An ordered clone bank for chromosome I of Saccharomyces cerevisiae.

Chromosome I of Saccharomyces cerevisiae DC5 rho 0 was dissected into segments with an average size of 14.0 kb and cloned into lambda phage vectors. The physical maps of the resultant clones, totaling 205.9 kb, were used to construct an ordered clone bank of this chromosome.     

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.     

Centromeric DNA from chromosome VI in Saccharomyces cerevisiae strains

The functional sequence from the centromere in chromosome VI ( CEN6 ) of Saccharomyces cerevisiae was narrowed down to a stretch of 500 bp by a Bal31 deletion approach. The DNA sequence in this region shows three long stretches, 40 bp, 96 bp, and 63 bp of 85% and more AT pairs and a pyrimidine purine bias in the individual single strands. We assume that the CEN6 functional sequences encompass these AT-rich stretches because this part shows striking similarities to sequence elements common to CEN3 and CEN11 DNA. A strain comparison revealed that CEN6 DNA sequences are confined to the Saccharomyces genus and probably only to the S. cerevisiae species. CEN6 is not highly conserved within S. cerevisiae strains because EcoRI and HindIII restriction site variants are found with high frequency.     

Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae.

We have reported the isolation of linking clones of HindIII and EcoRI fragments, altogether spanning a 230-kb continuous stretch of chromosome VI. The presence or absence of autonomously replicating sequence (ARS) activities in all of these fragments has been determined by using ARS searching vectors containing CEN4. Nine ARS fragments were identified, and their positions were mapped on the chromosome. Structures essential for and/or stimulative to ARS activity were determined for the ARS fragments by deletions and mutations. The organization of functional elements composed of core and stimulative sequences was found to be variable. Single core sequences were identified in eight of nine ARSs. The remaining ARS (ARS603) essential element is composed of two core-like sequences. The lengths of 3'- and 5'-flanking stimulative sequences required for the full activity of ARSs varied from ARS to ARS. Five ARSs required more than 100 bp of the 3'-flanking sequence as stimulative sequences, while not more than 79 bp of the 3' sequence was required by the other three ARSs. In addition, five ARSs had stimulative sequences varying from 127 to 312 bp in the 5'-flanking region of the core sequence. In general, these stimulative activities were correlated with low local delta Gs of unwinding, suggesting that the low local delta G of an ARS is an important element for determining the efficiency of initiation of replication of ARS plasmids.     

In vivo analysis of chromosome condensation in Saccharomyces cerevisiae

Although chromosome condensation in the yeast Saccharomyces cerevisiae has been widely studied, visualization of this process in vivo has not been achieved. Using Lac operator sequences integrated at two loci on the right arm of chromosome IV and a Lac repressor-GFP fusion protein, we were able to visualize linear condensation of this chromosome arm during G2/M phase. As previously determined in fixed cells, condensation in yeast required the condensin complex. Not seen after fixation of cells, we found that topoisomerase II is required for linear condensation. Further analysis of perturbed mitoses unexpectedly revealed that condensation is a transient state that occurs before anaphase in budding yeast. Blocking anaphase progression by activation of the spindle assembly checkpoint caused a loss of condensation that was dependent on Mad2, followed by a delayed loss of cohesion between sister chromatids. Release of cells from spindle checkpoint arrest resulted in recondensation before anaphase onset. The loss of condensation in preanaphase-arrested cells was abrogated by overproduction of the aurora B kinase, Ipl1, whereas in ipl1-321 mutant cells condensation was prematurely lost in anaphase/telophase. In vivo analysis of chromosome condensation has therefore revealed unsuspected relationships between higher order chromatin structure and cell cycle control.     

高效表达内切葡聚糖酶酿酒酵母工程菌的构建

内切葡聚糖酶 (EG) 是纤维素酶的重要组分,在纤维素降解酶系中发辉重要作用。由于天然微生物来源的内切葡聚糖酶产量低,极大地制约了其生产和应用,所以对内切葡聚糖酶进行高效异源表达是解决这一问题的有效途径。为了获得高效内切葡聚糖酶酿酒酵母工程菌,本研究从纤维梭菌中克隆了内切葡聚糖酶 (EG) 基因,全长1 996 bp,编码440个氨基酸,并与来源于酿酒酵母的PGK启动子序列、来源于pPIC9K质粒的α-信号肽序列以及来源于pSH65质粒的CYC1终止子序列通过重叠延伸PCR法构建完整表达盒 (PαEGC),通过整合rDNA的方法构建内切葡聚糖酶酿酒酵母的表达载体,在酿酒酵母中进行内切葡聚糖酶的随机多拷贝表达。利用微滴数字PCR鉴定内切葡聚糖酶拷贝数,并探索拷贝数与蛋白表达量之间的关系。通过rDNA整合法获得了拷贝数为1、3、4、7、9、11、15、16、19、21、22、23的内切葡聚糖酶酿酒酵母工程菌,结果表明当拷贝数为15时,酶活性最高,为351 U/mL。本研究成功构建了内切葡聚糖酶酿酒酵母工程菌,为其他工业酶异源高效表达提供参考和借鉴。     

Construction of xylose-assimilating Saccharomyces cerevisiae

The xylose reductase gene originating from Pichia stipitis was subcloned on an expression vector with the enolase promoter and terminator from Saccharomyces cerevisiae. The transformants of S. cerevisiae harboring the resultant plasmids produced xylose reductase constitutively at a rate about 3 times higher than P. stipitis, but could not assimilate xylose due to the deficient conversion of xylitol to xylulose. The xylitol dehydrogenase gene was also isolated from the gene library of P. stipitis by plaque hybridization using a probe specific for its N-terminal amino acid sequence. The gene transferred into S. cerevisiae was well expressed. Furthermore, high expressions of the xylose reductase and xylitol dehydrogenase genes in S. cerevisiae were achieved by introducing both genes on the same or coexisting plasmids. The transformants could grow on a medium containing xylose as the sole carbon source, but ethanol production from xylose was less than that by P. stipitis and a significant amount of xylitol was excreted into the culture broth.     

Meiotic chromosome condensation and pairing in Saccharomyces cerevisiae studied by chromosome painting

Non-isotopic high resolution in sity hybridization was applied to cytological preparations of sporulating yeast cells. Ribosomal DNA (rDNA) and chromosome V-specific recombinant lambda clones were used to tag individual chromosomes and chromosome subregions. This allowed the study of chromosome behaviour during early meiotic prophase. It was found that chromatin becomes condensed and homologous DNA sequences then appear to become aligned prior to synaptonemal complex formation.by E.R. Schmidt     

产虫草素酿酒酵母工程菌株的构建与发酵优化

虫草素作为药用真菌蛹虫草的主要活性成分,具有抗肿瘤、抗病毒等多种生理功能。现阶段虫草素主要通过蛹虫草液体发酵生产,但发酵周期长、生产强度低,制约了其大规模开发利用。文中在酿酒酵母Saccharomyces cerevisiae S288C中异源表达虫草素合成关键基因ScCNS1和ScCNS2,成功构建了产虫草素的酵母工程菌SHC16,发酵240 h虫草素产量可达67.32 mg/L;基因表达分析显示,发酵后期磷酸戊糖途径、嘌呤代谢及虫草素合成途径关键酶编码基因ZWF1、PRS4、ADE4、ScCNS1及ScCNS2表达水平显著上调。进一步地,通过优化发酵培养基组成,确定以50 g/L葡萄糖为初始底物结合一次补料、添加5 mmol/L Cu2+和1.0 g/L腺嘌呤为最适培养基组成。基于此在5 L搅拌发酵罐中开展补料分批发酵,144 h虫草素产量达到137.27 mg/L,生产强度达0.95 mg/(L·h),较未优化发酵体系提高240%。     

DNA-length polymorphisms of chromosome III in the yeast Saccharomyces cerevisiae

The chromosome-sized DNAs of sporulation-deficient mutants, which had been isolated by mutagenizing spores of a homothallic diploid strain (MT98a-3D) of Saccharomyces cerevisiae, were analyzed by pulsed-field gel electrophoresis. While the size of chromosome III DNA of the parent strain was 450 kb, some mutants had one or more chromosome III DNAs of 350 kb, 450 kb, 530 kb and 630 kb. No size variation was observed for other chromosomes. Chromosome III DNAs of laboratory-stock strains, except MT98a-3D, were in the neighborhood of 350 kb. Size variation of chromosome III was observed at a high frequency in spore clones derived from MT98a-3D strain. The results suggest that DNA-length polymorphisms of chromosome III are generated by the loss or addition of a specific DNA unit of approximately 100 kb.