Yeast
1097-0061
0749-503X
Anh Quốc
Cơ quản chủ quản: WILEY , John Wiley and Sons Ltd
Lĩnh vực:
GeneticsBioengineeringApplied Microbiology and BiotechnologyBiotechnologyBiochemistryMedicine (miscellaneous)
Phân tích ảnh hưởng
Thông tin về tạp chí
Yeast publishes original articles and reviews on the most significant developments of research with unicellular fungi, including innovative methods of broad applicability. It is essential reading for those wishing to keep up to date with this rapidly moving field of yeast biology. Topics covered include: biochemistry and molecular biology; biodiversity and taxonomy; biotechnology; cell and developmental biology; ecology and evolution; genetics and genomics; metabolism and physiology; pathobiology; synthetic and systems biology; tools and resources
Các bài báo tiêu biểu
Deletion of <i>PDE2</i>, the gene encoding the high‐affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in <i>Candida albicans</i> Abstract A role for the cAMP‐dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2 , the gene encoding the high cAMP‐affinity phosphodiesterase. Unlike wild‐type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild‐type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60–65 nm, compared to 80–85 nm in the wild‐type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans . Copyright © 2005 John Wiley & Sons, Ltd.
Tập 22 Số 4 - Trang 285-294 - 2005
Sequence, mapping and disruption of <i>CCC2</i>, a gene that cross‐complements the Ca<sup>2+</sup>‐sensitive phenotype of <i>csg1</i> mutants and encodes a P‐type ATPase belonging to the Cu<sup>2+</sup>‐ATPase subfamily Abstract We have isolated, sequenced, mapped and disrupted a gene, CCC2 , from Saccharomyces cerevisiae . This gene displays non‐allelic complementation of the Ca2+ ‐sensitive phenotype conferred by the csg1 mutation. Analysis of the CCC2p amino acid sequence reveals that it encodes a member of the P‐type ATPase family and is most similar to a subfamily thought to consist of Cu2+ transporters, including the human genes that mutate to cause Wilson disease and Menkes disease. The ability of this gene, in two or more copies, to reverse the csg1 defect suggests that Ca2+ ‐induced death of csg1 mutant cells is related to Cu2+ metabolism. Cells without CCC2 require increased Cu2+ concentrations for growth. Therefore CCC2p may function to provide Cu2+ to a cellular compartment rather than in removal of excess of Cu2+ . The sequence of CCC2 is available through GenBank under accession number L36317.
Tập 11 Số 3 - Trang 283-292 - 1995
Yeast/<i>E. coli</i> shuttle vectors with multiple unique restriction sites Abstract Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomuosly in Saccharomyces cerevisiae and in E. coli; (2) they contain the β‐lactamase gene and confer ampicillin resistance to E. coli ; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI , SacI , KpnI , SmaI , BamH1 , XbaI , SbaI , SalI , PstI , SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI , which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of β‐galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these grown under non‐selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for constructions of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for antunomous replication in yeast. The derivative plasmids YIp651 and YIp352 can therefore be used ti integrate specific sequences into yeast chromosomal DNA.
Tập 2 Số 3 - Trang 163-167 - 1986
Mass spectrometry‐based profiling of phospholipids and sphingolipids in extracts from <i>Saccharomyces cerevisiae</i> Abstract Lipids are rapidly moving to centre stage in many fields of biological sciences. Lipidomics, the systems‐level scale analysis of lipids and their interacting factors, is thus an emerging field which holds great promise for drug and biomarker discovery. Here we present a mass spectrometry‐based approach for profiling of polar lipids, in particular phospholipids and sphingolipids, in Saccharomyces cerevisiae . The first step includes semi‐quantitative surveys of lipids in an untargeted fashion, which is particularly powerful for detection of changes that cannot easily be anticipated. This leads to the identification of ions with increased or decreased signal intensities. Comprehensive theoretical calculation of the masses of yeast phospholipid and sphingolipid molecular species, based on fatty acyl and headgroup heterogeneity, is next used to tentatively assign ions of interest. Subsequent targeted analysis using tandem mass spectrometry allows for characterization and quantification of phospholipids and sphingolipids. Given the high degree of conservation in pathways of lipid metabolism between different organisms, it can be expected that this method will lead to the discovery of novel enzymatic activities and modulators of known ones, particularly when used in combination with genetic and chemogenetic libraries and screens. We validated the method using the EUROSCARF library of non‐essential deletion mutants. Mutants of SCS7 , a lipid hydroxylase, and SLC1 , a putative acyl transferase with unknown substrate specificity, were profiled for their phospholipid and sphingolipid content. The observed changes in lipid profiles are consistent with previous observations and extend our knowledge on in vivo substrate use under permissive growth conditions. Copyright © 2006 John Wiley & Sons, Ltd.
Tập 23 Số 6 - Trang 465-477 - 2006
Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeast <i>Saccharomyces cerevisiae</i> Abstract Methylglyoxal is associated with a broad spectrum of biological effects, including cytostatic and cytotoxic activities. It is detoxified by the glyoxylase system or by its reduction to lactaldehyde by methylglyoxal reductase. We show that methylglyoxal reductase (NADPH‐dependent) is encoded by GRE2 (YOL151w ). We associated this activity with its gene by partially purifying the enzyme and identifying by MALDI–TOF the proteins in candidate bands on SDS–PAGE gels whose relative intensities correlated with specific activity through three purification steps. The candidate proteins were then purified using a glutathione‐S ‐transferase tag that was fused to them, and tested for methylglyoxal reductase activity. The advantage of this approach is that only modest protein purification is required. Our approach should be useful for identifying many of the genes that encode the metabolic pathway enzymes that have not been associated with a gene (about 275 in S. cerevisiae , by our estimate). Copyright © 2003 John Wiley & Sons, Ltd.
Tập 20 Số 6 - Trang 545-554 - 2003
Four ARF GAPs in <i>Saccharomyces cerevisiae</i> have both overlapping and distinct functions Abstract Previous studies in yeast have revealed the presence of four proteins with a conserved, cysteine‐rich, ARF GAP domain that share the ability to suppress the conditional growth defect of the arf1‐3 mutant. Three of these proteins have been shown previously to be ADP‐ribosylation factor (ARF) GTPase‐activating proteins (GAPs). We now demonstrate that the fourth also exhibits in vitro ARF GAP activity and correlates the suppressor and ARF GAP activities for all four. Because the four ARF GAP proteins are quite diverse outside the ARF GAP domain, a genetic analysis was undertaken to define the level of functional cross‐talk between them. A large number of synthetic defects were observed that point to a high degree of functional overlap among the four ARF GAPs. However, several differences were also noted in the ability of each gene to suppress the synthetic defects of others and in the impact of single or combined deletions on assays of membrane traffic. We interpret these results as supportive evidence for roles of ARF GAPs in a number of distinct, essential cellular processes that include cell growth, protein secretion, endocytosis and cell cycling. The description of the specificities of the ARF GAPs for the different responses is viewed as a necessary first step in dissecting biologically relevant pathways through a functionally overlapping family of signalling proteins. Copyright © 2003 John Wiley & Sons, Ltd.
Tập 20 Số 4 - Trang 315-330 - 2003
Applications of high efficiency lithium acetate transformation of intact yeast cells using single‐stranded nucleic acids as carrier Abstract The 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, the LEU2 , HIS3 , URA3 , TRP1 and ARG4 genes were found among the primary transformations at a frequency of approximately 0·1%. The RAD4 gene, which is toxic to Escherichia 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 of the single event.
Tập 7 Số 3 - Trang 253-263 - 1991
An efficient transformation procedure enabling long‐term storage of competent cells of various yeast genera
Tập 7 Số 7 - Trang 691-692 - 1991
Parameters affecting the frequencies of transformation and co‐transfromation with synthetic oligonucleotides in yeast Abstract Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially fuctional iso‐1‐cytochrome c . Aproximately 3 × 104 transformanrs, constituting 0·1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogenous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double‐strand plasmid, was efficient in a rad52 − strain, ssuggesting that the pathway for transformation with oligonucleotides is different from that with linearized double‐strand plasmid. We describe a procedure of co‐transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amono acid alteration elsewhere in the iso‐1‐cytochrome c molecule; approximately 20% of the transformants obtained by co‐transformation contained these desired second alterations.
Tập 8 Số 11 - Trang 935-948 - 1992
Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure Abstract An improved lithium acetate (LiAc)/single‐stranded DNA (SS‐DNA)/polyethylene glycol (PEG) protocol which yields >1 × 106 transformants/μg plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS‐DNA/PEG transformation. The highest transformation efficiency was observed when 1 × 108 cells were transformed with 100 ng plasmid DNA in the presence of 50 μg SS carrier DNA. The yield of transformants increased linearly up to 5 μg plasmid per transformation. A 20‐min heat shock at 42°C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32 P‐labelled plasmid DNA than did double‐stranded (DS) carrier. The LiAc/SS‐DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS‐DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS‐DNA/PEG method is discussed.
Tập 11 Số 4 - Trang 355-360 - 1995