摘要:

AbstractThe endoplasmic reticulum (ER) and other secretory compartments ofSaccharomyces cerevisiaehave biochemical functions that closely parallel those described in higher eukaryotic cells, yet the morphology of the yeast organelles is quite distinct. In order to associate ER functions with the corresponding cellular structures, we localized several proteins, each of which is expected to be associated with the ER on the basis of enzymatic activity, biological function, or oligosaccharide content. These marker proteins were visualized by immunofluorescence or immunoelectron microscopy, allowing definition of theS. cerevisiaeER structure, both in intact cells and at the ultrastructural level. Each marker protein was most abundant within the membranes that envelop the nucleus and several were also found in extensions of the ER that frequently juxtapose the plasma membrane. Double‐labeling experiments were entirely consistent with the idea that the marker proteins reside within the same compartment. This analysis has permitted, for the first time, a detailed characterization of the ER morphology as yeast cells proceed through their growth and division cycle

ISSN:0749-503X    

DOI:10.1002/yea.320070902

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe allantoinase (DAL1) gene fromSaccharomyces cerevisiaehas been cloned, sequenced, and found to encode a 472 amino acid protein with a Mrof 52 028.DAL1is expressed in an inducer‐independent manner in strain M970 (∑1278b genetic background) and modestly responds to mutation of theda180locus. Expression was also sensitive to nitrogen catabolite repression (NCR). Correlated with these expression characteristics, the upstream region ofDAL1contained five copies of a sequence that is homolgous to theDAL UASNTRelement previously shown to be required for transcriptional activation and NCR sensitivity of theDAL5andDAL7genes. Missing from theDAL15′ flanking region were any sequences with significant homology to theDAL7 UISelement required for response to inducer. These observations further support the roles of UASNTRandDAL7 UISin the regulation of allantoin pathway gene expre

ISSN:0749-503X    

DOI:10.1002/yea.320070903

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractAnalyses by sodium dodecyl sulphate–polyacrylamide gradient‐gel electrophoresis and Western blotting of the proteins from boiled cell samples from all growth phases of yeast cultures, and from all stages of extract preparation indicate that the smaller subunit (s‐monomer), which is found in purified glycogen phosphorylase (EC 2.4.1.1) from baker's yeast, is not present in the living cell. It is observed in extracts ofSaccharomyces carlsbergensisandS. cerevisiaeafter incubation at ambient temperatures or even after storage in the frozen state at −25°C. Its formation is sensitive towards pepstatin A, and it is absent from extracts of several mutants ofS. cerevisiaethat do not contain active proteinase yscA (EC 3.4.33.6). When purified proteinase yscA‐deficient strains are grown with a reduced amount of complex nitrogen compounds, the slightly smaller sc‐monomer is formed in their extracts. This event must be attributed to a different proteinase, since it is sensitive towards p‐hydroxymercuriphenylsulphonate, but not towards pepstatin A. The N‐terminal amino acid of the sc‐monomer was found to be blocked, as in the case of the native 1‐monomer, b

ISSN:0749-503X    

DOI:10.1002/yea.320070904

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractIn order to isolate mutants with impaired uptake of branched‐chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched‐chain amino acid biosynthesis. Eighteen mutants of independent origin were found. Ten of them were assayed for branched‐chain amino acid uptake. Three of these were impaired in the uptake ofL‐valine,L‐isoleucine andL‐leucine, while the rest were unaffected in uptake of any of the three amino acids. Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis‐Menten kinetics. This analysis suggested that a high‐affinity system for all three amino acids is absent in the mutant, whereas low‐affinity uptake ofL‐isoleucine andL‐leucine by one or more systems remains unaffected. Moreover, medium‐affinity uptake components forL‐valine andL‐leucine, not originally seen in the wild type, were identified in the mutant. In the wild type, 10 mMof any of the amino acidsL‐alanine,L‐cysteine,L‐isoleucine,L‐leucine,L‐tryptophan andL‐valine reduce uptake of any of the three branched‐chain amino acids. We propose that a permease responsible for high‐affinity uptake of the branched‐chain amino acids in strain S288C is partially or completely inactive in the mutant. Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene. The wild‐type allele is denotedBAP1forbranched‐chainamino acidpermease. TheBAP1‐dependent syste

ISSN:0749-503X    

DOI:10.1002/yea.320070905

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractLC1, a YIP5‐derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids inSaccharomyces cerevisiae. ARS plasmids carried in yeast hosts are normally mitotically unstable. In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss. In the case ofS. cerevisiaecarrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10–20% or 30–50% respectively. We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker. Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70%. To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA. In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hydribization. These results suggest that plasmid is unable to replicate in these cells. Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium. Possible explanations for the phenomena observed are disc

ISSN:0749-503X    

DOI:10.1002/yea.320070906

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe 4·4 kbSphI DNA fragment (GSH1) that complements the γ‐glutamylcysteine synthetase‐deficient mutation (gsh1) ofSaccharomyces cerevisiaeYH1 was cloned into vector plasmid YEp24. Gene disruption of the cloned fragment confirmed that this segment was the same gene asgsh1. Mutant strain YH1 with this plasmid not only restored γ‐glutamylcysteine synthetase (GSH‐1) activity but the glutathione content and the growth rate. DNA sequence analysis of theSphIfragment showed that theGSH1structural gene contained 2034 bp and predicted a polypeptide of 678 amino acids. The deduced amino acid sequence had about a 45% homology to that of rat kidney GSH‐I, but a very low homology (about 26%) to that ofEscherichia coliGSH‐I. Northern analysis showed thatGSH1had been transcribed into an approximately 2·7 kb mRNA fragment. Southern analysis showed thatGSH1mapped

ISSN:0749-503X    

DOI:10.1002/yea.320070907

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractTheLEU2gene, coding for β‐isopropylmalate dehydrogenase, of the yeastKluyveromyces marxianuswas isolated and sequenced. An open reading frame, coding for a protein with a molecular weight of 38kDa was found. Comparison of the deduced amino acid sequence of theLEU2gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities. The cloned gene could complement aleuBmutation ofEscherichia coliand aleu2mutation ofSaccharomyces cerevisiae. Using orthogonal field alternation gel electrophoresis, the genomic copy of the gene was found to be located at chromosome VI or VII. Analysis of the 5′‐untranslated region indicated the presence of a putative binding site for the LEU3 protein, which is involved in the leucine‐specific regulation of transcription. We show that the cloned gene can be used for the construction of a non‐revertingK. marxianus

ISSN:0749-503X    

DOI:10.1002/yea.320070908

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe nucleotide sequence of a 1558 bp DNA fragment from the right arm of chromosome III ofSaccharomyces cerevisiaecontains an open reading frame of 954 nucleotides with coding potential for a protein with high similarity to the ubiquitous cyclophilins which are both peptidyl‐prolylcis‐transisomerases and cyclosporin A‐binding proteins. It should, therefore, represent the third gene (SCC3) of this kind fromS. cerevisiae.SCC3is present in a single copy in the genome ofS. cerevisiaeand results in a constitutively expressed 1·2 kb transcript during cell growth. Its putative protein product (Scc3) contains two hydrophobic cores, one at the amino terminal, 20 amino acids long, which could serve as a signal peptide, and the other one at the carboxyl end with a structure similar to a transmembrane helix. These findings suggest that Scc3 could be a secretory or, more likely, a transmembrane protein. The only cyclophilin with similar structure to that of Scc3 is ninaA fromDrosophila melanogaster, a transmembrane protein which seems to be implicated in the correct folding and/or intercalation of rhodopsin in the endoplasmic reticulum of the fly photoreceptors (Stamnes, M. A.et al.,Cell65, 219–227, 1991). In addition, the amino and the carboxy regions of Scc3 and ninaA share a significant level of homology, which suggests that they have a similar function, albeit for different target

ISSN:0749-503X    

DOI:10.1002/yea.320070909

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractWe report the sequence of a 6·3 kb segment of DNA mapping near the end of the right arm of chromosome III ofSaccharomyces cerevisiae. The sequence reveals a major open reading frame coding for a putative protein of 1047 amino acids with a striking similarity to the bacterial proteins involved in recognition of mismatched DNA base pairs. This is particularly interesting as the existence of a yeast mismatch repair system similar to that of bacteria has been postulated for some years, but a yeast protein homologous to the bacterial mismatch binding protein had not been identified.The results of a comparison of the putative yeast mismatch binding protein with the bacterial mismatch binding proteins and with two cognate mammalian sequences, support the idea that a similar mismatch repair system may be present also in mammalian cells. The possibility that all of these proteins may have evolved from a common ancestral gene is also discussed

ISSN:0749-503X    

DOI:10.1002/yea.320070910

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have isolated the gene encoding the 16 kDa vacuolar H+‐ATPase fromSchizosaccharomyces pombe. On the basis of RNA splicing signals and amino acid sequence homology with other 16 kDa H+‐ATPases, the genomic DNA sequence indicated the 16 kDa protein is encoded by five exons. The C‐terminal 50 amino acids has more than 90% homology with vacuolar H+‐ATPases of mammalia

ISSN:0749-503X    

DOI:10.1002/yea.320070911

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY