A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

基于DNA芯片的细菌基因表达谱技术的建立与评价

目的建立和优化基于全基因组DNA芯片的细菌基因表达谱技术，并用实时定量RT-PCR对此技术平台进行评价。方法提取并纯化鼠疫耶尔森菌总RNA，以六核苷酸随机引物（N6）和／或基因组特异引物（GDP）逆转录合成cDNA，用CY染料直接或间接标记后，与包括鼠疫耶尔森菌4005个ORF的全基因组芯片杂交，通过芯片扫描仪获得表达谱分析结果，归一化后进行分析。结果用实时定量PCR技术验证。结果采用N6＋GDP混合引物以间接法标记获得了较为理想的结果，表达谱技术与实时定量PER技术所得结果高度相关，证明了此技术的可靠性和实验设计与数据分析的合理性。结论合理的设计和归一化的分析方法是基于DNA芯片的细菌基因表达谱分析成功的关键，该技术的建立为细菌功能基因组研究奠定了基础。     

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。     

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene

Summary Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc⁺ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc⁺ colonies by UV irradiation. It is postulated that these UV-induced cdc⁺ colonies arise as the result infidelity in DNA replication.     

A mechanism of palindromic gene amplification in Saccharomyces cerevisiae

Selective gene amplification is associated with normal development, neoplasia, and drug resistance. One class of amplification events results in large arrays of inverted repeats that are often complex in structure, thus providing little information about their genesis. We made a recombination substrate in Saccharomyces cerevisiae that frequently generates palindromic duplications to repair a site-specific double-strand break in strains deleted for the SAE2 gene. The resulting palindromes are stable in sae2Delta cells, but unstable in wild-type cells. We previously proposed that the palindromes are formed by invasion and break-induced replication, followed by an unknown end joining mechanism. Here we demonstrate that palindrome formation can occur in the absence of RAD50, YKU70, and LIG4, indicating that palindrome formation defines a new class of nonhomologous end joining events. Sequence data from 24 independent palindromic duplication junctions suggest that the duplication mechanism utilizes extremely short (4-6 bp), closely spaced (2-9 bp), inverted repeats to prime DNA synthesis via an intramolecular foldback of a 3' end. In view of our data, we present a foldback priming model for how a single copy sequence is duplicated to generate a palindrome.     

The DNA repair gene PSO3 of Saccharomyces cerevisiae belongs to the RAD3 epistasis group

Summary The mutant allele pso3-1 of Saccharomyces cerevisiae confers sensitivity to treatment with UV_365nm (UVA) light-activated mono- and bi-functional psoralens. When pso3-1 is combined in double mutants with selected rad and pso mutant alleles and subjected to 8-MOP+UVA treatment, epistatic interaction with regard to survival is observed with pso1, pso2, and rad3. With the same treatment the combination of pso3-1 with rad6 and rad52 leads to synergistic interaction. For the monofunctional agent 3-carbethoxypsoralen (3-CPs) the analysis of double mutants yields the same results as with the bifunctional 8-methoxypsoralen (8-MOP) with the exception of the pso1-1pso3-1 double mutant. Here we find an additive interaction, i.e., the sensitivities of both parental strains are summed in the double mutant, which indicates a different substrate specificity of the repair activity encoded by the PSO1 and PSO3 genes.     

Control of the expression of the ADE2 gene of the yeast Saccharomyces cerevisiae

The ADE2 gene encodes AIR-carboxylase which catalyzes the sixth step of the purine biosynthetic pathway in Saccharomyces cerevisiae. We have analyzed the effect of deletions in the promoter region of this gene on the expression of the enzyme using a fusion of the ADE2 gene promoter to the bacterial lacZ gene. Adenine added to the growth medium repressed the expression of the fusion at the level of mRNA. The ADE2-lacZ fusion expression can be slightly activated in response to amino-acid starvation, but only in Gcn4 ⁺ strains and in an adenine-supplemented medium. In the absence of adenine in the medium ADE2 gene expression is derepressed, and neither starvation for histidine nor a gcd1 general control regulatory mutation leads to additional derepression. Our experiments indicate that the ADE2 gene of the purine biosynthetic pathway is under both specific adenine control and the general amino-acid control system. The cis-acting promoter elements mediating both modes of regulation overlap each other and are located around the proximal TGACTC sequence.     

The REV2 gene of Saccharomyces cerevisiae: cloning and DNA sequence

Summary The REV2 gene of Saccharomyces cerevisiae was cloned and sequenced; it contains an open reading frame of 1985 bp with a coding potential of 662 amino acids. Interruption of the chromosomal REV2 gene by integrating the URA3 gene coupled with partial deletion of the 3 terminal region produced viable haploid rev2 mutants. This indicates that the REV2 gene is non-essential for growth. The rev2 mutant is slightly more UV-sensitive than strains carrying various rev2 alleles (rev2-1, rev2x, rad5-1, rad5-8). The putative Rev2 protein is probably a globular protein containing a highly conserved nucleotide-binding site and two zinc-finger domains.     

Mechanisms of mitochondrial DNA escape to the nucleus in the yeast Saccharomyces cerevisiae

The transfer of organelle nucleic acid to the nucleus has been observed in both plants and animals. Using a unique assay to monitor mitochondrial DNA escape to the nucleus in the yeast Saccharomyces cerevisiae, we previously showed that mutations in several nuclear genes, collectively called yme mutants, cause a high rate of mitochondrial DNA escape to the nucleus. Here we demonstrate that mtDNA escape occurs via an intracellular mechanism that is dependent on the composition of the growth medium and the genetic state of the mitochondrial genome, and is independent of an RNA intermediate. Isolation of several unique second-site suppressors of the high rate of mitochondrial DNA-escape phenotype of yme mutants suggests that there are multiple independent pathways by which this nucleic acid transfer occurs. We also demonstrate that the presence of centromeric plasmids in the nucleus can reduce the perceived rate of DNA escape from the mitochondria. We propose that mitochondrial DNA-escape events are manifested as unstable nuclear plasmids that can interact with centromeric plasmids resulting in a decrease in the number of observed events. Received: 21 April / 7 June 1999     

A cytoplasmic factor involved in the expression of resistance to C8-ATC in Saccharomyces cerevisiae

A dozen mutants of Saccharomyces cerevisiae, resistant to C8-ATC have been characterized. C8-ATC was previously established as a biologically toxic compound. Frequency of mutants (10(-7)) was typical for spontaneous mutations. One very stable mutant was characterized extensively. The genetical analysis revealed that resistance in this mutant was determined by single-gene mutation. The rho 0 cells, obtained by ethidium bromide (EB) mutagenesis of the resistant strain, were completely devoid of resistance. A large percentage of rho- cells, obtained by a moderate EB treatment of resistant cells were still able to express resistance to C8-ATC. Therefore we hypothesized that, in our particular strain, a cytoplasmic factor is involved in nuclear determination of resistance.     

Requirement of the Saccharomyces cerevisiae APN1 gene for the repair of mitochondrial DNA alkylation damage

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1Δ) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1‐dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose‐dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild‐type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1Δ strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1Δ cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1Δ mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

Physical mapping of the MEL gene family in Saccharomyces cerevisiae

Nine members, MEL2–MEL10, of the MEL gene family coding for -galactosidase were physically mapped to the ends of the chromosomes by chromosome fragmentation. Genetic mapping of the genes supported the location of all the MEL genes in the left arm of their resident chromosomes.