X-ray enhances mating type switching in heterothallic strains of Saccharomyces cerevisiae

Summary A procedure for the determination of the frequency of mating type switching in heterothallic strains of Saccharomyces cerevisiae was worked out. In cell populations irradiated with X-rays the frequencies of switching were enhanced in a dose-dependent manner. The possible implication of this finding for understanding the mechanism of carcinogenesis is discussed.     

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.     

DNA damage induced mating type switching in Saccharomyces cerevisiae.

Haploid cells of the yeast Saccharomyces cerevisiae are able to undergo a differentiation-like process: they can switch their mating type between the a and the alpha state. The molecular mechanism of this interconversion of mating types is intrachromosomal gene conversion. It has been shown in a variety of studies that mating type switching in heterothallic strains can be induced by DNA damaging agents, and that different DNA damaging agents differ in the length of incubation after treatment required for induction. Because X-rays induce switching immediately after irradiation and because the DNA double-strand break repair pathway is required for switching, the event initiating heterothallic mating type switching is likely to be a DNA double-strand break. Therefore the assay for heterothallic mating type switching may screen for the induction of DNA double-strand breaks. Several aspects indicating a relationship of mating type switching to mechanisms associated with carcinogenesis are discussed.     

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.     

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.     

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.     

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.     

A new disruption vector (pDHO) to obtain heterothallic strains from both Saccharomyces cerevisiae and Saccharomyces pastorianus

Yeasts are responsible for several traits in fermented beverages, including wine and beer, and their genetic manipulation is often necessary to improve the quality of the fermentation product. Improvement of wild-type strains of Saccharomyces cerevisiae and Saccharomyces pastorianus is difficult due to their homothallic character and variable ploidy level. Homothallism is determined by the HO gene in S. cerevisiae and the Sc-HO gene in S. pastorianus. In this work, we describe the construction of an HO disruption vector (pDHO) containing an HO disruption cassette and discuss its use in generating heterothallic yeast strains from homothallic Saccharomyces species.     

Homothallic switching of Saccharomyces cerevisiae mating type genes by using a donor containing a large internal deletion.

Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.     

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.     

Directional bias during mating type switching in Saccharomyces is independent of chromosomal architecture

Haploid Saccharomyces cells have the remarkable potential to change mating type as often as every generation, a process accomplished by an intrachromosomal gene conversion between an expressor locus MAT and one of two repositories of mating type information, HML or HMR. The particular locus selected as donor is dictated by the mating type of the cell, a bias that ensures productive mating type interconversion. Here we use green fluorescent protein tagging of the expressor and donor loci on chromosome III to show that this preference for donor locus does not result from a predetermined organization of chromosome III: HML and MAT as well as HMR and MAT remain separated in cells of both mating types. In fact, cells in which the inappropriate donor locus is artificially tethered to MAT still predominantly select the correct donor. We find, though, that initiation of switching leads to a rapid association of the correct donor locus with MAT. Thus, in mating type switching in Saccharomyces, donor preference is imposed at commitment to recombination rather than at physical contact of interacting DNA strands.     

酵母交配过程的极性生长及其调控机制

酿酒酵母单倍体细胞能够与相反交配型的单倍体细胞发生交配。交配时酿酒酵母放弃原有出芽位点,根据信息素的浓度梯度,重新选择生长位点,向相反交配型细胞伸出突起进行极性生长。交配因子受体指导选择交配突起的位点,通过G蛋白激活Ste20p,将信号经由Ste11p、Ste7p和Fus3p组成的MAPK模块传递到Far1p和Ste12p等因子,调控相关基因的转录,抑制原有的出芽位点,选择新的生长位点,并使细胞周期停止在G1期,G蛋白与Cdc24p、Cdc42p和Bem1p等蛋白作用,聚集在细胞,使得肌协蛋白细胞骨架在交配突起处聚集,呈极性化分布,使细胞发生极性生长。     

Degradation of mating factor by alpha-mating type cells of Saccharomyces cerevisiae.

The change of the mating factor activity during the culture of Saccharomyces cerevisiae X-2180 1B, an alpha-mating type haploid strain, were followed. The activity increased rapidly during the exponential phase of growth, reached a maximum during the early stationary phase and then decreased. Oligopeptides comprising partial sequences of the mating factor were isolated from the culture fluids at various phases of cell growth. We concluded that the mating factor, a tridecapeptide, was degraded during culture into two peptides, Trp-His-Trp-Leu-Gln-Leu and Lys-Pro-Gly-Gln-Pro-Met-Tyr, by cleavage of the peptide bond between Leu-6 and Lys-7 of the mating factor. A dodecapeptide lacking the N-terminal Trp residue was not detected at any stage of cell growth examined.     

Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.

When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.     

Site of action of mating factor in a-mating type cell of Saccharomyces cerevisiae.

The process of the entry of FITC-conjugated mating factor into $\text{a}$-mating type cells of $\text{Saccharomyces}\text{cerevisiae}$ and its concentration into the nucleus were observed. But, when $\text{α}$-mating type cells or diploid cells of $\text{S.}\text{cerevisiae}$ were incubated with the FITC-conjugated mating factor, its adsorption to the cell surface of the test organisms and its incorporation into the cell did not occur. The peptides formed by the cleavage of mating factor by $\text{α}$-mating type cells of $\text{S.}\text{cerevisiae}$ were not adsorbed onto $\text{a}$-mating type cells.     

Nutritional and physiological factors affecting germination of heterothallic Saccharomyces cerevisiae ascospores.

Saccharomyces cerevisiae strain AP-3 was examined with respect to those nutritional requirements and physiological conditions which influence its germination rate. It was found that glucose as a carbon source supported the most rapid rate of germination for this heterothallic strain. In contrast, strain AP-3 spore germination was supported the least by the carbon sources potassium acetate and lactose. Of the nitrogen sources tested in culture medium containing glucose, the complex nitrogen sources peptone and casein hydrolysate appeared to be capable of stimulating germination better than a control culture containing ammonium sulphate. None of the amino acids screened were found to stimulate strain AP-3 germination compared with ammonium sulphate. The optimal culture medium pH for ascospore germination was 4.5 although spore germination could still be initiated by glucose between pH 3.0 and pH 7.5. Germination initiation by glucose was observed over a temperature range from 25 degrees C to 50 degrees C, but the optimal temperature appeared to be 40 degrees C.     

Mechanisms that ensure monogamous mating in Saccharomyces cerevisiae

Haploid cells of the budding yeast Saccharomyces cerevisiae communicate using secreted pheromones and mate to form diploid zygotes. Mating is monogamous, resulting in the fusion of precisely one cell of each mating type. Monogamous mating in crowded conditions, where cells have access to more than one potential partner, raises the question of how multiple-mating outcomes are prevented. Here we identify mutants capable of mating with multiple partners, revealing the mechanisms that ensure monogamous mating. Before fusion, cells develop polarity foci oriented toward potential partners. Competition between these polarity foci within each cell leads to disassembly of all but one focus, thus favoring a single fusion event. Fusion promotes the formation of heterodimeric complexes between subunits that are uniquely expressed in each mating type. One complex shuts off haploid-specific gene expression, and the other shuts off the ability to respond to pheromone. Zygotes able to form either complex remain monogamous, but zygotes lacking both can re-mate.