酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究, 我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因, 并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3为标记基因, 在不含尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株, 该菌株在低K+培养基中导入拟南芥K+转运体基因AtKuP1可恢复正常生长。     

酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究,我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因,并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3力标记基因,在不舍尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株,该菌株在低K 培养基中导入拟南芥K 转运体基因AtKuP1可恢复正常生长.     

Mitotic Recombination and Genetic Changes in Saccharomyces cerevisiae during Wine Fermentation

Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3 homozygotes were detected at a rate of 1 × 10⁻⁵ to 3 × 10⁻⁵ per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis.     

Recombination factors of Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has been an excellent genetic and biochemical model for our understanding of homologous recombination. Central to the process of homologous recombination are the products of the RAD52 epistasis group of genes, whose functions we now know include the nucleolytic processing of DNA double-stand breaks, the ability to conduct a DNA homology search, and the capacity to promote the exchange of genetic information between homologous regions on recombining chromosomes. It is also clear that the basic functions of the RAD52 group of genes have been highly conserved among eukaryotes. Disruption of this important process causes genomic instability, which can result in a number of unsavory consequences, including tumorigenesis and cell death.     

Multiple Pathways for Homologous Recombination in Saccharomyces Cerevisiae

The genes in the RAD52 epistasis group of Saccharomyces cerevisiae are necessary for most mitotic and meiotic recombination events. Using an intrachromosomal inverted-repeat assay, we previously demonstrated that mitotic recombination of this substrate is dependent upon the RAD52 gene. In the present study the requirement for other genes in this epistasis group for recombination of inverted repeats has been analyzed, and double and triple mutant strains were examined for their epistatic relationships. The majority of recombination events are mediated by a RAD51-dependent pathway, where the RAD54, RAD55 and RAD57 genes function downstream of RAD51. Cells mutated in RAD55 or RAD57 as well as double mutants are cold-sensitive for inverted-repeat recombination, whereas a rad51 rad55 rad57 triple mutant is not. The RAD1 gene is not required for inverted-repeat recombination but is able to process spontaneous DNA lesions to produce recombinant products in the absence of RAD51. Furthermore, there is still considerably more recombination in rad1 rad51 mutants than in rad52 mutants, indicating the presence of another, as yet unidentified, recombination pathway.     

天蓝色链霉菌中分化发育基因参与同源性重组

在天蓝色链霉菌中,ＳＣＰ２＾＊质粒接合转移频率的快增长,ＳＣＰ２＾＊质粒介导的质粒同源性重组频率的快增长与气生菌丝的形成同步。在１株ｂｌｄ基因突变株中,３株ｗｈｉ基因突变株中,测定ＳＣＰ２＾＊质粒接合转移及其介导的质粒同源性重组,结果表明,除ｗｈｉＡ基因突变导致质粒接合转移频率不稳定外,其余３个基因突变对质粒接合转移不产生影响,但在所测定的４个基因突变株中,质粒同源性重组的频率降低超过１０掊。     

Telomere Recombination Accelerates Cellular Aging in Saccharomyces cerevisiae

Telomeres are nucleoprotein structures located at the linear ends of eukaryotic chromosomes. Telomere integrity is required for cell proliferation and survival. Although the vast majority of eukaryotic species use telomerase as a primary means for telomere maintenance, a few species can use recombination or retrotransposon-mediated maintenance pathways. Since Saccharomyces cerevisiae can use both telomerase and recombination to replicate telomeres, budding yeast provides a useful system with which to examine the evolutionary advantages of telomerase and recombination in preserving an organism or cell under natural selection. In this study, we examined the life span in telomerase-null, post-senescent type II survivors that have employed homologous recombination to replicate their telomeres. Type II recombination survivors stably maintained chromosomal integrity but exhibited a significantly reduced replicative life span. Normal patterns of cell morphology at the end of a replicative life span and aging-dependent sterility were observed in telomerase-null type II survivors, suggesting the type II survivors aged prematurely in a manner that is phenotypically consistent with that of wild-type senescent cells. The shortened life span of type II survivors was extended by calorie restriction or TOR1 deletion, but not by Fob1p inactivation or Sir2p over-expression. Intriguingly, rDNA recombination was decreased in type II survivors, indicating that the premature aging of type II survivors was not caused by an increase in extra-chromosomal rDNA circle accumulation. Reintroduction of telomerase activity immediately restored the replicative life span of type II survivors despite their heterogeneous telomeres. These results suggest that telomere recombination accelerates cellular aging in telomerase-null type II survivors and that telomerase is likely a superior telomere maintenance pathway in sustaining yeast replicative life span.     

The Frequency of Homologous Recombination in Human ALT Cells

Instead of telomerase, some immortal cells use the alternative lengthening of telomeres pathway (ALT) to maintain their telomeres. There is good evidence that homologous recombination contributes to the ALT mechanism. Using an inducible GFP reporter system to measure the frequency of homologous recombination, we asked whether or not ALT cells exhibited a general change of the recombination machinery. Our results show that the frequency of homologous recombination for non-telomeric sequences in ALT cells is identical to that in telomerase positive cells, irrespective of whether the reporter was present at an intra-chromosomal location or next to a telomeric sequence. We conclude that the underlying recombination defect in ALT cells is restricted to telomeric sequences.     

Homologous Recombination is Activated at Early Time Points Following Exposure to Cobalt Chloride Induced Hypoxic Conditions in Saccharomyces cerevisiae

DNA repair functions are essential for the maintenance of genetic integrity and are regulated in response to both environmental and chemical stressors in mammalian and yeast cells in culture. The inhibitory effect of limited O₂ availability on DNA repair functions in general and on homologous recombination (HR) in particular, correlates with increased chromosomal abnormalities in hypoxic cancer cells. Given the above, we have investigated the effects of CoCl₂,––a hypoxia mimetic agent on HR and genetic aberrations in Saccharomyces cerevisiae. Our studies demonstrate that both acute and chronic exposure to CoCl₂ activated HR and increased genetic aberrations in S. cerevisiae D7 cells. At early time points following addition of CoCl₂ to the growth media, cells were briefly arrested in the G1-S boundary concomitant with a transient increase in Rad52-GFP foci formation and induction of low levels of DNA damage. The mode of action of CoCl₂ is thus similar to that of DNA synthesis inhibitors, the later are known to induce HR and cause G1-S arrest. We propose that the activation of HR in the presence of the hypoxia mimetic agent may be attributed to the replication stress and/or DNA damage induced by the stressor.     

Resection Activity of the Sgs1 Helicase Alters the Affinity of DNA Ends for Homologous Recombination Proteins in Saccharomyces cerevisiae

The RecQ helicase family is critical during DNA damage repair, and mutations in these proteins are associated with Bloom, Werner, or Rothmund-Thompson syndromes in humans, leading to cancer predisposition and/or premature aging. In the budding yeast Saccharomyces cerevisiae, mutations in the RecQ homolog, SGS1, phenocopy many of the defects observed in the human syndromes. One challenge to studying RecQ helicases is that their disruption leads to a pleiotropic phenotype. Using yeast, we show that the separation-of-function allele of SGS1, sgs1-D664Δ, has impaired activity at DNA ends, resulting in a resection processivity defect. Compromising Sgs1 resection function in the absence of the Sae2 nuclease causes slow growth, which is alleviated by making the DNA ends accessible to Exo1 nuclease. Furthermore, fluorescent microscopy studies reveal that, when Sgs1 resection activity is compromised in sae2Δ cells, Mre11 repair foci persist. We suggest a model where the role of Sgs1 in end resection along with Sae2 is important for removing Mre11 from DNA ends during repair.     

Subcellular Sites Involved in Lipid Synthesis in Saccharomyces cerevisiae

When the crude ribosomal fraction of Saccharomyces cerevisiae was separated into "light" and "heavy" fractions, fatty acid synthetase was concentrated in the former, whereas acetyl-Coenzyme A synthetase, fatty acid "desaturase," and squalene oxidocyclase were found in the latter. The "desaturase" sedimented with the ribosomal material and was not solubilized by low concentrations of sodium deoxycholate (DOC). The other two systems found in the "heavy" fraction sedimented with the membranes, but, upon solubilization of the membranes by DOC, these enzyme systems remained as particles.     

酿酒酵母减数分裂的事件和特异性基因

减数分裂是生物体重要的有性生殖方式,它提供来自母本和父本的基因信息,产生具有生物多样性的子代,使其能够适应环境的变化而不断进化。本文简述了现已阐明的酿酒酵母减数分裂的重要事件如同源染色体配对、联会、基因重组、染色体分裂和特异性基因。在同源染色体配对的过程中现已发现了2条途径,一条由Rad51独立完成,另一条有Dmc1、Hop2、Rad51和Mnd1参与,同时Rad51也可能参与。Red1、Hop1和Zip1是联会复合体的组成成分,而联会也要求其他减数分裂的特异性基因如Hop2的参与。基因重组是减数分裂中最重要的事件,它为子代提供了新的遗传信息,是生物多样性的基础之一。Spo11、Rad52组、Dmc1、Mnd1、Msh4、Msh5、Mek1、Red1和Hop1参与了基因重组。Spo11是发现和研究得最早的启动基因重组的基因之一;Rec8、Spo13和Sgo1参与了染色体分裂的过程。     

拟南芥组蛋白分子伴侣AtHIRA参与体细胞同源重组及盐胁迫响应

HIRA是组蛋白H3.3的特异分子伴侣, 在组蛋白H3.3掺入染色质的过程中发挥重要作用。研究表明, HIRA在哺乳动物胚胎发育和DNA损伤修复过程中不可或缺。而目前人们对于植物中HIRA同源基因功能的研究相对较少。该研究主要关注拟南芥(Arabidopsis thaliana) AtHIRA基因在植物体细胞同源重组以及减数分裂同源重组过程中的功能。将体细胞同源重组和减数分裂同源重组报告系统分别导入野生型和hira-1突变体后统计同源重组频率, 结果表明在正常生长条件下及在伯莱霉素(bleomycin)或UV-C处理条件下, hira-1突变体体细胞的分子内和分子间同源重组频率均低于野生型。而在正常生长条件下, 野生型与hira-1突变体花粉母细胞间的减数分裂同源重组频率没有明显差异, hira-1突变体的DNA损伤水平与野生型接近。qRT-PCR结果表明, DNA损伤修复相关基因RAD51和RAD54在hira-1突变体中的表达水平均高于野生型。此外, 盐胁迫处理实验表明, hira-1突变体对于高盐胁迫更加敏感。综上, AtHIRA在拟南芥体细胞同源重组及盐胁迫响应过程中发挥了一定作用。     

Strep-tag II and Twin-Strep Based Cassettes for Protein Tagging by Homologous Recombination and Characterization of Endogenous Macromolecular Assemblies in Saccharomyces cerevisiae

Peptide sequences fused to a gene of interest facilitate the isolation of proteins or protein complexes from cell extracts. In the case of fluorescent protein tags, the tagged protein can be visually localized in living cells. To tag endogenous genes, PCR-based homologous recombination is a powerful approach used in the yeast Saccharomyces cerevisiae. This approach uses short, homologous DNA sequences that flank the tagging cassette to direct recombination. Here, we constructed a set of plasmids, whose sequences were optimized for codon usage in yeast, for Strep-tag II and Twin-Strep tagging in S. cerevisiae. Some plasmids also contain sequences encoding for a fluorescent protein followed by the purification tag. We demonstrate using the yeast pyruvate dehydrogenase (PDH) complex that these plasmids can be used to purify large protein complexes efficiently. We furthermore demonstrate that purification from the endogenous pool using the Strep-tag system results in functionally active complexes. Finally, using the fluorescent tags, we show that a kinase and a phosphatase involved in regulating the activity of the PDH complex localize in the cells’ mitochondria. In conclusion, our cassettes can be used as tools for biochemical, functional, and structural analyses of endogenous multi-protein assemblies in yeast.