摘要:

AbstractWe have constructed 2‐μm‐based yeast expression vectors containing a copy of the metallothionein (CUP1) gene ofSaccharomyces cerevisiaeas a semi‐dominant, selectable marker. When used for the expression of the thrombin inhibitor hirudin, originally derived from the leechHirudo medicinalis, these vectors displayed the following characteristics. (1) In the presence of copper salts, they were mitotically more stable than similarly designed control vectors lacking theCUP1gene. In copper‐sensitive host strains, the apparent plasmid stability was 100%, even in complex media and during fed‐batch fermentation for an extended period of time. (2) Use of theCUP1‐stabilized plasmids improved the production of hirudin by both copper‐sensitive and copper‐resistant hosts. The highest hirudin titers were obtained with a ΔCUP1host. (3) Copper selection resulted in a moderate increase in average plasmid copy numbers (up to two‐fold) as assessed by measuring hirudin expression from a constitutive promoter (GAPFL). This effect was most noticeable if the vector showed an asymmetric segregation pattern (i.e., high rates of plasmid loss in the absence of copper). (4) TheCUP1marker proved particularly useful in combination with aCUP1‐promoter‐controlled expression cassette on the same plasmid. In such a set‐up, the rates of transcription of the heterologous protein and that of the selectable marker are tightly linked. Therefore, an increase in selective pressure directly provokes an increase in product yields. In a copper‐sensitive host strain, this plasmid design allowed for the production of very high amounts of biologically active hirudin. Our results clearly establish the utility of theCUP1marker in the construction of stab

ISSN:0749-503X    

DOI:10.1002/yea.320110102

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe purine‐cytosine permease (PCP) of the yeastSaccharomyces cerevisiaewas detected by immunological methods. Using antibodies directed against synthetic peptides, whose sequences were derived from the primary structure of the PCP, immunoprecipitation of [35S]methionine‐labelled PCP was achieved either from cellular extracts or fromin vitrotranslation mixtures. Non‐labelled PCP was also detected on Western blots of membrane proteins. Similar migration rates were observed for PCP originating both from immunoprecipitated cellular extracts and fromin vitrotranslation mixtures. Hence, post‐translational processing, if any, only slightly affects the size of the protein. Also no evidence was found for N‐linked core‐glycosylation: identical migration rates were observed when immunoprecipitated PCP molecules were extracted from cells labelled for 10 min with [35S]methionine, pretreated or not with tunicamycin.On the other hand, the suppresion of the two potential N‐linked glycosylation sequences in the DNA did not lead to inactivation of the transport activity, confirming that N‐linked glycosylation is not required for the pe

ISSN:0749-503X    

DOI:10.1002/yea.320110103

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractIn order to characterize new yeast genes regulating cell proliferation, a number of overexpression‐sensitive clones have been isolated from aSaccharomyces cerevisiaecDNA library in a multicopy vector under the control of theGAL1promoter, on the basis of growth arrest phenotype under galactose‐induction conditions. Thirteen of the independent clones isolated in this way correspond to previously known genes (predominantly coding for morphogenesis‐related proteins or for multifunctional transcriptional factors), while the remaining 11 independent clones represent new genes with unknown functions. The more stringent conditions employed in this screening compared with previous ones that also employed a dominant genetics approach to isolate overexpression‐sensitive genes has allowed us to extend the number of yeast genes that exhibit this phenotype. The effect of overexpression ofMCM1(whose product participates in the regulation of a number of apparently unrelated cellular functions) has been studied in more detail. Galactose‐induced overexpression ofMCM1leads to rapid growth arrest at the G1or S cell cycle stages, with many morphologically‐abnormal cells. Several of the other clones also exhibit a G1arrest terminal phenotype when ov

ISSN:0749-503X    

DOI:10.1002/yea.320110104

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe demonstrate that serine instead of leucine is specified by the CUG codon in the yeastCandida maltosa. Evidence for this deviation from the universal genetic code was obtained by means ofin vitrotranslation experiments. Depending on the cell‐free system used, either serine, in theC. maltosasystem, or leucine, in the control with the conventional wheat germ system, was found to be incorporated into the translation products of artificial CUG‐containing mRNAs. Moreover, we were able to transfer the non‐universal decoding of CUG to the wheat germ system by adding a tRNA fraction isolated fromC. maltosa. This finding indicates the presence inC. maltosaof an unusual serine tRNA that recognizes CUG. As a consequence of the altered genetic code, expression inSaccharomyces cerevisiaeofC. maltosacytochrome P450 genes required an exchange of their CTG triplets by TCT encoding serine in order to produce the authentic proteins. In contrast, heterologous expression of the originalC. maltosagenes resulted in the formation of still active but unstable enzymes probably subject to selective proteolysis in the host

ISSN:0749-503X    

DOI:10.1002/yea.320110105

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractAn alkane‐assimilating yeastCandida maltosahad been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine inC. cylindracea. The same altered codon usage had also been suggested byin vitroexperiments in someCandidayeasts which are phylogenetically closely related toC. maltosa.In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine inC. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene ofC. maltosacontaining a CTG codon was expressed inC. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNASerCAG gene ofC. albicanscould be isolated from the genome ofC. maltosa; (3) theSaccharomyces cerevisiae URA3gene, which has one CTG codon, could not complement theura3mutation ofC. maltosaas itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement theura3mutation.The last result is the first example of succeeding in functional expression of a heterologous gene inCandidaspecies having an altered codon usage by changing the CTG codon in the gene to another codon.The nucleotide sequence datum reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the Accession Number D2607

ISSN:0749-503X    

DOI:10.1002/yea.320110106

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractA set ofGAL2+yeast strains that are isogenic to strain S288C have been constructed. They contain non‐reverting mutations in genes commonly used for selection for recombinant plasmids. Strains from this collection are being used for the European Union Yeast Genome Sequencing Programme. Representative strains from this collection have been deposited with the ATC

ISSN:0749-503X    

DOI:10.1002/yea.320110107

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe have sequenced a continuous segment of 17 137 bp on chromosome X. Sequence analysis of this stretch revealed 14 open reading frames (ORFs) at least 100 amino acids long. One gene, encoding the mitochondrial 60S ribosomal protein L8, had already been sequenced. Four ORF products show weak homologies with known protein sequences. The nine remaining ORF products have no homologies with sequences in data banks. The nucleotide sequence of the 17·1 kb fragment is available through the EMBL data library under Accession Number Z34288

ISSN:0749-503X    

DOI:10.1002/yea.320110108

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 cosmid (pIX338) containing the centromere region of yeast (Saccharomyces cerevisiae) chromosome IX. The complete nucleotide sequence of 33·8 kb was obtained by using an efficient directed sequencing strategy in combination with automated DNA sequencing on the A.L.F. DNA sequencer. Sequence analysis revealed the presence of 17 open reading frames (ORFs), four of them previously known yeast genes (sly12, pan1, sts1andprl1), a tRNA gene and the centromere motif. Exhaustive database searches detected sequence homologues of known function for as many as 14 of the 17 ORFs. These include a mammalian tyrosine kinase substrate; theEscherichia colicell cycle protein MinD; the human inositol polyphosphate‐5‐phosphatase (gene OCRL) involved in Lowe's syndrome, a developmental disorder; and helicases, for which the new yeast member defines a distinct DEAD/H‐box subfamily. A surprisingly large fraction of the ORFs (at least six out of 17) in the centromeric region are apparently involved in RNA or DNA binding.The nucleotide sequence reported here has been submitted to the EMBL data library under the accession number

ISSN:0749-503X    

DOI:10.1002/yea.320110109

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe nucleotide sequence of a fragment of 7200 base pairs ofSaccharomyces cerevisiaechromosome II has been determined. The sequence contains three open reading frames (ORFs). Two genes for galactose metabolism,GAL7and part of theGAL10coding region, are localized on the fragment. Comparison to the previously published sequence data showed several differences, leading to changes in the amino acid sequences ofGAL7andGAL10.One new ORF, YBR0224, was detected, coding for a protein with 918 amino acids. Comparison to the DNA and protein data bases showed no significant homologies. The protein has some interesting features pointing to a function involved in transcription regulation: a leucine zipper motif, a highly acidic region, possibly involved in transcription activation and a putative nuclear localization signal. Deletion analysis showed that the gene is essential when deleted in strain W303. Spores could germinate and form microcolonies, but efforts to propagate the colonies failed. Deletion of this gene in a different genetic background (strain M5) led to very poor‐growing mutant strains with cells showing aberrant cellular morphologies.The nucleotide sequence of the fragment has been deposited in the EMBL‐database under the Accession Number X81

ISSN:0749-503X    

DOI:10.1002/yea.320110110

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe nucleotide sequence of two adjacentClaI fragments from the left arm ofSaccharomyces cerevisiaechromosome XIV has been determined. Analysis of the 13,520 bp DNA segment reveals nine open reading frames (ORFs) longer than 300 bp. N1302 contains the consensus sequence for a phosphate‐binding loop common to ATP‐ and GTP‐binding proteins and a strictly conserved ‘SRC’ sequence of unknown function present in all accessory proteins of replicative polymerases. N1306 shares homologies with serine/threonine phosphatases.N1310encodes RAP1 (TUF or SBF‐E), a transcription regulator.N1330is theMER1gene required for chromosome pairing and genetic recombination. Two ORFs show no homology with proteins in the databases and no particular features.N1311is not likely to be expressed as it is located on the complementary strand ofN1310. The sequence has been submitted to the EMBL data library under Accession Nu

ISSN:0749-503X    

DOI:10.1002/yea.320110111

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY