XV. Yeast sequencing reports. Isolation and DNA sequence of the STE13 gene encoding dipeptidyl aminopeptidase

We have isolated a mutant which exhibits partial constitutivity for a -specific gene expression in α cells. The wild-type gene was cloned and demonstrated to be allelic to the STE13 gene, which encodes the dipeptidyl aminopeptidase involved in processing of the α-factor prepropheromone. Thus, the mating defect of the ste13 mutations in α cells may result both from the production of incompletely processed α-factor and from the increased expression of a -specific genes. The STE13 open reading frame of 931 amino acids contains a putative membrane-spanning segment near its amino terminus and is 31% identical to a second yeast dipeptidyl aminopeptidase (DAP2). A null mutant of STE13 has been constructed: it is viable and sporulation-proficient. The sequence has been deposited in the GenBank data library under Accession Number L21944.     

Homolog of a-factor receptor gene in Saccharomyces exiguus

Homologs of Saccharomyces cerevisiae STE3, a -factor receptor gene were detected from S. exiguus NFRI 3539 by low stringency Southern hybridization. This strain might have at least two types of homolog. One of these homologs, designated as e-STE3 was cloned. Its nucleotide sequence revealed 60% identity to STE3. The putative protein coding region consisted of 453 amino acid residues. The amino acid sequence identity between STE3 and e-STE3 was 62%, and that of the N-terminal 303 amino acid residues considered to be the pheromone binding domain was 79%. The e-STE3 sequence submitted to the DDBJ/EMBL/GenBank data libraries is available under Accession Number AB003086. © 1998 John Wiley & Sons, Ltd.     

XIV. Yeast sequencing reports. Sequence analysis of a 30 kb DNA segment from yeast chromosome XIV carrying a ribosomal protein gene cluster,the genes encoding a plasma membrane protein and a subunit of replication factor C,and a novel putative serine/threonine protein kinase gene

We have determined the nucleotide sequence of a 30 kb fragment of chromosome XIV of Saccharomyces cerevisiae. The sequence revealed the presence of 19 open reading frames (ORFs) longer than 300 bp. NO422 and NO425 correspond to the split ribosomal protein genes encoding S16A and rp28, respectively, NO450 displays a striking similarity with serine/threonine protein kinase genes, in particular with STE20, and therefore may encode a novel member of this protein family. NO453 is the longest ORF in this DNA segment, having a size of 4908 bp, but its function is not yet known. NO530 encodes the plasma membrane protein Mid1p and NO533 corresponds to the gene coding for a 40 kDa subunit of replication factor C. The remaining ORFs show weak or no homology with proteins in the data bases. The sequence has been submitted to the EMBL data library under Accession Number U23084.     

Sequence and analysis of a 33 kb fragment from the right arm of chromosome XV of the yeast Saccharomyces cerevisiae

We have determined the nucleotide sequence of a cosmid (pEOA423) from chromosome XV of Saccharomyces cerevisiae. Analysis of the 33,173 bp sequence reveals the presence of 20 putative open reading frames (ORFs). Five of them correspond to previously known genes (MGM1, STE4, CDC44, STE13, RPB8). The previously published nucleotide sequences are in perfect agreement with our sequence except for STE4 and MGM1. In the latter case, 59 amino acids were truncated from the published protein at its N-terminal end due to a frameshift. The putative translation products of six other ORFs exhibit significant homology with protein sequences in public databases: O50 03 and O50 17 products are homologs of the ANC1 and MIP1 proteins of S. cerevisiae, respectively; O50 05 product is similar to that of a protein of unknown function from Myxococcus xanthus; O50 12 product is probably a new ATP/ADP carrier; O50 13 product shows homology with group II tRNA synthetases; and the O50 16 product exhibits strong similarity with the N-terminal domain of the NifU proteins from several prokaryotes. The remaining nine ORFs show no significant similarity. Among these, two contiguous ORFs (O50 19 and O50 20) are very similar to each other, suggesting an ancient tandem duplication. The 33,173 bp sequence has been submitted to EMBL (Accession Number: X92441).     

XIII. Yeast sequencing reports. Cloning and sequencing of the NES24 gene of Saccharomyces cerevisiae

We have cloned NES24 using a temperature-sensitive nes24-1 mutant as a host and sequenced a 3162 bp XhoI-EcoRI DNA fragment containing the NES24 gene. Computer analysis revealed that this segment contains a 1806 bp open reading frame which is needed for complementation of the nes24-1 mutation. We found SUP8 in the region upstream of the NES24 gene, placing the NES24 gene on chromosome XIII. A protein homology search indicated that NES24 encodes a new protein. The disruption of the NES24 gene resulted in temperature-sensitive growth. The sequence has been deposited in DDBJ/EmBL/GenBank data bases under Accession Number D15052.     

Analysis of the rad3-101 and rad3-102 mutations of saccharomyces cerevisiae: Implications for structure/function of rad3 protein

The mutations rad3-101 and rad3-102 (formerly rem1-1 and rem1-2) of the essential RAD3 gene of Saccharomyces cerevisiae confer a phenotype of semidominant enhancement of spontaneous mitotic recombination and mutation frequencies, but not extreme sensitivity to ultraviolet (UV) light. These properties differ from the previously published observations of other rad3 mutations, which are very UV-sensitive but do not alter recombination frequencies significantly. We have located the position of DNA sequence changes from wild-type RAD3 to the rad3-101 and rad3-102 mutations and have demonstrated that these sequence changes are necessary and sufficient to confer the (Rem^?) mutant phenotype when transferred into otherwise wild-type RAD3 plasmids. The Rem^? mutations are not located in the same region. It is possible that the two regions of the gene in which these mutations map define portions of the molecule which are in contact when folded in the native configuration. To begin to test this hypothesis, we have constructed two double mutant alleles, one with rad3-101 and rad3-102, and one with the UV-sensitive rad3-1 mutation and rad3-102. We find that plasmids carrying these double mutant alleles of RAD3 are no longer able to confer a hyper-recombinational phenotype and do not complement the UV-sensitivity of the excision-defective rad3-2 allele. We conclude that the double mutant alleles are non-functional for excision repair, and may be null. We have also constructed new rad3 alleles by oligonucleotide-directed mutagenesis and have tested their effects on spontaneous mutation and mitotic recombination and on UV repair.     

XII. Yeast sequencing reports. Sequence,mapping and disruption of CCC1, a gene that cross-complements the Ca2+-sensitive phenotype of csg1 mutants

We have isolated, sequenced, mapped and disrupted a novel gene, CCC1, from Saccharomyces cerevisiae. This gene displays non-allelic complementation of the Ca²⁺-sensitive phenotype conferred by the csg1 mutation. The ability of this gene, in two copies per cell, to reverse the csg1 defect suggests it may have a role in regulating Ca²⁺ homeostasis. The sequence of CCC1 indicates that it encodes a 322 amino acid, membrane-associated protein. The CCC1 gene is located on the right arm of chromosome XII. The sequence has been deposited in the GenBank data library under Accession Number L24112.     

Isolation and DNA sequence of the STE14 gene encoding farnesyl cysteine: Carboxyl methyltransferase

We isolated a mutant defective in C-terminal farnesyl cysteine:carboxyl methyltransferase activity from a screen for mutations causing a -specific sterility. A genomic fragment was cloned from a yeast multi-copy library that restored mating. Both the cloned gene and the sterile mutation were allelic to the STE14 gene. A ste14-complementing 2·17 kb BamHI fragment subclone was sequenced and found to encode a 239 amino acid protein with a molecular weight of 27,887 Daltons. The hydrophobicity profile of the methyltransferase reveals the presence of at least five potential transmembrane domains. In comparisons of the C-terminal methyltransferase amino acid sequence with those in the PIR and Swiss protein databases, no significantly similar sequences were found nor were conserved regions from other methyltransferases present.     

Identification of YHR019 in Saccharomyces cerevisiae chromosome VIII as the gene for the cytosolic asparaginyl-tRNA synthetase

Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs⁻-1, was amplified from genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenylate recognition. The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine. © 1998 John Wiley & Sons, Ltd.     

XIII. Yeast mapping reports. MTF1, encoding the yeast mitochondrial RNA polymerase specificity factor,is located on chromosome XIII

MTF1 is a nuclear gene that encodes the promoter recognition factor of the yeast mitochondrial RNA polymerase. The MTF1 gene was physically mapped to chromosome XIII. Genetic mapping data indicate that the gene is closely linked to RNA1.     

Release and partial characterization of cell-envelope proteinases fromLactococcus lactis subsp.lactis IFPL 359 andLactobacillus casei subsp. casei IFPL 731 isolated from raw goat,s-milk cheese

Different methods of releasing the cell-envelope proteinase (CEP) fromLactococcus lactis IFPL 359 (Lc-CEP) andLactobacillus casei IFPL 731 (Lb-CEP) have been tested. Release of Lc-CEP was higher in Ca^2+-free buffer than in the presence of lysozyme and Ca^2+-. Lb-CEP was not soluble in Ca^2+--free buffer, making necessary the use of chelating agents such as ethylenediaminetetraacetate (EDTA) to attain release yields of 15–20%. Solubilizing the cell wall oflb.casei using lysozyme and mutanolysin improved CEP release yields, even in the presence of Ca^2+-. Two differently charged chromophoric peptides were degraded by whole cells and the soluble fractions studied at different hydrolysis rates in both the strains considered. Based on the specificity of these CEPs for the different substrates, the two proteinases can be placed in the same class as the CEP_I/III mixed-type variants that have been identified in lactococcal proteinases. In both strains ß-casein was hydrolysed more rapidly than _s-cascin.     

Analysis of a 23 kb region on the left arm of yeast chromosome IV

We have determined the complete nucleotide sequence of a 23 kb segment from the left arm of chromosome IV, which is carried by the cosmid 1L10. This sequence contains the 3′ coding region of the STE7 and RET1 (COP1) genes, and 13 complete open reading frames longer than 300 bp, of which ten correspond to putative new genes and three (CLB3, MSH5 and RPC53) have been sequenced previously. The sequence from cosmid 1L10 was obtained entirely by a combined subcloning and walking primer strategy.     

Alkaline Degradation of Monosaccharides Part VIII. A13C NMR Spectroscopic Study

The analysis of alkaline degradation products of monosaccharides has been performed by ¹³C NMR spectroscopy. The ¹³C chemical shifts of several carboxylic acids produced by alkaline degradation of monosaccharides have been determined. The application of these data to the identification and quantification of products in an alkaline degradation mixture of D -glucose is presented. Carboxylic acid products up to 6 carbon atoms have easily been identified in such complex reaction mixtures. Furthermore, information on the different functional groups present in the oligomeric reaction products has been obtained from the alkaline degradation of 1-¹³C-D -glucose.     

Characterization of Na+/H+-antiporter gene closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii

In order to clarify the relationship between salt-tolerance of Zygosaccharomyces rouxii and the function of Na⁺/H⁺-antiporter, a gene was isolated from Z. rouxii which exhibited homology to the Na⁺/H⁺-antiporter gene (sod2) from Schizosaccharomyces pombe. This newly isolated gene (Z-SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz. pombe homologue. The predicted amino-acid sequence of Z-Sod2p was highly homologous to that of the Sz. pombe protein, but included an extra-hydrophilic stretch in the C-terminal region. The expression of Z-SOD2 was constitutive and independent of NaCl-shock. Z-SOD2-disruptants of Z. rouxii did not grow in media supplemented with 3 M -NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z-SOD2 was closely related to the salt-tolerance of Z. rouxii. Several genes are also compared and discussed in relation to the salt-tolerance of Z. rouxii. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number: D43629.     

Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae

The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read‐through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage‐dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains’ tolerance of a number of transition metal salts. Copyright © 2015 John Wiley & Sons, Ltd.     

Localization of the FAR3 gene: Genetic mapping and molecular cloning using a chromosome walk-‘n’-roll strategy

FAR3 is a newly-discovered yeast gene required specifically for pheromone-mediated cell cycle arrest. I have used strains harboring the far3-1 mutation to map the gene to the right arm of chromosome XIII, establishing the gene order CEN13-LYS7-MCM1-FAR3. I cloned the FAR3 gene based on its genetic map position using a strategy that combined chromosome walking and a related technique termed ‘chromosome rolling’. In addition to the genetic and physical localization of FAR3, I present data that suggest corrections to the tentative map positions of VAN1 and ARG80.     

X–XI. Yeast mapping reports. The use of random-breakage mapping to locate the genes APN1 and YUH1 in the Saccharomyces genome,and to determine gene order near the left end of chromosome XI

We have used the previously described technique of random-breakage mapping to locate the two yeast genes APN1 and YUH1. The APN1 locus is located ～235 kb from the left telomere of chromosome XI, and shows weak (～53 cM) genetic linkage to ura1. The YUH1 locus is located ～140 kb from the right telomere of chromosome X, and genetically maps 3·6 cM distal to cdc11. In addition, we show by random-breakage mapping that TRP3 is located ～45 kb from the left telomere of chromosome XI, whereas FAS1 is ～110 kb from the same telomere. This supports a gene order on the left distal portion of chromosome XI that agrees with other physical reports but is inverted with respect to Edition 11 of the published genetic map. This report confirms that random-breakage mapping is a rapid and convenient method of locating cloned genes.     

Saccharomyces cerevisiae mata mutant cells defective in pointed projection formation in response to α-factor at high concentrations

We have isolated Saccharomyces cerevisiae MAT a mutant cells that do not form a pointed projection but elongate in response to α-factor at high concentrations. Complementation tests defined three genes, PPF1, PPF2, and PPF3 (for pointed projection formation), necessary for pointed projection formation. Allelism tests with genes known to be needed for projection formation revealed that PPF1 is identical to SPA2, while PPF2 and PPF3 are not allelic to SST2, STE2, SPA2, BEM1 or SLK1/SSP31/BCK1. The morphology of MAT a ppf mutants treated with high concentrations of α-factor is similar to that of MAT a PPF cells treated with α-factor at low concentrations. Quantitative mating tests showed that PPF2 and PPF3 are not essential for mating in either MAT a or MATα background. Monitoring of division arrest and expression of an α-factor-inducible gene revealed that mutations in the PPF genes do not affect the responses of MAT a cells to low concentrations of α-factor. Unlike wild-type cells, the ppf mutants exhibited early recovery from α-factor-induced division arrest. Furthermore, vegetatively growing ppf3-1 cells are slightly defective in cell separation of mother and daughter cells and in selection of the correct bud sites in all cell types. These results indicate that PPF2 and PPF3 are involved in the response to α-factor at high concentrations and that PPF3 is also required for proper establishment of polarity in vegetative growth.