The Mating System in Saccharomyces

Many, but not all, Saccharomyces species are heterothallic.The mating types in heterothallic species are determined bytwo allelic genes. The mating-type genes occasionally mutatefrom one to the other. Vegetative cells of opposite mating typewere found in some single ascospore colonies. They show conjugationtubes, stimulated by the proximity of cells of the other matingtype. The cultures alao show zygotes, which on sporulation yieldplus(+) and minus(–) progenies. The zygotes bud off diploid vegetative cells which grow fasterthan the original haploid cells and so tend to replace them.If mutation occurs early, replacement is complete and the culturegives no mating reaction but sporulates. If it occurs late,the culture is a mixture of haploid cells giving a mating reactionand diploid cells that will sporulate.     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

Induction of trehalase activity on a nitrogen-free medium: a sporulation-specific event in the fission yeast, Schizosaccharomyces pombe

Summary Kinetic experiments with synchronously sporulating cultures of a homothallic h⁹⁰ strain of Schizosaccharomyces pombe showed that trehalase activity abruptly increased in the late sporulation process, coinciding with the appearance of visible spores. Trehalase activity was absent in vegetative cells. A set of strains different in genetic constitution at the mating type loci was tested for induction of trehalase on nitrogen-free sporulation medium. The appearance of trehalase activity on the sporulation medium was observed only in sporulating cultures; cultures of homothallic strains (h⁹⁰) and diploid strains heterozygous for mating type (h⁺/h^–), and mixed cultures of heterothallic h⁺ and h^– strains. Trehalase activity was not induced in nonsporogenic strains: heterothallic haploid strains (h⁺ and h^–), diploid strains homozygous for mating type (h⁺/h⁺ and h^–/h^–) and the homothallic strain harboring the mutation in the mat2 gene, which was unable to undergo the first meiotic division. Trehalose accumulation on the sporulation medium was observed solely in the sporulating cultures. These results led us to conclude that the induction of trehalase activity as well as the accumulation of trehalose in the medium lacking nitrogen sources was a sporulation-specific event under the control of the mating type genes.     

Mating-Type Functions for Meiosis and Sporulation in Yeast Act through Cytoplasm

Given a nutritional regime marked by a low nitrogen level and the absence of fermentable carbon sources, conventional a/α diploid cells of Saccharomyces cerevisiae exhibit a complex developmental sequence that includes a round of premeiotic DNA replication, commitment to meiosis and the elaboration of mature tetrads containing viable ascospores. Ordinarily, haploid cells and diploid cells of genotype a/a and α/α fail to display these reactions under comparable conditions. Here, we describe a simple technique for sporulation of α/α and a/a cells. Cells of genotype α/α are mated to haploid a cells carrying the kar1 (karyogamy defective) mutation to yield heterokaryons containing the corresponding diploid and haploid nuclei. The kar1 strains mate normally, but nuclei in the resultant zygotes do not fuse. When heterokaryotic cells are inoculated into sporulation media, they produce asci with six spores. Four spores carry genotypes derived from the diploid nucleus and the other two possess the markers originating from the haploid nucleus, i.e., the diploid nucleus divides meiotically while the haploid nucleus apparently divides mitotically. Similarly, the a/a genome is "helped" to sporulate as a consequence of mating with α kar1 strains. The results allow us to conclude that the mating-type functions essential for meiosis and sporulation are communicated and act through the cytoplasm and that sporulation can be dissociated from typical meiosis. This procedure will facilitate the genetic analysis of strains that are otherwise unable to sporulate.     

Premeiotic DNA synthesis in fission yeast

Sporulating and various non-sporulating strains of S. pombe, especially several mutants deficient in conjugation or meiosis, were compared with respect to DNA synthesis under sporulation conditions. Meiosis and sporulation were induced by a transfer to nitrogen-free medium. As synchronized mitotic division was observed in all the strains as a first response to the shift, reducing the DNA amount per cell from the replicated state in G2 to the unreplicated state in the G1 phase of the cell cycle. Cells of the heterothallic wild-type strains ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{+}}$ or $\text{h}{}^{\mathrm{?}}\text{h}{}^{\mathrm{?}}$) accumulated in G1 with respect to DNA synthesis when they were incubated separately. In a mixed culture of these strains a period of enhanced DNA synthesis was observed after the start of zygote formation. This period of synthesis was absent in mutant fus1, where only prezygotes accumulated. Hence we conclude that in zygotic meiosis the premeiotic DNA synthesis is confined to zygotes after conjugation has been completed. In the diploid sporulating wild-type strain ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{?}}$), capable of azygotic meiosis without prior conjugation, premeiotic DNA synthesis occurred between $\text{2}\text{1}\text{2}$ and 5 h after the shift to the sporulation medium. There was no significant premeiotic DNA synthesis observed in diploid cells of the meiosis-deficient mutants mei1 or mei3, whereas premeiotic DNA synthesis proceeded normally in mutant mei4, which is blocked at a stage after commitment to meiosis in opposition to both the other mutants.     

Sequential Logic of Polarity Determination during the Haploid-to-Diploid Transition in Saccharomyces cerevisiae

In many organisms, the geometry of encounter of haploid germ cells is arbitrary. In Saccharomyces cerevisiae, the resulting zygotes have been seen to bud asymmetrically in several directions as they produce diploid progeny. What mechanisms account for the choice of direction, and do the mechanisms directing polarity change over time? Distinct subgroups of cortical “landmark” proteins guide budding by haploid versus diploid cells, both of which require the Bud1/Rsr1 GTPase to link landmarks to actin. We observed that as mating pairs of haploid cells form zygotes, bud site specification progresses through three phases. The first phase follows disassembly and limited scattering of proteins that concentrated at the zone of cell contact, followed by their reassembly to produce a large medial bud. Bud1 is not required for medial placement of the initial bud. The second phase produces a contiguous bud(s) and depends on axial landmarks. As the titer of the Axl1 landmark diminishes, the third phase ultimately redirects budding toward terminal sites and is promoted by bipolar landmarks. Thus, following the initial random encounter that specifies medial budding, sequential spatial choices are orchestrated by the titer of a single cortical determinant that determines whether successive buds will be contiguous to their predecessors.     

Characterization of the genetic system of the xylose-fermenting yeast Pichia stipitis

High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid. Sporulation ability of these clones pointed to a homothallic life cycle. Mating was induced by cultivation under nutritionally poor conditions on malt extract medium. Conjugation was followed immediately by sporulation. However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started. Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate. The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads. Received: 29 February 1996 / Accepted: 29 March 1996     

Cytoduction as a new tool in studying the cytoplasmic heredity in yeast

Summary When crossing the haploid cells of genetically marked yeast strains we observed the appearance of both normal diploid zygotes and haploid nuclear cytoplasmic hybrids. The latter had the nuclear markers of one and the cytoplasmic marker (rho⁺) of the other parent. The autonomous cytoplasmic factor transfer was termed as cytoduction. Cytoduction is supposed to be the abortive form of yeast cell mating. Only about 1% of cytoductants is usually observed.Cytoduction can be used as a simple test on cytoplasmic determination of some characters. We observed the transfer into cytoductant cells of not only rho⁺ marker but of resistance factors to antibiotics (erythromycin, neomycin) and killer factor as well. Cytoduction can be applied towards constructing strains having the identical nucleus genotype with mitochondria and other cytoplasmic factors of different origin.In crossing strains with doubly marked mitochondria recombination of mitochondrial markers in cytoductant haploid cells was observed, the pattern of which was similar to that of mitochondrial recombination in normal zygotes.     

The G protein β subunit Gpb1 ofSchizosaccharomyces pombe is a negative regulator of sexual development

ASchizosaccharomyces pombe homolog of mammalian genes encoding G proteinβ subunits,gpb1 ⁺, was cloned by the polymerase chain reaction using primer pairs that correspond to sequences conserved in several Gβ genes of other species followed by screening of genomic and cDNA libraries. Thegpb1 gene encodes 317 amino acids that show 47% homology with human Gβ ₁ and Gβ ₂ and 40% homology withSaccharomyces cerevisiae Gβ protein. Disruption of thegpb1 gene indicated that this gene is not required for vegetative cell growth. However,gpb1-disrupted haploid cells mated and sporulated faster than wild-type cells, both in sporulation (MEA) and in complex medium (YE): when examined 23 h after transfer to sporulation medium, 35% ofgpb1-disrupted haploid pairs had undergone conjugation and sporulation, whereas only 3–5% of wild-type haploid pairs had done so. Overexpression of thegpb1 gene suppressed this facilitated conjugation and sporulation phenotype ofgpb1-disrupted cells but did not cause any obvious effect in wild-type cells. Co-disruption of one of the twoS. pombe Gα-subunit genes,gpa2, in thegpb1-disrupted cells did not change the accelerated conjugation and sporulation phenotype of thegpb1 ^? cells. However, co-disruption of theras1 gene abolished thegpb1 ^? phenotype. These results suggest that Gpbl is a negative regulator of conjugation and sporulation that apparently works upstream of Ras1 function inS. pombe. The possible relationship of Gpbl to two previously identified, putative Gα proteins ofS. pombe is discussed.     

A genetic study of x-ray sensitive mutants in yeast

A set of 64 mutants of Saccharomyces cerevisiae that confer sensitivity to X-ray inactivation were analyzed genetically to determine the number of genetic loci involved. The mode of interaction of various combinations of mutants was also determined. A minimum of 17 genes, when mutant, increase X-ray sensitivity of yeast, primarily by eliminating the resistance of budding haploid cells and by removing the shoulder on the survival curves of diploid cells. Eight mutant loci affect principally X-ray sensitivity while the remaining genes also control sensitivity to ultraviolet. Some of the genes when homozygous block sporulation or result in partial or complete sterility. Examination of the survival responses of multiple-mutant strains indicated a minimum of two pathways in the repair of X-ray damage. A number of the mutants have been mapped and these were found to be dispersed over the genome.     

New Heterothallic species of Hansenula

Summary As the genusHansenula is at present constituted,H. fabianii appears to be the first species of its phylogenetic line to have become independent of trees and bark beetles. It is also the last species of its line to occur in nature with the haploid, or basic, number of chromosomes. So near is it to the diploid level of evolution that a sporulation medium rich in malt extract favors the development of diploid vegetative cells in the period between conjugation of opposite sexes and the onset of sporulation. The diploid form is easily isolated from the mixture and, as long as sporulation is prevented, may be kept in pure state. Media that favor vegetative development of the species favor its occurrence as haploid vegetative cell.H. fabianii is most closely related toHansenula subpelliculosa Bedford, which occurs in nature as the diploid form. Both species have been isolated almost exclusively as contaminants of fermentations, and both have been used industrially in Oriental fermentations to make alcoholic beverages or foods.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Rare-Cell Fusion Events between Diploid and Haploid Strains of the Sexually Agglutinative Yeast HANSENULA WINGEI

Diploids of the yeast Hansenula wingei are nonagglutinative and do not form zygotes in mixed cultures with either sexually agglutinative haploid mating type. However, a low frequency of diploid x haploid cell fusions (about 10^-3) is detectable by prototrophic selection. This frequency of rare diploid x haploid matings is not increased after the diploid culture is induced for sexual agglutination. Therefore, we conclude that genes that repress mating are different from those that repress sexual agglutination.——Six prototrophs isolated from one diploid x haploid cross had an average DNA value (µg DNA per 10⁸ cells) of 6.19, compared to 2.53 and 4.35 for the haploid and diploid strains, respectively. Four prototrophs were clearly cell-fusion products because they contained genes from both the diploid and the haploid partners. However, genetic analysis of the prototrophs yielded results inconsistent with triploid meiosis; all six isolates yielded a 2:2 segregation for the mating-type alleles and linked genes.——Mitotic segregation of monosomic (2n-1) cells lacking one homolog of the chromosome carrying the mating-type locus is proposed to explain the rare production of sexually active cells in the diploid cultures. Fusion between such monosomic cells and normal haploids is thought to have produced 3n-1 cells, disomic for the chromosome carrying the mating-type locus. We conclude that in the diploid strain we studied, the physiological mechanisms repressing sexual agglutination and conjugation function efficiently, but events occuring during mitosis lead to a low frequency of genetically altered cells in the population.     

Genetic analysis of the non-sporulating phenotype of brewer's yeasts

The ploidies and sporulation abilities of six brewer's yeasts were examined. One (YB11-1) out of the six was triploid and sporulating, another (IFO2031) was haploid, and the others (IFO1167, IFO2003, S341 and YB3-7) were diploid and non-sporulating. The five non-sporulating strains did not have the premeiotic DNA synthesis. Their non-sporulating phenotypes were genetically analyzed by examining the sporulation abilities of hybrids between brewer's yeasts and standard genetic strains of Saccharomyces cerevisiae. All non-sporulating brewer's yeasts complemented 32 sporulation-deficient mutations (spoT–spoT23, spo1–spo5, spo7, spo8, spo10, and spo11). Hybrids between brewer's yeasts and haploid or diploid strains homozygous for the mating-type locus had poor or no sporualtion. On the contrary, hybrids between brewer's yeasts and diploid strains heterozygous for the mating-type locus sporulated at a high frequency. These results indicated that the non-sporulating phenotype of brewer's yeasts was caused by a deficiency of the mating-type genes rather than by mutations of sporulation genes. The Southern hybridization probed with the MATa gene showed polymorphisms in mating-type genes of brewer's yeasts.     

Adaptation mechanism of yeast to extreme environments: Construction of salt-tolerance mutants of the yeast Saccharomyces cerevisiae

A salt-tolerant mutant was derived from a haploid yeast, Saccharomyces cerevisiae DKD-5D-H, through repeated mutations with ethyl methanesulfonate. The mutant showed decreased cell size and could grow in the presence of NaCl up to 10%. A similar mutation method was also used to induce salt-tolerance in a diploid yeast, S. cerevisiae Kyokai no. 7. The mutant of the strain showed drastically decreased cell size, almost comparable with that of bacteria, and was resistant to NaCl up to 18%. These two mutation studies on haploid and diploid yeast strains indicated that the salt-tolerance was acquired through the decrease in yeast cell size.     

Lipid Synthesis During Sporulation of Saccharomyces cerevisiae

Lipid synthesis was studied in both sporulating (diploid) and nonsporulating (haploid) cells of Saccharomyces cerevisiae. Two phases of lipid synthesis occur in diploid cells transferred to sporulation medium. Phase I, which occurs during the first 12 h of exposure to sporulation medium, was also observed in the haploid strains. Phase II, occurring from the 20th to the 25th h, coincided with the appearance of mature asci and was observed only in the diploid cells. The majority of phospholipid synthesis took place during period I, whereas neutral lipid synthesis occurred during both periods. Phospholipid synthesis was virtually identical in both type and quantity in the sporulating and nonsporulating strains.     

Improvement of a Wine Saccharomyces cerevisiae Strain by a Breeding Program

Hybridization by spore conjugation was used to develop new and improved wine yeasts of Saccharomyces cerevisiae. The procedure was achieved with diploid, homothallic strains with high sporulation frequency and high spore viability. The method was verified by crossing flocculent and non-H₂S-forming strains. Single-spore descendants of the hybrids were studied by tetrad analysis with regard to the aforementioned characters and the other two winemaking traits, i.e., ethanol production and fermentation rate. A highly flocculent, non-H₂S-forming wine yeast strain with a high fermentation rate and high ethanol production was obtained.     

Characterization of industrial strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exhibiting filamentous growth induced by alcohols and nutrient deprivation

Summary The use of microorganisms in biotechnology is an important economic area of interest in Brazil, especially the use of Saccharomyces cerevisiae in the baking and alcohol fermentation industries. Dimorphism in S. cerevisiae (cell morphology alterations from budding cells to filamentous structures) has been observed in conditions of nitrogen and carbon deprivation and in the presence of fusel alcohols. This can be described as a defense mechanism that allows the yeast to forage for nutrients through cell elongation, hyphal formation and invasive growth. In this work fifteen industrial strains of S. cerevisiae (including haploid and diploid strains) isolated from the fermentative process for alcohol production were characterized for filamentation on solid culture media under growth conditions of carbon- and nitrogen-deprivation and in the presence of fusel alcohols. The majority of strains showed filamentation induced by isoamyl alcohol, butanol, isopropanol and isobutanol, but not by methanol. In rich medium (YEPD), both haploid and diploid strains showed invasive growth, although this kind of filamentous growth was more common in haploid strains. Similar results were observed when fructose or mannose was used as the sole carbon source. In nitrogen-deficient medium (SLAD) the strains did not filament. The results obtained indicate that the filamentation induced by higher alcohols and carbon deprivation (specially carbon) is a common process in industrial strains of S. cerevisiae contributing towards their maintenance/survival in adverse conditions.     

Mutants of the fission yeast Schizosaccharomyces pombe which alter the shift between cell proliferation and sporulation

Summary Mutants of S. pombe have been isolated which undergo conjugation and sporulation in rich medium, conditions which are normally inhibitory for these processes. Two of these mutants are also able to sporulate from the haploid state in the absence of heterozygosity at the mating type locus. These recessive mutants define a single nuclear gene called ran1 which is unlinked to mating type. It is proposed that the ran1 gene codes for an inhibitor in the control of the initiation of conjugation and sporulation. In wild type cells the inhibitory effect is released by nutritional starvation and heterozygosity at the mating type locus. This allows the cells to proceed to sporulation. The ran1 mutants are unusual in that they attempt to undergo a reductional meiotic division from the haploid state. They are also genetically unstable and generate extragenic suppressors at high frequency.     

Fidelity of conjugation in Saccharomyces cerevisiae.

Summary An efficient method for the production of synchronous zygotes in Saccharomyces cerevisiae is described. Cells were synchronised under defined conditions in either an a, mixed culture or by incubation of each mating type in cell-free medium in which cells of the opposite mating type had been grown. Synchronised cells were allowed to fuse under defined conditions on filter membranes. This method was used to test the fidelity of conjugation in S. cerevisiae. Under conditions where cells of a or mating type were in contact with up to 6 cells of each of two strains of opposite mating type, less than 1 multiple mating in 10⁴ diploid matings occurred. It is concluded that in sexual conjugation in S. cerevisiae some process distinct from cell contact restricts cell fusion to paired combinations of conjugant cells.     

Sexual co-flocculation by heterothallic cells of the fission yeast Schizosaccharomyces pombe modulated by medium constituents

Novel simple synthetic media for inducing sexual co-flocculation in a short time after mixing heterothallic fission-yeast (Schizosaccharomyces pombe) cells of h^- and h⁺ were devised; The most effective of these, mannose synthetic medium (MSM), contains 0.4% mannose as a carbon source in addition to galactose, KH₂PO₄ (pH4.0) and 4 vitamins. The addition of galactose to the medium suppressed the asexual self-flocculation but rather promoted the sexual co-flocculation. By transferring and mixing h^- and h⁺ cells grown in malt-extract broth plus galactose into MSM, these heterothallic strains were revealed to be sexually ready through a long period of the log to stationary phases. Furthermore, a variety of C sources and NH₄Cl at various concentrations in various media were examined for their effects upon sexual co-flocculation, conjugation and sporulation; it was found that the sugar concentration strictly affected the progress of the sequence of sexual reproduction at 26°C but not 30°C and that sexual co-flocculation of the heterothallic strains was induced only under lower concentrations of C and N source than that for the homothallic one.