摘要:

AbstractIn order to develop DNA probes for rapid, sensitive and specific detection of the pathogenic yeast speciesCandida albicans, we carried out comparative sequence analysis of the two internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA (rDNA) units ofC. albicansand the closely related pathogenic speciesC. tropicalis. While overall sequence similarity between the two species was considerable (65–75%), both ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific target sites for the identification ofC. albicans. On the basis of these results one ITS1‐derived (ANAB1) and two ITS2‐derived (ANAB2andANAB3) oligonucleotides were selected, chemically synthesized, and used as hybridization probes. Their specificity and reliability were evaluated in dot‐blot hybridization experiments with total genomic DNA from 13 strains of medically importantCandidaspecies, six strains of other yeast genera associated with man and animals, and ten strains previously identified asC. albicansby phenotypic criteria. Under well‐defined hybridization conditions the three probes hybridized exclusively with DNA derived from strains belonging to the speciesC. albicans, thus demonstrating their potential clinical usefulness. The failure of four of the (presumed)C. albicansstrains to show hybridization to the ITS probes sheds doubt upon their taxonomic classification, which is reinforced by other phenotypic aspects of thes

ISSN:0749-503X    

DOI:10.1002/yea.320100603

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractA yeast strain with a point mutation in the nuclear gene for the core subunit of mitochondrial RNA polymerase was used to isolate new extragenic suppressors. Spontaneously occurring phenotypical revertants were analysed by crosses with the wild‐type and tetrad dissection. One of the new nuclear suppressor mutants was characterized by temperature‐sensitive growth on non‐fermentable carbon sources. This mutant was transformed with a genomic yeast library. Two independent types of DNA clones were isolated which both complemented the temperature‐sensitive defect. Subcloning and DNA sequencing identified two novel yeast genes which code for proteins with the characteristic features of transcription factors. Both factors exhibit highly structured protein domains consisting of runs and clusters of asparagine and glutamine residues. One of the proteins contains in addition zinc‐finger domains of the C2H2‐type. Therefore the genes are proposed to be namedAZF1(asparagine‐richzinc‐ffinger protein) andPGD1(polyglutaminedomain protein). Gene disruption of both reading frames has no detectable influence on the vegetative growth on complete glucose or glycerol media, indicating that the genes may act as high copy number suppressors of the mutant defect. Additional transformation experiments showed thatAZF1is also an efficient suppressor for the original defect in the core subunit of mitochondrial RNA polymerase. The DNA sequences for theAZF1andPGD1genes were submitted to the EMBL data base (Accession Numbers: Z26

ISSN:0749-503X    

DOI:10.1002/yea.320100604

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe cloned and sequenced aZygosaccharomyces cidri MELgene with a view to investigating the structure and regulation of yeastMELgenes. The amino acid sequence deduced from the nucleotide sequence showed 78·6% and 78·2% similarity toSaccharomyces cerevisiaeandSaccharomyces pastorianusα‐galactosidases, respectively. The expression of theMELgene in severalZygosaccharomycesstrains was induced by galactose.An electrophoretic karyotype of severalZygosaccharomycesspecies was obtained using contour‐clamped electric field gel electrophoresis. The minimum number of chromosomes was five forZ. cidri, six forZ. fermentati, three forZ. florentinus, and four forZ. microellipsoides. The sizes of the chromosomes were generally larger than those ofS. cerevisiae, the smallest containing approximately 0·4 megabase.TheMELgene was located, using theZ. cidri MELgene as a probe, on the largest chromosome of theZ. cidristrains. In addition, a smaller chromosome (600 kb) inZ. cidristrain CBS4575 showed hybridization to the homologousMELprobe. This chromosome was absent inZ. cidristrain CBS5666. The probe hybridized to the largest chromosome of Mel+Z. fermentatistrains but failed to hybridize to any chromosome of Mel+Z. mrakiiorZ. florentinusstrains. These results suggest the existence of a polymorphicMELgene family in the yeastZygosaccharomyces.The sequence has been deposited in the EMBL Data Library under Accession Number

ISSN:0749-503X    

DOI:10.1002/yea.320100605

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractYeast exo‐1,3‐β‐glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(β‐D‐glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of theEXG1gene product ofSaccharomyces cerevisiaeand its homologous gene inCandida albicansto function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions inS. cerevisiae. This technique has the advantage over other reporter gene systems—such as β‐galactosidase fusions—that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells—by sorting—after f

ISSN:0749-503X    

DOI:10.1002/yea.320100606

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractTranscription of themat1‐Pmgene ofSchizosaccharomyces pombecontrolling entry into meiosis is stimulated by the mating pheromone,M‐factor. We have studied its expression by monitoring β‐galactosidase activity in cells carrying a plasmid‐bornemat1‐Pm/lacZfusion construct. Stimulation required theM‐factor receptor (Map3) and other proteins (Gpa1, Byr1, Byr2 and Spk1) thought to be involved in propagating the pheromone signal within the cell. Mutational activation ofgpa1encoding an α subunit of the receptor‐coupled heterotrimeric G protein causes full expression ofmat1‐Pmeven in the absence of pheromone, suggesting that Gpa1 is a key signal transmitter. Furthermore, an activatedras1val17mutant exhibited a much stronger level of induction than wild‐type cells, though full expression needsM‐factor treatment. Deletion analysis of themat1‐Pmpromoter region identified a stretch of 21 bp that is shown to play a critical role in controlling expression. This region lies just upstream of a TATA‐like box and contains a TR‐box (TTCTTTGTTY) motif which is the recognition site of a putative transcription factor Ste11. Point mutations in the TR‐box motif abolished the expression ofmat1‐Pm/lacZ. Almost no expression ofmat1‐Pmwas detected in aste11deletion mutant, whereas overproduction of Ste11

ISSN:0749-503X    

DOI:10.1002/yea.320100607

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have used a random selection protocol to define the consensus and range of binding sites for theSaccharomyces cerevisiaeREB1 protein. Thirty‐five elements were sequenced which bound specifically to a GST‐REB1p fusion protein coupled to glutathione–Sepharose under conditions in which more than 99·9% of the random sequences were not retained. Twenty‐two of the elements contained the core sequence CGGGTRR, with all but one of the remaining elements containing only one deviation from the core. Of the core sequence, the only residues that were absolutely conserved were the three consecutive G residues. Statistical analysis of a nucleotide‐use matrix suggested that the REB1p binding site also extends into flanking sequences with the optimal sequence for REB1p binding being GNGCCGGGGTAACNC. There was a positive correlation between the ability of the sites to bindin vitroand activate transcriptionin vivo; however, the presence of non‐conformants suggests that the binding site may contribute more to transcriptional activation than simply allowing protein binding. Interestingly, one of the REB1p binding elements had a DNAse 1 footprint appreciably longer than other elements with similar affinity. Analysis of its sequence indicated the potential for a second REB1p binding site on the opposite strand. This suggests that two closely positioned low‐affinity sites can function together as a highly active site. In addition, database searches with some of the randomly defined REB1p binding sites suggest that related elements are commonly found within ‘TATA

ISSN:0749-503X    

DOI:10.1002/yea.320100608

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe sequence of theCandida albicansinositol biosynthetic gene,CaINO1, and its flanking regions is determined in this study. The largest open reading frame has a coding sequence of 1560 base pairs, corresponding to a predicted protein of 521 amino acids. Three primary transcriptional start sites are found 64, 57 and 52 base pairs upstream of the ATG translational start site at position 1374. Five stop codons exist in a cluster at the end of the coding region. Within the upstream region TATA and CAAT eukaryotic regulatory sequences are identified along with regions corresponding to a 10 base pair inositol/choline responsive element consensus sequence. Computer analysis of the DNA sequence shows strong homology to theSaccharomyces cerevisiae INO1gene. A comparison of the deduced amino acid sequence of theC. albicans INO1gene product, inositol‐1‐phosphate synthase, with its homolog inS. cerevisiaeshows 64% identity and 77% similarity. The differences between the two proteins are most prominent in the N‐terminal regions. The sequence has been deposited in the GenBank/EMBL data library under Accession Number L

ISSN:0749-503X    

DOI:10.1002/yea.320100609

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have isolated a mutant which exhibits partial constitutivity fora‐specific gene expression in α cells. The wild‐type gene was cloned and demonstrated to be allelic to theSTE13gene, which encodes the dipeptidyl aminopeptidase involved in processing of the α‐factor prepropheromone. Thus, the mating defect of theste13mutations in α cells may result both from the production of incompletely processed α‐factor and from the increased expression ofa‐specific genes.TheSTE13open 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 ofSTE13has been constructed: it is viable and sporulation‐proficient. The sequence has been deposited in the GenBank data library under Access

ISSN:0749-503X    

DOI:10.1002/yea.320100610

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe COR region, a gene cluster located on chromosome X ofSaccharomyces cerevisiaeand including genesCYC1, UTR1, UTR3, OSM1, tRNAGlyandRAD7, was sequenced within the framework of the European Union genome systematic sequencing project. It was compared with previously published sequences to be found in GenBank under the acronym YSCCORA. While some of the discrepancies observed can be readily ascribed to polymorphism, others most probably result from sequencing errors. A revised version of the sequence of the COR cluster is given. The sequence has been deposited in the EMBL Data Library under Accession Number L26347.

ISSN:0749-503X    

DOI:10.1002/yea.320100611

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThis paper reports the DNA sequence and analysis of an 11·7 kb segment localized on the right arm ofSaccharomyces cerevisiaechromosome II. This fragment contains one incomplete and five long and non‐overlapping open reading frames (ORFs) designated from centromere to telomere‐proximal side as: YBR1406, 1409, 1410, 1411, 1412 and 1413. YBR1406 corresponds to the 5′ end toPGI1encoding phosphoglucoisomerase. YBR1410 encodes a polypeptide of 798 amino acids whose C terminus contains five repeats (WD‐40 repeat) similar to those found in the β‐subunits of G proteins and different yeast proteins such as Tup1, Prp4 and Cdc4. The higher similarity score is obtained with dTAFII80, a component of the RNA polymerase II transcriptional complex TFIID. YBR1411 encodes a polypeptide of 464 amino acids which belongs to the family of α‐mannosyltransferases:KRE2/MNT1, KTR1, KTR2, YUR1and the product of previously sequenced ORF YBR1445. YBR1412 corresponds toBEM1. The two ORFs, YBR1409 and YBR1413, which do not exhibit significant similarity with any known coding sequences, define new genes. The sequence has been deposited in the EMBL Data Library under Accession

ISSN:0749-503X    

DOI:10.1002/yea.320100612

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY