Identification and characterization of amidase- homologous AMI1 genes of bottom-fermenting yeast

It has been proposed that a bottom-fermenting yeast strain of Saccharomyces pastorianus is a natural hybrid between S. cerevisiae and S. bayanus and possesses at least two types of genome. In the process of conducting expressed sequence tag (EST) analysis, we isolated bottom-fermenting yeast-specific (BFY) genes that have no significant homology with sequences in the S288C database. One of the BFY genes, AMI1, encodes a protein with homology to an amidase conserved among plants, Bacillus subtilis, Neurospora crassa, Schizosaccharomyces pombe and Saccharomyces species, with the exception of S. cerevisiae S288C. In the bottom-fermenting yeast, three alleles of AMI1 (one AMI1-A and two AMI1-B alleles) were found on different chromosomes. AMI1-A on chromosome XIII is most homologous to the S. bayanus AMI1 gene, while AMI1-B on chromosome X is most homologous to the Saccharomyces paradoxus AMI1 gene. Overproduction of AMI1 in S. cerevisiae resulted in a slow-growth phenotype. Although a hydropathy plot shows that Ami1p has a putative signal sequence, it was located in the cell, not secreted into the medium. By metabolome analysis of intracellular compounds, the amount of histidine and arginine is increased, and the amount of threonine, lysine and nicotinic acid is decreased in the Ami1p-overproducing strain as compared with the control, suggesting that Ami1p may hydrolyse some amides related to amino acid and niacin metabolism in the cell.     

A new approach to species determination for yeast strains: DNA microarray-based comparative genomic hybridization using a yeast DNA microarray with 6000 genes

DNA-DNA hybridization is known as the superior method in the elucidation of relationships between closely related taxa, such as species and strain. For species determination we propose a new DNA-DNA hybridization method: the DNA microarray-based comparative genomic hybridization (CGH) method, using a yeast DNA microarray with approximately 6000 genes. The genome from a yeast strain as a sample strain (Sample) was labelled with Cy3-dye and hybridized to a single DNA microarray, together with the Cy5-labelled genome of S. cerevisiae S288C as a reference strain (Reference). The log2 ratio values [log2[Cy3(Sample)/Cy5(Reference)]: Ratio] of signal intensities of all the gene spots were estimated and divided into the following groups: Ratio < or = -1; -1 < Ratio < 1; 1 < or = Ratio. The hybridization profiles of the genomes of type strains belonging to the genus Saccharomyces were significantly different from that of S. cerevisiae S288C. The Ratio-based grouping allowed us to discriminate between some species from S. cerevisiae more clearly. Furthermore, cluster analysis discriminated between closely related species and strains. Using this method, we were able to not only perform species determination but also to obtain information on alternation in gene copy number of such gene amplifications and deletions with single-gene resolution. These observations indicated that DNA microarray-based CGH is a powerful system for species determination and comparative genome analysis.     

Molecular cloning of the RPS0 gene from Candida tropicalis.

The Saccharomyces cerevisiae RPS0 A and B genes encode proteins essential for maturation of the 40S ribosomal subunit precursors. We have isolated a homologue of the RPS0 gene from Candida tropicalis, which we named CtRPS0. The C. tropicalis RPS0 encodes a protein of 261 amino acid residues with a predicted molecular weight of 28.65 kDa and an isoelectric point of 4.79. CtRps0p displays significant amino acid sequence homology with Rps0p from C. albicans, S. cerevisiae, Neurospora crassa, Schizosaccharomyces pombe, Pneumocystis carinii and higher organisms, such as human, mouse and rat. CtRPS0 on a high copy number vector can complement the lethal phenotype linked to the disruption of both RPS0 genes in S. cerevisiae. Southern blot analysis suggests that CtRPS0 is present at a single locus within the C. tropicalis genome.     

Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.     

Molecular cloning of the CRM1 gene from Candida albicans

In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.     

Genomic differences between Candida glabrata and Saccharomyces cerevisiae around the MRPL28 and GCN3 loci

We report the sequences of two genomic regions from the pathogenic yeast Candida glabrata and their comparison to Saccharomyces cerevisiae. A 3 kb region from C. glabrata was sequenced that contains homologues of the S. cerevisiae genes TFB3, MRPL28 and STP1. The equivalent region in S. cerevisiae includes a fourth gene, MFA1, coding for mating factor a. The absence of MFA1 is consistent with C. glabrata's asexual life cycle, although we cannot exclude the possibility that a-factor gene(s) are located somewhere else in its genome. We also report the sequence of a 16 kb region from C. glabrata that contains a five-gene cluster similar to S. cerevisiae chromosome XI (including GCN3) followed by a four-gene cluster similar to chromosome XV (including HIS3). A small-scale rearrangement of gene order has occurred in the chromosome XI-like section.     

CYS3基因的敲除及其对Saccharomyces cerevisiae3-甲硫基丙醇合成代谢的影响

研究胱硫醚γ-裂解酶基因CYS3敲除对S. cerevisiae 3-甲硫基丙醇合成代谢的影响。将编码胱硫醚-γ-裂解酶的CYS3基因和抗性标记基因Zeocin克隆,构建敲除组件CYS3Δ:Zeocin,醋酸锂法将其转化导入S. cerevisiae S288C表达,构建CYS3基因敲除的工程菌。结果表明：摇瓶发酵120 h时,工程菌S. cerevisiae C3和S. cerevisiae S288C的3-甲硫基丙醇生成量分别为0.60 g/L和0.94 g/L,S. cerevisiae C3较野生型S288C的3-甲硫基丙醇生成量降低36.2%。说明CYS3基因敲除对S. cerevisiae的3-甲硫基丙醇有较大影响,并呈现负调节作用。     

酿酒酵母表达侧耳源单基因生物合成麦角硫因

运用生物信息学方法对侧耳属蘑菇（平菇、杏鲍菇和白灵菇）的麦角硫因合成酶基因Egt 1进行挖掘,对其蛋白结构和功能进行预测,并构建酿酒酵母表达载体,在酿酒酵母EC 1118中进行表达研究。结果表明：克隆得到侧耳属平菇、杏鲍菇和白灵菇麦角硫因合成基因PoEgt 1、PeEgt 1和PtEgt 1,三者核苷酸序列同源性为97.03%,氨基酸序列同源性为97.93%,联合菌体破碎法并通过体外酶促反应后检测到麦角硫因,产量为（2.5±0.08）mg/L,证明了该基因具有单基因合成麦角硫因的活性。     

Polymorphism of the MPR1 gene required for toxic proline analogue resistance in the Saccharomyces cerevisiae complex species

We recently discovered, on the chromosome of Saccharomyces cerevisiae sigma 1278b, novel MPR1 and MPR2 genes required for resistance to a toxic analogue of L-proline, L-azetidine-2-carboxylic acid. The MPR genes, which were absent in the S. cerevisiae genome project strain S288C, encoded a novel acetyltransferase of 229 amino acids that detoxifies the analogue by acetylating it. The MPR1 gene homologue found in Schizosaccharomyces pombe was also shown to encode a similar acetyltransferase. To further analyse the origin and the physiological role of the yeast novel gene, we report here the comparative analysis of the MPR1 gene in the S. cerevisiae complex spp. which belong to the Saccharomyces sensu stricto group. Only the type strain of S. paradoxus exhibited resistance and acetyltransferase activity to L-azetidine-2-carboxylic acid. PCR was then used to isolate the new MPR1 homologue (Spa MPR1) from S. paradoxus with the primers based on the sequence of the MPR1 gene. Gene expression and enzymatic analysis showed that the cloned Spa MPR1 gene encodes an L-azetidine-2-carboxylic acid acetyltransferase of 231 amino acids, which has 87% identity to the MPR1 protein. We also found in the protein databases that S. bayanus contains a DNA fragment that is partly homologous to the MPR1 gene. However, the gene product was considered to lose the enzymatic activity, possibly due to the gene truncation or the base substitution(s) at the important region for catalysis. Further, genomic PCR analysis showed that most of the S. cerevisiae complex spp. have the sequence highly homologous to the MPR1 gene.     

Negative regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by a Candida albicans orthologue of OPI1

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/choline-responsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.     

New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region

The use of PCR-based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR-amplified functional analysis (FA)-cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3'-ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C-terminal GFP-tagging were generated by adding GFP-sequences flanked at the 5'-end by a (Gly-Ala)3-linker and at the 3'-end by the S. cerevisiae URA3-terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3'-ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene-specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future.     

Two genes encoding putative mitochondrial alcohol dehydrogenases are present in the yeast Kluyveromyces lactis

Four structural genes encoding isozymes of the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis have been identified by hybridization to ADH2 DNA probes from Saccharomyces cerevisiae. In this paper we report on the isolation of KlADH4 and the complete sequencing of KlADH3 and KlADH4, two genes which show high homology to KlADH1, the ADH gene previously isolated in K. lactis, and to the ADH genes of S. cerevisiae. When compared with KlADH1, both KlADH3 and KlADH4 encode amino-terminal extensions which show the characteristics of the mitochondrial targeting sequences. These extensions are poorly conserved both at the nucleotide and the amino acid level. Surprisingly, the KlADH4 extension shows a higher identity at the amino acid level to the one encoded by ADH3 of S. cerevisiae than to the KlADH3 presequence. KlADH3 and KlADH4, in contrast to the ADH3 gene of S. cerevisiae, show a strong bias in the choice of codons.     

Genome-wide identification of fungal GPI proteins

Glycosylphosphatidylinositol-modified (GPI) proteins share structural features that allow their identification using a genomic approach. From the known S. cerevisiae and C. albicans GPI proteins, the following consensus sequence for the GPI attachment site and its downstream region was derived: [NSGDAC]-[GASVIETKDLF]-[GASV]-X(4,19)-[FILMVAGPSTCYWN](10)>, where > indicates the C-terminal end of the protein. This consensus sequence, which recognized known GPI proteins from various fungi, was used to screen the genomes of the yeasts S. cerevisiae, C. albicans, Sz. pombe and the filamentous fungus N. crassa for putative GPI proteins. The subsets of proteins so obtained were further screened for the presence of an N-terminal signal sequence for the secretion and absence of internal transmembrane domains. In this way, we identified 66 putative GPI proteins in S. cerevisiae. Some of these are known GPI proteins that were not identified by earlier genomic analyses, indicating that this selection procedure renders a more complete image of the S. cerevisiae GPI proteome. Using the same approach, 104 putative GPI proteins were identified in the human pathogen C. albicans. Among these were the proteins Gas/Phr, Ecm33, Crh and Plb, all members of GPI protein families that are also present in S. cerevisiae. In addition, several proteins and protein families with no significant homology to S. cerevisiae proteins were identified, including the cell wall-associated Als, Csa1/Rbt5, Hwp1/Rbt1 and Hyr1 protein families. In Sz. pombe, which has a low level of (galacto)mannan in the cell wall compared to C. albicans and S. cerevisiae, only 33 GPI candidates were identified and in N. crassa 97. BLAST searches revealed that about half of the putative GPI proteins that were identified in Sz. pombe and N. crassa are homologous to known or putative GPI proteins from other fungi. We conclude that our algorithm is selective and can also be used for GPI protein identification in other fungi.     

Osmoresistant yeast Zygosaccharomyces rouxii: the two most studied wild-type strains (ATCC 2623 and ATCC 42981) differ in osmotolerance and glycerol metabolism

The yeast Zygosaccharomyces rouxii is known for its high tolerance to osmotic stress, which is thought to be caused by sets of specific genes. Relatively few Z. rouxii genes have been identified so far, all of them having homologues in Saccharomyces cerevisiae; none of them was Z. rouxii-specific. Most of the known Z. rouxii genes were isolated from two wild-type strains, ATCC 2623 and ATCC 42981. In this study, we compared these two strains with regard to some of their morphological, physiological and genomic properties. Important differences were found in their salt tolerance and assimilation of glycerol and karyotype; slight differences were also present in their cell morphology. The ATCC 42981 strain showed a higher resistance to salts, higher glycerol production and, unlike ATCC 2623, was able to assimilate glycerol. Under conditions of osmotic stress, the glycerol production in both Z. rouxii strains was much lower than in a S. cerevisiae S288c culture, which suggested the presence of a system that efficiently retains glycerol inside Z. rouxii cells. The karyotype analysis revealed that ATCC 42981 cells contain more chromosomes and have a bigger genome size than those of ATCC 2623.     

Genetic homology between Saccharomyces cerevisiae and its sibling species S. paradoxus and S. bayanus: electrophoretic karyotypes.

Chromosomal DNAs of many monosporic strains of the biological species Saccharomyces cerevisiae, S. paradoxus and S. bayanus were analysed using contour-clamped homogeneous electric field electrophoresis. Southern blot hybridization with eight cloned S. cerevisiae genes (ADC1, CUP1, GAL4, LEU2, rDNA, SUC2, TRP1 and URA3) assigned to different chromosomes was used to study homology and chromosomal location of the genes in the three sibling species. A comparative study of Ty1, Ty2 and telomere-associated Y' sequences having multiple chromosomal location was also done. Chromosome length polymorphism was found in cultured strains of S. cerevisiae. Wild S. cerevisiae and S. paradoxus strains yielded chromosome banding patterns very similar to each other. The karyotype pattern of S. bayanus was readily distinguishable from that of S. cerevisiae and S. paradoxus. Southern blot analysis revealed a low degree of homology between the S. cerevisiae genes studied and the corresponding S. paradoxus and S. bayanus genes. The number of chromosomes appears to be 16 in all three species.     

高糖胁迫下槲皮素对酿酒酵母胞内损伤的保护作用与机制

以酿酒酵母S288c为模型,分析高糖胁迫下槲皮素对其胞内损伤的保护作用及机制。结果表明:与对照相比,高糖胁迫不影响酵母胞内活性氧(reactive oxygen species,ROS)水平,但显著降低了胞内酶比活力(P0.05);槲皮素处理后,与对照组和高糖组相比,酿酒酵母胞内ROS水平、超氧化物歧化酶和过氧化氢酶活力均显著下降(P0.05),而过氧化物酶(peroxidase,POD)比活力极显著升高(P0.01),说明POD比活力对高糖耐受性反应更为灵敏,可作为衡量高糖胁迫应激机制的重要生理指标,槲皮素可通过调节胞内POD比活力来提高机体的抗氧化能力。另外,实时荧光定量聚合酶链式反应结果表明高质量浓度葡萄糖显著抑制了酵母中GPD2和SUC2的表达水平(P0.05),并极显著提高了HXT1的表达水平(P0.01),而对GUT1的表达影响不显著;槲皮素处理后,高糖胁迫下酵母中GPD2、SUC2和HXT1的表达水平显著提高(P0.05),而GUT1无显著变化,说明槲皮素可能通过高渗透甘油途径、菊糖水解途径和己糖转运途径等来促进葡萄糖的分解代谢,从而达到保护机体细胞免受伤害的作用。结果表明槲皮素对高糖诱导的酿酒酵母胞内损伤具有保护作用,其作用机制可能与自身的抗氧化作用以及利用调节机体内高渗透甘油途径与糖的分解和转运途径存在一定的关联性。     

Studies of yeast Kluyveromyces lactis mutations conferring super-secretion of recombinant proteins

We have isolated mutants responsible for a super-secretion phenotype in Kluyveromyces lactis using the gene coding for a Bacillus amyloliquefaciens alpha-amylase as a marker for secretion. These mutations defined two groups, dominant and recessive. The recessive mutant strain, which secreted the heterologous protein in five-fold excess compared to the wild-type strain, was used for the cloning of genes, restraining the super-secreting phenotype. In screening for genes affecting super-secreting phenotype, we found that multiple copies of 10 different independently isolated DNA sequences suppressed the super-secreting phenotype. The first among the genes characterized, named KlSEL1 ('secretion lowering') showed homology to Saccharomyces cerevisiae ORF YML013w. The KlSEL1 gene is predicted to encode a polypeptide of 620 amino acid residues containing a putative transmembrane domain and UBX domain, characteristic for the ubiquitin-regulatory proteins. We demonstrated that the disruption of the SEL1 orthologues in K. lactis and S. cerevisiae conferred the super-secreting phenotype. SEL1 isolated from S. cerevisiae suppressed the super-secretion phenotype in K. lactis klsel1 strain, likewise homologous KlSEL1. No other phenotypic features for strains lacking the SEL1 gene were noticed except for the S. cerevisiae mutant growth being notably slower than in a wt strain. No growth changes were observed in the K. lactis klsel1 mutant. The set of genes (suppressors of over-secreting phenotype) could be attractive for further analysis of gene functions, super-secreting mechanisms and construction of new strains. This collection could be useful for the expedient construction of reduced yeast genomes, optimized for heterologous protein secretion.