Sequencing of a 9·2 kb telomeric fragment from the right arm of Saccharomyces cerevisiae chromosome XIV

We report the complete sequence of a 9·2 kb fragment next to and including the right telomere of Saccharomyces cerevisiae chromosome XIV. Four open reading frames (ORFs) longer than 100 amino acids were observed in the sequenced segment. One ORF (378 codons) does not show any significant homology with proteins in the databases and corresponds to a putative new gene. Two ORFs are almost identical to the known YCR007/YKL219 and PAU1-like hypothetical protein families already identified on several S. cerevisiae chromosomes. These ORFs, whose function is unknown, are generally associated with sub-telomeric regions of chromosomes. The fourth one shows significant identities with bacterial mannitol dehydrogenases. It could be a yeast gene implicated in the metabolism of mannitol (or a related substrate). The sequence has been deposited in the EMBL data library under Accession Number X86790.     

X. Yeast sequencing reports. Sequence analysis of a 40·2 kb DNA fragment located near the left telomere of yeast chromosome X

We have sequenced on both strands a 40,257 bp fragment located near the left telomere of chromosome X of Saccharomyces cerevisiae. The sequenced segment contains 21 open reading frames (ORFs) at least 100 amino acids long. Five of the ORFs correspond to known amino acid sequences: two hypothetical proteins in the subtelomeric Y′ repeat region of 65·4 and 12·8 KDa, the cytochrome B pre-mRNA processing CBP1 protein, the mitochondrial nuclease NUC1 and the CRT1 protein. Of the 16 remaining ORFs, eight show highest homologies with the S. cerevisiae hexose transporters family (two ORFs), the yeast α-glucosidase (two ORFs), the yeast PEP1 precursor, the Escherichia coli galactoside O-acetyltransferase, the S. cerevisiae 137·7 KDa protein located in the Y′ region and a protein of unknown function of Schizosaccharomyces pombe. Finally, eight of the ORFs exhibit no significant similarity with any amino acid sequences described in data banks. DNA sequence comparison has revealed the presence of different repeated elements characteristic of yeast chromosome ends. Disruption studies have been performed on two ORFs encoding putative proteins of unknown function. The sequence has been entered in the EMBL Data Library under Accession Number Z34098.     

Sequence of the open reading frame of the FL01 gene from Saccharomyces cerevisiae

The cloned part of the flocculation gene FLO1 of Saccharomyces cerevisiae (Teunissen, A.W.R.H., van den Berg, J.A. and Steensma, H.Y. (1993). Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae, Yeast, in press) has been sequenced. The sequence contains a large open reading frame of 2685 bp. The amino acid sequence of the putative protein reveals a serine- and threonine-rich C-terminus (46%), the presence of repeated sequences and a possible secretion signal at the N-terminus. Although the sequence is not complete (we assume the missing fragment consists of repeat units), these data strongly suggest that the protein is located in the cell wall, and thus may be directly involved in the flocculation process.     

A method for efficient quantitative analysis of genomic subtelomere Y′ element abundance in yeasts

Subtelomere Y′ elements get amplified by homologous recombination in sustaining the survival and division of the budding yeast Saccharomyces cerevisiae. However, current method for measurement of the subtelomere structures uses Southern blotting with labelled specific probes, which is laborious and time-consuming. By multiple sequence alignment analysis of all 19 subtelomere Y′ elements across the 13 chromosomes of the sequenced S288C strain deposited in the yeast genome SGD database, we identified 12 consensus and relative longer fragments and 14 pairs of unique primers for real-time quantitative PCR analysis. With a PAC2 or ACT1 located near the centromere of chromosome V and VI as internal controls, these primers were applied to real-time quantitative PCR analysis, so the relative Y′ element intensity normalised to that of wild type (WT) cells was calculated for subtelomere Y′ element copy numbers across all different chromosomes using the formula: 2^[−((CT_{mutant Y′} − CT_{mutant control}) − (CT_{WT Y′} − CT_{WT control}))]. This novel quantitative subtelomere amplification assay across chromosomes by real-time PCR proves to be a much simpler and more sensitive way than the traditional Southern blotting method to analyse the Y′ element recombination events in survivors derived from telomerase deficiency or recruitment failure.     

Characterization of MEL genes in the genus Zygosaccharomyces

We cloned and sequenced a Zygosaccharomyces cidri MEL gene with a view to investigating the structure and regulation of yeast MEL genes. The amino acid sequence deduced from the nucleotide sequence showed 78·6% and 78·2% similarity to Saccharomyces cerevisiae and Saccharomyces pastorianus α-galactosidases, respectively. The expression of the MEL gene in several Zygosaccharomyces strains was induced by galactose. An electrophoretic karyotype of several Zygosaccharomyces species was obtained using contour-clamped electric field gel electrophoresis. The minimum number of chromosomes was five for Z. cidri, six for Z. fermentati, three for Z. florentinus, and four for Z. microellipsoides. The sizes of the chromosomes were generally larger than those of S. cerevisiae, the smallest containing approximately 0·4 megabase. The MEL gene was located, using the Z. cidri MEL gene as a probe, on the largest chromosome of the Z. cidri strains. In addition, a smaller chromosome (600 kb) in Z. cidri strain CBS4575 showed hybridization to the homologous MEL probe. This chromosome was absent in Z. cidri strain CBS5666. The probe hybridized to the largest chromosome of Mel⁺ Z. fermentati strains but failed to hybridize to any chromosome of Mel⁺ Z. mrakii or Z. florentinus strains. These results suggest the existence of a polymorphic MEL gene family in the yeast Zygosaccharomyces. The sequence has been deposited in the EMBL Data Library under Accession Number L24957.     

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.     

HST1, a new member of the SIR2 family of genes

A novel Saccharomyces cerevisiae gene, HST1, was identified from among anonymous cDNAs and the complete corresponding genomic clone was isolated and sequenced. HST1 is very closely related to SIR2, showing 71% sequence identity over 84% of its length. Polymerase chain reaction with degenerate primers on S. cerevisiae DNA identified three additional SIR2-related genes designated HST2, HST3 and HST4. The sequences of HST2, HST3 and HST4 correspond to sequences previously released by the S. cerevisiae genome sequencing project as U33335, NCBI gi:965078; X87331, NCBI gi:829135; and Z48784, YD9346.03, respectively. Disruption of HST1 has shown no phenotype with respect to mechanisms in which SIR2 has a role, namely, regional silencing of HMLα, or in rDNA recombination. The sequence of HST1 has been deposited in the DDBJ, EMBL and GenBank at NCBI database under Accession Number L47120.     

Isolation and Characterization of the Candida albicans PFY1 Gene for Profilin

We have isolated the Candida albicans gene for profilin, PFY1. Degenerate oligonucleotide primers based on regions of high homology were utilized to obtain a polymerase chain reaction-amplified copy of the gene. This was then used as a probe to isolate the gene from a C. albicans genomic library. Our studies indicate that the full-length gene is unstable in Escherichia coli. Several clones were sequenced, and the predicted amino acid sequence demonstrated homology with profilin proteins from other organisms, most notably Saccharomyces cerevisiae. Northern analysis revealed that the gene is expressed in C. albicans. Attempts to express the gene in S. cerevisiae cells were unsuccessful until the C. albicans promoter was replaced with an S. cerevisiae promoter. Functional complementation of the gene was demonstrated in S. cerevisiae profilin-requiring cells. Antibodies raised to isolated C. albicans profilin protein recognized a protein of the predicted molecular weight when the gene was expressed in S. cerevisiae cells. The sequence of the C. albicans PFY1 gene has been deposited in the Genome Sequence database under Accession Number L3783. © 1997 John Wiley & Sons, Ltd.     

Simultaneous visualisation of the complete sets of telomeres from the MmeI generated terminal restriction fragments in yeasts

Telomere length is measured using Southern blotting of the chromosomal terminal restriction fragments (TRFs) released by endonuclease digestion in cells from yeast to human. In the budding yeast Saccharomyces cerevisiae, XhoI or PstI is applied to cut the subtelomere Y′ element and release TRFs from the 17 subtelomeres. However, telomeres from other 15 X-element-only subtelomeres are omitted from analysis. Here, we report a method for measuring all 32 telomeres in S. cerevisiae using the endonuclease MmeI. Based on analyses of the endonuclease cleavage sites, we found that the TRFs generated by MmeI displayed two distinguishable bands in the sizes of ~500 and ~700 bp comprising telomeres (300 bp) and subtelomeres (200–400 bp). The modified MmeI-restricted TRF (mTRF) method recapitulated telomere shortening and lengthening caused by deficiencies of YKu and Rif1 respectively in S. cerevisiae. Furthermore, we found that mTRF was also applicable to telomere length analysis in S. paradoxus strains. These results demonstrate a useful tool for simultaneous detection of telomeres from all chromosomal ends with both X-element-only and Y′-element subtelomeres in S. cerevisiae species.     

Kluyveromyces marxianus small DNA fragments contain both autonomous replicative and centromeric elements that also function in Kluyveromyces lactis

Two fragments containing both an autonomous replicating sequence (ARS) and a centromere have been isolated and sequenced from the yeast Kluyveromyces marxianus. The ARS and centromeric core sequences are only 500 bp apart, but ARS activity could be separated from the centromeric sequences. Centromeric sequences are organized in a similar way to those of budding yeasts: two well-conserved elements: CDEI (5′ TCACGTG 3′) and CDEIII (5′ TNTTCCGAAAGTWAAA 3′), are separated by a 165 bp AT-rich (± 90%) CDEII element whose length is twice that of Saccharomyces cerevisiae CDEII but almost identical to that of K. lactis. The ARS-core consensus sequence (5′ TTTATTGTT 3′) is also similar to that of K. lactis. Both ARS and centromeric elements function in this strain, albeit inefficiently, but not in S. cerevisiae. A third ARS-containing fragment with a different organization has been isolated and sequenced. The nucleotide sequences of DNA fragments reported in this paper will appear in the EMB data library under the accession numbers: Z31562, Z31563, Z31564.     

Yeast sequencing reports. CAN1, a gene encoding a permease for basic amino acids in Candida albicans

The first gene coding for an amino-acid permease of Candida albicans was sequenced. The DNA fragment complementing the lysine-permease deficiency was 3385 bp long. An open reading frame of 1713 nucleotides was found encoding a protein of 571 amino acids, with a calculated molecular weight of 63 343. Analysis of the deduced primary structure revealed ten membrane spanning regions and three potential N-glycosylation sites. The protein sequence is strongly homologous to both permeases for basic amino acids (Can1 and Lyp1) of Saccharomyces cerevisiae. C-terminal part of another ORF (105 aa), highly homologous to the gene HAL2 of S. cerevisiae, was found 133 bp downstream, and in tail-to-tail orientation to the permease gene. The sequence data will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries under the accession number X76689.     

Potassium uptake systems of Candida krusei

Candida krusei is a pathogenic yeast species that is phylogenetically outside both of the well-studied yeast groups, whole genome duplication and CUG. Like all other yeast species, it needs to accumulate high amounts of potassium cations, which are needed for proliferation and many other cell functions. A search in the sequenced genomes of nine C. krusei strains revealed the existence of two highly conserved genes encoding putative potassium uptake systems. Both of them belong to the TRK family, whose members have been found in all the sequenced genomes of species from the Saccharomycetales subclade. Analysis and comparison of the two C. krusei Trk sequences revealed all the typical features of yeast Trk proteins but also an unusual extension of the CkTrk2 hydrophilic N-terminus. The expression of both putative CkTRK genes in Saccharomyces cerevisiae lacking its own potassium importers showed that only CkTrk1 is able to complement the absence of S. cerevisiae's own transporters and provide cells with a sufficient amount of potassium. Interestingly, a portion of the CkTrk1 molecules were localized to the vacuolar membrane. The presence of CkTrk2 had no evident phenotype, due to the fact that this protein was not correctly targeted to the S. cerevisiae plasma membrane. Thus, CkTrk2 is the first studied yeast Trk protein to date that was not properly recognized and targeted to the plasma membrane upon heterologous expression in 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 62 kb DNA sequence of chromosome X reveals 36 open reading frames and a gene cluster with a counterpart on chromosome XI

We have sequenced a 61,989 bp stretch located between genes RAD7 and FIP1 of Saccharomyces cerevisiae chromosome X. This stretch contains 36 open reading frames (ORFs) of at least 100 codons. Fourteen of these correspond to sequences previously published as HIT1, CDC8, YAP17, CBF1, NAT1, RPA12, CCT5, TOR1, RFC2, PEM2, CDC11, MIR1, STE18 and GRR1. The proteins deduced from four ORFs (YJR059w, YJR065c, YJR075w, YJR078w) have significant similarity to proteins of known function from yeast or other organisms, including S. cerevisiae serine/threonine-specific protein kinase, Schizosaccharomyces pombe Act2 protein, S. cerevisiae mannosyltransferase OCH1 protein and mouse indoleamine 2,3-dioxygenase, respectively. Four of the remaining 18 ORFs have similarity to proteins with unknown function, six are weakly similar to other known sequences, while another eight exhibit no similarity to any known sequence. In addition, three tRNA genes have been recognized. Three genes clustered within 22 kb (YJR059w, YJR061w and TOR1) have counterparts arranged within 15 kb on the left arm of chromosome XI. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L47993.     

Composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay

There is a lack of knowledge about the composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay cultivars. The objectives were to determine the relative abundance of indigenous and commercial S. cerevisiae strains in spontaneous fermentations at three wineries from the two cultivars and to compare the composition of the S. cerevisiae strains between cultivars and wineries. Three fermentation vessels were sampled at three stages of fermentation for each cultivar at each winery. Isolates were identified to the strain level using seven microsatellite loci. Commercial S. cerevisiae strains were isolated at a frequency higher than that of the indigenous strains at each winery for both cultivars. The composition of S. cerevisiae strains was different for each cultivar and at each winery. Our results illustrate the clear influence of inoculated commercial active dry yeast strains on the composition of S. cerevisiae strains in spontaneous fermentations at wineries conducting both inoculated and spontaneous fermentations.     

Sequence Analysis of 203 Kilobases from Saccharomyces cerevisiae Chromosome VII

The nucleotide sequences of five major regions from chromosome VII of Saccharomyces cerevisiae have been determined and analysed. These regions represent 203 kilobases corresponding to approximately one-fifth of the complete yeast chromosome VII. Two fragments originate from the left arm of this chromosome. The first one of about 15·8 kb starts approximately 75 kb from the left telomere and is bordered by the SKI8 chromosomal marker. The second fragment covers the 72·6 kb region between the chromosomal markers CYH2 and ALG2. On the right chromosomal arm three regions, a 70·6 kb region between the MSB2 and the KSS1 chromosomal markers and two smaller regions dominated by the KRE11 marker and another one in the vicinity of the SER2 marker were sequenced. We found a total of 114 open reading frames (ORFs), 13 of which were completely overlapping with larger ORFs running in the opposite direction. A total of 44 yeast genes, the physiological functions of which are known, could be precisely mapped on this chromosome. Of the remaining 57 ORFs, 26 shared sequence homologies with known genes, among which were 13 other S. cerevisiae genes and five genes from other organisms. No homology with any sequence in the databases could be found for 31 ORFs. Furthermore, five Ty elements were found, one of which may not be functional due to a frame shift in its Ty1B amino acid sequence. The five chromosomal regions harboured five potential ARS elements and one sigma element together with eight tRNA genes and two snRNAs, one of which is encoded by an intron of a protein-coding gene. © 1997 by John Wiley & Sons, Ltd.