摘要:

AbstractSchizosaccharomyces pombewas treated with either cycloheximide or anisomycin at levels sufficient to inhibit>95% of protein synthesis for periods upon to 3 h, equivalent to one cell cycle. Treatment for as little as 1 h caused significant loss of the Golgi apparatus by both immunofluorescence and electron microscopy. The loss was quantitated by stereology on electron micrographs. Nearly 90% of the stacked Golgi was lost over a 3 h period. No other intracellular membrane compartment seemed to be affected. Measurement of enzyme activities confirmed these observations. The activity of a resident of the Golgi apparatus, α‐1,2 galactosyltransferase, was reduced over this time, whereas the endoplasmic reticulum marker, BiP, and the cytoplasmic enzyme, hexokinase, were unaffected. The morphological changes associated with cycloheximide addition were reversed on its removal, though there was a lag before cells recommenced growth or secretion of the enzyme, acid phosphata

ISSN:0749-503X    

DOI:10.1002/yea.320100102

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe mutationsrad3‐101andrad3‐102(formerlyrem1‐1andrem1‐2) of the essentialRAD3gene ofSaccharomyces cerevisiaeconfer a phenotype of semidominant enhancement of spontaneous mitotic recombination and mutation frequencies, butnotextreme sensitivity to ultraviolet (UV) light. These properties differ from the previously published observations of otherrad3mutations, which are very UV‐sensitive but do not alter recombination frequencies significantly. We have located the position of DNA sequence changes from wild‐typeRAD3to therad3‐101andrad3‐102mutations and have demonstrated that these sequence changes are necessary and sufficient to confer the (Rem−) mutant phenotype when transferred into otherwise wild‐typeRAD3plasmids. The Rem−mutations are not located in the same region. It is possible that the two regions of the gene in which these mutations map define portions of the molecule which are in contact when folded in the native configuration. To begin to test this hypothesis, we have constructed two double mutant alleles, one withrad3‐101andrad3‐102, and one with the UV‐sensitiverad3‐1mutation andrad3‐102. We find that plasmids carrying these double mutant alleles ofRAD3are no longer able to confer a hyper‐recombinational phenotype and do not complement the UV‐sensitivity of the excision‐defectiverad3‐2allele. We conclude that the double mutant alleles are non‐functional for excision repair, and may be null. We have also constructed newrad3alleles by oligonucleotide‐directed mutagenesis and have tested their effects on spontaneous mutation

ISSN:0749-503X    

DOI:10.1002/yea.320100103

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractLevansucrase, aBacillus subtilisextracellular enzyme, was not secreted in the culture medium when produced in yeast. The protein accumulated inside the cell in its precursor form which represented 0·3% of total proteins. The absence of any post‐translational modifications, such as signal sequence cleavage or addition of N‐linked sugars, indicated that this protein did not enter the reticulum secretion pathway.Direct observation of the cells by confocal laser scanning microscopy showed that levansucrase was associated with the cytoplasmic membrane. Subcellular fractionation experiments revealed that levansucrase precursor form is associated with membranes through weak ionic interactions. The purified precursor displayed the same catalytic properties as levansucrase secreted byB. subtilis. Thus yeast could be used as a source of levansucrase precursor allowing its isolation as a pure form on a milligram s

ISSN:0749-503X    

DOI:10.1002/yea.320100104

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have tested the clones used in the European Yeast Chromosome III Sequencing Programme for possible artefacts that might have been introduced during cloning or passage throughEscherichia coli. Southern analysis was performed to compare theBamHI,EcoRI,HindIII andPstI restriction pattern for each clone with that of the corresponding locus on chromosome III in the parental yeast strain. In addition, further enzymes were used to compare the restriction maps of most clones with the map predicted by the nucleotide sequence (Oliveret al., 1992). Only four of 506 6‐bp restriction sites predicted by the sequence were not observed experimentally. No significant cloning artefacts appear to disrupt the published sequence of chromosome III. The restriction patterns of six yeast strains have also been compared. In addition to two previously identified sites of Ty integration on chromosome III (Warmingtonet al., 1986; Stuckaet al., 1989; Newlonet al., 1991), a new polymorphic site involving Ty retrotransposition (the Far Right‐Arm transposition Hot‐Spot, FRAHS) has been identified close toCRY1. On the basis of simple restriction polymorphisms, the strains S288C, AB972 and W303‐1b are closely related, while XJ24‐24a and J178 are more distant relatives of S288C. A polyploid distillery yeast is heterozygous for many polymorphisms, particularly on the right arm of the c

ISSN:0749-503X    

DOI:10.1002/yea.320100105

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe constitutive transport of hexoses in yeast has been re‐examined with a new radioactive experimental approach devised to distinguish between association or independence of the transport step with phosphorylation of the sugar substrate. The approach takes advantage of the fact that the label of [2‐3H]mannose disappears once it has been phosphorylated by the yeast, due to its conversion to fructose‐6‐phosphate. Our results with wild‐type yeast and this fermentable sugar support the view that the transport of hexoses in yeast does not involve phosphorylation of the substrate. Other features of the transport process have been examined using this experimental procedure and are also

ISSN:0749-503X    

DOI:10.1002/yea.320100106

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractYarrowia lipolyticaDO613, carrying thexpr6‐13mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys‐Arg at the end of the pro‐region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys‐Arg of an artificial substrate. TheXPR6gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pairSalI‐SphIXPR6fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110 016). The deduced amino acid sequence had significant homology toSaccharomyces cerevisiaeKex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of theXPR6gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolatedxpr6alleles were due to leaky mutations. Second, mating typeBstrains carrying the disruptedXPR6gene did not mate, but mating typeAstrains did. Third, theXPR6disruption strains grew poorly on rich media at pH 5·5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon‐like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardle

ISSN:0749-503X    

DOI:10.1002/yea.320100107

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractSPTgenes are suppressors of mutations induced by the retrotransposon Ty inSaccharomyces cerevisiae. AllSPTgenes isolated to date suppress Ty‐induced mutations by altering transcription.SPT23was identified as a multicopy suppressor of the Ty‐induced promoter mutationshis4‐912δ andlys2‐61. Multicopy expression ofSPT23suppresses a variety of Ty‐induced promoter mutations, including theMAT‐regulated alleleshis4‐917(480) andlys2‐173R2. Here, we report the initial characterization of theSPT23gene, including its nucleotide sequence and location in the yeast genome. TheSPT23gene contains a 1854 base pair open reading frame. Searches of the current data bases show no homology betweenSPT23and previously described genes or proteins. TheSPT23gene is located betweenRAM2andMAK11on the left arm of chromosome XI. Tn10‐LUKinsertional mutagenesis of theSPT23gene indicates thatSPT23is not essential for vegetative growth andspt23mutations do not confe

ISSN:0749-503X    

DOI:10.1002/yea.320100108

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractRearrangements within plasmid DNA are commonly observed during transformation of eukaryotic cells. One possible cause of rearrangements may be recombination between repeated sequences induced by some lesions in the plasmid. We have examined the mechanisms of transformation‐associated recombination in the yeastSaccharomyces cerevisiaeusing a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmids contain homologous or diverged (19%) repeats of theURA3genes (fromS. cerevisiaeorS. carlsbergensis) separated by the genetically detectableADE2colour marker. Recombination during transformation for covalently closed circular plasmids was over 100‐fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single‐strand breaks that are ligatable, as well as double‐strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation‐associated recombination between repeat DNAs is under the influence of theRAD52and

ISSN:0749-503X    

DOI:10.1002/yea.320100109

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractIn this paper are described a set of new high‐copy‐number yeast vectors, which are specially designed for the conditional expression of epitope‐tagged proteinsin vivo. One of the major advantages of these plasmids is that they allow polymerase chain reaction‐amplified open reading frames to be automatically fused in frame with the epitope‐coding sequence, avoiding longer procedures such as site‐directed mutagenesis. This heterologous construction can be realized either at the 5′‐end of the coding sequence, in the pYeF1 vector, or at its 3′‐end, in pYeF2, generating N‐ or C‐terminal tagged proteins, respectively. Moreover, to increase the usefulness of the method, derivatives of the two basicURA3‐borne pYeF1 and pYeF2 were constructed, carrying either theHIS3orTRP1gene as a marker of selection. These vectors could be of use for the purpose of functional analysis of the newly discovered genes resulting from the systematic sequencing of the yeast genome. Here, we present results showing the functional expression and the efficient immunoprecipitation of the epitope‐tagged Rna15 protein, which is involved inSaccharomyc

ISSN:0749-503X    

DOI:10.1002/yea.320100110

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have isolated two yeast genes,KIN1andKIN2, by their homology to the protein kinase family of viral oncogenes. Previous studies have identified the yeastKIN1gene product (pp145KIN1) as a 145 kilodalton (kDa) phosphoprotein with serine/threonine‐specific protein kinase activity. To identify and biochemically characterize theKIN2gene product, antibodies were raised against a bacterial β‐galactosidase/KIN2 fusion polypeptide.In vivo, theKIN2gene product is a 145 kDa phosphoprotein, pp145KIN2. In immune complexes, pp145KIN2demonstrates serine/threonine protein kinase activity, transferring phosphate from [γ‐32P]ATP to either itself or the exogenously added substrates α‐casein, acid‐denatured enolase, or phosvitin.In vitro, kinase activity is dependent on either Mn2+or Mg2+ions. Both enzymes, pp145KIN1and pp145KIN2, prefer ATP over GTP as their phosphoryl donor. Since a new class of yeast protein kinases has been identified which are serine/tyrosine‐specific, we analysed a wide range of substrates to see if any could be phosphorylated by pp145KIN1or pp145KIN2on tyrosine residues. Both enzymes phosphorylate α‐casein, acid‐denatured enolase, and phosvitin on serine and threonine residues. Neither enzyme could phosphorylate tyrosine residues even though good substrates for tyrosine‐specific kinases such as enolase, angiotensin II, and the synthetic polymer GLU80TYR20were used. The biochemical analysis of KIN2 kinase activity shows remarkable similarity to that of its most closely related yeast kinase, KIN1. It remains to be seen if these two yeast protein kinases share any functional relationships

ISSN:0749-503X    

DOI:10.1002/yea.320100111

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY