Elp3基因缺失对酿酒酵母咖啡因耐受性的影响

过量咖啡因(Caffeine, CAF)对真核细胞有毒害作用.Elp3是组蛋白乙酰转移酶Elongator复合物的催化亚基,其缺失会影响Elongator的功能,进而使酵母产生对CAF敏感表型.为研究酿酒酵母CAF耐受性,以不同浓度CAF处理野生型、elp3⊿及转入完整和部分缺失Elp3的elp3⊿菌株,检测其敏感性,并通过RT-qPCR和ChIP实验研究了热激胁迫下SSA3的表达.结果发现常温下各菌株生长均随CAF浓度的增加而减缓,且elp3⊿细胞CAF耐受性下降更显著;高温处理使elp3⊿菌株对CAF更加敏感;在elp3⊿菌株中转入两保守功能域缺失的Elp3,其咖啡因耐受性并无明显变化;无论热激与否,elp3⊿菌株SSA3表达均显著低于野生型对照,Elp3直接参与热激胁迫下SSA3的转录延伸.研究表明Elp3基因缺失和高温处理均可使酿酒酵母的CAF耐受性降低;Elp3两保守功能域为其参与CAF胁迫反应所必需;高温协同处理下elp3⊿菌株CAF耐受性的显著降低可能与此条件下SSA3等应激基因表达受阻有关.     

ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae

The oncogenes of Harvey and Kirsten murine sarcoma viruses (v-rasH and v-rasK) and their cellular homologues (c-rasH and c-rasK) constitute two members of the ras gene family. Each functional member of the ras gene family encodes a 21,000 molecular weight protein (p21ras). ras genes have been detected in a wide variety of vertebrate species, including Xenopus laevis (R. E. Steele, personal communication), and in Drosophila melanogaster. We report here the detection of ras-related genes in the yeast Saccharomyces cerevisiae, and the isolation of two ras-related molecular clones, c-rassc-1 and c-rassc-2, from the DNA of Saccharomyces. Both c-rassc-1 and c-rassc-2 hybridize specifically to probes prepared from mammalian ras DNA. Sequencing of c-rassc-1 reveals extensive amino acid homology between the protein encoded by c-rassc-1 and the p21 encoded by c-rasH. Our studies suggest that these clones can be used to elucidate the normal cellular functions of ras-related genes in this relatively simple eukaryotic organism.     

Expression of the SOD gene from Trichoderma harzianum in Saccharomyces cerevisiae

Superoxide dismutases are metalloproteins which play a major role in defense against oxygen radicalmediated toxicity in aerobic organisms. Such proteins are important endogeneity cytoprotection factor involving defence. A 751-bp full-length cDNA sequence of an SOD gene was isolated from the Trichoderma harzianum. The full-length cDNA of the SOD gene consists of one 465-bp open reading frame nucleotide, which encodes a 15.7-kDa polypeptide consisting of 154 amino acid residues. Sequence analysis revealed that SOD gene has more than 72%-86% amino acid sequence homology with those of other fungi. The SOD gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2-SOD. SOD expressed by pYES2-SOD was induced by galactose. We test whether SOD could offer abiotic stress resistance when it was introduced into yeast ceils. A transgenic yeast harboring T. harzianum SOD was generated under the control of a constitutively expressed GAL promoter. The results indicated that SOD yeast transformants had significantly higher resistance to salt and drought stress.     

An initiation site for meiotic gene conversion in the yeast Saccharomyces cerevisiae

An initiation site for meiotic gene conversion has been identified in the promoter region of the ARG4 gene of Saccharomyces cerevisiae. The chromosome on which initiation occurs is the recipient of genetic information during gene conversion.     

Sensitivity to cyclosporin A is mediated by cyclophilin in Neurospora crassa and Saccharomyces cerevisiae

Cyclosporin A, a cyclic fungal undecapeptide produced by Tolypocladium inflatum, is a potent immunosuppressive drug originally isolated as an antifungal antibiotic. Cyclosporin A (CsA) is widely used in humans to prevent rejection of transplanted organs such as kidney, heart, bone marrow and liver. The biochemical basis of CsA action is not known: its primary cellular target has been suggested to be calmodulin, the prolactin receptor or cyclophilin, a CsA-binding protein originally isolated from the cytosol of bovine thymocytes. Cyclophilin has been shown to be a highly conserved protein present in all eukaryotic cells tested and to be identical to peptidyl-prolyl cis-trans isomerase, a novel type of enzyme that accelerates the slow refolding phase of certain proteins in vitro. We demonstrate that in the lower eukaryotes N. crassa and S. cerevisiae, cyclo philin mediates the cytotoxic CsA effect. In CsA-resistant mutants of both organisms, the cyclophilin protein is either lost completely or, if present, has lost its ability to bind CsA.     

Th2CysPrx基因提高酿酒酵母多种胁迫耐受性研究

【目的】过氧化还原蛋白是生物体调节体内氧化还原系统的一种重要蛋白质,在植物生长代谢中起重要作用。前期研究发现过表达刚毛柽柳Th2CysPrx基因的烟草和柽柳中4种抗氧化酶基因(ThGSTZ1、ThGPX、ThSOD和ThPOD)的表达水平上调, 转基因植株的NaCl胁迫耐受性提高。本实验探究Th2CysPrx基因除了响应NaCl胁迫应答,是否还参与其他盐胁迫和其他胁迫的应答。 【方法】将Th2CysPrx构建到pYES2酵母表达载体上,转化到酿酒酵母INVSC1细胞中,分析比较重组酵母(pYES2-Th2CysPrx)和对照酵母(pYES2)细胞在各种非生物胁迫后的生长情况。【结果】过表达Th2CysPrx基因的重组酵母细胞在低温、KCl和LiCl胁迫后存活率与对照相比差异不显著;但H₂O₂、NaCl或NaHCO₃胁迫后酵母细胞的存活率显著提高。此外,高于0.5 mol/L山梨醇或高温(达到44 ℃),或低于质量分数0.007% CdCl₂胁迫后,过表达Th2CysPrx基因能显著提高重组酵母的细胞存活率。【结论】Th2CysPrx基因可以显著增强酵母细胞对NaCl和NaHCO₃胁迫的耐受能力,以及特定的渗透胁迫、高温胁迫、氧化胁迫和重金属胁迫的耐受能力,但对低温、KCl和LiCl胁迫耐受能力无明显影响。     

Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase

Thiamine pyrophosphate 1 is an essential cofactor in all living systems. Its biosynthesis involves the separate syntheses of the pyrimidine 2 and thiazole 3 precursors, which are then coupled. Two biosynthetic routes to the thiamine thiazole have been identified. In prokaryotes, five enzymes act on three substrates to produce the thiazole via a complex oxidative condensation reaction, the mechanistic details of which are now well established. In contrast, only one gene product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae). Here we report the preparation of fully active recombinant wild-type THI4p, the identification of an iron-dependent sulphide transfer reaction from a conserved cysteine residue of the protein to a reaction intermediate and the demonstration that THI4p is a suicide enzyme undergoing only a single turnover.     

The role of heteroduplex correction in gene conversion in Saccharomyces cerevisiae

Two different models have been proposed to explain the relative frequencies of the non-mendelian allelic segregations which are detected by tetrad analysis after meiosis in fungi. The first model maintains that 6:2 type tetrads result from correction of heteroduplexes containing mismatched sites and 5:3 type tetrads result from failure to correct mismatched sites. The second model suggests that 6:2 segregations result from the filling-in of double-strand gaps using information obtained from both strands of a homologous duplex. In this model 5:3 type tetrads result if the allele is included in the heteroduplex regions flanking the gap and the resulting mismatched nucleotides are not corrected. We have studied the correction of heteroduplex plasmid DNA in pms1 mutant strains of Saccharomyces cerevisiae, which are known to exhibit higher frequencies of 5:3 type tetrads and lower frequencies of 6:2 tetrads than wild-type strains. Our results suggest that the pms1 mutation causes a defect in mismatch correction, supporting the hypothesis that meiotic gene conversion in wild-type yeast cells often results from the correction of heteroduplex DNA.     

Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae.

An unusual protein splicing reaction joins the N-terminal segment (A) and the C-terminal segment (C) of the 119K primary translation product (ABC) of the yeast VMA1 gene to yield a 69K vacuolar H(+)-ATPase subunit (AC) and an internal 50K polypeptide (B). This 50K protein is a site-specific DNA endonuclease that shares 34% identity with the homothallic switching endonuclease. The site cleaved by the VMA1-derived endonuclease exists in a VMA1 allele that lacks the derived endonuclease segment of the open reading frame. Cleavage at this site only occurs during meiosis and initiates 'homing', a genetic event that converts a VMA1 allele lacking the endonuclease coding sequence into one that contains it.     

Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae

The migration of genetic information from ancestral prokaryotic endosymbionts into eukaryotic nuclei is thought to have had an important role in the evolution of mitochondria and chloroplasts. Here we describe an assay for the detection of movement of DNA between mitochondria and the nucleus in yeast. Because recombinant plasmid DNA replicates after transformation into mitochondria of yeast strains lacking endogenous mitochondrial DNA we were able to propagate the nuclear genetic marker URA3 in mitochondria. As expected, the wild-type URA3 gene in mitochondria failed to complement the uracil auxotrophy (Ura-) caused by a nuclear ura3 mutation. But selection of Ura+ prototrophs from a Ura- strain carrying URA3 on a plasmid in its mitochondria enabled us to detect plasmid movement to the nucleus. Conversely, as the plasmid used also contained the mitochondrial gene COX2 required for respiratory growth, we were able to set up corresponding selections to detect migration of DNA from the nucleus to the mitochondria. Our results show that, in yeast, DNA escapes from mitochondria and appears in the nucleus at a surprisingly high frequency (approximately 2 x 10(-5) per cell per generation). But the rate at which DNA makes the journey in the opposite direction--nucleus to mitochondria--is apparently at least 100,000 times less.     

Functional profiling of the Saccharomyces cerevisiae genome

Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.     

A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity

Despite the sequencing of the human and mouse genomes, few genetic mechanisms for protecting against autoimmune disease are currently known. Here we systematically screen the mouse genome for autoimmune regulators to isolate a mouse strain, sanroque, with severe autoimmune disease resulting from a single recessive defect in a previously unknown mechanism for repressing antibody responses to self. The sanroque mutation acts within mature T cells to cause formation of excessive numbers of follicular helper T cells and germinal centres. The mutation disrupts a repressor of ICOS, an essential co-stimulatory receptor for follicular T cells, and results in excessive production of the cytokine interleukin-21. sanroque mice fail to repress diabetes-causing T cells, and develop high titres of autoantibodies and a pattern of pathology consistent with lupus. The causative mutation is in a gene of previously unknown function, roquin (Rc3h1), which encodes a highly conserved member of the RING-type ubiquitin ligase protein family. The Roquin protein is distinguished by the presence of a CCCH zinc-finger found in RNA-binding proteins, and localization to cytosolic RNA granules implicated in regulating messenger RNA translation and stability.     

Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo

Transactivator-promoter complexes are essential intermediates in the activation of eukaryotic gene expression. Recent studies of these complexes have shown that some are quite dynamic in living cells owing to rapid and reversible disruption of activator-promoter complexes by molecular chaperones, or a slower, ubiquitin-proteasome-pathway-mediated turnover of DNA-bound activator. These mechanisms may act to ensure continued responsiveness of activators to signalling cascades by limiting the lifetime of the active protein-DNA complex. Furthermore, the potency of some activators is compromised by proteasome inhibition, leading to the suggestion that periodic clearance of activators from a promoter is essential for high-level expression. Here we describe a variant of the chromatin immunoprecipitation assay that has allowed direct observation of the kinetic stability of native Gal4-promoter complexes in yeast. Under non-inducing conditions, the complex is dynamic, but on induction the Gal4-promoter complexes 'lock in' and exhibit long half-lives. Inhibition of proteasome-mediated proteolysis had little or no effect on Gal4-mediated gene expression. These studies, combined with earlier data, show that the lifetimes of different transactivator-promoter complexes in vivo can vary widely and that proteasome-mediated turnover is not a general requirement for transactivator function.