J. Mol. Hd. (1991) 218, 543-556

A General Approach to the Isolation of Cell Cycle-regulated Genes in the Budding Yeast, Saccharomyces cerevisiae Clive Price, Kim Nasmyth and Tillman Schuster? Institute

of Molecular Pathology Dr. Bohrgasse, 7 A 1030, Wien Austria

(Received 31 October 1990; accepted 28 November

1990)

We describe a general approach to the isolation of cell cycle-dependently regulated transcripts in Saccharomyces cerevisiae. This approach is based on the physical identification of cell cycle-regulated transcripts by Northern hybridization using as probes yeast DNA isolated from an ordered S. cerevisiae genomic library. The purpose of this is twofold; first, to assess the importance of transcriptional regulation in cell cycle control; and second, to identify novel genes that may have important roles in the eukaryotic cell cycle. We report the isolation of two previously uncharacterized genes that are transcribed at points in the cell cycle to which specific transcriptional activation has not been assigned: namely, mitosis and early GI phase. It is argued that these transcripts serve as important landmarks for cell cycle events that are not readily distinguished by either morphological or cytological criteria. The cell cycle-dependent transcription of the RNRl and CLNl genes is also described and the implications for cell cycle control, in G,, are discussed with reference t’o these two genes.

1. Introduction Recently, a convincing and universal model for the control of mitosis in eukaryotic cells was put forward (for a review, see Murray & Kirschner, 1989; Murray, 1989; Nurse, 1990; Lewin, 1990). Central t,o this model is the activity of a serine/ threonine kinase known as p34cdcZ. This protein is the product of the ClX’28 gene of Saccharomyces eerevisiae and of the cdc2 gene of Schizosaccharomyces pombe. Homologues of ~34’~” have been identified in a wide variety of organisms, ranging from frogs and starfish to mammals (Dunphy et al., 1988; Gautier et al., 1988; Labbe et al., 1989b; Lee & Nurse, 1987). Moreover, these homoloas well as structurally gues are functionally conserved. ~34”~“’ is one component of maturationpromoting factor (MPFS) an activity involved in mitotic induction, first identified in studies of fro oocytes. In both S. pombe and S. cerevisiae ~34’~’ 5 is an absolute requirement for mitosis. A second component of MPF has been identified as cyclin (Draetta et aZ.. 1989; Gautier et al., 1999; Labbe et t Author t)o whom correspondence should be addressed. 1 Abbreviations used: MPF, maturation-promoting fartor; kb. IO3 bases; kbp. lo3 base-pairs.

al., 1989a; Meijer et aZ., 1989). The current model states that mitosis is initiated following complex formation between ~34”~“’ and cyclin and that t,his association is accompanied by dephosphorylation of p34cdc2, leading to activation of the protein kinase activity. In order to complete mitosis this activity must then be destroyed, and it is likely that t’his is effected by specific degradation of the cyclin moiety (Murray et al., 1989). Cyclin also becomes phosphory?ated during mitosis but as yet no functional significance can be attributed to t’his event (Pondaven et al., 1990). This model is post-translational, involving both protein modification and degradation. It is likely, however, that additional levels of control exert an important influence on the correct ordering of the discontinuous processes of the cell cycle. One hypothesis is that cell cycleregulated transcriptional activation might, play a role in governing the timing and ordering of the events of the cell cycle. Many genes are known to be regulated in a cell cycle-dependent manner at the transcriptional level; however, it is not known to what extent this regulation is important for cell cycle control. Comparison of t,he regulation of the DNA ligase genes of S. cerevisiae and S. pombe indicates that cell cycledependent expression is not necessary (White et aZ.,

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1986). The s’. cerevisiae gene, CDCS, is cell cycleregulated whereas the S. pombe gene, cdcl?‘, is not.

Extrapolation of this evidence suggests that this would be true for most DNA replication enzymes, i.e. that cell cycle-dependent transcriptional regulation is not an essential feature of the individual genes. Global constitutive expression of the DNA replication enzymes might however severely disrupt normal cell cycle control. The question remains as to whether there are cell cycle-regulated genes whose transcriptional regulation is rate limiting in the control of cell cycle progression. In order to assess the importance of periodic gene expression in the eukaryotic cell cycle we have undertaken a comprehensive search for cell cycle-dependently regulated transcripts in the yeast S. cerevisiae. A second important reason for our approach is to attempt to identify novel CDC (cell division cycle) genes. It is probable that conventional genetic analyses will have failed to identify many important genes involved in cell cycle control. This reasoning is based on two arguments. First, it is evident that it is difficult to isolate conditional mutations in many essential loci in S. cerevisiae. Genetic analysis of chromosome I of S. cerevisiae involving saturation mutagenesis with either ethylmethanesulphonate or N-methyl-N’-nitro-N-nitrosoguanidine, revealed only three essential loci. All three of these loci had been identified in separate studies (Kaback et al., 1984). These data suggest that conventional mutagenic analyses will have failed to identify many important genes. A second drawback to the conventional genetic approach is caused by the problem of redundancy. Redundancy is illustrated by the histone loci of S. cerevisiae, each of the core histone genes being present twice per haploid genome (Hereford et al., 1979; Smith & Murray, 1983). Consequently, the histone genes were never identified genetically. In order to overcome these two problems, we have chosen to use a physical approach to identify genes transcriptionally regulated in a cell cycle-dependent manner. We expect that this will enable us to identify novel genes that define functions important for normal cell cycle progression and growth, irrespective of whether or not the transcriptional regulation is important for cell cycle control. The experimental approach that we have adopted relies upon the existence of an ordered S. cerevisiae genomic library of minimal overlap, cloned in bacteriophage lambda (Olson et al., 1986). In the primary screen, individual genomic fragments are isolated from the recombinant phage and used as probes in Northern blots (Thomas, 1980) against cell cycle stage-specific RNA samples. Transcripts identified as being cell cycle-regulated in this screen are then further analysed by Northern hybridization, to establish whether or not they exhibit genuine periodicity in synchronized cultures. This paper describes the preliminary results of our analysis. We define two new Cla88e8 of cell cycleregulated transcripts, which are detectable at points in the cell cycle at which specific transcript periodi-

city has not previously been observed. We have also identified GLNl, a redundant gene that is thought to play an important role in controlling the G, to S phase transition in S. cerevisiae (Hadwiger et al., 1989). Finally, we describe the gene encoding the large subunit of ribonucleotide reductase, an essential DNA metabolism enzyme, which had not been isolated as a CDC gene by conventional genetic analyses.

2. Materials and Methods (a) yeast strains Strain

Genotype

H745

MATaIMATalpha ADE2/ade2-1 TRPljtrpl-I CANl/cunI-100 LEiJ2/leu2-3,112 HIS3/his3-11,15 URAS/uraS GAL2/ga1/2 MATa gal2 bar1 : : URA3 MATa gal2 MATa hmla HMRa HO:: URS2del a&2-1 canl-100 trpl-1 leu2-3 ura3 cdc15-2

Y1170 Y818 Y838

(b) Primary

screen

The strain Y 1170 was grown in YEPD (1 y0 yeast extract, 2% Bacto peptone, 2% glucose) medium at 30°C to an A,,, of 1.0. Cells were harvested by centrifugation, washed once with ice-cold water, centrifuged once more and immediately frozen in liquid nitrogen. RNA isolated from these cells represents the control cycling haploid sample. Cells for the G,-arrested sample were grown as above to an A,,, of 1.0, at which point alpha factor was added to a concentration of 100 rig/ml. Cells were held in the presence of alpha factor for 90 min until > 95 y0 of the cells were arrested as schmoos. Cells were then harvested as above. Cells for the G,/S transition sample were treated as for the alpha factor-arrested population to the 90-min point following addition of alpha factor. At this point cells were collected by filtration, using a 1.2 pm pore diameter, and washed with 2 vols of prewarmed YEPD. The cells were then resuspended in an equal volume of fresh pre warmed YEPD and grown for a further 50 min, at which point >90% of the cells were budding. The cells were then harvested as above. Cells for the mitotic sample were again grown to an A,,-,, of 1.0 in YEPD at 30°C. Dimethyl sulphoxide was then added to 1 O/o and the cells were allowed to continue growth for a further 2 h before nocodazole was added to a concentration of 20 &ml. The cells were maintained in the presence of nocodazole for 3 h, at which point all cells were arrested in late mitosis, and then harvested as above. The diploid strain H745 was grown to an A,,, of 30 in minimal media at 30°C. The cells were then pumped onto the centrifugal elutriator (Beckman JE-50) and allowed to equilibrate in fresh prewarmed medium at 3000 revs/min for 30 min. At this point, 8 500-ml fractions were collected, after gradual increase of the pump speed, and approximately 30% (v/v) of ice was added. These fractions were then harvested by centrifugation as above. The cells were pooled and represent the diploid G, sample; the pooled cells showed less than 5% budding. The cells remaining in the rotor were then collected and harvested as above to form the diploid control population. (c) Alpha factor synchronization. The strain Y1170 was grown to an A,,, of 1.0 at 30°C in YEPD. Alpha factor was then added to a concentration

Yeast Cell Cycle-dependent Transcription of 100 rig/ml. The cells were maintained at 30°C with aeration for 150 min, then collected by filtration, as above. and washed with an equal volume of prewarmed YEPD. The cells were then resuspended in an equal volume of fresh YEPD, additionally supplemented with 0.1 vol. of preconditioned medium. Preconditioned medium was obtained by growing strain Y818 overnight in YEPD and the cells were removed by filtration. Y818 is the parental strain to Y 1170 and is wild-type for the BAR1 locus. The product of this gene is a secreted proteaw t,hat degrades alpha factor (Manney, 1983). Samples were taken every 10 min for 3 h and harvested by centrifugation, as above; @5 ml samples were removed at the same time points for analysis by in situ immun(~fluoresCellCe. (d) edcl5 synchronization The strain Y838 was grown to an A,,, of 2.5 at room temperature and the cells were harvested by centrifugation, again at room temperature. The cells were then resuspended in 05 vol. of YEPD prewarmed to 37°C and maintained at’ this temperature with aeration for 3 h. An equal volume of YEPD at 15°C was then added and the cells were allowed to grow at room temperature for a furt,her 270 min. Samples were taken every 15 min throughout this time course and the cells collected by centrifugation as above. As for the alpha factor synchrony, ti5 ml samples of cells were removed at the same time points for it2 situ immunofluorescence analysis. (e) Elutriation The prot,ocol used for t’his experiment was similar to that described above except that cells were grown only to an A,,, of 1.5 prior to loading ont.0 the rotor, the same diploid strain H745 was used. Twelve fractions were collected, after gradually increasing the pump speed; 0.1 ml samples of each fraction were fixed in 3.7 y. formaldehyde and the numbers of budded cells were counted. Agiin the cells remaining in the rotor served as the control sample. (t] RR’A isolation and electrophoresis RNA was prepared by the hot phenol method (Domdey mRNA was isolated rt al.. 1984). Polyadenylated by poly(U)-Sepharose (Pharmacia) chromatography according to the manufacturer’s recommendations. The RNA was resolved on 1.2 y0 agarose-formaldehyde vertical gels for the primary screen and on 1% agaroseformaldehyde horizontal gels for all other analyses. The gels and RNA samples were prepared according to the method of Maniatis et al. (1982). (g) Northern Matting RNA was transferred to Genescreen (NEN) nylon membranes by the capillary method (Maniatis et al., 1982), in 10 x SSC (SSC is 0.15 M-NaCl, 0.015 M-sodium citrate. pH 7.0) without any pretreatment of the gel. The transfer was left overnight. the filters were then washed briefly in 50 mm-sodium phosphate (pH 7.2), baked at 80°C for 30 min in a vacuum oven and then the RNA was rrosslinked to the filters with ultraviolet light (Church & Gilbert. 1984). (h) Phage DNA isolation and probe preparation The recombinant phage were isolated by a modification of the method of Zabarovsky & Turina (1988). The

polyethylene glycol precipitation step was omitted and the DNA was purified by extraction with phenol after pelletting of the phage particles by centrifugation at 18,500 revs/min for 3 h in a Sorvall SS34 rotor. The Escherichia coli host strain for phage growth was C600 (Appleyard, 1954). Liquid lysates were grown in NZYDT broth (Gibco) at 37°C. The DNA was cleaved with EcoRI and Hind111 and resolved on 1 o/o low melting point agarose gels in 1 x TAE buffer (Maniatis et al.. 1982). Individual fragments were excised from the gels and labelled according to the method of Feinberg & Vogelstein (1984). The control probes and subcloned fragments were isolated using Geneclean (Bio 101) from l”/; agarose gels run in 1 x TAE buffer and again labrlled by the hexamer primer method. (i) Hybridizat%on Hybridization was done according to the Southern protocol described by Nasmyth (1982). Following overnight hybridization at 65”C, the filters were washed twice in 2 x SSC, @l% SDS at 65’C for 30 min and then twice in @2 x SSC. @l o/o SDS at 65°C. Filters were air dried at room temperature and exposed to Kodak X Omat film at -80°C in the presence of an intensifying screen. (j) In situ immunojluorr~scencu In situ immunofluorescence was carried out by a modification of the method of Kilmartin & Adams (1984) and was described by Nasmyth et nl. (1990). The rat antitubulin monoclonal antibody YOL l/34 was provided b> J. Kilmartin. (k) DNA manipulation All subcloning procedures were performed by standard methods described by Maniatis et al. (1982). In all cases the plasmid pCT1 was used as a vector: this plasmid is a derivative of pUC18 (Yannisch-Perron et al.. 1985) with an altered polylinker carrying the following restrict>ion sites; NotI, EcoRI, Nrul. HindlIT, YotJ. DNA sequencing was performed using the dideoxynucleotide chain termination method (Sanger et al., 1977) on double-stranded plasmid DNA templates (Hattori & Sakaki. 1986). Plasmid DNA was isolated by the alkaline l.ysis method (Ish-Horowiz & Burke, 1981). The E. coli stram DH 1 was used in all cloning experiments and transformed according to the method of Hanahan (1983).

3. Results (a) Primary

screen

The experimental approach that we have adopted is dependent upon the existence of an ordered yeast genomic library constructed in bacteriophage lambda (Olson et al., 1986). The yeast genomic DNA within this library has been mapped and ordered by double digestion with the restriction endonucleases EcoRI and HindIII. Individual EcoRI/tjindIII fragments from the recombinant phages are isolated from low melting point agarose gels, radioactively labelled by random priming (Feinberg & Vogelstein, 1984), and used as probes in Northern blots (Thomas, 1980) of polyadenylated RNA isolated at, various stages of the cell cycle, listed below: exponentially growing (1) an asynchronous, control sample; (2) a haploid G, sample of cells

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arrested in late G, prior to START with the pheromone alpha factor; (3) a synchronous G1/S phase haploid sample of cells arrested with alpha factor and then released into fresh medium and grown for a further 50 minutes; (4) a synchronous haploid, sample of cells arrested in mitosis with the antitubulin agent, nocodazole; (5) an asynchronous exponentially growing diploid sample; (6) a population of small unbudded diploid G1 cells isolated by centrifugal elutriation. Figure 1 shows schematically how the RNA samples are combined to produce filters for hybridization with the individual probes. The library consists of 855 phages each containing an average of nine mapped EcoRI/HindIII fragments. It is necessary to use individual fragments as probes due to the gene density of the S. cerevisiae genome. We have estimated this to be one transcriptional unit per 2.8 kb. As the average insert size of the phage library is 15 kb, each phage would identify at least five transcripts. Thus a complete screen of the library will require approximately 8000 hybridizations. It is the high gene density that has led us to undertake the approach we describe here, as opposed to more obvious strategies based on differ-

d4pLwltwe

Gl

1 1

Transfer onto GeneScreen Cut membranes into strips 1

2

3

4

5

6

ential hybridization with cDNA generated from different cell cycle stages. We believe that any differential hybridization approach would be less sensitive and more difficult to interpret than our chosen method, due entirely to the high gene density. Constitutively expressed transcripts would always predominate in any cDNA sample and would prevent the identification of transcripts differentially expressed within the cell cycle, if the cDNA was used to probe the phage library. Furthermore, it is likely that very few genes would exhibit sufficiently large cell cycle-dependent variation in transcript abundance t’o permit a successful use of differential hybridization and, in addition. many interesting genes may well be expressed at very low levels. To date we have screened 450 of t’he phages in the primary analysis. Analysis of the data derived from 300 phages has identified 20 transcripts that in the secondary screen (see below) are clearly cell cycle-regulated. We are currently analysing a further 120 candidate transcripts in the secondary screen, by analogy with our early data, we would expect at least 60 of these 120 to be genuinely cell cycle-regulated. Assuming that such transcripts are randomly distributed in the genome we predict that there would exist approximately 250 loci that direct the synthesis of cell cycle-regulated transcripts. This value compares with a recent review listing 11 known cell cycle-regulated transcripts in addition to the histone loci (Johnston. 1990). Figure 2 shows the results obtained in the primary screen for three control genes, the constitutively expressed lJRA3 gene and the cell cycleregulated histone H2A gene (Hereford et al., 1981; Osley et al., 1986) and SWZ5 (Nasmyth et al., 1987). As expected the IJTRA3 transcript is visible in all six RNA samples, ruling out the possibility t,hat any apparent fluctuation in transcript abundance is caused by gel-loading errors. The histone H2A transcript, the lower of the two visible transcripts in lanes 1, 3 and 5, is present in both the asynchronous control samples (lanes 1 and 5) and the G,/S transition sample (lane 3), but not in the haploid G,. mitotic or diploid G, samples (lanes 2, 4 and 6). which is the predicted result. The H2A transcript is known to be activated at a point in late G, (Osley et al., 1986). The upper transcript is derived from the gene encoding Protein 1 (Hereford et al., 1979). The SW15 transcript is present in all the haploid samples except

Figure 1. A diagramatic representation of the preparation of filters for the primary screen. A 100 pg portion of polysdenylated RNA for each sample was loaded onto separate, 1.2o/Oagarose-formaldehyde gels. The gels were run in parallel and the RNA was then transferred to nylon membranes (Genescreen, DuPont). After baking and ultraviolet cross-linking, the filters were cut into 2 mm strips and 1 strip from each RNA sample combined to produce a single filter for the primary screen.

for the G, sample

(lane 2) and this is

in agreement with the published data (Nasmyth et al., 1987). It is, however, only barely detectable in the diploid control sample (lane 5) but clearly absent in diploid G, cells (lane 6). These results demonstrate that cell cycle-dependent transcripts are readily identified by the method we have employed.

It

should

be noted

that,

as the

RNA

samples are run on separate gels and then combined for hybridization, the transcripts appear at different gel mobilities in each of the six samples. Figure 2 also presents the results derived

four uncharacterized

from

DNA fragments isolated from

Yeast Cell Cycle-dependent Transcription

12345

URA3

547

6

H2A

C

0

Figure 2. Results of control and experimental Piorthern hybridizations that constitute the primary screen. Lanes 1 to (i correspond to the different RNA samples described in both Fig. 1 legend and the text. The control hybridizations are labriled. URA3, H2A and SWT5. The transcripts A, B, C and D represent examples of putative cell vyclr-dependent expression and are discussed in the text.

library that appear to identify cell cycle-regulated transcripts. The transcript labelled as A is present in the asynchronous control samples (lanes 1 and 5) as well as the G,/S transition sample (lane 3) but absent in both the haploid G, and the mitotic

the

samples (lanes :! and 4). It is, however, detectable in the diploid G, sample (lane 6). These data suggest that this transcript is activated at a point late in G, and expression continues into early S phase. The apparent. discrepancy between thr results obtained

C. Price et al.

548

with the two G, samples is attributed to the method of synchronization and not to differences in ploidy. The diploid sample contains a wider spectrum of G, cells in comparison to the haploid G1 sample, i.e. the haploid G, cells are arrested at a point prior to the onset of transcription of this gene. A second possibility is that transcription of this gene is specifically repressed by the action of alpha factor. In contrast, the appearance of transcript B, the lower of the two visible transcripts, is restricted to the haploid control and mitotic samples (lanes 1 and 4). However, it is also present in both diploid samples (lanes 5 and 6), which is difficult to correlate with the results from the haploid cells. This result emphasizes the need to use additional screens in order to establish that a transcript is genuinely cell cycle-regulated. Tentatively, we conclude that expression of t’his gene may be mitosis specific and that, the transcript may remain detectable in early G, cells but is not present at the point of alpha factor arrest. Transcript C is restricted to the haploid and diploid asynchronous control and G, samples (lanes 1, 2, 5 and 6), suggesting that expression of this gene is G, specific. Transcript D shows similar regulation to A. We conclude from the above data that transcripts A and D are expressed in late G ,/early S phase, transcript C in early G,, as it is present in both haploid samples but not the G1/S transition sample, and transcript B is mitosis specific. (b) Secondary Screen The results of the primary analysis presented above suggest that the transcripts A, B, C and D are present only at specific points in the cell cycle. In order to confirm the above data it is important to establish that the transcripts exhibit temporal periodicity in exponentially growing synchronous cultures. The secondary screen analyses the behaviour of the previously identified transcripts in cells that are first arrested at a specific point in the cell cycle and then released from the cell cycle block. Samples are then removed from these cultures at successive time points and the RNA is analysed by Northern blotting. Two different synchronization procedures were used, the first synchronizes cells in G, and the second in late mitosis. Figure 3 shows the results obtained following synchronization of the cells in late G,. This is done by first arresting the cells with the pheromone alpha factor followed by removal of the pheromone and subsequent growth

in fresh, pheromone-free media. The graph illustrates the degree of synchrony that was obtained by assessing the number of cells with anaphase spindles at each time point. Total cellular RNA from each time point was loaded. The URA3 transcript is constitutively expressed throughout the time course of this experiment and establishes that there is little variation in the amount of RNA loaded for each time point. Transcripts A and D behave in a similar manner, being first detectable 10 minutes after release from the alpha factor arrest and peaking between 20 and 40 minutes post-release. Thereafter. the two transcripts decrease in abundance as cells proceed through the cell cycle until reaching a second peak between 80 and 110 minutes. This second peak coincides with a sharp decline in the number of anaphase cells and this behaviour is repeated in the next cell cycle. The fact that these transcripts are detected in the 80 minute sample. when the number of anaphase cells reaches a maximum, is attributed to the fact that the degree of synchrony is only 600/b and thus a significant number of cells will have already completed mitosis and entered G1. Comparison with the H2A control establishes t’hat the onset of transcription of these two genes occurs in G, at a point prior to that of the histone H2A gene, and in all probability ceases in early S phase. It has previously been established that histone H2A transcription is initiated in late G, and persists into early S phase (Osley et aZ.. 1986). These data are consistent with the earlier conclusion, based on the primary screen, that transcripts A and D are activated in the G, phase of the cell cycle. That these two transcripts exhibit genuine variation in abundance in a cell cycledependent manner is strongly suggested by the finding that they exhibit the same behaviour in the second and third cell cycles. Transcript B also exhibits clear cell cycle-dependent changes in amount., but in this case the peaks closely parallel the index of anaphase cells. The transcript is first detectable at 60 minutes following release, the point at which the first anaphase cells are apparent, and peaks between 70 and 100 minutes. The transcript behaves identically in the next cell cycle. These data support the earlier conclusions that transcript B is mitosis specific and that it remains detectable in early G,, as it is present in the lOO-minute sample when the minimum number of anaphase cells are seen. Transcript C is detectable in the arrested sample and in the first sample following release: it) decreases in abundance t’hereafter, before

Figure 3. Cell cycle-dependent expression in synchronized culture. Cells were arrested in G, by treatment with alpha factor, and subsequently released. The zero time point was taken immediately prior to the release and subsequent. samples were removed every 10 min up to 180 min. The graph plots the synchrony, determined by counting the number of cells at each time point, exhibiting extended anaphase spindles. A 10 pg sample of total RNA isolated from each time point was electrophoresed on 1 To agarose-formaldehyde gels and transferred to nylon membranes. The filters were then probed with the DNA fragments defined in the primary screen. The control transcripts are labelled, URA3 and H2A, and transcripts A, B, C and D are those described in Fig. 2 legend and in the text. Synchrony was determined by scoring for the presence of anaphase spindles following in situ immunolluorescence with rat anti-tubulin monoclonal antibodies (see Materials and Methods).

549

Yeast Cell Cycle-dependent Transcription

URA3

H2A

D

C

D

80

60

40

20

0

.!_;,r ..... ... 0

20

40

60

80

100120140160180

Time of release Fig. 3.

:

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reaching a second peak between 80 and 110 minutes, coincident with the decline in numbers of anaphase cells. This behaviour is observed in the following cell cycle. The first peak in abundance of transcript C precedes that of transcripts A and D, which supports the notion that this transcript is G, specific and that the onset of transcription is a very early G, event. It should be noted that the degree of synchrony within the population decreases with time; therefore, the peaks in transcript abundance show considerable overlap in the second cycle. The second method of synchronization employs a thermoreversible ts allele of the CDCl5 gene. Conditional mutations in this gene cause cell cycle arrest in late anaphase (Cullotti & Hartwell, 1971). Exponentially growing cells are first uniformally arrested in late anaphase by a temperature shift from the permissive to the restrictive temperature; the temperature shift is subsequently reversed and samples of the culture are removed at successive time points. There are two reasons for using this second synchronization. First, the degree of synchrony achieved in this manner is considerably higher than that obtained after synchronization with alpha factor, as can be seen by comparison of the two indices of anaphase cells (Figs 3 and 4). That the synchrony is much better using this second procedure can be attributed to the fact that the cells exhibit very little lag in recovery upon shifting to the permissive temperature. Second, it is important to confirm the data derived from alpha factor synchronization in order to rule out the possibility that the observed transcriptional behaviour is an artefact of the method of synchronization. Figure 4 shows the results obtained for the same transcripts, A, B, C and D, using this second synchronization procedure. The data are broadly similar to those obtained from the alpha factor synchronization. The peak of detection of transcripts A and D follows immediately after the decline in the number of anaphase cells within the culture in both the first and second cycles. Furthermore, the peak in abundance of these two transcripts again precedes that of the histone H2A gene. This result supports the previous conclusion that the onset of transcription of these two genes is G, specific. The peak in abundance of transcript B is coincident with the presence of anaphase cells within the culture in both cycles and it is detectable at t)he point of cdc75-mediated arrest. These data lead us to conclude that transcription of this gene occurs exclusively during mitosis and is perhaps specific to anaphase. Transcript, C peaks within 30 minutes of release, prior to the maximal levels of transcripts A and D, consistent with the conclusion that the onset of transcription of this gene is a very early G, event.

Under these conditions the disappearance of transcript B and appearance of transcript C are seen as reciprocal events, in the first cycle subsequent to release. This suggests that, despite the fact that anaphase spindles are visible in all cells at 30 minutes following release, the cells have already entered Gi. The URA3 transcript is only barely detectable at the zero time point; however, as both the Protein 1 transcript, in the H2A panel, and transcript B are visible in this sample we do not attribute this to gel-loading errors. Further, all of the above hybridizations have been performed at least twice on different filters, with the same result each time. (c) Identi$cation

of the genes

The next stage of the project is to begin to characterize those genes that are clearly cell cycledependently expressed. It is important to note that, as a result of the colinearity of the physical and genetic maps, it is often possible to eliminate previously identified cell cycle-regulated genes. We have, for example, identified the histone HTBl locus on chromosome IV and the TOP2 gene on chromosome XIV. The HTBl gene was eliminated by Southern hybridization with a defined HTBl probe and the TOP2 gene by subcloning and partial DNA sequencing (data not shown). The most rapid and powerful approach to the identification of genes mapping to previously uncharacterized loci is to subclone the appropriate EcoRI/HindIII fragments and to derive partial DNA sequence. By taking this approach it has been possible to identify two of the four transcripts described above. Transcript A was detected by three adjacent fragments derived from two phages from the yeast genomic library. These fragments were subcloned and subjected to partial DNA sequence analysis, as indicated in Figure 5(a). Computer comparisons of the predicted amino acid sequences derived from the DNA sequence of these subclones revealed significant homology to the murine ribonucleotide reductase large subunit (data not shown). Recently, another group has identified two S. cerevisiae genes with similar homologies, namely the RNRl and RNR3 genes (Elledge & Davis, 1990). They have also established that the RNRl gene is regulated at the t’ranscriptional level in a cell cycle-dependent manner, whereas the RNR3 gene is only expressed following DNA damage. Comparison of our data with theirs leads us to conclude that transcript A is derived from the RNRl gene. This gene maps to the right arm of chromosome V. The second transcript, B, was detected by a single 3 kbp Hind111 fragment mapping to the right arm

Figure 4. Cell cycle-dependent expression in cdcl5 synchronized cultures. Exponentially growing cells were arrested in mitosis by incubation at 37°C and subsequently released by growth at 25°C. Samples were taken every 15 min up to 270 min; the zero time point was taken immediately prior to the release from temperature arrest. Synchrony was determined as described for Fig. 3. Again. 10 pg of total RNA from each time point was electrophoresed on 1 o/0 agarose formaldehyde gels and the probes were the same as those described in Figs 2 and 3.

Yeast Cell Cycle-dependent Transcription

,551

URA3

H2A

A

B

C

D

80

.-c -u) u” ;5 8

60 40 20 0 0

30

60

90

120150

180

Time of release Fig. 4.

210240270

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RNRI R I

I3 - -

s I

HR I I -- ----a

H

(cl) CLN I R

R I cc -

4

H I

b lkb

(b) Figure 5. Identification of the genes corresponding to transcripts A and D. (a) The restriction map of the locus that determined transcript A with the regions for which the DNA sequence w&9 established. As indicated, this corresponds to the RNRl gene. (b) The same information for the gene encoding transcript D, which was determined to be the CLNl gene. Restriction sites: B, BgaI; H, HindHI; R, EcoRI; S, SphI.

of chromosome II. This fragment wa subcloned and sequenced (W. Rossoll, unpublished results). The fragment contained one complete open reading frame that was shown to be colinear with the regulated transcript (W. Rossoll, unpublished results). Computer analysis of the predicted amino acid sequence of the cell cycle-regulated gene revealed no significant homology within the current databases. However, there is considerable overall structural similarities to known membrane proteins, e.g. bacteriorhodopsin (data not shown). We have named this gene MST1 (mitosis specific transcript). This gene is under further analysis in our laboratory. Transcript C mapped to a 2-l kbp Hind111 fragment from the right arm of chromosome V. This fragment was again subcloned and sequenced. Analysis of the single open reading frame revealed no significant homologies within the current databases (data not shown). We have named this gene EGTl (early G, transcript). The final transcript, D, was detected by two adjacent fragments mapping to the right arm of chromosome XIII. Figure 5(b) shows a restriction map of this region and indicates the area of partial DNA sequence. Analysis of both the derived amino acid sequence and the DNA sequence revealed that this gene had previdusly been identified as CLNl (Hadwiger et al., 1989; Hadwiger & Reed, 1990). (d) G l-speci$c transcription Analysis of the data presented above suggested that the onset of transcription of the RNRl, CLNl

and EGTl genes were all G, specific. However, the appearance of the RNRl and CLNl transcripts was restricted to late G,/early S phase, whereas the EGTl transcript appears much earlier, in all likelihood at a point prior to START. START is defined as the point in G, beyond which cells are irreversibly committed to mitotic division (Pringle & Hartwell, 1981). In an effort to substantiate this, we undertook the following experiment. An asynchronous culture of diploid cells was grown in minimal medium and then size fractionated by centrifugal elutriation. The earliest fractions contained a population of small, unbudded daughter cells, the majority of which were in the G1 phase of the cell cycle, prior to START, whereas the later fractions contained increasing numbers of budded cells. Total RNA was then isolated from each fraction and analysed by Northern blotting. Figure 6 shows the results of this analysis as well as the budding index for each fraction. The first three fractions contained either none or very small numbers of budded cells and we therefore believe that the majority of cells in these fractions are indeed at a point in G, prior to START. The EGTI transcript is clearly present in all fractions, whereas both the RNRl and CLNl transcripts only appear once the number of budded cells begins to increase. Again the URA3 transcript serves as a control for gel-loading artefacts. We conclude from these data that the EGTl gene is indeed transcribed at a very early point in G1, prior to START, whereas the RNRl and CLNl genes are not transcribed until later in G 1.

4. Discussion We describe a general approach for the identification of S. cerevisae genes regulated at the transcriptional level in a cell cycle-dependent manner. We assume that these genes are transcriptionally activated in such a manner, although we have not distinguished between activation and repression and it is entirely possible that both mechanisms operate in tandem, as is the case for the histone H2A/H2R gene locus in S. cerevisae (Osley et al., 1986). It is also likely that post-transcriptional mechanisms play an important role in controlling the levels of cell cycle-regulated transcripts (Hereford et al., 1979; Lycan et al., 1987; Xu et al., 1990). We have chosen four transcripts to illustrate our approach and to underline our reasons for undertaking this project. First, we report the isolation of two previously unidentified genes, EGTl and MSTI, which are expressed in early G, and mitosis, respectively. This represents the first report of cell cycle-dependent transcriptional activation at these two points in the cell cycle. The onset of EGTl transcription should serve as an important landmark event within the cell cycle, for entry into the G1 phase. At present there is no good cytological marker for the transition from mitosis to G,. The behaviour of this transcript in the CDClS synchronization experiment supports this contention. The transcript level peaks 30 minutes after the release

Yeast Cell Cycle-dependent Transcription

5.53

URA3

Fraction

1

2

3

4

5

6

7

8

9 10 11 12

C

( % Budding ) Figure 6. G,-specific transcription. Cells were size fractionated by centrifugal elutriation and total RXA was isolated from each fraction. A 10 ,ug sample of RNA from each fraction was electrophoresed on 19/bagarostt-formaldehyde gels and transferred to nylon membranes. The probes used are identified in the text: URA3 served as a control. The budding index for each fraction is indicated. The fraction labelled C corresponds to RNA isolated from the cells remaining in t,he rotor following the removal of the 12 different size fractions. and serves as a control.

from cell cycle arrest at a time when the cells are morphologically and cytologically indistinguishable from their appearance at the previous two time points, when the transcript is not present. This indicates t)hat the cells have undergone a physiological change of state that allows the transcription of EGTl but is not reflected

onset of by mor-

phological or cytological alterations. This conclusion is furt’her supported by the results obtained by

the analysis of MST2 transcription in t,he same experiment. In this case a physiological change is indicated by the loss of the MST1 transcript at exactly the same point in time. Detailed analysis of the transcriptional control of this gene should provide useful insights into this event. It, will also be of interest to establish whether or not the cont,rol of transcription of the MST1 gene is linked to the known controls governing the entry int’o and exit

554

C. Price et al.

from mitosis (Nurse, 1990). At present we are investigating these two genes in more detail and the results of these studies will be reported elsewhere. The third gene, RNRl, encoding the large subunit of ribonucleotide reductase, had not been described for S. cerevisine at the time at which we initiated this project, although subsequently its isolation has been described (Elledge & Davis, 1990). The main point of interest of this gene, in relation to this work, is that its product is the target for the action of hydroxyurea, a drug that leads to cell cycle arrest in early S phase. Thus it should be an ideal candidate CDC gene and might have been expected to be identified by traditional mutagenic screens in S. cerevisiae (see Pringle & Hartwell, 1981, and references therein). However, this was not the case despite the fact that in S. probe the cdc22 gene probably encodes the same function (FernandezSarabia & Fantes, 1990). This emphasizes the point that many important genes will have remained unidentified by more traditional genetic approaches. An argument that is further supported by evidence derived from a combined physical and genetic analysis of chromosome I of S. cerevisiae (Kaback et al., 1984) and further data relating to the isolation of the CDC42 and CDC43 genes of S. cerevisiae (see Adams et al., 1990, and references therein). This work demonstrates that different genes exhibit clear differences in susceptibility to conditional mutation by conventional mutagenic approaches. We therefore believe that our approach will allow us to detect genes that have not been identified by mutagenesis. The fourth gene that we describe is the CLNl gene: first isolated as a multicopy supressor of the cd&%-4 allele (Hadwiger et al., 1989). This gene belongs to a family of three genes that exhibit homologies to cyclins (Evans et al., 1983; Nash et al., 1988; Cross, 1988; Hadwiger et al., 1989) and are thought to play an important role in cell cycle control acting at START in the Gi phase of the cell cycle (Nash et al., 1988; Cross, 1988; Hadwiger et al., 1989). Of these three genes only the CLN3 gene was uncovered by traditional genetic means and was termed either WHIl (Sudbery et al., 1980) or DAlr’l (Cross, 1988). The importance of the CLNl gene in reference to the work herein is twofold. First, it demonstrates that our screen identified a putative cell cycle-regulating gene that is transcriptionally regulated in a cell cycle-dependent manner. It is thus likely that we will be able to identify further such genes and it strengthens the arguments for an important role for cell cycle-dependent transcription in control of the eukaryotic cell cycle. Second, the finding that these three genes exhibit redundancy (Hadwiger et al., 1989) underlines the fact that a physical approach to the identification of genes is able to circumvent a major obstacle inherent in traditional genetic screens. The transcriptional behaviour of CLNl closely parallels that of the RNRl gene in all of the experiments presented here. This raises an important question, as the onset of transcription of the RNRl gene is

thought to be dependent upon the completion of START (Elledge & Davis, 1990). In contrast it has been proposed that the product of the CLNI gene is required for START (Hadwiger et al., 1989). One model of G1 cyclin activity is that the genes are transcribed upon entry into the G, phase of the cell cycle; the gene products then accumulate to a critical level that leads to commitment to the mitotic cycle. However, if transcription of CLNl is indeed restricted to the same point in the cell cycle as RNRl then this model does not hold, at least for the CLNl gene. The simplest explanation of this apparent contradiction is that we are unable to detect a small but significant temporal difference in the onset of transcription of these two genes and that CLNl is in fact t’ranscribed first. Indeed, in the first cycle following CDC15 synchronization of the CLNI transcript is detected prior to RNRI. It is, however, clear from the elutriation experiment (Fig. 6), that CLNl transcription is not activated as cells enter Gi, in contrast to EGTI. One possible explanation for these results is that there exists a positive feedback mechanism, monitoring the G, phase of the cell cycle. This model predicts that the full transcriptional activation of the CLNI gene (and CLN2, unpublished results) would be dependent

upon

a threshold

level

of

protein

kinase

complexes between the products of the three G, cyclin genes and the CDC28 gene product. Once this threshold level is achieved the CLNl and CLN2 genes are fully activated, thus further increasing the amount

of kinase

complexes.

It is then this higher

level of kinase activity that is required for the cell to become committed to the mitotic cycle, and START is defined as this point of commitment. Tt is not inconceivable that transcription of the RNRl gene is regulated by the same mechanism and that transcriptional activation of this gene is commensurate with START. We believe, on the basis of the data and arguments presented above, that our approach will lead of many novel genes exerting to the identification important functions at specific points in the cell cycle. In addition it is probable that the transcripts that we identify will serve as useful molecular markers of cell cycle events and that the study of the transcriptional properties of these genes will

ultimately controlling

provide important insight into events of the eukaryotic cell cycle.

the

We are greatly indebted to Maynard Olson and his colleagues Jim Dutchik and Linda Riles. Without their generosity in supplying us with the ordered phage library and unpublished mapping data this project would not have been possible. We also thank Wilfried Ross011 and Robert Fritsche for technical assistance, Robert Kurzbauer and Sissi Ender for DNA sequencing. Finally, we thank Gustav Ammerer, Denise Barlow and Beverley Errede for comments on the manuscript.

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A general approach to the isolation of cell cycle-regulated genes in the budding yeast, Saccharomyces cerevisiae.

We describe a general approach to the isolation of cell cycle-dependently regulated transcripts in Saccharomyces cerevisiae. This approach is based on...
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