摘要:

AbstractThis publication marks the beginning of the construction of a gene–protein index that relates proteins which are resolved on the two‐dimensional protein map ofSaccharomyces cerevisiaewith their corresponding genes. We report the identification of 36 novel polypeptide spots on the yeast protein map. They correspond to the products of 26 genes. Together with the polypeptide spots previously identified, this raises to 41 the number of genes whose products have been identified on the protein map. The proteins identified here are concerned with four major areas of yeast cellular physiology: carbon metabolism, heat shock, amino acid biosynthesis and purine biosynthesis. Given the molecular weight and isoelectric point of the identified proteins, and the codon‐usage bias of the corresponding genes, it can be estimated that 25 to 35% of all the soluble yeast proteins are detectable under the labelling and running gel conditions used in this

ISSN:0749-503X    

DOI:10.1002/yea.320110702

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe ORF5 ofKluyveromyces lactiskiller plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One‐step gene disruption of ORF5, viain vivohomologous recombination between native plasmid k2 and a transfer vector employing theSaccharomyces cerevisiae LEU2gene fused to the k2 UCS5 element, yielded Leu+transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by theLEU2marker, termed rk2, in addition to the wild‐type plasmids k1 and k2. Northern analysis detected a plasmid‐dependentLEU2transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+selective growth; however rk2 was displaced by k2 during non‐selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over‐expression of an epitope‐tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predic

ISSN:0749-503X    

DOI:10.1002/yea.320110703

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractIn order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient ‘recombinationin vivo’ technique. Two sets of vectors were developed. The first set, called ‘expression vectors’, contains an expression cassette with a yeast promoter and thePGKterminator separated by a polylinker, and anEscherichia colireplicon. Subcloning in these vectors of a DNA fragment generates a ‘transfer vector’ which is compatible with the second set ofE. coli–yeast shuttle vectors. This set of ‘recombination vectors’ contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid‐bearing host. Plasmid copy numbers can be modulated through the use ofURA3orURA3‐das the selective marker together with anARS/CENand the 2 μm replicon.Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast orE. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single‐stranded DNA inE. colifor sequencing or site‐directed mutagenesis.The sequence presented (Figure 1a) has been entered in the EMBL data library und

ISSN:0749-503X    

DOI:10.1002/yea.320110704

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractA distributive model has been constructed to describe the maintenance of the native 2 μm and 2 μm‐based plasmids in the yeastSaccharomyces cerevisiae. This model includes elements which represent the influence of selection, segregation, replication and amplification on plasmid stability. A computer program has been written in TURBO PASCAL to implement the model and a number of simulation experiments have been carried out. These simulations permitted the choice of a form of the model which is compatible with the available experimental evidence. The form chosen involves an amplification system in which theRAFgene product binds to the Rep1/Rep2 dimer to prevent the latter acting to repress the activity of theFLPgene. At the same time an upper limit (or ‘ceiling’) was imposed on the number of plasmid molecules able to replicate. Maternal bias was accommodated by ‘tagging’ a small proportion of molecules for inheritance by the mother nucleus and these tags being removed (or ‘cleared’) by the Rep1/Rep2 dimers. This final form of the model makes specific predictions about the stability of 2 μm and YEp plasmids in yeast populations and about the distribution of plasmid copy number between cells in such populations. The predictions on stability have been subjected to experimental test and results provide good suppo

ISSN:0749-503X    

DOI:10.1002/yea.320110705

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractFor the sequencing of the left arm of chromosome XII ofSaccharomyces cerevisiae, we fine‐mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere. Total yeast DNA in agarose blocks was digested with I‐PpoI, which exclusively cuts once in each repeat unit of the rDNA. The resulting fragment was isolated from pulsed‐field gels, together with the equally sized chromosome IX. A cosmid library of some 30‐fold chromosome coverage was generated from this material, with the cloning efficiency being around 20 000 clones per microgram genomic DNA. The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes. For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures. The ordered library serves as a tool for the physical mapping of genetic markers. Also, a minimal set of 15 clones was selected that covers the entire fragment. This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromos

ISSN:0749-503X    

DOI:10.1002/yea.320110706

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe report the DNA sequence of a segment located on the right arm of chromosome II fromSaccharomyces cerevisiaeS288C near the subtelomeric sequences. The sequence was determined using a random cloning strategy followed by an oligonucleotide‐directed sequencing. The segment contains four non‐overlapping open reading frames (ORFs) YBR297w, YBR298c, YBR299w and YBR301c, and two overlapping ones (YBR300c and YBR300w). Three of them—YBR297w, YBR298c and YBR299w—are theMAL3R(transcriptional regulatory protein),MAL3T(maltose permease) andMAL3S(maltase) genes of theMAL3locus previously localized. The three other ORFs are unidentified. AnotherMALlocus (MAL1) has been localized on chromosome VII. The Mal−phenotype of strain S288c cannot be explained by telomeric silencing. The sequences have been submitted to the EMBL data library under Accession Numbers Z36166; Z36167; Z36168; Z36169 a

ISSN:0749-503X    

DOI:10.1002/yea.320110707

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe report the nucleotide sequence of a 32·8 kb DNA segment from the right arm ofSaccharomyces cerevisiaechromosome IV. The sequence contains 20 open reading frames (ORFs) longer than 300 bp as well as the 240 bp gene coding for the essential SSS1 secretory protein. Nine ORFs previously totally or partially sequenced (TPS2, PPH3, RAD55, SED1, PDC2, AFR1, SSS1, SLU7andD4478) are presented, as well as the transmembrane protein D4405, the leucine zipper containingD4495and a new tRNA for arginine.D4456andD4461are separated by a single in‐frame stop codon only. The other five ORFs show no particular features or significant homology. The sequence is recorded in EMBL database under Accession Number X820

ISSN:0749-503X    

DOI:10.1002/yea.320110708

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractIn the framework of the European BIOTECH project for sequencing theSaccharomyces cerevisiaegenome, we have determined the nucleotide sequence of the left part of the cosmid clone 232 and the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 33,099 base pairs of sequence derived from the left arm of chromosome X of strain S288C. This sequence reveals 17 open reading frames (ORFs) with more than 299 base pairs, including the published sequences forARG3, LIGTR/LIG1, ORF2, ACT3andSCP160. Two other ORFs showed similarity withS. cerevisiaegenes: one with theCAN1gene coding for an arginine permease, and one with genes encoding the family of transcriptional activators containing a fungal Zn(II)2‐Cys6binuclear cluster domain like that found in Ppr1p or Gal4p. Both putative proteins contain a leucine zipper motif, the Can1p homologue has 12 putative membrane‐spanning domains and a putative α2‐SCB‐α2 binding site. In a diploid disruption mutant of ORFJ0922coding for the transcriptional activator homologue, no colonies appeared before 10 days after transformation and then grew slowly. In contrast, haploid disruption mutants showed a growth phenotype like wild‐type cells. One ORF showed weak similarity to therad4gene product ofSchizosaccharomyces pombeand is essential for yeast growth. Five ORFs showed similarity to putative genes on the right arm of chromosome XI ofS. cerevisiae. Two of them have similarity to each other and belong to a family of extracellular proteins that groups mammalian SCP/Tpx‐1, insects Ag3/Ag5, plants PR‐1 and fungi Sc7/Sc14. Three small ORFs are completely contained in the larger ORFs on the corresponding complementary strand and thus probably do not represent real genes. The DNA sequence has been deposited in the EMBL data library under Accessio

ISSN:0749-503X    

DOI:10.1002/yea.320110709

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractFAR3is a newly‐discovered yeast gene required specifically for pheromone‐mediated cell cycle arrest. I have used strains harboring thefar3‐1mutation to map the gene to the right arm of chromosome XIII, establishing the gene orderCEN13‐LYS7‐MCM1‐FAR3. I cloned theFAR3gene based on its genetic map position using a strategy that combined chromosome walking and a related technique termed ‘chromosome rolling’. In addition to the genetic and physical localization ofFAR3, I present data that suggest corrections to the tentative map positions

ISSN:0749-503X    

DOI:10.1002/yea.320110710

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractTheACC1/FAS3gene has been mapped to the right arm of chromosome XIV by both genetic and physical methods. The gene is closely linked toRNA2and is allelic to theABP2gene of chromosome XIV.

ISSN:0749-503X    

DOI:10.1002/yea.320110711

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY