIGS. 6A and 6B (cdc25A), 7A and 7B (cdc25B), and 8A and 8B (cdc25C) demonstrate the effect of expressing a human cdc25 in a yeast "wee" background. Each of FIGS. 6A, 7A and 8A show that at the permissive temperature of 25� C. (wee1 is expressed) the cells are able to proliferate. However, as illustrated by FIGS. 6B, 7B and 8B, shifting the temperature to the non-permissive temperature of 37� C. results in mitotic catastrophe. Microscopic analysis of the yeast cells present on the 37� C. plates revealed that the expression of a human cdc25 in a yeast wee background resulted in mitotic catastrophe for the cells.
EXAMPLE 5 To provide a more stable transformant and uniform expression of the human cdc25 gene, each of the resulting ura4-cdc25 fragments of Examples 1-3 was used to transform a ura4+S. pombe strain. As in Example 4, each of the S. pombe strain carried a thermosensitive allele of its own cdc25 gene, such as the cdc25-22 phenotype, so that at non-permissive temperatures the exogenous cdc25 is principally responsible for activation of cdc2. In one embodiment, the S. Pombe wee1-50 cdc25-22 ura4+ strain was transformed with a ura4-cdc25 fragment of Examples 1-3. This particular strain is generally viable at 25� C. as well as the restrictive temperature of 37� C. as the loss of endogenous cdc25 activity is recovered by the concomitant loss of wee1 function at 37� C. However, integration and over expression of the human cdc25, as demonstrated in Example 4, can result in a mitotic catastrophic phenotype at 37� C. as the wee1 checkpoint is impaired.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 6(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2420 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 460..2031(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CGAAAGGCCGGCCTTGGCTGCGACAGCCTGGGTAAGAGGTGTAGGTCGGCTTGGTTTTCT60GCTACCCGGAGCTGGGCAAGCGGGTGGGGAGAACAGCGAAGACAGCGTGAGCCTGGGCCG120TTGCCTCGAGG CTCTCGCCCGGCTTCTCTTGCCGACCCGCCACGTTTGTTTGGATTTAAT180CTTACAGCTGGTTGCCGGCGCCCGCCCGCCCGCTGGCCTCGCGGTGTGAGAGGGAAGCAC240CCGTGCCTGTGTCTCGTGGCTGGCGCCTGGAGGGTCCGCACACCCGCGCGGCCGCGC CGC300TTTGCCCGCGGCAGCCGCGTCCCTGAACCGCGGAGTCGTGTTTGTGTTTGACCCGCGGGC360GCCGGTGGCGCGCGGCCGAGGCCGGTGTCGGCGGGGCGGGGCGGTCGCGCGGGAGGCAGA420GGAAGAGGGAGCGGGAGCTCTGCGAGGCCGGGCG CCGCCATGGAACTGGGCCCG474MetGluLeuGlyPro15AGCCCCGCACCGCGCCGCCTGCTCTT CGCCTGCAGCCCCCCTCCCGCG522SerProAlaProArgArgLeuLeuPheAlaCysSerProProProAla101520TCGCAGCCCGTCGTGAAGGCGCTAT TTGGCGCTTCAGCCGCCGGGGGA570SerGlnProValValLysAlaLeuPheGlyAlaSerAlaAlaGlyGly253035CTGTCGCCTGTCACCAACCTGACCGTC ACTATGGACCAGCTGCAGGGT618LeuSerProValThrAsnLeuThrValThrMetAspGlnLeuGlnGly404550CTGGGCAGTGATTATGAGCAACCACTGGAGGTG AAGAACAACAGTAAT666LeuGlySerAspTyrGluGlnProLeuGluValLysAsnAsnSerAsn556065CTGCAGATAATGGGCTCCTCCAGATCAACAGATTCAGGTTT CTGTCTA714LeuGlnIleMetGlySerSerArgSerThrAspSerGlyPheCysLeu70758085GATTCTCCTGGGCCATTGGACAGTAAAGAAAACCTTG AAAATCCTATG762AspSerProGlyProLeuAspSerLysGluAsnLeuGluAsnProMet9095100AGAAGAATACATTCCCTACCTCAAAAGCTGTTGGGA TGTAGTCCAGCT810ArgArgIleHisSerLeuProGlnLysLeuLeuGlyCysSerProAla105110115CTGAAGAGGAGCCATTCTGATTCTCTTGACCATGACATC TTTCAGCTC858LeuLysArgSerHisSerAspSerLeuAspHisAspIlePheGlnLeu120125130ATCGACCCAGATGAGAACAAGGAAAATGAAGCCTTTGAGTTTAA GAAG906IleAspProAspGluAsnLysGluAsnGluAlaPheGluPheLysLys135140145CCAGTAAGACCTGTATCTCGTGGCTGCCTGCACTCTCATGGACTCCAG 954ProValArgProValSerArgGlyCysLeuHisSerHisGlyLeuGln150155160165GAGGGTAAAGATCTCTTCACACAGAGGCAGAACTCTGCCCAGCTCGGA 1002GluGlyLysAspLeuPheThrGlnArgGlnAsnSerAlaGlnLeuGly170175180ATGCTTTCCTCAAATGAAAGAGATAGCAGTGAACCAGGGAATTTCATT 1050MetLeuSerSerAsnGluArgAspSerSerGluProGlyAsnPheIle185190195CCTCTTTTTACACCCCAGTCACCTGTGACAGCCACTTTGTCTGATGAG 1098ProLeuPheThrProGlnSerProValThrAlaThrLeuSerAspGlu200205210GATGATGGCTTCGTGGACCTTCTCGATGGAGACAATCTGAAGAATGAG1146 AspAspGlyPheValAspLeuLeuAspGlyAspAsnLeuLysAsnGlu215220225GAGGAGACCCCCTCGTGCATGGCAAGCCTCTGGACAGCTCCTCTCGTC1194GluGluTh rProSerCysMetAlaSerLeuTrpThrAlaProLeuVal230235240245ATGAGAACTACAAACCTTGACAACCGATGCAAGCTGTTTGACTCCCCT1242MetA rgThrThrAsnLeuAspAsnArgCysLysLeuPheAspSerPro250255260TCCCTGTGTAGCTCCAGCACTCGGTCAGTGTTGAAGAGACCAGAACGT1290Ser LeuCysSerSerSerThrArgSerValLeuLysArgProGluArg265270275TCTCAAGAGGAGTCTCCACCTGGAAGTACAAAGAGGAGGAAGAGCATG1338SerGln GluGluSerProProGlySerThrLysArgArgLysSerMet280285290TCTGGGGCCAGCCCCAAAGAGTCAACTAATCCAGAGAAGGCCCATGAG1386SerGlyAlaSe rProLysGluSerThrAsnProGluLysAlaHisGlu295300305ACTCTTCATCAGTCTTTATCCCTGGCATCTTCCCCCAAAGGAACCATT1434ThrLeuHisGlnSerLeuS erLeuAlaSerSerProLysGlyThrIle310315320325GAGAACATTTTGGACAATGACCCAAGGGACCTTATAGGAGACTTCTCC1482GluAsnIleLeuAsp AsnAspProArgAspLeuIleGlyAspPheSer330335340AAGGGTTATCTCTTTCATACAGTTGCTGGGAAACATCAGGATTTAAAA1530LysGlyTyrLeuPhe HisThrValAlaGlyLysHisGlnAspLeuLys345350355TACATCTCTCCAGAAATTATGGCATCTGTTTTGAATGGCAAGTTTGCC1578TyrIleSerProGluIl eMetAlaSerValLeuAsnGlyLysPheAla360365370AACCTCATTAAAGAGTTTGTTATCATCGACTGTCGATACCCATATGAA1626AsnLeuIleLysGluPheValI leIleAspCysArgTyrProTyrGlu375380385TACGAGGGAGGCCACATCAAGGGTGCAGTGAACTTGCACATGGAAGAA1674TyrGluGlyGlyHisIleLysGlyAlaVal AsnLeuHisMetGluGlu390395400405GAGGTTGAAGACTTCTTATTGAAGAAGCCCATTGTACCTACTGATGGC1722GluValGluAspPheLeuLeuLysLys ProIleValProThrAspGly410415420AAGCGTGTCATTGTTGTGTTTCACTGCGAGTTTTCTTCTGAGAGAGGT1770LysArgValIleValValPheHisCy sGluPheSerSerGluArgGly425430435CCCCGCATGTGCCGGTATGTGAGAGAGAGAGATCGCCTGGGTAATGAA1818ProArgMetCysArgTyrValArgGluA rgAspArgLeuGlyAsnGlu440445450TACCCCAAACTCCACTACCCTGAGCTGTATGTCCTGAAGGGGGGATAC1866TyrProLysLeuHisTyrProGluLeuTyrVal LeuLysGlyGlyTyr455460465AAGGAGTTCTTTATGAAATGCCAGTCTTACTGTGAGCCCCCTAGCTAC1914LysGluPhePheMetLysCysGlnSerTyrCysGluProPro SerTyr470475480485CGGCCCATGCACCACGAGGACTTTAAAGAAGACCTGAAGAAGTTCCGC1962ArgProMetHisHisGluAspPheLysGluAspLeuLy sLysPheArg490495500ACCAAGAGCCGGACCTGGGCAGGGGAGAAGAGCAAGAGGGAGATCTAC2010ThrLysSerArgThrTrpAlaGlyGluLysSerLysA rgGluIleTyr505510515AGTCGTCTGAAGAAGCTCTGAGGGCGGCAGGACCAGCCAGCAGCAGCC2058SerArgLeuLysLysLeu520CAAGCTTCCCTC CATCCCCCTTTACCCTCTTTCCTGCAGAGAAACTTAAGCAAAGGGGAC2118AGCTGTGTGACATTTGGAGAGGGGGCCTGGGACTTCCATGCCTTAAACCTACCTCCCACA2178CTCCCAAGGTTGGAGACCCAGGCCATCTTGCTGGCTACGCCTCTTCTGTCCCTGTTAG AC2238GTCCTCCGTCCATTACAGAACTGTGCCACAATGCAGTTCTGAGCACCGTGTCAAGCTGCT2298CTGAGCCACAGTGGGATGAACCAGCCGGGGCCTTATCGGGCTCCAGCATCTCATGAGGGG2358AGAGGAGACGGAGGGGACTAGAGAAGTTTACACAG AAATGCTGCTGGCCAAATAGCAAAG2418AG2420(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 523 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetGluLeuGlyProSerProAlaProArgArgLeuLeuPheAlaCys151015SerProProProAlaSerGlnProValValLysAlaLeu PheGlyAla202530SerAlaAlaGlyGlyLeuSerProValThrAsnLeuThrValThrMet354045AspGlnLe uGlnGlyLeuGlySerAspTyrGluGlnProLeuGluVal505560LysAsnAsnSerAsnLeuGlnIleMetGlySerSerArgSerThrAsp6570 7580SerGlyPheCysLeuAspSerProGlyProLeuAspSerLysGluAsn859095LeuGluAsnProMetArgArgIleHisS erLeuProGlnLysLeuLeu100105110GlyCysSerProAlaLeuLysArgSerHisSerAspSerLeuAspHis1151201 25AspIlePheGlnLeuIleAspProAspGluAsnLysGluAsnGluAla130135140PheGluPheLysLysProValArgProValSerArgGlyCysLeuHis145 150155160SerHisGlyLeuGlnGluGlyLysAspLeuPheThrGlnArgGlnAsn165170175SerAlaGlnLeuGlyMe tLeuSerSerAsnGluArgAspSerSerGlu180185190ProGlyAsnPheIleProLeuPheThrProGlnSerProValThrAla195200 205ThrLeuSerAspGluAspAspGlyPheValAspLeuLeuAspGlyAsp210215220AsnLeuLysAsnGluGluGluThrProSerCysMetAlaSerLeuTrp 225230235240ThrAlaProLeuValMetArgThrThrAsnLeuAspAsnArgCysLys245250255LeuPhe AspSerProSerLeuCysSerSerSerThrArgSerValLeu260265270LysArgProGluArgSerGlnGluGluSerProProGlySerThrLys275 280285ArgArgLysSerMetSerGlyAlaSerProLysGluSerThrAsnPro290295300GluLysAlaHisGluThrLeuHisGlnSerLeuSerLe uAlaSerSer305310315320ProLysGlyThrIleGluAsnIleLeuAspAsnAspProArgAspLeu325330 335IleGlyAspPheSerLysGlyTyrLeuPheHisThrValAlaGlyLys340345350HisGlnAspLeuLysTyrIleSerProGluIleMetAlaSerValLeu 355360365AsnGlyLysPheAlaAsnLeuIleLysGluPheValIleIleAspCys370375380ArgTyrProTyrGluTyrGluGlyGly HisIleLysGlyAlaValAsn385390395400LeuHisMetGluGluGluValGluAspPheLeuLeuLysLysProIle405410 415ValProThrAspGlyLysArgValIleValValPheHisCysGluPhe420425430SerSerGluArgGlyProArgMetCysArgTyrValArgGl uArgAsp435440445ArgLeuGlyAsnGluTyrProLysLeuHisTyrProGluLeuTyrVal450455460LeuLysGlyGlyTyr LysGluPhePheMetLysCysGlnSerTyrCys465470475480GluProProSerTyrArgProMetHisHisGluAspPheLysGluAsp485 490495LeuLysLysPheArgThrLysSerArgThrTrpAlaGlyGluLysSer500505510LysArgGluIleTyrSerArgLeuLysLys Leu515520(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2886 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 73..1773 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:CTGCCCTGCGCCCCGCCCTCCAGCCAGCCTGCCAGCTGTGCCGGCGTTTGTTGGTCTGCC60GGCCCCGCCGCGATGGAGGTGCCCCAGCCGGAGCCCGCGCCAGGCTCG108MetGluValProGlnPro GluProAlaProGlySer1510GCTCTCAGTCCAGCAGGCGTGTGCGGTGGCGCCCAGCGTCCGGGCCAC156AlaLeuSerProAlaGlyValCysGlyGlyAlaG lnArgProGlyHis152025CTCCCGGGCCTCCTGCTGGGATCTCATGGCCTCCTGGGGTCCCCGGTG204LeuProGlyLeuLeuLeuGlySerHisGlyLeuLeuGly SerProVal303540CGGGCGGCCGCTTCCTCGCCGGTCACCACCCTCACCCAGACCATGCAC252ArgAlaAlaAlaSerSerProValThrThrLeuThrGlnThrMetHis45505560GACCTCGCCGGGCTCGGCAGCCGCAGCCGCCTGACGCACCTATCCCTG300AspLeuAlaGlyLeuGlySerArgSerArgLeuThrHisLeuSe rLeu657075TCTCGACGGGCATCCGAATCCTCCCTGTCGTCTGAATCCTCCGAATCT348SerArgArgAlaSerGluSerSerLeuSerSerGluSerSerG luSer808590TCTGATGCAGCTCTCTGCATGGATTCCCCCAGCCCTCTGGACCCCCAC396SerAspAlaAlaLeuCysMetAspSerProSerProLeuAspPro His95100105ATGGCGGAGCAGACGTTTGAACAGGCCATCCAGGCAGCCAGCCGGATC444MetAlaGluGlnThrPheGluGlnAlaIleGlnAlaAlaSerArgIle 110115120ATTCGAAACGAGCAGTTTGCCATCAGACGCTTCCAGTCTATGCCGGTG492IleArgAsnGluGlnPheAlaIleArgArgPheGlnSerMetProVal125 130135140AGGCTGCTGGGCCACAGCCCCGTGCTTCGGAACATCACCAACTCCCAG540ArgLeuLeuGlyHisSerProValLeuArgAsnIleThrAsnSerGln 145150155GCGCCCGACGGCCGGAGGAAGAGCGAGGCGGGCAGTGGAGCTGCCAGC588AlaProAspGlyArgArgLysSerGluAlaGlySerGlyAlaAlaSer 160165170AGCTCTGGGGAAGACAAGGAGAATGATGGATTTGTCTTCAAGATGCCA636SerSerGlyGluAspLysGluAsnAspGlyPheValPheLysMetPro17 5180185TGGAACCCCACACATCCCAGCTCCACCCATGCTCTGGCAGAGTGGGCC684TrpAsnProThrHisProSerSerThrHisAlaLeuAlaGluTrpAla190 195200AGCCGCAGGGAAGCCTTTGCCCAGAGACCCAGCTCGGCCCCCGACCTG732SerArgArgGluAlaPheAlaGlnArgProSerSerAlaProAspLeu205210 215220ATGTGTCTCAGTCCTGACCCGAAGATGGAATTGGAGGAGCTCAGCCCC780MetCysLeuSerProAspProLysMetGluLeuGluGluLeuSerPro225 230235CTGGCCCTAGGTCGCTTCTCTCTGACCCCTGCAGAGGGGGATACTGAG828LeuAlaLeuGlyArgPheSerLeuThrProAlaGluGlyAspThrGlu240 245250GAAGATGATGGATTTGTGGACATCCTAGAGAGTGACTTAAAGGATGAT876GluAspAspGlyPheValAspIleLeuGluSerAspLeuLysAspAsp255 260265GATGCAGTTCCCCCAGGCATGGAGAGTCTCATTAGTGCCCCACTGGTC924AspAlaValProProGlyMetGluSerLeuIleSerAlaProLeuVal270275 280AAGACCTTGGAAAAGGAAGAGGAAAAGGACCTCGTCATGTACAGCAAG972LysThrLeuGluLysGluGluGluLysAspLeuValMetTyrSerLys285290295 300TGCCAGCGGCTCTTCCGCTCTCCGTCCATGCCCTGCAGCGTGATCCGG1020CysGlnArgLeuPheArgSerProSerMetProCysSerValIleArg30531 0315CCCATCCTCAAGAGGCTGGAGCGGCCCCAGGACAGGGACACGCCCGTG1068ProIleLeuLysArgLeuGluArgProGlnAspArgAspThrProVal320325 330CAGAATAAGCGGAGGCGGAGCGTGACCCCTCCTGAGGAGCAGCAGGAG1116GlnAsnLysArgArgArgSerValThrProProGluGluGlnGlnGlu335340 345GCTGAGGAACCTAAAGCCCGCGCTCTCCGCTCAAAATCACTGTGTCAC1164AlaGluGluProLysAlaArgAlaLeuArgSerLysSerLeuCysHis350355360GATGAGATCGAGAACCTCCTGGACAGTGACCACCGAGAGCTGATTGGA1212AspGluIleGluAsnLeuLeuAspSerAspHisArgGluLeuIleGly365370375 380GATTACTCTAAGGCCTTCCTCCTACAGACAGTAGACGGAAAGCACCAA1260AspTyrSerLysAlaPheLeuLeuGlnThrValAspGlyLysHisGln385390 395GACCTCAAGTACATCTCACCAGAAACGATGGTGGCCCTATTGACGGGC1308AspLeuLysTyrIleSerProGluThrMetValAlaLeuLeuThrGly400405 410AAGTTCAGCAACATCGTGGATAAGTTTGTGATTGTAGACTGCAGATAC1356LysPheSerAsnIleValAspLysPheValIleValAspCysArgTyr415420425C CCTATGAATATGAAGGCGGGCACATCAAGACTGCGGTGAACTTGCCC1404ProTyrGluTyrGluGlyGlyHisIleLysThrAlaValAsnLeuPro430435440CTGGAACGC GACGCCGAGAGCTTCCTACTGAAGAGCCCCATCGCGCCC1452LeuGluArgAspAlaGluSerPheLeuLeuLysSerProIleAlaPro445450455460TGTAGC CTGGACAAGAGAGTCATCCTCATTTTCCACTGTGAATTCTCA1500CysSerLeuAspLysArgValIleLeuIlePheHisCysGluPheSer465470475TCTGA GCGTGGGCCCCGCATGTGCCGTTTCATCAGGGAACGAGACCGT1548SerGluArgGlyProArgMetCysArgPheIleArgGluArgAspArg480485490GCTGTCA ACGACTACCCCAGCCTCTACTACCCTGAGATGTATATCCTG1596AlaValAsnAspTyrProSerLeuTyrTyrProGluMetTyrIleLeu495500505AAAGGCGGCTAC AAGGAGTTCTTCCCTCAGCACCCGAACTTCTGTGAA1644LysGlyGlyTyrLysGluPhePheProGlnHisProAsnPheCysGlu510515520CCCCAGGACTACCGGCCCATG AACCACGAGGCCTTCAAGGATGAGCTA1692ProGlnAspTyrArgProMetAsnHisGluAlaPheLysAspGluLeu525530535540AAGACCTTCCGCCTCAA GACTCGCAGCTGGGCTGGGGAGCGGAGCCGG1740LysThrPheArgLeuLysThrArgSerTrpAlaGlyGluArgSerArg545550555CGGGAGCTCTGTAGCC GGCTGCAGGACCAGTGAGGGGCCTGCGCCAGTCC1790ArgGluLeuCysSerArgLeuGlnAspGln560565TGCTACCTCCCTTGCCTTTCGAGGCCTGAAGCCAGCTGCCCTATGGGCCTGCCGGGCTGA 1850GGGCCTGCTGGAGGCCTCAGGTGCTGTCCATGGGAAAGATGGTGTGGTGTCCTGCCTGTC1910TGCCCCAGCCCAGATTCCCCTGTGTCATCCCATCATTTTCCATATCCTGGTGCCCCCCAC1970CCCTGGAAGAGCCCAGTCTGTTGAGTTAGTTAAGTTGGGT TAATACCAGCTTAAAGTCAG2030TATTTTGTGTCCTCCAGGAGCTTCTTGTTTCCTTGTTAGGGTTAACCCTTCATCTTCCTG2090TGTCCTGAAACGCTCCAGAGCTAAACTCCTTCCTGGCCTGAGAGTCAGCTCTCTGCCCTG2150TGTACTTCCCGGGCCAG GGCTGCCCCTAATCTCTGTAGGAACCGTGGTATGTCTGCCATG2210TTGCCCCTTTCTCTTTTCCCCTTTCCTGTCCCACCATACGAGCACCTCCAGCCTGAACAG2270AAGCTCTTACTCTTTCCTATTTCAGTGTTACCTGTGTGCTTGGTCTGTTTGACTTTACGC 2330CCATCTCAGGACACTTCCGTAGACTGTTTAGGTTCCCCTGTCAAATATCAGTTACCCACT2390CGGTCCCAGTTTTGTTGCCCCAGAAAGGGATGTTATTATCCTTGGGGGCTCCCAGGGCAA2450GGGTTAAGGCCTGAATCATGAGCCTGCTGGAAGCCCAGCC CCTACTGCTGTGAACCCTGG2510GGCCTGACTGCTCAGAACTTGCTGCTGTCTTGTTGCGGATGGATGGAAGGTTGGATGGAT2570GGGTGGATGGCCGTGGATGGCCGTGGATGCGCAGTGCCTTGCATACCCAAACCAGGTGGG2630AGCGTTTTGTTGAGCAT GACACCTGCAGCAGGAATATATGTGTGCCTATTTGTGTGGACA2690AAAATATTTACACTTAGGGTTTGGAGCTATTCAAGAAGAAATGTCACAGAAGCAGCTAAA2750CCAAGGACTGAGCACCCTCTGGATTCTGAATCTCAATATGGGGGCAGGGCTGTGCTTGAA 2810GGCCCTGCTGAGTCATCTGTTAGGGCCTTGGTTCAATAAAGCACTGAGCAAGTTGAGAAA2870AAAAAAAAAAAAAAAA2886(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 566 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:MetGluValProGlnProGluProAlaProGlySerAlaLeuSerPro151015AlaGly ValCysGlyGlyAlaGlnArgProGlyHisLeuProGlyLeu202530LeuLeuGlySerHisGlyLeuLeuGlySerProValArgAlaAlaAla35 4045SerSerProValThrThrLeuThrGlnThrMetHisAspLeuAlaGly505560LeuGlySerArgSerArgLeuThrHisLeuSerLeuSe rArgArgAla65707580SerGluSerSerLeuSerSerGluSerSerGluSerSerAspAlaAla8590 95LeuCysMetAspSerProSerProLeuAspProHisMetAlaGluGln100105110ThrPheGluGlnAlaIleGlnAlaAlaSerArgIleIleArgAsnGlu 115120125GlnPheAlaIleArgArgPheGlnSerMetProValArgLeuLeuGly130135140HisSerProValLeuArgAsnIleThr AsnSerGlnAlaProAspGly145150155160ArgArgLysSerGluAlaGlySerGlyAlaAlaSerSerSerGlyGlu165170 175AspLysGluAsnAspGlyPheValPheLysMetProTrpAsnProThr180185190HisProSerSerThrHisAlaLeuAlaGluTrpAlaSerAr gArgGlu195200205AlaPheAlaGlnArgProSerSerAlaProAspLeuMetCysLeuSer210215220ProAspProLysMet GluLeuGluGluLeuSerProLeuAlaLeuGly225230235240ArgPheSerLeuThrProAlaGluGlyAspThrGluGluAspAspGly245 250255PheValAspIleLeuGluSerAspLeuLysAspAspAspAlaValPro260265270ProGlyMetGluSerLeuIleSerAlaPro LeuValLysThrLeuGlu275280285LysGluGluGluLysAspLeuValMetTyrSerLysCysGlnArgLeu290295300PheA rgSerProSerMetProCysSerValIleArgProIleLeuLys305310315320ArgLeuGluArgProGlnAspArgAspThrProValGlnAsnLysArg 325330335ArgArgSerValThrProProGluGluGlnGlnGluAlaGluGluPro340345350LysAlaArgAlaLeuArg SerLysSerLeuCysHisAspGluIleGlu355360365AsnLeuLeuAspSerAspHisArgGluLeuIleGlyAspTyrSerLys370375 380AlaPheLeuLeuGlnThrValAspGlyLysHisGlnAspLeuLysTyr385390395400IleSerProGluThrMetValAlaLeuLeuThrGlyLysPheSer Asn405410415IleValAspLysPheValIleValAspCysArgTyrProTyrGluTyr420425430GluGlyG lyHisIleLysThrAlaValAsnLeuProLeuGluArgAsp435440445AlaGluSerPheLeuLeuLysSerProIleAlaProCysSerLeuAsp4504 55460LysArgValIleLeuIlePheHisCysGluPheSerSerGluArgGly465470475480ProArgMetCysArgPheIleArgGluArgAsp ArgAlaValAsnAsp485490495TyrProSerLeuTyrTyrProGluMetTyrIleLeuLysGlyGlyTyr500505 510LysGluPhePheProGlnHisProAsnPheCysGluProGlnAspTyr515520525ArgProMetAsnHisGluAlaPheLysAspGluLeuLysThrPheArg530 535540LeuLysThrArgSerTrpAlaGlyGluArgSerArgArgGluLeuCys545550555560SerArgLeuGlnAspGln 565(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2062 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 211..1631(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:CAGGAAGACT CTGAGTCCGACGTTGGCCTACCCAGTCGGAAGGCAGAGCTGCAATCTAGT60TAACTACCTCCTTTCCCCTAGATTTCCTTTCATTCTGCTCAAGTCTTCGCCTGTGTCCGA120TCCCTATCTACTTTCTCTCCTCTTGTAGCAAGCCTCAGACTCCAGGCTTGAGCTA GGTTT180TGTTTTTCTCCTGGTGAGAATTCGAAGACCATGTCTACGGAACTCTTCTCATCC234MetSerThrGluLeuPheSerSer1 5ACAAGAGAGGAAGGAAGCTCTGGCTCAGGACCCAGTTTTAGGTCTAAT282ThrArgGluGluGlySerSerGlySerGlyProSerPheArgSerAsn101520 CAAAGGAAAATGTTAAACCTGCTCCTGGAGAGAGACACTTCCTTTACC330GlnArgLysMetLeuAsnLeuLeuLeuGluArgAspThrSerPheThr253035 40GTCTGTCCAGATGTCCCTAGAACTCCAGTGGGCAAATTTCTTGGTGAT378ValCysProAspValProArgThrProValGlyLysPheLeuGlyAsp4550 55TCTGCAAACCTAAGCATTTTGTCTGGAGGAACCCCAAAATGTTGCCTC426SerAlaAsnLeuSerIleLeuSerGlyGlyThrProLysCysCysLeu606570GATCTTTCGAATCTTAGCAGTGGGGAGATAACTGCCACTCAGCTTACC474AspLeuSerAsnLeuSerSerGlyGluIleThrAlaThrGlnLeuThr758085ACT TCTGCAGACCTTGATGAAACTGGTCACCTGGATTCTTCAGGACTT522ThrSerAlaAspLeuAspGluThrGlyHisLeuAspSerSerGlyLeu9095100CAGGAAGTGCAT TTAGCTGGGATGAATCATGACCAGCACCTAATGAAA570GlnGluValHisLeuAlaGlyMetAsnHisAspGlnHisLeuMetLys105110115120TGTAGCCC AGCACAGCTTCTTTGTAGCACTCCGAATGGTTTGGACCGT618CysSerProAlaGlnLeuLeuCysSerThrProAsnGlyLeuAspArg125130135GGCCATA GAAAGAGAGATGCAATGTGTAGTTCATCTGCAAATAAAGAA666GlyHisArgLysArgAspAlaMetCysSerSerSerAlaAsnLysGlu140145150AATGACAAT GGAAACTTGGTGGACAGTGAAATGAAATATTTGGGCAGT714AsnAspAsnGlyAsnLeuValAspSerGluMetLysTyrLeuGlySer155160165CCCATTACTACTGTT CCAAAATTGGATAAAAATCCAAACCTAGGAGAA762ProIleThrThrValProLysLeuAspLysAsnProAsnLeuGlyGlu170175180GACCAGGCAGAAGAGATTTCAGA TGAATTAATGGAGTTTTCCCTGAAA810AspGlnAlaGluGluIleSerAspGluLeuMetGluPheSerLeuLys185190195200GATCAAGAAGCAAAGGTGA GCAGAAGTGGCCTATATCGCTCCCCGTCG858AspGlnGluAlaLysValSerArgSerGlyLeuTyrArgSerProSer205210215ATGCCAGAGAACTTGAAC AGGCCAAGACTGAAGCAGGTGGAAAAATTC906MetProGluAsnLeuAsnArgProArgLeuLysGlnValGluLysPhe220225230AAGGACAACACAATACCAGAT AAAGTTAAAAAAAAGTATTTTTCTGGC954LysAspAsnThrIleProAspLysValLysLysLysTyrPheSerGly235240245CAAGGAAAGCTCAGGAAGGGCTTATG TTTAAAGAAGACAGTCTCTCTG1002GlnGlyLysLeuArgLysGlyLeuCysLeuLysLysThrValSerLeu250255260TGTGACATTACTATCACTCAGATGCTGGAGGAAG ATTCTAACCAGGGG1050CysAspIleThrIleThrGlnMetLeuGluGluAspSerAsnGlnGly265270275280CACCTGATTGGTGATTTTTCCAAGGTATGT GCGCTGCCAACCGTGTCA1098HisLeuIleGlyAspPheSerLysValCysAlaLeuProThrValSer285290295GGGAAACACCAAGATCTGAAGTATGTCAAC CCAGAAACAGTGGCTGCC1146GlyLysHisGlnAspLeuLysTyrValAsnProGluThrValAlaAla300305310TTACTGTCGGGGAAGTTCCAGGGTCTGATTGA GAAGTTTTATGTCATT1194LeuLeuSerGlyLysPheGlnGlyLeuIleGluLysPheTyrValIle315320325GATTGTCGCTATCCATATGAGTATCTGGGAGGACACA TCCAGGGAGCC1242AspCysArgTyrProTyrGluTyrLeuGlyGlyHisIleGlnGlyAla330335340TTAAACTTATATAGTCAGGAAGAACTGTTTAACTTCTTTCTGAAG AAG1290LeuAsnLeuTyrSerGlnGluGluLeuPheAsnPhePheLeuLysLys345350355360CCCATCGTCCCTTTGGACACCCAGAAGAGAATAATCATCGTG TTCCAC1338ProIleValProLeuAspThrGlnLysArgIleIleIleValPheHis365370375TGTGAATTCTCCTCAGAGAGGGGCCCCCGAATGTGCCGCTG TCTGCGT1386CysGluPheSerSerGluArgGlyProArgMetCysArgCysLeuArg380385390GAAGAGGACAGGTCTCTGAACCAGTATCCTGCATTGTACTACC CAGAG1434GluGluAspArgSerLeuAsnGlnTyrProAlaLeuTyrTyrProGlu395400405CTATATATCCTTAAAGGCGGCTACAGAGACTTCTTTCCAGAATATATG 1482LeuTyrIleLeuLysGlyGlyTyrArgAspPhePheProGluTyrMet410415420GAACTGTGTGAACCACAGAGCTACTGCCCTATGCATCATCAGGACCAC1530G luLeuCysGluProGlnSerTyrCysProMetHisHisGlnAspHis425430435440AAGACTGAGTTGCTGAGGTGTCGAAGCCAGAGCAAAGTGCAGGAAGGG15 78LysThrGluLeuLeuArgCysArgSerGlnSerLysValGlnGluGly445450455GAGCGGCAGCTGCGGGAGCAGATTGCCCTTCTGGTGAAGGACATGAGC1 626GluArgGlnLeuArgGluGlnIleAlaLeuLeuValLysAspMetSer460465470CCATGATAACATTCCAGCCACTGGCTGCTAACAAGTCACCAAAAAGACACTGCAG1681PROAAACCCTGAGCAGAAAGAGGCCTTCTGGATGGCCAAACCCAAGATTATTAAAAGATGTCT1741CTGCAAACCAACAGGCTACCAACTTGTATCCAGGCCTGGGAATGGATTAGGTTTCAGCAG1801AGCTGAAAGCTGGTGGCCAGAGTCCTGGAGCTGGCTCTA TAAGGCAGCCTTGAGTGCATA1861GAGATTTGTATTGGTTCAGGGAACTCTGGCATTCCTTTTCCCAACTCCTCATGTCTTCTC1921ACAAGCCAGCCAACTCTTTCTCTCTGGGCTTCGGGCTATGCAAGAGCGTTGTCTACCTTC1981TTTCTTTGTATTTTCC TTCTTTGTTTCCCCCTCTTTCTTTTTTAAAAATGGAAAAATAAA2041CACTACAGAATGAGAAAAAAA2062(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 473 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:MetSerThrGluLeuPheSerSerThrArgGluGluGlySerSerGly151015SerGlyProSerPheArgSerAsn GlnArgLysMetLeuAsnLeuLeu202530LeuGluArgAspThrSerPheThrValCysProAspValProArgThr3540 45ProValGlyLysPheLeuGlyAspSerAlaAsnLeuSerIleLeuSer505560GlyGlyThrProLysCysCysLeuAspLeuSerAsnLeuSerSerGly65 707580GluIleThrAlaThrGlnLeuThrThrSerAlaAspLeuAspGluThr859095GlyHisLeuAsp SerSerGlyLeuGlnGluValHisLeuAlaGlyMet100105110AsnHisAspGlnHisLeuMetLysCysSerProAlaGlnLeuLeuCys115 120125SerThrProAsnGlyLeuAspArgGlyHisArgLysArgAspAlaMet130135140CysSerSerSerAlaAsnLysGluAsnAspAsnGlyAsnLeuVal Asp145150155160SerGluMetLysTyrLeuGlySerProIleThrThrValProLysLeu165170175A spLysAsnProAsnLeuGlyGluAspGlnAlaGluGluIleSerAsp180185190GluLeuMetGluPheSerLeuLysAspGlnGluAlaLysValSerArg195 200205SerGlyLeuTyrArgSerProSerMetProGluAsnLeuAsnArgPro210215220ArgLeuLysGlnValGluLysPheLysAspAsn ThrIleProAspLys225230235240ValLysLysLysTyrPheSerGlyGlnGlyLysLeuArgLysGlyLeu245250 255CysLeuLysLysThrValSerLeuCysAspIleThrIleThrGlnMet260265270LeuGluGluAspSerAsnGlnGlyHisLeuIleGlyAspPheSerLys275280285ValCysAlaLeuProThrValSerGlyLysHisGlnAspLeuLysTyr290295300ValAsnProGluThrValAlaA laLeuLeuSerGlyLysPheGlnGly305310315320LeuIleGluLysPheTyrValIleAspCysArgTyrProTyrGluTyr325 330335LeuGlyGlyHisIleGlnGlyAlaLeuAsnLeuTyrSerGlnGluGlu340345350LeuPheAsnPhePheLeuLysLysProIleValPro LeuAspThrGln355360365LysArgIleIleIleValPheHisCysGluPheSerSerGluArgGly370375380ProArgMetCy sArgCysLeuArgGluGluAspArgSerLeuAsnGln385390395400TyrProAlaLeuTyrTyrProGluLeuTyrIleLeuLysGlyGlyTyr40 5410415ArgAspPhePheProGluTyrMetGluLeuCysGluProGlnSerTyr420425430CysProMetHisHisGlnAspHisL ysThrGluLeuLeuArgCysArg435440445SerGlnSerLysValGlnGluGlyGluArgGlnLeuArgGluGlnIle450455460 AlaLeuLeuValLysAspMetSerPro465470
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS5294538 *May 5, 1992Mar 15, 1994Cold Spring Harbor Labs.Enzyme inhibitorWO1992020796A2 *May 18, 1992Nov 26, 1992Cold Spring Harbor LabD-type cyclin and uses related theretoWO1993006123A1 *Sep 16, 1992Apr 1, 1993Hutchinson Fred Cancer ResHuman cyclin e* Cited by examinerNon-Patent CitationsReference1Andreassen et al. "2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells" PNAS 1992, vol. 89, pp. 2272-2276.2 *Andreassen et al. 2 Aminopurine overrides multiple cell cycle checkpoints in BHK cells PNAS 1992, vol. 89, pp. 2272 2276.3Andreassen et al., "Induction of partial mitosis in BHK cells by 2-aminopurine" Journal of Cell Science 1991, vol. 100, pp. 299-310.4 *Andreassen et al., Induction of partial mitosis in BHK cells by 2 aminopurine Journal of Cell Science 1991, vol. 100, pp. 299 310.5Brizuela et al., "P13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase" The EMBO Journal 1987, vol. 6, No. 11, pp. 3507-3514.6 *Brizuela et al., P13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase The EMBO Journal 1987, vol. 6, No. 11, pp. 3507 3514.7Coleman et al., "Negative Regulation of the Wee1 Protein Kinase by Direct Action of the Nim1/Cdr1 Mitotic Inducer" Cell 1993, vol. 72. pp. 919-929.8 *Coleman et al., Negative Regulation of the Wee1 Protein Kinase by Direct Action of the Nim1/Cdr1 Mitotic Inducer Cell 1993, vol. 72. pp. 919 929.9 *D Urso et al., Cell Cycle Control of DNA Replication by a Homologue from Human Cells of the p34cdc2 Protein Kinase Science Research Articles 1990, vol. 250, pp. 786 791.10Ducommun et al., "Cdc2 phosphorylation is required for its interaction with cyclin" The EMBO Journal 1991, vol. 10, No. 11, pp. 3311-3319.11 *Ducommun et al., Cdc2 phosphorylation is required for its interaction with cyclin The EMBO Journal 1991, vol. 10, No. 11, pp. 3311 3319.12Dunphy et al. "The cdc25 Protein Conyains an Intrinsic Phosphatase Activity" Cell 1991, vol. 67, pp. 189-196.13 *Dunphy et al. The cdc25 Protein Conyains an Intrinsic Phosphatase Activity Cell 1991, vol. 67, pp. 189 196.14D'Urso et al., "Cell Cycle Control of DNA Replication by a Homologue from Human Cells of the p34cdc2 Protein Kinase" Science Research Articles 1990, vol. 250, pp. 786-791.15Enoch et al., "Mutation of Fission Yeast Cell Cycle Control Genes Abolishes Dependence of Mitosis on DNA Replication" Cell 1990, vol. 60, pp. 665-673.16 *Enoch et al., Mutation of Fission Yeast Cell Cycle Control Genes Abolishes Dependence of Mitosis on DNA Replication Cell 1990, vol. 60, pp. 665 673.17Galactionov et al., "Specific Activation of cdc25 Tyrosins Phosphatases by B-Type Cyclins: Evidence for Multiple Roles of Mitotic Cyclins" Cell 1991, vol. 67, pp. 1181-1194.18 *Galactionov et al., Specific Activation of cdc25 Tyrosins Phosphatases by B Type Cyclins: Evidence for Multiple Roles of Mitotic Cyclins Cell 1991, vol. 67, pp. 1181 1194.19Gautier et al., "Cdc25 is a Specific Tyrosine Phosphatase That Directly Activates p34cdc2" Cell 1991, vol. 67, pp. 197-211.20 *Gautier et al., Cdc25 is a Specific Tyrosine Phosphatase That Directly Activates p34cdc2 Cell 1991, vol. 67, pp. 197 211.21Hoffmann et al., "Phosphorylation and activation of human cdc25-C by cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis" The EMBO Journal 1993, vol. 12, No. 1, pp. 53-63.22 *Hoffmann et al., Phosphorylation and activation of human cdc25 C by cdc2 cyclin B and its involvement in the self amplification of MPF at mitosis The EMBO Journal 1993, vol. 12, No. 1, pp. 53 63.23Lau et al., "Mechanism by which caffeine potentiates lethality of nitrogen mustard" PNAS 1982, vol. 79, pp. 2942-2946.24 *Lau et al., Mechanism by which caffeine potentiates lethality of nitrogen mustard PNAS 1982, vol. 79, pp. 2942 2946.25Lundgren et al., "Mik1 and wee1 Cooperate in the Inhibitor Tyrosine Phosphorylation of cdc2" Cell 1991, vol. 64, pp. 1111-1122.26 *Lundgren et al., Mik1 and wee1 Cooperate in the Inhibitor Tyrosine Phosphorylation of cdc2 Cell 1991, vol. 64, pp. 1111 1122.27 *Millar et al., Cell 68:407 410 (1992).28Millar et al., Cell 68:407-410 (1992).29Murray, A. W., "Creative blocks: cell-cycle checkpoints and feedback controls" Nature Review Article 1992, vol. 359, pp. 599-604.30 *Murray, A. W., Creative blocks: cell cycle checkpoints and feedback controls Nature Review Article 1992, vol. 359, pp. 599 604.31Osmani et al., "Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint" The EMBO Journal 1991, vol. 10, No. 9, pp. 2669-2679.32 *Osmani et al., Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint The EMBO Journal 1991, vol. 10, No. 9, pp. 2669 2679.33Parker et al., "Inactivation of the p34cdc2-Cyclin B Complex by the Human WEE1 Tyrosine Kinase" Science 1992, vol. 257, pp. 1955-1957.34 *Parker et al., Inactivation of the p34cdc2 Cyclin B Complex by the Human WEE1 Tyrosine Kinase Science 1992, vol. 257, pp. 1955 1957.35Railet et al., "A New Screening Test for Antimitotic Compounds Using the Universal M Phase-Specific Protein Kinase, p34cdc2/cyclin Bcdc13, Affinity-Immobilized on p13suc1-Coated Microtitration Plates" Anticancer Research 1991, vol. 11, pp. 1581-1590.36 *Railet et al., A New Screening Test for Antimitotic Compounds Using the Universal M Phase Specific Protein Kinase, p34cdc2/cyclin Bcdc13, Affinity Immobilized on p13suc1 Coated Microtitration Plates Anticancer Research 1991, vol. 11, pp. 1581 1590.37Rowley et al., "The wee1 protein kinase is required for radiation-induced mitotic delay" Nature Letters to Nature, 1992, vol. 356, pp. 353-355.38 *Rowley et al., The wee1 protein kinase is required for radiation induced mitotic delay Nature Letters to Nature, 1992, vol. 356, pp. 353 355.39Russell et al., "The Mitotic Inducer nim1+ Functions in a Regulatory Network of Protein Kinase Homologs Controlling the Initiation of Mitosis" Cell 1987, vol. 49, pp. 569-576.40 *Russell et al., Cell 45:145 153 (1986).41Russell et al., Cell 45:145-153 (1986).42 *Russell et al., The Mitotic Inducer nim1 Functions in a Regulatory Network of Protein Kinase Homologs Controlling the Initiation of Mitosis Cell 1987, vol. 49, pp. 569 576.43 *Sheldrick et al., Bioessays 15:775 782 (1993).44Sheldrick et al., Bioessays 15:775-782 (1993).45Steinmann et al., "Chemically induced premature mitosis: Differential response in rodent and human cells and the relationship to cyclin B synthesis and p34cdc2/cyclin B complex formation" PNAS 1991, vol. 88, pp. 6843-6847.46 *Steinmann et al., Chemically induced premature mitosis: Differential response in rodent and human cells and the relationship to cyclin B synthesis and p34cdc2/cyclin B complex formation PNAS 1991, vol. 88, pp. 6843 6847.47Subramani et al., "Checkpoint controls in Schizosaccharomyces pombe: rad1" The EMBO Journal 1992, vol. 11, No. 4, pp. 1335-1342.48 *Subramani et al., Checkpoint controls in Schizosaccharomyces pombe: rad1 The EMBO Journal 1992, vol. 11, No. 4, pp. 1335 1342.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS5856463 *Sep 18, 1996Jan 5, 1999Prydz; Hans Peter BlankenborgPSKH-1 ribozymesUS5861249 *Apr 23, 1996Jan 19, 1999Cold Spring Harbor LaboratoryAssays and reagents for identifying modulators of cdc25-mediated mitotic activationUS5932435 *Apr 20, 1995Aug 3, 1999Gene Shears Pty. Ltd.Screening antisense and ribozyme nucleic acids in schizosaccharomyces pombeUS5972697 *Nov 13, 1995Oct 26, 1999The Salk Institute For Biological StudiesNIMA interacting proteinsUS6037136 *Oct 24, 1994Mar 14, 2000Cold Spring Harbor LaboratoryInteractions between RaF proto-oncogenes and CDC25 phosphatases, and uses related theretoUS6048693 *Oct 16, 1997Apr 11, 2000Bittech, Inc.Screening for modulators of mammalian cell cycle regulatory proteins; administering a compound to a cell line and analyzing the expression of a reporter gene in the cell line, thereby determining whether the compound affects normalUS6248549 *Jun 22, 1998Jun 19, 2001Queen's University At KingstonMethods of modulating muscle contractionUS6251585 *Apr 24, 1995Jun 26, 2001Mitotix, Inc.Assay and reagents for identifying anti-proliferative agentsUS6322975 *Jan 15, 1999Nov 27, 2001Cold Spring Harbor LabGenetically engineered cells for detection of modulators of cell proliferation and differentiation associated with apoptosisUS6350747Feb 28, 2000Feb 26, 2002Glaxo Wellcome Inc.Protein serine/threonine kinase and protein tyrosine kinase enzyme inhibitors; angiogenesis inhibitors; antitumor and antiproliferative agentsUS6369086Mar 4, 1999Apr 9, 2002Smithkline Beecham CorporationSubstituted oxidole derivatives as protein tyrosine and as protein serine/threonine kinase inhibitorsUS6387919Sep 3, 1998May 14, 2002Glaxo Wellcome Inc.Substituted oxindole derivatives as protein tyrosine kinase and as protein serine/threonine kinase inhibitorsUS6489134Nov 28, 2000Dec 3, 2002The Regents Of The University Of CaliforniaKinesin motor modulators derived from the marine sponge AdociaUS6492398Mar 1, 2000Dec 10, 2002Smithkline Beechman CorporationThiazoloindolinone compoundsUS6521417May 10, 2000Feb 18, 2003Princeton UniversityIncubating permeabilized cells expressing mutant kinase with radiolabeled analog; cytolysis, separation by sodium dodecyl sulfate polyacrylamide gel electrophoresisUS6524850Mar 26, 1999Feb 25, 2003The Scripps Research InstituteKinase wee1 fusion protein compositions, nucleotide sequences, expression systems, and methods of useUS6541503Aug 8, 2001Apr 1, 2003Smithkline Beecham CorporationSubstituted oxindole derivatives as protein tyrosine kinase and as protein serine/threonine kinase inhibitorsUS6569878Oct 27, 1998May 27, 2003Agouron Pharmaceuticals Inc.Substituted 4-amino-thiazol-2-yl compounds as cyclin-dependent kinase inhibitorsUS6596848Mar 24, 1999Jul 22, 2003Salk Institute For Biological StudiesAntibodies to NIMA interacting proteinsUS6610483 *Jul 20, 2000Aug 26, 2003Princeton UniversityMethod for the identification of a pattern of changes in cellular responses induced by the selective inhibition of a signaling molecule, by determining the specific effects of a selective inhibitor on a mutant form of signaling molecule onUS6620818Mar 1, 2000Sep 16, 2003Smithkline Beecham CorporationAdministering indolin-2-one derivative as cyclin-dependent kinase inhibitorUS6624171Mar 3, 2000Sep 23, 2003Smithkline Beecham CorporationUseful as cyclin dependent kinase 11 inhibitors, for preventing/reducing the severity of epithelial cytotoxicity side-effects (e.g., alopecia, plantar- palmar syndrome, mucositis) induced by chemotherapy and/or radiation therapyUS6777200Nov 27, 2002Aug 17, 2004The Regents Of The University Of CaliforniaKinesin motor modulators derived from the marine sponge AdociaUS6815439Sep 23, 2003Nov 9, 2004Smithkline Beecham CorporationPyrrolo(2,3-b)pyridine derivatives as cyclin-dependent kinase inhibitorsUS6818632Sep 27, 2001Nov 16, 2004Smithkline Beecham CorporationProtein tyrosine kinase and protein serin/threonine kinase inhibitory activity; anticancer agents; treating chemotherapy induced alopeciaUS7105529Nov 30, 2001Sep 12, 2006Smithkline Beecham CorporationA novel compound for treating alopecia induced by cancer chemotherapy or radiotherapyUS7125677Oct 15, 2003Oct 24, 2006The Salk Institute For Biological StudiesNIMA interacting proteinsUS7125955Aug 25, 2003Oct 24, 2006The Salk Institute For Biological Studiesmitotic kinase, NIMA, encoded by the Aspergillus nimA gene; pure WW domain of a Pin1 polypeptide which interacts with NIMA; a Peptidyl-prolyl cis/trans isomerases (PPIase); drug target; Pin1 is a PPIase known to be essential for lifeUS7129253Dec 19, 2003Oct 31, 2006Smithkline Beecham CorporationCompoundsUS7148003Nov 17, 2003Dec 12, 2006The Salk Institute For Biological StudiesBioassay to determine inhibition of the peptidyl-prolyl isomerase activity of a Pin1 protein; antimitotic agentsUS7148216Dec 28, 2004Dec 12, 2006Combinatorx, Inc.Combinations of drugs for the treatment of neoplastic disordersUS7164012Jul 8, 2003Jan 16, 2007The Salk Institue For Biological StudiesNucleic acids, expression vectors and host cells exhibiting the same peptidyl-prolyl isomerase activity as a Pin protein fragment; antimitotic agentsUS7314926Apr 7, 2000Jan 1, 2008Antisoma Research LimitedInhibiting cell proliferation, breast cancer, prostate cancer, cervical cancer, lung cancer, or tumor cellsUS7348151Dec 29, 1999Mar 25, 2008Hansjoerg ForsterMethod for the cellular high-throughput-detection of nuclear receptor ligand interactionsUS7833736Apr 2, 2007Nov 16, 2010Cell Signaling Technology, Inc.determining whether a mammalian cancer is likely to be resistant or responsive to OSI-930; reduces well-known side effects and are often of limited effect since they fail to specifically target the underlying causes of the malignanciesUS7960540Oct 31, 2007Jun 14, 2011Advanced Cancer Therapeutics, LlcAntiproliferative activity of G-rich oligonucleotides and method of using same to bind to nucleolinUS8114850Oct 31, 2007Feb 14, 2012Advanced Cancer Therapeutics, LlcAntiproliferative activity of G-rich oligonucleotides and method of using same to bind to nucleolinUS8648051Oct 29, 2004Feb 11, 2014Advanced Cancer Therapeutics, LlcAntiproliferative activity of G-rich oligonucleotides and method of using same to bind to nucleolinWO1997011163A1 *Sep 18, 1996Mar 27, 1997Gaute BredePskh-1 ribozymes and uses in disease treatmentWO1997017986A1 *Oct 28, 1996May 22, 1997Salk Inst For Biological StudiNima interacting proteinsWO1999049061A1 *Mar 26, 1999Sep 30, 1999Michael N BoddyKinase wee1 fusion protein compositions, nucleotide sequences, expression systems, and methods of useWO2001009373A2 *Jul 31, 2000Feb 8, 2001Basf Biores CorpNative cdc25 substrates, compositions and uses related theretoWO2007127002A2 *Mar 29, 2007Nov 8, 2007Cell Signaling Technology IncProtein markers of responsiveness to type iii receptor tyrosine kinase inhibitors* Cited by examinerClassifications U.S. Classification435/29, 435/21, 435/254.2, 435/7.31International ClassificationA61K45/00, C12N1/15, A61P43/00, G01N33/50, C12N15/09, C12N9/16, G01N33/15, C12R1/645, A61K38/45, C12N1/19, C12Q1/18Cooperative ClassificationC12N9/16, G01N33/5011European ClassificationC12N9/16, G01N33/50D2BLegal EventsDateCodeEventDescriptionOct 21, 2003FPExpired due to failure to pay maintenance feeEffective date: 20030822Aug 22, 2003LAPSLapse for failure to pay maintenance feesMar 12, 2003REMIMaintenance fee reminder mailedMay 18, 2001ASAssignmentOwner name: GPC BIOTECH INC., MASSACHUSETTSFree format text: CHANGE OF NAME;ASSIGNOR:MITOTIX, INC.;REEL/FRAME:011783/0018Effective date: 20000510Owner name: GPC BIOTECH INC. 610 LINCOLN STREET WALTHAM MASSACOwner name: GPC BIOTECH INC. 610 LINCOLN STREETWALTHAM, MASSACFree format text: CHANGE OF NAME;ASSIGNOR:MITOTIX, INC. /AR;REEL/FRAME:011783/0018Feb 19, 1999FPAYFee paymentYear of fee payment: 4Dec 29, 1993ASAssignmentOwner name: MITOTIX, INC., MASSACHUSETTSFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DRAETTA, GIULIO;COTTAREL, GUILLAUME;DAMAGNEZ, VERONIQUE;REEL/FRAME:006817/0315Effective date: 19930604RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services