摘要:

AbstractIn the yeastSaccharomyces cerevisiaethePEL1gene is essential for the viability ofrho−/rhoopetitemutants, and its mutation in respiring cells results in a pleiotropic phenotype. Results of complementation analysis with different subclones of chromosomal DNA and re‐sequencing of the YCL4w‐YCL3w segment of chromosome III demonstrate that the coding region of thePEL1gene corresponds to 1467 bp. The size of thePEL1transcript in Northern blot analysis was estimated to be approximately 1·5 kb. Transcription initiation in wild‐type cells was found to occur at the position −9 relative to the ATG. ThePEL1gene was moderately expressed irrespective of the state of the mitochondrial genome and the nature of the carbon sources. Disruption of thePEL1gene was not lethal and resulted in the same phenotype as observed with thepel1mutant, i.e. the cells were not able to survive ethidium bromide mutagenesis, were thermosensitive for growth on glucose at 37°C and failed to grow on minimal glycerol medium. Although the Pel1 protein exhibits significant similarity to a family of phosphatidylserine synthases, the disruptedPEL1gene was not complemented by the multicopy plasmid‐borneCHO1gene encoding an essential yeast phosphatidylserine synthase. The Accession Number of thePEL1gene in the EMBL data libr

ISSN:0749-503X    

DOI:10.1002/yea.320111302

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractSaccharomyces cerevisiaecontains two genes (DOGR1andDOGR2) that are able to confer 2‐deoxyglucose resistance when they are overexpressed. These genes are very similar, sharing 92% identity at the protein level. They code for two isoenzymes with 2‐deoxyglucose‐6 phosphate (2‐DOG‐6P) phosphatase activity. These enzymes have been purified and characterized. DogR1p shows an optimum pH of 6, an optimum temperature of 30°C and aKMon 2‐DOG‐6P of 17 mM. DogR2p shows a similar optimum pH, but the optimum temperature is 40°C and it exhibits aKMon 2‐DOG‐6P of 41 mM. Both enzymes require 10 mM‐MgCl2for maximal activity and they are inhibited b

ISSN:0749-503X    

DOI:10.1002/yea.320111303

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractA system has been developed which allows the selection of integrative transformants with replacement of theHansenula polymorphamethanol oxidase gene (MOX) with expression cassettes carrying heterologous gene under the control of theMOXpromoter. The system is convenient for comparison of the expression levels of different constructs integrated into the same locus of theH. polymorphagenome. This system was used to compare the secretion levels of human urinary plasminogen activator, the secretion of which was directed by different signal sequences.

ISSN:0749-503X    

DOI:10.1002/yea.320111304

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractRNase MRP and RNase P ribonucleoproteins are structurally and functionally similar across a large evolutionary distance. To better characterize possible complex interrelationships between these two enzymes, we have employed the fission yeastSchizosaccharomyces pombe. UnlikeSaccharomyces cerevisiae, S. pombeis believed to harbour only one genetic locus for the RNA component of RNase P and does not contain a known mitochondrially encoded RNase P RNA. We have identified the single nuclear gene for the RNA component of RNase MRP inS. pombe, mrp‐1, by homology to vertebrate RNase MRP RNAs. Themrp‐1gene encodes an RNA of maximum mature length 400 nucleotides that shares a high degree of identity, in evolutionarily conserved regions, to both vertebrate RNase MRP RNAs andS. pombeRNase P RNA. Disruption ofmrp‐1in the diploid strain SP826 and sporulation of tetrads resulted in a 2 dead:2 viable segregation, consistent with the gene being essential. Lethality is rescued by a plasmid‐borne copy ofmrp‐1. Partially purified ribonucleoprotein RNase MRP activity correctly and efficiently processed all previously characterized heterologous mitochondrial RNA substrates. The compact mitochondrial genome ofS. pombecontains sequence elements with>50% identity to mammalian D‐loop CSBI and CSBII elements. The identification ofmrp‐1inS. pombeshould facilitate not only comparisons between the related ribonucleoproteins RNase MRP and RNase P, but should also provide an opportunity for genetic elucidation of RNase MRP function in a situation reflective of the a

ISSN:0749-503X    

DOI:10.1002/yea.320111305

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractEpitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre‐existing antibodies. We have designed vectors containing theURA3gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag‐URA3‐tag cassette such that the ends of the PCR fragments possess homology to the gene of interest.In vivorecombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura+phenotype. Finally, selection for Ura−cells on 5‐fluoro‐orotic acid plates yields cells where recombination between the repeated epitopes has ‘popped out’ theURA3gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag threeSaccharomyces cerevisiaeproteins, Cln1

ISSN:0749-503X    

DOI:10.1002/yea.320111306

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame inSaccharomyces cerevisiae.

ISSN:0749-503X    

DOI:10.1002/yea.320111307

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe sequence of a 13 kbp fragment located in the vicinity of the left telomere of chromosome XV (cosmid pEOA179) has been determined. Seven new open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB629, AOA342, AOC231, AOE555, AOE236, AOA236 and AOE1045). Three of them show no identity with proteins deposited in the data banks. ORF AOB629 (629 amino acids) has some similarity with previously described ferric reductases fromSaccharomyces cerevisiaeandSchizosaccharomyces pombe. ORF AOA342 encodes a polypeptide reminiscent of dihydroflavonol‐4‐reductases from a number of plant species. AOE236 displays a high level of identity when compared with peroxisomal membrane proteins previously cloned from the methylotrophic yeastCandida boidinii. Finally, AOE1045 encodes a large protein (1045 residues) with some identity with a hypothetical 147 kDa protein identified during the sequencing ofCaenorhabditis eleganschromosome 3. The complete nucleotide sequence of the 13 kbp fragment has been deposited at the EMBL data base (Accession Number Z482

ISSN:0749-503X    

DOI:10.1002/yea.320111308

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe have determined the nucleotide sequence of a 3·3 kb fragment containing the gene (ADE2) encoding phosphoribosylaminoimidazole carboxylase (AIRC) from the yeastSchwanniomyces occidentalis. Translation of a 1671 bp open reading frame predicts a protein of 557 amino acids which has significant homology to AIRC fromSaccharomyces cerevisiaeandSchizosaccharomyces pombe. The 5′ untranslated region of theS. occidentalisgene contains a sequence corresponding to the consensus binding site of theS. cerevisiaetranscription regulatory proteins GCN4, BAS1 and BAS2. TheADE2gene nucleotide sequence has received the GenBank Accession Number U232

ISSN:0749-503X    

DOI:10.1002/yea.320111309

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe have isolated a 3·7 kbEcoR1 fragment from a genomic library ofCandida albicanswhich displayed a 65% level of identity with thePRSgene family (PRS) ofSaccharomyces cerevisiae. ThePRSgene encodes a phosphoribosylpyrophosphate (PRPP) synthetase ofS. cerevisiae, which catalyses the synthesis of purines, pyrimidines, and amino acids such as histidine and tryptophan. By Northern analyses, we observed that the entire 3·7 kbEcoR1 fragment as well as a 1·1 kbKpnI‐SacI internal fragment of the 3·7 kbEcoR1 fragment hybridized to the same 1.4 kb transcript. An internal 2·6 kbKpnI fragment was subcloned and sequenced. A deduced sequence of 321 amino acids representing a polypeptide of 35·2 kDa was determined. A FASTA search indicated that theC. albicans PRS (Ca PRS1)had an overall homology at the amino acid level of 91% with theS. cerevisiae PRS3. Putative transcriptional start and termination sequences as well as a cation‐binding, PRPP synthetase signature sequence were identified.Ca PRS1was localized to chromosome 2 of theC. albicansgenome. Low stringency hybridizations indicates that the organism may possess multiplePRSgenes. The function of these genes in nitrogen signaling is discussed. TheCa PRS1 sequence submitted to the EMBL data library is available under Accession Numb

ISSN:0749-503X    

DOI:10.1002/yea.320111310

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe have determined the nucleotide sequence of a 30 kb fragment of chromosome XIV ofSaccharomyces cerevisiae. The sequence revealed the presence of 19 open reading frames (ORFs) longer than 300 bp.NO422andNO425correspond to the split ribosomal protein genes encoding S16A and rp28, respectively,NO450displays a striking similarity with serine/threonine protein kinase genes, in particular withSTE20, and therefore may encode a novel member of this protein family.NO453is the longest ORF in this DNA segment, having a size of 4908 bp, but its function is not yet known.NO530encodes the plasma membrane protein Mid1p andNO533corresponds 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.

ISSN:0749-503X    

DOI:10.1002/yea.320111311

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY