An engineered nonsense URA3 allele provides a versatile system to detect the presence, absence and appearance of the [PSI+] prion in Saccharomyces cerevisiae

Common methods to identify yeast cells containing the prion form of the Sup35 translation termination factor, [PSI+], involve a nonsense suppressor phenotype. Decreased function of Sup35p in [PSI+] cells leads to read-through of certain nonsense mutations in a few auxotrophic markers, e.g. ade1-14. This read-through results in growth on adenine-deficient media. While this powerful tool has dramatically facilitated the study of [PSI+], it is limited to a narrow range of laboratory strains and cannot easily be used to screen for cells that have lost the [PSI+] prion. Therefore we have engineered a nonsense mutation in the widely used URA3 gene, termed the ura3-14 allele. Introduction of the ura3-14 allele into an array of genetic backgrounds, carrying a loss-of-function URA3 mutation and [PSI+], allows for growth on media lacking uracil, indicative of decreased translational termination efficiency. This ura3-14 allele is able to distinguish various forms of the [PSI+] prion, called variants, and is able to detect the de novo appearance of [PSI+] in strains carrying the prion form of Rnq1p, [PIN+]. Furthermore, 5-fluoroorotic acid, which kills cells making functional Ura3p, provides a means to select for [psi-] derivatives in a population of [PSI+] cells marked with the ura3-14 allele, making this system much more versatile than previous methods.     

贝酵母ADR1基因的克隆和生物信息学分析

酿酒酵母ADR1基因是编码过氧化物酶蛋白质转录的正向调节基因,对乙醇代谢有正向调节作用。本文采用PCR技术首次在贝酵母基因组DNA中对ADR1基因序列进行全长克隆,利用在线分析工具ProtParam、ProtScale、TMHMM、PredictProtein、Swiss-Model等软件对其编码蛋白质的基本理化性质进行分析,同时预测了该基因所编码蛋白质的二级结构和三级结构。结果表明:该核苷酸序列含有一个长3960 bp的开放阅读框,可编码1319个氨基酸。编码的蛋白质为在细胞核中行使调控功能的亲水蛋白,含有17个丝氨酸(S)激酶潜在磷酸化位点、四个coil区和2个锌指结构域,与酿酒酵母ADR1基因所编码的蛋白质结构和性质极为相似,可初步认为贝酵母ADR1基因是乙醇脱氢酶的调控基因。     

In vivo evidence for non-universal usage of the codon CUG in Candida maltosa

An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa. In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNA ^SerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation. The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon. The nucleotide sequence datum reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the Accession Number D26074.     

Molecular cloning of CIF1, a yeast gene necessary for growth on glucose.

The cif1 mutation of Saccharomyces cerevisiae (Navon et al., Biochemistry 18, 4487-4499, 1979) causes inability to grow on glucose and absence of catabolite inactivation. We have cloned the CIF1 gene by complementation of function and located it in a 2.75 kb SphI-BstEII fragment situated at ca. 18 kb centromere distal of LYS2 and ca. 80 kb centromere proximal of TYR1 on chromosome II. Southern analysis demonstrated that CIF1 is present in a single copy in the yeast genome. Northern analysis revealed that the corresponding mRNA of 1.8 kb is more abundant in cells grown on galactose than in those grown on glucose. A protein of ca. 54 kDa was predicted from the open reading frame in the sequenced fragment. In strains carrying the cif1 mutation the intracellular concentration of ATP decreased immediately after addition of glucose while the intracellular concentration of cAMP did not increase. cAMP concentration increased in response to galactose or 2,4-dinitrophenol. Disruption of BCY1 or overexpression of CDC25 in a cif1 background did not restore growth on glucose, suggesting that the absence of cAMP signal is not the primary cause of lack of growth on glucose. Complementation tests showed that cif1 is not allelic to fdp1 although the two genes seem to be functionally related.     

Molecular cloning of the gamma-glutamylcysteine synthetase gene of Saccharomyces cerevisiae.

The 4.4 kb SphI DNA fragment (GSH1) that complements the gamma-glutamylcysteine synthetase-deficient mutation (gsh1) of Saccharomyces cerevisiae YH1 was cloned into vector plasmid YEp24. Gene disruption of the cloned fragment confirmed that this segment was the same gene as gsh1. Mutant strain YH1 with this plasmid not only restored gamma-glutamylcysteine synthetase (GSH-I) activity but the glutathione content and the growth rate. DNA sequence analysis of the SphI fragment showed that the GSH1 structural gene contained 2034 bp and predicted a polypeptide of 678 amino acids. The deduced amino acid sequence had about a 45% homology to that of rat kidney GSH-I, but a very low homology (about 26%) to that of Escherichia coli GSH-I. Northern analysis showed that GSH1 had been transcribed into an approximately 2.7 kb mRNA fragment. Southern analysis showed that GSH1 mapped at chromosome X.     

A single nucleotide change in the MNR1 (VCX1/HUM1) gene determines resistance to manganese in Saccharomyces cerevisiae

Several spontaneous Mn2+-resistant mutants were isolated from Saccharomyces cerevisiae strain W303-1b. All displayed an identical semidominant resistance phenotype. The gene responsible for this phenotype from one of these mutants, as well as that from the wild-type, was cloned and sequenced, which allowed the identification of a single nucleotide change in the former. Further sequencing work with the remaining mutants, as well as with others on the MNAR1 gene (Bianchi et al., 1981), indicated that all displayed the same mutation (mnr1 allele). In addition, MNR1 was shown to correspond to VCX1 and HUM1, which determine a vacuolar membrane protein. The nucleotide sequence has been deposited at EMBL, with Accession No. AJ001272.     

Stop codon decoding in Candida albicans: from non-standard back to standard

The human pathogen Candida albicans translates the standard leucine-CUG codon as serine. This genetic code change is mediated by a novel ser-tRNA(CAG), which induces aberrant mRNA decoding in vitro, resulting in retardation of the electrophoretic mobility of the polypeptides synthesized in its presence. These non-standard decoding events have been attributed to readthrough of the UAG and UGA stop codons encoded by the Brome Mosaic Virus RNA 4, which codes for the virion coat protein, and the rabbit globin mRNAs, respectively. In order to fully elucidate the behaviour of the C. albicans ser-tRNA(CAG) towards stop codons, we have used other cell-free translation systems and reporter genes. However, the reporter systems used encode several CUG codons, making it impossible to distinguish whether the slow migration of the polypeptides is caused by the replacement of leucines by serines at the CUG codons, readthrough, or a combination of both. Therefore, we have constructed new reporter systems lacking CUG codons and have used them to demonstrate that aberrant mRNA decoding in vitro is not a result from stop codon readthrough or any other non-standard translational event. Our data show that a single leucine to serine replacement at only one of the four CUG codons encoded by the BMV RNA-4 gene is responsible for the aberrant migration of the BMV coat protein on SDS-PAGE, suggesting that this amino acid substitution (ser for leu) significantly alters the structure of the virion coat protein. The data therefore show that the only aberrant event mediated by the ser-tRNA(CAG) is decoding of the leu-CUG codon as serine.     

啤酒酵母乙醛代谢关键酶相关性研究

乙醛是啤酒中的主要风味物质,其代谢主要来自酵母细胞。酵母中乙醇脱氢酶及乙醛脱氢酶是乙醛代谢的关键酶,对乙醛变化起着重要作用。跟踪啤酒酵母发酵过程中相对酶活力及乙醛变化,发现两种乙醇脱氢酶和乙醛脱氢酶的相对酶活力与发酵过程乙醛含量变化具有一定相关性。同时对低产乙醛啤酒酿酒酵母kb2-4与出发菌株啤酒酵母kb进行发酵试验,跟踪检测相对酶活力及乙醛含量,其乙醇脱氢酶Ⅰ和乙醇脱氢酶Ⅱ及乙醛脱氢酶相对酶活力均高于出发菌株,平均增幅分别为15.5%,11.6%和5%。3种酶活性的变化协同作用可以使乙醛含量降幅最大为33.8%。     

构建ADH2和THI3基因敲除酿酒酵母用于降低糯米酒中高级醇的研究

为了降低糯米酒高级醇含量,以酿酒酵母（Saccharomyces cerevisiae）菌株XF1的单倍体XF1a7和XF1α6为原始菌,采用Cre/loxP同源重组系统构建乙醇脱氢酶基因ADH2和类丙酮酸脱羧酶基因THI3缺失的单倍体酵母,再通过单倍体的杂交构建ADH2单基因缺失双倍体酵母XF1-A和ADH2与THI3双基因缺失的双倍体酵母XF1-AT。结果表明,重组菌XF1-A、XF1-AT与原始菌XF1的生长性能相似,菌株XF1-A和XF1-AT的基本发酵性能与菌株XF1无显著差异,菌株XF1-A酿造糯米酒中高级醇含量为522.16 mg/L,比菌株XF1低11.16%;菌株XF1-AT的高级醇含量为462.03 mg/L,比菌株XF1低21.39%。综上,ADH2和THI3基因敲除酿酒酵母能够有效降低糯米酒中高级醇生成量。     

IV. Yeast sequencing reports. Nucleotide sequence of the SAC2 gene of Saccharomyces cerevisiae

A temperature-sensitive mutation (act1-1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC2 gene. A cloned genomic DNA fragment that complements the cold-sensitive growth phenotype associated with such a suppressor mutation (sac2-1) was sequenced. The fragment contained an open reading frame that encodes a 641 amino acid predicted hydrophilic protein with a molecular weight of 74 445. No sequences with significant similarity to SAC2 were found in the GenBank and EMBL databases. A SAC2 disruption mutation was constructed which had phenotypes similar to the sac2-1 point mutation. A haploid SAC2 disruption strain failed to grow at low temperature and the disruption allele suppressed the temperature-sensitive act1-1 growth defect. The suppression phenotype was dependent on the strain background. The SAC2 sequence has been submitted to the EMBL data library (Accession Number Z29988).     

CRISPR/Cas9介导的酿酒酵母ADH2基因中断及反义RNA干扰GPD1的表达

CRISPR/Cas9是一个简单、高效的用于靶向目的基因和无标记的基因组工程的工具。本文通过构建酿酒酵母沉默组件PGK-SGPD1-CYC1,使甘油-3-磷酸脱氢酶I(Glycerol-3-phosphate dehydrogenase,GPD1)基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。应用CRISPR/Cas9基因编辑技术,在中断乙醇脱氢酶Ⅱ(alcohol dehydrogenase Ⅱ,ADH2)基因的同时,定点敲入GPD1基因的反义干扰组件,从而特定地干扰GPD1的表达。采用高效的酵母化学转化法将反应组件敲入酿酒酵母Y1H中,CRISPR/Cas9介导的同源重组效率达43.48%,由此获得了ADH2基因中断和GPD1反义干扰的酿酒酵母突变株。发酵实验结果表明,酿酒酵母突变菌株SG1-1与出发菌株Y1H相比,乙醇产率提高了9.07%,甘油产率下降了12.05%,乙酸产率下降了12.30%,结果表明通过中断ADH2基因及插入GPD1反义干扰组件,既能够中断ADH2基因的功能,减少乙醇转化为乙醛,同时也能在一定程度上干扰GPD1基因的表达,提高乙醇产率。