Changes in production of the mating-type-specific glycoproteins, agglutination substances in association with mating type interconversion in homothallic strains of the yeast, Saccharomyces cerevisiae

Summary Sexual activity in homothallic strains of Saccharomyces cerevisiae was investigated. We succeeded in culturing homothallic haploid cells without conjugation, by lowering the pH value of the culture medium. In spore cultures of a homothallic strain both a and pheromones were detected. Agglutination substances of a and mating types were detected in homothallic haploid cells from spore cultures in early logarithmic phase regardless of mating type information at the HML and HMR loci, but either a or agglutination substance was detected predominantly in homothallic haploid cells from spore culture in late logarithmic phase, depending on mating type information at the HML and HMR loci.A part of this work was supported by Grants-in-Aid from Ministry of Education, Science and Culture, Japan, to N.Y.     

Induction of mating type interconversion in a heterothallic strain of Saccharomyces cerevisiae by DNA damaging agents

Mating type interconversion of the yeast, Saccharomyces cerevisiae, is an example of a directed genome rearrangement leading to a change in gene expression and in the differentiation state of a cell. In heterothallic haploid cells this switching of the mating type from a to alpha or vice versa, which is accomplished by an intrachromosomal gene conversion mechanism, is a rare event, happening about once per 10(6) cells per generation. Those cells that have changed their mating type can be trapped as diploid colonies by making them mate with tester cells possessing complementary markers. We found that treating haploids with UV light or with chemical carcinogens before they could mate resulted in a significant and dose-dependent enhancement of the numbers of diploid colonies. By genetic as well as by DNA hybridization analyses, these diploid clones were proved to be descendants of haploids which had changed their mating type by the bona fide gene conversion process. Thus, the DNA damaging agents had caused the induction of a directed gene rearrangement. It is suggested that induction of genome rearrangements might be part of a general response to DNA damage, at least in yeast cells. If similar responses also took place in cell populations constituting multicellular organisms, induced gene rearrangements and a generally enhanced mobility of the genome as a consequence of DNA damage might play a determining role in chemical and radiation-induced carcinogenesis.     

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.     

Genetic analysis of inducible sexual agglutination ability in the yeast Saccharomyces cerevisiae

Genetic regulation of the inducibility of sexual agglutination ability in the yeast Saccharomyces cerevisiae was studied. Detailed analysis of the degree of sexual agglutination was carried out; it showed that a greater number of genes are involved in the regulation of inducible sexual agglutination in strain H1-0 than previously assumed. Although dominancy of inducible phenotype over constitutive was confirmed, the effectiveness of one gene changing the constitutive phenotype to the inducible seemed to be somewhat low. Quantity per cell of agglutination substances responsible for sexual agglutination increased as the agglutination ability became greater.     

A new gene affecting the efficiency of mating-type interconversions in homothallic strains of Saccharomyces cerevisiae

Homothallic strains of Saccharomyes cerevisiae are able to switch efficiently from one mating genotype to another. From a single haploid spore arise both a and mating type cells, which then self-mate to produce a colony consisting almost exclusively of nonmating a/ diploid cells. We have isolated a mutant homothallic strain that gives rise to colonies that show bisexual mating behavior. The mating reaction is always asymmetric, that is, in some colonies a mating is much stronger than mating, while others show greater than a mating.-This mating phenotype arises from the presence of three cell types in a colony: some a/ nonmating diploids and an unequal number of a and haploid cells. The predominant haploid type is that of the original cell that gives rise to the colony. This mixture of cell types arises from a very reduced efficiency of homothallic mating-type interconversions in the mutant strain.-The mutation, designated switch (swi1-1), behaves as a single genetic locus. The mutation is centromere linked, but not linked to the mating type locus or to any of the homothallism genes: HO, HMa and HM. The switch mutation does not affect the efficiency of self-mating, but rather directly affects the frequency of interconversion of mating types.     

The dynamics of homologous pairing during mating type interconversion in budding yeast

Cells repair most double-strand breaks (DSBs) that arise during replication or by environmental insults through homologous recombination, a high-fidelity process critical for maintenance of genomic integrity. However, neither the detailed mechanism of homologous recombination nor the specific roles of critical components of the recombination machinery—such as Bloom and Werner syndrome proteins—have been resolved. We have taken a novel approach to examining the mechanism of homologous recombination by tracking both a DSB and the template from which it is repaired during the repair process in individual yeast cells. The two loci were labeled with arrays of DNA binding sites and visualized in live cells expressing green fluorescent protein–DNA binding protein chimeras. Following induction of an endonuclease that introduces a DSB next to one of the marked loci, live cells were imaged repeatedly to determine the relative positions of the DSB and the template locus. We found a significant increase in persistent associations between donor and recipient loci following formation of the DSB, demonstrating DSB-induced pairing between donor and template. However, such associations were transient and occurred repeatedly in every cell, a result not predicted from previous studies on populations of cells. Moreover, these associations were absent in sgs1 or srs2 mutants, yeast homologs of the Bloom and Werner syndrome genes, but were enhanced in a rad54 mutant, whose protein product promotes efficient strand exchange in vitro. Our results indicate that a DSB makes multiple and reversible contacts with a template during the repair process, suggesting that repair could involve interactions with multiple templates, potentially creating novel combinations of sequences at the repair site. Our results further suggest that both Sgs1 and Srs2 are required for efficient completion of recombination and that Rad54 may serve to dissociate such interactions. Finally, these results demonstrate that mechanistic insights into recombination not accessible from studies of populations of cells emerge from observations of individual cells.     

Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae.

In homothallic cells of Saccharomyces cerevisiae, a or alpha mating type information at the mating type locus (MAT) is replaced by the transposition of the opposite mating type allele from HML alpha or HMRa. The rad52-1 mutation, which reduces mitotic and abolishes meiotic recombination, also affects homothallic switching (Malone and Esposito, Proc. Natl. Acad. Sci. U.S.A. 77:503-507, 1980). We have found that both HO rad52 MATa and HO rad52 MAT alpha cells die. This lethality is suppressed by mutations that substantially reduce but do not eliminate homothallic conversions. These mutations map at or near the MAT locus (MAT alpha inc, MATa-inc, MATa stk1) or are unlinked to MAT (HO-1 and swi1). These results suggest that the switching event itself is involved in the lethality. With the exception of swi1, HO rad52 strains carrying one of the above mutations cannot convert mating type at all. MAT alpha rad52 HO swi1 strains apparently can switch MAT alpha to MATa. However, when we analyzed these a maters, we found that few, if any, of them were bona fide MATa cells. These a-like cells were instead either deleted for part of chromosome III distal to and including MAT or had lost the entire third chromosome. Approximately 30% of the time, an a-like cell could be repaired to a normal MATa genotype if the cell was mated to a RAD52 MAT alpha-inc strain. The effects of rad52 were also studied in mata/MAT alpha-inc rad52/rad52 ho/HO diploids. When this diploid attempted to switch mata to MATa, an unstable broken chromosome was generated in nearly every cell. These studies suggest that homothallic switching involves the formation of a double-stranded deoxyribonucleic acid break or a structure which is labile in rad52 cells and results in a broken chromosome. We propose that the production of a double-stranded deoxyribonucleic acid break is the lethal event in rad52 HO cells.     

Stationary-phase mutations in proofreading exonuclease-deficient strains of the yeast Saccharomyces cerevisiae

In order to understand the role of yeast polymerases in spontaneous mutagenesis in non-growing cells we have studied the effects of mutations that impair the 3'--> 5' exonuclease function of polymerases delta (pol3-01) and epsilon (pol2-4) on the spontaneous reversion frequency of the frameshift mutation his7-2 in cells starved for histidine. We showed that for each exonuclease-deficient mutant the rate of reversion per viable cell per day observed in stationary-phase cells remained constant up to the 9th day of starvation (while the number of viable cells dropped), and was very similar to that observed in the same mutants during the growth phase. These data suggest that both DNA polymerases are involved in the control of mutability in non-growing cells.     

Physical monitoring of mating type switching in Saccharomyces cerevisiae.

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.     

A defect in the sterol : Steryl ester interconversion in a mutant of the yeast,Saccharomyces cerevisiae

A culture cycle dependent interconversion of sterols and steryl esters is disturbed in a mutant of Saccharomycescerevisiae. Independent extragenic suppressors to this mutant return the mutant's pleiotropic phenotype to that of the parental wild type. Concomitant with the alterations in interconversion, modifications were found in the yeast proteins that antigenically react with antibodies elicited against mammalian apolipoproteins. Suppressor mutations returned the aberrant immunoblot banding pattern of the mutant to that of the wild type in apolipoprotein B.     

Ste50p sustains mating pheromone-induced signal transduction in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the hetero-trimeric G protein transduces the mating pheromone signal from a cell-surface receptor. Free Gβγ then activates a mitogen-activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal-transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone-induced signal which leads cells to hormone-induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone-induced signal transduction, and overexpression of STE50 intensifies the pheromone-induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the α-pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two-hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae.     

The sexual agglutination substance is secreted through the yeast secretory pathway in Saccharomyces cerevisiae

Molecular Genetics and Genomics - A Saccharomyces cerevisiae a strain carrying the secretory mutation sec1, sec7 or sec18 showed no sexual agglutination ability when treated with α pheromone...     

Intermediates of recombination during mating type switching in Saccharomyces cerevisiae.

We have identified two novel intermediates of homothallic switching of the yeast mating type gene, from MATa to MAT alpha. Following HO endonuclease cleavage, 5' to 3' exonucleolytic digestion is observed distal to the HO cut, creating a 3'-ended single-stranded tail. This recision is more extensive in a rad52 strain unable to switch. Surprisingly, the proximal side of the HO cut is protected from degradation; this stabilization depends on the presence of the silent copy donor sequences. A second intermediate was identified by a quantitative application of the polymerase chain reaction (PCR). The Y alpha-MAT distal covalent fragment of the switched product appears 30 min prior to the appearance of the MAT proximal Y alpha junction. No covalent joining of MAT distal to HML distal sequences is detected. We suggested that the MAT DNA distal to the HO cut invades the intact donor and is extended by DNA synthesis. This step is prevented in a rad52 strain. These intermediates are consistent with a model for MAT switching in which only the distal side of the HO cut is initially active in strand invasion and transfer of information from the donor.     

Ethylene production by metabolic engineering of the yeast Saccharomyces cerevisiae

The non-ethylene producing yeast, Saccharomyces cerevisiae, was transformed into an ethylene producer by introducing the ethylene forming enzyme from the plant pathogenic bacterium Pseudomonas syringae. Cultivation of the metabolically engineered strain was performed in well-controlled bioreactors as aerobic batch cultures with an on-line monitoring of ethylene production. The highest productivity was obtained during the respiro-fermentative growth on glucose but there was also a significant rate of formation during the subsequent phase of ethanol respiration. Furthermore, investigations were performed whether substitution of the original nitrogen source, NH(4)(+), for glutamate could improve productivity and yield of ethylene even more. The rationale being that one of the substrates for the enzyme is 2-oxoglutarate and this compound can be formed from glutamate in a single reaction. Indeed, there was a substantial improvement in the rate of production and the final yield of ethylene was almost three times higher when NH(4)(+) was replaced by glutamate.