Meiotic Gene Conversion Mutants in SACCHAROMYCES CEREVISIAE . I. Isolation and Characterization of pms1-1 and pms1-2

The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.     

Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae

Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

Tests for recombinagens in fungi.

Three types of mitotic recombination can be studied in Aspergillus nidulans and Saccharomyces cerevisiae: (1) The classical type of reciprocal mitotic crossing-over which can be detected when it occurs between non-sister chromatids at the four-strand stage followed by co-segregation of a crossing-over and a non-crossing-over chromatid in the subsequent mitotic division. Consequently, mitotic crossing-over reflects cellular responses to primary genetic damage in the G2 phase of the cell cycle. (2) Mitotic gene conversion is a unidirectional event of a localized transfer of genetic information between non-sister chromatids which in yeast can extend to segments of up to 18 cM and even beyond 22 cM in Aspergillus nidulans. Mitotic gene conversion can also occur between unreplicated chromatids and lead to the expression of the newly created genotype without any need for a subsequent mitotic cell division. It reflects a cellular response in G1. (3) Mitotic sister-strand gene conversion can be studied in a recently constructed strain with the same technical ease as classical non-sister chromatid gene conversion. It can be induced by chemicals which do not induce mutation in the Salmonella system and non-sister chromatid gene conversion. Mitotic segregation in Saccharomyces cerevisiae results almost exclusively from crossing-over and gene conversion whereas mitotic chromosomal malsegregation contributes only very little. In contrast to this, in Aspergillus nidulans, both processes contribute considerably so that mitotic segregants always have to be tested for their mechanistic origin.     

The detection of mitotic and meiotic chromosome gain in the yeast Saccharomyces cerevisiae: effects of methyl benzimidazol-2-yl carbamate, methyl methanesulfonate, ethyl methanesulfonate, dimethyl sulfoxide, propionitrile and cyclophosphamide monohydrate

The diploid yeast strain BR1669 was used to study induction of mitotic and meiotic chromosome gain by selected chemical agents. The test relies on a gene dosage selection system in which hyperploidy is detected by the simultaneous increase in copy number of two alleles residing on the right arm of chromosome VIII: arg4-8 and cup1S (Rockmill and Fogel. 1988; Whittaker et al., 1988). Methyl methanesulfonate (MMS) induced mitotic, but not meiotic, chromosome gain. Methyl benzimidazol-2-yl carbamate (MBC) and ethyl methanesulfonate (EMS) induced both mitotic and meiotic chromosome gain. Propionitrile, a polar aprotic solvent, induced only mitotic chromosome gain; a reliable response was only achieved by overnight incubation of treated cultures at 0 degrees C. MBC is postulated to act by binding directly to tubulin. The requirement for low-temperature incubation suggests that propionitrile also induces aneuploidy by perturbation of microtubular dynamics. The alkylating agents MMS and EMS probably induce recombination which might in turn perturb chromosome segregation. Cyclophosphamide monohydrate and dimethyl sulfoxide (DMSO) failed to induce mitotic or meiotic chromosome gain.     

Dbf2 is essential for cytokinesis and correct mitotic spindle formation in Candida albicans

We have characterized the DBF2 gene, encoding a protein kinase of the NDR family in Candida albicans, and demonstrate that this gene is essential for cell viability. Conditional mutants were constructed by using the MET3 promoter to analyse the phenotype of cells lacking this kinase. The absence of Dbf2 resulted in cells arrested as large-budded pairs that failed to contract the actomyosin ring, a function similar to that described for its Saccharomyces cerevisiae orthologue. In addition to its role in cytokinesis, Dbf2 regulates mitotic spindle organization and nuclear segregation as Dbf2-depleted cells have abnormal microtubules and severe defects in nuclear migration to the daughter cell, which results in a cell cycle block during mitosis. Taken together, these results imply that Dbf2 performs several functions during exit from mitosis and cytokinesis. Consistent with a role in spindle organization, the protein localizes to the mitotic spindle during anaphase, and it interacts physically with tubulin, as indicated by immunoprecipitation experiments. Finally, DBF2 depletion also resulted in impaired true hyphal growth.     

A yeast strain for simultaneous detection of induced mitotic crossing over,mitotic gene conversion and reverse mutation

A diploid yeast strain is described which can be used to study induction of mitotic crossing over, mitotic gene conversion and reverse mutation.Mitotic crossing over can be detected visually as pink and red twin sectored colonies which are due to the formation of homozygous cells of the genotype ade240/ade240 (deep red) and ade-2-119/ade2-119 (pink) from the originally heteroallelic condition ade2-40/ade2-119 which forms white colonies.Mitotic gene conversion is monitored by the appearance of tryptophan non-requiring colonies on selective media. The alleles involved are tryp5-12 and trp5-27 derived from the widely used strain D4.Mutation induction can be followed by the appearance of isoleucine non-requiring colonies on selective media. D7 is homoallelic ilv1-92/ilv1-92. The isoleucine requirement caused by ilv1-92 can be alleviated by true reverse mutation and allele non-specific suppressor mutation.The effects of ethyl methanesulfonate (EMS), nitrous acid, ultraviolet light and hycanthone methanesulfonate were studied with D7 stationary phase cells. Mitotic crossing over as monitored by red/pink twin sectored colonies was almost equally frequent among normal and convertant cells. This showed again that mitotic recombination is not due to the presence fo a few cells committed to meiosis in an otherwise mitotic cell population.The dose-response curves for induction of mitotic gene conversion and reversion of the isoleucine requirement were exponential. In contrast to this, the dose-response curve for induction of twin sectored red and pink colonies reached a plateau at doses giving about 30% cell killing. This could partly be due to lethal segregation in the progeny of treated cells.None of the agents tested would induce only one type of mitotic recombination, gene conversion or crossing over. There was, however, some mutagen specificity in the induction of isoleucine prototrophs.     

The causes of instability of artificial mini-chromosomes in yeasts mutant for chl genes

In mutants chl2, chl3, chl5, and chl6, which control mitotic chromosome transmission, the behaviour of the centromeric plasmids with various genes was analyzed. The main cause of chromosome instability in chl2, chl5, and chl6 is chromosome loss during cell division (1:0 segregation). The main cause of chromosome instability in chl3. is nondisjunction (2:0 segregation). According to this, the chl3 mutant, but not other chl's, cannot maintain the mini-chromosome with SUP11 gene. This gene causes cell death in high copy number. Chromosome nondisjunction in chl3 is also confirmed by the data on the mini-chromosome carrying CUP1 gene responsible for copper-resistance in yeast. The copper resistancy level in chl3 transformants is much higher than in chl5 or wild type transformants. Elevated copper resistance of chl3 transformants is caused by the transit accumulation of CUP1-carrying mini-chromosome in part of the cell population as a result of segregation mistakes upon cell divisions. Thus, the CHL3 gene is a new gene that controls the process of mitotic chromosome disjunction in yeast.     

Characterization and mapping of an informational suppressor in Aspergillus nidulans

The present work was undertaken to characterize a suppressor gene present in a mutant strain of A. nidulans obtained with NTG (N-Methyl-N'-Nitro-N-Nitrosoguanidine). Analyses of this mutant have shown that this suppressor, designated suO1, induces phenotypic co-reversion of several auxotrophic mutations and makes the strain sensitive to aminoglycoside antibiotics and lower temperatures. suO1 has shown to be on linkage group VIII. The vegetative growth of the mutant strain is very unstable because the suppressor gene induces the production of prototrophic mitotic sectors. The strains bearing the suO1 gene produce cleistothecia containing a reduced number of viable ascospores during the sexual cycle. The segregation of the genetic markers has also been observed in the mutant strain self crossed. From the above results it may be concluded that suO1 is an informational suppressor.     

Long-Tract Mitotic Gene Conversion in Yeast: Evidence for a Triparental Contribution during Spontaneous Recombination

In Saccharomyces cerevisiae, spontaneous mitotic gene conversion at one site is statistically correlated with recombination at other loci. In general, coincident conversion frequencies are highest for tightly linked markers and decline as a function of intermarker distance. Paradoxically, a significant fraction of mitotic gene convertants exhibits concomitant nonreciprocal segregation for multiple and widely spaced markers. We have undertaken a detailed genetic analysis of this class of mitotic recombinants. Our results indicate that mitotic gene conversion in yeast is frequently associated with nonreciprocal segregation of markers centromere-distal to the selected site of conversion. In addition, distal markers are often found to be mosaic within the product colonies. These observations, and others described here, suggest that a percentage of gene conversion in vegetative yeast cells is coupled to a chromosome break and repair mechanism. This hypothesis was further tested using a strain trisomic for chromosome VII which was specially marked to detect homolog-dependent repair events. An association between mitotic gene conversion events and the production of broken chromosomes which are repaired by a homologous-pairing-copy mechanism was supported.     

EBNA1 partitions Epstein-Barr virus plasmids in yeast cells by attaching to human EBNA1-binding protein 2 on mitotic chromosomes

Epstein-Barr virus (EBV) episomal genomes are stably maintained in human cells and are partitioned during cell division by mitotic chromosome attachment. Partitioning is mediated by the viral EBNA1 protein, which binds both the EBV segregation element (FR) and a mitotic chromosomal component. We previously showed that the segregation of EBV-based plasmids can be reconstituted in Saccharomyces cerevisiae and is absolutely dependent on EBNA1, the EBV FR sequence, and the human EBNA1-binding protein 2 (EBP2). We have now used this yeast system to elucidate the functional contribution of human EBP2 to EBNA1-mediated plasmid partitioning. Human EBP2 was found to attach to yeast mitotic chromosomes in a cell cycle-dependent manner and cause EBNA1 to associate with the mitotic chromosomes. The domain of human EBP2 that binds both yeast and human chromosomes was mapped and shown to be functionally distinct from the EBNA1-binding domain. The functionality and localization of human EBP2 mutants and fusion proteins indicated that the attachment of EBNA1 to mitotic chromosomes is crucial for EBV plasmid segregation in S. cerevisiae, as it is in humans, and that this is the contribution of human EBP2. The results also indicate that plasmid segregation in S. cerevisiae can occur through chromosome attachment.     

RAD6+ gene of Saccharomyces cerevisiae codes for two mutationally separable deoxyribonucleic acid repair functions.

The response of two mutant alleles of the RAD6+ gene of Saccharomyces cerevisiae to the ochre translational suppressor SUQ5 was determined. Both the ultraviolet sensitivity phenotype and the deficiency in ultraviolet-induced mutagenesis phenotype of the rad6-1 allele were suppressed in a [psi+] background. For the rad6-3 allele, only the ultraviolet-sensitivity phenotype was suppressible in a [psi+] background. An SUQ5 rad6-3 [psi+] strain that was examined showed the normal rad6-3 deficiency in ultraviolet-induced mutagenesis. We propose that the RAD6+ gene is divided into two cistrons, RAD6A and RAD6B. RAD6A codes for an activity responsible for the error-prone repair of ultraviolet-induced lesions in deoxyribonucleic acid but is not involved in a cell's resistance to the lethal effects of ultraviolet light. RAD6B codes for an activity essential for error-free repair of potentially lethal mutagenic damage.     

MTBP plays a crucial role in mitotic progression and chromosome segregation

Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint.     

Genetic Analysis of Multiple Drug Cross Resistance in SACCHAROMYCES CEREVISIAE: a Nuclear-Mitochondrial Gene Interaction

A mutant of the yeast Saccharomyces cerevisiae, cross resistant to several antibiotics, was isolated in our laboratory and subjected to genetic analysis. Tetrad analysis of diploids obtained from crosses between the resistant mutant and a sensitive wild-type strain suggest that the multiple resistance to the five agents, oligomycin (OLI), rhodamine 6G (RHG), tetracycline (TCN), chloramphenicol (CAP) and cycloheximide (CHX) is determined by a single nuclear gene, ant1, and requires several cytoplasmic genes for expression of resistance to oligomycin, rhodamine 6G and tetracycline. --Vegetatively growing diploid clones derived from the cross ant1 [RHO+] X +[RHO+] show mitotic segregation of two phenotypic classes for the drugs OLI, RHG TCN. Diploids derived from the two reciprocal crosses, ant1 [RHO+] X +[RHO-] and ant1 [RHO-] X +[RHO+], fail to exhibit mitotic segregation. These results are consistent with our hypothesis concerning the involvement of cytoplasmic loci. They suggest, in addition, that these loci are associated with mitochondrial DNA (mtDNA). --Evidence for this association is provided by the demonstration of genetic linkage between the cytoplasmic loci involved in the interaction, RHG-1, TCN-1 and OLI-5, and two well-characterized mitochondrial loci, ERY and CAP. --We have mapped the nuclear ant1 locus 3.3 cM from the centromere-linked gene, leu1, on the same side of the centromere of chromosome VII as leu1. --In the light of these findings, we discuss the claims made by several authors of the episomal nature of mutations similar to the one described here, as well as of the possible involvement of yeast 2 mu DNA in such mutations.     

Reduction of ultraviolet-induced mitotic delay by caffeine in G2-phase irradiated plasmodia of Physarum polycephalum

Synchronously mitotic surface Plasmodia ofPhysarum polycephalum were ultra-violet-irradiated at different times during G2-phase (—4 h to —20 min with respect to metaphase), and treated immediately thereafter with varying concentrations of caffeine. It was observed that ultraviolet-induced mitotic delay is reduced significantly by this methylxanthine. In plasmodia irradiated between —4 and —1 h with respect to metaphase, the effect was concentration-dependent and the need for a certain threshold dose for obtaining the reduction in delay was apparent. However, higher doses than this were fairly toxic when applied at this part of the cycle and led to more mitotic delay than that obtained with UV alone. The most striking observation made during this study was the phase-specific precipitous effect seen in those plasmodia irradiated at about 20 min before mitosis which almost eliminated the long delay due to ultraviolet-irradiation. These results are discussed in the context of some of the known effects of ultraviolet and caffeine on a mitosis-promoting factor. It is proposed that the significant reduction of ultraviolet-induced mitotic delay reported here is due to the reactivation of the ultraviolet-inactivated mitosis-promoting factor by caffeine. Alternatively, it is possible that caffeine may prevent the inactivation of this factor by ultraviolet.