+

WO1998016660A1 - Essai phenotypique de la fonction cycline/cdk (kinase a dependance cycline) - Google Patents

Essai phenotypique de la fonction cycline/cdk (kinase a dependance cycline) Download PDF

Info

Publication number
WO1998016660A1
WO1998016660A1 PCT/US1997/018608 US9718608W WO9816660A1 WO 1998016660 A1 WO1998016660 A1 WO 1998016660A1 US 9718608 W US9718608 W US 9718608W WO 9816660 A1 WO9816660 A1 WO 9816660A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
cyclin
cdk
cell line
seq
Prior art date
Application number
PCT/US1997/018608
Other languages
English (en)
Inventor
Grant A. Bitter
Original Assignee
Bittech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bittech, Inc. filed Critical Bittech, Inc.
Priority to AU49038/97A priority Critical patent/AU4903897A/en
Publication of WO1998016660A1 publication Critical patent/WO1998016660A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/70—Vector systems having a special element relevant for transcription from fungi
    • C12N2830/702—Vector systems having a special element relevant for transcription from fungi yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • This invention relates to methods of identifying inhibitors and activators of mammalian cell cycle regulatory proteins, especially inhibitors and activators of cyclins, cyclin-dependent kinases (CDKs), cyclin/CDK complexes, cyclin kinase inhibitors (CKIs), and cyclin/CDK/CKI complexes, by a functional assay in vivo; to cell lines and vectors useful for these methods; and to cell cycle regulatory products identified by these methods.
  • CDKs cyclin-dependent kinases
  • CKIs cyclin kinase inhibitors
  • CKIs cyclin kinase inhibitors
  • CDKs Cyclin-dependent kinases
  • CDKs are serine/threonine protein phosphorylation enzymes that are activated through interaction with cyclins and that phosphorylate (and thus control the activity of) various molecules associated with cell growth and division, thereby controlling progression through various stages of the eukaryotic cell cycle (reviewed in Morgan, 1995, Nature 374: 131-134; Pines, 1993, Trends Biochem. Sci. 18: 195-197; Sherr, 1993, Cell, 73:1059-1965).
  • CDKs and their regulatory proteins have become a focus of great interest, as understanding their function sheds light on normal cellular growth controls as well as cellular proliferation defects associated with diseases such as cancer.
  • the family of known CDKs is characterized by similar size (35-40KDa), sequence homology ( >40 identity) and, primarily, a phosphorylating ability that is activated by cyclin.
  • the interacting cyclins are correspondingly characterized by the ability to bind and activate CDKs. This binding may be mediated via a relatively conserved, 100-amino-acid, CDK-interacting domain called the "cyclin box. " In some cases, the cyclin box constitutes the chief extent of homology among cyclins.
  • Gl first gap phase
  • S synthesis
  • G2 second gap phase
  • M mitosis
  • This checkpoint is termed the "restriction point" in mammalian cells and "start” in yeast.
  • the cell duplicates its genetic material during S phase and synthesizes necessary proteins in preparation for mitosis during G2.
  • Another decision point exists in G2 when cellular machinery determines if the DNA has been completely replicated and if adequate protein components have been produced to support mitosis. Progression through this checkpoint leads to mitosis, cell division and the next cell cycle.
  • the Gl/S phase transition and the G2/M phase transition accordingly serve as crucial checkpoints to monitor appropriate progression through the cell cycle. Further details on cellular activity for each stage of the cell cycle are set out below in connection with the various activities of CDKs and related molecules.
  • D-cyclins In mammalian cells, there are three cyclins referred to as "D-cyclins" which exhibit cell- type-specific expression. Most mammalian cells express cyclin D3 and either cyclin DI or D2.
  • the D cyclins associate with CDK4 and, in some cell types, also with CDK6 (not shown) and function in Gl phase to promote progression through the restriction point. Other cyclin types (with different reference letters) exhibit different cellular or functional specifications.
  • the cyclin E/CDK2 complex is believed to function after the D cyclins at the Gl to S transition to promote DNA replication.
  • the cyclin A/CDK2 and cyclin B/Cdc2 complexes function during S phase. Cdc2 continues to associate with cyclin B in G2 phase and subsequently with cyclins B and A in mitosis.
  • Cdc2 continues to associate with cyclin B in G2 phase and subsequently with cyclins B and A in mitosis.
  • Several mammalian CKIs have been identified, including pl5 (also known as pl5 /ABW *, INK4b and MTS2), pl6 (also known as ⁇ 6 INK4a , INK4a and MTS1), pl8 (also known as pl8 /MMc and INK4c), pl9 (also known as pl9TM and INK4d), p21 (also known as p21 ⁇ w , CIPl, SDIl and WAF1), p27 (also known as p21 ⁇ P1 and KIP1) and p57 (also known as p57 s >2 and KIP
  • cyclins and CDKs depicted in Figure 1 represent the major species identified to date. Additional species may be known or discovered in the future.
  • CDK activation is also regulated by feedback mechanisms which prevent entry of cells into the next phase of the cell cycle prior to completion of the appropriate macromolecular events (reviewed in Murray, 1992; Hunter and Pines, 1994, Cell 79:573-582; Hartwell and Kastan, 1994, Science 266: 1821-26; Elledge, 1996; Paulovich et al , 1997, Cell 88:315-321).
  • the first major checkpoint occurs in Gl and, in S. cerevisiae, requires that the cell achieve a minimum size.
  • Start is the major checkpoint of the cell cycle, and once this point is passed, the cell is committed to a mitotic division cycle.
  • the second checkpoint at the G2-to-M phase transition ensures that the cell has completed all DNA replication and DNA repair prior to initiating mitosis. While many of the molecular details of the checkpoint mechanisms are incompletely understood, they appear at least in part to involve cyclin-CDK activation.
  • cyclin/CDKs phosphorylate retinoblastoma (Rb) and other related proteins, resulting in activation of genes required for DNA synthesis (reviewed in Nigg, 1993, Trends Cell Biol. 3:296-301).
  • Cells do not pass the Gl checkpoint if they contain extensive DNA damage. This is due to p53-dependent induction of p21, a CKI which inhibits the cyclin E/CDK2 complex.
  • the G2/M checkpoint appears to directly affect cyclin/CDK activation. Blocks of unreplicated vertebrate DNA inhibit cyclin Bl/Cdc2 activation by preventing dephosphorylation of Cdc2, halting further progression through the cell cycle.
  • cell cycle regulation of cyclin and CKI expression controls the specificity and activity of CDKs, and this regulation is integrated with the major cell cycle checkpoints.
  • yeast appear to have one major CDK (CDC28 in S. cerevisiae, and CDC2 in S. pombe).
  • CDC28 interacts with cyclins CLNI or CLN2 during Gl and with CLB1, CLB2, CLB3 or CLB4 during G2.
  • the S. cerevisiae PCL1/PHO85 and PCL2/PHO85 cyclin/CDK complexes have also been shown to contribute to, but not be required for, cell cycle regulation (Measday et al.
  • CDC25B Overexpression of CDC25B was observed in 32% of human primary breast tumors tested.
  • TGF-0 which inhibits mammalian cell growth in a manner similar to that observed with contact inhibition in cell culture, prevents assembly and activation of cyclin E/CDK2 complexes (Koff et al , 1993, Science 260:536-539). It has also been speculated that the tumor suppressor gene p53 exerts part of its effect through cell cycle control. Over 50% of human cancers harbor a mutation in p53. As mentioned earlier, the wild type p53 protein stimulates expression of a 21 kilodalton protein (p21), a CKI which inhibits CDK2. CDK2 activity is required for a mammalian cell to pass the restriction point and begin DNA replication (El-Diery et al. , 1994, Cancer Res. 54: 1169-74; Harper et al. , 1993, Cell 75:805- 816; Xiong et al. , 1993, Nature 366:701-704).
  • CDK4, CDK6, PCNA, Rb or pl6 INK4a mimetic, or cyclin D/CDK4 inhibitor CDK4, CDK6, PCNA, Rb or pl6 INK4a mimetic, or cyclin D/CDK4 inhibitor
  • PCNA PCNA, Rb or pl6 INK4a mimetic, or cyclin D/CDK4 inhibitor
  • Table I summarizes the cell cycle related genes that have been documented to be defective in various cancers. These defects are either mutations in, or aberrant expression of, genes encoding cyclins, CDKs, CKIs or other proteins that affect the activity of these cell cycle regulatory proteins. For the majority of cancers exhibiting cell cycle defects, the loss of cellular growth control appears to reflect the failure to inhibit a particular CDK. Specifically CDK2, CDK4 or CDK6 appears to be inappropriately active due to the lack of normal CDK inhibition controls. Most cancer therapeutics currently in use are cytotoxic compounds which were frequently identified by their ability to kill rapidly growing tumor cells in culture. The strategy in utilizing these compounds for therapy has been to attempt to kill cancer cells while limiting the toxicity for the patient.
  • antineoplastic agents generally function by damaging DNA, inhibiting DNA replication or precursor synthesis, inhibiting DNA topoisomerases or by disrupting function of the mitotic apparatus.
  • Chemotherapeutic agents currently in use are effective against certain cancers but, in general, have been of limited therapeutic value. Since these cytotoxic compounds are frequently ineffective and generally have significant adverse effects on the patient, considerable need exists for the development of more efficacious therapies. To date, only limited technology has been described for identifying compounds that affect the activity of cell cycle regulatory proteins. Colas et al.
  • Draetta et al. U. S. Patent No. 5,443,962 disclose one method of identifying cell cycle regulatory proteins that inhibit CDC25 phosphatase. This screen only detects proteins acting through CDC25, and is dependent on the use of cells that have been manipulated to have a hypermitotic phenotype. As discussed earlier, the majority of cancer-associated cell cycle defects identified thus far involve a failure to inhibit the activity of a CDK. While specific cyclin/CDK inhibitors may thus be therapeutically indicated, identifying such inhibitors in cell-based screens poses a challenge in that these inhibitors are expected to exhibit the phenotype of arresting cell cycle progression.
  • the PH05 gene of S. cerevisiae encodes a secreted acid phosphatase. Transcription of the gene is repressed when yeast are grown in high concentrations of inorganic phosphate, and transcription of the PH05 gene is induced in response to phosphate starvation. The secreted acid phosphatase generates inorganic phosphate from extracellular nutrients, thus allowing the cells to grow in phosphate depleted medium. Genetic studies (reviewed in Oshima, 1982, In: The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, pp. 159-180; Johnston and Carlson, 1992, In: The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol.
  • PH02 and PH04 encode transcription factors that bind to an upstream activation sequence (UASp jjQ ⁇ ) in the PH05 expression control sequences to activate transcription (Senstag and Hinnen, 1987, Nucleic Acids Res. 15:233, 241; Berber and Hilger, 1988, Gene 66:307-314; Vogel and Hinnen, 1989, Mol. Cell. Biol. 9:2050-56; Yoshida et al. , 1989, Mol. Gen. Genet. 217:31-33).
  • UASp jjQ ⁇ upstream activation sequence
  • PH080 and PH085 are required for repression of PH05 gene transcription.
  • the PH085 gene sequence shows that the encoded protein has significant amino acid sequence homology to the major yeast CDK, CDC28p (Toh-e et al. , 1988, Mol. Gen. Genet. 2 ⁇ : 162-164).
  • the PH080 gene product is homologous to two yeast cyclins. Within a 120 amino acid cyclin homology region that contains residues conserved in all cyclins, PHO80p is 33% identical to both PCLlp (HCS26p; Ogas et al , 1991 , Cell 66: 1015-1020) and PCL2p (ORFDp; Frohlich et al.
  • PHO80p and PHO85p satisfy the biochemical definition of an interacting cyclin-CDK pair (Kaffman et al. , 1994, Science 263: 1153-1156).
  • PHO80p and PHO85p form a complex, and the purified PHO80p/PHO85p complex can interact with purified PHO4p in vitro.
  • the purified PHO80p/PHO85p complex phosphorylates PHO4p in vitro, creating a phosphorylation pattern similar to the in vivo phosphorylation pattern of PHO4p (O'Neill et al.
  • the positive regulator PH081 has been shown to encode a protein that binds to and inactivates the PHO80p/PHO85p complex (Schneider et al. , 1994, Science 266: 122-126. Transcription of PH081 is induced by phosphate starvation. Increased synthesis of PHO ⁇ lp, in conjunction with an apparent post-translational modification in low phosphate, results in inactivation of the PHO80p/PHO85p cyclin/CDK causing derepression of PH05 (Ogawa et al. , 1995, Mol. Cell. Biol. 15:997-1004).
  • the PHO81 encoded protein appears to function as a CKI, inactivating the PHO80p/PHO85p kinase and thereby preventing phosphorylation of PHO4p.
  • PHO80, PH085 and PH081 satisfy the biochemical definition of a cyclin, CDK and CKI, respectively, they do not appear be required for normal cell cycle control since the genes can be deleted individually or in combination without major effects on cell growth properties.
  • CDKs are a highly conserved family of proteins. For example, the major S. cerevisiae CDK, CDC28p, shares over 51 % amino acid homology with PHO85p (Toh-e et al. , 1988) .
  • pombe cdc2 gene product (Lee et al , 1987, Nature 344:503-508), while the human CDK2 gene can complement a S. cerevisiae temperature sensitive cdc28 mutation (Elledge and Spottswood, 1991, EMBO J. 10:2653-2659).
  • the cell cycle-dependent phosphorylation of human retinoblastoma protein (pRB) is faithfully reproduced in S. cerevisiae (Hatakeyama et al. , 1994, Genes Dev. 8: 1759-1771). This phosphorylation is dependent on yeast CLN3 and either CLN1 or CLN2.
  • yeast CLN2p and CLN3p in pRB hyperphosphorylation can be complemented by expression of human cyclin E and cyclin DI , respectively.
  • the function of native mammalian cyclins, CDKs and other cell cycle regulatory proteins may be measured in vivo by complementation of specific yeast mutations.
  • the convergence of cancer research and cell cycle research has presented the opportunity to develop novel cancer therapeutic agents by targeting specific proteins that are mutant or aberrantly regulated in cancer cells. This opportunity has highlighted the absence of adequate screening methods for identifying agents that specifically influence the activity of cell cycle regulatory proteins.
  • Cell cycle regulatory genes are genes that control and coordinate progression from one stage of a cell cycle to the next stage or to the next cell cycle. These genes include cyclins, cyclin-dependent kinases, cyclin-dependent kinase inhibitors, cyclin and/or CDK phosphatases, and genes whose protein products regulate the activity of these genes or proteins.
  • Transcription control factors are regulatory proteins that bind to DNA expression control sequences, RNA Polymerase or other transcription factors to modulate the activity of the RNA Polymerase complex and to control transcription of DNA into messenger RNA.
  • Promoter sequences are functionally defined DNA sequences that direct transcription of DNA. At a minimum, RNA Polymerase interacts with promoter sequences to initiate transcription of a gene.
  • Homologous is used to define two sequences that have substantial similarity to each other. Substantial similarity may be determined by using computer programs known in the art such as those provided by the Genetics Computer Group, Inc. For example, the homology of a given sequence with individual members of a sequence database may be established using the FastA program. For a group of related sequences, the PileUp program creates a multiple sequence alignment showing regions of homology. The Compare program identifies segments of homology between two sequences while the BestFit program determines the optimal alignment of two sequences. A preferred determination of homology compares two amino acid sequences over a stretch comprising at least 20 amino acids, and alignment reveals amino acid identity or amino acid conservation in at least 30% of the amino acid positions.
  • nucleotide sequence homology compares two nucleotide sequences over a stretch comprising at least thirty nucleotides, and alignment of the sequences reveals nucleotide identity in at least 30% of the nucleotide positions.
  • Hybrid gene is a gene comprising sequences from two or more original genes. Accordingly, hybrid proteins are the gene products encoded by hybrid genes.
  • Target gene is herein used to describe a mammalian cell-cycle regulatory gene, also referred to herein as the "second" gene, whose activity is measured by the methods of the current invention.
  • Chromosomal mutation is an alteration of a chromosomal gene that causes the gene to be incapable of producing a functional gene product; the alteration can be one or more point mutations, an insertional mutation such as a gene disruption, or the deletion of all or part of the gene.
  • a method of screening for a compound that affects cell cycle control of a target cell comprising administering a compound to a host cell line, wherein the host cell line comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated directly or indirectly through phosphorylation by a cyclin/CDK phosphorylation system; and a hybrid gene comprising a first coding region from a first gene native to the host cell line and a second coding region from a second target gene, wherein the first gene encodes a gene product
  • FIG. 1 is a diagram showing the major CDKs known to exist in mammalian and S. cerevisiae cells, the cyclins with which each CDK interacts, and the different phases of the cell cycle in which they act.
  • Figure 2 is a schematic representation of three CDK2-PHO85 hybrid proteins.
  • Figure 3 is a schematic representation of basic expression vectors pBTl, pBT3 and pBT6.
  • Figures 4A and 4B are schematic representations of the PHO5 expression control sequences and the plasmids pBTll, pBT12 and pBT13.
  • Figure 5 is a restriction map of the expression vectors, pBT16, pBT15 and pBT17.
  • Figure 6 is a graphical representation of comparative expression levels of reporter gene in different yeast strains.
  • Figures 7A and 7B are graphical representations of experiments relating to CK2- P85ml activity.
  • Figure 8 is a diagram of the proteins of the PH05 regulon controlling expression of various reporter genes.
  • Figure 9 is a graphical representation of ⁇ -galactosidase reporter gene activity produced in various cell lines containing a hybrid or a native CDK gene.
  • the present invention provides a number of related techniques for controlling cell cycle regulation for a variety of purposes, such as stimulating growth of cells (as in wound healing) or regulating excessive cell growth and division (as in cancer therapy).
  • aspects of the invention that can be separately practiced, such as processes useful for the discovery of compounds that will be effective in regulating cell growth (e.g., drug discovery), as well as the application of the discovered compounds to either up-regulate or down-regulate cell growth, so that it is not necessary (or even desirable) to practice all of the aspects of the invention at the same time. Routine testing is part of practicing the invention.
  • Cyclin/CDK complexes phosphorylate an array of proteins within a cell.
  • One class of substrates for these complexes comprises transcription control factors.
  • the genes regulated by these transcription factors owe their expression to strict control by one specific cyclin/CDK pair.
  • functionality of the specific cyclin/CDK pair can be studied in in vivo assays.
  • the present invention provides assays and reagents for identifying circumstances, including those involving genes, mutations and compounds, that affect the activity of mammalian cell cycle regulation gene products.
  • a cell line is provided in which functional sequences from a mammalian gene are substituted for sequences of a native yeast cell cycle regulatory gene that regulates transcription. Because the mammalian sequences impart the function of the native cell cycle regulatory gene sequences being replaced, agents that interfere with or alter the functionality of the peptides encoded by the mammalian gene sequences (and thus affect the cell cycle of the cell from which the mammalian DNA sequence is obtained) can be identified by analyzing expression of a reporter gene that is operably linked to the expression control sequences.
  • the invention uses a reporter gene whose activity will indicate the integrity of the cyclin/CDK phosphorylating system used in the assay.
  • the reporter gene can be any gene whose expression is detectable, for example by an enzymatic activity assay, a nutritional assay, a structural assay, as by an immunoassay, or by antibiotic resistance or other selection.
  • a variety of reporter genes are commonly utilized and well known in the art (See Example 1).
  • the reporter gene is selected from the group consisting of the E. coli LacZ (encoding ⁇ -galactosidase), the E. coli tn5 neo gene, the LEU2, URA3, HIS3, and LYS2 genes of 5.
  • LEU2, URA3, HIS3, and LYS2 are preferred for their ability to provide a positive selection for yeast cells expressing the reporter gene, where promoter activity confers the ability of cells that are mutant in the corresponding genes to grow in culture media lacking leucine, uracil, histidine or lysine, respectively.
  • LEU2 is used as a reporter gene in a system where phosphorylation by the cyclin/CDK inactivates a positively acting transcription factor
  • a cyclin kinase-inhibitor that antagonizes the cyclin/CDK will induce transcription of LEU2 under specific conditions.
  • leu2 ⁇ cells in leucine-depleted medium will only grow if the LEU2 reporter gene is caused to be expressed.
  • agents that inhibit the cyclin/CDK phosphorylation system can be identified by their ability to promote cell growth in leucine-depleted media.
  • the tn5 neo gene likewise is preferred as a reporter gene for conferring to bacterial, yeast or mammalian cells the ability to grow in medium containing the antibiotic G418 (neomycin or geneticin).
  • URA3 and LYS2 genes are more preferred for their additional ability to provide a positive selection for cells that do not appropriately activate transcription of the particular CDK-responsive promoter.
  • Yeast cells expressing the URA3 gene are not viable in media containing 5-fluoroorotic acid (FOA), while those expressing the LYS2 gene are not viable in media containing ⁇ j-aminoadipate (AAD). These reporter genes are thus useful to select for cells that acquire the ability to repress transcription from the particular CDK-responsive promoter.
  • FOA 5-fluoroorotic acid
  • AAD ⁇ j-aminoadipate
  • cyclin/CDK inactivates a positively acting transcription factor in the system and thus represses transcription of the reporter gene
  • a mutation or compound that incapacitates cyclin/CDK phosphorylation activity will allow transcription of the reporter gene under conditions where transcription would normally be repressed.
  • Cells expressing URA3 as a reporter gene will not be viable in a medium containing FOA.
  • cells expressing LYS2 as a reporter gene will not be viable in medium containing AAD. Therefore, by using URA3 or LYS2 as reporter genes, agents that compensate for the defect in cyclin/CDK activity can be identified by their ability to confer viability to cells grown in FOA or AAD, respectively.
  • this type of positive selection can be used with the above mentioned reporter genes to identify inhibitors of a CKI that inhibits a specific cyclin/CDK.
  • cells having a URA3 reporter gene will not grow in the presence of FOA, while cells having a LYS2 reporter gene will not grow in the presence of AAD.
  • Inhibitors of CKI will reinstate the repressed state and allow growth in FOA or AAD, respectively.
  • the LacZ gene, firefly luciferase gene, the jelly fish green fluorescent protein gene and the CAT gene encode proteins that allow optical quantitation of the activity of the cyclin/CDK-responsive promoter.
  • reporter genes can be monitored by optical sensors and are particularly suited for automated high throughput screening. It will be apparent to those skilled in the art that if the transcription factor that positively regulates reporter gene expression is activated, rather than repressed, by a specific cyclin/CDK, then the phenotypes conferred by the above reporter genes in this strain will reflect the opposite state of the cyclin/CDK (active or inhibited) than the examples above. Similarly, a transcriptional repressor whose activity is regulated by a cyclin/CDK will confer specific phenotypes from each reporter gene dependent on whether the cyclin/CDK activates or inhibits the transcriptional repressor.
  • Expression control sequences of the current invention comprise upstream activation sequences and promoter sequences. Transcription regulatory factors and RNA polymerase bind to these sequences to regulate the timing and extent of transcription of the downstream gene sequences.
  • the upstream activation sequences used in the present invention include various DNA regions that bind transcription factors, the activities of which are controlled, either directly or indirectly, by a cyclin/CDK phosphorylating complex. The complex can regulate transcription through directly phosphorylating a DNA-binding transcription factor, where the phosphorylation state of the transcription factor corresponds to its ability to activate or repress transcription.
  • the cyclin/CDK complex can regulate transcription indirectly through phosphorylating a protein that interacts with a DNA-binding transcription factor, where an interaction regulates the activity of the transcription factor.
  • upstream activation sequences are known in the art (Dynlacht et al. , 1995, Nature 374: 114).
  • the RNA polymerase itself can be regulated directly or indirectly by a cyclin/CDK phosphorylating complex (Liao et al. , 1995, Nature 374: 193-96).
  • the upstream activation sequences of the current invention are most preferably from the PH05 gene of S. cerevisiae.
  • preferred cell lines are strains of S. cerevisiae.
  • the PH05 gene encodes a secreted acid phosphatase, and regulation of PHO5p expression is dependent on concentrations of inorganic phosphate.
  • the PH05 expression control sequences are activated by the yeast transcription factor PHO4p, whose activity is regulated by the cyclin/CDK pair PHO80p/PHO85p.
  • the PHO80p/PHO85p complex phosphorylates PHO4p to a hyperphosphorylated, inactive state; when yeast are starved for inorganic phosphate, transcription of the CKI gene PH081 is induced, preventing phosphorylation of PHO4p and allowing PHO4p to activate the PH05 gene promoter.
  • Upstream activating sequences can be identified using methods well known in the art. Generally, these control sequences reside upstream of the gene coding region and bind to the transcription factors regulated by the relevant cyclin CDK complex. Minimally, these sequences are also characterized by their ability to confer the transcriptional regulation of the particular gene to a heterologous gene. The upstream activation sequences may be utilized in the same promoter context as found in the native gene.
  • the DNA region at - 405 to -10 relative to the PH05 initiation codon contains the full expression control sequence of the PH05 gene.
  • the current inventor has cloned this region into a plasmid; the region confers phosphate-dependent regulation to a reporter gene cloned downstream.
  • hybrid expression control sequences may be used in the present invention, wherein the minimal upstream activating sequence of a particular gene may be combined with a promoter sequence from a heterologous gene to regulate transcription of a reporter gene.
  • a wide array of heterologous gene promoters is available in the art.
  • PH05 UAS sequence extending from -405 to -206 relative to the initiation codon is sufficient to confer phosphate-dependent regulation to the TDH3 promoter downstream region, resulting in repression of reporter gene expression in high phosphate and efficient reporter gene expression when cells are grown in low phosphate (See Example 2).
  • the PH05 regulatory system, or regulon, in S. cerevisiae provides a number of advantages for assessing cyclin/CDK activity.
  • the in vivo phosphorylation substrate of the PHO80p/PHO85p kinase is known to be PHO4p, a positive transcriptional activator of PH05 gene transcription.
  • the PHO80p/PHO85p pair is not required for cell viability, nor does it appear to be required for normal cell cycle control (except in a clnlcln2 mutant background). These properties allow extensive genetic modification of these proteins without adverse effects on yeast viability and growth.
  • cell synchronization is not required in the screening system.
  • yeast cells mammalian cell cycle regulatory proteins can be introduced and studied in the absence of other mammalian proteins (albeit in the presence of homologous and, potentially, functionally similar yeast proteins).
  • yeast cyclin/CDK systems which are encompassed by the current invention.
  • Kuchin et al 1995, Proc. Natl. Acad. of Sci. 92:4006-10, have demonstrated that the S. cerevisiae SSN8 and SSN3 genes encode, respectively, cyclin and CDK homologs which also interact biochemically as a cyclin/CDK complex.
  • the SSN8p/SSN3p kinase contributes to transcriptional repression of a variety of genes.
  • This and other S. cerevisiae CDKs that regulate the activity of a specific transcription factor can also be used in the current invention.
  • Utilization of different cyclin/CDK systems expands the scope of native and hybrid proteins that can be synthesized and broadens the number of potential targets for screening for anti-cancer and anti- proliferation compounds or lead compounds.
  • the methods of the present invention may be applied to cyclin/CDK screening systems in S. pombe. It is appreciated that the method of the current invention can encompass use of expression control sequences native to mammalian genes and other desirable target gene systems.
  • the cell line of the current invention is preferably a mammalian cell line.
  • Various mammalian transcription factors have been shown to be regulated by a cyclin/CDK phosphorylation complex. Expression control sequences responsive to these transcription factors can be operably linked to reporter genes to monitor the activity of a cyclin/CDK complex.
  • the E2F mammalian transcription factors (also referred to as DRTF1) constitute a family of (at least) five related proteins.
  • E2F1 is the best characterized of these and, as a heterodimer with the homologous DP-1 protein, is capable of activating expression control sequences containing E2F binding sites (reviewed in La Thangue, 1994, Current Opinion in Cell Biology 6:443- 50).
  • a number of growth regulatory proteins have been shown to associate with E2F. These include Retinoblastoma protein (Rb), pi 07, cyclin E/CDK2 and cyclin A/CDK2.
  • Rb Retinoblastoma protein
  • pi 07 pi 07
  • cyclin E/CDK2 cyclin A/CDK2
  • E2F binds preferentially (both in vitro and in vivo) to the underphosphorylated form of the Rb protein and dissociates when Rb is phosphorylated by cyclin E/CDK2 (Dynlacht et al. , 1995).
  • cyclin E/CDK2 Phosphorylation of Rb in Gl phase by cyclin E/CDK2 allows E2F1 to dissociate, complex with DP-1 , bind to an E2F-responsive expression control sequence and activate transcription. Subsequently, cyclin A/CDK2 phosphorylates E2F1 in S phase and abolishes its ability to bind DNA and activate transcription. Since cyclin E/CDK2 is activated prior to DNA synthesis but cyclin A/CDK2 is not activated until S phase, an E2F-regulated expression control sequence will exhibit cell cycle dependent expression. A reporter gene operably linked to such a control sequence will be activated late in Gl phase.
  • This cell cycle dependent transcription can be measured in synchronized cell cultures, such as those synchronized by a double thymidine block.
  • reporter gene expression can be measured in asynchronous cultures where the fraction of cells transcribing the reporter gene will be equivalent to the fraction of the cell cycle in which cyclin E/CDK2 is active prior to the activation of cyclin A/CDK2.
  • reporter gene expression in asynchronous cultures will be proportionately less than that measured at the optimal time in synchronous cultures.
  • the assay system can be optimized (1) to be independent of cell cycle regulation and (2) to isolate the effect of a single cyclin/CDK pair.
  • cyclin E and/or E2F1 might be constitutively produced or overexpressed in the cell, generating cell lines in which regulation of the reporter gene promoter is independent of the cell cycle.
  • the E2F system can be exploited to provide an assay system that confers cyclin/CDK regulated transcription to an expression control sequence that originally exhibits no cyclin/CDK regulation.
  • a cell line can be provided with a reporter gene operably linked to an expression control sequence comprising a promoter and the upstream activation sequence for yeast GAL4p transcription factor.
  • Such expression control systems are activated by GAL4p or hybrid mammalian/yeast GAL4p molecules, expressed in mammalian cells.
  • the cells are further provided with a fusion gene encoding a hybrid protein comprising GAL4p DNA binding and transcription activation domains fused to the RB binding domain of E2F1.
  • the modular functions of transcription factors will allow the fusion gene product to retain the RB-mediated regulation of E2F1. That is, phosphorylation of RB by cyclin E/CDK2 will release RB from its interaction with a fusion gene product and allow the GAL4p domains of the fusion gene product to bind the GAL4 UAS. Reporter gene expression is thus rendered dependent on cyclin E/CDK2 phosphorylation of Rb, but expression of the fusion gene is independent of cyclin A/CDK2 mediated phosphorylation of E2F and inactivation.
  • the recently identified mouse DMPl is a myb-like transcription factor which activates promoters containing the sequences CCCG(G or T)ATGT.
  • DMPl binds to cyclin D in vitro and when coexpressed with cyclin D in insect cells and, furthermore, is phosphorylated by cyclin D/CDK4 under these conditions (Hirai and Sherr, 1966, Mol. Cell. Biol. 16:6457-6467). If the ability of DMPl to activate promoters is dependent on its phosphorylation state, then expression of reporter genes containing DMPl binding sites in the promoter may be used as an indirect measurement of cyclin D/CDK4 function.
  • Hybrid genes The invention can be practiced with hybrid gene products in order to achieve certain advantages.
  • the hybrid genes used with the current invention comprise a first coding region and a second coding region from different sources (discussed below) that are operably linked together.
  • the first coding region is derived from a first gene that is native, or endogenous, to the cell line being used in the screen and which affects phosphorylation by the cyclin/CDK phosphorylation system that regulates transcription of the reporter gene in the cell line.
  • the chromosomal copy of the first gene can be mutated or disrupted and expression of the reporter gene analyzed using techniques well known in the art (Kaiser et al.
  • the first gene has sequence homology to at least one gene known to be involved in cell cycle regulation. More preferably, the first gene encodes a protein selected from the group consisting of cyclins, CDKs and cyclin kinase inhibitors.
  • the second coding region of a hybrid gene of the current invention is from a second, non-native, target (often mammalian) gene, wherein the target gene displays homology to the first gene.
  • the coding regions from the first and second genes are chosen according to information available in the art, preserving the portions of the first gene that are required to exert its regulation over expression of the reporter gene. For example, genes encoding hybrids between yeast CDC28p and PHO85p have been constructed, providing preliminary data regarding residues of PHO85p that are required to retain PHO85p specific kinase function in a yeast hybrid protein (Santos et al , 1995).
  • the structure of amino acids in the catalytic site (Arg 33, Glu 51, Asp 145) is altered by cyclin A binding such that the ⁇ -y bond of ATP is moved into a position favorable for nucleophilic attack from a bound substrate protein.
  • the structure determination by Jeffrey et al. (1995) also reveals the regions of contact between human cyclin A and CDK2.
  • the cyclin makes primary contact to the PSTAIRE region of CDK2 and also binds to the T loop region.
  • the differences between the crystal structure of the cyclin A/CDK2 complex and that previously determined for free CDK2 illustrates the conformational changes in the CDK induced upon cyclin binding which confer catalytic activity to the complex.
  • the second, target gene of this invention is homologous to the first gene, thus the target gene preferably is or has sequence homology to a gene known to be involved in cell cycle regulation.
  • the second, target gene encodes a protein selected from the group consisting of cyclins, CDKs and cyclin kinase inhibitors.
  • the target is a mammalian gene and is cyclin A, cyclin E, a human D-type cyclin, CDK2, CDK4, CDK6, pl6 INK4a , p21 or p27.
  • the general strategy for constructing functional hybrid genes of the current invention is as follows.
  • a hybrid gene is constructed to include a region of a first, native gene that is deduced, based on structural and functional studies and assays available in the art, to be necessary for transcriptional control of the expression control sequences linked to the reporter gene.
  • the hybrid gene is expressed in a cell line harboring a reporter gene construct, where the chromosomal copy of the first, native gene is mutant or disrupted. Reporter gene expression is examined to determine whether the hybrid gene product has functionally substituted for the first gene to effect normal regulation of reporter gene expression. If the hybrid gene product properly regulates reporter gene transcription, it can further be determined whether the hybrid gene product utilizes the other components (cyclin/CDK/CKI) of the particular assay system or if the hybrid gene uses other homologous genes native to the cell line. If the hybrid gene product does not properly regulate transcription of the reporter gene, the hybrid gene can be expressed in the same yeast strain together with cDNAs encoding the cyclin/CDK/CKI components that natively associate with the target gene.
  • hybrid e.g., mammalian/yeast
  • the resulting phosphorylating system can be used according to the present invention.
  • Hybrid CDK proteins have previously been constructed between two homologous yeast CDKs, PH085 and CDC28 (Santos et al , 1995, Mol. Cell Biol. JL5:5482-91). Analyses of these hybrids determined that the critical regions of PHO85p required to retain transcription regulation function are residues 155 to 254. The current invention capitalizes on the new demonstration that a target/host, e.g.
  • hybrid protein can substitute for the function of an endogenous gene involved in cyclin/CDK regulation.
  • the hybrid gene has sequence homology to genes known to be involved in cell cycle regulation. More preferably, the hybrid gene encodes a hybrid protein homologous to proteins selected from the group consisting of cyclins, CDKs and cyclin kinase inhibitors.
  • the first, native gene is PH085 and the corresponding first coding region used in the hybrid gene encodes amino acids 155 to 302 of PH085; and the second, target gene is human CDK2 and the corresponding second coding region used in the hybrid gene encodes amino acids 1 to 151 of human CDK2 (CK2-P85#1).
  • the hybrid gene is comprised of a first coding region encoding amino acids 155 to 251 from PH085, a second coding region encoding amino acids 1 to 151 from human CDK2, and a third coding region encoding amino acids 256-298 of CDK2 (CK2- P85#2).
  • the particular coding regions are joined in a relative order consistent with their native positions within their respective genes, forming the hybrid genes depicted in Figure 2.
  • the structure of the hybrid CDKs constructed are depicted with regions derived from human CDK2 depicted as solid bars and regions derived from PHO85p represented as open bars. The amino acid position, from the native protein sequences, at the amino and carboxyl ends of each region of the hybrids is indicated.
  • hybrid CDKs In constructing hybrid CDKs, convenient restriction sites in the PH085 gene can be employed. Examples are Hindlll, which cleaves at codon 9 in the amino terminus; Bglll, which cleaves at codon 51 , just on the carboxy end of the PSTAIRE motif; and EcoRI, which cleaves at codon 80 between the amino and carboxyl lobes of the CDK.
  • genes encoding specific hybrid mammalian/yeast or other target/host CDKs can be assembled using two-step PCR procedures utilizing appropriately designed primers, as is demonstrated in Example 4.
  • the hybrid gene is comprised of one region of the first, native gene and one region of the second, target gene.
  • the hybrid gene can contain numerous discontinuous regions of each of the first and second gene.
  • selected regions of the protein may be identified which retain the sequences of the first gene necessary to preserve transcription regulatory function, while all remaining sequences can be replaced by sequences from a homologous target mammalian gene.
  • hCDK2 is a cell cycle regulation protein that is capable of alternately forming complexes with cyclin E or cyclin A.
  • a number of cancers have been identified in which mutations in p53 or failure to produce functional p21 alters regulation the cyclin E/CDK2 complex (reviewed in Hunter and Pines, 1994; Sherr, 1996).
  • agents that inhibit cyclin E/CDK2 function can be identified.
  • agents that inhibit cyclin E/CDK2 function such as p21 mimetics, can be identified.
  • several cancers are associated with defects in cyclin A, which affects cell cycle regulation through the cyclin A/CDK2 complex.
  • Manipulation of a yeast cell strain such that reporter gene expression is jointly dependent on cyclin A sequences and CDK2 allows identification of inhibitors of the cyclin A/CDK2 complex.
  • the human CKI and tumor suppressor pl6 INK4a is mutated or deleted in a variety of tumor cell lines and primary tumors.
  • the suppressor pl6 INK4a inhibits CDK4, and compounds that mimic the effect of pl6 1NK4a may be useful cancer therapeutic agents.
  • Suppressor pl6 INK4a exhibits significant homology to the yeast CKI PHO81p in an ankyrin repeat domain. The observation that the PHO81p ankyrin domain alone (amino acids 584 to 724) is capable of inhibiting PHO80p/PHO85p in vivo (Ogawa et al , 1995, Mol. Cell. Biol. 15:997-1004) indicates that a hybrid CKI gene including the PHO81p ankyrin domain can retain the functions necessary for appropriate transcriptional regulation of the PH05 expression control sequences.
  • the native pl6 INK4a protein may functionally replace the PHO81p CKI.
  • the hybrid gene is a CKI, where the first, native gene is PH081 and the second, target gene is pl6 IN 4a .
  • the ankyrin repeat domain of PH081 is used in the hybrid gene.
  • the functional hybrid gene is identified by its ability to inhibit the PHO80p/PHO85p phosphorylation of PHO4p transcription factor. Because phosphorylation leads to transcriptional repression, a pho81 disruption strain represses transcription in both high and low phosphate. A functional hybrid CKI gene restores this strain's ability to derepress transcription.
  • the hybrid gene is a CDK, where the first gene is PHO85p and the second gene is CDK4. Functional hybrids can be identified by the ability to confer PH05 promoter repression in high phosphate in a pho85 mutant host.
  • a mutation or deletion can be introduced in a hybrid gene, where the hybrid gene in the absence of the mutation provides a gene product effective to permit normal phosphorylation control of reporter gene transcription.
  • Example 4 illustrates such a mutation, CK2-P85ml , which has a single-codon deletion in the hybrid gene CK2-85#1.
  • the hybrid gene mutation or deletion preferably corresponds to a mutation associated with a disease state.
  • Use of the mutated hybrid gene in the screens of the current invention allows targeted screening for agents that overcome specific mutations in cell cycle regulatory proteins.
  • the assay system using a PHO85p/CDK4p hybrid gene can be used to screen for compounds which restore pl6 INK4a responsiveness to mutant CDK4.
  • novel inhibitors of the mutant CDK4 can be identified, which would be useful therapeutically to treat these melanomas.
  • a preferred embodiment of the current invention allows measurement of the activity of hybrid mammalian/yeast proteins.
  • native human cell cycle regulatory proteins can in some cases substitute for the first, native gene product that affects cyclin/CDK mediated transcriptional regulation of the reporter gene. This substitution, or complementation, can easily be tested using the same assay methods as described for testing hybrid genes. That is, the chromosomal copy of the first, native gene is disrupted, and the human cell cycle regulatory gene is expressed in a cell that harbors a reporter gene construct that is transcriptionally regulated by the first, native gene. Control of expression of the reporter gene can be analyzed to determine whether the human gene can complement a mutation in the first gene.
  • the hybrid and native mammalian genes of the current invention can be expressed from DNA incorporated into the chromosome.
  • these genes are expressed extrachromosomally, preferably from a plasmid.
  • the hybrid and native mammalian genes are preferably expressed from expression control sequences that are derived from the first, native gene, which is complemented by the hybrid or full-length mammalian gene.
  • use of expression control sequences from the first, native gene, which is homologous to the hybrid or full-length mammalian gene allows expression of hybrid and full-length mammalian genes at the levels and times similar to the native expression for the cyclin/CDK phosphorylation system being utilized.
  • a hybrid or full-length mammalian gene is preferably expressed from PH085 gene expression control sequences.
  • expression vectors are used which allow high-level constitutive expression of the hybrid or full-length mammalian gene; an especially preferred vector is pBT6, which uses the efficient TDH3 gene promoter. Expression vectors facilitating cloning and expression from appropriate expression control sequences are provided in the current invention and explained in detail in Example 3.
  • vectors are useful when the PH05 controlling cyclin/CDK regulon is utilized; the vectors contain expression control sequences from PH085 (pBT16), PHO80 (pBT15) and PH081 (pBT17) and are provided for expression, respectively, of CDKs, cyclins and CKIs.
  • a cell line can be manipulated to mutate or otherwise disrupt chromosomal genes. For example, when hybrid or full-length mammalian genes are assayed for whether they confer the transcription regulatory function of the first gene, the assays can be performed with haploid cells in which the chromosomal copy of the first gene is mutant or disrupted. Similarly, when a first, native gene is substituted for by a hybrid or full-length mammalian gene, the cell line is most preferably made mutant for the first, native gene. Genetic and molecular techniques for targeting chromosomal genes for disruption or mutation are well known in the art (Kaiser et al.
  • Mutant strains display the expected phenotype as explained herein. For example, a pho81 disruption strain fails to derepress reporter gene expression in low phosphate, and a pho85 and/or pho80 disruption strain fails to repress reporter gene expression in high phosphate. Phenotypic indications that the desired gene was disrupted are to be confirmed by molecular analysis.
  • the components of the current invention as described above provide an assay system that allows for selective growth, or positive selection, of cells exhibiting the desired cyclin/CDK transcriptional control.
  • cells with an intact, functional cyclin/CDK complex are used to select for agents that impair the functional cyclin/CDK activity.
  • cells with functional cyclin/CDK/CKI regulons are used to select for agents that impair the functional CKI activity.
  • Another embodiment allows selection for cells having mutations in cyclin or CDK that disrupt the normal regulation of phosphorylation by the complex.
  • Example 7 details two positive selection regimens encompassed by the current invention. Assays involving selection rather than screening afford the ability to assay the effects of a large number of candidate agents.
  • Candidate agents can be from any source, such as small molecule chemical libraries, combinatorial libraries, cDNA libraries, random peptide libraries, and random RNA libraries.
  • selections allow the purification (cloning), from a complex mixture, of DNA encoding the molecule with the desired biological activity.
  • libraries and methods of preparing them are available in the art (Freier et al., 1995, J. Medicinal Chem. 38: 344-52). Cell growth can be measured by visual inspection, by spectrophotometric analysis of cell density of liquid cultures, or by plating for single colonies on the appropriate selective plates.
  • sources for agents tested in selection regimens include cDNA libraries, random peptide libraries, and random RNA libraries.
  • multiple embodiments of the current invention utilize reporter genes whose gene products can be readily detected.
  • the screens of the current invention utilize reporter genes whose gene products are detectable by a spectrophotometric or colorimetric assay, such as genes encoding ⁇ -galactosidase, green fluorescent protein, luciferase and CAT. It is appreciated that these reporter genes allow rapid, automated screening of a wide range of candidate agents.
  • sources of candidate agents include extracts from natural sources, synthetic compounds, small molecule chemical libraries, combinatorial libraries, cDNA libraries, random peptide libraries, and random RNA libraries.
  • small molecule libraries and combinatorial libraries are applied to the screens of the current invention.
  • products of rational design programs can be assayed for activity in these screens.
  • the percentage of pharmacologically active compounds identified in a cell-based screen can be increased by maximizing cellular uptake and retention of test compounds.
  • the screening cell line's ability to import and/or retain test compounds is enhanced by altering one or more of the genes involved in transporting molecules across the plasma membrane, thus causing increased or decreased activity or levels of transport proteins.
  • polar molecules are transported by membrane proteins which are generally classified within three categories: channels, facilitators (also termed permeases, carriers or transporters) and pumps.
  • MDR muitidrug resistance
  • PDR pleiotropic drug resistance
  • inactivation of the yeast PDR1 and/or PDR3 genes decreases the levels of the PDR5p, SNQ2p, YORlp proteins (and possibly other ABC transporters).
  • strains optimized for screening libraries of particular chemical classes of compounds may be generated.
  • Secondary Screening Agents that demonstrate activity in a screening or selection assay of the current invention are subjected to secondary screens to confirm activity and specificity. For example, compounds identified as inhibitors of a cyclin and a hybrid CDK2/PHO85p complex are tested in a secondary screen using the native yeast PHO80p/PHO85p system. This secondary screen distinguishes compounds that are specific to the human CDK2 epitopes from those targeted to both CDK2 and PHO85p or specifically to PHO85p epitopes. Secondary screening may also be useful, when necessary, to demonstrate that the agent acts through the cyclin CDK complex and does not exert a direct effect on the transcription factor(s) in the assay. Experimental techniques used to conduct secondary testing utilize similar techniques as those described above and will be apparent to those of ordinary skill in the art.
  • the invention includes a method of screening for a compound that affects mammalian cell cycle regulatory proteins, comprising administering a compound to a cell line, wherein the cell line comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an Upstream Activation Sequence and a promoter, and the Upstream Activation Sequence comprises a DNA region that binds to a transcription control factor that is regulated through phosphorylation by a cyclin/CDK phosphorylation system; and an effector gene providing a gene product effective to permit normal cyclin/CDK regulation of the transcription control factor; and analyzing expression of the reporter gene in the cell line, thereby determining whether the compound affects the normal regulation.
  • Another method of the invention is a method of identifying a gene that affects mammalian cell cycle regulatory proteins, comprising providing a cell line that comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an Upstream Activation Sequence and a promoter, and the Upstream Activation Sequence comprises a DNA region that binds to a transcription control factor that is regulated through phosphorylation by a cyclin/CDK phosphorylation system; and an effector gene providing a gene product effective to permit normal cyclin/CDK regulation of the transcription control factor; introducing into the cell line expression of an exogenous gene; and analyzing expression of the reporter gene, thereby determining whether the exogenous gene affects the normal regulation.
  • An alternative embodiment of the invention is a method of identifying a gene that affects mammalian cell cycle regulatory proteins, comprising providing a cell line that comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an Upstream Activation Sequence and a promoter, and the Upstream Activation Sequence comprises a DNA region that binds to a transcription control factor that is regulated through phosphorylation by a cyclin/CDK phosphorylation system; and an effector gene providing a gene product effective to permit normal cyclin/CDK regulation of the transcription control factor; introducing a mutation in a chromosomal test gene; and analyzing expression of the reporter gene, thereby determining whether the test gene affects the normal regulation.
  • the effector gene is a hybrid gene comprising a first coding region from a gene native to the cell line and a second coding region from a second gene, wherein the native gene encodes a gene product that affects phosphorylation by the cyclin/CDK phosphorylation system, and the second gene is mammalian and is homologous to the native gene;
  • the cell line further comprises a chromosomal mutation in the native gene;
  • the effector gene is a mammalian gene; the mammalian gene is homologous to a native gene;
  • the mammalian gene is a cyclin-dependent kinase;
  • the mammalian gene encodes hCDK2; the mammalian gene encodes the amino terminus of hCDK2.
  • the invention also includes a method of screening for a compound that affects mammalian cell cycle regulatory proteins, comprising administering a compound to a cell line, wherein the cell line comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated, directly or indirectly, through phosphorylation by a cyclin/CDK phosphorylation system; and a hybrid gene comprising a first coding region from a gene native to the cell line and a second coding region from a second target gene, wherein the native gene encodes a gene product that is involved in phosphorylation by the cyclin/CDK phosphorylation system, and the second gene is mammalian and is homologous to the native gene, and the hybrid gene provides a gene product effective to permit normal cyclin/CDK regulation of the transcription control factor; and analyzing expression of the
  • Also part of the invention is a cell line comprising genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated, directly or indirectly, through phosphorylation by a cyclin CDK phosphorylation system; and a hybrid gene comprising a first coding region from a gene native to the cell line and a second coding region from a second gene, wherein the native gene encodes a gene product that affects phosphorylation by the cyclin/CDK phosphorylation system, and the second gene is mammalian and is homologous to the native gene, and the hybrid gene provides a gene product effective to permit normal cyclin/CDK regulation of the transcription control factor.
  • the gene expression control sequence comprises an upstream activation sequence and a promoter
  • the upstream activation sequence comprises a DNA region that binds to a transcription control
  • a different method of the invention provides a method of identifying a gene that affects mammalian cell cycle regulatory proteins, comprising providing a cell line that comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated, directly or indirectly , through phosphorylation by a cyclin/CDK phosphorylation system ; and a hybrid gene comprising a first coding region from a gene native to the cell line and a second coding region from a second gene, wherein the native gene encodes a gene product that affects phosphorylation by the cyclin/CDK phosphorylation system, and the second gene is mammalian and is homologous to the native gene, and the hybrid gene provides a gene product effective to permit normal cyclin/CDK regulation of the transcription control factor; and introducing into the cell line expression of an exogenous
  • Screening tests of the invention include a method of identifying a gene that affects mammalian cell cycle regulatory proteins, comprising providing a cell line that comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated, directly or indirectly, through phosphorylation by a cyclin/CDK phosphorylation system; and a hybrid gene comprising a first coding region from a gene native to the cell line and a second coding region from a second gene, wherein the native gene encodes a gene product that affects phosphorylation by the cyclin/CDK phosphorylation system, and the second gene is mammalian and is homologous to the native gene, and the hybrid gene provides a gene product effective to permit normal cyclin/CDK regulation of the transcription control factor; and introducing a mutation in a chromosomal test
  • Other screening methods include a method of screening for a compound that inhibits mammalian CDKs, comprising administering a compound to a cell line, wherein the cell line comprises genetic information comprising a reporter gene, wherein expression of the reporter gene confers to the cell line the ability to grow in certain media; the reporter gene is operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence is from the PH05 gene, wherein the gene expression control sequence is transcriptionally regulated by a cyclin/CDK phosphorylation system; and a hybrid gene comprising a first coding region from a gene native to the cell line and a second coding region from a second gene, wherein the native gene affects regulation by the cyclin/CDK phosphorylation system, and the second gene is mammalian and is homologous to the native gene, and the hybrid gene provides a gene product effective to permit normal cyclin CDK regulation of the transcription control factor; and growing cells
  • hybrid genes include those in which the hybrid gene is expressed from a plasmid; the hybrid gene is a cyclin-dependent kinase; the native gene is PH085; the second gene is hCDK2; the chromosomal PH085 gene is non-functional; the hybrid gene is selected from the group consisting of CK2-P85#1, CK2-P85#2, CK2-P85ml, CK2-P85 ⁇ C and CK4-P85#1 ; the cell line has a chromosomal mutation in the native gene; the hybrid gene is expressed from a PH085 promoter; the hybrid gene is expressed from the plasmid expression vector pBTl, pBT6, pYES2 or pBT16; the hybrid gene encodes a hybrid cyclin; the native gene is PHO80; the second gene is human cyclin A; the second gene is human cyclin E; the second gene
  • the invention also includes a method of screening for a compound that affects mammalian cell cycle regulatory proteins, comprising administering a compound to a cell line, wherein the cell line comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated, directly or indirectly, through phosphorylation by a cyclin/CDK phosphorylation system; and a mammalian gene homologous to a gene native to the cell line, wherein the native gene affects the regulation by the cyclin/CDK phosphorylation system and the mammalian gene is effective to permit normal phosphorylation control of the transcription factor; and analyzing expression of the reporter gene in the cell line, thereby determining whether the compound affects the normal regulation.
  • Also part of the invention is a cell line comprising genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated, directly or indirectly, through phosphorylation by a cyclin/CDK phosphorylation system; and a mammalian gene homologous to a gene native to the cell line, wherein the native gene affects the regulation by the cyclin/CDK phosphorylation system and the mammalian gene is effective to permit normal phosphorylation control of the transcription factor.
  • a different method of the invention provides a method of identifying a gene that affects mammalian cell cycle regulatory proteins, comprising providing a cell line that comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated, directly or indirectly, through phosphorylation by a cyclin/CDK phosphorylation system; and a mammalian gene homologous to a gene native to the cell line, wherein the native gene affects the regulation by the cyclin/CDK phosphorylation system and the mammalian gene is effective to permit normal phosphorylation control of the transcription factor; and introducing into the cell line expression of an exogenous gene; analyzing expression of the reporter gene, thereby determining whether the exogenous gene affects the normal regulation.
  • Screening tests of the invention include a method of identifying a gene that affects mammalian cell cycle regulatory proteins, comprising providing a cell line that comprises genetic information comprising a reporter gene operably linked to a gene expression control sequence, wherein the gene expression control sequence comprises an upstream activation sequence and a promoter, and the upstream activation sequence comprises a DNA region that binds to a transcription control factor that is regulated, directly or indirectly, through phosphorylation by a cyclin/CDK phosphorylation system; and a mammalian gene homologous to a gene native to the cell line, wherein the native gene affects the regulation by the cyclin/CDK phosphorylation system and the mammalian gene is effective to permit normal phosphorylation control of the transcription factor; and introducing a mutation in a chromosomal test gene; analyzing expression of the reporter gene, thereby determining whether the test gene affects the normal regulation.
  • the gene expression control sequence comprises sequences native to the cell line;
  • the reporter gene is selected from the group consisting of E. coli ⁇ - galactosidase, E. coli tn5 neo, the S. cerevisiae genes LEU2, URA3, HIS3, LYS2, A. victoria green fluorescent protein gene, the P.
  • the host cell has a chromosomal mutation in the native gene;
  • the host cell line is a yeast cell line;
  • the host cell line is a strain of Saccharomyces cerevisiae;
  • the cell line has a genetic alteration impairing export of molecules from the cell;
  • the cell line has a genetic alteration enhancing transport of molecules into the cell;
  • the cell line is a yeast cell line with a chromosomal mutation in the PDR5, SN22, YOR1, PDR1 or PDR3 genes;
  • the expression control sequence is from the PH05 gene of S. cerevisiae;
  • the upstream activation sequence is from the PH05 gene of S.
  • the CDK is PH085; the chromosomal PH085 gene is non-functional; the cell line is a mammalian cell line; the transcription control factor is selected from the group consisting of the E2F family of mammalian transcription factors; the CDK is CDK2; and selecting for strains that express or fail to express the reporter gene.
  • a compound or gene affecting mammalian cell cycle regulatory proteins that is obtained by a method described above is also an embodiment of the invention.
  • EXAMPLE 1 - CELL LINES, PLASMIDS, GENES AND EXPRESSION VECTORS. Strains and Culture Media. Constructed plasmids were cloned in E. coli HB101 or DH5G:, using ampicillin selection on LB plates. Saccharomyces cerevisiae strains used in these examples are CM-1 (MAT pep4-3 trplA ura3; Bitter et al , 1991, Mol. Gen. Genet.
  • YPH499 MAT a ura3-52 lys2-801 ade2-101 trpl-A63 his3-A200 Ieu2-Al; Sikorski and Hieter, 1989, Genetics 122: 19-27
  • YPH500 MAT ura3-52 lys2-801 ade2- 101 trpl-A63 his3-A200 Ieu2-Al; Sikorski and Hieter, 1989.
  • Selective yeast media is SD (0.67% yeast nitrogen base without amino acids, 2% dextrose) containing the appropriate nutritional supplements.
  • Plasmids pBTl, pBT3 and pBT5 contain a URA3 selectable marker while plasmids pBTll, pBT12 and pBT13 include a TRP1 selectable marker gene (below).
  • Yeast were transformed by the lithium acetate procedure (Ito et al, 1983, J. Bacteriol. 153:163-168).
  • Low and high phosphate media were prepared as follows. Phosphate was precipitated as MgNH 4 PO 4 from a 10X stock (1.7% w/v) of yeast nitrogen base without amino acids and without ammonium sulfate (YNB w/o aa and AS) as described by O'Connell and Baker (1992, Genetics 132:63-73).
  • Low phosphate media consisted of 0.17 % phosphate depleted YNB w/o aa and AS, 0.5% (NH 4 ) 2 SO 4 , 2% dextrose, any required nutritional supplements and 20 mg/L KH 2 PO 4 .
  • High phosphate media contained, instead, 1500 mg/L KH 2 PO 4 .
  • Yeast strain YBT1 containing a disruption of the chromosomal PH085 gene, was constructed as follows. The PH085 gene was PCR amplified as a -949 bp fragment (below), restricted and cloned into the BamHI site of pRS405 (Sikorski and Hieter, 1989) to generate the plasmid pRS405/PHO85.
  • the PCR amplified HIS3 gene fragment (below) was digested with BamHI and ligated into the Bglll site (codon 49 of the PH085 gene) in pRS405/PHO85 to generate pRS405/pho85 : : HIS3.
  • This plasmid was digested with BamHI to release the linear pho85 : :HIS3 gene disruption fragment and transformed into strain YPH500, selecting for histidine prototropy.
  • Strain YBT1 has the genotype MAT a ura3-52 lys2-801 ade2-101 trpl-A63 his3-A200 Ieu2-Al pho85 HIS3.
  • Yeast strain YBT3 containing a disruption of the chromosomal PH080 gene, was constructed as follows.
  • the PH080 gene was PCR amplified as a -922 bp fragment (below), restricted and cloned into the BamHI site of pRS403 ⁇ C (pRS403 in which the CM site was deleted by restriction, end-filling with Taq DNA polymerase, and religation) to generate the plasmid pRS403 ⁇ C/PH0S0.
  • ADE2 gene fragment (below) was digested with CM and ligated into the CM site (codon 101 of the PH080 gene) in ⁇ RS403 ⁇ C/PHO80 to generate pRS403 ⁇ C/pho80 : : ADE2.
  • This plasmid was digested with BamHI to release the linear pho80 : '.
  • ADE2 gene disruption fragment and transformed into strain YPH499, selecting for adenine prototropy.
  • Strain YBT3 has the genotype MAT a ura3-52 lys2-801 ade2-101 trpl-A63 his3-A200 Ieu2-Al pho80 ⁇ ADE2.
  • Yeast strain pdr5::URA3 is an improved cell line which, due to a disruption of the PDR5 gene encoding a yeast homologue of a mammalian multidrug resistance pump protein, has impaired ability to transport certain compounds out of the cell.
  • the PDR5 gene was disrupted as follows. The 5' end of the PDR5 gene was amplified from S. cerevisiae S288C DNA using the 5' primer, SEQ ID NO:42 and the 3' primer, SEQ ID NO:43. The 3' end of the PDR5 gene was PCR amplified with the 5' primer, SEQ ID NO:44, and the 3' primer, SEQ ID NO:45.
  • SEQ ID NO:44 is complementary to the 5' end of SEQ ID NO: 43.
  • An overlap extension reaction of the two PCR products thus results in a polynucleotide having 260bp of 5' flanking DNA plus the first four amino acid codons of the PDR5 gene fused to the last 8 amino acid codons of PDR5 plus 253bp of 3' flanking DNA.
  • Approximately equimolar amounts of each product were mixed and PCR amplified in the presence of terminal 5' primer, SEQ ID NO:42, and terminal 3' primer, SEQ ID NO:45.
  • the predominant PCR overlap extension product was the approximately 582 bp fusion polynucleotide which contains internal Xbal and Bglll sites.
  • the fragment was digested with Kpnl and Hindlll and cloned into pUC19 to generate the pUC19/PDR5 plasmid, which was subsequently digested with Xbal and Bglll. Then, the yeast URA3 gene was excised from pBTl as an approximately 1260 bp Xbal to Bglll fragment, gel purified, and cloned between the Xbal and Bglll sites within the PDR5 portion of the pUC19/PDR5 plasmid.
  • the pdr5::URA3 gene disruption fragment was excised with Kpnl and Hindlll and used to transform yeast strain YBT1 containing pBTll/Z, and uracil prototrophs were selected. Disruption of the chromosomal PDR5 gene was confirmed by PCR analysis of chromosomal DNA using appropriate primers. From the pdr5::URA3 strain, a strain was isolated which was resistant to 5- fluoroorotic acid due to an uncharacterized mutation in ura3. This pdr5::ura3 strain can be used for introduction of plasmids with a URA3 marker gene.
  • Yeast strain pdr ⁇ A which contains a null mutation of the PDR5 gene and lacks the URA3 gene was derived from the pdr5::URA3 strain as follows. ⁇ epdr5::URA3 strain was transformed with the 582 bp PDR5 overlap extension product which was excised from pUC19/pdr5 with Kpnl and Hindlll and selected on plates containing 5 -fluoroorotic acid. As a result of homologous recombination, 5 -fluoroorotic acid resistant cells have deleted the URA3 gene and flanking PDR5 DNA. Generation of the pdr5A null mutation was confirmed by PCR analysis of chromosomal DNA using appropriate primers.
  • Yeast strain pdrl::URA3 is an improved cell line which, due to a disruption of the PDR1 gene encoding a zinc finger protein required for expression of the PDR5, SNQ2 and YOR1 genes, has impaired ability to transport certain compounds out of the cell.
  • the PDR1 gene was disrupted as follows. The 5' end of the PDR1 gene was amplified from S. cerevisiae S288C DNA using 5' primer, SEQ ID NO:46, and 3' primer, SEQ ID NO:47. The 3' end of the PDR1 gene was PCR amplified with the 5' primer, SEQ ID NO:48, and the 3' primer, SEQ ID NO:49.
  • SEQ ID NO:48 is complementary to the 5' end of SEQ ID NO:47.
  • An overlap extension reaction of the two PCR products thus results in a polynucleotide having 273 bp of 5' flanking DNA plus the first seven amino acid codons of the PDR1 gene fused to the last nine codons of PDR1 plus 373 bp of 3' flanking DNA.
  • Approximately equimolar amounts of each product were mixed and PCR amplified in the presence of terminal 5' primer SEQ ID NO:46 and terminal 3' primer SEQ ID NO:49.
  • the predominant PCR overlap extension product was the approximately 730 bp fusion polynucleotide which contains internal Xbal and Bglll sites.
  • the fragment was digested with Kpnl and Hindlll and cloned into pUC19 to generate pUC19/PDRl , which was subsequently digested with Xbal and Bglll.
  • the yeast URA3 gene was excised from pBTl as an approximately 1260 bp Xbal to Bglll fragment, gel purified and cloned between the Xbal and Bglll sites within the PDR1 portion of the pUC19/PDRl plasmid.
  • the pdrl::URA3 gene disruption fragment is excised with Kpnl and Hindlll and used to transform yeast strain YBT1 containing pBTl l/Z, and uracil prototrophs were selected.
  • telomere pdrl::URA3 strain Disruption of the chromosomal PDR1 gene in the resulting pdrl::URA3 strain is confirmed by PCR analysis of chromosomal DNA using appropriate primers. From the pdrl::URA3 strain, a strain is isolated which is resistant to 5 -fluoroorotic acid due to an uncharacterized mutation in ura3. This pdrl::ura3 strain can be used for introduction of plasmids with a URA3 marker gene. Yeast strain pdrlA, which contains a null mutation of the PDR1 gene and lacks the URA3 gene is derived from the pdrl::URA3 strain as follows.
  • the pdrl::URA3 strain is transformed with the 730 bp PDR1 overlap extension product which was excised from pUC19/pdrl with Kpnl and Hindlll and selected on plates containing 5-fluoroorotic acid. Homologous recombination gives rise to 5-fluoroorotic acid resistant cells lacking the URA3 gene and flanking PDR1 DNA. Generation of the pdrlA null mutation is confirmed by PCR analysis of chromosomal DNA using appropriate primers.
  • yeast integrative plasmids pRS403 and pRS405 (Sikorski and Hieter, 1989) were purchased from Stratagene, the yeast expression vector pYES2 was purchased from Invitrogen and plasmid pCl-neo was purchased from Promega.
  • Yeast expression vectors pBTl , pBT3 and pBT5 were constructed from, respectively, pGPD(s), pGPD( ⁇ GPE) and pGP381 as follows. The parent vectors are identical except for the DNA sequence included in the promoter region (Bitter et al , 1991), and the same assembly strategy was used to generate each of the new expression vectors.
  • the — 599 bp Bglll to EcoRI fragment of the resulting vectors was replaced with the yeast ARS1 element, which was PCR amplified as a - 280 bp fragment from pGPD(s)/Z using the primers, SEQ ID NO:3 and SEQ ID NO:4.
  • This final step completed the construction of yeast expression vectors pBTl, pBT3 and pBT5.
  • Yeast expression vector pBT6 was constructed from pBTl by cloning the ohgonucleotide obtained by annealing SEQ ID NO:5 and SEQ ID NO:41, as a Bglll to BamHI fragment into the BamHI site and selecting a clone which regenerates the BamHI site adjacent to the PGK terminator region.
  • Figure 3 depicts several of these expression vectors.
  • Yeast expression vectors regulated by phosphate concentration were constructed as follows. The - 375 bp Kpnl to BamHI promoter fragment of pGP381 was replaced with the PCR amplified PH05 gene promoter region (below) to generate the vector pBTl 1.
  • the PCR amplified PH05 gene transcription termination region (below) was cloned into pBTl 1 , in the correct orientation relative to the promoter, as a BamHI to Bglll fragment to generate pBT12.
  • the UAS region from the PH05 gene promoter was PCR amplified (below) and ligated between the Kpnl and Sail sites of pGP381/Z (Bitter et al. , 1991) to generate the plasmid pBT13/Z.
  • the PH05 gene promoter (nucleotides -405 to -10 relative to the translation initiation site) was PCR amplified from S.
  • yeast PH05 gene promoter UAS (nucleotides -405 to -206) was PCR amplified from S. cerevisiae S288C DNA using the same 5' primer, SEQ ID NO:6, and the 3' primer, SEQ ID NO:8.
  • the yeast PH05 gene transcription termination region was PCR amplified from S. cerevisiae S288C DNA using the 5' primer, SEQ ID NO:9, and the 3' primer, SEQ ID NO:10.
  • yeast genes including the promoter regions, were PCR amplified for use as selectable markers on expression vectors or for gene disruptions (above).
  • the URA3 gene Rose et al, 1984, Gene 29:113-124 coding region plus -216 bp of 5' and -77 bp of 3' flanking sequence was PCR amplified from pYES2 with primers which introduced an Xbal site and Bglll site at the 5' and 3' ends, respectively.
  • the HIS3 gene coding region plus -495 bp of 5' and - 138 bp of 3' flanking sequence was PCR amplified from pYES2 (Invitrogen) with primers which introduced BamHI sites at the ends.
  • the ADE2 gene coding region (Stotz and Linder, 1990, Gene 95:91-98) plus -642 bp of 5' and - 128 bp of 3' flanking DNA was PCR amplified from S. cerevisiae S288C DNA with primers which introduced a C site at each end.
  • the following genes were PCR amplified and ligated into the above expression vectors as follows.
  • the yeast PH085 gene coding region (Uesono et al. , 1987, Nucl. Acids Res. 15: 10299-10309), including -20 bp of 5' and -20 bp of 3' untranslated region, was PCR amplified from S.
  • cerevisiae S288C genomic DNA (Promega) using the 5' primer, SEQ ID NO:ll, and the 3' primer, SEQ ID NO:12. After digestion with BamHI, the fragment was cloned into BamHI restricted pBTl to generate pBTl/PHO85 (correct orientation relative to promoter) and pBTl/PHO85R (reverse orientation).
  • the native S. cerevisiae PHO85p expressed in these studies corresponds to coding sequence 2 of GenBank Accession #Y00867, X13515 and encodes a 302 amino acid protein.
  • the yeast PH080 gene coding region (Madden et al. , 1988, Nucl. Acids Res. 16:2625- 2637), including -20 bp of 5' and — 18 bp of 3' untranslated region, was PCR amplified from S. cerevisiae S288C genomic DNA using the 5' primer, SEQ ID NO:13, and the 3' SEQ ID NO: 14.
  • the fragment was cloned into BamHI restricted pBTl to generate pBTl/PHO80 (correct orientation relative to promoter) and pBTl/PHO80R (reverse orientation).
  • the amplified fragment was also cloned into pBT3 to generate pBT3/PHO80 (correct orientation) and pBT3/PHO80R (reverse orientation).
  • the fragment was also cloned in the correct orientation in the BamHI site of pYES2 to generate pYES2/PHO80.
  • coli tn5 neo gene coding region including — 25 bp of 5' flanking and —23 bp of 3' flanking untranslated region, was PCR amplified from pCI-neo (Promega) using the 5' primer, SEQ ID NO:15, and the 3' primer, SEQ ID NO:16. After digestion with BamHI, the fragment was cloned in the correct orientation into BamHI digested pBT12 to generate pBT12/NEO.
  • the yeast LEU2 gene coding region (Andreadis et al , 1982, Cell 31:319-325), including approximately 27 bp of 5' and approximately 40 bp of 3' untranslated region, was PCR amplified from S.
  • the indicated expression vectors were transformed into strain CM-1 or YPH500 and grown to an OD 595 of approximately 1.0 in either low or high phosphate medium.
  • Cells were permeabilized and ⁇ -galactosidase assayed as described (Bitter et al , 1991). One unit equals an increase of 1 A 420 divided by the product of (minutes incubated, OD 595 of the permeabilized cell suspension, and mL of suspension assayed).
  • FIG. 4 A schematically depicts the 5' flanking region of the yeast PH05 gene.
  • Figure 4B depicts key features of the plasmid vectors pBTll, pBT12 and pBT13.
  • the E. coli LacZ gene was used as a reporter gene and was inserted into the unique BamHI site of pBTll or pBT13 (Example 1).
  • the native PH05 promoter incorporated in pBTl 1 is regulated by the P, concentration in the growth medium, being repressed in high 1 phosphate and induced by phosphate starvation (See Table II).
  • the hybrid
  • TDH3(UAS PH05 ) promoter in pBT13/Z is regulated by the phosphate concentration of the
  • the UAS PH05 functions as an enhancer with pBT13/Z
  • the level of ⁇ -galactosidase produced from pBTll/Z and pBT13/Z is 75-90% the level
  • Example 44 demonstrated in Example 2 is mediated by PHO85p and PHO80p, genetic experiments were
  • Haploid yeast strains were constructed containing disruptions of either the
  • vectors 5 allow cyclins, CDKs, and CKIs respectively to be expressed appropriately to restore high 6 phosphate repression and low phosphate derepression of the PH05 promoter to a cell 7 disrupted for a cyclin, CDK or CKI.
  • the Hindlll to BamHI fragment of vector pBT5 8 (identical to pBTl , except for the TDH3 promoter segment inco ⁇ orated) contains the GP3 ⁇ l 9 promoter. This fragment was replaced with the PHO80, PH085, or PH081 promoter.
  • the 0 PHO promoters were PCR amplified from S. cerevisiae S2 ⁇ C genomic DNA using the 1 primers indicated below.
  • Vector pBT16 includes approximately 613 bp from the 9 PH085 gene promoter (-616 to -4 relative to ATG initiation codon), isolated using the 5' 0 primer, SEQ ID NO:21, and the 3' primer, SEQ ID NO:22.
  • Vector pBT17 includes 1 approximately lOO ⁇ bp from the PH081 gene promoter (-1011 to -4 relative to the ATG 2 initiation codon), isolated using the 5' primer, SEQ ID NO:23, and the 3' primer, SEQ ID 3 NO:24. These vectors are useful for expressing, respectively, cyclins, CDKs or CKIs at levels and under similar regulation as the native PHO80, PH085 and PH08 genes.
  • Each of these vectors includes multiple cloning sites for genes to be inserted and expressed, and the restriction endonuclease map of each appears in Figure 5.
  • the native PH085 gene was expressed from pBT16 in strain YBT1
  • the native PHO80 gene was expressed from pBT15 in strain YBT3.
  • the results (Table IV) demonstrate complementation of the appropriate chromosomal disruption by each expression vector. Expression of the native PHO80 gene from pBT15 does not result in aberrant regulation as observed previously for high level expression of PHO80.
  • expression of PHO80 from pBT15 complements the chromosomal pho80 disruption, repressing the PH05 promoter of pBTll/Z in high phosphate and allowing derepression of the PH05 promoter in low 1 phosphate.
  • the CK2-P85#1 gene encodes amino acids 1 to 151 of hCDK2 0 fused to amino acids 155 to 302 of PHO85p.
  • the CK2-P85#2 gene encodes amino acids 1 1 to 151 of hCDK2 fused in frame to amino acids 155 to 251 of PHO85p which in turn is 2 fused in frame to amino acids 256 to 298 of hCDK2.
  • the CK2-P ⁇ 5ml gene is identical to 3 CK2-P ⁇ 5#l except for the deletion of the second alanine in the conserved GLARA motif (see 1 amino acid sequences of fusion regions at end of this Example). The first two gene fusions were expressed from vector pBT6, while CK2-P ⁇ 5ml was expressed from pYES2. Specific
  • the human CDK2 gene was obtained (ATCC #65967) in plasmid pSElOOO. This
  • yeast replicating plasmid with a URA3 marker has an approximately 1500 bp cDNA, ⁇ including all of the coding region of human CDK2, cloned downstream of the yeast GAL1 9 promoter (Elledge and Spottswood, 1991, EMBO J. , 10:2653-2659).
  • the 5' end of the PH085 5' primer is 6 complementary to the hCDK2 3' primer.
  • An overlap extension reaction of the two PCR 7 products thus results in a full length hybrid gene, including codons 1-151 of hCDK2 fused 8 in frame to codons 155-302 of PH085.
  • Approximately equimolar ratios of the two purified 9 PCR products were mixed and PCR amplified in the presence of the hCDK2 5' primer and 0 the PH085 3' primer.
  • the predominant amplification product in this second reaction was the 1 full length approximately 961 bp hCOK2-PH085 gene fusion.
  • the purified PCR reaction 2 products were digested with BamHI and Spel and cloned into vector pBT6, which had been 3 digested with BamHI and Spel to generate pBT6/CK2-P85# 1.
  • 4 5 Construction of CK2-P85#2 6 Codons 1 to 151 of hCDK2 were PCR amplified from pSElOOO DNA as described 7 for construction of CK2-P85#1 to isolate the amino terminal coding portion of hCDK2.
  • Codons 155 to 251 of PH085 were PCR amplified from S.
  • CK2-P85ml 2 A mutated version of CK2-P85#1, CK2-P ⁇ 5ml, was constructed as follows. Codons 3 I to 149 of hCDK2 were PCR amplified from pSElOOO using the 5' primer, SEQ ID NO:32, 4 and the 3' primer, SEQ ID NO:33.
  • Codons 154 to 302 of PH085 were PCR amplified from 5 S. cerevisiae S2 ⁇ C genomic DNA using the 5' primer, SEQ ID NO:34 and the 3' primer, 6 SEQ ID NO:28. 7 8 Fusion Junctions 9
  • the amino acid sequence of hCDK2 and PHO85p in the region of the first in frame fusion 0 is represented respectively by SEQ ID NO:35 and SEQ ID NO:36.
  • the amino acid 1 sequence at the first fusion junction is depicted in SEQ ID NO:37.
  • the amino acid sequence 2 of hCDK2 and PHO ⁇ 5p in the region of the second in frame fusion is represented, 3 respectively, by SEQ ID NO:38 and SEQ ID NO:39.
  • the amino acid sequence at the 4 second fusion junction is depicted in SEQ ID NO: 40.
  • 5 6 Construction of CK4-P85#1, a Hybrid of hCDK4 and PHO85 7 Codons 1-163 of human CDK4 (hCDK4) were PCR amplified from plasmid 8 pCMV/CDK4 (constructed by Sander van den Huevel, Massachusetts General Hospital and 9 obtained from Raymond Deshaies, California Institute of Technology) using the 5' primer, 0 SEQ ID NO-.50, and the 3' primer, SEQ ID NO:51.
  • Codons 154 to 302 of PH085 were 1 PCR amplified using the 5' primer, SEQ ID NO:52, and the 3' primer, SEQ ID NO:28.
  • SEQ ID NO:52 The 5' end of SEQ ID NO:52 is complementary to SEQ ID NO:51. Approximately 3 equimolar ratios of the two PCR products were mixed and PCR amplified in the presence of 1 primers SEQ ID NO:50 and SEQ ID NO:28. The predominant PCR overlap extension
  • Plasmid pYES2/CK4-P ⁇ 5#l ⁇ was transformed into strain YBTI containing plasmid pBTl 1/Z, and histidine, tryptophan and 9 uracil prototrophs were selected.
  • strain YBTI which contains a pho85::HIS3
  • the hybrid CK2-P85ml gene is also capable of complementing the pho85 disruption
  • strains expressed either the native yeast PH085 gene from vector pBTl, or the 1 hybrid CK2-P85ml gene from vector pYES2. Units /3-galactosidase produced in each strain
  • the cells are grown in glucose as the carbon source (repressing conditions for pYES2/CK2-
  • Codons 154 to 251 of PH085 were PCR amplified from S. cerevisiae S2 ⁇ C DNA
  • the amplification product includes an in-frame ⁇ ATG upstream of codon 154 of PH085. Codons 256 to 29 ⁇ of hCDK2 were PCR amplified 9 from pSElOOO using the 5' primer, SEQ ID NO:30, and the 3' primer, SEQ ID NO:31.
  • the PH085 3' primer is complementary to the 5' primer for the hCDK2 carboxy terminal
  • the amplification product has a termination codon after leucine at position 251.
  • Codons 1-151 of hCDK2 were PCR amplified from pSElOOO using the 5' primer,
  • Plasmid pBT6/CK2-N was also containing pBTll/Z selecting for uracil prototrophs. Plasmid pBT6/CK2-N was also
  • Example 5 demonstrated the ability of CK2-P ⁇ 5#2 to restore high-phosphate
  • the pBT6/CK2-N protein contains amino acids 1-151 of hCDK2 (which is
  • this truncated protein requires the yeast PH080 gene product for regulation l ⁇ of the PH05 promoter.
  • the first 151 amino acids of hCDK2 can substitute for
  • yeast LEU2 gene is involved in leucine biosynthesis.
  • Each gene was PCR
  • pBT12/LEU2 confers leucine prototropy to ⁇ 9- 33 93 % of YPH500 cells grown in low phosphate liquid medium and plated onto low phosphate 34 medium lacking leucine. Only 5-6% of the same pBT12/LEU2 transformed cells grow in 35 high phosphate medium lacking leucine.
  • 36 prolonged incubation (an additional three days) on high phosphate plates results in emergence 37 of additional colonies (Table Vllb), indicating that selection appears to be most effective 38 when carried out on or prior to the third day after plating.
  • Table Vllb additional colonies
  • the background gene expression detected under repressing conditions may be reduced through adjustments to the plating regime.
  • Pregrowth conditions 9 in liquid culture, plate media composition and incubation conditions may be modified to 0 reduce background.
  • the basal level of transcription under repressed conditions may 1 furthermore be reduced by modifying the reporter gene promoter and untranslated leader 2 region.
  • HIS3 TRPl ADE2 auxotrophs were 8 selected on SD plates containing lysine, leucine and uracil.
  • Strain YBT 13 containing 9 pBTll/Z was confirmed to be a pho85 '.HIS 3 pho80 ADE2 double disruptant by PCR 0 analysis of chromosomal DNA using appropriate primers (data not shown).
  • This strain 1 (YBT13; pBTll/Z) was transformed with either pBTl/PHO ⁇ 5 or pBT6/CK2-P ⁇ 5#2 2 (Examples 1 and 4) and selected for HIS3 TRPl ADE2 URA3 auxotrophs on SD plates 3 containing lysine and leucine.
  • Expression vector pBT6/CK2-P ⁇ 5#2 also requires the PHO ⁇ Op cyclin for activity.
  • control cells diluted into buffer without a test compound, are prepared for reference.
  • the screen is performed utilizing, as a host, a yeast strain which has a
  • the combinatorial library samples are 1 previously buffer exchanged or diluted into high phosphate medium selective for plasmid
  • the screen is performed utilizing, as a host, a yeast strain which has a
  • the source material utilized is a human cDNA bank constructed
  • 26 cDNA bank is constructed in a yeast expression vector such as p413-MET25 (Mumberg et).
  • Colonies are selected after at least 1 day, and less than 3 days, incubation at 30°C.
  • Colonies isolated in the above screen contain putative cDNA which encodes peptides capable of inhibiting the CK2-P ⁇ 5#2. These clones are subjected to secondary screens as follows. The cDNA in the p413MET25 vector in each isolated yeast clone is recovered by shuttling into E. coli and subsequently introduced into S. cerevisiae YBTI cells containing plasmids pBTll/LEU2 and pBTl/PHO85.
  • the isolated cDNA also inhibits PHO ⁇ 5p, and may be a general CDK inhibitor. If this secondary screen shows no effect of the expressed cDNA, then the 1 inhibitory activity is specific for the human CDK2 epitopes incorporated into CK2-P ⁇ 5#2.
  • the screen is performed utilizing, as a host, a yeast strain which has a disruption of the chromosomal pho81 gene. In this strain, the reporter gene remains repressed in low phosphate since a functional PHO ⁇ lp CKI is not produced. Therefore, the screen in this host is performed in phosphate depleted medium.
  • EXAMPLE 13 ⁇ SELECTION FOR PEPTIDES THAT INHIBIT CYCLIN/CDK FROM A RANDOM 8 PEPTIDE LIBRARY Procedure is the same as in Example 10, except random peptide coding bank is used 0 in the p413MET25 expression vector. This can be a free peptide bank or a structurally 1 constrained peptide bank as described in Colas et al. (1996) Nature 3 ⁇ 0, 54 ⁇ -550. 2 3 4 5 EXAMPLE 14 ⁇ USE OF E2F MAMMALIAN CELL SYSTEM 6 Standard recombinant DNA techniques are utilized to place the E.
  • coli LacZ gene 7 under control of the Chinese hamster ovary cell (CHO) dihydrofolate reductase gene (DHFR) ⁇ promoter.
  • CHO cells are stably transfected with this construct and clones selected by 9 standard techniques (Ausubel et al). Since the DHFR promoter is activated by E2F, 0 monitoring LacZ expression in this cell line directly measures the availability of functional 1 E2F and indirectly measures the activity of cell cycle regulatory proteins. E2F 2 heterodimerizes with RB and in this form is incapable of activating promoters.
  • 3 Phosphorylation by cyclin E/CDK2 in late Gl phase causes dissociation of the heterodimer 1 enabling E2F to activate responsive promoters. Subsequently, in S phase, cyclin A/CDK2
  • This cell line is utilized to screen for inhibitors of cyclin A/CDK2 as follows.
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI- SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO : 1 :
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:3:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:4:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO : 5 :
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO : 6 :
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO : 7 :
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 8:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI - SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:10:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 11:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 12:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:13:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO : 14 :
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 15:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:16:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 17:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:18:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 19:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 21:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:22:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:23:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:24:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 25:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:26:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:28:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 30:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 31:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:32:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:33:
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI - SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 52

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur un procédé de criblage de composés affectant chez les mammifères les protéines régulatrices du cycle cellulaire consistant: (A) à administrer un composé à une lignée de cellules, ladite lignée de cellules comportant des informations génétiques comprenant: (1) un gène marqueur fonctionnellement lié à une séquence de régulation de l'expression génique, laquelle comprend une séquence d'activation en amont et un promoteur, la séquence d'activation en amont comprend elle-même une région d'ADN se fixant à un facteur de régulation de transcription régulé par phosphorylation au moyen d'un système de phosphorylation par cycline/CDK (kinase à dépendance cycline); et (2) un gène hybride comprenant une première zone de codage d'un gène natif provenant de la lignée de cellules, et une deuxième zone de codage d'un deuxième gène, le premier gène codant pour un produit génique agissant sur la phosphorylation au moyen d'un système de phosphorylation par cycline/CDK, et le deuxième gène, de mammifère, étant l'homologue du gène natif, et le gène hybride fournissant un produit génique efficace pour permettre la régulation normale par cycline/CDK du facteur de régulation de transcription; et (B) à analyser l'expression du gène marqueur dans la lignée de cellules de manière à déterminer si le composé agit sur la régulation normale. L'invention porte également sur des lignées spécifiques de cellules et des méthodes spécifiques.
PCT/US1997/018608 1996-10-16 1997-10-16 Essai phenotypique de la fonction cycline/cdk (kinase a dependance cycline) WO1998016660A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU49038/97A AU4903897A (en) 1996-10-16 1997-10-16 Phenotypic assays of cyclin/cyclin-dependent kinase function

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US2912796P 1996-10-16 1996-10-16
US60/029,127 1996-10-16
US3196896P 1996-11-27 1996-11-27
US60/031,968 1996-11-27

Publications (1)

Publication Number Publication Date
WO1998016660A1 true WO1998016660A1 (fr) 1998-04-23

Family

ID=26704582

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/018608 WO1998016660A1 (fr) 1996-10-16 1997-10-16 Essai phenotypique de la fonction cycline/cdk (kinase a dependance cycline)

Country Status (2)

Country Link
AU (1) AU4903897A (fr)
WO (1) WO1998016660A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580736A (en) * 1992-10-30 1996-12-03 The General Hospital Corporation Interaction trap system for isolating novel proteins
US5667987A (en) * 1994-07-12 1997-09-16 Bristol-Myers Squibb Company P53 response genes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580736A (en) * 1992-10-30 1996-12-03 The General Hospital Corporation Interaction trap system for isolating novel proteins
US5667987A (en) * 1994-07-12 1997-09-16 Bristol-Myers Squibb Company P53 response genes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATURE, 09 March 1995, Volume 374, LIAO S.M. et al., "A Kinase-Cyclin Pair in the RNA Polymerase II Holoenzyme", pages 193-196. *
PROC. NATL. ACAD. SCI. U.S.A., April 1995, Volume 92, KUCHIN S. et al., "Cyclin-Dependent Protein Kinase and Cyclin Homologs SSN3 and SSN8 Contribute to Transcriptional Control in Yeast", pages 4006-4010. *

Also Published As

Publication number Publication date
AU4903897A (en) 1998-05-11

Similar Documents

Publication Publication Date Title
Strubin et al. Yeast and human TFIID with altered DNA-binding specificity for TATA elements
Vidal et al. RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes
Nasmyth et al. The role of SWI4 and SWI6 in the activity of G1 cyclins in yeast
Rubin-Bejerano et al. Induction of meiosis in Saccharomyces cerevisiae depends on conversion of the transcriptional repressor Ume6 to a positive regulator by its regulated association with the transcriptional activator Ime1
US6048693A (en) Phenotypic assays of cyclin/cyclin-dependent kinase function
Marshall et al. Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae
Althoefer et al. Mcm1 is required to coordinate G2-specific transcription in Saccharomyces cerevisiae
Means et al. Regulatory functions of calmodulin
US5955280A (en) Reverse two-hybrid system
Colwill et al. In vivo analysis of the domains of yeast Rvs167p suggests Rvs167p function is mediated through multiple protein interactions
EP1362913B1 (fr) Système de piégeage d'intéraction pour l'isolation de protéines
US5885779A (en) Repressed trans-activator system for characterization of protein-protein interactions
Ohi et al. Myb-related Schizosaccharomyces pombe cdc5p is structurally and functionally conserved in eukaryotes
Kirkman-Correia et al. Functional domains of the yeast STE12 protein, a pheromone-responsive transcriptional activator
US6509447B1 (en) G protein chimeras and methods of screening compounds
EP1053347B1 (fr) Procede de detection de peptides
McCarroll et al. SPO13 negatively regulates the progression of mitotic and meiotic nuclear division in Saccharomyces cerevisiae.
Hendrick et al. Yeast frameshift suppressor mutations in the genes coding for transcription factor Mbf1p and ribosomal protein S3: evidence for autoregulation of S3 synthesis
DOMINGUEZ et al. Structural and functional similarities between the central eukaryotic initiation factor (eIF) 4A-binding domain of mammalian eIF4G and the eIF4A-binding domain of yeast eIF4G
Howard et al. The Ras/PKA signaling pathway of Saccharomyces cerevisiae exhibits a functional interaction with the Sin4p complex of the RNA polymerase II holoenzyme
Nakazawa et al. AAR2, a Gene for Splicing Pre-mRNA of the MAT a 1 Cistron in Cell Type Control of Saccharomyces cerevisiae
WO1998016660A1 (fr) Essai phenotypique de la fonction cycline/cdk (kinase a dependance cycline)
Ni et al. Candida albicans Cdc37 interacts with the Crk1 kinase and is required for Crk1 production
Macpherson et al. A yeast taf17 mutant requires the Swi6 transcriptional activator for viability and shows defects in cell cycle-regulated transcription
Levine et al. Isolation and characterization of new alleles of the cyclin-dependent kinase gene CDC28 with cyclin-specific functional and biochemical defects

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载