Analysis of the genetic variability in the species of the Saccharomyces sensu stricto complex

Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis was applied to differentiate the sibling species Saccharomyces bayanus, S. cerevisiae, S. paradoxus and S. pastorianus, which constitute the most common strains of the Saccharomyces sensu stricto complex. Six decamer primers of arbitrary sequences were used to amplify the DNA of 58 strains. Species-specific (diagnostic) bands were obtained for each species. Two phylogenetic trees constructed by the neighbour-joining and maximum parsimony methods clearly showed that the delimitation of these related yeast species is possible by using RAPD analysis. Four groups of strains, corresponding to the species S. bayanus, S. cerevisiae, S. paradoxus and S. pastorianus, were obtained. Within the S. bayanus taxon, two groups of strains were observed. One includes the former type strain of S. uvarum, CECT1969(T), and closely related wine strains (S. bayanus var. uvarum), whilst the other contains S. bayanus type strain CECT1941(T) and strains CECT1991 and 10513 (S. bayanus var. bayanus). The heterogeneous S. paradoxus group was divided into three lineages, corresponding to different geographic origin, American, Japanese and European populations. In addition, due to the multilocus nature of the RAPD-PCR marker, this method is both useful and appropriate for the identification of the hybrid origin of S. pastorianus. The hybrid nature was deduced from the analysis of the fraction of bands shared by each hybrid strain and the parental species. Among the 58 strains analysed, six S. pastorianus strains were hybrids, although the fraction of genome coming from each parent varied depending on the strain.     

Isolation of GCR1, a major transcription factor of glycolytic genes in Saccharomyces cerevisiae, from Kluyveromyces lactis

To study the function of GCR1, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Kluyveromyces lactis gene that complements the growth defect of a S. cerevisiae Deltagcr1 mutant was isolated. Introduction of this gene into the Deltagcr1 mutant also restored the activities of glycolytic enzymes. DNA sequencing of KlGCR1 predicted an open reading frame of a 767 amino acid protein with an overall identity of 33% and similarity of 48% to Gcr1p from S. cerevisiae. Its DDBJ/EMBL/GenBank Accession No. is AB046391.     

An experimental system for the study of mutations in the HMR locus of Saccharomyces cerevisiae: the insertion of Ty into HMRa vs. the conversion of HMRa to HMRalpha

A cross between a sir4-11 strain (sir4-11 HMLalpha MATalpha HMRa, non-mating type) and an a-mating strain (SIR(+) HMLalpha MATa HMRa) of Saccharomyces cerevisiae forms diploid clones at a frequency of 5 x 10(-6), but the obtained diploid clones often (>70%) have altered forms of the HMRa-containing restriction fragment, designated @ HMRa'. We previously found that some HMRa's are associated with the conversion of HMRa to HMRalpha. In this report, we present evidence that another @ HMRa' associates with the insertion of Ty into Ya of HMR. We also found that the sir4-11 strain increased mating frequency by UV irradiation to a level of 9 x 10(-4), and that generation of HMRa' was completely prevented by disruption of RAD52 of the sir4-11 strain. Hence, we conclude that the mutations that cause generation of HMRa' occur in the sir4-11 strain prior to mating. Due to these mutations, the sir4-11 strain converts to alpha-mating type and readily mates with the a-mating strain. We discuss the usefulness of the sir4-11 strain for the study of mutations in the HMR locus of S. cerevisiae.     

Sequence comparison of the ARG4 chromosomal regions from the two related yeasts,Saccharomyces cerevisiae and Saccharomyces douglasii

A 3·6 kb DNA fragment from Saccharomyces douglasii, containing the ARG4 gene, has been cloned, sequenced and compared to the corresponding region from Saccharomyces cerevisiae. The organization of this region is identical in both yeasts. It contains besides the ARG4 gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81). The ARG4 and the YSD83 coding regions differ from their S. cerevisiae homologs by 8.1% and 12·5%, respectively, of base substitutions. The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2·8%) than in Ysc83 (12·4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83. The non-coding regions are less conserved, with small AT-rich insertions/deletions and 20% base substitutions. However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints. The Gcn4 binding site and the region where meiotic double-strand breaks occur, are fully conserved. The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged organisms.     

Analysis of a Ty1-less variant of Saccharomyces paradoxus: the gain and loss of Ty1 elements

Because Ty elements transpose through an RNA intermediate, element accumulation through retrotransposition must be regulated or offset by element loss to avoid uncontrolled genome expansion. Here we examine the fate of Ty sequences in Saccharomyces strain 337, a strain that is reported to lack Ty1 and Ty2 elements, but contains remnant solo long terminal repeats (LTRs). Although strain 337 was initially classified as Saccharomyces cerevisiae, our work indicates that this strain is more closely related to S. paradoxus. Several degenerate Ty1 and Ty2 LTRs were mapped to the same insertion sites as full-length Ty1 and Ty2 elements in S. cerevisiae, suggesting that this strain lost Ty elements by LTR-LTR recombination. Southern analysis indicates that strain 337 also lacks Ty4 and Ty5 elements. We estimated the rates of element gain and loss in this strain by introducing a single transposition-competent Ty1 element. The results indicate that Ty1 retrotransposition occurs at a much higher rate than elimination, suggesting that copy-number-dependent co-factors or environmental conditions contribute to the loss of Ty elements in this genome.     

Comparison of the structural characteristics of chromosome VI in Saccharomyces sensu stricto: The divergence,species-dependent features and uniqueness of saké yeasts

Previous studies have revealed that chromosome VI of saké yeasts is much larger than that of the other strains of Saccharomyces cerevisiae. Southern analysis using segments of chromosome VI of a laboratory strain as probes suggested that the nucleotide sequence of a major portion of this chromosome is conserved, but considerable diversity was found in the distal parts in the other strains. Physical maps also indicated that differences in length of chromosome VI were mainly due to differences in its ends. NotI was found to generate 9 kb and/or 16 kb fragments from the left telomere of chromosome VI in most saké yeasts, but no fragment in the case of AB972. SfiI produced one or two 30–50 kb fragments from the right end of this chromosome in all saké yeasts tested, but produced a 20 kb fragment in the case of AB972. All S. cerevisiae strains not employed in saké brewing were the same as AB972 in these respects. S. paradoxus had one NotI site in chromosome VI, while S. bayanus had two, one of which is possibly common to both species. The SfiI site mentioned above was present in chromosome VI of all species, while that of S. bayanus and S. paradoxus each had a second site distinct from the other. Chromosome VI of S. pastorianus was not distinguishable from that of S. bayanus. © 1998 John Wiley & Sons, Ltd.     

Characterization of Ypr1p from Saccharomyces cerevisiae as a 2-methylbutyraldehyde reductase

The metabolism of aldehydes and ketones in yeast is important for biosynthetic, catabolic and detoxication processes. Aldo-keto reductases are a family of enzymes that are able to reduce aldehydes and ketones. The roles of individual aldo-keto reductases in yeast has been difficult to determine because of overlapping substrate specificities of these enzymes. In this study, we have cloned, expressed and characterized the aldo-keto reductase Ypr1p from the yeast Saccharomyces cerevisiae and we describe its substrate specificity. The enzyme displays high specific activity towards 2-methylbutyraldehyde, as well as other aldehydes such as hexanal. It exhibits extremely low activity as a glycerol dehydrogenase. The enzyme functions over a wide pH range and uses NADPH as co-factor. In comparison to other mammalian and yeast aldo-keto reductases, Ypr1p has relatively high affinity for D,L-glyceraldehyde (1.08 mM) and hexanal (0.39 mM), but relatively low affinity for 4-nitrobenzaldehyde (1.07 mM). It displays higher specific activity for 2-methylbutyraldehyde than does yeast alcohol dehydrogenase and has a K(m) for 2-methyl butyraldehyde of 1.09 mM. The enzyme is expressed during growth on glucose, but its levels are rapidly induced by osmotic and oxidative stress. Yeast in which the YPR1 gene has been deleted possess 50% lower 2-methylbutyraldehyde reductase activity than the wild-type strain. This suggests that the enzyme may contribute to 2-methyl butyraldehyde reduction in vivo. It may therefore play a role in isoleucine catabolism and fusel alcohol formation and may influence flavour formation by strains of brewing yeast.     

Phylogenetic relationships among species of Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces determined from partial ribosomal RNA sequences

Species of the genera Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces were compared from their extent of divergence in three regions from small (18S) and large (25S) subunit ribosomal RNAs comprising a total of 900 nucleotides. With the exception of the closely related Saccharomyces bayanus and S. pastorianus, which appear to have identical sequences, all other species could be distinguished by nucleotide differences in a variable region of the large subunit, and genus-specific nucleotides were discernible in all three regions. The taxon D. tamarii differed markedly from other species and is excluded from Debaryomyces. By contrast, Schwanniomyces occidentalis showed few nucleotide differences with Debaryomyces spp. and its transfer to Debaryomyces is proposed. Schizosaccharomyces proved to be somewhat more divergent than Saccharomyces and Debaryomyces, but species differences appear insufficient for dividing the genus. Some of the factors influencing estimates of phylogenetic distances from rRNA sequences are discussed.     

Distribution and sequence analysis of a novel Ty3-like element in natural Saccharomyces paradoxus isolates

Little is known about the transposable elements of species closely related to Saccharomyces cerevisiae. We present a novel transposable element in Saccharomyces paradoxus, a close congener of S. cerevisiae. Sequence analysis of this element, designated Ty3-1p, indicates that it is a homologue of the S. cerevisiae Ty3 element. Ty3-1p shares 82% nucleotide identity with an S. cerevisiae Ty3 element and appears to be structured identically to Ty3, containing two overlapping open reading frames, six retroviral-like domains, a J domain, and flanking sigma-like elements. A sigma element from Ty3-1p is 75% identical to a Ty3 sigma element. There is no evidence of horizontal transfer of Ty3 in Saccharomyces sensu stricto. We assess the distributions of Ty3p and Ty3 element insertions in natural population samples of S. paradoxus and S. cerevisiae. The S. paradoxus population sample exhibits Ty3p insertions present at a variety of sites at low frequency; this suggests that Ty3p elements are active in the sampled population. The S. cerevisiae population sample exhibits a uniform Ty3 hybridization profile in which all element insertions appear to be fixed. We comment on the possible causes of these contrasting observed distributions (GenBank Accession Nos AY198186 and AY198187).     

Expression in Escherichia coli of a recombinant adenosine kinase from Saccharomyces cerevisiae: purification, kinetics and substrate analyses

The Saccharomyces cerevisiae ADO1 gene is known to encode a homologue of eukaryotic adenosine kinases. This gene was expressed in Escherichia coli as a recombinant protein fused to a polyhistidine tag by using the rhamnose-inducible bacterial promoter rhaB. The recombinant protein was purified to apparent homogeneity and its ability to phosphorylate different substrates was evaluated. Adenosine (Km 3 microM) is its primary substrate. In addition, it also phosphorylates, albeit less efficiently, 3'-deoxyadenosine (cordycepin; Km 1.84 mM) and 3'-amino-3'-deoxyadenosine (Km 0.26 mM). Other kinetic properties of the recombinant enzyme have also been determined.     

Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae

Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities.     

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.     

Molecular cloning and DNA analysis of a gene encoding alpha mating pheromone from the yeast Saccharomyces naganishii

A DNA fragment encoding the precursor peptide for alpha mating pheromone was isolated from the S. naganishii genome based on the amino acid sequence of the mature pheromone. The precursor peptide contains three copies of the pheromone. Hydrophobicity analysis of the precursor peptide revealed an N-terminal signal sequence for translocation into the lumen of the endoplasmic reticulum and several signals for a series of secretion-related processes. However, upstream regulatory sequences necessary for expression of the S. cerevisiae alpha mating pheromone gene were not found, suggesting the divergence of systems that regulate alpha mating pheromone gene expression in S. naganishii and S. cerevisiae. Hybridization of a probe corresponding to the S. naganishii alpha mating pheromone nucleotide sequence to S. naganishii chromosomal DNA revealed a single gene located on either chromosome VI or VII. The S. naganishii alpha mating pheromone sequence has been deposited in the DDBJ/EMBL/GenBank data library under Accession No. AB086431.     

贝酵母耐硫相关基因FZF1的克隆与分析

FZF1基因在酿酒酵母中对亚硫酸盐转运蛋白编码基因SSU1具有正向调节作用,但在贝酵母中尚无相关报道,本研究拟通过对该基因进行克隆和生物信息学分析为后续研究做出参考。首先对贝酵母FZF1基因进行克隆,并利用在线分析工具Prot Param、Prot Scale、TMHMM、Predict Protein、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900 bp的开放阅读框,可编码299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18个丝氨酸（S）激酶潜在磷酸化位点、一个Coil区和4个锌指结构域,与酿酒酵母FZF1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FZF1基因与细胞的耐硫性相关;而贝酵母FZF1基因所编码的蛋白质中仅有4个锌指,则可能是贝酵母硫耐受能力比酿酒酵母弱的重要原因。      

Consideration of the evolution of the Saccharomyces cerevisiae MEL gene family on the basis of the nucleotide sequences of the genes and their flanking regions

Analysis of the DNA sequences of new members of the Saccharomyces cerevisiae MEL1-MEL10 gene family showed high homology between the members. The MEL gene family, α-galactosidase-coding sequences, have diverged into two groups; one consisting of MEL1 and MEL2 and the other of MEL3-MEL10. In two S. cerevisiae strains containing five or seven MEL genes each, all the genes are nearly identical, suggesting very rapid distribution of the gene to separate chromosomes. The sequence homology and the abrupt change to sequence heterogeneity at the centromere-proximal 3′ end of the MEL genes suggest that the distribution of the genes to new chromosomal locations has occurred partly by reciprocal recombination at solo delta sequences. We identified a new open reading frame sufficient to code for a 554 amino acid long protein of unknown function. The new open reading frame (Accession number Z37509) is located in the 3′ non-coding region of MEL3-MEL10 genes in opposite orientation to the MEL genes (Accession numbers Z37508, Z37510, Z37511). Northern analysis of total RNA showed no hybridization to a homologous probe, suggesting that the gene is not expressed efficiently if at all.