Analysis of meiotic recombination events near a recombination hotspot in the yeast Saccharomyces cerevisiae

The region of yeast chromosome III between the HIS4 and LEU2 genes has an unusually high frequency of meiotic recombination. In order to determine the pattern of cross-over and gene conversion events, we constructed a strain with a number of heterozygous markers in this 25-kb interval. We found that very high levels of reombination are localized to regions of DNA near HIS4. In addition, analysis of the patterns of co-conversion of adjacent markers suggests that there is more than one initiation site contributing to recombination of HIS4.     

Gene conversion and reciprocal exchange in a Ty-mediated translocation in yeast

Summary A haploid yeast mutant carrying a reciprocal translocation was analyzed. Cloning and comparison of sequences involved in the translocation event in wildtype and mutant revealed that the crossover between non-homologous chromosomes has occured within Ty sequences. By DNA sequence analysis it could be demonstrated that the reciprocal recombination event is accompanied by a short segment of non-reciprocal exchange (gene conversion) in the immediate vicinity of the crossover. Analysis of the translocation mutant and revertant isolates also indicated that the regulatory effect of Ty elements on adjacent genes can be modified by discrete changes within a Ty element.     

Genetic effects of photoactivated psoralens during meiosis in DNA repair mutantpso3-1 ofSaccharomyces cerevisiae

The influence of the DNA repair genePSO3 on photoactivated psoralen-induced meiotic recombination, gene conversion, reverse mutation, and on survival, was assayed in diploid strains ofSaccharomyces cerevisiae homozygous for the wild-type or thepso3-1 mutant allele. Sporulation was normal in thepso3-1 diploid. Wild-type and mutant strains had the same sensitivity to photoactivated monofunctional psoralen (3-CPs+UVA) in meiosis-uncommitted and meiosis-committed stages. The mutant showed higher sensitivity to photoactivated bifunctional psoralen (8-MOP+UVA) during all stages of the meiotic cycle. Mutation induction by 3-CPs+UVA or 8-MOP+UVA in meiosis-committed cells revealed no significant differences between wild-type and thepso3-1 mutant. The status of thePSO3 gene has no influence on the kinetics of induction of gene conversion and crossing-over after 3-CPs+UVA treatment in meiosis-committed cells: gene conversion was blocked while recombination was induced. After treatment with 8-MOP+UVA gene conversion was also blocked in both strains while crossing-over could only be observed in meiosis-committed wild-type cells.     

FLP recombinase induction of the breakage-fusion-bridge cycle and gene conversion in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SM^R) allele of ILV2] as selectable markers, and the 2 m site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir ⁺] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir ⁺] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA^– cells continued to segregate for loss of SMR1 until stable URA^– SM^R or URA^–SM^S cells were produced. Gene conversion was identified in stable URA^–SM^R cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 m DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.     

Simultaneous detection of changes in chromosome number,gene conversion and intergenic recombination during mitosis of Saccharomyces cerevisiae: spontaneous and ultraviolet light induced events

Summary We have employed a hyperhaploid strain of Saccharomyces cerevisiae disomic for chromosome VII to monitor spontaneous and ultraviolet light induced restitution of haploidy (chromosomal loss and/or nondisjunction), mitotic gene conversion and mitotic intergenic recombination. The disomic chromosomal pair incorporates six heterozygous markers, including cyh2 ^r, distributed on both sides of the centromere. Cycloheximide resistant segregants of spontaneous origin were analyzed to calculate the spontaneous mitotic rates of restitution of haploidy, intergenic recombination and gene conversion that result in expression of the cyh2 ^r mutation. Restitution of haploidy was found to be the most common source of spontaneously arising cycloheximide resistant segregants. In contrast, those induced by ultraviolet light resulted most frequently from gene conversion of CYH2 ^s to cyh2 ^r. The chromosome VII hyperhaploid system provides a sensitive method to detect the aneugenic and recombinagenic effects of suspect chemical and physical agents.     

Further evidence of a disparity between conversion and restoration in the his1 locus of Saccharomyces cerevisiae

Summary Heteroduplex repair models of recombination predict that restoration of the parental genotype from heteroduplex will have the same frequency as conversion to the genotype of the homologue. We have reported previously (Savage and Hastings 1981) that the proportion of intragenic reciprocal events in the his1 locus of yeast is too low for this expectation. In this study, two classes of recombination event involving longer lengths of his1 are compared in a series of crosses using a constant right-hand marker. This comparison gives a value for conversion: restoration for the left-hand markers which is independant of the method used before. The values obtained by the two methods are significantly correlated, confirming that there is a disparity in the ratio of conversion to restoration, and that this disparity is seen as a deficiency of restoration for five alleles of his1.     

Chromosomal reorganization during meiosis of Saccharomyces cerevisiae baker's yeasts

The genomic constitution of two S. cerevisiae baker's yeasts and their meiotic products have been analyzed by pulsed-field gel-electrophoresis, hybridization with specific gene probes, marker segregation, and flow cytometry. The parental strains have chromosomal patterns substantially different from those of laboratory strains used as controls. This pattern is partly the result of there being more than one copy of homologous chromosomes of different size, as judged by Southern-blot hybridization carried out with specific gene probes. Flow cytometry indicated that the strains have a 2.7 C DNA content. Tetrad analysis showed disomy for some chromosomes and tetrasomy for others. When two complete tetrads were subjected to molecular analysis the results confirmed instances of segregation of homologous chromosomes of different size. However, the presence of chromosomal bands absent in the parentals and the disappearance of chromosomal bands present in the parental strains were frequently seen. This result was attributed to two different phenomena: (1) the presence of multiple Ty1 and Ty2 transposable elements which seem to undergo interchromosomal translocation together with amplification, giving rise to differences in chromosomal size; (2) the presence of multiple Y′ subtelomeric regions, giving rise to asymmetrical homologous recombination and, as a consequence, differences betwen the size of the recombinant chromosomes and the non-recombinant parental chromosomes. Chromosomal reorganization occurs with a very high frequency during meiosis. By contrast, mitosis is very stable, as judged by the reproducible electrophoretic karyotype shown by the parental strains in successive generations. Received: 13 June / 9 July 1997     

Identification of an Alu-mediated tandem duplication of exons 8 and 9 in a patient with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency

A tandem repeat of exons 8 and 9 was identified in the cDNA for mitochondrial acetoacetyl-CoA thiolase (T2) in a typical T2 deficient patient. Routine mutation analysis using PCR at the genomic level had failed to identify any mutations. Alu element-mediated unequal homologous recombination between an Alu-Jo in intron 7 and another Alu-Jo in intron 9 appears to be responsible for this duplication.     

Control of recombination within and between DNA plasmids of Saccharomyces cerevisiae

Summary [2 m⁺ and [2m°] yeast were transformed to stable leucine prototrophy with the hybrid yeast — E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 m yeast DNA plasmid in addition to the yeast nuclear LEU2 ⁺ gene and the Co1E1 derivative, pMB9. In the [2 m⁺] transformants, a new wholly yeast LEU2 ⁺ plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 m DNA. The plamid, pYX, in the absence of 2 m DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 m DNA portion of the plasmid. pJDB219 was found to require the presence of 2 m DNA to undergo this intramolecular recombination. The results suggest that 2, m DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.     

Determination of mating-type conversion rates of heterothallic Saccharomyces cerevisiae with the fluctuation assay

Summary The fluctuation assay of Luria and Delbrück was adapted for the exact determination of mating-type interconversion rates of semi-sterile heterothallic strains of Saccharomyces cereviae. These rates varied between 10^–7 and 10^–6, depending on the individual strain, and were enhanced in a dose-dependent manner for cells growing in the presence of increasing concentrations of sodium butyrate. Under our experimental conditions, the distribution of alpha cells over populations of a(ste2) cells corresponded to a Poissonian distribution (within sample errors); that over populations of a(ste14)-cells did not differ significantly from the distribution calculated by Lea and Coulsen.     

Characterization of the role played by the RAD59 gene of Saccharomyces cerevisiae in ectopic recombination

The RAD52 group of genes in the yeast Saccharomyces cerevisiae controls the repair of DNA damage by a recombinational mechanism. Despite the growing evidence for physical and biochemical interactions between the proteins of this repair group, mutations in individual genes show very different effects on various types of recombination. The RAD59 gene encodes a protein with similarity to Rad52p which plays a role in the repair of damage caused by ionizing radiation. In the present study we have examined the role played by the Rad59 protein in mitotic ectopic recombination and analyzed the genetic interactions with other members of the repair group. We found that Rad59p plays a role in ectopic gene conversion that depends on the presence of Rad52p but is independent of the function of the RecA homologue Rad51p and of the Rad57 protein. The RAD59 gene product also participates in the RAD1-dependent pathway of recombination between direct repeats. We propose that Rad59p may act in a salvage mechanism that operates when the Rad51 filament is not functional. Received: 3 February / 22 April 1999     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

Analysis of conversion tracts associated with recombination events at the am locus of Neurospora crassa

In cross B163, heteroallelic am ¹ am ⁶ and heterozygous for both conventional genetic flanking markers and closer molecular markers, we previously found that the majority on flanker exchanges were remote from events that generated prototrophic recombinants. We report here that natural polymorphisms distinguishing the parents of cross B163 also include sequences within and closely flanking am. Segregation of these markers in B163 prototrophs confirms that the majority of meiotic recombination events at am resulted from gene conversion. Conversion of am ⁶, the distal allele, is more frequent than conversion of am ¹. Twelve percent of tracts in am ⁶ convertants were discontinuous while 30% of continuous tracts converting am ⁶ extend less than 741 bp. Received: 8 September 1997 / 15 April 1998     

Glucose repression on RIM1, a gene encoding a mitochondrial single-stranded DNA-binding protein, in Saccharomyces cerevisiae: a possible regulation at pre-mRNA splicing

The mitochondrial single-stranded DNA-binding protein (SSB) encoded by a nuclear gene, RIM1, is a homolog of Escherichia coli SSB. The addition of glucose decreased the amount of RIM1-mRNA in cells growing in a glycerol medium, but increased the amount of the immature RIM1-mRNA. The changes in the amounts of both mature and immature RIM1-mRNAs were dependent on SRN1/REG1/HEX2, a gene relating to pre-mRNA-splicing and glucose repression. These observations suggest that the expression of the mitochondrial SSB is regulated, at least in part, by pre-mRNA splicing under the control of glucose repression. Received: 27 April / 14 September 1998     

Roles of SGS1, MUS81, and RAD51 in the repair of lagging-strand replication defects in Saccharomyces cerevisiae

Yeast cells lacking the SGS1 DNA helicase and the MUS81 structure-specific endonuclease display a synthetic lethality that is suppressed by loss of the RAD51 recombinase. This epistatic interaction suggests that the primary function of SGS1 or MUS81, or both genes, is downstream of RAD51. To identify RAD51-independent functions of SGS1 and MUS81, a synthetic-lethal screen was performed on the sgs1 mus81 rad51triple mutant. We found that mutation of RNH202, which encodes a subunit of the hetero-trimeric RNase H2, generates a profound synthetic-sickness in this background. RNase H2 is thought to play a non-essential role in Okazaki fragment maturation. Cells lacking RNH202 showed synthetic growth defects when combined with either mus81 or sgs1 alone. But, whereas the loss of RAD51 had little effect on rnh202 sgs1 double mutants, it strongly inhibited the growth of rnh202 mus81 cells. These data indicate that the primary function of SGS1, but not MUS81, is downstream of RAD51. SGS1 must have some RAD51-independent function, however, since the growth of rnh202 mus81 rad51cells was further compromised by the loss of SGS1. Consistent with these results, we show that rnh202 cells display a sensitivity to DNA-damaging agents that is exacerbated in the absence of RAD51 or MUS81. These data support a model in which defects in lagging-strand replication are repaired by the Mus81 endonuclease or through a pathway dependent on Rad51 and Sgs1.     

红色荧光蛋白在酿酒酵母中的表达特性研究

目的研究红色荧光蛋白编码基因Dsred在酿酒酵母菌中的快速克隆与表达。方法根据已发表基因序列设计引物,采用连续重叠PCR方法快速克隆获得全长Dsred基因,将其与pMD-18T载体连接后进行测序鉴定。通过In-fusion方法将鉴定正确的pMD-Dsred重组载体与酿酒酵母表达载体pYeDP60进行连接,测序后利用LiAc方法将鉴定正确的pYeDP60-Dsred重组表达载体转化至酿酒酵母菌W303-1B中,PCR扩增筛选阳性克隆,获得的W303B[pYeDP60-Dsred]工程菌经诱导培养后进行SDS-PAGE电泳分析和绿光激发荧光成像检测。将工程菌分别接种至YPD、YPG、SCG和SCD培养基,培养48、72、96、120和144h后分别测定吸光度(A600)值。取诱导表达后的工程菌(菌液组)进行离心(菌体组)、离心后加甘油(高渗组)处理,分别置于–70、–20、4、28和37℃条件培养,荧光显微镜观察其表达特性。结果连续重叠PCR扩增和测序鉴定结果表明,扩增获得的Dsred基因长为678bp,其序列与已发表的基因序列完全一致;Dsred基因已成功插入pYeDP60-Dsred重组表达载体,且受酵母诱导型启动子GAL10-CYC1调控表达。PCR扩增筛选和SDS-PAGE分析显示,pYeDP60-Dsred重组表达载体已成功导入酿酒酵母菌中,诱导表达产物相对分子质量与预期相符,且诱导后工程菌可在绿光激发下发出红色荧光。4种培养基内工程菌的菌体生长情况无明显差别,重组Dsred红色荧光蛋白表达不会抑制酿酒酵母菌体生长。荧光显微镜观察显示,工程菌经不同处理后,高渗组红色荧光蛋白成熟时间最短,菌液组时间最长;红色荧光蛋白在37℃条件下培养时成熟最早,但降解速度较快。结论成功构建了pYeDP60-Dsred重组酿酒酵母表达载体,实现了Dsred基因在酿酒酵母菌体内的异源表达。红色荧光蛋白表达对酿酒酵母菌体生长无明显影响,且离心保留菌体和加入甘油等缺水高渗处理都有利于红色荧光蛋白的成熟。     

A sequence-specific endonuclease,Endo.SceI,can efficiently induce gene conversion in yeast mitochondria lacking a major exonuclease

Endo.SceI most likely initiates homologous recombination of yeast mitochondrial genome through sequence-specific double-strand scission of DNA. According to the double-strand break-repair model for the mechanism of homologous recombination, DNA ends created by sequence-specific endonucleases have to be processed by exonucleases. The major mitochondrial exonuclease (encoded by NUC1) has been shown to greatly affect the length of conversion tracts at the 21S rRNA locus when site-specific gene conversion is induced by endonuclease. In order to examine the role of the NUC1 nuclease in the Endo.SceI-induced recombination, recombination frequencies were measured after crossing of parental strains either in the presence or absence of NUC1 nuclease activity. The frequency of gene conversion in the oli2 locus induced by Endo.SceI was not reduced by disruption of the NUC1 gene. This result strongly implicates the presence of multiple exonucleases for the processing of the DNA ends created by sequencespecific endonucleases.     

G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae

Summary Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36–48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.     

The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability,recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation

Summary The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT n+1 chromosome VII disomic strain LBW (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATaIMaT ree46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intergenic recombination resulting in prototrophy. When compared to MAT rec46-1 n+1 disomics however, MATa/MAT rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATaIMAT rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).