High levels of the mitochondrial large ribosomal subunit protein 40 prevent loss of mitochondrial DNA in null mmf1 Saccharomyces cerevisiae cells

Members of the YERO57c/YJGFc/UK114 protein family have been identified in bacteria and eukaryotes. The budding yeast Saccharomyces cerevisiae contains two different proteins of this family, Hmf1p and Mmf1p. We have previously shown that Mmf1p is a mitochondrial protein functionally related to its human homologue and able to influence the maintenance of mitochondrial DNA. Deletion of Mmf1 results in loss of the mitochondrial genome. Using a multicopy suppression approach, we have identified a protein of the mitochondrial large ribosomal subunit, MRPL40, which stabilizes mtDNA in Deltammf1 cells. Overexpression of MRPL40 did not prevent loss of mtDNA in a mutant strain lacking the mitochondrial protein Abf2p. Thus, MRPL40 does not have a general effect on mtDNA stability, but it may be specific for the mmf1-null strain. We also show that the Deltamrpl40 cells present a similar phenotype to the mmf1-null strain, having reduced mtDNA stability and growth rate. Furthermore, we observed that rho(+)Deltamrpl40 haploid cells can be obtained when tetrads are directly dissected on medium containing a non-fermentable carbon source. Thus, replication and segregation of the mtDNA can occur in the absence of MRPL40. We also show that another mitochondrial ribosomal protein, MRPL38, is able to overcome the Deltammf1-associated defect. Together, our results suggest a link between Mmf1p and the two mitochondrial ribosomal proteins.     

Effects of the S288c genetic background and common auxotrophic markers on mitochondrial DNA function in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Throughout its development as a research tool, several strain backgrounds have been utilized and different combinations of auxotrophic marker genes have been introduced into them, creating a useful but non‐homogeneous set of strains. The ade2 allele was used as an auxotrophic marker, and for ‘red–white’ screening for respiratory competence. his3 alleles that influence the expression of MRM1 have been used as selectable markers, and the MIP1[S] allele, found in the commonly used S228c strain, is associated with mitochondrial DNA defects. The focus of the current work was to examine the effects of these alleles, singly and in combination, on the maintenance of mitochondrial function. The combination of the ade2 and MIP1[S] alleles is associated with a slight increase in point mutations in mitochondrial DNA. The deletion in the his3Δ200 allele, which removes the promoter for MRM1, is associated with loss of respiratory competence at 37 °C in the presence of either MIP1 allele. Thus, multiple factors can contribute to the maintenance of mitochondrial function, reinforcing the concept that strain background is an important consideration in both designing experiments and comparing results obtained by different research groups. Copyright © 2009 John Wiley & Sons, Ltd.     

Mutagenic effect of freezing on nuclear DNA of Saccharomyces cerevisiae

Although fragmentation of DNA has been observed in cells undergoing freezing procedures, a mutagenic effect of sub‐zero temperature treatment has not been proved by induction and isolation of mutants in nuclear DNA (nDNA). In this communication we supply evidence for mutagenicity of freezing on nDNA of Saccharomyces cerevisiae cells. In the absence of cryoprotectors, cooling for 2 h at +4°C and freezing for 1 h at ?10°C and 16 h at ?20°C, with a cooling rate of 3°C/min, resulted in induction of frame‐shift and reverse mutations in microsatellite and coding regions of nDNA. The sub‐zero temperature exposure also has a strong recombinogenic effect, evidenced by induction of gene‐conversion and crossing‐over events. Freezing induces mutations and enhances recombination with a frequency equal to or higher than that of methylmethanesulphonate at comparable survival rates. The signals for the appearance of nDNA lesions induced by freezing are detected and transduced by the DNA damage pathway. Extracellular cryoprotectors did not prevent the mutagenic effect of freezing, while accumulation of trehalose inside cells reduced nDNA cryodamage. Freezing of cells is accompanied by generation of high ROS levels, and the oxidative stress raised during the freeze–thaw process is the most likely reason for the DNA damaging effect. Experiments with mitochondrial rho^– mutants or scavengers of ROS indicated that mutagenic and recombinogenic effects of sub‐zero temperatures can be decreased but not eliminated by reduction of ROS level. The complete protection against cryodamage in nDNA required simultaneous usage of intracellular cryoprotector and ROS scavenger during the freeze–thaw process. Copyright © 2012 John Wiley & Sons, Ltd.     

Sequence of a 17·1 kb DNA fragment from chromosome X of Saccharomyces cerevisiae includes the mitochondrial ribosomal protein L8

We have sequenced a continuous segment of 17 137 bp on chromosome X. Sequence analysis of this stretch revealed 14 open reading frames (ORFs) at least 100 amino acids long. One gene, encoding the mitochondrial 60S ribosomal protein L8, had already been sequenced. Four ORF products show weak homologies with known protein sequences. The nine remaining ORF products have no homologies with sequences in data banks. The nucleotide sequence of the 17·1 kb fragment is available through the EMBL data library under Accession Number Z34288.     

Identification of Saccharomyces cerevisiae in bakery products by PCR amplification of the ITS region of ribosomal DNA

 A non-conventional sensitive and specific method for the detection of Saccharomyces cerevisiae in food is proposed. Polymerase chain reaction (PCR) led to the identification of this yeast in some commercial bakery products. The comparative use of two methods of clean-up of DNA from Triticum spp., S. cerevisiae and some foods shows an improvement in extraction yield for the phenol-free method. Analysis of some sequences of ribosomal genes shows different amplified products for Triticum and S. cerevisiae. Internal transcribed spacer amplification (ITS₁/ITS₄) produced a single band of about 650 and 800 bp for Triticum and S. cerevisiae, respectively. ITS₁/ITS₂ universal fungal primers gave a single band of about 300 and 450 bp for Triticum and S. cerevisiae, respectively. Both amplification products are able to distinguish between the yeast and the DNA from T. aestivum and T. durum, showing polymorphism in the ITS₁ region. Amplification on two primers designed using the sequence of the ITS₁ region of S. cerevisiae rDNA (SC₁/SC₂) led to a single band of 300 bp, species-specific for the yeast. These primers enabled an unambiguous distinction between fermented foods containing S. cerevisiae and those leavened by baking powders to be made. We suggest that this PCR assay could be used for quality control and for the trace detection of S. cerevisiae as a potential food allergen. Received: 7 January 1999     

Barosensitivity in Saccharomyces cerevisiae is Closely Associated with a Deletion of the COX1 Gene

High hydrostatic pressure causes physical stress to microorganisms; therefore, this technology may be applied to food pasteurization without introducing the unfavorable effects of thermal denaturation. However, its application is limited to high‐value foods because the treatment requires a robust steel vessel and expensive pressurization equipment. To reduce these costs, we studied the pasteurization of Saccharomyces cerevisiae using relatively moderate high‐pressure levels. A mutant strain isolated by ultraviolet mutagenesis showed significant loss of viability under high‐pressure conditions. Gene expression analysis of the mutant strain revealed that it incurred a deletion of the COX1 gene. Our results suggest that the pressure‐sensitivity can readily be introduced into industrial/food microorganisms by complementing a COX1 deleted mitochondria.     

Studies on the ATP3 gene of Saccharomyces cerevisiae: presence of two closely linked copies,ATP3a and ATP3b,on the right arm of chromosome II

In this paper, we present evidence that there are two closely linked copies of the ATP3 gene coding for the gamma subunit of the F(1)F(0)-ATPase complex (EC3.6.1.34) in four laboratory strains of Saccharomyces cerevisiae, even though the yeast genome project has reported that ATP3 is a single-copy gene on chromosome II. We previously reported that the gene dosage (three copies) of ATP1 and ATP2 is coincident with the subunit number of F(1)-alpha and F(1)-beta, but that the gene dosage of ATP3 was not consistent with the subunit stoichiometry of F(1)F(0)-ATPase. By applying long PCR and gene walking analyses, we estimated that the two copies of ATP3 were approximately 20 kb apart, and we designated that which is proximal to the centromere ATP3a, while we named that which is distal ATP3b. The nucleotide sequences of the two copies of ATP3 were identical to the reported sequence in the W303-1A, W303-1B and LL20 strains, while only the DC5 strain had a single base substitution in its ATP3a. With the exception of this substitution, the other nucleotide sequences were identical to the upstream 860 bp and the downstream 150 bp. The differences between ATP3 with the single base substitution (Ser(308) to Phe) and ATP3 without the substitution on the complementation of the ATP3 disruptant and on the maintenance of the mitochondrial DNA were observed, suggesting that Atp3ap and Atp3bp in the DC5 strain might have different functions. However, it should not always be necessary for yeast cells to carry different types of ATP3 because the other three strains carry the same type of ATP3. It was also demonstrated that the disruption of the ATP3 genes basically leads to a loss of wild-type mtDNA, but the stability of the mtDNA is not dependent on the ATP3 alone.     

BAT基因改造对酿酒酵母高级醇生成量的影响

酿酒酵母BAT基因编码支链氨基酸转氨酶,其中BAT1和BAT2基因分别编码线粒体和细胞质氨基酸转氨酶,位于细胞不同的位置导致二者的生理功能有所差异,BAT1基因在线粒体中倾向催化α-酮酸合成氨基酸,细胞质中的BAT2基因将氨基酸转化为α-酮酸,通过敲除BAT2以减少α-酮酸合成,过表达BAT1以增加α-酮酸消耗达到降低酿酒酵母高级醇的合成的目的。本研究以酿酒酵母AY15单倍体α5为出发菌株,结合融合PCR技术构建重组质粒p UC-BABPB1K,获得BA-PGK-BAT1-BB重组盒,并利用醋酸锂转化法和同源重组技术筛选出缺失BAT2基因同时过表达BAT1基因的突变株B-8,将其和亲本菌株α5、BAT2基因缺失菌株α5ΔBAT2进行酒精发酵实验,发酵结束后进行发酵性能和高级醇的测定。实验结果表明,与亲本菌株相比,异丁醇降低了25%,异戊醇降低了15%,活性戊醇降低了30%;与α5ΔBAT2菌株相比,异丁醇提高了0.5倍,异戊醇增加了0.1倍,活性戊醇增加了0.3倍。     

基于NRPS基因筛选与鉴定葡萄附生真菌

以非核糖体多肽合成酶（nonribosomal peptide synthetase,NRPS）基因为筛选靶点,从葡萄中筛选出含有NRPS基因的附生真菌,以期发现具有抗菌作用的环肽类化合物。采用平板划线技术、斜面分离纯化技术分离葡萄附生真菌,从中筛选出一株含有NRPS基因的附生真菌（编号PTLM-1）。在此基础上,构建系统发育进化树,结合形态学方法,将该菌株鉴定为腐皮镰刀菌Fusarium solani。经过NRPS系统发育进化树分析,发现该菌株有产生抗菌环肽类化合物——环孢菌素的潜力。通过对该菌的发酵产物进行电喷雾质谱分析,进一步证实了该预测结果。该研究为定向筛选产生环肽类化合物的菌株提供了新的研究方法与理论依据。      

Sequencing and analysis of 51 kb on the right arm of chromosome XV from Saccharomyces cerevisiae reveals 30 open reading frames

We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.     

Invasive growth of Saccharomyces cerevisiae depends on environmental triggers: a quantitative model

In this contribution, the influence of various physicochemical factors on Saccharomyces cerevisiae invasive growth is examined quantitatively. Agar‐invasion assays are generally applied for in vitro studies on S. cerevisiae invasiveness, the phenomenon observed as a putative virulence trait in this clinically more and more concerning yeast. However, qualitative agar‐invasion assays, used until now, strongly limit the feasibility and interpretation of analyses and therefore needed to be improved. Besides, knowledge in this field concerning the physiology of invasive growth, influenced by stress conditions related to the human alimentary tract and food, is poor and should be expanded. For this purpose, a quantitative agar‐invasion assay, presented in our previous work, was applied in this contribution to clarify the significance of the stress factors controlling the adhesion and invasion of the yeast in greater detail. Ten virulent and non‐virulent S. cerevisiae strains were assayed at various temperatures, pH values, nutrient starvation, modified atmosphere, and different concentrations of NaCl, CaCl₂ and preservatives. With the use of specific parameters, like a relative invasion, eight invasive growth models were hypothesized, which enabled intelligible interpretation of the results. A strong preference for invasive growth (meaning high relative invasion) was observed when the strains were grown on nitrogen‐ and glucose‐depleted media. A significant increase in the invasion of the strains was also determined at temperatures typical for human fever (37–39 °C). On the other hand, a strong repressive effect on invasion was found in the presence of salts, anoxia and some preservatives. Copyright © 2010 John Wiley & Sons, Ltd.     

Nucleotide sequence analysis of a 32,500 bp region of the right arm of Saccharomyces cerevisiae chromosome IV

We have sequenced a region containing 32.5 kb of the right arm of chromosome IV of Saccharomyces cerevisiae. Twenty open reading frames (ORFs) greater than 100 amino acids could be identified in this region. Six ORFs correspond to known yeast genes, including DOA4, UBC5 and UBC3, the gene products of which are involved in ubiquitin metabolism. UBC5 is preceded by the two tRNA genes tRNA-Arg2 and tRNA-Asp. Six genes were discovered with homologies to non-yeast genes or with homologies to other yeast ORFs. One of these could be identified as ribosomal protein gene RPS13. The putative function of eight ORFs remains unclear because comparison to different DNA or protein databases revealed no significant patterns. The sequence from cosmid 2F21 was obtained entirely by a combined subcloning and walking primer strategy, and has been deposited in the EMBL data library under Accession Number X84162.     

Analysis of a 36·2 kb DNA sequence including the right telomere of chromosome VI from Saccharomyces cerevisiae

The nucleotide sequence of a 36·2-kb distal region containing the right telomere of chromosome VI was determined. Both strands of DNA cloned into cosmid clone 9965 and plasmid clone pEL174P2 were sequenced with an average redundancy of 7·9 per base pair, by both dye primer and dye terminator cycle sequencing methods. The G + C content of the sequence was found to be 37·9%. Eighteen open reading frames (ORFs) longer than 100 amino acids were detected. Four of these ORFs (9965orfR017, 9965orfF016, 9965orfR009 and 9965orfF003) were found to encode previously identified genes (YMR31, PRE4, NIN1 and HXK1, respectively). Six ORFs (9965orfR013, 9965orfF018, 9965orfF006, 9965orfR014, 9965orfF013 and 9965orfR020) were found to be homologous to hypothetical 121·4-kDa protein in the BCK 5′ region, Bacillus subtilis DnaJ protein, hypothetical Trp-Asp repeats containing protein in DBP3-MRPL27, putative mitochondrial carrier YBR291C protein, Salmonella typhimurium nicotinate-nucleotide pyrophosphorylase, and Escherichia coli cystathionine β-lyase, respectively. The putative proteins encoded by 9965orfF018, 9965orfR014 and 9965orfR020 were found to be, respectively, a new member of the family of DnaJ-like proteins, the mitochondrial carrier protein and cystathionine lyase. The nucleotide sequence reported here has been deposited in the DDBJ/GenBank/EMBL data library under Accession Number D44597.     

The sequence of a 24 152 bp segment from the left arm of chromosome XIV from Saccharomyces cerevisiae between the BNI1 and the POL2 genes

In the framework of the European Union programme for sequencing the genome of Saccharomyces cerevisiae we have determined the nucleotide sequence of a region of 24 152 bp located on the left arm of chromosome XIV between the BNI1 and the POL2 genes. The sequence was obtained by directed sequence analysis using a mixture of ExoIII and primer walking strategies. Subsequent analysis revealed 13 open reading frames (ORFs) including four small ORFs completely internal to, or partly overlapping with, other ORFs. Five of these ORFs have been described previously (BNI1, APL1, LYP1, PIK1, POL2) and thus 74·8% of the 24 152 bp were already present in the databases prior to this sequencing effort. Interestingly, all 13 identified ORFs are characterized by a low codon adaptation index (0·04–0·22). In addition, this region of chromosome XIV shows an unusually high gene density with about 88% of coding DNA. This amounts to one gene per 2177 bp, which is significantly above the average gene length (about 1500 bp). For eight ORFs considerable homologies to ‘Expressed Sequence Tags’ derived from human cDNAs located in the XREF database could be identified. The complete nucleotide sequence of the 24 152 bp segment has been deposited in the EMBL data library under the Accession Number X92494.     

The role of the Saccharomyces cerevisiae lipoate protein ligase homologue,Lip3, in lipoic acid synthesis

The covalent attachment of lipoate to the lipoyl domains (LDs) of the central metabolism enzymes pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) is essential for their activation and thus for respiratory growth in Saccharomyces cerevisiae. A third lipoate‐dependent enzyme system, the glycine cleavage system (GCV), is required for utilization of glycine as a nitrogen source. Lipoate is synthesized by extraction of its precursor, octanoyl‐acyl carrier protein (ACP), from the pool of fatty acid biosynthetic intermediates. Alternatively, lipoate is salvaged from previously modified proteins or from growth medium by lipoate protein ligases (Lpls). The first Lpl to be characterized, LplA of Escherichia coli, catalyses two partial reactions: activation of the acyl chain by formation of acyl–AMP, followed by transfer of the acyl chain to lipoyl domains (LDs). There is a surprising diversity within the Lpl family of enzymes, several of which catalyse reactions other than ligation reactions. For example, the Bacillus subtilis Lpl homologue LipM is an octanoyltransferase that transfers the octanoyl moiety from octanoyl‐ACP to GCV. Another B. subtilis Lpl homologue, LipL, transfers octanoate from octanoyl–GCV to other LDs in an amido‐transfer reaction. Study of eukaryotic Lpls has lagged behind studies of the bacterial enzymes. We report that the Lip3 Lpl homologue of the yeast S. cerevisiae has octanoyl‐CoA–protein transferase activity, and discuss implications of this activity on the physiological role of Lip3 in lipoate synthesis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae

A new complementation group of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae (ts26-1 and ts26-2) has been isolated and characterized. This mutation maps at 40.7 cM from arg8 and 48.9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene. The dumbbell-shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication. To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures. The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis. The gene has been cloned from an S. cerevisiae genomic library by rescue of the conditional lethality of the mutants. It is present as a single copy in the haploid genome. DNA-RNA hybridization of the gene has identified 1 kb RNA, which is under cell-division-cycle control. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25,055 (214 amino acids).     

Multi‐gene phylogenetic analysis reveals that shochu‐fermenting Saccharomyces cerevisiae strains form a distinct sub‐clade of the Japanese sake cluster

Shochu is a traditional Japanese distilled spirit. The formation of the distinguishing flavour of shochu produced in individual distilleries is attributed to putative indigenous yeast strains. In this study, we performed the first (to our knowledge) phylogenetic classification of shochu strains based on nucleotide gene sequences. We performed phylogenetic classification of 21 putative indigenous shochu yeast strains isolated from 11 distilleries. All of these strains were shown or confirmed to be Saccharomyces cerevisiae, sharing species identification with 34 known S. cerevisiae strains (including commonly used shochu, sake, ale, whisky, bakery, bioethanol and laboratory yeast strains and clinical isolate) that were tested in parallel. Our analysis used five genes that reflect genome‐level phylogeny for the strain‐level classification. In a first step, we demonstrated that partial regions of the ZAP1, THI7, PXL1, YRR1 and GLG1 genes were sufficient to reproduce previous sub‐species classifications. In a second step, these five analysed regions from each of 25 strains (four commonly used shochu strains and the 21 putative indigenous shochu strains) were concatenated and used to generate a phylogenetic tree. Further analysis revealed that the putative indigenous shochu yeast strains form a monophyletic group that includes both the shochu yeasts and a subset of the sake group strains; this cluster is a sister group to other sake yeast strains, together comprising a sake‐shochu group. Differences among shochu strains were small, suggesting that it may be possible to correlate subtle phenotypic differences among shochu flavours with specific differences in genome sequences. Copyright © 2017 John Wiley & Sons, Ltd.