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Diploid cells of the yeast Saccharomyces cerevisiae become committed to recombination at meiotic levels without becoming committed to the meiotic disjunction of chromosomes. These two events of the meiotic process can be separated by removing cells from a meiosisinducing medium and returning them to a medium that promotes vegetative cell division. Cells removed at an appropriate time remain diploid, revert to mitosis, and display recombination with meiotic-like frequencies. Those removed after this time are committed to the completion of meiosis. Diploids of three conditional sporulationdeficient mutants (spol-l, spo2-1, and spo3-1) have been examined for recombination at restrictive temperatures. All exhibit commitment to recombination without commitment to meiotic disjunction as in the wild type. Cells of spol-l/spol-l do not replicate the spindle pole body for meiosis I; thus, recombination ability can be acquired by cells that do not proceed beyond this cytological stage.Diploid cells of Saccharomyces cerevisiae can be induced to undergo meisosis and ascospore formation (sporulation) by transfer from growth medium to sporulation medium. Several hours after this transfer, cells become committed to sporulation and do not revert to vegetative cell division when they are returned to growth medium (1). Premeiotic DNA synthesis and commitment to intragenic recombination (gene conversion) are initiated before cells become committed to meiosis (2, 3).In this study we have examined both intragenic and intergenic recombination in a normal diploid strain and in three conditional sporulation-deficient mutants (spol-l, spo2-1, and spo3-1). We provide evidence that commitment to intergenic as well as intragenic recombination at meiotic levels precedes commitment to meiotic chromosome disjunction and ascospore formation. METHODSStrains. Homothallic strains of Saccharomyces cerevisiae were used in this study. The genotype of the normal strain (Z193) and the relevant map distances (4) The genotypes of the three mutant diploids are identical to that of Z193 except that each is homozygous for a temperature-sensitive mutation preventing sporulation at 340: C816, spol-l/spol-l; C817, spo2-1/spo2-1; and C819, 8po3-1/8po3-l.Procedures for sporulation, dissection, and testing of auxotrophic requirements have been reported (5, 6).Giemsa Staining and Counting of Asci. The appearance of asci was determined by hemocytometer counts of samples fixed in 4% formaldehyde. Giemsa staining was performed as described (7).Allelism Tests. Diploids were tested for their allelic composition at trp5 and Iys2 as follows. Strains were grown on solid nutrient medium, replica plated to sporulation medium, and incubated at 200 (a permissive temperature for 8pO diploids) for 5 days. The sporulation plate was then replica-plated to tryptophanless or lysineless medium, and growth was scored after 2 days at 300. Due to genetic recombination during sporulation, replicas of heteroallelic diploids (lys2-1/lys2-2 or trp5-R/trp5-20) yield 50-100 prototr...

ABSTRACr Spontaneous reciprocal mitotic recombination in the yeast Saccharomyces cerevisiae, associated with heteroallelic recombination, occurs almost exclusively at the twostrand stage and involves recombination of unduplicated chromosomes (i.e., during GI) or the unduplicated regions of chromosomes during the S phase of mitosis. The associated heteroallelic recombination frequently reflects the formation of symmetric Holliday structures, is not strongly polarized with respect to conversion at the heteroallelic trp5 sites studied, occasionally results in simultaneous conversion of widely separated genetic markers, and is positively correlated with recombination of flanking markers.The recent compelling evidence that genetic recombination involves the formation of Holliday structures (1) prompted a reexamination of the classical G2 model (Fig. 1) (2) of mitotic recombination. Fig. 2 illustrates that sectored colonies can result from Holliday structures formed at the two-strand stage (e.g., Gi) when the Holliday structure is not cleaved and is replicated during the subsequent round of DNA synthesis (S phase). This mechanism, like that of exchange at the four-strand stage, generates the chromosomal genotypes resulting in daughter cells homozygous for markers distal to the site of exchange.The purpose of the experiment described below was to determine whether spontaneous mitotic recombination in yeast occurs at the four-strand stage as in Fig. 1 or at the two-strand stage as in Fig. 2. The experimental data lead to the conclusion that the spontaneous mitotic recombination observed is due primarily to recombination between either unreplicated homologous chromosomes (during Gl) or unreplicated regions of homologous chromosomes (during S) and therefore occurs at the two-strand stage for the chromosomal regions undergoing exchange. Media and Genetic Techniques. The compositions of the glucose nutrient medium (YPD), synthetic media, and sporulation medium and the relevant genetic techniques have been reported (4). MATERIALS AND METHODSMeasurement of Recombination Rates. Liquid YPD cultures of OR1, each consisting of 6 ml, were inoculated at ca 103 cells + buds per ml, grown aerobically at 30 , harvested during the exponential phase of growth (ca 2 X 107 cells + buds per ml), washed twice with sterile distilled water, and plated on complete synthetic medium and tryptophanless synthetic medium. The recombination rates resulting in tryptophanindependent prototrophic colonies were calculated by the method of the median as described by Lea and Coulson (5). 4436The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Acetate utilization and macromolecule synthesis during sporulation (meiosis) of Saccharomyces cerevisiae were studied. When diploid cells are transferred from glucose nutrient medium to acetate sporulation medium at early stationary phase, respiration of the exogenously supplied acetate proceeds without any apparent lag. At the completion of ascospore development, 62% of the acetate carbon consumed has been respired, 22% remains in the soluble pool, and 16% is incorporated into lipids, protein, nucleic acids, and other cell components. Measurements of the rate of protein synthesis during sporulation reveal two periods of maximal synthetic activity: an early phase coincidental with increases in deoxyribonucleic acid, ribonucleic acid, and protein cellular content and a later phase during ascospore formation. Experiments in which protein synthesis was inhibited at intervals during sporulation indicate that protein synthesis is required both for the initiation and completion of ascus development.

A procedure for detection of mutants exhibiting either enhanced or reduced spontaneous mutation during mitosis and/or meiosis has been developed to probe the joint genic control of spontaneous mutation and recombination in yeast. A semidominant mutator, rem1-1, recovered by this technique, exhibits enhanced spontaneous mutation,intragenic recombination, and intergenic recombination during mitosis. Diploids homozygous for rem1-1 exhibit normal levels of meiotic intragenic and intergenic recombination and diminished ascospore viability.