CA2103161A1 - D-type cyclin and uses related thereto - Google Patents

Abstract

A novel class of cyclins, referred to as D-type cyclins, of mammalian origin, particularly human origin, DNA and RNA encoding the novel cyclins, and a method of identifying other D-type and non-D-type cyclins. Also disclosed are a method of detecting an increased level of a D-type cyclin and a method of inhibiting cell division by interfering with formation of the protein kinase-D-type cyclin complex essential for cell cycle start.

Description

WOg2/20796 . PCT/US92/~146 ::
2103161 :

D-TYPE CYCLIN AND USES RELATED THERETO
__________________________ _ _ .

~ Descri~tion __ ____ _ ___ ~ Back~round of the Invention ____ ______________________ A typical cell cycle of a eukaryotic cell includes 05 the M phase, which includes nuclear division (mitosis) and cytoplasmic division or cytokinesis and interphase, which begins with the Gl phase, proceeds into the S phase and ends with the G2 phase, which continues until mitosis begins, initiating the next M phase. .In the S phase, DNA
~0 replication and histone synthesis occurs, while in the Gl and G2 phases, 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 Gl phase called the restriction 15 point or start, beyond which a cell is committed to completing the S, G2 and M phases.
Onset of the M phase appears to be regulated by a common mechanism in all eukaryotic cells. A key element of this mechanism is the protein kinase p84cdC2, whose 20 activation requires changes in phosphorylation and interaction with proteins referred to as cyclins, which also have an ongoing role in the M phase after acti-vation.
Cyclins are proteins that were discovered due to 25 their intense synthesis following the fertilization o marine invertebrate eggs ~Rosenthal, E.T. et _1., Cell 20:487-494 (1980)). It was subsequently observed that the abundance of two types of cyclin, A and B, oscillated WOg2/20796 PCT/US92/04146

-2-during ~he early cleavage divisions due to abrupt proteo-lytic degradation of the polypeptides at ~itosis and thus, they derived their name (E~ans, T. et al., Cell 33:389-396 (1983); Swenson, K.I. et al., Cell 47:867-870 05 (1986); Standart, N. et al., Dev. Biol. 124:248 258 (1987)).
Active rather than passive involvement of cyclins in regulation of cell di~ision became apparent with the obser~ation that a clam cyclin mRNA could cause acti-vation of frog oocytes and entry of these cells into Mphase (Swenson, K.I. et al., Cell 47:867-870 (1986)).
Activation of frog oocytes is associated with elaboration of an M phase inducing factor known as MPF (Masui, Y. and C.L. Markert, J. Ex~. Zool. 177:129 146 (1971); Smith, L.D. and R.E. Ecker, Dev. Biol. 25:232-247 (1971)). MPF
is a protein kinase in which the catalytic s~bunit is the fro~ homolog of the cdc2 protein kinase (Dunphy, ~.G. et al., Cell 54:423-431 (1988); Gautier, J. et al., Cell 54:433-439 (1988); Arion, D. et al., Cell 55:371-378 (1988)) Three types of classes of cyclins have been identi-fied to date: B, A and CLN cyclins. The B-type cyclin has been shown to act in mitosis by serving as an integral subunit of the cdc2 protein kinase (Booher, R.
and D. Beach, EMB0 J. 6:3441-3447 (1987); Draetta, G. et al., Cell 56:829-838 (1989); Labbe, J.C. et al., Cell 57:253-263 (1989); Labbe, J.C. et al., EMBO_J. 8:3053-3058 (1989); Mei~er, L. et al., EMBO_J. 8:2275-2282 (1989); Gautier, J. e_ al., Cell 60:487-494 (1990)). The A-type cyclin also independently associates with the cdc2 W092/20796 PCT/US92/~146 ~3~ 2103161 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., EMB0 J. 9:2865-2875 (1990); Giordano, A. et al., Cell 58:981-990 (1989);
05 Pines, J. and T. Hunter, Nature 346:760-763 (1990)). The functional difference between these two classes of cyclins is not yet fully understood.
Cellular and molecular studies of cyclins in invertebrate and vertebrate embryos ha~e been accompanied by genetic studies, particularly in ascomycete yeasts.
In the fission yeast, the cdc13 gene encodes a B-type cyclin that acts in cooperation with cdc2 to regulate entry into mitosis (Booher, R. and D. Beach, ~MB0 J., 6:3441-3447 (1987); Booher, R. and D. Beach, EMB0 J.
7:2321-2327 (1988); Hagan, I. et al., J. Cell Sci.
91:587-595 (1988); Solomon, M., Cell 54:738-740 (1988);
Goebl, M. and B. Byers, Cell 54:433-439 (1988); Booher, R.N. et al., Cell 58:485-497 (1989)).
__ ___ ____ __ Genetic studies in both the budding yeast and fission yeast have revealed that cdc2 (or CDC28 in budding yeast) 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);
_____________ ___ Nurse, P. and Y. Bissett, Nature 292:558-560 {1981~;
Piggot, J.R. et al., Nature 298:391-393 (1982); Reed, S.I. and C. Wittenberg, Proc._Nat._Acad._Sci._USA
87:5697-5701 (1990)).
In budding yeast, the start function of the CDC28 protein also requires association of the-catalytic

3~ subunit of the protein kinase with ancillary proteins WO 92/20796 PCI`/USg2J04146 ~ ?

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., EMB0 J.
05 7:4335-4346 (1988); Hadwiger, J.A. et al., Prot._Natl.
Acad._Sci. USA 86:6255-6259 (1989); Richardson, H.E. et _1., Cell 59:1127-1133 (1989)). The CLN genes are_ ____ __ essential for execution of start and in their absence, cells become arrested in the Gl phase of the cell cycle.
10 T~e CLNl and CLN2 transcripts oscillate in abundance through the cell cycle, but the CLN3 transcript does not.
~' In addition, the Cln2 protein has been shown to oscillate in parallel with its mRNA (Nash, R. et al., EMBO_J.
7:4335-4346 (1988); Cross, F.R., Mol._Cell._Biol.
8:4675-4684 (1988); Richardson, H.E. et al., Cell 59:1127-1133 (1988); Wittenberg, et al., l9gO)).
Although the precise biochemical properties con-ferred on cdc2/CDC28 by association with different cyclins have not bee~ fully elaborated, genetic studies of cyclin mutants clearly establishes that they confer "Gl" and "G2" properties on the catalytic subunit (Booher, R. and D. Beach, EMB0 J. 6:3441-3447 (1987);
Nash, R. et al., EMBO_J. 7:4335-4346 (1988); Richardson, H.E. et al., Cell 56:1127-1133 (1989)).
cdc2 and cyclins have been found not only in embryos and yeasts, but also in somatic human cells. The function of the cdc2/cyclin B enzyme appears to be the same in human cells as in other cell types (Riabowol, K.
et al., Cell 57:393-401 (1989)). A human A type cyclin 30 has also been found in association with cdc2. No CLN

, ,?,1031~il 5 ,;

type cyclin has yet been described in mammalian cells. A
better unders~anding of the elements involved in cell cycle regulation and of their interactions would con-tribute to a better understancing of cell replication and 05 perhaps even alter or control the process.

Summary_of_the_Inve tion The present invention relates to a novel class of cyclins, referred to as D-type cyclins, which are of mammalian origin and are a n~w family of cyelins related 10 to, but distinct from, previously described A, B or CLN
type cyclins. In particulsr, it relates to human cyclins, encoded by genes shown to be able to replace a CLN-type gene essential for cell cycle star~ in yeast, which complement a deficiency of a protein essential for 15 cell cycle start and which, on the basis of protein structure, are on a different branch of the evolutionary tree from A, B or GLN type cyclins. Three members of the x new family of D-type cyclins, referred to as the human D-type gene family, are described herein. They encode 20 small (33-34 KDa) proteins which share an average of 57%
identity over the entire coding region and 78~ in the cyclin box. One member of this new cyclin family, cyclin Dl or CCNDl, is 295 amino acld residues and has an estimated molecular weight of 33,670 daltons (Da). A ~-25 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 pl3.
A third member, cyclin D3 or CCND3, is 292 amino acid residues and has an estimated molecular weight of 30 approximately 32,482 daltons. It has been mapped to W092/~0796 PCT/US92/04146 chromosome 6p band p21. The D-type cyclins described herein are the smallest cyclin proteins identified to date. All three cyclin genes described herein are interrupted by an intron at the same position. D-type 05 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. Thus, the present invention includes reccmbinant D-type cyclins, isolated or'~urified D-type cyclins and ~ynthetic D-type cyclins.
The present invention also relates to DNA or RNA
encoding a D-eype cyclin of mammalian origin, particu-larly 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 i~vention 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 therspeutic aspects. For ex-20 ample, it relates to a method in which the presenceand/or quantity of a D-type cyclin (or cyclins) in tissues or biological ~amples, such as blood, urine, feces, mucous or saliva, is determined, using a nucleic acid probe based on a D-type cyclin gene or genes des-25 cribed 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 ab-normally high rate (i.e., if cells are likely to be cancero~s), by determining whether their cyclin levels or 30 activity are elevated (elevated level of activity being - indicative of an increased probability that cells will VWD92/207g6 PCT/US9~/~146 210316i ``
- 7 - ; à

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 05 a D-type cyclin(s), a gene(s) encoding a D-type cyclin(s) or a transcription product(s) (RNA).
In addition, 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 act:ivity -~
or function of a D-type cyclin(s). In this therapeutic ~
method, function of D-type cyclin(s) is blocked (totally ~`
15 or partially) by interfering with its ability to activate -the protein kinase it would otherwise (normally) activate (e.g., p34 or a related protein kinase), by means of ~`~
agents which interfere with D-~ype 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 ~olecule 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. The subject invention also relates to agents (e.g., oligonucleotides, antibodies, peptides) useful in the isolation, diagnostic or thera-peutic ~ethods described.

~"~" ~ ` r~3 W092/20796 PCT/USg2/04146 . ~, . . . ~ . -21031~ 8-Brief Descri~tion of the Fi~ures ____________ ______________ ____ Figure 1 is a schematic representation of a genetic screen for human cyclin genes.
Figure 2 is the human cyclin Dl nucleic acid 05 sequence (SEQ ID No. l) 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.
Fig~re 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 aminc acid nu~bers are on the right, amino acid numbers are gi~en 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 sequence (SEQ ID No. 5) and a~ino acid sequence ~SEQ ID
No. 6), 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 5 shows the cyclin gene family.
Figure SA shows the amino acid sequence alignment of seven cyclin genes (CYCDl-Hs, SEQ ID No. 7; CYCA-Hs, SEQ
ID No. 8; CYCA-Dm, SEQ ID No. 9; CYCBl-Hs, SEQ ID No. 10;
CDC13-Sp, SEQ ID No. 11; CLNl-Sc, SE~ 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.
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.

` 2103161 g , .

Figure 6 shows alternative polyadenylation of the cyclin Dl gene transcript.
Figure 6A is a comparison of several cDNA clones isolated from different cell lines. Open boxes represent 05 the 1.7 kb small transcript containing the coding region of cyclin Dl gene. Shadowed boxes represent the 3' fragment present in the 4.8 kb long transcript.
Restriction sites are given above each cDNA clone to ~-indicate the alignment of these clones.
Figure 6B shows the nucleotide sequence surrou~ding the first polyadenylation site for several c~NA clones -(CYCDl-21, SEQ ID No. 14; CYCDl-H12, SEQ ID No. 15; -CYCDl-HO34, SEQ ID No. 16; CYCDl-T078, SEQ ID No. 17 and a genomic clone; CYCDl-GO68, SEQ ID No. 18).
Figure 6C is a summary of the structure and alter-native polyadenylation of the cyclin Dl gene. Open boxes represent the small transcript, the shadowed box repre-sents 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 (CYCDl-Hs, SEQ ID No. 19; ~-CYLl -Mm r 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; CYCRl-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 WOg2/20796 PCT/US92/~146 2i03161 -lO-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 05 the top of each clone.
Figure 9 is the nucleic acid sequence (SEQ ID No.
30) and amino acid sequence (S~Q ID No. 31) of a cyclin D2 pseudogene.
Figure 10 is the nucleic acid se~uence (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 ~o.
34) of 1.3 kb of human cyclin Dl 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 txanscription starts at approximately nucleotide -160.

Detailed Descri~tion of the Invention _______________ _____________________ As described herein, 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 ~hat they fill the role of a known cyclin protein by activatin~, a protein kinase whose 30 activation is essential for cell cycle start, an event in W092/20796 PCT/US92/~1~

the Gl phase at which a cell becomes committed to cell division. Specifically, human D-type cyclin proteins, as well as the genes which encode them, have been identi-fied, isolated and shown to be able to replace CLN type 05 cyclin known to be essential for cell cycle start in yeast. The chromosomal locations of CCND2 and CCND3 have also been mapped.
As a result, a new class of cyclins (D type) is available, as are DNA and RNA encoding the novel D-type ~;
cyclins, antibodies specific for (which bind to) D-type cyclins and methods of their use in the identification of additional cyclins, the detection of such proteins and oligonucleotides in biological samples, the inhibition of abnormally increased rates of cell division and the identification of inhibitor~ of cyclins.
The following is a description of the identification and characterization of human D-type cyclins and of the uses of these no~el cyclins and related products.
' Isolation and_Characterization_of Human Cyclin D2 and D3 _ __ ____ .
As represented schematically in Figure 1 and des-cribed in detail in Example 1, a mutant yeast strain in which two of the three CLN genes (CLNl and CLN2) were inactive and expression of the third was conditional, was 25 used to identify human cDNA clones which rescue yeast from CLN deficiency. A human glioblastoma cDNA library carried in a yeast expression ~ector (pADNS) was intro-duced into the mutant yeast strain. Two yeast transform-ants (pCYCDl-21 and pCYCDl-19) which ~rew despite the WO92!20796 PCT/US92/04146 !, L~

lack of function of all three CLN genes and were not revertants, were identified and recovered in E. coll.
Both rescued the mutant (CLN deficient) strain when reintroduced into yeast, although rescue was inefficient `~:
05 and the rescued strain grew relatively poorly.
pCYCDl-l9 and pCYCDl-21 were shown, by restriction mapping and partial DNA sequence analysis, to be inde~
pendent clones representing the same gene. A HeLa cDNA
library was screened for a full length cDNA clone, using the 1.2 kb insert of pCYCDl-21 as probe. Complete sequencing was done of the longest of~nine posi~i~e clones identified in this manner (pCYCDl-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 oligonucleotlde probes derived from three highly conserved regions of D-type cyclins, as descrihed in Example 4. As described, two 5' oligonucleotides and one 3' degenerate oligonucle-otide 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, lg91, under the terms of the Budapest Treaty.
Accession number 68620 has been assigned to the deposit.
Comparison of the CYCDl-H12-encoded protein se~uence with that of known cyclins (see Figure SA) showed that W092/20796 PCT/US92/~146 -13- ~103161 . . ., ~, there was homology between the new cyclin and A, B and CLN type cyclins, but also made it clear that CYCDl - differs from these existing classes.
An assessment of how this new cyclin gene and its 05 product might be related in an e~olutionary sense to other cyclin genes was carried out by a comprehensive comparison of the amino acid sequences of all known cyclins (Figure 5B and Example 1). Results of this comparison showed that CYCDl represents a new class of cyclin, designated herein cyclin D.
Expression of cyclin Dl gene in human cells was ' studied using Northern analysis, as described in Example 2. Results showed that levels of cyclin Dl expression were very low in se~eral cell lines. The entire coding region of the CYCDl 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 abunda~t. Most of the cDNA clonas isolated from various cDNA l~braries proved to be very si~ilar to clone ~CYCDl-H12 and, thus, it appears that `-the 1.7 kb transcript detected in ~orthern blots corres-ponds to the nucleo~ide sequenee 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 CYCDl (Figure 6).
Differential expression of cyclin Dl in different tissues and cell lines was also assessed, as described in Example 3. Screening of cDNA libraries to obtain full length CYCDl clones had demonstrated that the cDNA

~ 14-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 05 cDNA). Northern and Western blotting were carried out to determine whether cyclin Dl 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. Uestern blotting using anti-CYLl antibody readily detected the presence of a 34kd polypept~de in the glioblas~oma cells and demonstrated tha~ 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 CYCDl protein ex-pressed in E. coli. Thus, results demonstrated differ-ential occurrence of the cyclin Dl in the cell types analyzed, with the highest levels being in cells of neural origin.
As also described herein (Example 6), human genomic libraries were screened using cDNA probes and genomic clones of human D-type cyclins, specifically Dl, D2 and D3, have been isolated and characterized. Nucleic acid sequences of cyclin Dl, D2 and D3 promoters are repre-sented in Figures 11-13. Specifically, the entire 1.3 kb cyclin Dl cDNA clone was used as a probe to screen a normal human liver genomic library, resulting in identi-fication of three positive clones. One of these clones(G6) contained a DNA insert shown to contain 1150 bp of -W092/20796 PCT/US92/~146 -15- 21031~1 ~

upstream promoter sequence and a 198 bp exon, followed by an intron. Lambda genomic clones corresponding to the human cyclin D2 and lambda genomic clones corresponding to the human cyclin D3 were also isolated and character-- 05 ized, using a similar approach. One clone (~D2-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. One clone (G9) was shown to contain (Flgure 8C) 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.
Thus, as a result of the work described herein, a novel class of mammalian cyclins, designated cyclin D or D-type cyclin, has been identified and shown to be dis- -tinct, on the basis of structure of the gene (protein) ~
product, from previously-identified cyclins. Three ;
members of this new class, designated cyclin Dl or CCNDl, ~`
zo cyclin D2 or CCND2 and cyclin D3 or CCND3, have been -~
isolated and sequenced. They have been shown to fulfill the role of snother 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 25 cyclin Dl gene is expressed differentially in different cell types, with expression being highest in cells of neural origin.

Uses of the Invention It is possible, using the methods and materials 30 described herein, to identify genes (DNA or RNA) which ~`1 0 ~1`6-1 - 16-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 05 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 mutsnt 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) is intro-duced into a mutant yeast strain, such as the strain described herein (Example 1 and Experimental Procedures).
The strain used contains altered CLN genes. In the case of the specific strain descr~bed herein, insertional `~
mutations in the CLNl and CLN2 genes rendered them inactive and alteration of the CLN3 gene allowed for its ;~
conditional expression from a galactose-inducible, glucose-repressible promoter; as exemplified, this promoter is a galactose-inducible, glucose-repressible-promoter but others can be used.
Mutant yeast transformed with ~he cDNA library in the expression vector are screened for their ability to grow on glucose-containing medium. In medium containing galactose, the CLN3 gene is expressed and cell viability is maintained, despite the absence of CLNl and CLN2. In medium containing glucose, all CLN function is lost and the yeast cells arrest in the Gl phase of the cell cycle.
- Thus, the ability of a yeast transformant to grow on W092/20796 - PCT/US9~/04146 -17- ~103161 glucose-containing m~dium is an indication of the pre-sence in the transformant of DNA able to replace the function of a gene essential for cell cycle start.
Although not required, this can be confirmed by use of an 05 expression vector, such as 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 (~ntroduction of DNA which replaces the unexpressed or nonfunctional yeast gene(s) essential for cell cycle start). ~sing this method, cyclins of all types (D type, non-D type) can be identified by their ability to replace .
CLN3 function when transformants are grown on glucose.
15Screening of additional cDNA or genomic libraries eo 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 Dl, D2 or D3 cDNA sequences of Figures 2-4, respecti~ely, 20 or all or a portion of the corresponding genomic sequen- -ces described herein can be used as probes. The hybrid-ization conditions can be varied as desired and, as a result, the sequences identified will be of greater or les~er complementarity to the probe sequence (i.e., if higher or lower stringency conditions are used~. Addi-tionally, an anti-D type cyclin antibody, such as CYLl or another raised against Dl 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.

~103161 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 05 saliva. This has poeential 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 prolifer-ation. For example, several previous reports have suggested the oncogenic potential of altered human cyclin A function. The human cyclin A gene was found to be a ~, target for hepatitis B virus integration in a hepato-cellular carcinoma (Uand, J. et al., Nature 343:555-557 (1990)). Cyclin A has also been shown to associate with adenovirus ElA in virally infected cells (Giordano, A et al., Cell 58:981-990 (1989); Pines, J. and T. Hunter, Nature 34~-760-763 (1990)). Further, the PRADl gene, ______ ___ which has the same sequence as the cyclin Dl 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 abnormal-ities in chromosome llql3. In particular, identification of CCNDl (PRADl) as a candidate BCLl oncogene provides the most direct evidence for the oncogenic potential of cyclin genes. This also suggests that other members of the D-type cyclin family may be involved in oncogenesis.
In this context, the chromosomal locations of the CC~D2 and CCND3 genes have been mapped to 12pl3 and 6p21, respectively. Region 12pl3 contains sites of several translocations that are associated with specific immuno-phenotypes of disease, such as acute lymphoblastic W092/20796 PCT/US92/~146 - 19 - ~ .

leukemia, chronic myelomoncytic leukemia, and acute myeloid leukemia. Particularly, the isochromosome of the short arm of chromosome 12 [1(12p)] is one of a few known consistent chromosomal abnormalities in human solid 05 tumors and is seen in 90% of adult testicular germ cell tumors. Region 6p21, on the other hand, has been impli-cated in the manifestation of chronic lymphoproliferative disorder and leiomyoma. Region tp21, the locus of ~LA
complex, is also one of the best characterized regions of the human genome. Many diseases have been previously linked to the KLA complex, but the etiology of few of ~- these diseases is fully understood. Molecular cloning ;~
and chromosomal localization of cyclins D2 and D3 should make it possible to determine whether they are directly involved in these translocations, and if so, whether they are activated. If they prove to be involved, diagnostic and therapeutic methods described herein can be used to assess an individual's disease state or probability of developing a condition associated with or caused by such translocations, to monitor therapy effec~iveness (by assessing the effect of a drug or drugs on cell proliferation) and to provide treatment. ~`
The present invention includes a diagnostic method to detect altered expression of a cyclin gene, such as cyclin Dl, D2, D3 or another D-type cyclin. The method can be carried out to detect altered expression in cells or in a biological sample. As shown herein, there is high sequence similarity among cyclin D genes, which indicates that different members of D-type cyclins ~ay use similar mechanisms in regulating the cell cycle (e.g., association with the same catalytic subunit and W092/20796 PCT/US92/~146 ~ -20-acting upon the same substrates). The fact that there is cell-type-specific differential expression, in both mouse and human cells, makes it reasonable to suggest that different cell lineages or d~fferent tissues may use 05 different D-type cyclins to perform very similar func-tions and that altered tissue-specific expression of cyclin D genes as a result of translocation or other mutational events may contribute to abnormal cell proliferation. As described herein, cyclin Dl is ex-pressed differentially in tissues analyzed; in particu-lar, it has been shown to be expressed at the highest ~' levels in cells of neural origin (e.g., glioblastoma cells).
As a result of the work described herein, 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 ~arious cell types or of one or more of the types of D cyclins) can also be determined.
In a diagnostic method of ehe present invention, cells obtained from an individusl are processed in order to render nucleic acid sequences in them available for hybridization with complementary nucleic acid se~uences.
25 All or a portion of the Dl, 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;
such a portion must be of sufficient length to hybridize to complementary sequences in a sample and remain hybri-30 dized under the conditions used and will generally be atleast six nucleotides long. Hybridization is detected using known techniques (e.g., measurement of labeled W092/20796 PCT/US~2/~1~
-21- ~ 3 1 ~ I

hybridization complexes, if radiolabeled or fluorescently labeled oligonucleotide probed are used). The extent to which hybridi~ation occurs is quantitated; increased levels of the D-type cyclin gene is indicative of 05 increased potential for cell di~ision.
Alternatively, 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 tech-nlques and an antibody specific for the D-type cyclin(s).
10 In a third type of diagnostic method, 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 H1, as a substrate. Arion, D. et al., Cell, 55:371-378 (1988~.
15 In 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 20 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 prolifera~ion or oncogenesis or of the actual occurrence of abnoxmal prolifera~ion or 25 oncogenesis. It is also possible to detect more than one type of cyclin (e.g., A, B, and/or D) in a cell or tissue sample by using a set of probes (e.g., a set of nucleic acid probes or a set of antibodies), the members of which each recognize and bind to a selected cyclin and col-30 lectively provide information about twv or more cyclinsin the tissues or cells analyzed. Such probes are also ~ 22-2~03161 the sub;ect 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).
05 A method of inhibiting cell division, particularly cell division which would otherwise occur at an abnormal-ly high rate, is also possible. For example, 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. In ~' o~e embodiment, 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.
15 This can be carried out by introducing antisense oligo- -nucleotides into cells, in which they hybridize to the 'cyclin-encoding nucleic acid sequences, preventing their further processing. It is also possible to inhibit expression of the cyclin by interfering with an essential D-type transcription factor. There are reasons to believe that the regulation of cyclin gene transcription may play an important role in regulating the cell cycle and cell growth and oscillations of cyclin mRNA levels are critical in controlling cell division. The Gl phase 25 is the time at which cells commit to a new round of division in response to external and internal sequences and, thus, trar.scription factors which regulate expres-sion of Gl cyclins are surely important in controlling cell proliferation. Modulation of the transcription 30 factors is one route by which D-type cyclin activity can be influenced, resulting, in the case of inhibition or W092/20796 PCT/US92/~l~
-23- ~1~3161 prevention of function of the transcription factor(s), in reduced D-type cyclin activity. Alternatively, complex formation can be prevented indirectly by degrading the D-type cyclin(s), such as by in~roducing a protease or 05 substance which enhances cyclin breakdown into celIs. In either case, the effect is indirect in that less D-type cyclin is available than would otherwise be the case.
In another embodiment, protein kinase-D type cyclin complèx formation is prevented in a more direct manner 10 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 acti~lty 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 organlc compound which, like the endogenous D-type cyclin, b~nds the protein kinase, but whose binding does not result in activation of the enzyme or results in its being disabled or de-graded. 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 25 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. As described 30 herein, there is differential expression in tissues of W092/2~796 ~CT/US92/~146 ~ 03161 -24-D-type cyclins. Thus, it is possible to selectively decrease mitotic capability of cells by the use of an a~ent (e.g., an antibody or anti-sense or other nucleic acid molecule) which is designed to interfere with 05 (inhibit) the activity and/or level of expression of a selccted type (or types) of D cyclin. For example, in treating tumors in~olving the central nervous system or other non-hemotopoietic tissues, agents which selectively inhibit cyclin Dl might be expected to be particularly 10 useful, since Dl has been sh~wn to be differentially expressed (expressed at particularly high levels in cells of neural origin).
Antibodies specifically reacti~e with D-type cyclins of the present ~nvention can also be produc~d, using 15 known methods. For example, 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 ho.st animal after sufficient time for an~ibodies to have 20 been formed. Monoclonal antibodies can also be produced using known techniques. Sambrook, J. et al., Molecular Clonin~. _A_Laboratory Manual, Cold Spring ~arbor Laboratory, Cold Spring Harbor, NY (1989).
The present invention also includes a method of 25 screening compounds or molecules for their ability to inhibit or suppress the function of a cyclin, particu-larly a D-type cyclin. For example, mutant cells as described herein, in which a D-type cyclin such as Dl or D3, is expressed, can be used. A compound or molecule to 30 be assessed for its ability to inhibit a D-type cyclin is contacted with the cells, under conditions appropriate W092/20796 - PCT/US92/041~

-25- 21031Gl 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 di~ision in the presence of 05 the compound or ~olecule being assessed with cell division of an appropriate control (e.g., the same type of cells without added test drug> will demonstrate the ability or inability of the compound or molecule to inhibit the cyclin. Existing compounds or molecules (e.g., those present in a fermentation broth or a chemical "library"~ or those developed to inhibit the ~' cyclin activation of its protein kinase can be screened for their effectiveness using this method. Dr~gs which inhibit D-type cyclin are also the subject of this invention.
The present invention will now be illustrated by the following examples, which are not ~ntended to be limiting -in any way.

_XAMPLES______ Experimental procedurPs for Examples 1-3 are pre-sented after Example 3.

EXAMPLE 1 Identification of Human cDNA Clones that _.________ ________________________________________ Rescue CLN Deficiency ____________________ In S. cerevisiae, there are three Cln proteins.
_____________ 25 Disruption of any one CLN gene has little effect on growth, but if all three CLN genes are disrupted, ~he cells arrest in Gl (Richardson, H.E. et al , Cell 59:1127-1133 (1989)). A yeast strain was constructed, as W092~20796 PCT/US92/~l~

;: -26-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, 05 glucose-repressible promoter GALl (see Figure 1). The strain is designated 305-15d #21. In medium containing galsctose the CLN3 gene is expressed and despite the absence of both CLNl and CLN2, cell viability is retaîned (Fig. 1). In a medium containing glucose, all CLN
1~ function is lost and the cells arrest in the Gl phase of the cell cycle.
~' A human glioblastoma cDNA 11brary carried in the yeast expression vector pADNS (Colicelli, J. et al., Pro.
Natl. Acad. Sci. USA 86:3599-3603 (1989)) was introduced ____________________ __ 15 into the yeast. The vector pADNS has the LEU2 marker, the 2~ replication origin~ and the promoter and ter-minator sequences from the yeast alcohol dehydrogenase gene (Figure 1). Approximately 3 x 10 transformants were screened for the ability to grow on glucose con-20 taining medium. After 12 days of incubation, twelvecolonies were obtained. The majority of these proved to be re~ertants. However, in two cases, the ability to grow on glucose correlated with the maintenance of the LEU2 marker as assessed by plasmid stability tests.
25 These two yeast transformants carried plasmids designated pCYCDl-21 and pCYCD1-19 (see below). Both were recovered in E._coli. Upon reintroduction into yeast, the plasmids rescued the CLN deficient strain, although the rescue was inefficient and the rescued strain grew relatively 30 poorly.

W092/20796 PCT/US92/~146 -27- ~ 10~31 6 1 The restriction map and partial DNA sequence analy-sis revealed that pCYCDl-19 and pCYCDl-21 were indepen-dent clones representing the same gene. The 1.2 kb insert of pCYCDl-21 was used as probe to screen a human 05 HeLa cDNA library for a full length cDNA clone. Approxi-mately 2 million cDNA clones were screened and 9 posi-tives were obtained. The longest one of these clones, pCYCDl-Hl2 (1325 bp), was completely sequenced (Figure ~-2). The sequence exhiblts a very high GC content within the coding region (61%) and contains a poly A tail (69 A
residues). The estimated molecular weight of the predic-ted protein product of the gene is 33,670 daltons start-ing from the first in-frame AUG codon at nucleotide 145 H~
(Figure 2). The predicted protein is related to other syclins (see below) and has an unusually low pI of 4.9 (compared to 6.4 of human cyclin A, 7.7 of human cyclin B
and 5.6 of CLNl), largely contributed by the high concen-tration of acidic residues at its C-terminus.
There are nelther methionine nor s~op codons 5' to the predicted initiating methionine at nucleotide 145.
Because of this and also because of the apparent N-terminal ~runcation of CYCDl with respect to other cyclins (see below for more detail), four additional human cD~A libraries were further screened to see if the ~CYCDl-H12 clone might lack the full 5' region of the cDNA. Among more than 100 cDNA clones isolated from these screens, none was found that had a more extensive 5' region than that of ~CYCDl-Hl2. The full length coding capacity of clone H12 was later confirmed by Uestern blot analysis (see below).

092~20796 ~ PCT/US92/~146 r ; - 2 8 ~

CYCDl 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._cerevisi_e CLN1. By comparison with A and B type cyclins, the difference is 05 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., Nat_re 349:132-138 (1~91)).

Sequence_AnalysiS_of_Dl_an _ComEarison with Other Cyclins Sequence analysis revealed homology between the CYCDl-H12 encoded protein and other cyclins, How-e~er, it is clear that CYCD1 differs fro~ the three existing ciasses of cyclins, A, B and CLN. To examine how this new cyclin gene might be evolutionary related to other cyclins, a comprehensive amino acid sequence comparison of all cyclin genes was conducted. Fifteen previously publishea cyclin sequences as well as CYCDl were first aligned using a strategy described in detail by Xiong and Eickbush (Xiong, Y. and T.H. Eickbush, EMBO
J. 9:3353-3362 (1990)). Effort was made to reach the maximum similarity between sequences with the minimum introduction of insertion/deletions and to include as much sequence as possible. With the exception of CLN
cyclins, this alignment contains about 200 amino acids residues which occupies more than 70% of total coding region of CYCD1 (Figure 5A). There is a conserved domain and some scattered similarities between members of A and B type cyclins N-terminal to the aligned region (Glotzer, M. _t al., Nature 349:132-138 (1991)), but this is not___ ______ ___ W092/20796 PCT/US92/~l~

present in either CLN cyclins or CYCDl and CYLl and so they were not included in the alignment.
The percent divergence for all pairwise comparisons of the 17 aligned sequences was calculated and used to 05 construct an evolutionary tree of cyclin gene family using the Neighbor-Joining method (Saitou, N. and M. Nei, _ol. Biol. Evol. 4:406-425 (1987) and Experimental______________ _ Procedures~. Because of the lowest similarity of CLN
cyclins to ~he other three classes, the tree (Figure 5B) was rooted at the connection between the CLN cyclins and the others. It is ~ery clear from this evolutionary tree that CYC~Dl, CYCD2 and CYCD3 represent a distinct new class of cyclin, designated cyclin D.

EXAMPLE_2 Ex~ression_of_the Cyclin_Dl_Ge_e_i__Human Cells Express~on of cyclin Dl gene in human cells was studied by Northern analysis. Initial studies indicated that the level of cyclin Dl expression was very low in several cell lines. Poly (A)~RNA was prepared from HeLa cells and probed with the entire coding region of CYCDl 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 librar-ies are very similar to the clone ~CYCDl-H12 (Ei~ure 2).
Thus, it appears that the 1.7 kb transcript detected in Northern blots corresponds to nucleotide sequence in Figure 2.

W092~20796 - PCT/US92/04146 To understand the origin of the larger 4.8 kb transcript, both 5' and 3' end sub-fragments of the ~CYCDl-H12 clone were used to screen both cDNA and genomic libraries, to test whether there might be alter-05 na~ive transcription initiation, polyadenylstion and/ormRNA splicing. Two longer cDNA clones, ~CYCDl-H034 (1.7 kb) from HeLa cells and ~DYDCl-T078 (4.1 kb) from human teratocarcinoma cells, as well as several genomic clones were isolated and partially sequenced. Both ~CYCDl-H034 and ~CYCDl-T078 have identical sequences to ~CYCDl-H12 clone from their 5' ends (Figure 6). Both differ from ~CYCDl-H12 in having additional sequences at the 3' end, after the site of polyadenylation. These 3' sequences are the same in ~CYCDl-H034 and ~CYCDl-T078, but extend further in the latter clone (Figure 6). Nucleotide sequencing of a genomic clone within this reglon revealed colinearity between the cDNAs and the genomic D~A (Figure 6). There is a single base deletion (an A residue) in ~CYCDl-T078 cDNA clone. This ~ay be the result of polymorphis~, although it is not possible to exclude the possibility that some other mechanism is involved. The same 4.8 kb transcript, but not the 1.7 kb transcript, was detected using the 3' end extra fragment from clone T078 as a probe.
It appears that the two mRNAs detected in Northern blots arise by differential polyadenylation (Figure 6).
Strangely, there is no recognizable polyadenylation sequence (AAUAAA) anywhere within the sequence of clone ~CYCDl-H12, even though polyadenylation has clearly occurred (Figure 2). There is also no close variant of AAUAAA (nothing with less than two mismatches).

2l~)3l6l :

EXAMPLE 3 Differen~ial Ex~ression of Cyclin Dl Gene _________ _______________ ____________ ____________ i__Different_Cell Ty~es During the screening of cDNA libraries to obtain full length clones of CYCDl, it became evident that the 05 cDNA library derived from the human glioblastoma cell line (U118 MG) from which the yeast transformants were obtained gave rise to many more positi~es than the other four cDNA libraries. Northern and Western blotting were carried out to explore the possibility that cyclin Dl might be differentially expressed in different tissues or cell lines. Total RNA was isolated from U118 MG cells`
~-~ and analyzed by Northern blot using the CYCDl gene coding ~
region as probe. The level of transcript is 7 to 10 fold higher in the glioblastoma cells, compared to HeLa cells.
In both HeLa and U118 MG cells, both high and low molecular weight transcripts are observed.
To investigate whether the abundant CYCDl message in the U118 MG cell line is reflected at the protein level, cell extracts were prepared and Western blotting was `~
performed using anti-CYLl prepared against mouse CYLl (provided by Matsushime, H. et al.). This anti-CYLl antibody was able to detect nanogrsm quantities of recombinant CYCDl on Uestern blots ~data not shown), and was also able to detect CYCDl in the original yeast transformants ~y immunoprecipitation and Wes~ern analysis. Initial experiments using total cell extracts, from HeLa, 293 or U118 MG cells failed to detect any signal. However, if the cell extracts were immuno-precipitated with the serum before being subjected to 30 SDS-PAGE and immunoblotting, a 34 kd polypeptide was readily detected in Ull~ MG cells. The protein is far W092/20796 PCT/US92/~146 less abundant in HeLa cells and was not detectable in 293 cells. The molecular weight of the anti-CYCLl cross-reactive material from U118 MG and HeLa is exactly that of the h~man CYCDl protein expressed in E. coli. This 05 arg~es that the sequenced cDNA clones contain the entire open reading frame.

EXPERIMENTAL PROCEDURES

Strain Construction The parental strain was BF305-lSd (MATa leu2-3 leu2-112 his3-11 his3-lS ura3-52 trpl adel metl4 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 GALl promoter. A
15 0.75 kb Eco~I-BamHI fragment containing the bidirectional -~
GAL10-GALl promoters was fused to the 5' end of the CLN3 gene, such that the BamHI (GALl) end was attached 110 nucleotides upstream of the CLN3 start codon. An EcoRI
fragment stretching from the GAL10 promoter to the middle of CLN3 (Nash, R. e_ al., EMBO J. 7:4335-4346 (198B)) was then subcloned between the XhoI and EcoRI sites of pBF30 (Nash, R. et al., _MBO J. 7:4335-4346 (1988)). The ligation of the XhoI end to the EcoRI end was accom-plished by filling in the ends with Klenow, and blunt-end 25 ligating (destroying the EcoRI site). As a result, the GALl promoter had replaced the DNA normally found between -110 and -411 upstream of CLN3. Next, an EcoRI to SphI
fragment was excised from this new pBF30 derivative.
This fragment had extensive 5' and 3' homology to the CLN3 region, but contained the GALl 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.
05 In addition, average cell size was measured when the GAL1 promoter was induced or uninduced. Uhen the GALl promoter was induced by growing the cells in 1% raffinose and 1~ galactose, mode cell volume was about 25~m3 (com-pared to a mode volume of about 40~m3 for the parental strain) whereas when the promoter was not induced (raffinose alone), or was repressed by the presence of glucose, cell volume was much larger than for the wild-type strain. These experiments showed that CLN3 had been placed under control of the GAL1 pro~oter. It is ~m-portant to note that this GALl-controlled, glucose repressible gene is the only source of CLN3 protein in the cell.
Second, the CLNl gene was disrupted. A fragment of CLNl was obtained from I. Fitch, and used to obtain a ;~
full length clone of CLNl by hybridization, and this was subcloned into a pUC plasmid. A BamHI fragment carrying the-HIS3 gene was inserted into an NcoI site in the CLN1 open reading frame. A large EcoRI fragment with ex-tensive 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 YNB-his raffinose galactose plates. His~ clones were selected, and checked by Southern analysis.
Finally, the 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 W092/20796 PCT/USg2/~146 A

subcloned into a pUC plasmid. An EcoRI fragment carrying the TRPl gene was inserted into an SpeI site in the CLN2 open reading frame. A BamHI-KpnI fragment was excised and used to trsnsform the BF305-lSd GAL-CLN3 HIS3::clnl 05 strain described above. Transformation was done on YNB-trp raffinose galactose plates. Trp+ clones were selected. In this case, because the TRPl fragment included an ARS, many of the transformants contained ~
autonomously replicatlng plasmid rather than a disrupted ~;
CLN2 gene. Howe~er, several percent of the transformants were simple TRPl::cln2 disruptants, as shown by pheno-~' typic and Southern analysis.
One particular 305-15d GALl-CLN3 HIS3::clnl 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 over-expressor. When glucose was added, or when galactose wasremoved, cells accumulated in Gl phase, and cell division ceased, though cells continued to increase in mass and volume. After overnight incubation in the Gl-arrested state, essentially no budded cells were seen, and a large proportion of the cells had lysed due to their uncon-trolled increase in size.
When 305-15d #21 was spread on glucose plates, revertant colonies arose at a frequency of about 10-7.
The nature of these glucose-resistant, galactose-independent mutants was not investigated.

W092/2079~ PCT/US9~/~146 ~103161 Yeast_S~hero~lasts_Transformation S. cerevisae spheroplasts transformation was carried ______.______ out according to Burgers and Pe~ci~al and Allshire (Burgers, P.M.J. and K.J. Percival, Anal. Biochem.
05 163:391 397 (1987); Allshire, R.C., Proc._Natl._Acad.
Sci. USA 87:4043-4047 (1990)).

Cell Culture -__..__________ HeLa and 293 cells were cultured at 37-C either on plates or in suspension in Dulbecco's modified Eagle's 10 medium (DMEM) supplemented with 10% fetal calf serum. -~
~lioblastoma U118 MG cells were cultured on plates in DMEM supplemented with 15% fetal bovine serum and 0.1 mM
non-essential amino acid (GIBCO~.

Nucleic Acid Procedures Most molecular biology techniques were essentially the ~ame as described by Sambrook et al. (Sambrook, J. et al., Molecular Clonin~ A Laborat~ry Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).
~hagmid vectorc pUC118 or pUCll9 (Vieira, J. and 3.
Messing, Meth. E_z~mol. 153:3 11 (1987)) or pBlueScript (Stratagene) were used as cloning vectors. DNA sequences were determined either by a chain termination method (Sanger, F. et al., Proc. Natl. Acad. Sci._USA 74:5463-5467 (1977)) using Sequenase Kit (United States Biochem-ical) or on an Automated Sequencing System (373A, AppliedBiosystems).
Human HeLa cell cDNA library in ~ZAP II was pur-chased from Stratagene. Human T cell cDNA library in ~gtlO was a gift of M. Gillman (Cold Spring Harbor W092/20796- PCT/US92~04146 ~ -36-w103161 Laboratory). Human glioblastoma U118 MG and glioblastoma SW1088 cell cDNA libraries in ~ZAP II were gifts of M.
Wigler (Cold Spring Harbor Laboratory). Human terato- ~
carcinoma cell cDNA library ~gtlO was a gift of Skow- `
05 ronski (Cold Spring Harbor Laboratory). Normal human liver genomic library ~GEM-ll was purchased from Promega.
Total RNA from cell culture was extracted exactly according to Sambrook et al. (Sambrook, J. et al., Molecular Clonin~: A Laboratory Manual Cold Spring ________________ ______________ _______ Harbor Laboratory, Cold Spring Harbor, NY (1939)) using guanidium thiocyanate followed by centrirugation in CsCl ~, solution. Poly(A)+RNA was isolated from total RNA
preparation using Poly (A)~Quick push columns (Strata-gene). RNA samples were separated on a 1% agarose-15 formaldehyde-MOPS gel and transferred to a nitrocellulose filter. Northern hybridizations (as well as library screening) were carried out at 68-C in a solution con-taining 5 x Denhardt's solution, 2 x SSC, 0.1% SDS, 100 ~g/ml denatured Salmon sperm DNA, 25 ~M NaP04 (pH7.0) and 10% dextran sulfate. Probes were labelled by the random priming labelling method (Feinberg, A. and ~. Vogelstein, Anal. Biochem. 132:6-13 (1983)). A 1.3 kb Hind III
______________ ___ fragment of cDNA clone pCYCDl-Hl2 was used as codin~
region probe for Northern hybridization and genomic library screening, a 1.7 kb Hind III-EcoRI fragment from cDNA clone pCYCDl-T073 ~as used as 3' fragment probe.
To express human cyclin Dl gene in bacteria, a 1.3 kb Nco I-Hind II fra~ment of pCYCDl-H12 containing the entire CYCDl open reading frame was subcloned into a T7 expression vector (pET3d, Studier, F.W. e_ al., Methods in Enzymolo~y 185:60-89 (1990)). Induction of E. coli ______ ____ ___ __ ____ W092/20796 PCT/US9~/04146 ~37~ 2103161 strain BL21 (DE3) harboring the expression construct was according to Studier (Studier~ F.W. et al., Methods in Enzymolo~y 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 (CYCDl 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 CYCDl 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.

Se~uence Ali~nment and Formation of an Evolutionary Tree __ _________ __ --____ _ :
Protein sequence alignment was conducted vixtually by eye according to the methods described and discussed in detail by Xiong and Eickbush (Xiong, Y. and T.H. .
Eickbush, EMB0 J. 9:3353-3362 (1990)). Numbers within certain sequenc~s indicate the number of amino acid 20 residues omitted from the sequence as the result of insertion.
Numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of insertion (e.g., for CLNl, ...TUG25RLS...-25 indicates that 25 amino acids ha~e been omitted between Gand R). Sources for each sequence used in this alignment and in the construction of an evolutionary tree (Figure 5B) are as follows: C~CA-Hs, human A type cyclin (Wang, J. et al., Nature 343:555-557 (1990)); CYCA-Xl, Xenopus 30 A-type cyclin (Minshull, J. et al., EMBO_J. 9:2865-2875 W092/20796 - PCT/US92/~l~

~1990)); CYCA-Ss, clam A-type cyclin (Swenson, K.I. et al., Cell 47:867-870 (1986); CYCA-Dm, Drosophila A-type cyclin (Lehner, C.F. and P.H. O'Farrell, Cell 56:957-968 (1989)); CYCBl-Hs, human Bl-type cyclin (Pines, J. and T.
05 Hunter, Cell 58:833-846 (1989)); CYCBl-Xl and CYCB2-Xl, Xenopus Bl- and B2-type cyclin (Minshull, J. et al., Cell 56:947-956 (1989)); CYCB-Ss, clam B-type cyclin (Westendorf, J.M et al., J. Cell Biol., 108:1431-1444 (1989)); CYCB-Asp, starfish B-type cyclin (Tachibana, K.
et al., Dev. Biol. 140:241-252 (1990)); CYCB-Arp, sea urchin B-type cyclin (Pines, J. and T. Hunter, EMB0 J.
, S:2987-2995 (1987)); CYCB-Dm, Drosophila B-type cyclin (Lehner, C.F. and P.H. O'Farrell, Cell 61:535-547 (1990)); CDC13-Sp, S. po~be CDC13 (Booher, R. and D.
15 Beach, EMBO_J. 7:2321-2327 (1988)); CLNl-Sc and CLN2-Sc, S. cerevisiae cyclin 1 and 2 (Hadwiger, J.A. et al., Proc. Natl. Acad. Sci. USA 86:6255-6259 (1989)); CLN3-Sc, S. cerevisiae cyclin 3 (Nash, R. et al., EMB0 J. 7:4335-_____________ :
4346 (1988)).
A total of 17 cyclin sequences were aligned and two representive sequences from each class are presented in Figure 5A.
Percent divergence of all pairwise comparison of 17 sequences were calculated from 154 amino acid residues 25 common to all 17 sequences, which does no`t include the 50 residue segments loca~ed at N-terminal part of A, B and D-type cyclins because of its absence from CLN type cyclins. A ~ap/insertion was counted as one mismatch regardless of its size. Before tree construction, all 30 values were changed to distance with Poisson correction (d ~ -log S, where the S = sequence similarity (Nei, M., - W092/20796 PCT/US92i~146 ~,103161 ~
-39- :

Molecular Evolutionary Genetics pp. 287-326 Columbia ________ ____________ _________ ~
University Press, NY (1987)). Calculation of pairwise comparison and Poisson correction were conducted using computer programs developed at University of Rochester.
Evolutionary treec of cyclin gene fam~ly was generated by 05 the Neighbor-Joining program tSaitou, N. and M. Nei, Mol.
Biol. Evol. 4:406-567 (1987)). All calculations were ___________ _ conducted on VAX computer MicroVMS V4.4 of Cold Spring Harbor Laboratory. The reliability of the tree was e~aluated by using a subset sequence (e.g., A, B and D-type cyclins), including ~ore residues (e.g., the 50-~- residue segment located at C-terminal of A, B and D-type cyclins, ~igure 5A) or adding several other unpublished cyclin sequences. They all gave rise to the tree with the same topology as the one presented in Figure SB.

Immunopreci~itatio__and_Western_Blots Cells from 60 to 80% confluent 100 mm dish were lysed in 1 ml of 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) for 30 minutes on ice. Immunoprecipi~ation was carried out using 1 mg protein from each cell lysate at 4C for overnight. After equilibrated with the lysis buffer, 60 ~1 of Protein A-agarose (PIERCE) was added to each immunoprecipitation and incubated at 4C for 1 hour with constant rotating. The immunoprecipitate was washed three times with the lysis buffer and final resuspended in 50 ~1 2 x 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
Electroblotting System (Millipore) for 45 minutes at a W092r20796 PCT/US92/~1~

~103161 constant current of 400 mA. The filter was blocked for 2 to 6 hours with 1 x 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, 05 1 mM EDTA, 0.25% gelatin and 0.02 sodium azide), radio-labelled with l25I-Protein A for l 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.
The tree was constructed using the Neighbor-Joining method (Saitou, N. and M. Nei, Mol. Biol. Evol., 4:406-,, 425 (1987). The length of horizon~al line reflects the divergence. The branch len~th between the node con-necting the CLN cyclins and other cyclins was arbitrary 15 divided~

MATERIALS_AND NETHODS
The following m~terials and methods were used in the work described in Examples 4-6.

Molecular Clonin~
The human HeLa cell cDNA library, the human glio-blastoma cell U118 MG cDNA library, the normal human liver ~enomic 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 ou~ at 68 and 50C, respectively. To prepare template DNA for PCR
reactions, approximately 2 million lambda phages from each cDNA library were plated at a density of 105 PFU/
150-mm plate, and DNA was prepared from the plate lysate W092t20796 - PCT/US92/04146 21031~1 according to Sambrook, J. et _1., _olecul_r_Clo_in~. _A
L_borator~ _anual, 2nd ed., Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY, 1989.

EXAMPLE_4 Isolation of Human_Cyclin D2 an D3 cDNAs 05 To isolate human cyclin D2 and D3 cDNAs, two 5' oligonucleotides and one 3' degenerate oligonucleotide were deri~ed from three highly conserved regions of human CCNDl, mouse cyll, cyl2, and cyl3 D-type cyclins (Matsu-shime, H. et al., Cell 65:701-713 (1991); Xiong, Y.
et_al., Cell 65:691-699; Fig. 8). The first 5' oligo-nucleotide primer, HCNDll, is a 8192-fold degenerate 38-mer (TGGATG[T/C]TNGA[A/G]GTNTG[T/C]GA[A/C]GA[A/G~CA-lA/GlAAlA/G~TGlT/C)GAlA/G]~A)(SEQ ID No. 37), encoding 13 amino acids (UMLEVCEEQKCEE)(SEQ ID No. 33). The second 5' oligonucleotide primer, HCN~12, is a 8192-fold degen-erate 29-~er (GTNTT[T/C~CCN[T/C~TNGCNATGAA[T/C]TA[T/C]- ;
TNGA)(SEQ ID No. 39), encoding 10 amino acids (VFPLAMNYLD)(SEQ ID No. 40). The 3' primer, HCND13, is a 3072~fold degenerate 24-mer ([A/GlTCNGT[A/G]TA~A/G/T]AT-[A/G]CANA[A/G][T/C]TT-[T/C]TC)(SEQ ID No. 41), encoding 8 amino acids (EKLCIYTD)~SEQ ID No. 42). The PCR reactions were carried out for 30 cycles at 94~C for 1 min, 48C
for 1 min, and 72C for 1 min. The reactions contained 50 ~M KCl, 10 mM Tris-HCl(pH 8.3), 1~5 mM MgC12, 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. PCR products generated by HCNDll 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 ~ith the . ` ' ! !: ! ' '.( ' ' !; .,` .

W092/20796 PCT/US92tO414 ~ J

expec~ed size (200 bp between primer HCNDll and HCND13) were purified and subcloned into the SmaI site of phagmid vector pUC118 for sequencing.
To isolate full-length cyclin D3 cDNA, the 201-bp 05 fragment of the D3 PCR product was labeled with oligo-nucleotide primers HCNDll and HCND13 using a random-primed labeling technique (Feinberg, A. P. et _1., A__l.
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 ~D3-H34. All hybridi-~, zations for the screen of human cyclin D3 were carriedout at high str~ngency. The PCR clones corresponding to CCNDl and CCND3 have been repeatedly isolated from both cDNA libraries; CCND2 has not. To isolate cyclin D2, a l-kb EcoRI fragment derived fro~ mouse c~12 cDNA was used as a probe to screen a human genomic library. Under low-strin&ency conditions, this probe hybridized to both human cyclins Dl and D2. The cyclin Dl elones were eliminated through another hybridization with a human cyclin D1 probe at ~;
high stringency. Human CCND2 genomic clones were sub-sequently identified by partial sequencing and by com-paring the predicted protein sequence with ~hat of human cyclins Dl and D3 as well as mouse cyl2.
As described above, human CCNDl (cyclin Dl) was isolated by rescuing a triple Cl_ deficiency mutant of Saccharomyces cerevisiae using a genetic complementation _________ ___ __________ screen. Evolutionary proximity between hu~an and mouse, and the high sequence similarity among cyll, cyl2, and cyl3, suggested the existence of two additional D-type W092/20796 PCT/US92/~146 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 deri~ed from highly conserved regions of human CCNDl, 05 mouse c~ll, c~12, and cyl3. Using these primers, cyclin Dl and a 200-bp DNA fragment that appeared to be the human homolog of mouse _yl3 from both human HeLa cell and glioblastoma cell cDNA libraries was isolated. A human HeLa cell cDNA library was screened wlth 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, AD3-H34 (196~ bp), was completely sequenced ~Figure 4).
Because a putative human cyclin D2 cD~A was not detected by PCR, mouse cyl2 cDNA was used as a heterolo-gous probe to screen a human c~NA library at low str~n-gency. This resulted, initially, in isolation of 10 clones from the HeLa cell cDNA library, but all corres-ponded 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 library, based on the assumption that the representation of Dl and D2 should be approximately equal. Of the 18 positives obtained, 10 corresponded to human cyclin Dl and 8 appeared to contain human cyclin D2 sequences (see bel~w). A 0.4-kb BamHI restriction fragment derived from AD2-G1 1 of the 8 putative cyclin D2 clones, was ~hen used as probe to screen a human hippocampus cDNA library at high stringency to search for a full-length cDNA clone of the cyclin D2 gene. Nine positives were obtained wo 92~20796 - P~r/us92to4t46 210'~161 after screening of approximately 1 million cDNA clones.
The longest cDNA clone, ~D2-P3 (19ll bp), was completely sequenced (Figure 3). Neither ~D2-P3 nor ~D3-H34 con-tains a poly(A) sequence, suggesting that part of the 3' 05 untranslated region might be missing.
The DNA sequence of 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 analy-sis of ~D3-H34 revealed a 292-amino-acid open reading frame encoding a protein with a 32,482-Da calculated molecular weight. As in the case of human cyclin Dl, there is neither methionine nor stop codons 5' to the presumptive initiating methionine codon for both ~D2-P3 ~, (nucleotide position 22, Figure 3) and ~D3-H34 (nucleo-t$de position 101, Figure 4). On the basis of the protein sequence comparison with human cyclin Dl and mouse c~ll (Figure 7) and preliminary results of the RNase protection experiment, both ~D2-P3 and ~D3-H34 are believed to contain full-length coding regions.
The protein sequence of all ll mAmmalian cyclins identified to date were compared to assess their struc-tural and evolutionary relationships. This includes `~
cyclin A, cyclins Bl and B2, 5iX 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 compari-son. First, as noted pre~iously 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. Second, they all lack the so-called "destruction box" identified in the N-terminus of W092/20796 PCT/US92/~146 ~1i)3161 both A- and B-type cyclins, which targets it for ubi-quitin-dependent degradation (Glotzer, M. et al., Nature 349:132-138 (1991)). This sug~ests either that the D-type cyclins have evolved a different mechanism to 05 govern their periodic degradation during each cell cycle or that they do not undergo such destruction. Third, the three human cyclin D genes share ~ery high similarity over their entire coding re~ion:60~ between Dl and D2, 60~ between D2 and D3, snd 52~ between Dl and D3.
Fourth, members of the D-type cyclins are more closely related to each other tha~ 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 1~ cyclins occurred after that of cyclin Bl from B2.
Finally, using th~ well-characterized mitotic B-type cyclin as an index, the most closely related genes are cyclin A (average 51~), followed by the E-type (40~), D-type (2g~), and C-type cyclins (20%).

EXAMPLE_5 Chromosome Localization_of_CCND2_and _____ The chromosome localization of CCND2 and CCND3 was determined by fluoresoence in situ hybridization.
Chromosome i_ situ suppression hybridization and in situ ~5 hybridization banding were performed as described pre-viously (Lichter, T. et al., Science 247-64-69 (1990);
Baldini, A. et al., Genomics 9:770-774 (1991)). Briefly __ ___ ________ _ ~D2-G4 and ~D3-~9 lambda genomic DNAs containing inserts of 15 and 16 kb, respectively, were labeled with biotin-ll-dUTP (Sigma) by nick-translation (Brigatti, D. J. et ~,"

2lo3l6l al., Urolo~y 126:32 50 (1983); Boyle, A. L., In Current Protocols in Molecular Biolo~y, Wiley, New York, 1991).
Probe size ranged between 200 and 400 nucleotides, and unincorporated nucleotides were separated from probes 05 using Sephadex G-50 spin columns (Sambrook, J. et al., Molecular Clonin~: A Laboratory Manual, 2nd ed., Cold ________________ ______________ _______ Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
Metaphase chro~oso~e 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 DNase-treated 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.
is ,he in situ hybridization banding pattern used for __ ____ chromosome identification and visual localization of the ;~
probe was generated by cohybridizing the spreads with 40 ng of an Alu 48-mer oligonucleotide. This Alu oligo was chemically labeled with digoxigenin-ll-dUTP (Boehringer-20 Mannheim) and denatured before being applied to denatured chromosomes. Following 16-18 h of incubation at 37C and posthybridization wash, slides were incubated with blocking solution and detection reagent (Lichter, T. et al., Science 247:64-6g (1990)). Biotin-labeled DNA was __ _ ___ ____ __ _ 25 detected using fluorescence isothiocyanate (FITC)-conjugated avidin DCS (S ~g/ml) (Vector Laboratories);
digoxigenin-labeled DNA was detected using a rhodamine~
conjugated anti-digoxigenin antibody (Boehringer-Mannheim). Fluorescence signals were imaged separately 30 using a Zeiss Axioskop-20 epifluorescence microscope equipped with a cooled CCD camera (Photometrics CH220).

- W092/20796 PCT/US9~/04146 Camera control and image acquisition were performed using an Apple Macintosh IIX computer. The gray scale images were pseudocolored and merged electronically as described pre~iously (Baldini, A. et al., Ge_o_ics 9:770-774 - 05 (1991)~. Image processing was done on a.Macintosh IIci computer using Gene Join Maxpix (software by Tim Rand in the laboratory of n . Uard, Yale~ to merge FITC and rhodamine images. Photographs were taken directly from the computer monitor.
Chromosomal fluorescence in sit_ 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 hybr~dization using the biotin-labeled probe and the digoxigen-labeled Alu 48-mer oligonucleotide (Fig. 5).
The Al_ 48-mer R-bands, consistent with the con-ventional R-banding pattern, were imaged and merged with images genPrated from the D2-G4 and D3-G9 hybridized probes. The loci of D2-G4 and D3-G9 were visualized against the Al_ 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 posi-tive R-band 12pl3, while D3-G9 lies on the positive R-band 6p21.
Cross-hybridization W25 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 W092/20796 PCT/US~2/~146 ~ 48-;~1 03161 nucleotide sequences of pseudogenes have diverged from their ancestral active ~enes. .

EXAMPLE_6 Isolatio_ an_ Characteriza_io_ of Ge_omic Clones of Human D-Ty~e Cyclins 05 Genomic clones of human D-type cyclins were isolated and characterized to study the genomic structure and to ~
obtaln probes for chromosomal mapping. The entire 1.3-kb ~;
cyclin Dl cDNA clone was used as probe to screen a normal human liver genomic library. Five million lambda clones 10 were screened, and three positives were obtained. After ~, initial restriction mapping and hybridizations, lambda ;~
clone G6 was chosen for further analysis. A 1.7-kb BamHI
restriction fragment ~f ~Dl-G6 was subcloned into pUC118 and completely sequenced. Comparison with the cDNA
15 clones pre~iously isolated and RNase protection experi-ment results (~ithers, D.A. et al., Mol._Cell._Biol.
11:4846 4853 (1991)) indicated that this fragment corres-ponds to the 5' part of the cyclin Dl gene. As shown in Figure 8A, it contains 1150 bp of upstream promoter 20 sequence and a 198-bp exon followed by an intron.
Eighteen lambda genomic clones were isolated from a similar screening using mouse cyl2 cDNA as a probe under low-stringency hybridization conditions, as described above (Example 4). Because it was noted in previous cDNA
25 library screening that the mouse cyl2 cDNA probe can cross-hybridize with the hu~an Dl gene at low stringency, a dot-blot hybridization at high stringency was carried out, using the human Dl cDNA probe. Ten of the 18 clones hybridized with the human Dl probe and 8 did not. On the 30 basis of the restriction digestion analysis, the 8 lambda W092/20796 - PCT/US92/~146 clones that did not hybridize with the human Dl probe at high stringency fall into three classes respresented by ~D2-Gl, ~D2-G2, and ~D2-G4, respectively. These three lambda clones were subcloned into a pUC plasmid vector, 05 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 ~D2-Gl was subsequently used as a probe to screen a human hippocampus cell cDNA library at high stringency. ~`
10 Detailed restriction mapping and partial sequencing indicated that ~D2-Gl and ~D2-G2 were two different clones corresponding to the same gene, whereas ~D2-G4 appeared to correspond to a different gene. A 2.7-kb SacI-S~aI fragment from ~D2-G4 and 1.5-kb BclI-B~lII
15 fragment from ~D2-Gl have been completely sequenced.
Nucleotide sequence comparison revealed that the clone ~D2-G4 corresponds ~o the D2 cD~A clone ~D2-P3 (Figure 3). As shown in Figure 8A, the 2.7-kb SacI-SmaI fragment contains 1620 bp of sequence 5' to the presumptive 20 initiating methionine codon identified in D2 cDNA (Figure 3~ and a 195-bp exon followed by a 907-bp intervening sequence.
Lambda genomic clones corresponding to the human cyclin D3 were isolated from the same genomic library 25 using human D3 cDNA as a probe. Of four million clones screened, nine were positives. Two classes of clones, represented by lD3-G4 and ~D3-G9, were distinguished by restriction digestion analysis. A 2.0-kb Hi_dIII-Sc_I
restriction fragment from ~D3-G5 and a 3.7-kb SacI-30 Hi_dIII restriction fragment from ~D3-G9 were further subcloned into a p~C plasmid vector for more detailed ~103161 restriction mapping and complete sequencing, as they both ;
hybridized to the 5' cyclin D3 cDNA probe. As presented in Figure 9C, the 3.7-kb fragment from clone G9 contains 1.8 kb of sequence 5' to the presumptive initiating 05 methionine codon identified in D3 cDNA (Figure 4), a 198-bp exon 1, a 684-bp exon 2, and a 870-bp intron. ~-Comparison of the genomic clones of cyclins Dl, D2, and D3 revealed that the coding regions of all three human CCND genes are interrupted at the same position by 10 an intron (indicated by an arrow in Figure 8). This indicated that the intron occurred be~ore the separation of cyclin D genes.

EXAMPLE 7 Isolation and Characterizatio_ of T_o Cyclin D Pseudo~enes ______ ______ ____ The 1.5-kb BclI-B~lII fragment subcloned from clone ~2-Gl has been completely sequenced and compared with cyclin D2 c~NA clone ~D2-P3. As shown in Figure 10, it contains three internal stop codons (nucleotide positions 495, 956, and 1310, indicated by asterisks), two frame-20 shifts (position 1188 and 1291, slash lines), one insertion, and one deletion. It has also accumulated many missense nucleotide substitutions, some of which occurred at the positions that are conserved in all cyclins. For example, triplet CGT at position 277 to 279 2~ of D2 cDNA (Fi~ure 3) encodes amino acid Arg, which is an invariant residue in all cyclins ~see Figure 8). A
nucleotide change from C to T at the corresponding position (nucleotide 731) in clone D2-Gl (Figure 10) gave rise to a triplet TGT encoding Cys instead of Arg.
30 Sequencing of the 2.0-kb Hi_dIII-ScaI fragment from clone WOg2/20796 PCT/US~2/04t46 -51~ 3161 ~D3-G5 re~ealed a cyclin D3 pseudogene (Figure 11). In addition to a nonsense mutation (nucleotide position 1265), two frameshifts (position 1210 and 1679), a 15-bp internal duplication (underlined region from position 05 1361 to 1376?, and many missense mutations, a nucleotide change from A to G at position 1182 resulted in an amino ;~
acid change from the presumptive initiating methionine codon ATG to GTG encoding Val. On the basis of these analyses, we conclude that clones ~D2-Gl and ~D3-G5 10 contain pseudogenes of cyclins D2 and D3, respectively.

E~UIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equi~alents to the specific embodiments of the 15 invention described herein. Such equi~alents are in-tended to be encompassed by the following claims.

Claims (57)

-52-

1. Recombinant cyclin of mammalian origin which re-places a CLN-type protein essential for cell start in budding yeast.

2. Recombinant cyclin of Claim 1 which is D-type cyclin.

3. Recombinant cyclin of Claim 2 which is of human origin.

4. Recombinant D type cyclin of Claim 3 selected from the group consisting of: cyclin D1, cyclin D2 and cyclin D3.

5. Purified D-type cyclin of mammalian origin of approximate molecular weight 34 kD.

6. Purified D type cyclin of Claim 5 having the amino acid sequence of Figure 2, the amino acid sequence of Figure 3 or the amino acid sequence of Figure 4.

7. Purified D type cyclin of Claim 5 which is selected from the group consistin of: cyclin D1, cyclin D2 and cyclin D3.

8. Recombinant D-type cyclin of mammalian origin of approximate molecular weight 34 kD.

9. Recombinant D-type cyclin of Claim 8 having the amino acid sequence of Figure 2, the amino acid sequence of Figure 3 or the amino acid sequence of Figure 4.

10. Isolated DNA encoding D-type cyclin of mammalian origin of approximate molecular weight 34 kD.

11. Isolated DNA of Claim 10 having the nucleic acid sequence of Figure 2, the nucleic acid sequence of Figure 3 or the nucleic acid sequence of figure 4.

12. Isolated DNA encoding a D-type cyclin protein which replaces a CLN-type protein essential for cell cycle start in budding yeast.

13. A DNA probe which hybridizes to at least a portion of a nucleic acid sequence selected from the group consisting of: The nucleic acid sequence of Figure 2, the nucleic acid sequence of Figure 3 and the nucleic acid sequence of Figure 4.

14. A DNA probe of Claim 13 which is labelled.

15. A labelled DNA probe of Claim 14 wherein the label is selected from the group consisting of: radio-active labels, fluorescent labels, enzymatic labels and binding pair members.

16. An antibody which specifically binds D-type cyclin of mammalian origin of approximate molecular weight 34 kD.

17. An antibody of Claim 16 which is a labelled mono-clonal antibody.

18. A method of identifying DNA which replaces a gene essential for cell cycle start in yeast, comprising the steps of:
a) providing mutant yeast cells in which the gene essential for cell cycle start is conditionally expressed;
b) introducing into mutant yeast cells of (a) a yeast vector which contain DNA to be assessed for its ability to replace a gene essential for cell cycle start in yeast and which expresses the DNA in the mutant yeast cells; and c) selecting transformed mutant yeast cells produced in (b) on the basis of their ability to grow under conditions under which the gene essential for cell cycle start in the mutant yeast cells provided in (a) is not expressed, wherein ability to grow under the conditions of (c) is indicative of the presence in trans-formed mutant yeast cells of DNA which replaces a gene essential for cell cycle start.

19. The method of Claim 18 wherein the mutant yeast cells have inactive CLN1 and CLN2 genes and an altered CLN3 gene which is conditionally expressed from a glucose-repressible promoter; the yeast vector is pADNS and screening in (c) is carried out by assessing the ability of transformed mutant yeast produced in (b) to grow in the presence of glucose.

20. The method of Claim 19 wherein the DNA which re-places a gene essential for cell cycle start in yeast is a D-type cyclin.

21. The method of Claim 20 further comprising confirming that ability to grow in the presence of glucose is not the result of reversion by affirming stability of the yeast vector in transformed mutant yeast selected in (c).

22. A method of identifying DNA encoding cyclin which replaces a gene essential for cell cycle start in yeast, comprising the steps of:
a) providing mutant yeast cells in which the CLN1 gene and the CLN2 gene are inactive and the CLN3 gene is conditionally expressed;
b) introducing into mutant yeast cells of (a) the yeast vector pADNS containing DNA to be assessed for its ability to replace the CLN3 gene, thereby producing transformed mutant yeast cells;
c) maintaining transformed mutant yeast cells produced in (b) on glucose-containing medium;
and d) selecting transformed mutant yeast cells produced in (b) on the basis of their ability to grow on glucose-containing medium.

23. The method of Claim 22 further comprising confirming the stability of the yeast vector pADNS in trans-formed mutant yeast cells selected in (d).

24. The method of Claim 23 wherein the cyclin which replaces a gene essential for cell cycle start in yeast is a D-type cyclin.

25. A method of detecting DNA encoding a cyclin of mammalian origin in a cell, comprising the steps of:
a) processing cells to render nucleic acid sequences present in the cells available for hybridization with complementary nucleic acid sequences;
b) combining the product of (a) with DNA encoding a D-type cyclin of mammalian origin or DNA
complementary to DNA encoding a D-type cyclin of mammalian origin;
c) maintaining the product of (b) under conditions appropriate for hybridization of complementary nucleic acid sequences; and d) detecting hybridization of complementary nucleic acid sequences, wherein hybridization is indicative of the presence of DNA encoding a D-type cyclin of mammalian origin.

26. The method of Claim 25 wherein in (b) the product of (a) is combined with DNA selected from the group consisting of: DNA having the sequence of Figure 2;
DNA complementary to the sequence of Figure 2; DNA
having the sequence of Figure 3; and DNA comple-mentary to the sequence of Figure 3.

27. The method of Claim 26 wherein the cyclin is a D-type cyclin.

28. The method of Claim 27 further comprising comparing hybridization detected in (d) with hybridization detected in appropriate control cells, wherein if hybridization detected in (d) is greater than hybridization in the control cells, it is indicative of increased levels of the DNA encoding the D-type cyclin of mammalian origin.

29. A method of detecting a D-type cyclin in a bio-logical sample, comprising the steps of:
a) providing a biological sample to be assessed for D-type cyclin level;
b) combining the biological sample with an anti-body specific for a D-type cyclin; and c) detecting binding of the antibody of (b) with a component of the biological sample, wherein binding is indicative of the presence of a D-type cyclin.

30. The method of Claim 29 wherein the antibody specific for a D-type cyclin is labelled.

31. A method of detecting amplification of a D-type cyclin in a biological sample, comprising the steps of:
a) providing a biological sample to be assessed for D-type cyclin level;
b) combining the biological sample with an anti-body specific for a D-type cyclin;
c) determining the extent to which the antibody specific for a D-type cyclin binds to D-type cyclin in the biological sample; and d) comparing the results of (c) with the extent to which the antibody specific for a D-type cyclin binds to D-type cyclin in an appropriate control, wherein greater binding of the antibody to D-type cyclin in the biological sample than in the appro-priate control is indicative of amplification of the D-type cyclin.

32. The method of Claim 31 wherein the antibody specific for a D-type cyclin is labelled.

33. A method of detecting in a cell an increased level of a D-type cyclin of mammalian origin, comprising the steps of:
a) processing cells to be analyzed to render nucleic acids present in the cells available for hybridization with complementary nucleic acid sequences;
b) combining the product of (a) with DNA which hybridizes with DNA encoding a D-type cyclin of mammalian origin under the conditions used;
c) maintaining the combination of (b) under conditions appropriate for hybridization of complementary nucleic acid sequences;
d) detecting hybridization of complementary nucleic acid sequences; and e) comparing hybridization detected in (d) with hybridization in appropriate control cells, wherein hybridization is indicative of the presence of a D-type cyclin of mammalian origin and greater hybridization in (d) than in the control cells is indicative of increased levels of the D-type cyclin of mammalian origin.

34. A method of inhibiting cell division comprising introducing into a cell a drug which interferes with formation in the cell of the protein kinase-D type cyclin complex essential for cell cycle start.

35. The method of Claim 34 wherein the drug is selected from the group consisting of:
a) oligonucleotide sequences which bind DNA
encoding D-type cyclins;
b) antibodies which specifically bind D-type cyclins;
c) agents which degrade D-type cyclins; and d) oligopeptides.

36. A method of interfering with activation in a cell of a protein kinase essential for cell cycle start, comprising introducing into the cell a drug selected from the group consisting of:
a) oligonucleotides which bind DNA encoding D-type cyclins;
b) peptides which bind the protein kinase essential for cell cycle start but do not activate it;
c) antibodies which specifically bind D-type cyclins; and d) agents which degrade D-type cyclins.

WHAT IS CLAIMED IS:

37. Recombinant purified D-type cyclin selected from the group consisting of: cyclin D2 and cyclin D3.

38. Recombinant D-type cyclin having the amino acid sequence of Figure 3 or the amino acid sequence of Figure 4.

39. Purified D-type cyclin which is selected from the group consisting of: cyclin D2 and cyclin D3.

40. Purified D-type cyclin having the amino acid sequence of Figure 3 or the amino acid sequence of Figure 4.

41. Isolated DNA having a nucleic acid sequence encoding the same protein as the nucleic acid sequence of Figure 3 or the nucleic acid sequence of Figure 4.

42. A DNA fragment probe which uniquely hybridizes to a nucleic acid sequence selected from the group consisting of: the nucleic acid sequence of Figure 3 and the nucleic acid sequence of Figure 4.

43. The DNA fragment probe according to Claim 42 wherein said DNA fragment which is labeled.

44. The DNA fragment according to Claim 43 wherein the label is selected from the group consisting of:
radioactive labels, fluorescent labels, enzymatic labels and binding pair members.

45; An antibody which specifically binds to at least one of cyclin D2 or cyclin D3 of mammalian origin.

46. The antibody according to Claim 45 which is a labeled monoclonal antibody.

47. A method for identifying a gene which rescues a mutation in a gene essential for cell cycle start in yeast, comprising the steps of:
introducing into mutant yeast cells a yeast expression vector containing a test DNA sequence, said mutant yeast cells having a mutation in at least one gene essential for cell cycle start, said mutation causing arrest in a phase just prior to said cell cycle start when said mutant yeast cells are grown under conditions which inhibit expression of said gene or genes; and selecting said transformed yeast cells for the ability to grow under said conditions which inhibit expression of said gene or genes;
wherein transformed yeast cells are selected which express DNA test sequences which allow said transformed yeast cells to enter the cell cycle start.

48. The method according to Claim 47 wherein said yeast expression vector is pADNS and said mutant yeast cells carry an inactive CLN1 gene, an inactive CLN2 gene, and a mutant CLN3 gene, said mutant CLN3 gene being conditionally expressed from a glucose-repressible promoter and said conditions which inhibit expression are growth in the presence of a medium comprising glucose.

49. A method of detecting in a test cell the presence of cyclin genes which are homologous to cyclin D2 or cyclin D3 genes of mammalian origin, comprising the steps of:
individually combining nucleic acid from test cells and control cells with a DNA fragment probe, said fragment probe being derived from DNA having the sequence of Figure 3 or Figure 4, said combining being performed under conditions appropriate to form complexes of complementary nucleic acid sequences; and detecting the presence of said complexes in said test cells.

50. A method of detecting a D2 cyclin or a D3 cyclin in a biological sample, comprising the steps of:
combining a biological sample with an antibody specific for at least one of D2 cyclin or D3 cyclin; and detecting binding of said antibody;
wherein binding is indicative of the presence of D2 cyclin or D3 cyclin.

51. The method according to Claim 50 wherein said antibody is labeled.

52. A method of determining the level of expression of D2 cyclin or D3 cyclin in a biological sample, comprising the steps of:
combining a biological sample with an antibody specific for at least one of D2 cyclin or D3 cyclin;
detecting a level of binding of said antibody in said biological sample and in a control sample, said control sample expressing D2 cyclin or D3 cyclin at a base level;
and comparing the levels of binding of said antibody in said biological sample and said control sample to determine said level of expression.

53. The method according to Claim 52 wherein said antibody is labeled.

54. A method of detecting increased levels of transcription of cyclin D2 or cyclin D3 of mammalian origin in a test cell, comprising the steps of:
individually combining nucleic acid from test cells and control cells with a DNA fragment probe, said fragment probe being derived from DNA having the sequence of Figure 3 or Figure 4, said combining being performed under conditions appropriate to form complexes of complementary nucleic acid sequences;
detecting the amount of said complexes in each of said test cells and said control cells; and comparing the amount of said complexes detected in said test cells with the amount of complexes detected in said control cells;
wherein a greater amount of complexes in said test cells compared to said control cells is indicative of increased transcription of cyclin D2 or cyclin D3 in said test cells.

55. A method of inhibiting cell division comprising the step of:
introducing into a cell an agent which specifically interferes with formation of complexes of cyclin dependent protein kinase with either cyclin D2 or cyclin D3.

56. The method according to Claim 55 wherein said agent is selected from the group consisting of:
a) oligonucleotide sequences having the same sequence as the DNA sequence of Figure 3 or Figure 4;
b) antibodies which specifically bind at least one of cyclin D2 or cyclin D3;
c agents which degrade cyclin D2 or cyclin D3; and d) oligopeptides.

57. A method of interfering with activation in a cell of a protein kinase essential for cell cycle start, comprising the step of:
introducing into a cell an agent selected from the group consisting of:
a) oligonucleotide sequences having the same sequence as the DNA sequence of Figure 3 or Figure 4, b) peptides with binding specificity for a protein kinase essential for cell cycle start, said binding of peptide preventing formation of complexes of said protein kinase and cyclin D2 or cyclin D3, said binding of peptide failing to activate said protein kinase;
c) blocking antibodies which specifically bind at least one of cyclin D2 or cyclin D3; and d) agents which specifically degrade cyclin D2 or cyclin D3;