ISSN:0749-503X    

DOI:10.1002/yea.320070302

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for whichSaccharomyces cerevisiaecells are not permeable. Two mutations,srb1andts1, have been identified in VY1160 mutant, and previous studies (Kozhinaet al., 1979) have shownsrb1to be responsible for cell lysis. We now present evidence that thets1mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time‐, temperature‐ and energy‐dependent an consistent with a fluid‐phase mechanism of endocytosis. The rate of steady‐state accumulation of hte dye at 37°C is 145 ng/μg DNA per h for VY1160 mutant and 23 ng/μg DNA per h for S288C parental strain. Studies with isogenic strains having either thesrb1or thets1mutation, orSRB1 TS1wild‐type alleles have shown that onlyts1strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24°C and shifted at 38°C shows thatts1strains, but notsrb1and wild‐type strains, increase ten‐fold the internalization of lucifer yellow 2 h after the shift at 38°C. The analysis ofts1× wild‐type crosses provides evidence that the temperature‐sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might the generally increased permeabil

ISSN:0749-503X    

DOI:10.1002/yea.320070303

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues. TheKIN1locus fromSaccharomyces cerevisiaewas isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG. The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases. Using antibodies raised against a bacterialLacZ/KIN1fusion protein, we have identified by immunoprecipitation the yeastKIN1gene product as a 145 000 dalton protein (p145KIN1). In exponentially growing yeast cells, theKIN1protein is phosphorylated primarily on serine residues. The gene product ofKIN1was shown to be a serine/threonine‐specific protein kinase in immune complexes, as detrmined by the transfer of label from [γ‐32P]ATP to either pp145KIN1or to an exogenously added substrate, α‐casein. The optimal metal ion concentration in this assay was 20 mM‐MnCl2. Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues. There is no apparent difference between wild‐type cells and cells containing a disruptedKIN1gene. The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in detrmining the role of phosphorylation in cellular growth and

ISSN:0749-503X    

DOI:10.1002/yea.320070304

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractYeast (Saccharomyces cerevisiae) cytosol was examined for the presence of calcium‐dependent membrane‐ or lipidbinding proteins that might paly fundamental roles in membrane‐associated phenomena in stimulated cells. A complex group of proteins was isolated from late log phase cultures of yeast strain YP3 on the basis of calcium‐dependent association with yeast secretory vesicles isolated from the temperature‐sensitivesec6‐4secretory mutant. The masses of the major proteins in this group were 32, 35, 47, 51, 55, 60, and 120 kDa. A similar group of proteins was isolated by calcium‐dependent association with bovine brain lipids enriched in the predominant acidic phospholipids of the yeast secretory vesicles. The 47 kDa protein was highly purified when commerical yeast cake was used as the source of yeast cytosol. The 32 kDa and 60 kDa proteins were demonstrated to reassociate with lipids at calcium concentrations of 100 μMor higher, while no association was promoted by 2 mM‐magnesium. The 47 kDa protein could be removed from lipids by reducing the calcium concentration to between 1 and 32 μM. The sequences of peptides isolated from digests of several of these proteins indicate that they are novel proteins but are insufficient to judge the possible homology of these proteins with mammalian membrane‐binding proteins. The sequence data may be adequeate to permit isolation and modification of the corresponding genes in order to assess the possible funtion of this class of proteins

ISSN:0749-503X    

DOI:10.1002/yea.320070305

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractORF 10 of the K2 plasmid fromKluyveromyces lactisencodes a small basic protein (22·3% lysine). The function of its product has been investigated. Western blot analysis, using an antibody against MS2 RNA polymeras/ORF 10 fusion protein, reveals a protein band with an apparent molecular weight of 14 kDa. The protein can bind a DNA‐Sepharose column, and is eluted by 350 mM‐salt. Immunoprecipitation experiments show that the ORF 10 protein coprecipitates with the linear genomic DNAs of the two killer plasmids (K1 and K2). From Western/Southern blot data, it is possible to conclude that the interaction between protein and DNA occurs directly, rather than via other proteins(s). ORF 10 is easily detected by Western blot and its transcript is one of the most abundant of the K2 plasmid, suggesting that this protein may have a structural rather than a regulatory function. This possibility is also suggested by the observed sequeence homology between the ORF 10 protein and the family of histone‐like pr

ISSN:0749-503X    

DOI:10.1002/yea.320070306

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important need of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, theLEU2,HIS3,URA3,TRP1andARG4genes were found among the primary transformations at a frequency of approximately 0·1%. TheRAD4gene, which is toxic toEscherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0·18%. Non‐selective transformation using this transformation proctocol was shown to increase the frequency of gene disruption three‐fold. Co‐transformation showed that 30–40% of the transformation‐competent cells take up more than one DNA molecule which can be used to enrich for integration and delection events 30‐ to 60‐fold. Co‐transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2–5% the frequency

ISSN:0749-503X    

DOI:10.1002/yea.320070307

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe cell division cycle geneCDC 15is essential for the late nucler division in the yeastSaccharomyces cerevisiae. The amino acid sequence of the 974 amino acids/110 kDaCDC 15gene product, as deduced from the nucletide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/theonine kinases besides those for protein tyrosine kinases. Many protein kinases non‐essential for growth are known.CDC 15represents an essential protein kinase likeCDC 7andCDC 28. A carboxyterminal deletion of 32 amino acids renders the protein inactiv

ISSN:0749-503X    

DOI:10.1002/yea.320070308

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractA physical map of a contiguous DNA fragment of 60 kb, extending from the centromere toTRP5on the left arm of the chromosome VII ofSaccharomyces cerevisiae, strain IL 125‐2B, was established. Within a 31 kb region fromPMA1towardsTRP5, a total of 12 transcription products ranging from 0·6 to 3·6 kb were identified in cells grown exponentially on rich medium. Near 87% of the DNA investigated was transcribed and on average one transcript, of 2·3 kb average length, was detected every 2·7 kb of DNA. The physical and genetical distances between the markersCEN7,pma1,leu1,pdr1andtrp5were compared. A recombination frequency of 1 cM corresponds to an average distance of 3·3 kb between alleles in this region of chromoso

ISSN:0749-503X    

DOI:10.1002/yea.320070309

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

ISSN:0749-503X    

DOI:10.1002/yea.320070310

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

ISSN:0749-503X    

DOI:10.1002/yea.320070311

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY