ISSN:0749-503X    

DOI:10.1002/yea.320090703

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractStrains ofSaccharomyces cerevisiaeharbouring temperature‐sensitive mutations in theSEC1andSEC5genes exhibit an accumulation of post‐Golgi secretory vesicles at 37°C. We have cloned a fragment of yeast DNA which carries two distinct genes, one of which complements asec1mutation, and the other asec5mutation. Genetic tests confirm that thesec1‐complementing gene is indeedSEC1, and is essential for cell growth. Nucleotide sequence analysis reveals that the clonedSEC1gene is the same as a previously sequencedsec1‐complementing gene. TheSEC1sequence encodes a protein of 724 amino acids with a predicted molecular mass of 83 kDa. Antibodies purified from a polyclonal antiserum raised against the protein product of the cloned gene recognize a yeast protein of apparent molecular mass 78 kDa which is found in a detergent‐resistant association with a rapidly sedimenting yeast subcellular fraction, behaviour which is suggestive of an interaction with a component of the yeast cy

ISSN:0749-503X    

DOI:10.1002/yea.320090704

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractA rapid and convenient method of protein purification involves creating a fusion protein with glutathione S‐transferase (GST) (Smith and Johnson,Gene67, 31–40, 1988). In this report, we describe two vectors for the conditional expression of GST fusions inSaccharomyces cerevisiae. The parent plasmid is based on a high‐copy, galactose‐inducible shuttle vector previously described (Baldariet al., EMBO J.6, 229–243, 1987). We have demonstrated the use of this system by creating fusions between GST and the yeastRAS2gene. GST‐Ras2 fusion proteins undergo the post‐translational modifications required for Ras2p to become membrane localized. These vectors provide a useful system for the expression an dpurification of eukaryotic proteins requiring post‐translation

ISSN:0749-503X    

DOI:10.1002/yea.320090705

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractIndividual yeast cells can be successfully isolated and recultured on plates with a new isolation method making use of optical trapping with infrared laser light. The cells can be selected on morphological criteria by high resolution microscopy. The isolation device is constructed from two coverslips separated by spacers, in which selected cells are transferred to a plastic capillary, using the optical trap. To test the procedure, selection experiments were done with a mixture of twoSaccharomyces cerevisiaestrains, distinguishable both in fluorescence microscopy and on agar plates. These experiments showed that only selected cells were isolated, and close to 100% of the isolated stationary‐phase cells formed colonies on agar plates, indicating a high recovery. A lower recovery was obtained with exponential‐phase cells, possibly because of a higher sensitivity to laser irradiation. Applications for this method may include the isolation of mutants with altered morphology and the isolation of subpopulations of yeast cultures, for their separate investigation or for the initiation of pure cultu

ISSN:0749-503X    

DOI:10.1002/yea.320090706

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractA major 31 kDa integral peroxisomal membrane protein (PMP31) ofHansenula polymorphawas purified to homogeneity from isolated peroxisomal membranes by FPLC after solubilization by Triton X‐100. Biochemical analysis indicated that this protein, which showed cross‐reactivity with antibodies against the 31 kDa porin of the mitochondrial outer membrane ofSaccharomyces cerevisiae, had pore‐forming properties. Firstly, proteoliposomes composed of asolectin and purified PMP31 showed selective permeability, determined as the [14C]sucrose/[3H]dextran leakage ratios. Furthermore, the generation of a ΔΨ by potassium diffusion gradients was negatively affected by the presence of PMP31 in asolectin liposomes. A similar effect was observed in proteoliposomes containing purified cytochromecoxidase as a ΔΨ generating system. Control experiments confirmed that the observed leakage is significant and introduced by the incorporation of PMP31 protein. Selective sucrose leakage was abolished in samples pretreated with glutaraldehyde; an identical effect of glutaraldehyde was, however, not observed for the membrane potential mea

ISSN:0749-503X    

DOI:10.1002/yea.320090707

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractCells of the yeastCandida utilisgrown in medium with short‐chain mono‐, di‐ or tricarboxylic acids transported L(‐)malic acid by two transport systems at pH 3·0. Results indicate that probably a proton symport for the ionized form of the acid and a facilitated diffusion for the undissociated form were present. Dicarboxylic acids such as succinic, fumaric, oxaloacetic and α‐ketoglutaric acids were competitive inhibitors of the malic acid for the high‐affinity system, suggesting that these acids used the same transport system. In turn, competitive inhibition uptake studies of labelled carboxylic acid in the low‐affinity range indicated that this system was non‐specific and able to accept not only carboxylic (mono‐, di‐ or tri‐) acids but also some amino acids. Additionally, under the same growth conditions,C. utilisproduced two mediated transport systems for lactic acid: a proton symport for the anionic form which appeared to be a common monocarboxylate carrier and a facilitated diffusion system for the undissociated acid displaying a substrate specificity similar to that observed for the low‐affinity dicarboxylic acid transport. The mediated carboxylic acid transport systems were inducible and subjected to repression by glucose. In glucose‐grown cells the undissociated dicarboxylic acids entered the cells slowly by simple diffusion. Repressed glucose‐grown cells were only able to produce both transport systems if an inducer, at low concentration (0·5%, w/v), was present during starvation in buffer. This process was inhibited by the presence of cycloheximide indicating that induction requiresde novoprotein synthesis. If a higher acid concentration was used, only the low‐affinity transport system was detectable, showing that the high‐affinity system was also repressed by

ISSN:0749-503X    

DOI:10.1002/yea.320090708

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractMicrosatellites are repeats of very short sequences of DNA, interspersed in the genome. In this paper, the occurrence of the two‐base repeat microsatellites has been investigated in the DNA sequence of yeast chromosome III. Only AT‐repeats were found at a significantly high frequency. Some of the regions with the highest concentration of AT‐repeats were located and further analysed, showing a close association with the core consensus of autonomously replicating sequ

ISSN:0749-503X    

DOI:10.1002/yea.320090709

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractThe glycolytic pathway inSaccharomyces cerevisiaeis activated by fermentable sugars at several steps. Mutants with deletions of genes coding for enzymes of the upper part of glycolysis were used to characterize the triggering mechanisms. Synthesis of fructose‐2,6‐bisphophate is catalysed by two 6‐phosphofructo‐2‐kinase isoenzymes, one of which is activated by fermentable sugars while synthesis of the second enzyme is induced (Kretschmer and Fraenkel, 1991). Increase in the level of fructose‐2, 6‐bisphosphate is demonstrated to depend on an internal metabolite upstream of the phosphoglucose isomerase reaction. The signalling process correlates with distinct temporal changes in the concentration of glucose‐6‐phosphate but not with its absolute level, indicating an adaptational mechanism. It is independent of the uptake and phosphorylation systems used by different sugars. Interestingly, this increase, although delayed, could also be observed in strains lacking the rapid cAMP increase after sugar addition which is thought to be responsible for the activating process. Synthesis of glucose‐6‐P and fructose‐6‐P is needed for the complete induction of pyruvate kinase and inactivation of fructose‐1,6‐bisphosphatase. On the other hand, induction of pyruvate decarboxylase depends mainly on a signal in

ISSN:0749-503X    

DOI:10.1002/yea.320090710

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractTheLYP1gene ofSaccharomyces cerevisiaewas cloned by complementation in lysine‐permease‐deficint recipient yeast cells, and its nucleotide sequence was determined. An open reading frame of 1833 nucleotides was found encoding a polypeptide of 611 amino acids, with a calculated molecular weight of 68 118. Analysis of the deduced primary structure of the protein revealed ten membrane‐spanning regions and three potential N‐glycosylation sites. Analysis of the deduced sequence of protein LYP1 indicates homology with other yeast amino‐acid permeases, in particular with CAN1, and also the lysine‐specific permease ofEscherichia coli. The strain transformed by a multi‐copy plasmid harbouring theLYP1gene, showed a 20‐fold increase in the maximum velocity of lysine uptake over that in the wild type, with no changes in the affinity of the permease fo

ISSN:0749-503X    

DOI:10.1002/yea.320090711

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractFormaldehyde (FA), a chemical with low toxic potential, is used as sole selective agent for transformation in the yeastSaccharomyces cerevisiae. Neither stable auxotrophic markers in recipient cells nor defined synthetic media are needed when multicopy vector YFRp1, containing the yeastSFAgene, is employed for yeast transformation. TheSFAgene gives stability to the vector and its yeast (and other) passenger genes when transformants are propagated in complex media supplemented with 3–5 mM‐FA. Use of inexpensive FA and non‐synthetic, undefined media will lower the cost of yeast transformant propagation considerably and thus make feasible large‐volume industrial application of transformants containing YFRp1 deri

ISSN:0749-503X    

DOI:10.1002/yea.320090712

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY