Current Genetics

Current Genetics (1982) 5:29-31

© Springer-Verlag 1982

Thymineless Recombination in Saccharomyces cerevisiae is Independent of the Ability to Undergo Meiosis Bernard A. Kunz 1, J. Gerard Little 1 , Friederike Eckardt 2, and Robert H. Haynes 1 1 BiologyDepartment, York University,Toronto, Ontario, Canada M3J 1P3 2 Gesellschaftftir Strahlen- und Umweltforschung,D-8042 Neuherberg,Federal Republic of Germany

Summary. Thymine nucleotide starvation is recombinagenic in Saccharomyces cerevisiae and induces formation of the 'nuclear dense body', a structure characteristic of yeast cells in meiosis. Conceivably, thymineless recombination in yeast, presumed to be mitotic, might be meiotic in nature. We have tested this hypothesis and have found that thymineless recombination can be induced in strains incapable of meiotic exchange. Key words: Thymineless recombination - Meiosis

The in vitro methylation of deoxyuridylate to thymidylate (dTMP), by thymidylate synthetase from Saccharomyces cerevisiae, requires the methyl donor N s, N l°methylenetetrahydrofolate (Bisson and Yhorner 1977). In vivo, treatment of yeast with the folate antagonists sulfanilamide plus aminopterin or methotrexate abolishes dTMP biosynthesis by limiting the supply of endogenous tetrahydrofolate (Laskowski and Lehmann-Brauns 1973; F~ith et al. 1974; Little and Haynes 1979; Barclay et al. 1982). Previously we have demonstrated that antifolate-induced thymine nucleotide starvation is recombinagenic in S. cerevisiae but does not appear to be mutagenic for nuclear genes (Kunz et al. 1980; Kunz 1981; Kunz and Haynes 1981; Barclay et al. 1982). Recently, it was observed that in yeast dTMP deprivation causes the formation of the 'nuclear dense body', a structure normally present only during meiosis (Moens et al. 1981). Meiotic recombination frequencies can be 100-fold greater than spontaneous mitotic recombina* Offprint requests to." B. A. Kunz at his present address:

National Institute Of Environmental Health Sciences, Laboratory of Molecular Genetics, P.O. Box 12233, Research Triangle Park, NC. 27709 USA

tion frequencies (Prakash et al. 1980). Since thymineless conditions also induce enhanced recombination, it was possible that thymidylate depletion might be one of the signals that switches cells from mitotic to meiotic development under conditions conducive to sporulation. If so, the increased genetic exchange that occurs during dTMP starvation might be related to meiosis. To test this hypothesis thymidylate stress was induced in diploids that either lacked mitochondrial DNA or were homozygous for mating-type. Such strains are incapable of meiotic recombination (Friis and Roman 1968; Roth and Fogel 1971) but can undergo spontaneous and UV-induced mitotic recombination (Friis and Roman 1968; B. A. Kunz, unpublished observations). If the recombination induced by dTMP depletion failed to occur in these strains, it would support the hypothesis that such recombination is meiotic. The diploid strain D7-T2 is respiratory-competent and heterozygous for mating-type (Kunz et al. 1980). There are two complementing heteroalleles at theADE2 locus of D7-T2. Separately, these confer different degrees of adenine dependence and red pigmentation. Reciprocal crossing-over between ADE2 and its centromere produces red/pink twin-sectors (Zimmermann 1973). Other genetic events such as gene conversion and chromosome loss result in additional types of aberrant coloured colonies. There also are two non-complementing heteroalleles at the TRP5 locus. Gene conversion at this site leads to the eiiaergence of tryptophan-independent clones. A respiratory-deficient derivative of D7-T2 was selected following ethidium bromide treatment and it was determined by CsC1 buoyant density gradient centrifugation that this isolate (D7-T2[rho°]) lacked mitochondrial DNA (Kunz et al. 1980). Two additional derivatives of D7-T2 (D7-T2-3c¢~ and D7-Y2-5c~c0 were selected on the basis of homozygosity for the mating-type (Kunz 1981). Other parental markers were 0172-8083/82/0005/0029/$ 01.00

B.A. Kunz et al.: Thymineless Recombination in S. cerevisiae

30

Table 1. Sporulation and meiotic recombination in D7-T2 and its derivatives. Stationary phase cells were suspended in sporulation medium containing 1% potassium acetate, 0.1% yeast extract and 0.05% dextrose (2 x 107 cells per ml) and incubated with shaking at 32 °C. At regular intervals aliquots were withdrawn, cell titres were determined by Coulter counter and washed cells were spread on synthetic media containing 0.67% yeast nitrogen base without amino acids, 2% dextrose and appropriate supplements to detect survivors and tryptophan prototrophs (trp+). Surviving fractions (viable cells per ml divided by Coulter counts per ml) and frequencies of gene conversion were determined after 5 days incubation at 30 °C. Asci were scored microscopically prior to plating ceils. Numbers in parentheses are the numbers of asci or colonies scored Strain

Time of incubation (h)

Surviving fraction

D7-T2

0 6 12 24 48

1.00 (1,231) 1.18 (1,447) 1.16 (1,383) 1.14 (1,300) 0.92 (1,237)

3.7 (273) 17 (6,680) 103 (8,561) 107 (8,361) 136 (10,109)

0 (0/200) 0 (0/200) 0 (0/200) 15 (34/226) 36 (74/208)

D7-T2[rho°]

0 6 12 24 48

1.00 (960) 1.20 (1,i48) 1.04 (1,022) 1.16 (1,310) 0.56 (702)

0.7 (42) 0.7 (51) 1.0 (59) 1.7 (137) 2.1 (87)

0 (0/200) 0 (0/200) 0 (0/200) 0 (0/200) 0 (0/200)

DT-T2-3aa

0 6 12 24 48

1.00 (342) 1.02 (552) 1.17 (605) 1.52 (810) 1.14 (651)

0.6 (31) 0.7 (37) 0.5 (29) 0.5 (43) 0.5 (35)

0 (0/200) 0 (0/200) 0 (0/200) 0 (0/200) 0 (0/200)

D7-T2-5aa

0 6 12 24 48

1.00 (470) 1.11 (534) 1.16 (679) 1.26 (701) 1.18 (749)

0.3 0.4 0.4 0.3 0.5

0 (0/200) 0 (0/200) 0 (0/200) 0 (0/200) 0 (0/200)

unaffected by the selection procedures. When incubated in sporulation medium, D7-T2[rho°], D7-T2-3aa and D7-T2-5aa were unable to form asci or to carry out meiotic gene conversion to tryptophan prototrophy (Table 1). On the other hand, D7-T2 spomlated and underwent meiotic recombination. To provoke thymine nucleotide starvation in D7-T2 and its derivatives, the diploids were incubated in nutrient medium containing the folate antagonists sulfanilamide and methotrexate (Kunz et al. 1980; Barclay et al. 1982). For each strain the antifolate-induced thymidylate stress was lethal and increased the frequencies of gene conversion and formation of aberrant coloured colonies (Table 2), including red/pink twin-sectors (not scored separately because of low numbers). D7-T2[rho ° ] was somewhat more resistant to the folate antagonists than the other derivatives and also exhibited lower frequencies of induced recombination. The reasons for these differences are not entirely clear although the decreased levels of genetic exchange may be related to the reduced cell killing in this strain [we have found a direct relation between the severity of cell killing caused

trp + per 105 survivors

(23) (23) (26) (21) (39)

% Asci

by thymidylate depletion and the degree of associated recombination in several radiation-sensitive mutants (Kunz and Haynes 1981)]. Nevertheless, it is apparent that drug-induced dTMP deprivation stimulates recombination in diploid cells incapable of meiotic exchange. This finding indicates that recombination observed as a consequence of thymidylate starvation is not associated with meiosis. Elsewhere, we have demonstrated that cdc21, which causes a temperature-sensitive dTMP auxotrophy (Game 1976) and prevents commitment to meiotic recombination at the restrictive temperature (Schild and Byers 1978), enhances exchange in mitotic cells growing under non.permissive conditions (Kunz et al. 1980). Premeiotic DNA synthesis is blocked in respiratory-deficient cells and in diploids homozygous for mating-type or for cdc21 (Croes 1967; Roth and Lusnak 1970; Schild and Byers 1978). Thus, is could be suggested that meiotic recombination might occur in these strains if thymidylate depletion induced a switch to meiotic development during mitotic DNA synthesis. This seems doubtful in view of the fact that when vegetatively

B. A. Kunz et al.: Thymineless Recombination in S. cerevisiae

31

Table 2. Antifolate-induced recombination in D7-T2 and derivatives incapable of sporulation. Log phase cells were suspended in a 1% yeast extract, 2% peptone, 2% dextrose broth (2 x 106 cells per ml) containing sulfanilamide (5 mg/ml) and methotrexate (100 ~g/ml) and incubated with shaking at 34 °C. At regular intervals aliquots were withdrawn and treated as described in the legend to Table 1 except that cells also were plated to measure the formation of aberrant coloured colonies. Numbers in parentheses are the numbers of colonies scored, trp+: tryptophan prototrophs Strain

Time of incubation (h)

Surviving fraction

D7-T2

0 3 6

1.00 (985) 0.28 (385) 0.02 (606)

D7-T2[rho °]

0 3 6

1.00 (667) 0.94 (1,484) 0.15 (331)

D7-T2-3e~

0 3 6

D7-T2-5~c~

0 3 6

trp + per 105 survivors

Aberrant colonies per 104 survivors

0.4 (42) 8.4 (129) 26 (31)

6.1 (6) 88 (42) 660 (40)

0.5 (36) 1,3 (38) 7,7 (51)

4.5 (3) 8.8 (13) 230 (76)

1.00 (847) 0.40 (617) 0.03 (454)

0.3 (29) 6.7 (83) 37 (34)

5.9 (5) 84 (52) 617 (28)

1.00 (818) 0.53 (801) 0.04 (560)

0.2 (19) 3.7 (59) 27 (30)

4.9 (4) 45 (36) 679 (38)

growing cells are transferred to spomlation medium, ongoing mitotic DNA synthesis is completed prior to the initiation of premeiotic DNA synthesis (Simchen et al. 1972). However, it remains a formal - if unlikely - possibility that certain meiotic processes such as recombination could occur in mitotic cells without the full complement of gene products required for normal meiotic development. Apart from the observation reported in this paper, other circumstantial findings also are inconsistent with the hypothesis that thymidylate deprivation provokes meiotic recombination. First, spontaneous mitotic exchange occurs more frequently in centromere-proximal intervals than does meiotic recombination, creating a relatively large region of map expansion around the centromere (Malone et al. 1980). Induction of recessive homozygosis by dTMP starvation in a cdc21 diploid also shows this 'mitotic' expansion (J. C. Game, personal communication). Second, diploids homozygous for radSO are capable of pre-meiotic DNA synthesis but not of meiotic recombination (Game et al. 1980) and display increased frequencies o f genetic exchange during thymidylate depletion (Kunz and Haynes 1981). Third, the rad54 mutation, which greatly suppresses induced mitotic recombination (Saeki et al. 1980) but permits sporulation (Game and Mortimer 1974) and meiotic gene conversion (B. A. Kunz, unpublished observations), abolishes completely the recombinagenicity of dTMP deprivation (Kunz and Haynes 1981). Thus, on the basis o f our results, and other findings discussed here, we conclude that recombination provoked by starvation for thymine nucleotides in yeast is independent o f the cells ability to undergo meiosis.

Acknowledgement. This work was supported by NSERC grants to R. H. Haynes and J. G. Little.

References Bisson L, Thorner J (1977) J Bacteriol 132:44-50 Barclay BJ, Kunz BA, Little JG, Haynes RH (1982) Can J Biochem (in press) Croes AF (1967) Planta 76:209-226 F/ith WW, Brendel M, Laskowski W, Lehmann-Brauns E (1974) Mol Gen Genet 132:335-345 Friis J, Roman H (1968) Genetics 59:33-36 Game JC (1976) Mol Gen Genet 146:313-315 Game JC, Mortimer RK (1974) Mut Res 24:281-292 Game JC, Zamb TJ, Braun RJ, Resnick MA, Roth RM (1980) Genetics 94:51-68 Kunz BA (1981) Ph.D. Thesis York University Toronto Canada Kunz BA, Haynes RH (1981) Mut Res (in press) Kunz BA, Barclay BJ, Game JC, Little JG, Haynes RH (1980) Proc Natl Acad Sci USA 77:6057-6061 Laskowski W, Lehmann-Brauns E (1973) Mol Gen Genet 125: 275-277 Little JG, Haynes RH (1979) Mol Gen Genet 168:141-151 Malone RE, Golin JE, Esposito MS (1980) Curt Genet 1:241248 Moens PB, Barclay BJ, Little JG (1981) Chromosoma 82:333340 Prakash S, Prakash L, Burke W, Monteleone BA (1980) Genetics 94:31-50 Roth R, Fogel S (1971) Mol Gen Genet 112:295-305 Roth R, Lusnak K (1970) Science 168:493-494 Saeki T, Machida I, Nakai S (1980) Murat Res 73:251-265 Schild D, Byers B (1978) Chromosoma 70:109-130 Simchen G, Pifion R, Salts Y (1972) Exp Cell Res 75:207-218 Zimmermann FK (1973) Mutat Res 21:263-269 Communicated b y B. Cox Received December 1, 1981

Thymineless recombination in Saccharomyces cerevisiae is independent of the ability to undergo meiosis.

Thymine nucleotide starvation is recombinagenic in Saccharomyces cerevisiae and induces formation of the 'nuclear dense body', a structure characteris...
264KB Sizes 0 Downloads 0 Views