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Current Genetics

Genetics (1983)7:309-312

© Springer-Verlag 1983

Mating Factor Dependence of G1 Cell Cycle Mutants of Saccharomyces cerevisiae B. M. Connolly, V. C. Bugeja 1, J. R. Piggot, and B. L. A. Carter 2 Department of Genetics, University of Dublin, Dublin 2, Ireland

Summary. Mutants in four G1 cdc strains of Saccharomyces cerevisiae were isolated which failed to show division arrest in the presence of a-factor. The cell cycle properties, terminal arrest morphology and mating competence of these mutants at the restrictive temperature were examined. The G1 specific arrest of the cdc 36 and cdc39 mutants is dependent upon the availability of an intact mating factor response system in Mat a cells. Cdc28 and cdc37 mutants exert their cell cycle blocks independently of the mating factor pathway. It is likely that the nature of the primary growth defect in cdc36 and cdc39 mutants is such that the a-factor pathway is activated in the absence of the pheromone at the restrictive temperature and that G1 arrest is a secondary consequence of a non-cycle specific event in such mutants. Key words: Saccharomyces cerevisiae - G1 cdc mutants - a-factor

Introduction The cdc28 mutation in Saccharomyces cerevisiae is known to cause cell-cycle specific arrest in the G1 phase of the cell cycle at the restrictive temperature (Hartwell 1973a). Cell cycle (Hereford and Hartwell 1974) and ultrastructural (Byers and Goetsch 1974) studies have demonstrated that the point of cell cycle arrest in cdc28 mutants is identical to that seen in a mating 1 Present address: St. Patrick's College, Maynooth, Co. Kildaire,

Ireland

type cells exposed t o the a-factor pheromone. Reid and Hartwell (1977) established that cdc28 mutants are unique among cdc mutants in being able to mate at the restrictive temperature. Reciprocal mating-factor induced arrest of cells of opposite mating type in G1 has been shown to be a prerequisite for conjugation of a and a mating type ceils (Hartwell 1973b). The G1 arrest seen in cdc28 mutants at the restrictive temperature and in a mating type cells exposed to the a-factor results in a superficially similar terminal arrest morphology. Reed (1980) has utilized the ability of cdc28 mutants to mate at the restrictive temperature to select new mutants in genes other than cdc28 which result in a similar phenotype. Of three such mutants isolated by Reed, one, cdc37, is apparently phenotypically identical with cdc28, although genetically unlinked, while two, cdc36 and cdc39 are unlike cdc28 in that they do not show homogeneous cell cycle arrest in a/a diploids homozygous for the mutant gene. Here we show that G1 specific arrest of the cdc36 and cdc39 mutants is dependent upon the availability of an intact mating factor response system in a haploids cells, while cdc28 and cdc37 mutants appear to exert their cell cycle blocks independently of the mating factor pathway.

Materials and Methods Organism. Saccharomyces cerevisiae haploid strains ST-10 (Mat a, ede28-6, met2, tyrl, lys2, his7, cyh2), SR661-2 (Mat a, cdc3616, trpl, ural), SR672-1 (Mata, cdc37-1, ural, cyh2) and SR6651 (mat a, cdc39-1, met2, tyrl, cyh2) were obtained from Dr. S. Reed (University of California, Santa Barbara). VB13c (Mat a, cdc39-1, met2, tyrl, argl) was derived from SR665-1. Other haploid strains used were JJ1C (mat a, argl, thrl) and X2180-1B (Yeast Genetics Stock Centre).

2 Present address: G. D. Searle and Co. Ltd, High Wycombe,

Bucks, England Offprint requests to: B. M. Connolly

Media. YEPD: 2% (w/v) glucose, 2% (w/v) bactopeptone, 1%

(w/v) yeast extract. MV: 2% (w/v) glucose, 0.5% (w/v) ammonium

310

B.M. Connolly et al.: Mating Factor and G1 Mutants of Yeast

Fig. I A - E . Terminal arrest m o r p h o l o g y o f Mat a cells arrested by exposure to c~-factor at 24 °C A and by the cdc37 B, c d c 3 7 a F r C, cdc36 D and c d c 3 6 a F r E m u t a t i o n s at t h e restrictive temperature. Note the areas o f relatively intense superficial staining with Calcofluor which occurs in a-factor arrested wild t y p e cells (A) and cdc36 cells at t h e restrictive temperature (arrowed in D). Such areas are absent in b o t h cdc28 and cdc37 arrested cells (B). The bar represents 10 ~ m

sulphate, 0.17% (w/v) yeast nitrogen base (without a m i n o acids). For solid media 2% (w/v) agar was included.

a-factor, a-factor was prepared from a culture filtrate o f X21801B cells and assayed as described by Bucking-Thr6m et al. (1973).

Mutagenesis. Cells were grown to mid-exponential phase in liquid YEPD at 24 °C. 106 cells, spread onto YEPD plates containing 32 units m1-1 o f a-factor were subjected to UV irradiation (50%

killing) and incubated in the dark at 24 °C. After 48 h dividing colonies were picked off and grown overnight at 24 °C on ~factor-free YEPD. Subsequently cells were re-streaked onto YEPD plates containing 8 units ml 1 a-factor and after 24 h at 24 °C single colonies were retained as a-factor resistant m u t a n t s .

Determination of Mating Competency. Strains were grown to mid-exponential phase in liquid YEPD at 24 °C. Each culture was then split and half was incubated at the restrictive tempera-

B. M. Connolly et al.: Mating Factor and G1 Mutants of Yeast ture (37 °C) for 3 h; the other half was left at 24 °C. Equal numbers (as determined by Coulter counter) of Mat a and Mat ~ cells were mixed and pelleted together. After 2.5 h incubation at 24 ° or 37 °C the pellet was resuspended in sterile distilled water to a cell density of 2 x 107 cells/ml. Dilutions were made and plated onto MV plates and incubated at 24 o or 37 °C for several days. The number of diploids was scored as the number ofprototrophie colonies per plate.

Photomicroscopy. Photomicrographs were taken on Kodak tri-X pan black and white film on a Laboval 18 1_11microscope fitted with a 200 watt UV source and fluorescein wavelength excitation filter (Carl Zeiss, Jena). Calcofluor staining was accomplished by resuspending cells from 1 ml of liquid culture in 0.1 ml of Calcofluor (American Cynamid Co.) at a concentration of 10 mg/ ml in distilled water.

Results All four cdc mutants discussed belowhave a predominantly (>85%) unbudded morphology at the restrictive temperature in a or a mating type haploid strains (Reed 1980; Fig. 1 B, 1D). Mutants in Mat a cells o f the four start cdc mutants, cdc28, cdc36, cdc37 and cdc39, were isolated which failed to show division arrest in the presence o f afactor at 24 °C ( a F r mutants). The double mutants, grown overnight on YEPD plates at the permissive temperature, were replica plated onto YEPD at the restrictive temperature. After 3 h incubation the temperature sensitivity and terminal (arrest) phenotype o f the mutants was scored microscopically. Of the two hundred cdc28aF r and the two hundred c d c 3 7 a F r mutants examined in this way all were temperature sensitive and none had altered terminal phenotypes as compared with the a-factor sensitive ( a F s) parent cdc m u t a n t (Fig. 1B, IC). Of the three hundred and fifty c d c 3 6 a F r and the 150 cdc39aF r mutants examined all were temperature sensitive but, in contrast to the c d c 2 8 a F r and c d c 3 7 a F r mutants, all failed to arrest homogeneously in G1 but arrested at all stages o f the cell cycle at the restrictive temperature (Fig. 1E). Liquid cultures o f the c d c 3 6 a F r mutants shifted from 24 o to 37 °C arrested with 60% b u d d e d cells while, under similar conditions the c d c 3 6 a F s parent arrested with less than 10% budded cells. Five a F r mutants derived from each cdc strain were examined in liquid culture at the restrictive temperature. As with the a F s parent cdc strain, cell volume, as determined b y an increase in the mean value o f the size distribution curve on the Coulter chanelizer, increased in the absence o f cell division in all cases. The apparent continuation o f growth at the restrictive temperature seen in b o t h the cdc36 and cdc39 a-factor resistant strains, unaccompanied b y cell cycle progress is puzzling. At present we can only say that we have failed to detect any homogeneity o f arrest morphology in these mutants. Thirty cdcaF r and twenty cdc37aF r mutants were examined for their ability to mate with a Mat a tester

311 strain (JJ1C) at the permissive and restrictive temperatures for growth (24 o and 37 °C respectively). All o f the double mutants were found to be sterile at b o t h temperatures; the frequency o f diploid formation was less than 0.01 o f that o f the a F s cdc parent strain. Similarily none o f the eighty cdc36aF r or the twenty c d c 3 9 a F r mutants examined for mating competence were found to be comp e t e n t at either temperature.

Discussion The cdc28, cdc36, cdc37 and cdc39 mutants all undergo G1 specific cell cycle arrest at the restrictive temperature at a point in the cell cycle indistinguishable from that at which a-factor exerts its inhibitory effect (Reed 1980). Our results suggest, however, that the cell cycle block imposed b y the cdc28 and cdc37 mutants is produced b y a mechanism which is distinct from that involved in a-factor arrest. The "interdependence" o f the cell cycle blocks imposed b y cdc28 and a-factor reported b y Hereford and Hartwell (1974), therefore seems to reflect only the fact that b o t h types o f arrest occur at the same point in the cell cycle, although arrest in each case is due to effects in functionally independent systems. The cdc36 and cdc39 mutants, on the other hand, seem to require an intact mating factor response pathway in order to show G1 specific cell cycle arrest, although they do show a non-specific division defect in a-factor resistant mutants (this study) and in a/a homozygous diploids (Reed 1980). It seems likely, therefore, that the nature o f the primary growth defect in cdc36 and cdc39 mutants is such that the a-factor response pathway is activated in the absence o f the pheromone at the restrictive temperature and that G1 arrest is a secondary consequence o f a non-cell cycle specific event in such mutants. This view is reinforced b y the similarity of Calcofluor staining patterns in the cdc36 and cdc39 mutants compared with a-factor arrested cells (Fig. 1A, 1D). Hartwell (1980) has identified eight complementation groups associated with a-factor response among 239 independenfly isolated a-factor resistant mutants. The possibility exists that sterile mutants belonging to one or more of these complementation groups will not show the suppression o f G1 arrest morphology which we rep o r t here for 350 cdc36aF r and 150 cdc39aF r mutants. It is also reasonable to propose that the cdc36 and cdc39 mutants should suppress the sterility o f certain a-factor resistant strains, although no such suppression was detected during this study.

Acknowledgements. This research was supported by grants from the Irish Cancer Society and the National Board of Science and Technology.

312 References Bucking-Throm E, Duntze W, Hartwell LH, Manney TR (1973) Exp Cell Res 7 6 : 9 9 - 1 1 0 Byers B, Goetsch L (1973) Cold Spring Harbour Symp Quant Biol 38:123-131 Hartwell LH (1973a) J Bacteriol 1 1 5 : 9 6 6 - 9 7 4 Hartwell LH (1973b) Exp Cell Res 7 6 : 1 1 1 - 1 1 7

B.M. ConnoUy et al.: Mating Factor and G1 Mutants of Yeast Hartwell LH (1980) J Cell Biol 85: 811 - 822 Hereford LM, Hartwe11 LH (1974) J Mol Biol 84:445-461 Reed SI (1980) Genetics 95:561-577 Reid BJ, Hartwell LH (1977) J Cell Biol 7 5 : 3 5 5 - 3 6 5 C o m m u n i c a t e d b y B. S. Cox Received December 14, 1982

Mating factor dependence of G1 cell cycle mutants of Saccharomyces cerevisiae.

Mutants in four G1 cdc strains of Saccharomyces cerevisiae were isolated which failed to show division arrest in the presence of α-factor. The cell cy...
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