摘要:

AbstractThe presence of adenylate cyclase activity was first demonstrated in membrane fractions from the budding yeastKluyveromyces marxianus. The enzyme showed a Mn2+‐ and Mg2+‐dependent activity, with optimal pH at around 6 as observed in other yeast species. As inSaccharomyces cerevisiae, where adenylate cyclase is regulated by RAS1 and RAS2, we detected a guanyl nucleotide‐dependent activity. Interestingly Y13–259 monoclonal antibody, raised against mammalian p21Ha‐ras, inhibited Mg2+plus GTP‐γ‐S‐dependent cAMP production, suggesting that the GTP binding proteins involved in adenylate cyclase regulation could be Ras proteins. The same antibody recognized on Western blot and immunoprecipitated a 40 kDa polypeptide fromK. marxianuscrude membranes. This polypeptide was not detected by an anti‐RAS2 polyclonal antibody raised againstS. cerevisiaeRAS2 protein, suggesting that Ras proteins from the two species could be stru

ISSN:0749-503X    

DOI:10.1002/yea.320100802

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe activity of poly(ADP‐ribose) polymerase (PADPRP), a chromatin‐associated enzyme present in most eukaryotic cells, is stimulated by DNA strand breaks, suggesting a role for the enzyme in the cellular response to DNA damage. However, the primary function of PADPRP remains unknown. We have selectedSchizosaccharomyces pombeas a simple eukaryotic system in which to study PADPRP function because this fission yeast shares with mammalian cells important cellular features possibly associated with poly‐(ADP‐ribos)ylation pathways. We investigated the existence of an endogenous yeast PADPRP by DNA and RNA hybridization to mammalian probes under low‐stringency conditions and by PADPRP activity assays. Our data indicate that fission yeasts are naturally devoid of PADPRP. We therefore isolatedS. pombestrains expressing PADPRP by transformation with a human full‐length PADPRP cDNA under the control of the SV40 early promoter. The human PADPRP construct was transcribed and translated inS. pombe, generating a major transcript of the same size (3.7 kb) as that detected in mammalian cells and a 113‐kDa polypeptide, identical in size to the native human PADPRP protein. Yeast recombinant PADPRP was enzymatically active and was recognized by antibodies to human PADPRP.S. pombecells expressing PADPRP (SPT strains) showed a stable phenotype that was characterized by: (i) cell cycle retardation as a result of a specific delay at the G1phase, (ii) decreased cell viability in stationary cultures, (iii) enhanced rates of spontaneous and radiation‐inducedade6‐ade7mutations, and (iv) increased sensitivity to radiation. SPT strains may prove efficient tools with which to investigate PADPRP functions in

ISSN:0749-503X    

DOI:10.1002/yea.320100803

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractTo study cell wall assembly, a simple screening method was devised for isolating cell wall mutants. Mutagenized cells were screened for hypersensitivity to Calcofluor White, which interferes with cell wall assembly. The rationale is that Calcofluor White amplifies the effect of cell wall mutations. As a result, the cells stop growing at lower concentrations of Calcofluor White than cells with normal cell wall. In this way, 63 Calcofluor White‐hypersensitive (cwh), monogenic mutants were obtained, ordered into 53 complementation groups.The mannose/glucose ratios of the mutant cell walls varied from 0.15 to 3.95, while wild‐type cell walls contained about equal amounts of mannose and glucose. This indicates that both low‐mannose and low‐glucose cell wall mutants had been obtained. Further characterization showed the presence of three low‐mannose cell wall mutants with amnn9‐like phenotype, affected, however, in different genes. In addition, four new killer‐resistant (kre) mutants were found, which are presumably affected in the synthesis of β1,6‐glucan. Most low‐glucose cell wall mutants were not killer resistant, indicating that they might be defective in the synthesis of β1,3‐glucan. Elevencwhmutants were found to be hypersensitive to papulacandin B, which is known to interfere with β1,3‐glucan synthesis, and fourcwhmutants were temperature‐sensitive and lysed at the restrictive temperature. Finally, ninecwhmutants were hypersensitive to caffeine, suggesting that these were affected in signal transduction rel

ISSN:0749-503X    

DOI:10.1002/yea.320100804

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractSaccharomyces cerevisiaecontains at least fourPRSgenes, all of which have been cloned and sequenced. Each of the four derived amino acid sequences have more than 60% similarity to the corresponding polypeptides of man, rat,Escherichia coliandSalmonella typhimurium. ThePRS1gene maps on chromosome XI,PRS2on chromosome V,PRS3on chromosome VIII andPRS4on chromosome II. One member of this gene family,PRS1, contains a region of non‐homology (NHR) shown by cDNA cloning and sequencing not to be an intron. The results presented here suggest that the presence of this NHR is not detrimental to the function of the gene. To date the possibility of protein splicing can be neither proven nor disputed. The sequences submitted to the EMBL data library are available under the following accession numbers:PRS1(X70069),PRS2(X74414) andPRS3(X74415

ISSN:0749-503X    

DOI:10.1002/yea.320100805

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

ISSN:0749-503X    

DOI:10.1002/yea.320100806

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractAddition of a nitrogen‐source to glucose‐repressed, nitrogen‐starved G0 cells of the yeastSaccharomyces cerevisiaein the presence of a fermentable carbon source induces growth and causes within a few minutes a five‐fold, protein‐synthesis‐independent increase in the activity of trehalase. Nitrogen‐activated trehalase could be deactivatedin vitroby alkaline phosphatase treatment, supporting the idea that the activation is triggered by phosphorylation. Yeast strains containing only one of the threeTPKgenes (which encode the catalytic subunit of cAMP‐dependent protein kinase) showed different degrees of nitrogen‐induced trehalase activation. The order of effectiveness was different from that previously reported for glucose‐induced activation of trehalase in glucose‐derepressed yeast cells. Further reduction ofTPK‐encoded catalytic subunit activity by partially inactivating point mutations in the remainingTPKgene further diminished nitrogen‐induced trehalase activation, while deletion of theBCY1gene (which encodes the regulatory subunit) in the same strains resulted in an increase in the extent of activation. Deletion of theRASgenes in such atpkw1bcy1strain had no effect. These results are consistent with mediation of nitrogen‐induced trehalase activation by the free catalytic subunits alone. They support our previous conclusion that cAMP does not act as second messenger in this nitrogen‐induced activation process and our suggestion that a novel nitrogen‐induced signaling pathway integrates with the cAMP pathway at the level of the free catalytic subunits of protein kinase A. Western blot experiments showed that the differences in the extent of trehalase activation were not due to differences in trehalase expression. On the other hand, we cannot completely exclude that protein kinase A influences the nitrogen‐induced activation mechanism itself rather than acting directly on trehalase. However, any such alternative explanation requires the existence of an additional, yet unknown, mechanism for activation of trehalase besides the well‐establi

ISSN:0749-503X    

DOI:10.1002/yea.320100807

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractPXP‐18 is a 14‐kDa major peroxisomal protein of the yeastCandida tropicalisand a homologue of the non‐specific lipid‐transfer protein (nsLTP) of mammals. Mammalian nsLTP is thought to facilitate the contact of membranes, to stimulate lipid‐transfer between them. If PXP‐18 functions like nsLTP, it must be present on organelle membranes. Immunoelectron microscopy ofC. tropicaliscells indicated that gold particles, which visualized PXP‐18, localized exclusively in the matrix of peroxisomes. Subcellular fractionation followed by Western blotting revealed the association of PXP‐18 with peroxisomes inC. tropicaliscells. An enzyme‐linked immunosorbent assay revealed that almost all the PXP‐18 associated with peroxisomes was detectable after the solubilization of the organelle but not before, implying the predominance of PXP‐18 inside peroxisomes. This differential assay was applied to the intracellular import of the intact and truncated PXP‐18s expressed inSaccharomyces cerevisiaecells. Most of the intact PXP‐18 was shown to be imported into the matrix of host‐cell peroxisomes, whereas the truncated PXP‐18, which lacked the C‐terminal tripeptide Pro‐Lys‐Leu, no longer targeted peroxisomes. These results are consistent with the view that PXP‐18 is the matrix protein of peroxisomes and must function in a sys

ISSN:0749-503X    

DOI:10.1002/yea.320100808

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe previously described a screen for thiamine‐repressible genes inSchizosaccharomyces pombeand reported on one such gene,nmt1, required for thiamine biosynthesis. Here we describe a second gene,nmt2, recovered in the same screen. Disruption ofnmt2also resulted in thiamine auxotrophy, indicating a role for thenmt2gene product in thiamine biosynthesis. Both genes are highly transcribed in minimal medium and repressed in medium containing thiamine, and nuclear ‘run‐on’ experiments confirm that expression in both cases is controlled by the rate of transcription initiation. The virtually identical kinetics of induction and repression suggest that the two genes are co‐ordinately regulated. Sequence comparison of the two promoters reveals a canonical TATA box, downstream of which is a perfectly conserved 11 bp element. Transcript mapping experiments show that transcription initiation of both genes is centred on thi

ISSN:0749-503X    

DOI:10.1002/yea.320100809

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have previously shown that mutations in the yeastKNR4gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1→3)‐β‐glucanase. In this report, we further demonstrated thatknr4mutant cells were more permeable to a chromogenic substrate, X‐GAL, suggesting that the mutant cell walls were leakier to certain non‐permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali‐insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a ‘knr4disrupted’ tetrad demonstrated that mutant cell walls contained much less (1→3)‐β‐glucan and (1→6)‐β‐glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild‐type strains. The data suggested that theknr4mutant cell walls were dramatically weakened, which may explain t

ISSN:0749-503X    

DOI:10.1002/yea.320100810

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractA 7·4 kb segment of chromosome II was sequenced and analysed. This segment is part of the 25 kb insert of cosmid clone α1004.10 which is located on the left arm of chromosome II. Sequence analysis revealed four open reading frames (ORFs), of which two had been characterized previously (SSA3, AAR2) and one was not identified. The other ORF was precisely 600 bp long and the deduced protein sequence predicted a very basic protein (pI=11·1; molecular weight=22·5 kDa). Evidence was found that the ORF is the S40 ribosomal protein gene (RPG)S8. Consensus splice signals were found in the 5′ leader sequence and also potential RPG‐specific sequences. Chromoblot analysis revealed a second copy of theS8RPG on chromosome IV or VIII. This copy is also closely linked to an hsp70 protein gene,SSA4. The sequence has been deposited in the EMBL data library under accession number

ISSN:0749-503X    

DOI:10.1002/yea.320100811

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY