CA2183254A1 - Mammalian cell cycle protein - Google Patents

Abstract

A novel mammalian cell cycle protein, p55CDC, DNA sequences encoding p55CDC, and a method for producing the protein are described. Also described are methods for detecting p55CDC and methods for modulating cell division by compounds which control the level or activity of p55CDC or p55CDC-associated protein complexes.

Description

Wo 95/2i917 21~ 3 ~ 5 4 r~
MAMMALIAN CELL CYCLE PROTEIN
The invention relates to a I l i ~n cell cycle protein, p55CDC, DNA sequences encoding same, antibodies specific for the protein, a method for producing the protein and methods for - - 1 At 1 n~ cell division by controlling the levels or activity of p55CDC
or p55CDC-associated protein complexes.
R~ l~l;K~IU~I~ OF TEIE INVENTION
The eukaryotic cell cycle has a growth phase and a reproductive phase, the latter composed of the chromosome cycle and the centrosome cycle which intersect in the establishment of the mitotic apparatus (for review, see 47). The profound morphologic changes which result in mitosis are ac~ od by a cascade of phosphorylation and dephosphorylation events. In - l; ~n cells, different complexes of kinases and their associated regulatory proteins control ~loyL-2s~1On through discrete steps of the cell cycle (for review, see 6~, 67) . While all eukaryotic cells use similar ~ ;~n; qmq to regulate progreSSiOn through the stages of the cell cycle, it is clear that unique, ~ n;~ ~ ons of regulatory cyclins, kinases and E~hnsrh~qes are rPsrnnq;hle for cell- and organism-specific patterns of cell division (18,51,52).
A variety of kinases have been identified which control the crucial transitions through the cell ---cycle. The most well characterized is the p34cdc2 protein, which has b~en if 1Pn~ ~ f; P~ in all eukaryotic WO9S/21917 ~183~S 4 r~
cells which have been ~xAmln~ (3, 16, 20, 28, 40, 41, 42, 59, 76). In addition, many other kinases which have homology to p34cdc2 and, like p34cdc2 fluctuate in activity during the cell cycle, have been described 5 ~48, 60) . Other types of kinases have also been shown to vary in activity at different stages of the cell cycle, and have been proposed to play a role in control of cell division, although they share little or no homology with p34cdc2. These include the M~P kinases, and the MEK
10 kinases which regulate MAP kinase activity (for review, see 11). In addition, a novel kinase has been identified in the fungus Asperg~llus n~dulans, the NIMA
kinase, which is rer~uired to initiate mitosis (53-55).
A -l;An kinase, Nekl, which has homology to the NIMA
15 kinase, has been found in mouse, where it is expressed at high levels in gonadal tissues and may be required for meiosis (43).
As mentioned above, the activity of many of 20 these kinases is regulated by their association with one or more cyclins. The cyclins are homologous with one another within a conserved region termed the cyclin box (44). The fluctuations in activity of the cyclin dt~ d~:llL kinases during the cell cycle result from

2~ differential ~csorlAt;nn with newly synth~sl7~ cyclins, which are then degraded at specific transition points in the cell cycle However, not all cyclins demonstrate the same degree of fluctuation during the cell cycle;
for example, levels of the D type cyclins do not 30 ~sr1 l l Ate as dramatically during the cell cycle as the A
and B type cyclins. In addition, a recently described cyclin, the mcs2 cyclin of S. po~oe, shows no variation in level during the cell cycle, nor does the novel kinase activity associated with the mcs2 cyclin 35 oscillate ( 4 9 ) .

g~
Wo 95/21917 PcrNSs~/01806 ~xperiments in yeast have defined a number of other cell division cycle (Cdc) proteins which are also crucial for the orderly progression of the cell cycle, although the fi~nctions of many of these proteins have 5 not been precisely defined (34). ~mwO of these proteins, the products of the CDC20 and CDC4 genes, have been proposed to be elements of the mitotic spindle or segr-o~At1~nAl apparatus (32). The cdc20 t aLu sensitive mutants arrest in mitosis at the non-permissive temperature, after the formation of a complete short spindle and nuclear migration to the neck between the mother cell and a large bud (6). It has been proposed that the Cdc20 protein is directly required for chromosomal v. (56). In addition, the Cdc20 protein is required for modulation of microtubule structure, either by promoting microtubule disassembly (1, 65) or by altering the surface of the microtubules, and is also required for microtubule-dependent processes other than mitosis (65).
The CDC4 gene of S. cerevislae (33), is essential for the initiation of D~ synthesis. Cells carrying a conditional-lethal, temperature-sensitive mutation in cdc4 arrest division at the non-permissive temperature, and the cells have a t~rm~nAt~on phenotype of multiple buds, a single nucleus, and duplicated spindle pole bodies rr~nn.~te~ by a bridge structure (6).
CDC4 also appears to be required for kâryogamy and sporulation (21, Ç8, 71) . Wnile the - -hAnl ~m of action of the Cdc4 protein is still unknown, subcellular localization studies in yeast have demonstrated that it is associated with the nucleo~k.~lPt~n (7). The appearance of the duplicated spindle pole bodies has been proposed to indicate that the CDC4 gene product is required for separation of the bodies and formation of the completed spindle (~, 75) . It has recently been 21832~4 Wo 95111917 demonstrated that removal of the centrosome (the equivalent of the spindle pole body in higher eukaryotes) from - l; An cells uncouples the growth cycle from the reproductive cycle, ~nrlicAtin~ that cell 5 division requires the presence of centrosomes to establish the bipolar mitotic spindle ~45).
It is an ob~ect of the present invention to identify one or more proteins involved in regulation of l0 the cell cycle, wherein said proteins may be targets for compounds which modulate the cell cycle. A novel protein, termed p55CDC has been identified. mRN~
encoding p55CDC was ubiquitously present in all cell lines ~Amin~d, as well as in embryonic tissue, placenta 15 and adult hematopoietic tissues, but was not ~l~tec~ri in cells induced to differentiate and cease cell division.
The deduced amino acid sequence of human p55CDC
demonstrates regions of homology with the S. cerev~s~ae Cdc20 and Cdc4 proteins within the G3-repeats found in 20 the carboxy terminal hal f of these three proteins .
Expression of p55CDC appears to be crucial for cell division in ~ n cells. p55CDC is phosphorylated in cycling cells. Lmmune complexes precipitated by a polyclonal antiserum to p55CDC have a kinase activity 25 which fluctuates during the cell cycle, although p55CDC
itself does no~ appear to be an endogenous substrate of the kinase activity.
~IJM~RY OF TE}E IZ~VENTIO~I
The invention relates to a novel 1~ Arl protein, p55CDC, which is essential for cell division.
It has been found that p55CDC is expressed in actively 35 pro~ rA~;nSr cells while expression is not ~lPtP~t~d in slowly dividing or quiescent cells. ~ransfection of ., . . _ _ _ _ _ _ _ _ _ wogs/2l917 21 83~5 ~
antisense p55CDC cDNA into CHO cells resulted in the isolation of only those cells having a compensatory increase in p55CDC rnRNA having the sense orientation.
DNA sequences encoding biologically active 5 p55CDC are also provided by the invention. DNA
sequences include rat (SEQ. ID NO: 1) and human (SEQ. ID
NO: 3) p55CDC and DNA hybridi2ing to rat or human p55CDC, or to a fragment thereof, wherein the hybridizing DNA encodes biologically active p55CDC.
Also provided for are vectors cl nt~;n;n~ p55CDC DNA
sequences and host cells transformed or transfected with said vectors. A method of producing a p55CDC
polypeptide comprising culturing transformed or transfected host cells such that p55CDC is expressed is also included p55CDC polypeptides of the invention will preferably form a complex with one or more host proteins such that the complex has cell-cycle ~ nf~nt kinase activity. The kinase activity of p55CDC complexes will f~ til~te during the cell cycle.
A method for modulating cell division is also f,n~ -qqed by the invention, wherein the method comprises introducing into a cell (e.g. a tumor cell) a compound which modulates the kinase activity of p55CDC
complexes. Mo~i11at;t~n of p55CDC associated kinase activity may involve an increase or decrease in activity at certain periods during the cell cycle which in turn may lead to alterations in timing or specificity of p55CDC-associated kinase activity. In a preferred embodiment, cell division is inhibited by exposure to compounds which interfere with p55CDC complex formation.

WO 95/~1917 ~ 2 5 4 r~

DESCRIPTION OF T~E FIG~REg Fig. l. Northern Analysis of p55CDC.
(A) Total RNA (30 ~Lg) from a variety of rat 5 tissues at dif~erent dev~ l stages was probed with a rat genomic 0.26 kb Pst I fragment.
~ ) PolyA+ RNA (2.5 ~lg) from human tissues was probed with a [32p] l~hPllPri p55CDC cDNA from rat.
(C) PolyA+ RNA (2 . 5 llg) from human l0 hematopoietic cell lines was analyzed with the same probe as in Fig. lB. The signal obtained with a rat actin cDNA probe is shown for comparison.
(D) Total RNA 30 ~lg) prepared from cell lines that were induced to differentiate as described in ~5 Experimental Procedures and from control cells was probed with the same probe as in Fig. lB. The ethidium bromide stain for 285RNA is shown for comparison. All details for RNA; c~ n and Northern blot hybridization are described in Experimental Procedures.
Fig. 2 Rat and Human p55CDC DNA sequence.
The compiled sequence from two rat cDNA clones is shown. The open reading frame of the human cDNA is shown only where it difiers from the rat sequence.
25 Nucleotide base pair numbers are shown to the left and amino acids, deduced Erom the nucleotides, are numbered at the right. Two in frame stop codons upstream of the initiation methionine are underlined and a polyadenylation signal downstream of the stop codon is 30 boxed.
Fig. 3. p55CDC has seven Gl~-repeats and shows homology to the S. cerev~s~ae Cdc20 and Cdc4 proteins.
(A) The alignment of the seven rat p55CDC
35 repeats was manually constructed following pairwise comparisons using the GCG BESTFIT program. Gaps were W0 95J21917 2 ~ 8 ~ b ~ ~ P~
introduced to obtain optimal alignment and are represented by spaces. Identical or highly conserved resldues which occur at a frequency of 4 times or greater are shown as white on black. Highly 5 conservative substitutions are defined as Ile, Leu or Val, Ser or Thr, and Ala or Gly.
(B) Alignment of the G~-repeats of human p55CDC with the Cdc20 and Cdc4 repeats was obtained using the GCG BESTFIT program followed by visual optimisation. Gaps were introduced to obtain optimal alignment and are represented by spaces . Irlon~ ~ c~ 1 residues are shown as white on black and the highly conserved residues are boxed. Highly conservative substitutions are defined as Ile, Leu or Val, Ser or Thr, Ala or Gly, Tyr or Phe, Asp or Glu and Arg, Lys or His .
Fig. 4. Southern Analysis of Genomic DNA from various species.
Genomic DNA (10 ~g) from several species was digested with Hind III and separated on 196 agarose gels.
The filter was probed with rat p55CDC cDNA under medium stringency conditions as defined in Materials and Methods .
Fig. 5. Overexpression of p55CDC cDNA in the sense or antisense orientation in CHO cells results in alteration of growth profiles.
(A) CHOd- cells were transfected with (~) PMT, (o) PMTp55s or (O) PMTp55as DNA and amplified as described in Materials and Methods. Cells were plated at a starting density of 0.5x106 cells/60mm dish and counted at the times shown. Arrows indicate the days when media was changed. Each point ~ es~ Ls the mean of duplicate counts from parallel cultures which usually varied from 2-14P6 from the plotted mean.
. . . .. .. _ . _ . . . . _ _ . . .... . . ,, ,, .. , . , _, _ _ _ WO95/21917 ~~54 PCr/US9S~01806 (B) Flow cytometry analysis of fixed and propidlum iodide stained PMTp55s (_) and PMTp55as (--) cells was performed as described in M;lt~r;~l ~ and Methods .
Fig. 6. Immune complexes ~ert~i by antibodies agalnst p55CDC.
(A) Cell lysates from 35S-labelled cells in log phase (250 ~Lg on lanes 1,2,3,6,7,8,11,12,13, or 500 llg on lanes 4, 5, 9,10, and 14) were immuno-precipitated with various antibodies. Immune complexes obtained with 10 1ll p34cdc2 M~b (lanes 1, 6 and 11), p55CDC competed antiserum (8.4 llg/lane 2,4,7,9,12) and affinity purified p55CDC antiserum (1 ~g/lane

3,5,8,10,13,14) were analyzed on 10% SDS-PAGE gels. The dried gel was exposed for autoradiography for 21 hours.
(B) Cell lysates from 35S-labelled cells in stationary phase (250 llg on lanes 1, 2, 3, 6, 7, 8, 11, 12, 13, or 500 ~lg on lanes 4,5,9,10,14,15) were 20 immunoprecipitated with various antibodies. Immune complexes obtained with 10 111 p34cdc2 ~b (lanes 1, 6,11), p55CDC competed antiserum (8.4 llg/lane 2, 4, 7, 9,12,14) or affinity purified p55CDC antibody (1 llg/lane 3,5,8,10,13,15) were analyzed on 10% SDS-PAGE
25 gels. Autoradiography was performed for 1 week.
Fig. 7. Histone Hl kinase activity of p55CDC
immune complexes and phosphorylation of p55CDC.
(A) 1ysates of CHO cell lines transfected 30 with vector (PMT), vector with sense transcript (PMTp55s), and vector with antisense transcript (PMTp55as) were immunoprecipltated with affinity purified p55CDC antibody. Immune complexes were assayed for histone H1 kinase activity as described in Materials 35 and Methods.

WO 95/21917 ~ 4 g (B) CHO cells were labelled with [32p]-orthoph~s~hAtP as detailed in Materlals and Methods.
Immune complexes obtained from 900 llg lysate precipitated with 1 llg of affinity purified p55CDC
5 antibody (lane 1) or 28 llg of p55CDC ~ ~P~ antiserum (lane 2) were analyzed by SDS-PAGE.
Fig. 8. Immune complexes detected by p55CDC
antibodies in Rat 1 and HeLa cells and their kinase 10 activity against a variety of substrates at different stages of the cell cycle.
(A) Lysates (250 llg) from exponentially growing Rat 1 and HeLa cells were immunoprecipitated with p55CDC competed antiserum (lanes 1 and 3), affinity 15 pllr~ f~ p55CDC antibody (lanes 2 and 4), and two different monoclonal antibodies against retinoblastoma protein (lanes 5 and 6).
(B) Lysates (200 llg) from HeLa cells prepared as described in Materials and Methods were 20 immunoprecipitated with either control p55CDC competed antiserum shown in the first lane of each substrate or with affinity purified p55CDC antibody. Kinase assays were performed as described in Materials and Methods with decreasing exogenous substrate concentrations shown 25 from left to right . The histone H1 ~ n~ nt rAt ~ r~n c in these assays was 0 . 4 mg/ml, 0 . 2 mg/ml and 0 .1 mg/ml .
Myelin basic protein ~MBP) and c~-casein c~n~ntrAt~ons decreased from 0 . 4 mg/ml to 0 .1 mg/ml . The control assay was always performed using the highest substrate 30 concentration.
(C) Lysates (200 ~Lg) prepared from HeLa cells as described in Materials and Methods were immunoprecipitated with increasing amounts of affinity purified p55CDC antibody (0.07 llg, 0.28 ,ug and 1.12 llg).
35 The negative control was done using 4.2 llg of the p55CDC
competed antiserum. Kinase assays were performed as .

WO 9S/21917 ~ ~ &~ F~~ " ~ - --described in Materials and Methods using 0. 4 mg/ml MBP
as the substrate.
(D) Lysates (200 ~Lg) were prepared from HeLa cells at various stages of the cell cycle as described in Materials and Methods and immuno-precipitated with either 8 . 4 llg p55CDC competed antiserum (lanes 1,8 and 9) or 1.0 Ilg of affinity purified p55CDC antibody (lanes 2-7) . Kinase assays were ~eLr ~~ using 0.4 mg/ml Hl, 0.4 mg/ml MBP or 0.4 mg/ml ~-casein as exogenous substrates.
~E) The excised bands from the dried gel obtained in Fig. 7D were counted. The control values (Fig. 8D lanes 1, 8 and 9) were subtracted from the experimental values (Fig. 8D lanes 2-7) and the results graphed.
Fig. 9. Cycling cells actively translate p55CDC and show high levels of associated ~-casein kinase activity when compared to quiescent cells.
(A) Growing and c~uiescent Ratl cells were labelled for one hour with 35S-Translabel as described in Materials and Methods. Lysates (100 llg) were immunoprecipitated with various antibodies. Immune complexes obtained with 10 ~1 of p34cdc2 M~b (lanes 1 and 7, p55CDC competed antiserum (8.4 ~Lg/ lanes 2 and 8) and affinity purified p55CDC antibody (0 . 035 ~lg/ lane 3, 0.14 ~Lg/lanes 4 and 9, 0.56 ,Ug/ lanes 5 and 10, 1.12 ,ug/lanes 6 and 11) were analyzed by SDS-PAGE.
(B) Lysates (100 ~lg) were prepared from 30 growing and quiescent P~atl cells as described in Materials and Methods. Immune complexes were obtained with 10 ~Ll p34cdc2 M~b, 8.4 ~g of p55CDC ~ ~Prl antiserum and 1.12 llg of affinity purified p55CDC
antibody. Kinase assays were performed as described in 35 ~aterials and Methods using 0.4 mg/ml a-casein as substrate .

;54 WO 95/21917 I ~ "~

D~TATT~r~ DESCRIPTION OF THE INVENTION
The present invention relates to a polypeptide ,lPc;~n~te~ p55CDC which is involved in s~;sn cell division. mRNA transcripts encoding p55CDC were expressed in embryonic tissues and adult hematopoietlc tissues whlch comprlse populations of proliferating 10 cells, but were not detected in adult tissues lacking actively dividinq cells. Moreover, human hematopoetlc cell lines which were induced to differentiate with chemical agents also demonstrated loss of p55CDC
transcript as cell division ceased. It has been 15 observed that polypeptides encoded by the rat p55CDC DNA
sequence (Figure 2 and SEQ ID NO :1 ) and human p55CDC DNA
sequence (Figure 2 and SEQ ID NO:3) have extensive amino acid sequence homology to portions of the cdc4 and cdc20 proteins from Sacchromyces cerevlsiae ~Figure 3). As 20 cdc4 and cdc20 are both known to be involved in mitosis and cell division, this homology has suggested involvement of p55CDC in these processes as well.
Additional evidence implicating p55CDC in cell division is presented in Example 2. It was shown that down-25 regulation of p55CDC expression by transfecting hostcells with a rat p55CDC anti-sense clone resulted in surviving cells which overproduced sense transcripts, apparently to compensate for the loss of p55CDC mRNA.
In additlon, rat p55CDC appears to be synthesized at 30 high levels in actively growing cells, but not in quiescent cells (Example 5).
p55CDC appears to modulate mitosis and cell division through the formatlon of a complex with at 35 least one other host cell protein. rl 1PSrPC c~nt~1n~ng p55CDC were precipitated by p55CDC antisera from Ratl Wo 95/21917 - 12 PCT~S95101806 cells, HeLa cells, and CHO cells tr~ne~ected with a rat p55CDC clone. A presumed host cell protein was observed to be assoclated with p55CDC in each of these cell lines (Examples 3 and 4 ) . The p55CDC complexes from these cell lines displayed kinase activity which fluctuated during the cell cycle. The klnase activity of the p55CDC complex can be dist i n~ll I qh~d from the activities of other known cell cycle-associated kinases, including cyclin A/CDK2, cyclin E/CDK2 and cyclin B/p34cdc2 complexes, in the following ways: (l) p55CDC complexes had kinase activity against a number of substrates, including histone Hl, myelin basic protein and c~-casein rather than against a single substrate; and (2) a decrease in p55CDC-associated kinase activity was observed at the Gl/S transition and at the G2/M
transition. This profile of cell cycle kinase activity has not been previously observed.
The invention provides for an isolated DNA
encoding a biologically active p55CDC polypeptide wherein the DNA is selected from the group consisting of: ~
a) DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2;
b) DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4; and c) DNA having a nucleotide sequence which hybridizes with the DNA of (a) or (b), or with a fragment thereof, wherein the hybridizing DNA encodes a polypeptide having the biological activity of p55CDC.
DNA of the present invention will prefPrPnt1~11y hybridize to DNA sequences encoding p55CDC under appropriate conditions of t ~LaLu~ and salt. Est~hl;ql of appropriate hyhr~ 7~t;on conditions is ~ell within the ability of one skilled in the art using published protocols (see e.g., 63). As an 21~3~5 example, hybridizations may be performed at 42 in gO%
f~rr-ml~ and 5xSSPE for at least 12 hours, followed by three washes in 2XSSC, 0 . l % SDS at 50 and one wash in 0.5xSSC, 0.1% SDS for 30 minutes. Sequences which hybridize with p55CDC DNA will be related by .IPlPt~9tl, insertion, point mutation, fL ~hl ft, alternative open reading frame, or mRNA splice variant. Hybridizing ~eq~Pn~oc may also be ~nt; C~nce nucleic acids (DNA or RNA) which bind to p55CDC DNA or RNA so as to modulate the expression of p55CDC. Antisense nucleic acids may target the p55CDC coding region or regulatory sequences involved in transcription and/or translation of p55CDC.
DNA sequences hybridizing to p55CDC DNA will preferably encode for a polypeptLde having the biological activity of p55CDC. As shown in Examples 3 and 4, p55CDC associates with one or more host proteins to form a complex wherein said complex has cell cycle-~ rf~n~l~nt kinase actlvity. The biological activity of p55CDC, as described herein, refers to a complex-associated kinase activity which is active on various substrates such as histone Hl, a-casein and myelin basic protein, and wherein the kinase activity on one or more substrates is modulated during the cell cycle. For example, the kinase activity of p55CDC complexes on a-casein is rl~mln1ch~d during the Gl/S and G2/M
transitions of the -11 iln cell cycle.
The invention also relates to a p55CDC
polypeptide as the product of procaryotic or eucaryotic expression of an exogenous DNA sequence, that is, p55CDC
is preferably rec~ `; n~nt p55CDC . Exogenous DNA
encoding p55CDC may be genomic DNA, cDNA, or may be partially or completely synthetic DNA. In one o~ , p55CDC DNA includes one or more codons which are preferred for expression in procaryotic host cells, WO 95/21917 218 3 ~ ~ 4 r ~

~re ~ ~ l l y E- col ~ host cells . Synthesis of DNA
rL, c for assembly into sequences for p55CDC
expression ls accomplished using synthetic methods which are readily avallable to one skllled ln the art, such as 5 those described in Engels et al. (Angew. Chem. Intl. Ed.
28, 716-734 ~1989) ) .
Also provided by the invention are plasmids and host cells for the expression of p55CDC protein.
10 p55CDC expression may be accomplished in procaryotic or eucaryotic hosts (e.g., mammalian, plant or insect cells, yeast or bacterial cells) . Preferred host cells include l ~ An cells, such as Chinese Hamster Ovary (CHO) cells, or b?'-tP~l hosts such as l~scher~chia 15 coli. p55CDC may be expressed from a variety of plasmid or viral vectors which are appropriate for the host cell being used. The use of vector pMT for the expression of rat p55CDC in CHO cells is described in Example 2.
However, other Yectors that are suitable for p55CDC
20 expression in other host cells may also be used.
Expression of p55CDC in transgenic animals may be obtained using expression vectors and DNA trAncfec~ ~ nn procedures available to one skilled in the art.
A method for producing a p55CDC polypeptide is also included. The method comprises culturing a procaryotic or eucaryotic host cell into which an expression vector ~nnt~n~n~ a p55CDC DNA sequence has been transformed or transfeeted such that a p55CDC
polypeptide is expressed.
An isolated p55CDC polypeptide ls ~n~ qed by the present invention. Such polypeptides may be produced by expression of DNA molecules encoding p55CDC, or they may be produced by chemical synthesis of peptides using procedures available to one skilled in WO 95/21917 2 1 8 3 2 ~ ~ r~

the art. p55CDC polypeptides produced by the afo r ~ nt ' on.o~l biological or chemical methods are isolated using purification techniques which are known to one skilled in the art. p55CDC polypeptides may be - 5 analogs of rat or human polypeptides shown in SEQ ID NO:
2 or SEQ ID NO: 4, respectively, wherein said analogs comprise the substitution, deletion or insertion of one or more amino acids. In addition, rh~m~c~l synthesis of p55CDC polypeptides allows the tnrl~lclnn of non-naturally occurring amino acids (e . g ., D-amino acids) at selected positions. Amino acid residues within the p55CDC polypeptide which are required for activity are determined by generating analogs and testing said analogs for activity, such as the ability to ~orm a complex having cell cycle associated kinase activity, or the ability to advance a host cell through the cell cycle. Protein kinase assays .lDq~ r~ hl~rl in Materials and Methods can be used to test for the biological activity of p55CDC analogs. Selected regions of a p55CDC
polypeptide, such as those which show homology to the cell division proteins cdc4 and cdc20 (see Figure 3), may be used to design biologically active p55CDC analogs or peptide rL - - ~t C ~ These regions are referred to as G13 repeats and are likely to be important in the structure and/or function of p55CDC.
Antibodies specifically binding p55CDC
polypeptides of the invention are also provided.
Antibodies may be polyclonal or monoclonal and may recogni~e fragments, analogs and fusion polypeptides of p55CDC as well as the intact protein. Mouse anti-p55CDC
antibodies may be produced by techniques available to one skilled in the art and may be modified to form chimeric or l ~ 7~d antibodies. Anti-p55CDC
antibodies are useful in assays described below for WO 9~21917 2 ~ PCrNS95/01806 quantitating p55CDC and p55CDC complexes that are present in biological samples.
A complex comprising p55CDC and at least one 5 other host cell protein is also provided. Example 3 describes an immune complex from tranfected CHO cells having p55CDC and an associated 210 kDa protein wherein the complex has cell cycle associated kinase activity.
Example 4 describes immune complexes from rat l and Hela 10 cells which have p55CDC associated with a second polypeptide and exhibit kinase activity. A llO kDa protein was ~ nt; ~ in rat immune complexes and a lO0 kDa protein was identified in Hela immune complexes.
The ability of p55CDC to associate with at least one 15 other polypeptide such that the resulting complex phosphorylates various host cell molecules appears to correlate with the ability of p55CDC to modulate the cell cycle. Also ~ ~qPcl by the invention are complexes comprising a p55CDC analog and at least one 20 other host cell protein. In a preferred F~mho~
p55CDC complexes will have cell cycle ~ n~ L kinase activity such as that described in Example 4.
The invention also relates to a method ~or 25 detecting levels of p55CDC in biological samples. The method comprises; nf-llh;~t; ng an antibody specifically binding p55CDC, or a fragment, analog, or fusion polypeptide thereof, with a sample under conditions suitable for Eorming a complex between the antibody and 30 p55CDC and detecting the presence of a p55CDC-antibody complex. The antibody may also bind to p55CDC when p55CDC is ~ 1 with other host cell proteins.
Therefore, the method also ~n~ ~ ses the rlPtprt; r~n of p55CDC complexes. Since p55CDC is present in actively 35 dividing cells, but not in ouiescent cells, it is anticipated that a diagnostic assay for p55CDC will be WO 95/21917 2 ~ s ~ 9! ~

most useful in identifying those samples having elevated levels of cell divislon.
Methods for modulating cell division are also 5 provided. It will be appreclated by one skilled in the art that compounds which modulate p55CDC activity will modulate cell cycle activity as well. Compounds which modulate the synthesis of p55CDC and/or modulate the ability of p55CDC to form a complex having cell cycle l0 associated kinase activity may be identified using the procedures rlt~SCr~ hP~l for determining p55CDC activity .
Mtlt~ t l on of p55CDC kinase activity may involve an increase or decrease in activity at certain periods during the cell cycle which may lead to alterations in 15 the timing or sre~-~ f~ ty of p55CDC complex activity, Compounds which in turn may be used to control cell division include, but are not limited to, the following:
(l) compounds which lncrease or decrease the levels of p55CDC synthesis; ~2) compounds which bind to p55CDC so 20 as to interfere with formation of a p55CDC complex having kinase activity; (3) compounds which compete with p55CDC for complex formation and themselves form inactive complexes; and (4) compounds which promote the formation of the p55CDC complex or stabilize said 25 complex from dissociation by lncreasing the half-life.
13xamples include nucleic acid - lt~r~le~ which bind to p55CDC DNA or p55CDC polypeptides, antibodies, peptides, organic molecules, and carbohydrates. Such ~ ~ul~ds are identified by screening large repetoires, or 30 libraries, comprisin~ nucleic acids, peptides or small organic molecules derived from chemical synthesis or natural sources (e.g., bacteria, fungi, plants).
Considerable literature exists on the synthesis, char~ctt~rl7at~n and screening of very large natural or 35 synthetic libraries of molecules or polymers. One skilled in the art would appreciate that such libraries wos~msl7 P~,l/-)_.'l ~$~

can be screened for compounds that modulate p55CDC
activity .
Compounds which inhibit the biosynthesis or 5 activity of p55CDC are useful in inhibiting the growth of those tumor-cells having increased levels o$ p55CDC
or increased levels of cell cycle dependent kinase activity ~c50r~tP~l with p55CDC as compared to normal, ~lnnr~nrProus cells. Compounds useful as 10 chemotherapeutic agents include, but are not limited to, the following: (1) compounds which decrease the levels of p55CDC synthesis; (2) Iuu~lds which bind to p55CDC
so as to interfere with formation of a p55CDC-host cell protein complex having kinase activity; and (3) 15 compounds which compete with p55CDC for association with one or more host cell proteins involved in complex formation and themselves form inactive complexes. Tumor cells which grow more rapidly than normal, nnnr~nrprous cells perhaps by virtue of increased p55CDC activity may 20 be more responsive to p55CDC-inhibiting agents. Such agents would be expected to have less effect on p55CDC
activity in normal cells.
A method of chemotherapy comprising treating a 25 mammal with an amount of a compound which is effective in reducing or inhibiting p55CDC activity in a ph~r~n-rF-ut;cally effective ad~uvant is also provided.
Compounds which reduce or inhibit p55CDC activity are ; ~lPnt; ~; erl by screening appropriate sources for activity 30 against p55CDC using assays for p55CDC activity as described herein. A dosage which is effective in reducing or inhibiting p55CDC activity may be determined by one skilled in the art taking into account such factors as the condition being treated and 35 administration regimen. Important considerations include the type and location of the tumor being treated ..... . . _ . _ _ _ _ _ _ WO 95/21917 ~ ~ ~ 3 ~ ~ ~ r~

and whether the route of A~' ~n~.ctratlon is by in~ection ~intravenous, intramuscular, or subcutaneous) or by oral or nasal intake. Compounds of the present invention are mixed with a ~hArr-^e~lt ~ cally acceptable ad~uvant which 5 may include any suitable buffer, so1 llh; l ~ 7er, preservative, carrier or anti-oxidant. Preferably, the adjuvant will not decrease the p55CDC-inhibiting activity of the - ~1 An extensive survey of phArr--ellt 1 cally acceptable a~uvants is found in 10 Remington's Pharmaceutical Sciences, 18th ed. A.R.
Gennaro, ed. Mack, Easton, PA (1990).

lL~ CATION AND t'T~ t'~T7~'rION OF p55CDC
The p55t~nC Cr~'n~
The gene encoding p55CDC was ~ ~IPnt; f ~ ed serendipitously during an attempt to identify novel 20 glycosyltransferase enzymes by low stringency screening of a rat genomic library with cDNA encoding the rat ~2, 6 sialyltransferase (57,73). During one round of screening, a genomic clone was isolated. Restriction map analysis first narrowed the hybridizing region to a 25 2 kb ~3gl II fragment. The cross hybridizing region of this fragment was further narrowed to a 0.26 kb Pst I
fragment which was used for Northern analysis of various embryonic, n~nAtAl and adult rat tissues.. This nt~f~d a tissue which could be used as a source of 30 RNA for construction of a cDNA library.
Northern analysis revealed tissue-specific and devell_, ' Al ly-regulated expression of a uni~lue transcript (Fig. lA). A 2 kb mRNA was abundant in RNA
35 from total rat embryo, and this transcript was ~nr~ ~-hF.
in embryonic rat liver. However, in two day old wo 95/21917 ~ 2 ~ ~ pcrNs95lol8o6 neonatal rats, the level of message in the liver decreased precipitously. The transcript was still abundant in spleen from 2 day old rats, and a small amount was present in kidney. In 16 day old rats, the 5 transcript was still abundant in spleen and thymus, but was barely ~l~tPrtAhl e in liver and kidney. The transcript was 1~lot detectable in any adult tissues, although a longer exposure of a blot ~ntA;n~n~ more RNA
did reveal a faint band in the spleen sample. The 10 presence of the transcript in hematopoietic tissues, such as neonatal liver, thymus and spleen, suggested that expression oi' this novel gene was highest in tissues in whic1~ cell proliferation was occuring.
A cDN~ library was constructed using polyA+
RNA from two day old rat spleen. Using the Pst I
genomic fragment as a probe, several positive plaques were ~IPnt~f~ , at a frequency of approximately l :15, 000 . The two largest cDNA inserts were subcloned 20 and sequenced. The nucleotide sequence (Fig. 2 and SEQ
ID N0: l) coded for a protein of 499 amino acids, with a predicted molecular mass of 55 kDa. However, this sequence did not appear to encode a classical glycosyltransferase enzyme, since there was no evidence 25 of an amino-terminal hydrophobic - n~ spanning signal-anchor domain, which is f~cc~nt ~ Al for glycosyltrAncf~rases to be properly oriented in the Golgi (57).
The human p55CDC gene was isolated from an HTl080 cell line cDNA library by the following procedures. The nucleotide sequence and deduced amino acid serlu~nc~ are shown in Fig. 2 and in SEQ ID NO: 3.
A comparison of the open reading frames of the rat and human sequences showed an 87% identity at the nucleotide level, which increased to 95% at the amino acid level.

W09~i/21917 - 21 -Differences in the human nucleotide sequence are shown above the rat sequence, and differences in the human amino acid sequence are shown below. The human sequence diverged considerably from the rat upstream of the ATG
5 start site, and also in the 3 ' untranslated region.
Homolo~v of p55Cr~c to cell cycle protelnc A search of the genEMBL database revealed that rat and human p55CDC proteins had seven regions of 10 homology with the WD-40 repeat of the B subunit of G
proteins (27) ~Fig. 3A), and to a number of proteins which contain this imperfect repeat motif (for review, see 12,72). These ~nr~ the products of the S.
cereYisiae genes CDC20 (65) and CDC4 (77), l'UPl/AE~2 (78), PRP4 (58), and MSI1 (62), as well as the products of the D. melanogas~er gene Espl, the D. discoidum gene AAC3 (66), the Arabidopsis thaliana gene COPl (13) and the dTAF1180 subunit of Drosoph~la TFllD (22). The highest degree of homology, illustrated in Fig. 3B, was 20 seen between p55CDC and the two S. cerevisiae cell division cycle proteins, Cdc20 (519 amino acids) and Cdc4 (779 amino acids). The BESTFIT analysis revealed a 45% identity between amino acids 172-407 of p55CDC and amino acids 249-479 of the Cdc20 protein, which 25 increased to 59% when highly conserved substitutions were ~ nr-l ~ d This was the only protein in which a high degree of similarity was found with the degenerate internal Gl~-repeats in p55CDC. The Cdc4 protein was the only protein which showed strong homology with all seven 30 repeats found in p55CDC, using the first seven of the nine repeats found in the Cdc4 protein (Fig. 3B). The alignment of the highly degenerate WD-40 repeats in these two proteins required the introduction of 16 gaps over 300 amino acid residues . This comparison ~ nrii c~ted 35 that 289~ of the residues in this region were identical, and 41% were identical or highly conserved. Notably, . . , . _ _ _ _ _ _ _ _ _ . _ _ _ _ . .

Wo 9S/21917 r~
2 ~ ~ 3 ~ 5 ~

the S. cerevis~ae Cdc20 and Cdc4 protelns each displayed a greater degree of homology to the mammalian p55CDC
protein than they did to each other.
Recently a clone isolated from a xenopus oocyte cDNA library by virtue of its ability to suppress the temperature sensitive defect of the S. Cerevisiae cdcl~ mutation, was shown to encode a protein of 518 amino acids that has seven GB-repeats in its carboxy terminal half t69). This protein called BTrCP (B-transducin repeat rnntA~n~ng protein) was not a fllnrt~nn~l homolog of CDC20, though overexpression of both these genes is capable of suppressing the cdc15 ~tlrn (1,69). Both BTrCP and p55CDC have seven GB-repeats and show 24% identity over this region.
The only protein that showed sl~n1 f~ rAnt homology to p55CDC ~xtPnrl1 nq beyond the GB- repeats was the S. cerev~siae MSI1 protein, which is a negative regulator of the RA" -~ Atrrl; n~lllrt ~ nn of cAMP levels (62). The MSI1 protein (422 amino acids) was 24%
identical to p55CDC, and this increased to 28% when only the amino terminal 178 residues of p55CDC were compared to the amino terminal 148 residues of MSI1.
Cross species homolo~v of ~55cnc A higll degree of evolutionary conservation within the p55CDC open reading frame was seen when genomic DNA from a variety of l ~ An species, chicken, D. melanogaster and S. cerevlsiae were Py~m;n~d by Southern blot analysis, using the rat cDNA probe (Fig. 4). A cross-hybridizing species was rlpt~rtAhle in all the l l ;~n and the avian species, although no bands were seen in the lanes rnnt;l~n~n~ s. cerevi-7iae and D. 1 7Ann~A~ter DNA. These results also in~l~r~AtP
that the gene encoding p55CDC is a single copy gene, with no closely related genes in the species ~YAml ned.

WO 95~21917 r~".J.. ~ -2183;2~4~

Es~}?ression of p55CDC in tissues an-l cell l; nes The pattern of expression of pS5CDC mRNA in embryonic and n~--n~t~l rat tissues, and the apparent relationship between p55CDC and the S. cerevisiae Cdc20 5 and Cdc4 proteins suggesting a possible role for p55CDC
in cell division, prompted us to examine other developing 1 ~n tissues for p55CDC expression.
Northern analysis of human tissues demonstrated a pattern of expression similar to that seen in the rat, 10 with high levels of expression in fetal liver and juvenile thymus, but no expression seen in fetal lung, adult lung or liver, or adult buffy coat, which is primarily comprised of non-dividing white blood cells ~Fig. lB) . A second Northern analysis ~ m~ n~ nrJ polyA+
15 RNA from adult human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas demonstrated expression of p55CDC in only one tissue, the placenta, which contains actively dividing cells; a similar pattern of expression has been described for p34cdc2 20 (48).
A number of human cell lines also expressed the p55CDC transcript. The transcript was ~hllnri~nt in all leukemia cell lines examined, ~ nr~ nrJ the T cell 25 lines MOLT 4f and CEM, the B cell lines Ra~i and Ramos, the monocytic cell line U937 and the myeloerythroid cell line K562 (Fig. lC). Indeed, we observed expression of the p55CDC transcript in every cell line f~ m~ n~ at log phase of growth, regardless of lineage.
To examine whether expression of the p55CDC
transcript was related to the ability of cells to divide, we took advantage of the unique properties of two leukemia cell lines, ~-562 and E~1-60. K-562 cells 35 can be induced by treatment with sodium butyrate to undergo erythroid differentiation with no significant WO 95/21917 2 1 8 ~
-- 2~ --effect on growth rate (2). In contrast, treatment of K-562 cells with the phorbol ester TPA causes monocytic differentiation A~ 1 ed by growth arrest (5) .
TL~' ' ' of HL-60 cells with TPA also causes monocytic differentiation, with arrest of DNA synthesis and cell division (61) . We ~YAm; nf~d the effects of these agents on the level of expression of p55CDC mRNA in these two cell lines (Fig. lD). The p55 transcript was easily detectable in both mock-treated cell lines. For both K-562 and ~L-60, treatment of the cells with TPA resulted in loss of p55CDC mRNA expression. In the K-562 cells treated with sodium butyrate, in which differentiation is not ;3~ ~ ed by growth arrest, the level of p55CDC
transcript was roughly equal to that found in the mock-treated cells. These results indicate that p55CDC mRNA
is synthesized only in dividing cells.

EFFECTS OF p55CDC ON CELL PROLIFERaTIO~
To explore possible functions of the p55CDC
protein, CHOd- cells were transfected with plasmid containing the cDNA encoding the rat p55CDC in either 25 the sense (PMTp55s) or antisense (PMTp55as) or~nt~t~n.
A 1. 8 kb fragment of rat cDNA was inserted downstream of the metallothionein promoter in the pMTOlQ/A+ 11 An expression vector (9). This vector also contains two dominant selectable markers, the bacterial neo gene and 30 the mouse DNFJ~ gene, driven by the SV40 promoter.
Control cells were transfected with vector alone (PMT).
Following amplification with methotrexate, the three pools of cells were plated at a density of 0.5xl06 cells per 60 mm plate in the presence of 0 . 05 mM zinc, and the 35 growth profiles plotted for four~een days (Fig. 5A).
Pools of transfected cells were studied, rather than Wo 95/21917 ~ 5 ~ PCr/USs5l0l806 individual clones, to m~n~m~7e the effect of any clonal variation in the CHOd- cells. Initially, llttle difference in the growth rates were observed among the three pools of transfected cells, although the PMTp55as `
cells were significantly larger than the PMTp55s or control cells as shown by forward scatter analysis determined by flow cytometry and visual observation under the microscope. Moreover, a DNA content analysis as measured by propidium iodide uptake showed that PMTp55as cells had an increased amount of DNA/cell, indicating that these cells were hyperdiploid (Fig. 5B).
As the plates began to reach confluence, dramatic differences in the growth profiles were observed. The PMTp55 as cells reached confluence first, at a lower cell number, consistent with their layer size. After reaching rrnflllonre the PMTp55as cells crnt;nllPd to divide slowly. The smaller PMTp55s cells r-rnt1n~led to divide at a faster rate after reaching confluence. The PMTp55s cells reached a density of 24X106 cells/plate by day 14, compared to 6X106 cells/plate for the PMTp55as cells. The growth profile of the PMT cells fell midway between those of the PMTp55s and PMTp55as cells.
Since the cells transfected with the vector encoding an antisense transcript continued to survive, although with an altered phenotype, we examined the pools of transfected cells for the presence of sense and antisense p55CDC mRNA transcripts using an RNAse protection assay (70). As shown in Table 1, the PMT
cells had an average of 166 copies of sense mRNA per cell, while, as expected, the PMTp55s cells had an increased average of 734 copies of sense mRNA per cell.
Surprisingly, the PMTp55as cells also had an increased number of copies of sense mRNA, with an average of 714 copies per cell. In addition, the PMTp55as cells had only a moderate amount of antisense mRNA, with an WO 95/21917 ?,~3~

average of 205 copies per cell, despite having been transfected with cDNA encodlng the antisense transcript.
This same pattern was observed when clonal cell lines isolated from the pools of cells were analyzed. Each of 5 the four PM~p55as clonal lines made elevated amounts of the sense transcript; in all lines, this amount was at least five times the amount of the antisense transcript.
As ~ ect~fl, in the control PMT cells, the average number of copies of sense transcript per cell declined 10 considerably in confluent cells.

p55CDC mRNA copy number in both sense and antisense orientation in various cell lines

4 8 hours 7 days Cell 1ine mRNA co ies/cella mRNA co- ies/cell~
Sense Anti-sense Sense Anti-sense PMT166 i 6 - 4 i 7 PMTp55s 734 i 40 21 i 7 240 i 9 16 i 5 PMTp55as 714 i 10 205 i 10 263 i 10 96 i 10 PMTp55A2s 771 i 12 2 NDb ND
PMTp55B6s 4136 i 66 117 i 4 ND ND
PMTp55B12as 706 i 20 126 i 3 ND ND
PMTp55G6as 1176 :t lO 213 i 14 ND ND
PMTp55Hsas 928 i 17 157 i 2 ND ND
PMTp55H11as 1149 i 21 128 i 7 ND ND
a. Values were ~ t~rm n.od as described in experimental procedures .
15 b. Not Determined Genomic DNA analysis of all six clonal cell lines demonstrated that the elevated expression of sense Wo 95/21917 ~ Pcr/USs5/0l806 transcripts was not due to amplification of the endogenous gene. The two clonal isolates expressing sense ori~ntAt;~n transcripts were dlstinct from one another. In contrast, it is likely that all four of the

5 PMTp55as clones we isolated derived from the f~ rAnc{,~n of only one transfected cell in the original pool of cells, since restriction map analyses of the four clonal PMTp55as cell lines using two different restriction enzymes and two different probes, to detect either 10 plasmid or p55CDC se~ PnC^c, demonstrated ~A,~nt~Al banding patterns. The results indicated that inhibition of p55CDC expression by Ant; C-~nce transcripts was c~ c~t-~ for by overexpression of sense transcripts.
This data suggested that p55CDC was essential for 15 maintenance of cell proliferation in culture.

IMMI~NE COMPLEXES CONTAINING p55CDC
Polyclonal rabbit antiserum was raised against a fusion protein r~rlnc1 ct; n~ of p55CDC and glutathione S-transferase. Both the original antiserum and an affinity purified antibody preparation precipitated a 25 protein of Mr 55 kDa from an ~n v~tro transcription/tr~nCl At ~ on reaction ~lntA; n; n~ p55CDC
cDNA, consistent with the predicted mass of the polypeptide .
To examine the level of p55CDC production in the transfected cell lines, immunoprecipit2tions were performed on extracts of 35S-lAh^l l~rl cells in log phase, using the affinity purified antibody. As shown in Fig. 6A, the PMTp55s and PMTp55as cells had increased levels of p55CDC compared to the PMT cells, consistent with the demonstration of increased numbers of Wo 9~21917 2 1 8 ~ 2 ~ ~ PCrlUSss/01806 transcripts encoding p55CDC in these cells. In the ~MTp55s cells, there was a strong band of 31 kDa which probably ~ S~ Ls a degradation product of p55CDC, since this band was also ~.ot~rtf~ on immunoblot analyses 5 of cell extracts using the polyclonal antibody preparation. This 31 kDa band was also observed when cell lysates were prepared without protease inhlbitors, and no intact p55CDC was detected in the absence of protease inhibitors. This peptide was not p34cdc2, 10 since no p34cdc2 protein was detected in any of the immune complexes.
Immunoprecipitates o~ p55CDC also rnnt;l~n~1 a protein of Mr 2l0 kDa. The amount of p210 ~l~tected in 15 the immunoprecipitates was roughly proportional to the amount of p55CDC. When this experiment was repeated on cells in stationary phase, seven days after plating, a significant decrease in the amounts of both p55CDC and p210 is observed (Fig. 6B~; in Fig. 6s, a one week 20 exposure of the autoradiogram was re~uired to detect p55CDC, compared to a 21 hour exposure in Fig. 6~.
These results indicate that production of p55CDC is highest in proliferating cells.

EXAMPI,E 4 ~CINASE ACTIVITY OF p55CDC IM~NE COMPLEXES
Since many events in the cell cycle are 30 controlled by various kinases, it was of interest to determine whether p55CDC immune complexes had any kinase activity . All immune complexes ~ m~ n~tl for protein kinase activity were precipitated under conditions identical to those used in Fig. 6. The immunoprecipiptation buI'fer was fonm~ ted (196 NP-40, 1%
deoxycholate and 0.1% SDS) to m;n;m;ze non-specific WO 95/21917 ~ 1 8 3 ~ 3 ~ C

protein assoclation. A number of cell division kinases can phosphorylate histone H1, so this substrate was assayed first. As shown in Fig. 7A, lmmune complexes precipitated with p55CDC antibody phosphorylated histone 5 Hl. Immune complexes prepared from lysates of the PMT, PMTp55s and PMlp55as cells all demonstrated kinase activity against histone H1. The highest levels of phosphorylation were seen in the PMTp55s and PMTp55as cells which have increased expression of p55CDC. In the 10 negative controls, using competed antiserum, a small amount of residual activity is seen. In reactions performed without addition of exogenous substrates, no phosphorylated proteins were detected, indicating that none of the proteins in the immune complex are 15 endogenous substrates of the kinase activity. However, when all three pools of transfected cells were labelled with [32p]- orthophosphate and p55CDC was immunoprecipitated, SDS-PAGE analysis revealed that p55CDC was phosphorylated (Fig. 7B). Thus, p55CDC is a 20 substrate of another endogenous kinase in the CHO cells.
In the PMTp55s cells, no 32P-labelled 31 kDa band was detected (see Fig. 6A and B, lanes 8 and 10), indicating that the 31 kDa degradation fragment of p55CDC is either not phosphorylated, or is dephosphorylated prior to 25 degradation.
ne wished to examine whether p55CDC in different cell lines was associated with other proteins in immune complexes and whether these complexes also had 30 kinase activity. The Ratl fibroblast and He1a cell lines were chosen for this analysis. Proliferating Ratl and HeLa cells were lysed, and immune complexes precipitated with the affinity purified p55CDC antibody (Fig. 8A, lanes 2 and 4) . SDS-PAGE analysis of the 35 immune complexes did not reveal the 210 kDa band seen in CHO cells, but did reveal other discrete bands which WO 9S/21917 2 ~ r appeared to be cell-specific. In the Ratl cells, a protein of llO kDa was present in the p55CDC imm. une complexes, while a protein of 100 kDa was seen in pS5CDC
immune complexes in HeLa cells.
The p55CDC immune complexes from He1a cells were ~rAm~ nP~l for kinase activity against a number of different substrates ~Fig. 8B). Kinase activity was detected with histone Hl, myelin basic protein and a-lO casein, with maximal activity detected with myelin basicprotein. B-casein was also P~Am~ nP~ but minimal activity was detected with B-casein as a substrate (data not shown). The level of kinase activity correlated with p55CDC concentration, since increasing the amount 15 of antibody used for the immunoprecipitation resulted in increased phosphorylation of myelin basic protein (Fig.
8C) .
To determine whether the p55CDC-AssorJ ~ted 20 kinase activity fl~rtl~tPcl during the cell cycle, as has been r~P~rr~ hPd for the cyclin-~PrPn~lpnt kinases, cells were arrested at various points in the cell cycIe and immune complexes precipitated from cell lysates were examined for kinase activity. A distinct pattern of a 25 cell cycle related fl-lrtllAt~on in kinase activity was detected with only one of the three substrates examined, a-casein (Figs. 8D and E). Kinase activity against a-casein was present in He1a cells and in cells blocked in Gl by serum starvation. The level of activity 30 against a-casein dropped approximately four-fold in cells arrested at Gl /s and returned to the higher levels in cells harvested during S phase. Kinase activity remained constant in cells in the G2 stage of the cell cycle and decreased six-fold in cells at the G2/M
35 transition. Kinase activity against histone Hl by p55CDC immune complexes was stable throughout the cell Wo 95/21917 ~ Pcr/uSssl01806 cycle (Fig. 8E). Backgound levels of histone Hl kinase activity in the G2/M cells (Fig. 8D, lane 8) was most likely due to residual p34cdc2 kinase activity in these samples. Kinase activity against myelin basic protein 5 was also relatively constant throughout the cell cycle, with the exception of the G2/M tr~nq;t~n~, where a two-fold decrease in activity was observed. While p55CDC
was difficult to detect by; t~hl ~tting of cell lysates prepared from cells at any of the various 10 stages, the amount of p55CDC present in cells did not appear to fluctuate during the cell cycle, in contrast to the fluctuation in kinase activity observed with p55CDC immune complexes.

EXaMPLE 5 p55CDC EXPRESSION AND KINASE ACTIVITY IN GROWING
AND Q~IESCENT CELI,S
The expression of p55CDC and the associated kinase actlvity in growing and quiescent populations of cells was compared by exploiting the ability of Ratl cells to arrest growth under limiting serum conditions.
As shown in Fig. 9A, exponentially growing Ratl cells actively 5ynth~c; ~Pd labelled p55CDC (lanes 3-6), while the quiescent population showed minimal production of p55CDC within the one hour labelling period (lanes 9-11). To rule out that the kinase activity we observed was precipated non-spP~-;fi~ lly from the cell lysates, increasing amounts of p55CDC antibody were used in the immunoprecipitations. As shown in lanes 3-6, increasing the amount of p55CDC antibody resulted in the precipitation of increasing levels of p55CDC. This result is consistent with the result observed in Fig.
8C, where increasing the amount of antibody used for preclpitation increased the level of p55CDC kinase .. _ . . . ... . .. _ _ _ _ _ _ ~ _ WO9s/2l9l7 ~ 2~ ~ P~

activity ~ t~ctf~l . The production of 1 Ah.ol 1 ~rl p34cdc2 is also substantially reduced in the quiescent population (compare lanes l and 7), although the amount of total p34cdc2 in the two samples was virtually 5 equivalent, as detected on Coomassie blue stained gels of immunoprecipitated material. We also ~ m;nPrl the p55CDC associated kinase activity under these two conditions and compared it to that observed for p34cdc2 immune complexes as a control. A higher level of l0 activity was observed with the p55CDC complexes using a-casein as a substrate, since a-casein is a poor substrate fior the p34cdc2 kinase (Fig. 9B) . Both the p34cdc2 kinase and the p55CDC associated kinase showed a decrease in activity in the quiescent cells. As seen in 15 the HeLa cells, when myelin basic protein was used as a substrate, no significant change in the p55CDC
associated kinase activity was observed.
M2~'1'1;'11T~T.~: AND ~ET~ODS
RNA A~A1 vsis Total RNA was prepared from freshly dissected rat tissues, human thymus and buffy coat, by the method 25 of Chomczynski ~nd Sacchi (8). mRNA from human cell lines was prepared by the Fastrack kit (Invitrogen).
Gel electrophoresis o~ total RNA (30 ~g/lane) was done in 1% agarose gels c~ntAin;ng formaldehyde and Northern hybridizations were performed as report~d earlier (73).
30 Radiol AhPl 1 .-~i probes were generated using the ~ rchAm Multiprime DNA labelling system R~N. 1601. mRNA size was ~t~rm; nf~rl by comparing with commercial RNA standards (~Pth~s-lA Research Laboratories, Gaithersburg, MD). mRNA
from other human tissues was purchased from Clontech as 35 was a multiple human tissue Northern blot.

wo gsm917 2 ~ $ ~ 2 ~ ~ P~ 5/~ -To generate the rlboprobes for the RNAse protection assay, the gel purified p55CDC cDNA fragment was subcloned into Bluescript (Stratagene, LaJolla, Q) in both the sense and antisense orientation relative to the T7 promoter. All subsequent steps were performed as described previously (70). Briefly, cells (lxlO6/ml) were washed in phosphate buffered saline (PBS) and lysed by incubation at room temperature for 20 minutes in lOmM
Tris pEI8.0, lmM EDTA, 20mM dithiothreitol, 100 llg/ml proteinase K and 0 . 2% SDS . Lysed samples were added to hybridization mix with the labelled riboprobe and ;nrllh~ted at 84- for 2 hours. Following RNAse digestion for 20 minutes at 37- using RNAse A and RNAse Tl, the sample was loaded onto a Sephacryl S200 Superfine gel filtration column (Sigma, St. Louis, MO) and the voLd volume fraction cont~n;ng the protected probe was counted. The quantity of gene sr,or~ f~ t' RNA was calculated from a standard curve. All assays were performed in duplicate.
DNA ~n~lys; ~
Genomic Southerns and restriction map analysis were performed using standard molecular biology techniques (63). Genomic DNA from various species was purchased from Clontech (Palo Alto, Q). Medium stringency hybrirl;7~t~0nc were performed at 42- in 40%
formamide. All hybridizations were performed at a salt concentration of 5xSSPE. Following overnight hybridizations the filters were washed three times in 2XSSC, 0.1% SDS at 50-. The final wash was done in 0.5XSSC, 0.196 SDS for 30 minutes. DNA ser~uence was ~l~trrm~ n~l using Sequenase (U. S . Biochemical), following the manufacturer's protocol. Serllenr;ng was also performed on the Applied Biosystems 373A automated DNA
sequencer using the Tag Dye Deoxy Terminator kit according to suggested protocol. The comparative _ _ _ _ _ _ . _ wo g5,2l9l, 2 ~: 8 ~ 2 ~ ~ F~~

percent identity values between the genes carrying the GJ~ motif were obtained using the GCG B13STFIT program with gap weight set at 2 . 0 and length weight at 0 . 05 .
5 onNA Cll-n~na of ~t ~55rnc A rat genomic library made from a partial E~coRI digest ligated into Charon 4A (rl ~nterh) was SrrPPnP~l at low stringency (hybridizations peLrO ~ in 43% formamide at 37 ) with a 435 base pair cDNA probe ~n~ -qqing amino acid rPc;f~1lPq 141-286 of the a2, 6 sialytransferase gene (73). Restriction map analysis of the; qol ~tPd genomic clone revealed a 2 kb Bgl II
fragment that hybridized to the probe. This fragment was subcloned into a pUC vector and further analysis 15 narrowed the hybridizing region to a 0.26 kb Pst 1 fragment which was used in all q~lhse~lPnt analysis.
PolyA+ RNA from newborn rat spleen was selected by two cycles of binding to oligo (dT)-cellulose type 2 (Collaborative Research). A cDNA library was 20 constructed us$ng the /?h;~rr-~~; ;I cDNA synthesis kit followed by ligation into the lgtlO vector. This was packaged using the Gigapack II Gold cloning kit (Stratagene). An initial packaging reaction gave 3.3 x 106 pfu and 1 x 106 pfu were screened using the 0.26 kb 25 Pst I fragment~ as probe.
~nN~ Cloni nq of H - - p55CDC
The human HT1080 cell line cDNA library was constructed in the pSPORT-l plasmid vector (BRL Life 30 Technologies, Inc. ) . DNA from 44 pools of approximately 5000 colonies each was linearized with Not I and screened by Southern blot, using the rat p55CDC cDNA as a probe. ~laaue and colony purification of the clones with the longest inserts was done using standard 35 techniques ( 63 ) .

WO9~/21917 21~ r~l"
~11 r~lllture Syn~hrr~n~zation an~l TAhelllnçr HL60 and K562 cells were grown in RPMI1640 ~Irvine Scientific, Irvine, CA) supplemented with 10 mM
HEPES and 15% fetal calf serum. Cells were seeded at a 5 c~nC~ntr~tion of 0.2 x 106 cells/ml media. Cells treated with lmM sodium butyrate were grown in 75 cm2 flasks for three days. Cells induced with phorbol ester were grown in the presence of 30 ng/ml 12-O-teradecanoyl-phorbol 13-acetate ~TPA) for three 10 days. Cells were lysed by guanidine thiocyanate and total RNA prepared as described ~). CHOd- cells were r-~nt~;n~1 in Dulbecco's modified Eagle's medium ~DMEM) supplemented with 5% fetal calf serum, glutamine, non-essential amino acids and hypt7x~nth~n-. Rat 1 cells 15 were maintained in DMEM ~ nnt~inin~ 1096 serum and glutamine and HeLa cells in a Minimum Essential Medium supplemented with 1096 serum, glutamine and non-~csontlAl amino acids.
HeLa cells were synchronized at the beginning of S phase (G1/S) by the double thymidine/aphidLcolin block described by Heintz et al (35) . Cells harvested 4 hours later were in S phase (59) . Synchr~n; 7~t ~ cm at the G2/M transition was achieved by growth in the presence of 0.5 llg/ml nocodazole for a period of 12-14 hours . The media was carefully Sll('`t i ~n~d off and the non-adherent mitotic cells were harvested by gently pipetting buffer onto the monolayer. The adherent cells were washed with PBS and then lysed. This rorl~ ~t ~ on is not mitotic and is PL~ ' inZlntly in G2 (36).
Asynchronous ~rr~nl~nt i A 1 1 y growing cells were grown in methionine and cysteine free media c~nti2~n~n~
29~ dialyzed serum for one hour followed by two hours in the same media cr~nt~nlng 100 llCi/ml media of 35S
Translabel ~ICN Biomedicals, Irvlne, CA). [32p]-WO 95/21917 2 I g ~ 2 ~ 5 orthnrilnsrh;lte (ICN Bi~ r~l s) labelling was performed for 3 hours following a one hour pro~ nrllh~t; f~n in phosphate rll~f~r~nt media.
Ratl cells were growth arrested by rinsing the plates with PBS, followed by rinsing in media rnnt~n~nr 0.1% fetal calf serum. The cells were grown in the low serum media fo~ 48 hours to obtain a riuiescent population. Labelling with 35S-Translabel was performed as described above, with the exception that the dialyzed serum concentration was r~ntA~ned at 0.1% and the label was incorporated over one hour . For the expnn~nt ~ ~1 1 y growing population of Ratl cells, the dialyzed serum concentration was r~lnt~n~d at 2% during the course of the labelling.
For flow cytometry analysis, 1 x l06 cells were washed in PBS and fixed in 70% ethanol, 2.0% Triton X-l00 for one hour. Fixed cells were washed in PBS and stained in a solution of 50 ~Lg/ml propidium iodide (PI) and 20 ~Lg/ml RNAse A. The cells were analysed for DNA
content (fl-lnr~Crf~nt intensity) and cell size (forward scatter) using the FACScan (Becton Dickinson, Mountain View, CA).
Tr~n~fection of CHOd- cells A 1. 8 kb cDNA obtained from the newborn rat spleen library was cloned into the Bam H1 site of the pMT0l0/A+ mammalian expression vector ~9). The cDNA was inserted downstream of the metallothionein promoter in both the sense (PMTp55s) and antisense (PMTp55as) orientations. These plasmids, as well as vector alone as control, were transfected into cells using Lipofectin (BRL Life Technologies) following the suggested protocol. Initial selection by Geneticin at 400 ~lg/ml in media without hypoxanthine was followed by stepwise ~ W09S/21917 283~ P~
amplification by methotrexate to a final concentration of 2 llM. Growth curves were performed in media rrnt~1n~ng 0.05mM zinc to induce the metallothionein promoter .

Ant~hndy Prel?~ration A p55CDC cDNA clone lacking the first 10 codons was inserted into the EcoR1 site of the pGEX-3X
vector (pll~rr-r~ ~ GST gene fusion system) . Competent 10 XL-l cells (Stratagene) were transformed and a colony harboring the r~ n~nt plasmid was isolated.
Cultures were induced with lsopropyl ~-D-thiogalactopyranoside (final concentration 0.1 m~) for growth of the fusion protein. A 76 kDa insoluble fusion 15 protein was obtained which stayed with the pellet following sonication and solubilization with 1% Triton CF-54. The pellet obtained from lysed and sonicated cells was washed twice with PBS containing 1% Triton CF-54 and the resulting pellet PXtr~rt~d with 10M urea.
20 All attempts to extract the fusion protein in anything short of lOM urea failed. The urea extract was dialyzed overnight against PBS and the resulting suspension stored in aliquots at -80-C until further pur~f~c~t~on by SDS-PAGE electrophoresis. The pellets were 25 resuspended in SDS sample buffer and separated in 10%
SDS gels. The region between the visible standard markers of 55 kDa and 80 kDa was excised out and the protein recovered by electroelution ~Biorad Model 422 ElectroEluter). This preparation was mixed with 30 Freund's complete ad~uvant and used for 1 ~7~tinn of rabbits. A booster in~ection was given after four weeks using Freund's incomplete adjuvant. Animals were bled 10-14 days following a booster in~ection. To obtain an affinity column for purification of antiserum, the crude 35 insoluble fusion protein pellet was resuspended ln coupling buffer (O.lM NaHCO3 pH8.3, 0.5M NaCl, 0.5% SDS) Wo 95/21917 ~ ~ g ~ ~ S 4 and coupled to cyanogen bromide-activated Sepharose, according to the manufacturer's instructions ~Ph;~rr~
Piscataway, NJ). A coupling efficiency of approximately 0 . 4 mg protein/ml gel was achieved. The antiserum was first absorbed against an unrelated insoluble fuslon protein to remove any antibodies reactive against glutathione S-transferase or c~nt~m1n~ting E. col~
proteins. This partially purified antiserum was applied to the affinity column. The column was washed with 5x column volumes of PBS and the affinity purified antibodies were eluted with 3M sodium thiocyanate.
Pooled antibody fractions were 1 ~ t~l y dialyzed against PBS and stored at -80 C. The flowthrough fraction from this column was used as ~_ _tP
antiserum.
o~recil~itations And Protein E~; n~ qe Acsays In vitro translation was performed using a nuclease treated rabbit reticulocyte lysate ~Promega, Madison, WI) and [3H]-leucine ~Amersham TRK683). The mRNA template was produced by using the Stratagene in vitro transcription kit and the p55CDC cDNA subcloned into the Bluescript vector as substrate. Cell lysates were prepared as described (59) after rinsing the plates twice with PBS. Cells were lysed in modified radioimmunoprecipitation assay (RIPA) buffer with additional proteases ~150 mM NaCl, 1.0% NP-40, 1.0%
sodium deoxycholate, 0.1% SDS, 2mM EDTA, 6mM Na2HPO4, 4mM NaH2P04 50mM NaF, 200 llM Na3VO4, 20 ~Lg/ml aprotinin, 1 ~Lg/ml leupeptin, 10 llg/ml soybean trypsin inhibitor and 50 ~Lg/ml phenylmethyl sulfonyl fluoride). ~ll protease inhibitors were purchased from Sigma. Protein concentrations were estimated uslng the P~ir;n~h~n;n;c acid reagent ~Pierce) . For 250 llg lysate in a final volume of 700 ~11 RIPA buffer, we used 7 ~11 of affinity purified p55QC antibodies ~140 ~Lg protein/ml) or 12 ~Ll W095121917 21 ~ r~ s~

of p55CDC competed antiserum (700 ,ug protein/ml) which gave an equivalent level of immunoglobulin for both preparations. Immunoprecipitation of p34cdc2 complexes was done using 10 111 of the p34cdc2 mouse monoclonal 5 antibody 17 (Santa Cruz Biotechnology, Santa Cruz, CA).
Other antibodies used in this study were Rb (lF8), a mouse monoclonal IgG against a Rb-B galactoside fusion protein (Santa Cruz Biotechnology) and Rb (Ab-1), another monoclonal antibody against retinoblastoma protein 10 (Oncogene Science, Uniondale, NY). The immune complexes were routinely; nrllh~ted overnight on ice and col 1 er next morning with 30 111 of a 5096 slurry of Protein G-Sepharose (ph~r~r~). The washed pellets were assayed for histone Hl kinase activity as described in (59).
15 All reactions were performed for 30 minutes at 30 .
Assays were also performed using a variety of kinase substrates at the indicated concentrations, using the same assay conditions. Histone H1 was purchased from Boehringer M~nnh~;m while myelin basic protein (MBP), i~-20 casein and a- casein were all purchased from Sigma. The reaction products were r~uantitated by excising the stained bands irom the dried gel and counting.

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* * *
While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is ~ n~n~lPf~ that the ~rr~n~L8~ claims cover all such e~Luivalent variationS
which come within the scope of the invention as claimed.

WO 95/21917 ~; t ~ ~ 2 ~ ~ P~

SEQUENCE LISTING
(1) GENERAL INFORMATION:
~i) APPLICANT: Wp;n~tp~nl Jaaminder ~ii) TITLE OF INVENT}ON: Novel ~ammalian Cell Cycle Protein ~iii) NUMBER OF SEQUENCES: 4 ~iv) ~ ADDRESS:
AI ADDRESSEE: Amgen Inc./Patent OrPr~;nn~/RBW
l3l STREET: 1840 Dehavilland Drive I C I CITY: Thou3and Oak3 IDI STATE: r7/llfr~rn~A
E I COUNTRY: USA
~F) ZIP: 91320--1789 ~v) COMPUTER READA~3LE FORM:
(A) MEDIU~ TYPE: Floppy di3k ~B) COMPUTER: IBM PC 71~;hlP
~C) OPERATING SYSTEM: PC--DOS/MS--DOS
~D) soFTnARE: PatentIn Relea3e #1.0, Ver3ion #1.25 ~vi) CURRENT APPLICATION DATA:
~A) APPLICATION NU~IBER:
~B) FILING DATE:
~C) CLASSIFICATION:
~2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE rP~p~
(A) LENGTH: 1767 ba3e pair3 (B) TYPE: nucleic acid ( C ) S TP ~ r ---c c unknown (D) TOPOLOGY: unknown (ii) ~OLECULE TYPE: cDNA
ix ) FEATURE:
~A) NAME/}~EY: CDS
(B) LOCATION: 193..1692 (~ci) SEaUENCE DESCRIPTION: SEQ ID NO:1:
bb~ C.L TAGCCGGTCA rAAAAr.AAr~ CATTCGGCAC TTCTACAGAC rjrArTrA~r.~ 60 GTCAGGGATT 1~ 7bb AGAGGTTTAC r~ArArrTr,r l~bl~ bL~ CGAACTGTGG 120 rAr~rArAr.r CCAGGAGTCC TGCGAGGTCC TGAGTTTGGT CGCCTCTCAC f (`~ 180 rr.TArArrrr. CC ATG GCG CAG TTC GTG TTC GAG AGC GAT TTG CAT TCA 228 ~et Ala Gln Phe Val Phe Glu Ser Aap Leu Hi3 Ser WO95/21917 2 1~ 4 r~

Leu Leu Gln Leu Asp Ala Pro Ile Pro A8n Ala Pro Ile Ala Arg Trp QG CGC AaA GQ AAA GAA GCC AQ GGC CQ GCC CCC TCG CCT ATG CGG 324 Gln Arg Lys Ald Lys Glu Ald Thr Gly Pro Ala Pro Ser Pro Xet Arg Ald Ala Asn Arg Ser His S~r Ala Gly Arg Thr Pro Gly Arg Thr Pr Gly Lys Ser Asn Ser Lya Val Gln Thr Thr Pro Ser Lys Pro Gly Gly Glu Arg Tyr Ile Pro Gln Arg Ser Ala Ser Gln Met Glu Val Ala Ser Phe Leu Leu Ser Lya Glu Aan Gln Pro Glu Aap Gly Gly Thr Pro Thr Lys Lya Glu His Gln Lys Ala Trp Ala Arg A~n Leu Asn Gly Phe A~

GTG GAG GAA GCC AAG ATC CTC AGG CTC AGT GGA AaA CCT QG AAT GCC 612 Val Glu Glu Ala Lys Ile Leu Arg Leu Ser Gly Lys Pro Gln Asn Ala CQ GAA GGC TAC QG AAC AGA TTG AaA GTA CTC TAC AGC QG AAA GCC 660 Pro Glu Gly Tyr Gln Aan Arg Leu Lya Val Leu Tyr Ser Gln Lys Ala Thr Pro Gly Ser Ser Arg Ly3 Ala Cys Arg Tyr Ile Pro Ser Leu Pro A~p Arg Ile Leu Asp Ala Pro Glu Ile Arg Asn Asp Tyr Tyr Leu Asn Leu Val A p Trp Ser Ser Gly A:~n Val Leu Ala Val Ala Leu Asp A~n Ser Val Tyr Leu Trp Asn Ala Gly Ser Gly Asp Ile Leu Gln Leu Leu QA ATG GAG QG CCT GGG GAC TAC ATA TCA TCC GTG GCC TGG ATC AaA 900 Gln Met Glu Gln Pro Gly Asp Tyr Ile Ser Ser Val Ald Trp Ile Lys -WO 95/21917 ~ 4 PCT/US9~/01806 Glu Gly Aan Tyr Leu Ala Val Gly Thr Ser A9n Ala Glu Val Gln Leu Trp Asp Val Gln Gln Gln Lys Arg Leu Arg Asn ~et Thr Ser His Ser Ala Arg Val Ser Ser Leu Ser Trp Asn Ser Tyr Ile Leu Ser Ser Gly Ser Arg Ser Gly His Ile His His Hi~ Asp Val Arg Val Ala Glu Hia His Val Ala Thr Leu Ser Gly His Ser Gln Glu Val Cys Gly Leu Arg Trp Ala Pro Asp Gly Arg ~is Leu Ala Ser Gly Gly Asn Asp Asn Ile Val Asn Val Trp Pro Ser Gly Pro Gly Glu Ser Gly Trp Val Pro Leu Gln Thr Phe Thr Gln Hia Gln Gly Ala Val Lys Ala Val Ala Trp Cys Pro Trp Gln Ser Asn Ile Leu Ala Thr Gly Gly Gly Thr Ser Asp Arg His Ile Arg Ile Trp Asn Val Cy9 Ser Gly Ala Cya Leu Ser Ala Val Asp Val His Ser Gln Val Cya Ser Ile Leu Trp Ser Pro Hi3 Tyr Lys Glu Leu Ile Ser Gly Hi3 Gly Phe Ala Gln A~n Gln Leu Val Ile Trp AAG TAC CCA ACC ATG GCC A~G GTG GCA GAG CTC AAA GGT CAC ACA GCC 1524 Lys Tyr Pro Thr ~et Ala Lys Val Ala Glu Leu Lya Gly His Thr Ala Arg Val Leu Ser Leu Thr Mct Ser Pro Asp Gly Ala Thr Val Ala Ser WO 95121917 ~ ~ ~ 3 ~ ~ ~ r~ s - ~

Al2 Ala Ala Aap Glu Thr Leu Arg Leu Trp Arg Cya Phe Glu Leu A~p Pro Ala Leu Arg Arg Glu Arg Glu Ly~ Ala Ser Thr Ser Lya Ser Ser CTC ATC CAC CAA GGC ATC CGG TGAAAGACAA C~.~,LLL~.LLL L~i -,LL~,~L~ 1719 Leu Ile His Gln Gly Ile Arg ATTTTGTTGT TGTTTATTTT TTTCTAATAA AGTTC~LTATC TTCCTTTC 1767 (2) INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE rl~A~Af~.~Tq~rTrc ~A) LENGTH: 499 amino acida ~) TYPE: amino ~cid ~D) TOPOLOGY: llnear ~ii) MOLECULE TYPE: proteln (xi) SED~ENCE DESCRIPTION: S~Q ID NO:2:
et Ala Gln Phe Val Phe Glu Ser Aap Leu Hia Ser Leu Leu Gln Leu ap Ala Pro Ile Pro Aan Ala Pro Ile Ala Arg Trp Gln Arg Ly3 Ala ya Glu Ala Thr Gly Pro ~la Pro Ser Pro Met Ary Ala A12 A3n Arg Ser Hi~ Ser Ala Gly Arg Thr Pro Gly Arg Thr Pro Gly Lya Ser Aan Ser Lya Val Gln Thr Thr Pro Ser Ly3 Pro Gly Gly Glu Arg Tyr Ile ro Gln Arg Ser Ala Ser Gln Met Glu Val Ala Ser Phe Leu Leu Ser y:s Glu A3n Gln Pro Glu A3p Gly Gly Thr Pro Thr Ly~ Ly3 Glu His ln Ly3 Ala Trp Ala Arg A3n Leu Aan Gly Phe A3p Val Glu Glu Ala Ly3 Ile Leu Arg Leu Ser Gly Ly3 Pro Gln Asn Ala Pro Glu Gly Tyr Gln Aan Arg Leu Lya Val Leu T~r Ser Gln Ly3 Ala Thr Pro Gly Ser Ser Arg Lya Ala Cya Arg Tyr Ile Pro Ser Leu Pro Aap Arg Ile Leu Wo9s/2l9l7 ~ 5~ ,IIL ~ ' sp Ala Pro Glu Ile Arg Asn Asp Tyr Tyr Leu Asn Leu Val ASp Trp Ser Ser Gly Asn Val Leu Ala Val Ala Leu Asp Asn Ser Val Tyr Leu Trp Asn Ala Gly Ser Gly Asp }le Leu Gln Leu Leu Gln Met Glu Gln Pro Gly ASp Tyr Ile Ser Ser Val Ala Trp Ile Lys Glu Gly Asn Tyr eu Al2 Val Gly Thr Ser Asn Ala Glu Val Gln Leu Trp Asp Val Gln ln Gln Lys Arg Leu Arg Asn Met Thr Ser His Ser Ala Arg Val Ser Ser Leu Ser Trp Asn Ser Tyr Ile Leu Ser Ser Gly Ser Arg Ser Gly Hi~ Ile Hi~ Hia His Asp Val Arg Val Ala Glu His His Val Ala Thr Leu Ser Gly His Ser Gln Glu Val Cys Gly Leu Arg Trp Ala Pro A~-p ly Arg His Leu Ala Ser Gly Gly Asn Asp Asn Ile Val Asn Val T

ro Ser Gly Pro Gly Glu Ser Gly Trp Val Pro Leu Gln Thr Phe Thr Gln His Gln Gly Ala Val Lys Ala Val Ala Trp Cys Pro Trp Gln Ser Asn Ile Leu Ala Thr Gly Gly Gly Thr Ser Asp Arg His Ile Arg Ile Trp Asn Val Cys Ser Gly Ala Cys Leu Ser Ala Val Asp Val His Ser ln Val Cys Ser Ile Leu Trp Ser Pro His Tyr Lys Glu Leu Ile Ser 40s 410 415 ly His Gly Phe Ala Gln Asn Gln Leu Val Ile Trp Lys Tyr Pro Thr et Ala Ly~ Val Ala Glu Leu Lys Gly His Thr Ala Arg Val Leu Ser - 435 440 44s Leu Thr Met Ser Pro Asp Gly Ala Thr Val Ala Ser Ala Ala Ala Asp Glu Thr Leu Arg Leu Trp Arg Cys Phe Glu Leu Asp Pro Ala Leu Arg WO 95/2191? 2 18 3 ~ ~ 4 .. . , .. ~

Arg Glu Arg Glu Lya Ala Ser Thr Ser Ly3 Ser Ser Leu Ile Hi3 Gln Gly Ile Arg ~2) INFORI~ATION FOR SEQ ID NO:3:
(i) SEQUENCE rRAl~DrTRRT.cTIcs (A) LENGTH: 1700 baJe pairs (3) TYPE: nucleic ~cid (C) STllD~ n~Ecs: unknown (D) TOPOLOGY: unknown (ii) ~OLECULE TYPE: cDNA
FEATURE:
(A) NA~E/REY: CDS
(E) LOCATION: 117..1616 (Yi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GTCGACCCAC .,~ . r~TA~rcrArr Cf TaTTDAD(: CCGGTCGGDA ~,L~ I.A~=A 60 rfirrDrr~r~ Tf rf TDrfirD rrDDrTrrAA GGACCCCTCC f ~ _17- ' GCTCCC 116 l~et Ala Gln Phe Ala Phe Glu Ser A9p Leu Hia Ser Leu Leu Gln Leu 5 10 lS
GAT GCA CCC ATC CCC AAT GCA CCC CCT GCG CGC TGG CAG CGC Af~A GCC 212 Asp Ala Pro Ile Pro Aan Ala Pro Pro Ala Arg Trp Gln Arg Lys Ala Lya Glu Ala Ala Gly Pro Ala Pro Ser Pro ~let Arg Ala Ala Asn Arg Ser His Ser Ala Gly Arg Thr Pro Gly Arg Thr Pro Gly Lys Ser Ser 50 55 ÇO
TCC AAG GTT CAG ACC ACT CCT AGC AD,A CCT GGC GGT GAC CGC TAT ATC 356 Ser Lys Val Gln Thr Thr Pro Ser Ly3 Pro Gly Gly A3p Arg Tyr Ile CCC CAT CGC AGT GCT G r~AG ATG GAG GTG GCC AGC TTC CTC CTG AGC 4 0 4 Pro Hi~ Arg Ser Ala Ala Gln ~et Glu Val Ala Ser Phe Leu Leu Ser Lys Glu Asn Gln Ser Glu Asn Ser Gln Thr Pro Thr Lys Ly3 Glu Hi~

WO 95121917 ~ 5 ~ PCTIUS9~101806 CAG AAA GCC TGG GCT TTG AAC CTG AAC GGT~TTT GAT GTA GAG GAA GCC 500 Gln Lys Ala Trp Ala Leu A5n Leu Asn Gly Phe Asp Val Glu Glu Ala Lys Ile Leu Arg Leu Ser Gly Lys Pro Gln Asn Ala Pro Glu Gly Tyr CAG AAC AGA CTG AhA GTA CTC TAC AGC CAA AAG GCC ACT CCT GGC TCC 596 Gln Asn Arg Leu Lys Val Leu Tyr Ser Gln Lya Ala Thr Pro Gly Ser Ser Arg Lys Thr Cys Arg Tyr Ile Pro Ser Leu Pro Asp Arg Ile Leu Asp Ala Pro Glu Ile Arg Asn Asp Tyr Tyr Leu Asn Leu Val Asp Trp Ser Ser Gly Asn Val Leu Ala Val Ala Leu Asp Asn Ser Val Tyr Leu Trp Ser Ala Ser Ser Gly Asp Ile Leu Gln Leu Leu Gln Met Glu Gln Pro Gly Glu Tyr Ile Ser Ser Val Ala Trp Ile Lys Glu Gly Asn Tyr Leu Ala Val Gly Thr Ser Ser Ala Glu Val Gln Leu Trp Asp Val Gln CAG CAG A~A CGG CTT CGA AAT ATG ACC AGT CAC TCT GCC CGA GTG GGC 932 Gln Gln Lys Arg Leu Arg Asn ~Set Thr Ser His Ser Ala Arg Val Gly Ser Leu Ser Trp A3n Ser Tyr Ile Leu Ser Ser Gly Ser Arg Ser Gly lli9 Ile His !~ia ~ia Asp Val Arg Val Ala Glu ~is l~is Val Ala Thr Leu Ser Gly His Ser Gln Glu Val Cys Gly Leu Arg Trp Ala Pro Asp Gly Arg l~i9 Leu Ala Ser Gly Gly Asn Asp Asn Leu Val Asn Val Trp WO 95121917 ~ 5 4 r Pro Ser Ala Pro Gly Glu Gly Gly Trp Val Pro Leu Gln Thr Phe Thr Gln Hi~ Gln Gly Ala Val Lys Ala Val Ala Trp Cya Pro Trp Gln Ser Aan Val Leu Ala Thr Gly Gly Gly Thr Ser A~p Arg Hi3 Ile Arg Ile Trp Aan V21 Cya Ser Gly Ala Cy~ Leu Ser Ala Val Aap Ala Hi~ Ser 385 390 395 ~ 400 Gln Val Cys Ser Ile Leu Trp Ser Pro Hia Tyr Lys Glu Leu Ile Ser Gly His Gly Phe Ala Gln A~n Gln Leu Val Ile Trp Lys Tyr Pro Thr ATG GCC AAG GTG GCT GAA CTC AaA GGT CAC ACA TCC CGG GTC CTG AGT 1460 Met Ala Lys Val Ala Glu Leu Lys Gly Hia Thr Ser Arg Val Leu Ser Leu Thr Met Ser Pro Asp Gly Ala Thr Val Ala Ser Ala Ala Ala A~p Glu Thr Leu Arg Leu Trp Arg Cy5 Phe Glu Leu Aap Pro Ala Arg Arg Arg Glu Arg Glu Lys Ala Ser Ala Ala Lys Ser Ser Leu Ile Hia Gln GGC ATC CGC l~r.~ArArr~A CCCATCACCT CAGTTGTTTT TTATTTTTCT 1653 Gly Ile Arg AATAAAGTCA l~:.L~_L~ ,LL CATGTTTTTT TTTTTAaAAA AAaAAAA 1700 (2) INFOR~IATION FOR SEQ ID NO:4:
i ) S EQUENCE r 1:1 A R A r 'I' R T~ T c T I CS:
(A) LENGTI~: 499 amino acida (B) TYPE: Dmino acid (D) TOPOLOGY: linear (ii) MOLECULE T'LPE: protein WO95~/21917 2I$32~4 P~ S -(xi~ SEQUENCE L1~eh~ N: SEO ID NO:4:
et Ala Gln Phe Ala Phe Glu Ser ABp Leu ~is Ser Leu Leu Gln Leu sp Ala Pro Ile Pro A3n Ala Pro Pro Ala Arg Trp Gln Arg Lya Ala ys Glu Ala Ala Gly Pro Ala Pro Ser Pro ~let Arg Ala Ala Asn Arg Ser Nis Ser Ala Gly Arg q'hr Pro Gly Arg Thr Pro Gly Lys Ser Ser Ser Lya Val Gln Thr Thr Pro Ser Lya Pro Gly Gly Asp Arg Tyr Ile ro His Arg Ser Ala Ala Gln ~let Glu Val Ala Ser Phe Leu Leu Ser ys Glu Asn Gln Ser Glu Asn Ser Gln Thr Pro Thr Lys Lys Glu liis Gln Lys Ala Trp Ala Leu Asn Leu Asn Gly Phe Asp Val Glu Glu Ala Lys Ile Leu Arg Leu Ser Gly Lys Pro Gln Asn Ala Pro Glu Gly Tyr Gln Asn Arg Leu Lys Val Leu Tyr Ser Gln Lys Ala Thr Pro Gly Ser er Arg Ly3 Thr Cys Arg Tyr Ile Pro Ser Leu Pro Aap Arg Ile Leu sp Ala Pro Glu Ile Arg Asn Asp Tyr Tyr Leu Asn Leu Val Asp Trp 180 ~85 190 Ser Ser Gly Asn Val Leu Ala Val Ala Leu AYP Aan Ser Val Tyr Leu Trp Ser Ala Ser Ser Gly Asp Ile Leu Gln Leu Leu Gln ~et Glu Gln Pro Gly Glu Tyr Ile Ser Ser Val Ala Trp Ile Lys Glu Gly Asn Tyr eu Al~ V21 Gly Thr Ser Ser Ala Glu Val Gln Leu Trp Asp Val Gln ln Gln Lys Arg Leu Arg Asn ~et Thr Ser ~118 Ser Al~ Arg Val Gly Ser Leu Ser Trp Asn Ser Tyr Ile Leu Ser Ser Gly Ser Arg Ser Gly His Ile }~is His E~is Asp Val Arg Val Ala Glu }~i8 l~is Val Ala Thr WO95/21917 ~3~ r~

Leu Ser Gly Hi3 Ser Gln Glu Val Cys Gly Leu Arg Trp Ala Pro Aap Gly Arg Hi~ Leu Al2 Ser Gly Gly A3n Aap Aan Leu Val Aan Val Trp ro Ser Ala Pro Gly Glu Gly Gly Trp Val Pro Leu Gln Thr Phe Thr Gln Hi~ Gln Gly Ala Val Lya Ala Val Ala Trp Cya Pro Trp Gln Ser Aan Val Leu Ala Thr Gly Gly Gly Thr Ser Aap Arg Hia Ile Arg Ile Trp Aan Val Cys Ser Gly Ala Cya Leu Ser Ala Val Aap Ala Hia Ser Gln Val Cya Ser Ile Leu Trp Ser Pro Hia Tyr Lya Glu Leu Ile Ser ly Hia Gly Phe Ala Gln Aan Gln Leu V~l Ile Trp Lya Tyr Pro Thr ~et Ala Lys Val Ala Glu Leu Lya Gly HiY Thr Ser Arg Val Leu Ser Leu Thr Met Ser Pro A~p Gly Ala Thr Val Ala Ser Ala Ala Ala As G1U Thr Leu Ary Leu Trp Arg Cy5 Phe Glu Leu A8p Pro Ala Arg A

Arg Glu Arg Glu Lya Ala Ser Ala Ala Ly~ Ser Ser Leu Ile H' Gly Ile Arg

Claims (23)

WHAT IS CLAIMED IS:

1. An isolated DNA encoding a biologically active p55CDC polypeptide selected from the group consisting of:
a) DNA having a nucleotide sequence encoding the amino acid sequence of SEQ. ID. NO: 2;
b) DNA having a nucleotide sequence encoding the amino acid sequence of SEQ. ID. NO: 4; and c) DNA having a nucleotide sequence which hybridizes with the DNA of (a) or (b), or with a fragment thereof, wherein the hybridizing DNA encodes a polypeptide having the biological activity of p55CDC.

2. An isolated DNA of Claim 1 which is cDNA, genomic DNA or synthetic DNA.

3. An isolated DNA of Claim 1 which includes one or more codons preferred for expression in E. coli host cells.

4. A biologically functional plasmid or viral DNA vector including DNA of Claim 1.

5. A procaryotic or eucaryotic host cell stably transformed or transfected with a DNA vector of Claim 4.

6. An isolated p55CDC polypeptide.

7. A polypeptide of Claim 6 having the amino acid sequence of SEQ. ID. NO: 4.

8. A polypeptide of Claim 6 characterized by being the product of procaryotic or eucaryotic expression of an exogenous DNA sequence.

9. A polypeptide having an amino acid sequence encoded by the DNA of Claim 1.

10. A polypeptide according to Claim 9 having the ability of forming a complex having cell-cycle dependent kinase activity.

11. A method of producing a p55CDC
polypeptide comprising culturing the host cell of Claim 5 to allow the host cell to express the p55CDC
polypeptide.

12. An antibody specifically binding a polypeptide encoded by the DNA of Claim 1.

13. An antibody of Claim 12 which is a monoclonal antibody.

14. A complex of two or more proteins wherein one of the proteins is p55CDC and wherein the complex has cell cycle dependent kinase activity.

15. A method of modulating cell division comprising introducing into a cell a compound which modulates the cell cycle-dependent kinase activity of the complex of Claim 14.

16. A method according to Claim 15 wherein the compound is selected from the group consisting of:
a) compounds which increase or decrease the levels of p55CDC synthesis;
b) compounds which interfere with the formation of a p55CDC complex having cell cycle dependent kinase activity; and c) compounds which promote the formation of a p55CDC complex or stabilize the complex.

17. A method according to Claim 16 wherein the compounds are nucleic acid molecules, polypeptides, peptides, antibodies, carbohydrates and organic molecules.

18. A method according to Claim 15 wherein cell division is inhibited by introducing into a cell a compound in an amount sufficient to inhibit the cell cycle-dependent kinase activity.

19. A method according to Claim 18 wherein the compound is selected from the group consisting of:
a) compounds which decrease the levels of p55CDC synthesis; and b) compounds which interfere with the formation of a p55CDC complex having cell cycle dependent kinase activity.

20. A method according to Claim 18 wherein the cell is a tumor cell.

21. A method of chemotherapy comprising treating a mammal with a compound in an amount sufficient to inhibit the kinase activity of the complex of Claim 14 in a pharmaceutically effective adjuvant.

22. A method for detecting the level of p55CDC in biological fluids comprising the steps of incubating an antibody specific for p55CDC with said fluid under conditions suitable for forming a complex between the antibody and p55CDC and detecting the presence of the antibody-p55CDC complex.

23. A method for detecting the level of a complex according to Claim 14 in biological fluids comprising the steps of incubating an antibody specific for p55CDC with said fluid under conditions suitable for antibody binding to p55CDC and detecting the presence of antibody bound to the complex.