Interconversion of Yeast Mating Types

The HO gene promotes interconversion between a and a mating types. As a consequence, homothallic diploid cells are formed by mating between siblings descended from a single crH0 or a HO spore. In order to determine the frequency and pattern of the mating-type switch, we have used a simple technique by which the mating phenotype can be assayed without losing the cell to the mating process itself. Specifically, we have performed pedigree analysis on descendants of single homothallic spores, testing these cells for sensitivity to a-factor. The switch from a to a and vice versa is detectable after a minimum of two cell divisions. 50% of the clones tested showed switching by the four-cell stage. Of the four cells descended from a single cell, only the oldest cell and its immediate daughter are observed to change mating type. This pattern suggests that one event in the switching process has occurred in the first cell division cycle. Restriction of the switched mating-type to two particular cells may reflect the action of the homothallism system followed by nonrandom segregation of DNA strands in mitosis. The mating behavior of cells which have sustained a change in mating type due to the HO gene is indistinguishable from that of heterothallic strains. T H E mating type locus of Saccharomyces cerevisiae exists in two states, a olr a, which control the ability of yeast cells to mate (MORTIMER and HAWTHORNE 1969). Cells which are a (or a/a diploids) can mate efficiently with Q (or a/a diploids), whereas cells of like mating-type mate with each other only rarely (HAWTHORNE 1963a). Ability of strains to mate is associated with the ability to produce and respond to diffusible, extracellular mating factors (MACKAY and MANNEY 1974a,b). In particular, a cells (but not a or a/a cells excrete an oligopeptide pheromone ‘‘a factor” which causes a cells to stop in the cell cycle just before the initiation of DNA synthesis (BUCKING-THROM et al. 1973; HEREFORD and HARTWELL 1974). The structure of the mating-type locus, i.e., the number of genes and controlling sites, is unknown, although mutations inseparable from the mating-type locus which block mating have been described (MACKAY and MANNEY 1974a,b; HAWTHORNE, personal communication). It is likely that the Present address: Section of Genetics, Development and Physiology, Cornell University, Ithaca, New York 14853. Genetics 83: 245-258 June, 1976. 246 J. B. HICKS A N D I. HERSKOWITZ mating-type locus specifies control functions for both mating and for sporulation. Indirect support for this view comes from the identification of genes via isolation of mating-deficient mutants (MACKAY and MANNEY 1974a,b). Different classes of mutants carried mutations unlinked to the mating type locus which affect only a cells, only a cells, or both a and a. It has been proposed that the mating-type locus regulates expression of these mating-specific genes ( MACKAY and MANNEY 1974a,b). When an a/a diploid is induced to sporulate, an individual spore gives rise to a culture of haploid cells either of mating type a or mating type a, none of which can be induced to sporulate. Some yeast strains (“homothallic” strains) behave differently from the above described “heterothallic” strains in that cultures grown from a single haploid spore contain diploid cells capable of sporulating (MORTIMER and HAWTHORNE 1969). These sporulating cells are formed from siblings descended from a single spore which have mated with each other (HAWTHORNE 196313; TAKANO and OSHIMA 1967). The genes responsible for homothallism have been in some cases derived from crosses between Saccharomyces cereuisiae and other yeasts (HAWTHORNE 196313; TAKANO and OSHIMA 1967) or derived from heterothallic strains by mutation (HOPPER and HALL 1975). In the case studied in this paper, homothallic and heterothallic strains differ by a single Mendelian gene, HO, which is unlinked to the mating-type locus (OSHIMA and TAKANO 1971; this paper). The sequence of events leading to the formation of the diploid is reported to occur as follows (HAWTHORNE 1963b; OSHIMA and TAKANO 1971) : A single spore is either of genotype a HO or a HO. After a number of cell divisions, the mating phenotype of some cells changes, and neighboring cells mate to form a diploid of constitution a/a HO/HO. Such a diploid is able to sporulate and gives rise to four spores each able to repeat the process. The change in mating phenotype promoted by the HO gene appears to be a change at the mating-type locus itself. When the HO gene is crossed out of strains which have sustained a change in mating type, the new mating type is maintained (TAKANO and OSHIMA 1970a; this paper). In addition, the new mating type appears to be stable since it segregates normally in the absence of the homothallism genes (TAKANO and OSHIMA 1970b). The homothallism genes therefore cause a stable inherited change at the mating-type locus. OSHIMA and TAKANO (1971) have proposed that the homothallism genes are concerned with a “controlling element,” analogous to those of maize (MCCLINTOCK 1956), which can reversibly associate with the mating-type locus. We have undertaken the present work in order to show in as simple a manner as possible the behavior of homothallic strains, with a goal of understanding the mechanism by which mating types are interconverted. We present here a pedigree analysis of descendants of single homothallic spores, in which the individual cells are tested for sensitivity to the mating pheromone, a factor ( SCHERER, HAAG and DUNTZE 1974). We have thus been able to determine the mating-type behavior of each cell easily and without losing the cell to the mating process itself. In addition, we demonstrate the stability of the mating-type locus in strains with changed mating type. YEAST M A T I N G T Y P E INTERCONVERSION 247 MATERIALS A N D METHODS Media. YEPD broth is 1% yeast extract, 2% peptone, and 2% glucose (added after autoclaving). YM-1 broth is 1% succinic acid, .6% NaOH, .5% yeast extract, 1% peptone, 57% Yeast Nitrogen Base (Difco), 2% glucose (added after autoclaving), and adenine and uracil (final concentration 10 pg/ml). YEPD plates contain in addition 2% agar. SD (minimal medium) plates for scoring nutritional requirements are .67% Yeast Nitrogen Base (Difco), 2% glucose (added after autoclaving), 2% agar, and the following additions as necessary: histidine, methionine, tryptophan, adenine, uracil, arginine (20 pg/ml) ; leucine, lysine, tyrosine (30 pg/ml) ; phenylalanine (50 pg/ml) ; threonine (200 pg/ml). SPOR plates (for sporulation) contain 1.5% potassium acetate, .25% yeast extract, and .1% glucose supplemented with additions as needed. Agar slabs for ascus dissection and pedigree analysis contain 4% glucose, 2% peptone, 1% yeast extract, and 3% agar. Strains. All strains used are described in Table I. The HO strain (X10-1B) used in most experiments was derived by a series of backcrosses between homothallic strain Z140-9A and heterothallic a strain X5-39. Diploids were formed by spore to cell matings between spores from a sporulated culture of Z140-9A and a vegetative culture of X5-39. Diploids were sporulated, and HO segregants identified by testing colonies for sporulation and mating (by the prototroph complementation assay). The latter test takes advantage of the fact that colonies derived from a homothallic spore contain primarily a /a (hence non-mating) diploids. Spores from an HO segregant were mated with an X5-39 cell. The resultant diploid was sporulated, and an HO spore again mated with X5-39. Strain X10-1B is a homothallic segregant obtained from this last sporulation. Table 2 shows the behavior of the HO gene in representative crosses, and demonstrates that homothallic behavior (HO) and heterothallic behavior (ho) segregate 2:2. The homothallic strain Z140-9A, supplied by G. Fink, carries the D (“Diploidizer”) gene which confers homothallism (WINGE and ROBERTS 1949; HAWTHORNE 1963b; ESPOSITO et al. 1970; ESPOSITO et al. 1972). Work by HARASHIMA, NOGI and OSHIMA (1974) indicates that this strain carries genes HO HMa HMa. Since crosses between Z140-9A and standard heterothallic strain X5-39 show 2 homothallic: 2 heterothallic spores, we believe that X5-39 is ho HMa HMa (see also ESPOSITO et al. 1972).