Biochimica et Biophysica Acta, 1132 (1992) 35-42 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

35

BBAEXP 92399

Isolation and characterization of a cdc2 c D N A from Dictyostelium discoideum Christine Michaelis a,c and G e r a l d W e e k s a,b Departments of ~ Microbiology and b Medical Genetics, Uniuersity of British Columbia, Vancouuer (Canada) and '" Institut fiir Biologie lIl der Albert-Ludwigs Uni~,ersitdt Freiburg, Freiburg (Germany) (Received 23 January 1992)

Key words: Differentiation; Ceil cycle; Protein kinase

A cdc2 homologous sequence was amplified from Dictyostelium discoideurn by the polymerase chain reaction and used to isolate several cDNA clones. The amino acid sequence encoded by these cDNAs exhibited approx. 60% identity to the Cdc2 proteins of other species. A cDNA containing the entire coding sequence complemented the temperature sensitive cdc28 mutant of Saccharamyces cereHsiae, although growth of the transformants was slow and limited. Southern blot analysis of restriction digests under high stringency conditions provided evidence that Dictyostelium contains a single cdc2 gene, although at lower stringency multiple fragments were detected, suggesting the existence of a cdc2 gene family. Northern blot analysis of RNA from different stages of Dictyostelium development showed that cdc2 mRNA levels increased during aggregation and then decreased to low levels by the pseudoplasmodial stage of development. By contrast, cdc2 mRNA levels remained relatively constant as cells passed from exponential growth to the stationary phase. Introduction In response to starvation Dictyostelium discoideum amoebae undergo a simple differentiation process that ultimately leads to the formation of either stalk or spore cells. Initially, the individual starving cells aggregate in response to pulses of cyclic A M P and eventually form a multicellular pseudoplasmodia. Within the pseudoplasmodia, prespore and prestalk cells are present in a precisely regulated spatial pattern and during the subsequent morphogenesis these cells mature into the stalk and spore cells of the final fruiting body. A variety of low molecular weight factors have been shown to influence the formation of both stalk and spore cells and these molecules have been proposed as morphogens responsible for generating and maintaining the spatial pattern. In most pattern formation models, it has been proposed that cells are directed to either a stalk or spore fate by chemical discontinuities that lead

Correspondence to: G. Weeks, Departments of Microbiology and Medical Genetics, University of British Columbia, Vancouver, BC, V6Y 1Z3, Canada. The sequence data reported in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession number M80808.

to the establishment of morphogen gradients (for review see Ref. 1). Evidence has been accumulating to indicate that cell type determination during differentiation is also influenced by the position of a cell in the cell cycle at the time of starvation [2-6]. It is possible therefore that the initial heterogeneity within the population is established by the cell cycle and that morphogenetic gradients are then responsible for the regulation of the final spatial pattern. There is some disagreement as to the precise way in which the cell cycle is connected to cell fate, but these differences may result in part from difficulties associated with cell synchronization and in part from different interpretations of the continued progression of the cell cycle during development. The underlying mechanism that determines the relationship between the two p h e n o m e n a is not understood, but may involve variations in the activities of cell cycle regulatory components. During the last few years considerable information has accumulated regarding the molecular mechanisms involved in the cell cycle of eucaryotic cells and these mechanisms appear to be conserved in organisms as diverse as yeast and humans [7]. At the center of all models of cell cycle function is the Cdc2 kinase, a protein that shares a high level of sequence identity in all species thus far studied. The activation and inactivation of Cdc2 kinase is believed to regulate the passage through the key steps of the cell cycle.

36 In order to better understand the relationship between the cell cycle and cell type determination in Dictyostelium, we have initiated analysis of the molecular constituents of the cell cycle. In this report we describe the isolation and sequencing of a cdc2 c D N A and its expression during growth and development. Materials and Methods

Growth and differentiation of D. discoideum Strain V12-M2 was grown in association with Enterobacter aerogenes on rich nutrient plates until the bacterial lawn had cleared. Cells were harvested in Bonnet's salts solution [8] and separated from residual bacteria by several centrifugations at 700 x g for 5 rain. 2- 10 ~ washed cells were set up to differentiate on 10 cm diameter non-nutrient agar plates containing Bonnet's salts and harvested into Bonner's salts buffer, at the indicated times of development. Strain Ax-2 cells were grown in shake culture in a rich nutrient media, HL-5, at 22°C and harvested at the indicated stages of growth.

D N A was sequenced by the chain termination method [12] using the M13 universal primer and primers that matched the sequence of the PCR fragment.

Isolation and sequencing of cdc2 cDNA A Agtll c D N A library, prepared from m R N A isolated from D. discoideum 8 h after starvation, and Azap libraries, prepared from m R N A from D. discoideum 0 h and 15 h after starvation, were plated and transferred onto nitrocellulose filters as described by Maniatis et al. [10]. The filters were prehybridized in 1.5 x SSPE (10), 1% SDS, 0.5% dried milk powder (Carnation) at 60°C and then probed by the addition of a 531 nucleotide PCR product (nucleotides 418 to 949, see Fig. 1) that had been labelled using the mixed oligonucleotide method [13]. Filters were washed for 30 rain at 65°C with 2 x SSC [10], 0.1% SDS followed by a second wash for 30 rain at 65°C with 1 x SSC, 0.1% SDS and then exposed to X-ray films. Five positive clones were isolated from the 8 h library and one positive clone was isolated from the 15 h library. D N A was isolated from liquid lysates of the positive clones and subcloned into pTZ18U for sequencing by the chain termination method [12].

Isolation of DNA and RNA Nuclei were isolated from vegetative cells of strain V12-M2 as described by Cocucci and Sussman [9] and genomic D N A extracted [10]. Total R N A was extracted with an acid guanidinium thiocyanate mixture [11].

Polymerase chain reaction (PCR) amplification of the cdc2 gene Single stranded c D N A was made by a primer extension reaction using 0.1 p~g of d T T - I primer (5'-TCT CCG A A T T C T A G A T T T T T T T T T T T T T T T - 3 ' ) and 1 p.g of total vegetative cell R N A as template. The extension reaction was carried out in 50 mM Tris-C1 (pH 8.4), 50 mM KC1, 8 mM MgC12, 2.5 mM DTT, 0.8 mM of each of dATP, dCTP, dGTP, dTTP and 200 U of M-MLV reverse transcriptase. The PCR reaction was carried out using the c D N A as template, dTT-2 primer (5'-TCT CCG A A T T C T A G A T T T T T T - 3 ' ) and the primer 5 ' - G G T G A A G G T ACI T A T / C GG3', representing the consensus amino acid sequence G E G T Y G . The reaction contained 120 ng of each of the two primers, 200 # M of each of dATP, dCTP, dGTP, dTFP, 0.025 U of Taq polymerase, 50 mM Tris-Cl (pH 8.0), 1.5 mM MgC12, 0.05% Tween 20, 0.05% NP40, in a final volume of 50 p.l. After an initial cycle of denaturation at 94°C for 1 rain, annealing at 50°C for 2 rain and extension at 72°C for 3.5 min, 30 additional cycles were carried out at 94°C for 15 s, 50°C for 40 s and 72°C for 90 s using an Ericomp thermocycler. The PCR products of the predicted length were isolated by agarose electrophoresis, purified and cloned into pTZ18U. Single stranded plasmid

Southern and Northern blot analysis For Southern blots, 5 # g of genomic D N A was digested with several restriction enzymes, size fractionated on a 0.8% agarose gel and transferred to nylon membranes. Membranes were hybridized as described above using the 531 nucleotide PCR product as probe and then washed for 30 rain at 45°C using 2 × SSC, 0.1% SDS to provide low stringency conditions. After exposure to X-ray film, the filters were rewashed for 30 rain at 65°C, 0.1 x SSC, 0.1% SDS followed by 30 rain at 65°C with 0.1 x SSC, 0.1% SDS to provide high stringency conditions and re-exposed to X-ray film. For Northern blots, R N A samples were denatured in 50% formamide, 6% formaldehyde, 40 mM 3(Nmorpholino)propane sulfonic acid (pH 7.0), 10 mM sodium acetate and 1 mM E D T A (pH 8.0) for 10 rain at 65°C. The R N A was size fractionated on a 1.25% formaldehyde agarose gel and then transferred to nylon membranes. Blots were hybridized using the 531 nucleotide PCR product as probe as described above, washed at 60°C for 30 rain with 2 x SSC, 0.1% SDS and then rewashed at 60°C for 30 rain with 0.1 x SSC, 0.1% SDS.

Complementation of S. cereuisiae cdc28 S. cereuisiae, strain 4072-28-1; MATa, cdc28-4, his3, leu2, ura3-52, canR is temperature sensitive for growth due to a deficient cdc28 gene product. The cdc2-cl c D N A was cloned in both the foreward and reverse orientations into the YeDP 1/8-2 plasmid. This plasmid consists of a GAL10-CYC1 promoter, a P G K

37 terminator, an ampicillin resistance gene, URA3 for uracil selection and a 2/x ori for replication in yeast. The temperature sensitive mutant was transformed by electroporation with the plasmids containing the 'sense' and 'anti-sense' Dictyostelium cdc2 cDNAs and with the parental plasmid. S. ceret,isiae strain YM608; MATa, ura3-52, his3-200, ade2-101, lys2-801, trpl-901, tyrl, met, canR, was transformed with the parental plasmid as a 'wild type' control. Colonies containing the plasmids were selected on a minimal medium containing 2% glucose, but lacking uracil [14]. The transformants were grown overnight at 30°C in minimal media containing 2% glucose and then harvested by centrifugation at 2000 rpm for 5 min. The cell pellets were resuspended in minimal media containing 2% glycerol. After 3 h growth at 30°C, galactose was added to a final concentration of 2% and growth was allowed to continue for 1 h. The cells were spread on YPG agar plates [15] and incubated at 36°C. After 3 days at 36°C the plates were switched to 30°C for 1 day. Results

Isolation of a cdc2-homologous tyostelium discoideum by PCR

sequence from Dic-

A degenerate oligonucleotide that corresponds to the highly conserved Cdc2 amino acid sequence GEG-

TYG was used as one of the primers for the PCR reaction with the dTT-2 oligonucleotide as the second primer and mRNA derived cDNA as template. The reaction yielded several products including one of approx. 900 nucleotides, which is the size anticipated if the product of the Dictyostelium cdc2 gene is the same size as the corresponding proteins from other organisms. The gel purified, approx. 900 nucleotide PCR product was cloned into pTZ18U and 34 clones were selected and partially sequenced. The derived amino acid sequence of three clones was identical and exhibited a high percentage identity to the Cdc2 proteins of other species. The derived amino acid sequence of a fourth clone (data not shown) exhibited a high percentage identity to the KIN28 gene product of S. cererisiae [16].

Isolation and characterization of cdc2 cDNA The cdc2-homologous PCR product was used to probe three cDNA libraries derived from mRNA of cells at various stages of development. Five clones were isolated from an 8 h (aggregation stage) library and all had sequences identical to that of the PCR product. One of the clones, cdc2-cl, was 1062 nucleotides in length and contained a complete coding sequence, 128 nucleotides of 5' noncoding sequence and 44 nucleotides of 3' noncoding sequence (Fig. 1). An addi-

~CAAAAAAATAAATAC~TC~TA~

38

ATTAAAAATATTA~AAAATA~AT~AAAATT~T~ATTTGATTTTTTTTTC~T~AT~GAAAAAAAAA~TATAAAATTTAGAAA

128

M E S D G G L S R Y Q K L E K L G E G T Y G K V Y K A K E K ATGGAATCAGATGGAGGTTTATCAAGATATc~AAATTAGAGAAATTAGGTGAAGGTACcTATGGT~AGTATATAAAGcAAAAGAAA~

30 218

A T G R M V A L K K I R L E D D G V P S T A L R E I S L L K GcAAcTGGTAGAATGGTTGCAcTT~A~GATTAGATTAG~GATGATGGTGTTcc~GTAc~CTTTAcG~AAATcTCTTTATTAAAA

60 308

E V P H P N V V S L F D V L H C Q N R L Y L V F E Y L D Q D G~GTACCAcATcCA~TGTTGTTAGTTTATTTGA~TAcTTCATTGTCAAAATAGAcTTTATT~GTATT~TATTTAGATC~GAT

90 398

L K K Y M D S V P A L C P Q L I K S Y L Y Q L L K G L A Y S I 2 0 TTAAAGAAATATATGGATTccGTAccAGcATTATGTccAC~TTGATAAAGAGTTAcCTTTATC~TTATTG~GGGTTTAGCATACAGT

488

H G H R I L H R D L K P Q N L L I D R Q G A L K L A D F G L I S 0 CATGGTCATAGAATTTTAcATAGAGATTTGAAACCAcAAAATcTTTT~TCGATcGTC~GGTGCAT~AAATTGGc~ATTT~GTTTA

578

A R A V S I P V R V Y T H E I V T L W Y R A P E V L L G S K GcTcGTGcAGTTAGTATTcCAGTAcGTGTTTACAcTcAcGAGATTGT~CccTT~GTATcG~CAccAGAGGTcTTAT~GGTTCAAAA

180 668

S Y S V P V D M W S V G C I F G E M L N K K P L F S G D C E 2 1 0 AGTTAcTcTGTAccAGTcGATATGTGGTCTGTTGGT~TATCTTTGG~AAA~TTAAAcAAAAAACCAT~TTTAG~G~AT~

758

I D Q I F R I F R V L G T P D D S I W P G V T K L P E Y V S 2 4 0 AT~ATcA~TCTTTAG~TcTTTAGAGTTTTAGGTACTCcAGATGATTc~TT~GCCAGGTGTCACTAAACTTCCAG~TATGTTTCA

848

T F P N W P G Q P Y N K I F P R C E P L A L D L I A K M L Q 2 7 0 AcTTTCcc~ATTGGCcAGGTck~ccATAT~TAAAATTTTccC~GATGTG~CCACT~cATTAGATTT~T~CTAAAA~CTTC~

938

Y E P S K R I S A K E A L L H P Y F G D L D T S F F * TATGAA••ATCAAAGAGAATTT•AG•TAAAGAA••A•TT•TT•A•••ATATTTTGGTGAT•TTGATAcTAGTTTTTT•TAAGAAATAGAA

296 1028

CTTATCAACATATCATCATTTAAAAAAAAAATCTA

1062

Fig. 1. Nucleotide and deduced amino acid sequence of Dictyosteliurn discoideum cdc2-cl cDNA. The cDNA sequence comprises 1062 nucleotides and includes an open reading frame that encodes a protein of 296 amino acids. The amino acids are shown in one letter code above the cDNA sequence. The star indicates the stop codon TAA.

38 tional clone, isolated from a 15 h library (pseudoplasmodial stage) also had a sequence identical to that of the PCR product. A comparison of the derived amino acid sequence with those of the Cdc2 proteins of S. cereL,isiae [17], S. pombe [18], human [19] and Zea mays [20] is shown in Fig. 2. The encoded Dictyostelium protein is 61% identical to the Cdc2 protein of S. cereti~iae, 61% identical to that of S. pornbe, 61.5%, identical to that of human and 64% identical to that of Z. mays. Several gaps had to be inserted to maximize the alignment. Most noticeably, the Dictyostelium protein had six additional amino acids in its N-terminal region relative to the proteins of S. pombe, human and maize, although only two additional amino acids relative to the S. ceredsiae protein. Despite these differences, the similarities between the Dictyostelium protein and the other Cdc2 proteins are sufficient to tentatively designate the encoding gene, cdc2.

Southern blots of genomic D N A restriction digests were probed using a PCR product. The EcoRI and HindlII digests contained a single hybridizing fragment, whereas Clal and HinclI digests contained two hybridizing fragments (Fig. 3). These results suggest the existence of a single Dictyostelium cdc2 gene, since the c D N A sequence contains single CTaI and Hincll sites and no E c o R I and Hindlll sites. At lower stringencies several additional fragments were detectable, suggesting the existence of a gene family. One of these additional fragments hybridized under high stringency conditions when the same Southern blot was probed with the KIN 28 homologous eDNA, that had been isolated as one of the original PCR products (data not shown). Since the S. cereti~iae KIN 28 gene product is highly related to the Cdc2 proteins, this result is consistent with the interpretation that the fragments that hybridize at low stringency with cdc2-cl are members of a cdc2 kinase gene family. However. the possibility

cdc2 cdc28

Dd Zm Hs Sp Sc

MESDGGLSRYQKLEKLGEGTYGKVYKA -.Q .E-V-.I ...... --D -T-I--I ...... -.N -.-V-.I ...... .S .E.AN.KR...V ......

cdc2 cdc2 cdc2 cdc2 cdc28

Dd Zm Hs Sp Sc

STALREISLLKEVP ---I ........ ---I ........ ---I ......... ---I ........

cdc2 cdc2 cdc2

Dd Zm Hs

VPA C-EF I-

cdc2 cdc28

Sp Sc

ISETGATS.D.R.VQKFT..-VN-VNFC.SR--I I-K DQP.GADIV.KFMM--C..I--C.S

cdc2 cdc2 cdc2 cdc2 cdc28

Dd Zm Hs Sp

ADFGLARAVSIPVRVYTHEIVTLWYRAPEVLLGSKSYSVPVDMWSVGCIFGEM ........ FG .... TF...V ......... I...ARQ..T...V ....... ........ FG.-I ...... V .............. AR..T---I..I-T--A.L ....... SFGV-L-N ................... RH..TG--I .......

Sc

G .......

cdc2 cdc2 cdc2 cdc2 cdc28

Dd Zm Hs Sp Sc

LNKKPLFSGDCEIDQIFRIFRVLGTPDDSIWPGVTKLPEYVSTFPNWPGQPYN V.Q .... P..S...EL.K.--I .... NEQS .... SC..DFKTA..R.QA-DLA AT ..... H.-S .... L ..... A .... NNEV..E.ES.QD-KN-..K-KPGSLA .RRS-..P.-S---E--K-.Q ..... NEEV ..... L-QD.K .... R.KRMDLH C.R.-I .... S ...... K ........ NEA...DIVY..DFKPS--Q-RRKDLS

cdc2 cdc2 cdc2 cdc2 cdc28

Dd Zm Hs Sp Sc

KIFPRCEPLALDLIAKMLQYEPSKRISAKEALLHPYFGDLDTSFF* TVV.NLD.AG...LS.--R ....... T.RQ..E.E..K..EVVQ* SHVKNLDENG...LS---I-D-A .... G.M--N .... N...NQIKKM* .VV.NG.ED-IE--SA..V.D.AH ..... R..QQN-LR-FH* QVV.SLD.RG .... D.L.A-D-IN .... RR-AI .... QES*

cdc2 cdc2 cdc2

KEKATGRMVALKKIRLE V .... LD.TANETI ........ V.--G RH.T-.QV--M ...... V .... RH-LS..I--M ...... V .... LDLRPGQGQ-V .........

HPNVVSLFDVLHCQN MN .G.I.R-H..V-SEK LR ...I..-Q-..MQDS NDENNRS-C.R-L-I..AES LKDDN I.R-Y.IV.SDAHK

RLYLVFEYLDQDLKKYMDS .I ........ L .... F--.... I--F-SM ..... L-K ...... F-.M ....... R ...... F..L...R-.EG

LCPQLIKSYLYQLLKGLAYSHGHRILHRDLKPQNLLIDRQGA AKN-T ........ I.H.V--C.S--V .............. PGQYMDSS.V ...... I.Q.IVFCHSR-V .............

FGV.L.A

..................

DDGVP QE-E--SEEE--DESE--SE-E---

............ ...............

LKL RTNA..DK-T I-KE.N NKD-N

G-Q..TG..T--I

-----

A-A..

.... A--

Fig. 2. Comparison of the amino acid sequence of Dictyostelium discoideum (Dd)Cdc2 with those of the Cdc2 homologs from Zea mays (Zm). human (Hs), Schizosaccharomyces pombe (Sp) and the Cdc28 gene product of Saccharornyces cerecisiae (Sc). The amino acid sequence of the Dictyosteliurn Cdc2 is shown in single letter code. Amino acids of the homologous gene products that are identical from other species are indicated by a dash, and those that are different are indicated by the single letter code. Gaps have been introduced where necessary to maximize the alignment.

39 that some of the fragments that hybridize at low stringency are due to repetitive D N A that is present at higher molar concentrations can not be precluded.

Complementation of a yeast cdc28 mutant T h e S. ceret,isiae C D C 28 gene product is the functional h o m o l o g of the Cdc2 proteins of other organisms. S. cerecisiae strain 4072-28-1 is t e m p e r a t u r e sensitive for growth due to a m u t a t e d C D C 28 gene and it was, therefore, transformed using the cdc2 c-1 c D N A in o r d e r to d e m o n s t r a t e that this D N A encodes a functional Cdc2 protein. T h e presumptive Dictyostelium cdc2 c D N A was cloned into the yeast vector in both the forward and reverse orientation and indep e n d e n t transformants containing each one of these constructs or the parental vector were isolated and tested for their ability to grow at the non-permissive t e m p e r a t u r e of 36°C. After 3 days of growth, only the transformants containing the cdc2 c D N A in the correct orientation formed colonies, although the colonies were very small relative to those of the wild-type con-

A 1

2

B 3

4

1

-

23.1

-

-

9.4

-

-

6.5

-

-

4.3

-

-

2.3

-

-

2.0

-

2

3

4

Fig. 4. Complementation of a Saccharomyces cerecisiae temperature sensitive mutant. S. cerecisiae, strain 4072-28-1, transformed with the plasmid YePD1/8-2 containing Dictyostelium cdc2 (bottom) or with the YePD1/8-2 plasmid alone (top) was plated on YPG agar, incubated for 3 days at 36°C and then shifted to 30°C for 1 day.

trol strain subjected to identical conditions (data not shown). Incubation for longer than 3 days did not lead to an increase in the size of the cdc2 transformant colonies. W h e n the plates were shifted to 30°C after 3 days at 36°C, however, the cdc2 transformant colonies grew to normal size, but there were still no visible colonies for the cells transformed with the parental plasmid (Fig. 4) or with the cdc2 c D N A in the reverse orientation (data not shown). These results indicate that the Dictyostelium cdc2 gene is capable of supporting the growth of a cdc28 mutant, but growth is slow and limited.

Expression of the cdc2 gene during growth and differentiation of Dictyostelium

Fig. 3. Southern blot analysis. 5 /xg of genomic DNA from D.

discoideum strain V12-M2 was digested with EcoRI (lane 1), ClaI (lane 2), HindIII (lane 3), HincII (lane 4) and the digests fractionated on a 0.8% agarose gel. The fragments were transferred to a nylon membrane and hybridized with the labeled 531 bp PCR fragment. Filters were washed under low stringency conditions (A) and high stringency conditions (B) as described under Materials and Methods and exposed to X-ray film for 12 h. Molecular sizes (kilobases) are indicated between the autoradiograms.

R N A was isolated from V12-M2 and Ax-2 cells at various stages of differentiation and size fractionated on agarose gels. T h e gels were blotted onto nylon m e m b r a n e s and hybridized with the cdc2 P C R p r o d u c t as probe. A single m R N A of approx. 1.4 kb was detected (Fig. 5). The a m o u n t of cdc2 m R N A increased significantly during the early stages of development and then declined to very low levels by the migrating pseudoplasmodial stage (16 h development). T h e a m o u n t of cdc2 m R N A in vegetative cells of V12-M2 was somewhat variable although always less than the level observed after 4 h of differentiation (data not shown). Since these cells were grown on bacteria, it is possible that the variability in the cdc2 m R N A levels

40 and remained constant as cells progressed into stationary phase (Fig. 6).

A 0

4

8

12 16 20

Discussion

1.4 ~"

B 0

4

8

12 16 2 0

1.4 ~"

Fig. 5. Northern blot analysis of cdc2 gene expression during D. discoideum development. Total RNA (20 p.g), isolated from cells of (A) V12-M2 and (B) Ax-2 at the indicated times after the onset of differentiation (h), was separated on a 1.25% formaldehyde-agarose gel, transferred to a nylon membrane and probed with the 531 bp PCR fragment of cdc2. Filters were washed under high stringency conditions as described under Materials and Methods and exposed to X-ray film. The estimated size of the transcript is indicated by the arrowhead.

in the vegetative cells are due to differences in the growth phase of the cells at the time of harvesting. To test the possibility that cdc2 mRNA levels vary with growth phase, RNA was isolated at various stages of growth of the axenic strain Ax-2 and subjected to the same Northern blot procedure. The relative level of cdc2 mRNA was constant during exponential growth

1

2

3

4

5

6

7

8

1.4 •

Fig. 6. Northern blot analysis of cdc2 mRNA from different growth stages. Total RNA (20 #.g) of D. discoideum from Ax-2 cells harvested at different stages of growth, was treated as described in the legend to Fig. 5. Cells were harvested at the following cell densities: 1.106 cells/ml (lane 1), 2.106 cells/ml (lane 2), 3-106 cells/ml (lane 3), 4.106 cells/ml (lane 4), 5-106 cells/ml (lane 5), 6.106 cells/ml (lane 6), 1.5.107 cells/ml (lane 7), harvested 18 h after the previous sample (lane 8).

Dictyostelium discoideum expresses a gene whose encoded product is approx. 60% identical to other Cdc2 proteins reaffirming the conservation of this important cell cycle gene in all organisms. Southern blot analysis at high stringency indicates that Dictyostelium contains a single cdc2 gene. At low stringency additional fragments were detected and these may represent members of a cdc2-1ike gene family, similar to those that have been described in both yeast [16,21-23] and mammalian cells [24-26]. Preliminary results (data not shown) indicate that the most prominant fragment that hybridizes at low stringency is highly conserved relative to the KIN28 gene of yeast [16]. The Dictyosteliurn cdc2 cDNA was capable of complementing a temperature sensitive cdc28 mutant of S. cerevisiae, although the transformants grew slowly and the extent of growth was limited. The slow growth characteristics of the transformants might be due to the fact that several of the Dictyostelium amino acids are different from those in the highly conserved sequences of other Cdc2 proteins. In particular, the conserved isoleucine in the PSTAIRE consensus sequence is substituted by leucine in the Dictyostelium protein and in the ATP binding domain the consensus G E G T Y G V V Y K becomes G E G T Y G K V Y K in Dictyostelium (Fig. 2). A more likely explanation is that the limited growth of the S. cerel:isiae transformants is due to inefficient transcription of the highly AT-rich Dictyostelium cdc2 cDNA [27]. However, it should also be noted, that the cdc2 genes from Drosophila [28] and Arabidopsis [15] complement but fail to restore complete wild type behaviour to cdc28 mutants of yeast, and that cdc2-1ike genes from Drosophila [28] and Xenopus [29] do not complement the defect. A functional homolog of the human cdc2 gene has recently been isolated [30,31]. This gene (cdc2) encodes a product that shares 65% identity to the human cdc2 gene product [30,31] and is capable of rescuing a cdc28 mutant of S. ceret'isiae. The Cdc2-1ike protein of Xenopus [29] is 89% identical to Cdk2, suggesting the possibility that Xenopus also encodes two cell cycle specific protein kinases. Vertebrates, therefore, appear to express two cell cycle specific protein kinases and it has been suggested that the Cdc2 kinase is important for the G2 to M phase transition and that the Cdk2 kinase is involved in the G1 to S transition. In contrast, in S. pombe and S. ceret'isiae the single Cdc2 (Cdc28) kinase is required for both G2 to M and G1 to S transitions, indicating a difference in cell cycle regulatory complexity between higher and lower eucaryotes. The available evidence indicates that Dictyostelium

41 encodes a single Cdc2 kinase that is 61.5% identical to the human Cdc2 kinase and 61.8% identical to the human Cdk2 kinase, suggesting that, like yeast, a single kinase is responsible for both G 2 / M and G 1 / S transitions. In addition, G1 phase is either very short or non-existent in Dictyostelium [3,32] and it is possible that G1 to S phase control does not occur in this organism. cdc2 mRNA levels do not decrease as Dictyostelium cells pass from the exponential growth into stationary phase. Thus, although the stationary phase cells are in G2 arrest [33] and have no apparent requirement for Cdc2 kinase activity, the cdc2 m R N A levels are maintained. In S. pombe, cdc2 gene expression does not vary during the cell cycle and does not decrease during G1 arrest [34] and it has been suggested that the important determinant in regulating Cdc2 kinase activity during the-cell cycle is protein phosphorylation. In contrast, when non-growing mammalian tissue culture cells are serum stimulated levels of cdc2 mRNA increase [35]. However, the accompanying increase in Cdc2 protein phosphorylation that occurs during serum stimulation is far more dramatic [35], again suggesting that phosphorylation is the key regulating event during the mammalian cell cycle. A decrease in cdc2 mRNA levels in quiescent cells may be of more importance in the terminally differentiated cells [35] and the finding that cdc2 gene expression is lowest in the non-proliferating cells of adult tissues of multicellular plants [20] and animals [36] is consistent with this idea. The dramatic decrease in cdc2 m R N A as Dictyostelium cells progress through the later stages of differentiation resulting in very low levels in terminally differentiated cells is therefore similar to the situation found in the non-dividing cells of adult tissues of multicellular organisms. The increase in cdc2 mRNA levels as cells pass through the aggregation phase of development is surprising. However, it has been shown that cell cycle progression occurs during differentiation [6,37,38] and the continued synthesis of Cdc2 protein might be essential for cells to pass through the cell cycle prior to aggregation. However, whether the increase in cdc2 m R N A level is due to increased gene expression or to differentially stable m R N A remains to be determined. In Dictyostelium there is accumulating evidence for a connection between the cell cycle and cell type determination during differentiation. Thus, the cell cycle state at the onset of differentiation may specify subsequent cell type determination [3,33,5,6]. Alternatively, it is possible that cells initiate differentiation only from a specific point in the cell cycle, and that the time at which cells initiate differentiation determines there subsequent cell fate [2]. The isolation of the Dictyostelium cdc2 gene will provide a new approach to distinguish between these and other possibilities.

Acknowledgments This research was supported by a grant from the National Science and Engineering Research Council of Canada. We wish to Shank Chi-Hung Siu for providing the 8 h Agtll library and Juliet Daniel the 0 h and 15 h Azap libraries. Ivan Sadowski provided S. cerevisiae strain 4072-28-1 and valuable advice on the yeast experiments, and Dennis Dixon, Patrick Rebstein and Andreas Meinke critically read the manuscript.

References 1 Weeks, G. and Gross, J.D. (1991) Biochem. Cell Biol. 69, 608-613. 2 Maeda, Y., Ohmori, T., Abe, T., Abe, F. and Amagai, A. (i989) Differentiation 41, 169-175. 3 McDonald, S.A. and Durston, A.J. (1984) J. Cell Sci. 66, 195-204. 4 Weijer, C.J., Duschl, G. and David, C.N. (1984) J. Cell Sci. 70, 133-145. 5 Gomer, R.H. and Firtel, R.A. (1987) Science 237, 758-762. 6 Sharpe, D.T. and Watts, D.J. (1985) Mol. Cell Biochem. 67, 3-9. 7 Lewin, B. (1990) Cell 61,743-752. 8 Bonner, J.T. (1947) J. Exp. Zool. 106, 1-26. 9 Cocucci, S.M. and Sussman, M. (1970) J. Cell Biol. 45, 399-407. 10 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. 11 Chomozynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 12 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467. 13 Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 14 Sherman, F. (1991) in Guide to Yeast Genetics and Molecular Biology (Guthrie, C. and Fink, G.R., eds.), Academic Press, New York. 15 Ferreira, P.C.G., Hemerly, A.S., Villaroel, R., Montag, M.V. and Inze, D. (1991) Plant Cell 3, 531-540. 16 Simon, M., Seraphin, B. and Faye, G. (1986) EMBO J. 5, 26972701. 17 Lorincz, A.T. and Reed, S.I. (1984) Nature 307, 183-185. 18 Hindley, J. and Phear, G.A. (1984) Gene 31, 129-134. 19 Lee, M.G. and Nurse, P. (1987) Nature 327, 31-35. 20 Colasanti, J., Tyers, M. and Sundaresan, V. (1991) Proc. Natl. Acad. Sci. USA 88, 3377-3381. 21 Ueseno, Y., Tanaka, K. and Toh-e, A. (1987) Nucleic Acids Res. 15, 10299-10309. 22 Courehesne, W.E., Kunisawa, R. and Thorner, J. (1989) Cell 58, 1107-1119. 23 Elion, E.A., Grisafi, P.L. and Fink, G.R. (1990) Cell 60, 649-664. 24 Hanks, S.K. (1987) Proc. Natl. Acad. Sci. USA 84, 388-392. 25 Matushushime, H., Jinno, A., Takagi, N. and Shibuya, M. (1990) Mol. Cell Biol. 10, 2261-2268. 26 Johnson, K.W. and Smith, K.A. (1991) J. Biol. Chem. 266, 34023407. 27 Romanos, M.A., Makoff, A.J., Fairweather, N.F., Beesley, K.M., Slater, D.E., Rayment, F.B., Payne, M.M. and Clare, J.J. (1991). Nucleic Acids Res. 19, 1461-1467. 28 Lehner, C.F. and O'Farrell, P.H. (1990) EMBO J. 9, 3573-3581. 29 Paris, J. LeGuellee, R., Coutourier, A., Le Guellec, K., Omilli, F., Camonis, J., MacNeill, S. and Phillipe, M. (1991) Proc. Natl. Acad. Sci. USA 88, 1039-1043. 30 Elledge, S.J. and Spottswood, M.R. (1991) EMBO J. 10, 26532659.

42 31 Tsai, L.-H., Harlow, E. and Meyerson, M. (1991) Nature 353, 174-177. 32 Weijer, C.J., Duschl, G. and David, C.N. (1984) J. Cell Sci. 70, 111-131. 33 Soil, D.R., Yarger, J. and Mirick, M. (1976) J. Cell Sci. 20, 513-523. 34 Durkacz, B., Carr, A. and Nurse, P. (1986) EMBO J. 5, 369-373.

35 Lee, M.G., Norbury, C.J., Spurr, N.K. and Nurse, P. (1988) Nature 333, 676-679. 36 Krek, W. and Nigg, E.A. (1989) EMBO J. 8, 31)71-3078. 37 Zada-Hames, I.M. and Ashworth, J.M. (1978) Dev. Biol. 63, 307-32O. 38 Durston, A.J. and Volk, F. (1978) Exp. Cell Res. 115, 455-457.

Isolation and characterization of a cdc 2 cDNA from Dictyostelium discoideum.

A cdc2 homologous sequence was amplified from Dictyostelium discoideum by the polymerase chain reaction and used to isolate several cDNA clones. The a...
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