摘要:

AbstractSelectable markers integrated by the ‘gamma’ deletion method (Sikorski and Hieter, 1989) can be efficiently replacedin vivowith other markers by transformation with homologous plasmids. Transformation frequencies in experiments designed to replace original selectable markers with an alternate marker were high and molecular analysis confirmed that all transformants that exhibited the expected phenotypes (loss of the original prototrophy and gain of the alternate prototrophy) resulted from homologous recombination between plasmid sequences at the target locus. This technique involves no plasmid construction and greatly facilitates the generation of yeast cells containing multiple gene disrupti

ISSN:0749-503X    

DOI:10.1002/yea.320100202

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have measured the content of ribosomes, the rate of synthesis of ribosomal RNA, and the level of the mRNA for ribosomal proteins as a culture ofSaccharomyces cerevisiaepasses through the growth cycle. The transcription of both ribosomal RNA and ribosomal protein genes disappears at an unexpectedly early stage in the growth cycle, accompanied by a decline in the total RNA content of the culture by nearly 50% and a decline in the number of ribosomes per cell to less than 25% of the maximum value. During this time the cells continue to grow through more than two doublings, initially at the normal log growth rate, which then decline gradually for several hours. The data suggest that the cell can sense an unfavorable change within the medium and responds by employing regulation of both synthesis and degradation of its ribosomes. We conclude that the cell regulates ribosome synthesis and content according to its estimate of thepotentialfor growth.

ISSN:0749-503X    

DOI:10.1002/yea.320100203

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe report the development of a homologousin vitroassay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeastSchizosaccharomyces pombe. Our protocol for preparing anS. pombeextract capable of translating natural messenger RNAs was modified from a procedure previously used forSaccharomyces cerevisiae, in which cells are lysed in a bead‐beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER‐targeted proteins, pre‐acid phosphatase fromS. pombeand prepro‐α‐factor fromS. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane‐enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A–Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate thatS. cerevisiaeprepro‐α‐factor can be post‐translationally imported into the fission yeast ER, whileS. pombepre‐acid phosphatase crosses the membrane only by a co

ISSN:0749-503X    

DOI:10.1002/yea.320100204

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractIn order to characterize the morphological steps defined by sporulation (spo) genes during the formation of ascospores in the fission yeastSchizosaccharomyces pombe, we performed an electron microscopic study of the ultrastructure of the spindle pole body (SPB) and of the development of the forespore membrane during the second meiotic division (meiosis II) in sporulation‐deficient (spo) mutants (spo4,spo5,spo14andspo18). No difference was found in terms of the function and the structure of the SPB during the first meiotic division (meiosis I) between the four mutants and wild‐type cells. However, during meiosis II, thespo4andspo18mutants underwent nuclear division but in neither case were the SPBs modified nor were forespore membranes formed. The SPBs of thespo18mutant diminished in size after meiosis II and eventually disappeared after 18 h in sporulation medium. By contrast, the SPBs of thespo4mutant remained unchanged even after an 18‐h incubation. The outer plaques of SPBs ofspo5andspo14mutants were sufficiently modified to allow them to initiate development of the forespore membrane, but the membrane had an abnormally expanded lumen and did not enclose the nuclei during meiosis II. Thespo5mutant produced anucleate spore‐like bodies while thespo14mutant formed unorganized structures with irregular peripheries which, presumably, contained spore‐wall precursors, instead of anucleate spore‐like bodies. We conclude that the modification of the SPB is essential for the formation of ascospores and at least two genes (spo5andspo14) participate in the development of the forespore membrane. The defective phenotypes define discrete steps in the development of ascospores, which proceeds via steps defined by the mutantspo4,spo18,spo14andspo5genes respectively. Our observations provide further substantial evidence that the SPB plays a pivotal role in the normal development of ascospore

ISSN:0749-503X    

DOI:10.1002/yea.320100205

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe pattern of energy metabolism of different types of yeasts (obligate aerobes and facultative anaerobes) in aerobic chemostat cultures has been evaluated and interpreted on the basis of a coupling of metabolic fluxes between glycolytic and oxidative components.A model has been formulated which defines glycolytic and oxidative subunits through which the substrate C‐flux (gram‐atom g−1h−1) is calculated, stating that a relative imbalance between glycolytic flux and subsequent oxidative steps alone is sufficient to account for the onset of oxidoreductive metabolism in any type of yeast, irrespective of the maximum respiratory capacity. The model is able to reproduce the patterns of behaviour reported for the different types of yeasts, and the individual features of each strain are explained on the basis of metabolic differences which are defined by a set of normalized parameters. The model can be applied to different substrates and conditions, providing a methodological basis for more detailed studies of the steps controlling yeast energy met

ISSN:0749-503X    

DOI:10.1002/yea.320100206

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThepfk3mutation ofSaccharomyces cerevisiaecauses glucose‐negativity in apfk1genetic background, the mutant is temperature‐sensitive for growth and homozygous diploids do not sporulate. It fails to accumulate trehalose, and has an altered glycogen accumulation profile under glucose‐starvation conditions.pfk3–6, one of the alleles ofpfk3, has an altered morphology, forming long chain‐like structures at 36°C. ThePFK3gene was cloned by complementation of the mutant phenotypes. Integrative transformation demonstrated that the complementing fragment encoded the authenticPFK3gene. The disruption of the gene does not affect viability. Like the EMS‐inducedpfk3mutant, the disruptants are temperature‐sensitive and in apfk1genetic background are also glucose‐negative. ThePFK3transcript is induced by heat‐shock. Partial DNA sequence shows thatPFK3is identical toTPS2(De Virgilioet al., 1993). We demonstrate that, apart from being a structural determinant of trehalose 6‐phosphate phosphatase,PFK3(TPS2) is required for PFKII synthesis and normal regulation ofS. cerevisiaeresponse to nutrient

ISSN:0749-503X    

DOI:10.1002/yea.320100207

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe DNA sequence of the flocculation geneFLO1ofSaccharomyces cerevisiae, which is located on chromosome I (Watariet al., 1989) was determined. The sequence contains a large open reading frame (ORF) of 2586 bp and codes for a protein of 862 amino acids. However, further study (genomic Southern and polymerase chain reaction analyses) indicated that the gene we cloned was not the intactFLO1gene but a form with an approximately 2 kb deletion in the ORF region. The intactFLO1gene was then cloned and its nucleotide sequence determined. The sequence revealed that the ORF of the intact gene is composed of 4611 bp which code for a protein of 1537 amino acids. A remarkable feature of the putative Flo1 protein is that it contains four families of repeated sequences composed of 18, 2, 3 and 3 repeats and that it has a large number of serines and threonines. In the deletedFLO1form, a large part of these repeated sequences was missing. The N‐ and C‐terminal regions are hydrophobic and both contain a potential membrane‐spanning region, suggesting that the Flo1 protein is an integral membrane protein and a cell wall comp

ISSN:0749-503X    

DOI:10.1002/yea.320100208

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThis paper reports the 1890‐bp sequence located upstream of theHEM2gene ofSaccharomyces cerevisiae. The following potential regulatory protein‐binding motifs were found: ABF1‐binding site, yAP1‐binding site, two REB1‐binding sites, a cyclic AMP‐responsive element, RAP1‐binding site, and several HAP2‐HAP3‐HAP4 binding sites, implicating a complex regulatory mechanism governing expressio

ISSN:0749-503X    

DOI:10.1002/yea.320100209

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe report the sequence of an 18,002 bp DNA fragment from the right arm ofSaccharomyces cerevisiaechromosome XI. This segment contains nine complete open reading frames (ORFs), YKR401 to YKR409, and part of another ORF, YKR400, covering altogether 87·2% of the entire sequence. One of them, YKR400, encodes an NAD‐dependent 5,10‐methylene‐tetrahydrofolate dehydrogenase. YKR404, YKR405 and YKR406 correspond to the previously characterizedHBS1,MRP‐L20andPRP16genes, coding for a translation elongation factor, a mitochondrial ribosomal protein and an ATP‐binding protein, respectively. The putative product of YKR407 contains the zinc‐binding region signature of neutral zinc metallopeptidases. The five other ORFs do not show significant homology to any known protein. The sequence data reported here have been assigned EMBL accession n

ISSN:0749-503X    

DOI:10.1002/yea.320100210

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe report the DNA sequence analysis of a region on the left arm of chromosome XI ofSaccharomyces cerevisiaeextending over 10 kb. The region contains five open reading frames (ORFs) of greater than 100 amino acids which do not show significant overlap with other ORFs. YKL408 contains a sequence with strong similarity to the RNA helicase pre‐mRNA splicing factorsPRP2,PRP16andPRP22(Burgesset al., 1990; Companyet al., 1991; Rubyet al., 1991). YKL409 corresponds to the geneSMY1, the sequence of which was previously reported by Lillie and Brown (1992). YKL410 is identical to ATPase subunit C (Beltranet al., 1992) except for an N‐terminal extension. YKL406 and YKL407 show no significant identity with any sequences in the databases searched. The sequence has been entered in the EMBL Data Library under Accession Number X75

ISSN:0749-503X    

DOI:10.1002/yea.320100211

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY