摘要:

AbstractThe recently published sequence of yeast chromosome III (YCIII) provides the longest continuous stretch of a eukaryotic DNA molecule sequenced to date (315 kb). The sequence contains 116 distinct AUG‐initiated open reading frames of at least 200 codons in length, more than 50 of which had not been described previously nor bear significant similarity to known proteins. We have analysed the YCIII known and putative‐protein sequences with respect to significant statistical features which might reflect on structural and functional characteristics. The YCIII proteins have striking similarities and differences in their sequence attribute distributions compared to the corresponding distributions for all available yeast sequences and other protein collections. Nine examples of YCIII proteins with distinctive sequence features are discussed in det

ISSN:0749-503X    

DOI:10.1002/yea.320091202

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractExpression/shuttle vectors for the yeastSaccharomyces cerevisiaehave usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number inEscherichia coli, and all have the yeastTRP1gene, and the 2 μm origin includingREP3sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximalEcoRI site and a unique promoter distalXhoI site, allowing for directional cloning and expression of ‘ZAP’‐type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters,ADH2, PGK, GAL10and SV40 were compared for their relative activity, both inE. coliand in yeast. All yeast promoters showed substantial activity inE. coliwithADH2showing the highest activity.ADH2also was well‐regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site‐directed m

ISSN:0749-503X    

DOI:10.1002/yea.320091203

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractA series ofSaccharomyces cerevisiae/Escherichia coliλ/plasmid expression vectors have been constructed which allow easy excision of the plasmid sequences from λ. Features of six are described, and two designated λPG15 and λAD5, are characterized in detail. Transcription of cloned sequences is controlled by the alternative promoters,ADH2, PGK, GAL10and SV40 early, and by theCYC1transcriptional terminator. UniqueEcoRI andXhoI restriction sites in the intervening polylinker make these λ vectors compatible for directional cloning of ‘ZAP’‐synthesized cDNAs. Inserted DNAs have been previously shown to have high levels of the genetic activity in bothS. cerevisiaeandE. coli, allowing these vectors to be used for genetic complementation in both species. Plasmid recovery from the λ vector is mediated by the activity of thecre‐encoded enzyme uponloxsequences flanking the plasmid and adjoining the λ arms. The plasmids contain the yeast 2 μm origin andE. colipBR322 origin, theURA3orTRP1yeast selectable markers, and ampicillin‐resistance marker inE. coli. The usefulness of the λPG15 and the λAD5 cloning vectors was demonstrated by constructing largeNeurospora crassacDNA libraries. The λPG15–N. crassalibrary was used to infectpurE,purCandtrpCmutants ofE. coli, and complemented and/or suppressed prototrophic colonies were selected. The flexibility and power of this system for cloning

ISSN:0749-503X    

DOI:10.1002/yea.320091204

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractMitochondrial mRNAs in yeast arise by processing of polygenic primary transcripts at a conserved dodecamer sequence (5′‐AAUAAPyAUUCUU‐3′). Previous results indicated that processing at dodecamer sites interrupted the sequence implying that it functioned primarily as a signal for 3′ end formation of mRNAs. We have determined the precise cleavage site for RNAs processed at the dodecamer sequences associated with theoli1gene and the ω intron of the 21S rRNA gene. In both cases cleavage occurred two bases downstream of the site. Hydrolysis left the PO4group attached to the 3′ terminus of the cleavage products. These results demonstrate for the first time that mature mitochondrial mRNAs terminate with an intact dodecamer sequence. In light of the recent identification of a protein complex within mitochondria that binds to RNAs terminating with an intact dodecamer sequence, these results support the idea that the dodecamer sequence functions not only within pre‐mRNAs as a processing site, but within mature mRNAs as well, possibly for the stabilization and/

ISSN:0749-503X    

DOI:10.1002/yea.320091205

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractWe demonstrate the successful adaptation to other yeast species of a protocol previously described for production of transcriptionally active whole cell extracts fromSaccharomyces cerevisiae(Woontner and Jaehning, 1990,J. Biol. Chem.265, 8979–8982). Extracts prepared fromSchizosaccharomyces pombe, Kluyveromyces lactisandCandida glabratawere all capable of initiating transcription from a template containing theS. cerevisiae CYC1TATA box fused to a G‐less cassette. Transcription in all of the extracts was sensitive to inhibition by α‐amanitin, indicating that it was catalysed by RNA polymerase II, and was dramatically stimulated by the chimeric activator GAL4/VP16. The different extracts used different subsets of a group of three initiation

ISSN:0749-503X    

DOI:10.1002/yea.320091206

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractMutants defective in O‐acetylhomoserine sulfhydrylase (OAH‐SHLase) were obtained in five yeast strains representative of different yeast genera:Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombeandTrichosporon cutaneum. In vitro, in all five strains, the enzyme also had O‐acetylserine (OAS) sulfhydrylase activity so it is a ‘bifunctional’ OAH/OAS‐SHLase (Yamagata, 1989). The enzyme was only found to be essential inS. cerevisiae(OAH SHLase‐negative mutants are auxotrophs). Its impairment inK. lactiscaused a slower growth rate and a decrease of the sulfur amino acid pool. InT. cutaneumonly the pool was affected whereas inY. lipolyticaandS. pombethe lesion caused no change in the growth rate nor in the pool. In all strains where OAH SHLase‐negative mutants were prototrophs, a monofunctional OAS sulfhydrylase was detected. The results indicate that OAH SHLase may play different physiological roles in

ISSN:0749-503X    

DOI:10.1002/yea.320091207

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have sequenced two segments containing a total of 51·6 kb of the left arm from chromosome XI ofSaccharomyces cerevisiae. The first segment of 38·5 kb contains 18 open reading frames (ORFs) of more than 100 amino acid residues. Five ORFs encode known yeast genes, including the fatty acid synthase gene (FAS1). Three new yeast genes were discovered with homologies to non‐yeast genes and ten new genes without homologies to any known sequences. The second segment of 13 kb contains five ORFs with two known yeast genes and three unknown genes. The sequences from cosmid pUKG041 were obtained entirely with the walking primer strategy resulting in a very low overall sequence redundancy of 2·8 and an average reading length of 443 b

ISSN:0749-503X    

DOI:10.1002/yea.320091208

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractAs part of the EEC yeast genome program, a fragment of 15 820 bp from the right arm ofSaccharomyces cerevisiaechromosome XI has been sequenced. This fragment corresponds roughly to the centromere‐distal half of cosmid pUKG046 and to a small fragment of cosmid pUKG096, which are located approximately 150 kb from the centromere. It contains four open reading frames (ORFs) which encode potential proteins of more than 100 amino acid residues, as well as theUBI2gene which carries an intron and does not show up as an ORF in the sequence analysis programs. One of the putative proteins, YKR412, is very rich in serine and has significant homology at the carboxyl end to Nopp140 phosphoprotein. YKR413 has several predicted transmembrane domains. YKR15, which has been recently cloned as theMPL1gene, encodes a polypeptide that shows homologies to myosin heavy chain and to the cytoskeleton protein Uso

ISSN:0749-503X    

DOI:10.1002/yea.320091209

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractWe report here the DNA sequence of a segment (α1006.13: YBLO5) of chromosome II ofSaccharomyces cerevisiae, extending over 32·5 kb. The segment contains 26 open reading frames (ORFs) from YBLO501 to YBLO526. YBL0505 corresponds to theSEC17gene and YBL0521 to theKIP1gene. YBL0516 contains an intron, YBL0513 shows homology with the RAT protein phosphatase and YBL0526 contains a zinc‐finger motif. Disruption of 14 genes by insertion of aURA3cassette has been performed and these mutants were analysed for their mating and sporulation ability, and for their growth on different carbon sources. YBL0515 and YBL0526 ORFs seem to be involved in the sporulation proc

ISSN:0749-503X    

DOI:10.1002/yea.320091210

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractA low‐affinity glucose transporter gene ofSaccharomyces cerevisiaewas cloned by complementation of therag1mutation in a strain ofKluyveromyces lactisdefective in low‐affinity glucose transport. Gene sequence and effects of null mutation inS. cerevisiaewere described. Data indicated that there are multiple genes for low‐affinity glucose tran

ISSN:0749-503X    

DOI:10.1002/yea.320091211

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY