EP0584264A1 - Cyclines du type d et leurs utilisations - Google Patents

Cyclines du type d et leurs utilisations

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Publication number
EP0584264A1
EP0584264A1 EP92913100A EP92913100A EP0584264A1 EP 0584264 A1 EP0584264 A1 EP 0584264A1 EP 92913100 A EP92913100 A EP 92913100A EP 92913100 A EP92913100 A EP 92913100A EP 0584264 A1 EP0584264 A1 EP 0584264A1
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Prior art keywords
cyclin
type
dna
cell
type cyclin
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German (de)
English (en)
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David H. Beach
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Cold Spring Harbor Laboratory
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Cold Spring Harbor Laboratory
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • a typical cell cycle of a eukaryotic cell includes the M phase, which includes nuclear division (mitosis) and cytoplasmic division or cytokinesis and interphase, which begins with the G1 phase, proceeds into the S phase and ends with the G2 phase, which continues until mitosis begins, initiating the next M phase.
  • M phase nuclear division (mitosis) and cytoplasmic division or cytokinesis and interphase
  • S phase DNA replication and histone synthesis occurs
  • no net DNA synthesis occurs, although damaged DNA can be repaired.
  • There are several key changes which occur during the cell cycle including a critical point in the G1 phase called the restriction point or start, beyond which a cell is committed to completing the S, G2 and M phases.
  • Cyclins are proteins that were discovered due to their intense synthesis following the fertilization of marine invertebrate eggs (Rosenthal, E.T. et al . , Cell 20:487-494 (1980)). It was subsequently observed that the abundance of two types of cyclin, A and B, oscillated during the early cleavage divisions due to abrupt proteolytic degradation of the polypeptides at mitosis and thus, they derived their name (Evans, T. et al., Cell 33:389-396 (1983); Swenson, K.I. et al., Cell 47:867-870 (1986); Standart, N. et al., Dev. Biol. 124:248-258
  • MPF M phase inducing factor
  • B Three types of classes of cyclins have been identified to date: B, A and CLN cyclins.
  • the B-type cyclin has been shown to act in mitosis by serving as an
  • the A-type cyclin also independently associates with the cdc2 kinase, forming an enzyme that appears to act earlier in the division cycle than mitosis (Draetta, G. et al., Cell 56:829-838 (1989); Minshull, J. et al., EMBO J. 9:2865-2875 (1990); Giordano, A. et al., Cell 58:981-990 (1989); Pines, J. and T. Hunter, Nature 346:760-763 (1990)).
  • the functional difference between these two classes of cyclins is not yet fully understood.
  • invertebrate and vertebrate embryos have been accompanied by genetic studies, particularly in ascomycete yeasts.
  • the cdcl3 gene encodes a B-type cyclin that acts in cooperation with cdc2 to regulate entry into mitosis (Booher, R. and D. Beach, EMBO J.,
  • cdc2 acts at two independent points in the cell cycle: mitosis and the so-called cell cycle "start" (Hartwell, L.H., J. Mol. Biol., 104:803-817 (1971);
  • the start function of the CDC28 protein also requires association of the catalytic subunit of the protein kinase with ancillary proteins that are structurally related to A and B-type cyclins.
  • This third class of cyclin has been called the Cln class, and three genes comprising a partially redundant gene family have been described (Nash, R. et al., EMBO J.
  • the CLN genes are essential for execution of start and in their absence, cells become arrested in the G1 phase of the cell cycle.
  • the CLN1 and CLN2 transcripts oscillate in abundance through the cell cycle, but the CLN3 transcript does not.
  • the Cln2 protein has been shown to o s cillate in parallel with its mRNA (Nash , R . et al . , EMBO J.
  • cdc2 and cyclins have been found not only in embryos and yeasts, but also in somatic human cells.
  • the present invention relates to a novel class of cyclins, referred to as D-type cyclins, which are of mammalian origin and are a new family of cyclins related to, but distinct from, previously described A, B or CLN type cyclins. In particular, it relates to human
  • cyclins encoded by genes shown to be able to replace a CLN-type gene essential for cell cycle start in yeast, which complement a deficiency of a protein essential for cell cycle start and which, on the basis of protein structure, are on a different branch of the evolutionary tree from A, B or CLN type cyclins.
  • cyclin D1 or CCND1 One member of this new cyclin family, cyclin D1 or CCND1, is 295 amino acid residues and has an estimated molecular weight of 33,670 daltons (Da).
  • a second member, cyclin D2 or CCND2 is 289 amino acid residues and has an estimated molecular weight of 33,045 daltons. It has been mapped to chromosome 12p band p13.
  • a third member, cyclin D3 or CCND3 is 292 amino acid residues and has an estimated molecular weight of
  • D-type cyclins of the present invention can be produced using recombinant techniques, can be synthesized chemically or can be Isolated or purified from sources in which they occur naturally.
  • the present invention includes recombinant D-type cyclins, isolated or purified D-type cyclins and synthetic D-type cyclins.
  • the present invention also relates to DNA or RNA encoding a D-type cyclin of mammalian origin, particularly of human origin, as well as to antibodies, both polyclonal and monoclonal, specific for a D-type cyclin of mammalian, particularly human, origin.
  • the present invention further relates to a method of isolating genes encoding other cyclins, such as other D-type cyclins and related (but non-D type) cyclins. It also has diagnostic and therapeutic aspects. For example, it relates to a method in which the presence and/or quantity of a D-type cyclin (or cyclins) In tissues or biological samples, such as blood, urine, feces, mucous or saliva, is determined, using a nucleic acid probe based on a D-type cyclin gene or genes described herein or an antibody specific for a D-type cyclin.
  • This embodiment can be used to predict whether cells are likely to undergo cell division at an abnormally high rate (i.e., if cells are likely to be cancerous), by determining whether their cyclin levels or activity are elevated (elevated level of activity being Indicative of an Increased probability that cells will undergo an abnormally high rate of division).
  • the present method also relates to a diagnostic method in which the occurrence of cell division at an abnormally high rate is assessed based on abnormally high levels of a D-type cyclin(s), a gene(s) encoding a D-type cyclin(s) or a transcription product(s) (RNA).
  • the present invention relates to a method of modulating (decreasing or enhancing) cell division by altering the activity of at least one D-type cyclin, such as D2, D2 or D3 in cells.
  • the present invention particularly relates to a method of inhibiting increased cell division by interfering with the activity or function of a D-type cyclin(s).
  • function of D-type cyclin(s) is blocked (totally or partially) by interfering with its ability to activate the protein kinase it would otherwise (normally) activate (e.g., p34 cdc2 or a related protein kinase), by means of agents which interfere with D-type cyclin activity, either directly or indirectly.
  • Such agents include anti-sense sequences or other transcriptional modulators which bind D cyclin-encoding DNA or RNA; antibodies which bind either the D-type cyclin or a molecule with which a D-type cyclin must interact or bind in order to carry out its role in cell cycle start; substances which bind the D-type cyclin(s); agents (e.g., proteases) which degrade or otherwise inactivate the D-type cyclin(s); or agents (e.g., small organic molecules) which interfere with association of the D-type cyclin with the catalytic subunit of the kinase.
  • agents e.g., oligonucleotides, antibodies, peptides
  • Figure 1 is a schematic representation of a genetic screen for human cyclin genes.
  • Figure 2 is the human cyclin D1 nucleic acid sequence (SEQ ID No. 1) and amino acid sequence (SEQ ID No. 2), in which nucleotide numbers and amino acid numbers are on the right, amino acid numbers are given with the initiation methionine as number one and the stop codon is indicated by an asterisk.
  • Figure 3 is the human cyclin D2 nucleic acid sequence (SEQ ID No. 3) and amino acid sequence (SEQ ID No. 4) in which nucleotide numbers and amino acid numbers are on the right, amino acid numbers are given with the Initiation methionine as number one and the stop codon is Indicated by an asterisk.
  • Figure 4 is the human cyclin D3 nucleic acid
  • amino acid sequence SEQ ID No. 5
  • amino acid sequence SEQ ID No. 6
  • nucleotide numbers and amino acid numbers are on the right
  • amino acid numbers are given with the Initiation methionine as number one and the stop codon is indicated by an asterisk.
  • Figure 5 shows the cyclin gene family.
  • Figure 5A shows the amino acid sequence alignment of seven cyclin genes (CYCD1-Hs, SEQ ID No. 7; CYCA-Hs, SEQ ID No. 8; CYCA-Dm, SEQ ID No. 9; CYCB1-Hs, SEQ ID No. 10; CDC13-Sp, SEQ ID No. 11; CLN1-Sc, SEQ ID No. 12; CLN3-Sc, SEQ ID No. 13), in which numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of Insertion.
  • CYCD1-Hs SEQ ID No. 7
  • CYCA-Hs SEQ ID No. 8
  • CYCA-Dm SEQ ID No. 9
  • CYCB1-Hs SEQ ID No. 10
  • CDC13-Sp SEQ ID No. 11
  • CLN1-Sc SEQ ID No. 12
  • CLN3-Sc SEQ ID No. 13
  • Figure 5B is a schematic representation of the evolutionary tree of the cyclin family, constructed using the Neighbor-Joining method; the length of horizontal line reflects the divergence.
  • Figure 6 shows alternative polyadenylation of the cyclin D1 gene transcript.
  • Figure 6A is a comparison of several cDNA clones isolated from different cell lines. Open boxes represent the 1.7 kb small transcript containing the coding region of cyclin D1 gene. Shadowed boxes represent the 3' fragment present in the 4.8 kb long transcript.
  • Figure 6B shows the nucleotide sequence surrounding the first polyadenylation site for several cDNA clones (CYCD1-21, SEQ ID No. 14; CYCD1-H12, SEQ ID No. 15;
  • CYCD1-HO34 SEQ ID No. 16
  • CYCD1-TO78 SEQ ID No. 17
  • a genomic clone CYCD1-G068, SEQ ID No. 18
  • Figure 6C is a summary of the structure and alternative polyadenylation of the cyclin D1 gene. Open boxes represent the small transcript, the shadowed box represents the 3' sequence in the large transcript and the filled boxes indicate the coding regions.
  • Figure 7 shows the protein sequence comparison of eleven mammalian cyclins (CYCD1-Hs, SEQ ID No. 19;
  • CYL1-Mm SEQ ID No. 20
  • CYCD2-Hs SEQ ID No. 21;
  • CYCL2-Mm SEQ ID No. 22
  • CYCD3-Hs SEQ ID No. 23;
  • CYL3-Mm SEQ ID No. 24; CYCA-Hs, SEQ ID No. 25; CYCB1-Hs, SEQ ID No. 26; CYCB2-Hs, SEQ ID No. 27; CYCC-Hs, SEQ ID No. 28; CYCE-Hs, SEQ ID No. 29).
  • Figure 8 is a schematic representation of the genomic structure of human cyclin D genes, in which each diagram represents one restriction fragment from each cyclin D gene that has been completely sequenced. Solid boxes indicate exon sequences, open boxes indicate intron or 5' and 3' untranslated sequences and hatched boxes represent pseudogenes. The positions of certain
  • restriction sites, ATG and stop codons are indicated at the top of each clone.
  • Figure 9 is the nucleic acid sequence (SEQ ID No. 30) and amino acid sequence (SEQ ID No. 31) of a cyclin D2 pseudogene.
  • Figure 10 is the nucleic acid sequence (SEQ ID No. 32) and the amino acid sequence (SEQ ID No. 33) of a cyclin D3 pseudogene.
  • Figure 11 is the nucleic acid sequence (SEQ ID No. 34) of 1.3 kb of human cyclin D1 promoter; the sequence ends at Initiation ATG codon and transcription starts at approximately nucleotide -160.
  • Figure 12 is the nucleotide sequence (SEQ ID No. 35) of 1.6 kb of human cyclin D2 promoter; the sequence ends at initiation ATG codon and transcription starts at approximately nucleotide -170.
  • Figure 13 is the nucleotide sequence (SEQ ID No. 36) of 3.2 kb of human cyclin D3 promoter; the sequence ends at initiation ATG codon and transcription starts at approximately nucleotide -160.
  • D-type cyclins a new class of mammalian cyclin proteins, designated D-type cyclins, has been identified, isolated and shown to serve as a control element for the cell cycle start, in that they fill the role of a known cyclin protein by activating a protein kinase whose activation is essential for cell cycle start, an event in the G1 phase at which a cell becomes committed to cell division.
  • human D-type cyclin proteins, as well as the genes which encode them have been identified, isolated and shown to be able to replace CLN type cyclin known to be essential for cell cycle start in yeast.
  • the chromosomal locations of CCND2 and CCND3 have also been mapped.
  • D type cyclins
  • DNA and RNA encoding the novel D-type cyclins
  • pCYCD1-19 and pCYCD1-21 were shown, by restriction mapping and partial DNA sequence analysis, to be independent clones representing the same gene.
  • a HeLa cDNA library was screened for a full length cDNA clone, using the 1.2 kb insert of pCYCD1-21 as probe. Complete sequencing was done of the longest of nine positive clones identified in this manner (pCYCD1-H12; 1325 bp).
  • the sequence of the 1.2 kb insert is presented in Figure 2; the predicted protein product of the gene is of approximate molecular weight 34,000 daltons.
  • Cyclin D2 and cyclin D3 cDNAs were isolated using the polymerase chain reaction and three oligonucleotide probes derived from three highly conserved regions of D-type cyclins, as described In Example 4. As described, two 5' oligonucleotides and one 3' degenerate oligonucleotide were used for this purpose.
  • the nucleotide and amino acid sequences of the CCND2 gene and encoded D2 cyclin protein are represented in Figure 3 and of the CCND3 gene and encoded D3 cyclin protein are represented in Figure 4.
  • a deposit of plasmid pCYC-D3 was made with the American Type Culture Collection (Rockville, MD) on May 14, 1991, under the terms of the Budapest Treaty.
  • cyclin D1 gene in human cells was studied using Northern analysis, as described in Example 2. Results showed that levels of cyclin D1 expression were very low in several cell lines. The entire coding region of the CYCD1 gene was used to probe poly(A)+ RNA from HeLa cells and demonstrated the presence of two major transcripts, one approximately 4.8 kb and the other approximately 1.7 kb , with the higher molecular weight form being the more abundant. Most of the cDNA clones isolated from various cDNA libraries proved to be very similar to clone ACYCD1-H12 and, thus, it appears that the 1.7 kb transcript detected in Northern blots corresponds to the nucleotide sequence of Figure 2. The origin of the larger (4.8 kb) transcript was unclear. As described in Example 2, it appears that the two mRNAs detected (4.8 kb and 1.7 kb ) arose by differential polyadenylation of CYCD1 ( Figure 6).
  • cyclin D1 Differential expression of cyclin D1 in different tissues and cell lines was also assessed, as described in Example 3. Screening of cDNA libraries to obtain full length CYCD1 clones had demonstrated that the cDNA library from the human glioblastoma cell line (U118 MG) used to produce yeast transformants produced many more positives than the other three cDNA libraries (human HeLa cell cDNA, human T cell cDNA, human teratocarcinoma cell cDNA). Northern and Western blotting were carried out to determine whether cyclin D1 is differentially expressed.
  • human glioblastoma cell line U118 MG
  • Northern and Western blotting were carried out to determine whether cyclin D1 is differentially expressed.
  • results showed (Example 3) that the level of transcript is 7 to 10 fold higher in the glioblastoma (U118 MG) cells than in HeLa cells, and that in both HeLa and U118 MG cells, the high and low molecular weight transcripts occurred.
  • Western blotting using anti-CYLl antibody readily detected the presence of a 34kd polypeptide in the glioblastoma cells and demonstrated that the protein is far less abundant in HeLa cells and not detectable in the 293 cells.
  • the molecular weight of the anti-CYCLl cross reactive material identified in U118 MG and HeLa cells is exactly that of the human CYCD1 protein expressed in E. coli.
  • results demonstrated differential occurrence of the cyclin D1 in the cell types analyzed, with the highest levels being in cells of neural origin.
  • Lambda genomic clones corresponding to the human cyclin D2 and lambda genomic clones corresponding to the human cyclin D3 were also isolated and characterized, using a similar approach.
  • One clone (7D2-G4) was shown to contain ( Figure 8B) a 2.7 kb SacI SmaI fragment which includes 1620 bp of sequence 5' to the presumptive initiating methionine codon identified in D2 cDNA ( Figure 3) and a 195 bp exon followed by a 907 bp intervening sequence.
  • cyclin D a novel class of mammalian cyclins, designated cyclin D or D-type cyclin, has been identified and shown to be distinct, on the basis of structure of the gene (protein) product, from previously-identified cyclins.
  • Three members of this new class, designated cyclin D1 or CCND1, cyclin D2 or CCND2 and cyclin D3 or CCND3 have been isolated and sequenced. They have been shown to fulfill the role of another cyclin (CLN type) in activation of the protein kinase (CDC28) which is essential for cell cycle start in yeast. It has also been shown that the cyclin D1 gene is expressed differentially in different cell types, with expression being highest in cells of neural origin.
  • genes which encode other cyclins (DNA or RNA which replaces a gene essential for cell cycle start).
  • This method can be used to identify additional members of the cyclin D class or other (non-D type) cyclins of either human or nonhuman origin. This can be done, for example, by screening other cDNA libraries using the budding yeast strain conditional for CLN cyclin expression, described in
  • Example 1 or another mutant in which the ability of a gene to replace cyclin expression can be assessed and used to identify cyclin homologues.
  • This method is carried out as described herein, particularly in Example 1 and as represented in Figure 1.
  • a cDNA library carried in an appropriate yeast vector e.g., pADNS
  • pADNS pADNS
  • the strain used contains altered CLN genes.
  • Mutant yeast transformed with the cDNA library in the expression vector are screened for their ability to grow on glucose-containing medium.
  • medium containing galactose the CLN3 gene is expressed and cell viability Is maintained, despite the absence of CLN1 and CLN2.
  • medium containing glucose all CLN function is lost and the yeast cells arrest in the G1 phase of the cell cycle.
  • the ability of a yeast transformant to grow on glucose-containing medium is an indication of the presence in the transformant of DNA able to replace the function of a gene essential for cell cycle s tart .
  • thi s can be conf irmed by use of an expression vector, such as pADNS , which contains a selectable marker (the LEU2 marker is present in pADNS).
  • an expression vector such as pADNS
  • pADNS which contains a selectable marker (the LEU2 marker is present in pADNS).
  • Assessment of the plasmid stability shows whether the ability to grow on glucose-containing medium is the result of reversion or the presence of DNA function
  • cyclins of all types can be identified by their ability to replace CLN3 function when transformants are grown on glucose.
  • Screening of additional cDNA or genomic libraries to identify other cyclin genes can be carried out using all or a portion of the human D-type cyclin DNAs disclosed herein as probes; for example, all or a portion of the D1, D2 or D3 cDNA sequences of Figures 2-4, respectively, or all or a portion of the corresponding genomic sequences described herein can be used as probes.
  • the hybridization conditions can be varied as desired and, as a result, the sequences identified will be of greater or lesser complementarity to the probe sequence (i.e., if higher or lower stringency conditions are used).
  • an anti-D type cyclin antibody such as CYL1 or another raised against D1 or D3 or other human D-type cyclin, can be used to detect other recombinant D-type cyclins produced in appropriate host cells transformed with a vector containing DNA thought to encode a cyclin. Based on work described herein, it is possible to detect altered expression of a D-type cyclin or Increased rates of cell division In cells obtained from a tissue or biological sample, such as blood, urine, feces, mucous or saliva. This has potential for use for diagnostic and prognostic purposes since, for example, there appears to be a link between alteration of a cyclin gene expression and cellular transformation or abnormal cell proliferation.
  • the PRAD1 gene which has the same sequence as the cyclin D1 gene, may play an important role in the development of various tumors (e.g., non-parathyroid neoplasis, human breast carcinomas and squamous cell carcinomas) with abnormalities in chromosome 11q13.
  • identification of CCND1 (PRAD1) as a candidate BCL1 oncogene provides the most direct evidence for the oncogenic potential of cyclin genes.
  • CCND1 PRAD1
  • the chromosomal locations of the CCND2 and CCND3 genes have been mapped to 12p13 and 6p21, respectively.
  • Region 12p13 contains sites of several translocatlons that are associated with specific immunophenotypes of disease, such as acute lymphoblastic leukemia, chronic myelomoncytic leukemia, and acute myeloid leukemia.
  • the isochromosome of the short arm of chromosome 12 [1(12p)] is one of a few known consistent chromosomal abnormalities in human solid tumors and is seen in 90% of adult testicular germ cell tumors.
  • Region 6p21 has been implicated in the manifestation of chronic lymphoproliferative disorder and leiomyoma.
  • Region tp21 the locus of HLA complex, is also one of the best characterized regions of the human genome.
  • the present invention includes a diagnostic method to detect altered expression of a cyclin gene, such as cyclin D1, D2, D3 or another D-type cyclin.
  • the method can be carried out to detect altered expression in cells or in a biological sample.
  • a cyclin gene such as cyclin D1, D2, D3 or another D-type cyclin.
  • the method can be carried out to detect altered expression in cells or in a biological sample.
  • cyclin D genes there is high sequence similarity among cyclin D genes, which indicates that different members of D-type cyclins may use similar mechanisms in regulating the cell cycle
  • cyclin D1 Is expressed differentially in tissues analyzed; in particular, it has been shown to be expressed at the highest levels in cells of neural origin (e.g., glioblastoma cells).
  • D-type cyclin expression can be detected and/or quantitated and results used as an Indicator of normal or abnormal (e.g., abnormally high rate of) cell division.
  • Differential expression (either expression in various cell types or of one or more of the types of D cyclins) can also be determined.
  • cells obtained from an individual are processed in order to render nucleic acid sequences in them available for hybridization with complementary nucleic acid sequences.
  • All or a portion of the D1, D2 and/or D3 cyclin (or other D-type cyclin gene) sequences can be used as a probe(s).
  • Such probes can be a portion of a D-type cyclin gene;
  • Hybridization is detected using known techniques (e.g., measurement of labeled hybridization complexes, if radiolabeled or fluorescently labeled oligonucleo tide probed are used). The extent to which hybridization occurs is quantitated; increased levels of the D-type cyclin gene is indicative of
  • the extent to which a D-type cyclin (or cyclins) is present in cells, in a specific cell type or in a body fluid can be determined using known techniques and an antibody specific for the D-type cyclin(s).
  • complex formation between the D-type cyclin and the protein kinase with which it normally or typically complexes is assessed, using exogenous substrate, such as histone HI, as a substrate. Arion, D. et al., Cell, 55:371-378 (1988).
  • each diagnostic method comparison of results obtained from cells or a body fluid being analyzed with results obtained from an appropriate control (e.g., cells of the same type known to have normal D-type cyclin levels and/or activity or the same body fluid obtained from an individual known to have normal D-type cyclin levels and/or activity) is carried out.
  • Increased D-type cyclin levels and/or activity may be indicative of an increased probability of abnormal cell proliferation or oncogenesis or of the actual occurrence of abnormal proliferation or oncogenesis.
  • cyclin e.g., A, B, and/or D
  • a set of probes e.g., a set of nucleic acid probes or a set of antibodies
  • Such probes are also the subject of the present invention; they will generally be detectably labelled (e.g., with a radioactive label, a fluorescent material, biotin or another member of a binding pair or an enzyme).
  • a method of Inhibiting cell division particularly cell division which would otherwise occur at an abnormally high rate, Is also possible.
  • increased cell division is reduced or prevented by introducing into cells a drug or other agent which can block, directly or indirectly, formation of the protein kinase-D type cyclin complex and, thus, block activation of the enzyme.
  • complex formation is prevented in an indirect manner, such as by preventing transcription and/or translation of the D-type cyclin DNA and/or RNA. This can be carried out by introducing antisense oligonucleotides into cells, in which they hybridize to the cyclin-encoding nucleic acid sequences, preventing their further processing.
  • the G1 phase is the time at which cells commit to a new round of division in response to external and internal sequences and, thus, transcription factors which regulate expression of G1 cyclins are surely Important in controlling cell proliferation. Modulation of the transcription factors Is one route by which D-type cyclin activity can be influenced, resulting, in the case of inhibition or prevention of function of the transcription factor(s), in reduced D-type cyclin activity.
  • complex formation can be prevented indirectly by degrading the D- type cyclin(s), such as by introducing a protease or substance which enhances cyclin breakdown into cells.
  • the effect is indirect in that less D-type cyclin is available than would otherwise be the case.
  • protein kinase-D type cyclin complex formation is prevented in a more direct manner by, for example, introducing into cells a drug or other agent which binds the protein kinase or the D-type cyclin or otherwise interferes with the physical association between the cyclin and the protein kinase it activates (e.g., by intercalation) or disrupts the catalytic activity of the enzyme.
  • This can be effected by means of antibodies which bind the kinase or the cyclin or a peptide or low molecular weight organic compound which, like the endogenous D-type cyclin, binds the protein kinase, but whose binding does not result in activation °f the enzyme or results in its being disabled or degraded.
  • Peptides and small organic compounds to be used for this purpose can be designed, based on analysis of the amino acid sequences of D-type cyclins, to include residues necessary for binding and to exclude residues whose presence results in activation. This can be done, for example, by systematically mapping the binding site(s) and designing molecules which recognize or otherwise associate with the site(s) necessary for activation, but do not cause activation.
  • agents which selectively inhibit cyclin D1 might be expected to be particularly useful, since D1 has been shown to be differentially expressed (expressed at particularly high levels in cells of neural origin).
  • Antibodies specifically reactive with D-type cyclins of the present invention can also be produced, using known methods.
  • anti-D type cyclin antisera can be produced by injecting an appropriate host (e.g., rabbits, mice, rats, pigs) with the D-type cyclin against which anti sera is desired and withdrawing blood from the host animal after sufficient time for antibodies to have been formed.
  • Monoclonal antibodies can also be produced using known techniques. Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
  • the present invention also includes a method of screening compounds or molecules for their ability to inhibit or suppress the function of a cyclin, particularly a D-type cyclin.
  • mutant cells as described herein, in which a D-type cyclin such as D1 or D3, is expressed can be used.
  • a compound or molecule to be assessed for its ability to inhibit a D-type cyclin is contacted with the cells, under conditions appropriate for entry of the compound or molecule into the cells. Inhibition of the cyclin will result in arrest of the cells or a reduced rate of cell division. Comparison of the rate or extent of cell division in the presence of the compound or molecule being assessed with cell
  • a yeast strain was constructed, as described below, which contained insertional mutations in the CLN1 and CLN2 genes to render them inactive.
  • the remaining CLN3 gene was further altered to allow for conditional expression from the galactose-inducible, glucose-repressible promoter GAL1 (see Figure 1).
  • the strain is designated 305-15d #21.
  • medium containing galactose the CLN3 gene is expressed and despite the absence of both CLN1 and CLN2 , cell viability is retained (Fig. 1).
  • a medium containing glucose all CLN function is lost and the cells arrest in the G1 phase of the cell cycle.
  • a human glioblastoma cDNA library carried in the yeast expression vector pADNS (Colicelli, J. et al., Pro. Natl. Acad. Sci. USA 86:3599-3603 (1989)) was introduced into the yeast.
  • the vector pADNS has the LEU2 marker, the 2 ⁇ replication origin, and the promoter and terminator sequences from the yeast alcohol dehydrogenase gene ( Figure 1). Approximately 3 ⁇ 10 transformants were screened for the ability to grow on glucose containing medium. After 12 days of incubation, twelve colonies were obtained. The majority of these proved to be revertants. However, in two cases, the ability to grow on glucose correlated with the maintenance of the LEU2 marker as assessed by plasmid stability tests.
  • the estimated molecular weight of the predicted protein product of the gene is 33,670 daltons starting from the first in-frame AUG codon at nucleotide 145 ( Figure 2).
  • the predicted protein is related to other cyclins (see below) and has an unusually low pi of 4.9
  • CYCD1 encodes the smallest (34 kd) cyclin protein identified so far, compared to the 49 kd human cyclin A, 50 kd human cyclin B and 62 kd S . cerevisiae CLN1.
  • a and B type cyclins By comparison with A and B type cyclins, the difference is due to the lack of almost the entire N-terminal segment that contains the so called "destruction box" identified in both A and B type cyclins (Glotzer, M. et al., Nature 349:132-138 (1991)).
  • cyclin D1 gene expression was studied by Northern analysis. Initial studies indicated that the level of cyclin D1 expression was very low in several cell lines. Poly (A)+RNA was prepared from HeLa cells and probed with the entire coding region of CYCD1 gene. Two major transcripts of 4.8 kb and 1.7 kb were detected. The high molecular weight form was the most abundant. With the exception of a few cDNA clones, which were truncated at either the 5' or 3' ends, most of the cDNA clones isolated from various different cDNA libraries are very similar to the clone ACYCD1-H12 ( Figure 2).
  • the 1.7 kb transcript detected in Northern blots corresponds to nucleotide sequence in Figure 2.
  • both 5' and 3' end sub-fragments of the ACYCD1-H12 clone were used to screen both cDNA and genomic libraries, to test whether there might be alternative transcription initiation, polyadenylation and/or mRNA splicing.
  • Two longer cDNA clones, ACYCD1-H034 (1.7 kb) from HeLa cells and ADYDC1-T078 (4.1 kb) from human teratocarcinoma cells, as well as several genomic clones were isolated and partially sequenced.
  • Both ACYCD1-H034 and ACYCD1-T078 have identical sequences to ACYCD1-H12 clone from their 5' ends ( Figure 6). Both differ from ACYCD1-H12 in having additional sequences at the 3' end, after the site of polyadenylation. These 3' sequences are the same in ACYCD1-H034 and ACYCD1-T078, but extend further In the latter clone ( Figure 6). Nucleotide sequencing of a genomic clone within this region revealed colinearity between the cDNAs and the genomic DNA ( Figure 6). There is a single base deletion (an A residue) in ACYCD1-T078 cDNA clone.
  • the parental strain was BF305-15d (MATa leu2-3 leu2-112 his3-11 his3-15 ura3-52 trp1 ade1 met14 arg5,6) (Futcher, B. and J. Carbon, Mol. Cell. Biol. 6:2213-2222 (1986)).
  • the strain was converted into a conditional cln- strain in three steps. First, the chromosomal CLN3 gene was placed under control of the GAL1 promoter. A 0.75 kb EcoRI-BamHI fragment containing the bidirectional GAL10-GAL1 promoters was fused to the 5' end of the CLN3 gene, such that the BamHI (GAL1) end was attached 110 nucleotides upstream of the CLN3 start codon.
  • This fragment had extensive 5' and 3' homology to the CLN3 region, but contained the GAL1 promoter and a URA3 marker just upstream of CLN3.
  • Strain BF305-15d was transformed with this fragment and Ura+ transformants were selected. These were checked by Southern analysis. In addition, average cell size was measured when the GAL1 promoter was induced or uninduced.
  • mode cell volume was about 25 ⁇ m 3 (compared to a mode volume of about 40 ⁇ m 3 for the parental strain) whereas when the promoter was not induced
  • CLN1 gene was disrupted.
  • a fragment of CLN1 was obtained from I. Fitch, and used to obtain a full length clone of CLN1 by hybridization, and this was subcloned into a pUC plasmid.
  • a BamHI fragment carrying the HIS3 gene was inserted into an Ncol site in the CLN1 open reading frame.
  • a large EcoRI fragment with extensive 5' and 3' homology to the CLN1 region was then excised, and used to transform the BF305-15d GAL-CLN3 strain described above. Transformation was done on
  • CLN2 gene was disrupted.
  • a fragment of CLN2 was obtained from I. Fitch, and used to obtain a full length clone of CLN2 by hybridization, and this was subcloned into a pUC plasmid.
  • An EcoRI fragment carrying the TRP1 gene was inserted into an SpeI site in the CLN2 open reading frame.
  • a BamHI-Kpnl fragment was excised and used to transform the BF305-15d GAL-CLN3 HIS3::cln1 strain described above. Transformation was done on
  • Trp+ clones were selected. In this case, because the TRP1 fragment included an ARS , many of the transformants contained autonomously replicating plasmid rather than a disrupted CLN2 gene. However, several percent of the transformants were simple TRP1::cln2 disruptants, as shown by phenotypic and Southern analysis.
  • TRPl::cln2 transformant called clone #21 (referred to hereafter as 305-15d #21) was analyzed extensively. When grown In 1% raffinose and 1% galactose, it had a doubling time indistinguishable from the CLN wild-type parental strain. However, it displayed a moderate Wee phenotype (small cell volume), as expected for a CLN3 overexpressor. When glucose was added, or when galactose was removed, cells accumulated in G1 phase, and cell division ceased, though cells continued to increase in mass and volume. After overnight incubation in the G1-arrested state, essentially no budded cells were seen, and a large proportion of the cells had lysed due to their uncontrolled increase in size.
  • HeLa and 293 cells were cultured at 37°C either on plates or in suspension in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.
  • DMEM Dulbecco's modified Eagle's medium
  • Glioblastoma U118 MG cells were cultured on plates in DMEM supplemented with 15% fetal bovine serum and 0.1 mM non-essential amino acid (GIBCO).
  • Phagmid vectors pUC118 or pUCH9 (Vieira, J. and J.
  • Human HeLa cell cDNA library in ⁇ ZAP II was purchased from Stratagene. Human T cell cDNA library in ⁇ gt10 was a gift of M. Gillman (Cold Spring Harbor Laboratory). Human glioblastoma U118 MG and glioblastoma SW1088 cell cDNA libraries in ⁇ ZAP II were gifts of M. Wigler (Cold Spring Harbor Laboratory). Human teratocarcinoma cell cDNA library ⁇ gt10 was a gift of Skowronski (Cold Spring Harbor Laboratory). Normal human liver genomic library ⁇ GEM-11 was purchased from Promega.
  • RNA from cell culture was extracted exactly according to Sambrook et al. (Sambrook, J. et al.,
  • RNA samples were separated on a 1% agaroseformaldehyde-MOPS gel and transferred to a nitrocellulose filter.
  • Northern hybridizations (as well as library screening) were carried out at 68oC in a solution containing 5 ⁇ Denhardt's solution, 2 ⁇ SSC, 0.1% SDS, 100 ⁇ g/ml denatured Salmon sperm DNA, 25 ⁇ M NaPO 4 (pH7.0) and 10% dextran sulfate.
  • Probes were labelled by the random priming labelling method (Feinberg, A. and B. Vogelstein, Anal. Biochem. 132:6-13 (1983)).
  • a 1.3 kb Hind III fragment of cDNA clone pCYCD1-H12 was used as coding region probe for Northern hybridization and genomic library screening, a 1.7 kb Hind III-EcoRI fragment from cDNA clone pCYCD1-T078 was used as 3' fragment probe.
  • Neo I-Hind II fragment of pCYCD1-H12 containing the entire CYCD1 open reading frame was subcloned into a T7 expression vector (pET3d, Studier, F.W. et al., Methods in Enzymology 185:60-89 (1990)).
  • pET3d Studier, F.W. et al., Methods in Enzymology 185:60-89 (1990)
  • Induction of E. coli strain BL21 (DE3) harboring the expression construct was according to Studier (Studier, F.W. et al., Methods in Enzymology 185:60-89 (1990)).
  • Bacterial culture was lysed by sonication in a lysis buffer (5 mM EDTA, 10% glycerol, 50 mM Tris-HCL, pH 8.0, 0.005% Triton X-100) containing 6 M urea (CYCD1 encoded p34 is only partial soluble in 8 M urea), centrifuged for 15 minutes at 20,000 g force. The pellet was washed once in the lysis buffer with 6 M urea, pelleted again, resuspended in lysis buffer containing 8 M urea, and centrifuged. The supernatant which enriched the 34 kd CYCD1 protein was loaded on a 10% polyacrymide gel. The 34 kd band was cut from the gel and eluted with PBS containing 0.1% SDS.
  • a lysis buffer 5 mM EDTA, 10% glycerol, 50 mM Tris-HCL, pH 8.0, 0.005% Triton X-100
  • Numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of insertion (e.g., for CLN1, ...TWG25RLS...- indicates that 25 amino acids have been omitted between G and R).
  • Sources for each sequence used in this alignment and in the construction of an evolutionary tree are as follows: CYCA-Hs, human A type cyclin (Wang, J. et al., Nature 343:555-557 (1990)); CYCA-X1, Xenopus A-type cyclin (Minshull, J. et al., EMBO J.
  • CYCA-Ss clam A-type cyclin (Swenson, K.I. et al., Cell 47:867-870 (1986); CYCA-Dm, Drosophila A-type cyclin (Lehner, CF. and P.H. O'Farrell, Cell 56:957-968 (1989)); CYCBl-Hs, human Bl-type cyclin (Pines, J. and T. Hunter, Cell 58:833-846 (1989)); CYCB1-Xl and CYCB2-X1,
  • lysis buffer 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 0.5% NP-40, 0.5% Nadeoxycholate, 1 mM PMSF
  • Immunoprecipitation was carried out using 1 mg protein from each cell lysate at 4°C for overnight.
  • 60 ⁇ l of Protein A-agarose PIERCE was added to each immunoprecipitation and incubated at 4°C for 1 hour with constant rotating.
  • the immunoprecipitate was washed three times with the lysis buffer and final resuspended in 50 ⁇ l 2 ⁇ SDS protein sample buffer, boiled for 5 minutes and loaded onto a 10% polyacrymide gel. Proteins were transferred to a nitrocellulose filter using a SDE Elec troblo tting System (Millipore) for 45 minutes at a constant current of 400 mA.
  • the filter was blocked for 2 to 6 hours with 1 ⁇ PBS, 3% BSA and 0.1% sodium azide, washed 10 minutes each time and 6 times with NET gel buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 0.25% gelatin and 0.02 sodium azide), radiolabelled with 125 I-Protein A for 1 hour in blocking solution with shaking. The blot was then washed 10 minutes each time and 6 times with the NET gel buffer before autoradiography.
  • NET gel buffer 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 0.25% gelatin and 0.02 sodium azide
  • the tree was constructed using the Neighbor-Joining method (Saitou, N. and M. Nei, Mol. Biol. Evol., 4:406- 425 (1987).
  • the length of horizontal line reflects the divergence.
  • the branch length between the node connecting the CLN cyclins and other cyclins was arbitrary divided.
  • the human HeLa cell cDNA library, the human glioblastoma cell U118 MG cDNA library, the normal human liver genomic library, and the hybridization buffer were the same as those described above.
  • a human hippocampus cDNA library was purchased from Stratagene, Inc. High- and low-stringency hybridizations were carried out at 68° and 50°C, respectively.
  • To prepare template DNA for PCR reactions approximately 2 million lambda phages from each cDNA library were plated at a density of 10 5 PFU/150-mm plate, and DNA was prepared from the plate lysate according to Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.
  • the first 5' oligonucleotide primer, HCND11 is a 8192-fold degenerate 38-mer (TGGATG [T/C] TNGAf A/G] GTNTG [T/C] GA [ A/C] GA[ A/G ] CA[A/G]AA[A/G]TG[T/C]GA[A/G]GA) (SEQ ID No. 37), encoding 13 amino acids (WMLEVCEEQKCEE) (SEQ ID No. 38).
  • the second 5' oligonucleotide primer, HCND12 is a 8192-fold degenerate 29-mer (GTNTT[T/C]CCN[T/C]TNGCNATGAA[T/C]TA[T/C]TNGA)(SEQ ID No. 39), encoding 10 amino acids
  • the 3' primer, HCND13 is a 3072-fold degenerate 24-mer ([A/G]TCNGT[A/G]TA[A/G/T] AT[A/G]CANA[A/G][T/C ] TT - [ T/C ] TC ) ( S EQ ID No . 41 ) , encoding 8 amino ac i ds (EKLCIYTD) (SEQ ID No. 42).
  • the PCR reactions were carried out for 30 cycles at 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min.
  • the reactions contained 50 mM KCl, 10 mM Tris-HCl(pH 8.3), 1.5 mM MgCL, 0.01% gelatin, 0.2 mM each of dATP, dGTP, dCTP, and dTTP, 2.5 units of Taq polymerase, 5 ⁇ M. of oligonucleotide, and 2-10 ⁇ g of template DNA.
  • HCND11 and HCND13 were verified in a second-round PCT reaction using HCND12 and HCND13 as the primers. After resolution on a 1.2% agarose gel, DNA fragments with the expected size (200 bp between primer HCND11 and HCND13) were purified and subcloned into the Imal site of phagmid vector pUC118 for sequencing.
  • the 201-bp fragment of the D3 PCR product was labeled with oligonucleotide primers HCND11 and HCND13 using a randomprimed labeling technique (Feinberg, A. P. et al., Anal. Biochem. 132:6-13 (1983)) and used to screen a human HeLa cell cDNA library.
  • the probe used to screen the human genomic library for the CCND3 gene was a 2-kb EcoRI fragment derived from cDNA clone AD3-H34. All hybridizations for the screen of human cyclin D3 were carried out at high stringency.
  • PCR clones corresponding to CCND1 and CCND3 have been repeatedly isolated from both cDNA libraries; CCND2 has not.
  • cyclin D2 a 1-kb EcoRI fragment derived from mouse cy12 cDNA was used as a probe to screen a human genomic library. Under low- stringency conditions, this probe hybridized to both human cyclins D1 and D2.
  • the cyclin D1 clones were eliminated through another hybridization with a human cyclin D1 probe at high stringency.
  • Human CCND2 genomic clones were subsequently identified by partial sequencing and by comparing the predicted protein sequence with that of human cyclins D1 and D3 as well as mouse cy12.
  • human CCND1 (cyclin D1) was isolated by rescuing a triple Cln deficiency mutant of Saccharomyces cerevisiae using a genetic comp lementati on screen . Evo lutionary proximity between human and mouse, and the high sequence similarity among cyll , cy12, and cy13, suggested the existence of two additional D-type cyclin genes in the human genome.
  • the PCR technique was first used to isolate the putative human cyclin D2 and D3 genes. Three degenerate oligonucleotide primers were derived from highly conserved regions of human CCND1, mouse cy11, cy12, and cy13.
  • cyclin D1 and a 200-bp DNA fragment that appeared to be the human homolog of mouse cy13 from both human HeLa cell and glioblastoma cell cDNA libraries was isolated.
  • a human HeLa cell cDNA library was screened with this PCR product as probe to obtain a full-length D3 clone. Some 1.2 million cDNA clones were screened, and six positives were obtained. The longest cDNA clone from this screen,
  • mouse cy12 cDNA was used as a heterologous probe to screen a human cDNA library at low stringency. This resulted, initially, in isolation of 10 clones from the HeLa cell cDNA library, but all corresponded to the human cyclin D1 gene on the basis of restriction mapping. Presumably, this was because cyclin D2 in HeLa cells is expressed at very low levels. Thus, the same probe was used to screen a human genomic
  • AD2-P3 (1911 bp) The longest cDNA clone, AD2-P3 (1911 bp), was completely sequenced ( Figure 3). Neither AD2-P3 nor AD3-H34 contains a poly(A) sequence, suggesting that part of the 3' untranslated region might be missing.
  • AD2-P3 revealed an open reading frame that could encode a 289 -amino-acid protein with a 33,045-Da calculated molecular weight.
  • a similar analysis of AD3-H34 revealed a 292-amino-acid open reading frame encoding a protein with a 32,482-Da calculated molecular weight.
  • human cyclin D1 there is neither methionine nor stop codons 5' to the presumptive initiating methionine codon for both AD2-P3 (nucleotide position 22, Figure 3) and AD3-H34 (nucleotide position 101, Figure 4).
  • both AD2-P3 and AD3-H34 are believed to contain full-length coding regions.
  • cyclin A The protein sequence of all 11 mammalian cyclins identified to date were compared to assess their structural and evolutionary relationships. This includes cyclin A, cyclins B1 and B2, six D-type cyclins (three from human and three from mouse), and the recently identified cyclins E and C ( Figure 7). Several features concerning D-type cyclins can be seen from this comparison. First, as noted previously for cyclin D1, all three cyclin D genes encode a similar small size protein ranging from 289 to 295 amino acid residues, the shortest cyclins found so far.
  • D-type cyclins have evolved a different mechanism to govern their periodic degradation during each cell cycle or that they do not undergo such destruction.
  • members of the D-type cyclins are more closely related to each other than are members of the B-type cyclins, averaging 78% for three cyclin D genes in the cyclin box versus 57% for two cyclin B genes. This suggests that the separation (emergence) of D-type cyclins occurred after that of cyclin Bl from B2.
  • chromosome localization of CCND2 and CCND3 was determined by fluorescence in situ hybridization.
  • Metaphase chromosome spreads prepared by the standard technique (Lichter, T. et al., Science 247:64-69 (1990)) were hybridized in situ with biotin-labeled D2-G4 or
  • D3-G9 Denaturation and preannealing of 5 ⁇ g of DNasetreated human placental DNA, 7 ⁇ g of DNased salmon sperm DNA, and 100 ng of labeled probe were performed before the cocktail was applied to Alu prehybridized slides.
  • Biotin- labeled DNA was detected using fluorescence isothiocyanate (FITC)-conjugated avidin DCS (5 ⁇ g/ml) (Vector Laboratories); digoxigenin-labeled DNA was detected using a rhodamine-conjugated anti-digoxigenin antibody (BoehringerMannheim).
  • FITC fluorescence isothiocyanate
  • avidin DCS 5 ⁇ g/ml
  • Fluorescence signals were imaged separately using a Zeiss Axioskop-20 epifluorescence microscope equipped with a cooled CCD camera (Photometries CH220). Camera control and image acquisition were performed using an Apple Macintosh IIX computer. The gray scale images were pseudocolored and merged electronically as described previously (Baldini, A. et al., Genomics 9:770-774
  • Chromosomal fluorescence in situ hybridization was used to localize D2-G4 and D3-G9.
  • the cytogenetic location of D2-G4 on chromosome 12p band 13 and that of D3-G9 on chromosome 6p band 21 were determined by direct visualization of the two-color fluorescence in situ
  • the Alu 48-mer R-bands consistent with the conventional R-banding pattern, were imaged and merged with images generated from the D2-G4 and D3-G9 hybridized probes.
  • the loci of D2-G4 and D3-G9 were visualized against the ALi banding by merging the corresponding FITC and rhodamine images. This merged image allows the direct visualization of D2-G4 and D3-G9 on chromosomes 12 and 6, respectively.
  • the D2-G4 probe lies on the positive R-band 12p13, while D3-G9 lies on the positive
  • Cro s s - hybr idiz at ion was not detected with either pseudogene cyclin D2 or D3, presumably because the potentially cross-hybridizing sequence represents only a sufficiently small proportion of the 15- and 16-kb genomic fragments (nonsuppressed) used as probe, and the nucleotide sequences of pseudogenes have diverged from their ancestral active genes.
  • Genomic clones of human D-type cyclins were isolated and characterized to study the genomic structure and to obtain probes for chromosomal mapping.
  • the entire 1.3-kb cyclin D1 cDNA clone was used as probe to screen a normal human liver genomic library.
  • Five million lambda clones were screened, and three positives were obtained.
  • lambda clone G6 was chosen for further analysis.
  • a 1.7-kb BamHI restriction fragment of XD1-G6 was subcloned into pUC118 and completely sequenced. Comparison with the cDNA clones previously isolated and RNase protection experiment results (Withers, D.A. et al., Mol. Cell. Biol.
  • the 8 lambda clones that did not hybridize with the human D1 probe at high stringency fall into three classes respresented by AD2-G1, AD2-G2, and AD2-G4, respectively.
  • These three lambda clones were subcloned into a pUC plasmid vector, and small restriction fragments containing coding region were identified by Southern hybridization using a mouse cyl2 cDNA probe.
  • a 0.4-kb BamHI fragment derived from AD2-G1 was subsequently used as a probe to screen a human hippocampus cell cDNA library at high stringency.
  • AD2-G1 and AD2-G2 were two different clones corresponding to the same gene, whereas AD2-G4 appeared to correspond to a different gene.
  • a 2.7-kb 2acl-2mal fragment from AD2-G4 and 1.5-kb BclI-BglII fragment from AD2-G1 have been completely sequenced.
  • Lambda genomic clones corresponding to the human cyclin D3 were Isolated from the same genomic library using human D3 cDNA as a probe. Of four million clones screened, nine were positives. Two classes of clones, represented by AD3-G4 and AD3-G9, were distinguished by restriction digestion analysis. A 2.0-kb HindIII-SacI restriction fragment from AD3-G5 and a 3.7-kb SacI- HindIII restriction fragment from AD3-G9 were further subcloned into a pUC plasmid vector for more detailed restriction mapping and complete sequencing, as they both hybridized to the 5' cyclin D3 cDNA probe.
  • the 3.7-kb fragment from clone G9 contains 1.8 kb of sequence 5' to the presumptive initiating methionine codon identified in D3 cDNA ( Figure 4), a 198-bp exon 1, a 684-bp exon 2, and a 870-bp intron.
  • the 1.5-kb BclI-BglII fragment subcloned from clone AD2-G1 has been completely sequenced and compared with cyclin D2 cDNA clone AD2-P3. As shown in Figure 10, it contains three internal stop codons (nucleotide positions 495, 956, and 1310, indicated by asterisks), two frameshifts (position 1188 and 1291, slash lines), one
  • triplet CGT at position 277 to 279 of D2 cDNA Figure 3 encodes amino acid Arg, which is an invariant residue in all cyclins (see Figure 8).

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Abstract

Nouvelle classe de cyclines, appelées cyclines du type D, d'origine mammifère, et notamment d'origine humaine, ADN et ARN codant pour ces nouvelles cyclines, et procédé d'identification d'autres cyclines du type D et du type non D. L'invention se rapporte également à un procédé de détection d'un niveau accru d'une cycline de type D et à une méthode d'inhibition de division cellulaire en empêchant la formation du complexe protéine kinase - cycline du type D essentiel au démarrage du cycle cellulaire.
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