CHEMICAL NATURE OF YEAST SEXUAL HORMONES

From culture media of yeast cells of the two mating types, a and α, hormones, which made cells of the opposite mating type expand, were extracted with methylene chloride. They were then isolated by either thin layer chromatography or vacuum distillation. The isolated hormones showed color reactions of steroidal compounds. According to Rf values and UV absorption spectra, the two yeast hormones apparently are different from testosterone and estradiol, which have activity similar to that of the yeast sexual hormones excreted by a and α type cells, respectively.     

Rapid identification and isolation of zygotes and the kinetics of mating in Saccharomyces cerevisiae

Summary Flow cytometry has been used in the selection of fusion products of the yeast Saccharomyces cerevisiae. Parental cells of opposite mating type were stained with different fluorescent dyes, permitting rapid identification of zygotes from natural matings based on dual-color flow cytometry. This procedure was then used to study the kinetics of mating in yeast and the physical and biological parameters that affect these kinetics, such as cell concentration, parental ratios, and parental strain growth rate.     

The sxa2-dependent inactivation of the P-factor mating pheromone in the fission yeast Schizosaccharomyces pombe

Haploid cells of the fission yeast Schizosaccharomyces pombe exist in one of two mating types, referred to as M and P. Conjugation occurs between cells of opposite mating type and is controlled by the reciprocal action of diffusible pheromones. Loss of function of the sxa2 gene in M cells causes hypersensitivity to the P-factor mating pheromone and a reduction in mating efficiency. Here we demonstrate the secretion of an sxa2-dependent carboxypeptidase that inactivates P-factor by removal of the C-terminal leucine residue.     

The trans action of HMRa in mating type interconversion

Summary HML and HMR are the sites of cryptic mating type genes in the yeast Saccharomyces cerevisiae. In the presence of the HO gene, the information from HML or HMR (an a or cassette) is transferred to the mating type locus (MAT). HML, HMR, and MAT are located on chromosome III, yet are widely separeted. Similarly, in other yeasts, at least some of the genes involved in mating type interconversion are linked to the mating type locus. We demonstrate here that a cassette donor (HMR) and the cassette target (MAT) need not be physically linked for successful mating type interconversion. In particular, we show that HMR a on one chromosome can donate an a cassette to the mating type locus on a homologous chromosome III.     

Transmission and recombination of mitochondrial genes in Saccharomyces cerevisiae after protoplast fusion

Summary Protoplasts of auxotrophic strains of Saccharomyces cerevisiae of opposite and identical mating types carrying different mitochondrial drug-resistance markers, with both homosexual and heterosexual mitochondrial backgrounds, were induced to fuse by polyethylene glycol. After selective regeneration of prototrophic fusion products, the transmission and recombination frequencies of mitochondrial genes in populations of cells were determined and compared with those obtained in mating processes. The frequencies obtained in the fusion experiments proved very similar to those found in the zygote clones. The behavior of mitochondrial genes was apparently affected neither by nuclear mating type background nor by the method of transfer of mitochondrial genomes (i.e., protoplast fusion or mating), making possible mitochondrial genetic studies by protoplast fusion irrespective of the mating type barrier of yeast strains.     

Pre-release feeding on yeast hydrolysate enhances sexual competitiveness of sterile male Queensland fruit flies in field cages

Recent laboratory studies of mass‐reared flies in small cages have found that periods of just 24‐ or 48‐h access to yeast hydrolysate can substantially enhance mating performance of mass‐reared male Queensland fruit flies, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) (‘Q‐flies’). Using field cage tests that provide a better approximation of nature, we here investigated whether access to yeast hydrolysate for 48 h after adult emergence improves the ability of male and female mass‐reared, sterile Q‐flies to compete sexually with wild‐type flies that had been provided continuous access to yeast hydrolysate. Mating probability of sterile males was significantly increased by 48‐h access to yeast hydrolysate; sterile males provided 48‐h access to yeast hydrolysate had mating probability similar to that of wild males provided continuous access to yeast hydrolysate, whereas sterile males deprived of access to yeast hydrolysate had much lower mating probability. Unlike males, access to yeast hydrolysate for 48 h did not increase mating probability of sterile female Q‐flies. We instead found that wild females provided continuous access to yeast hydrolysate had higher mating probability than sterile females that did or did not have 48‐h access to yeast hydrolysate. This result raises the possibility that a bisexual Q‐fly strain might operate essentially as a male‐only release when the flies are given access to yeast hydrolysate during a 48‐h pre‐release holding period. Sterile males given access to yeast hydrolysate for 48 h mated significantly earlier in the evening than wild males and, as in other recent studies, this tendency was associated with an increased tendency to mate on the trees rather than the cage walls. There was no evidence of sexual isolation in this study, as wild and sterile mass‐reared flies showed no evidence of preferential mating with their own kind. Further studies are now needed to assess the potential for pre‐release access to yeast hydrolysate to improve sexual performance and longevity of sterile, mass‐reared, Q‐flies in the field.     

Sex pheromones of the yeast Hansenula wingei and their relationship to sex pheromones in Saccharomyces cerevisiae and Saccharomyces kluyveri

The yeast, Hansenula wingei has two mating types designated 5 and 21. Cells of each mating type were found to produce mating type-specific sex pheromone which induces sexual agglutinability of the opposite mating type. Crude fractions of these pheromones were prepared by using an Amberlite CG 50 (H⁺ type) column. The agglutinability-inducing action of the pheromones required glucose as carbon source, but no external nitrogen source. The action of the pheromones was inhibited by 5 g/ml cycloheximide. The optimum pH for the pheromone action was 4.0. Pheromones of Saccharomyces cerevisiae and Saccharomyces kluyveri induced sexual agglutinability of 5 mating type cells but did not that of 21 mating type cells. a Pheromones of the Saccharomyces yeasts had no effect on both 5 and 21 mating type cells. The sex pheromones of H. wingei had no effect on the sexual agglutinability of inducible a cells of S. cerevisiae. From the experimental results obtained so far, we propose to call 5 and 21 mating types in H. wingei a and mating types, respectively.     

Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica

Summary The mating type gene MA TA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.     

The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is only expressed in M cells and the gene product is responsible for the secretion of the mating pheromone, M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe. Received: 20 December 1996 / Accepted: 29 January 1997     

A link between very long chain fatty acid elongation and mating-specific yeast cell cycle arrest

Ceramides and sphingolipid intermediates are well-established regulators of the cell cycle. In the budding yeast Saccharomyces cerevisae, the complex sphingolipid backbone, ceramide, comprises a long chain sphingoid base, a polar head group, and a very long chain fatty acid (VLCFA). While ceramides and long chain bases have been extensively studied as to their roles in regulating cell cycle arrest under multiple conditions, the roles of VLCFAs are not well understood. Here, we used the yeast elo2 and elo3 mutants, which are unable to elongate fatty acids, as tools to explore if maintaining VLCFA elongation is necessary for cell cycle arrest in response to yeast mating. We found that both elo2 and elo3 cells had severely reduced mating efficiencies and were unable to form polarized shmoo projections that are necessary for cell-cell contact during mating. They also lacked functional MAP kinase signaling activity and were defective in initiating a cell cycle arrest in response to pheromone. Additional data suggests that mislocalization of the Ste5 scaffold in elo2 and elo3 mutants upon mating initiation may be responsible for the inability to initiate a cell cycle arrest. Moreover, the lack of proper Ste5 localization may be caused by the inability of mutant cells to mobilize PIP₂. We suggest that VLCFAs are required for Ste5 localization, which is a necessary event for initiating MAP kinase signaling and cell cycle arrest during yeast mating initiation.     

Interconversion of Yeast Mating Types II. Restoration of Mating Ability to Sterile Mutants in Homothallic and Heterothallic Strains

The two mating types of the yeast Saccharomyces cerevisiae can be interconverted in both homothallic and heterothallic strains. Previous work indicates that all yeast cells contain the information to be both a and α and that the HO gene (in homothallic strains) promotes a change in mating type by causing a change at the mating type locus itself. In both heterothallic and homothallic strains, a defective α mating type locus can be converted to a functional a locus and subsequently to a functional α locus. In contrast, action of the HO gene does not restore mating ability to a strain defective in another gene for mating which is not at the mating type locus. These observations indicate that a yeast cell contains an additional copy (or copies) of α information, and lead to the "cassette" model for mating type interconversion. In this model, HMa and hmα loci are blocs of unexpressed α regulatory information, and HMα and hma loci are blocs of unexpressed a regulatory information. These blocs are silent because they lack an essential site for expression, and become active upon insertion of this information (or a copy of the information) into the mating type locus by action of the HO gene.     

Pheromone communication in the fission yeast Schizosaccharomyces pombe

Conjugation between two haploid yeast cells is generally controlled by the reciprocal action of diffusible mating pheromones, cells of each mating type releasing pheromones that induce mating-specific changes in cells of the opposite type. Recent studies into pheromone signalling in the fission yeast Schizosaccharomyces pombe have revealed significant parallels with processes in higher eukaryotes and could provide the opportunity for investigating communication in an organism that is amenable to both biochemical and genetic manipulation.     

Genomic organization of multiple genes coding for rhodotorucine A,a lipopeptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides

Rhodotorucine A, a lipopeptide mating pheromone, is secreted from mating type A cells of Rhodosporidium toruloides and induces sexual differentiation of the opposite mating type a cells. Genome of A-type cells contains three homologous genes (RHA1, RHA2, and RHA3) encoding rhodotorucine A. Genomic Southern blot analysis using RHA1 DNA as a probe showed that RHA1 strongly hybridize with A-type genomic DNA but weakly with a-type, suggesting that the sequences of RHA genes were dissimilar in the opposite a-type genome. The range of dissimilar regions in a-type genome was searched using RHA-flanking DNA segments as probes. The result suggests that a-type genome lacks sequences coding for rhodotorucine A and its 5 upstream but contains its 3 non-coding sequences. The absence of mating pheromone genes in the opposite mating type genome suggests that the expression of mating-type-specific genes in R. toruloides is not controlled trans-criptionally, as shown in the yeast Saccharomyces cerevisiae.     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Dominance at a Fungus Mating Type Locus

THE action of the two alleles which control mating type in fungi with bipolar heterothallism is uncertain. A diffusible oligopeptide, produced by the α mating type of Saccharomyces cerevisiae, blocks division and induces copulatory processes in cells of a mating type^2,3 and in some yeasts the mating type locus may be induced to mutate by other genes in the same cell^4,5. These observations in yeast, however, may not be representative of the other fungi with bipolar heterothallism².     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected. An erratum to this article can be found at     

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.     

The Genetic Control of Cell Growth and Development in Yeast Saccharomyces cerevisiae: Disturbed Sporulation in Diploids with a Decreased Activity of the Ras/cAMP Signal Transduction Pathway

Seven haploid strains (four with the MAT mating type and three with the MATa mating type) were selected from the Peterhof genetic collection of yeast. Previous phenotypic analysis assigned six of these strains to a physiological group of strains with changed activity of the Ras/cAMP signal transduction pathway. The haploids were crossed, and the resulting 12 diploids showed higher glycogen accumulation, tolerance to heat shock and nitrogen starvation, and sporulation in complete media. Ten of the diploids expressed the hypersporulation phenotype (higher sporulation efficiency). The phenotypic characters of these ten diploids suggested a reduced activity of the Ras/cAMP pathway. All 12 diploids were tested for sporulation and production of two groups of asci (those with one or two spores and those with three or four spores) as dependent on culture conditions (21, 30, or 34°C; standard sporulation medium or a complete medium containing potassium acetate or glycerol in place of glucose). Sporulation proved to depend on temperature and medium composition. The results are collated with the data on yeast phenotypes associated with a lower activity of the Ras/cAMP signal transduction pathway.     

Induced Production of a Sexual Hormone in Yeast

We have already described that α hormone excreted by α type cells expands cells of α type specifically and α hormone excreted by α type cells expands α type cells in Saccharomyces terevisiae. Both substances are steroidal compounds. In the present experiment the third hormonal substance has been found. a Hormone induced excretion of a methylene chloride-insoluble substance which expands a type cells specifically, when added to a type cells. Testosterone showed the same type of action as that of a hormone. On the other hand, a hormone did not show an action to induce excretion of methylene chloride-insoluble hormonal substance, when added to a type cells. Relation among three hormonal substances described above seems to be important to make clear the mating process of the yeast.     

Genetic and physiological variants of yeast selected from palm wine

Genetic screening of 1200-palm wine yeasts lead to the selection of fourteen isolates with various genetic and physiological properties. Nine of the isolates were identified as Saccharamyces species, three as Candida species, one as Schizosaccharomyces species and one as Kluyveromyces species. Five of the isolates were wild type parents, two were respiratory deficient mutants (rho) and nine were auxotrophic mutants. Four isolates were heterozygous diploid (αa) and two were homozygous diploid (aaα α) for the mating a mating types were further identified on mating with type loci. Four Mat α and four Mat a types were further identified on mating with standard haploid yeast strains. Forty-five percent sporulated on starvation medium producing tetrads. Fifty-two percent of the four-spored asci contained four viable spores. Maximum specific growth rate [μ_max] of the fourteen isolates range from0.13–0.26, five isolates were able to utilize exogenous nitrate for growth. Percentage alcohol production range between 5.8–8.8% for palm wine yeast, 8.5% for bakers’ yeast and 10.4% for brewers yeast. The palm wine yeast were more tolerant to exogenous alcohol but had a low alcohol productivity. Hybridization enhanced alcohol productivity and tolerance in the palm wine yeasts. This revised version was published online in June 2006 with corrections to the Cover Date.