Written in English
|Statement||by Alfonso Martinez-Arias.|
|LC Classifications||Microfilm 83/476 (Q)|
|The Physical Object|
|Pagination||viii, 123 leaves|
|Number of Pages||123|
|LC Control Number||83199631|
Gene Disruptions-The LEU2 gene of S. cerevisiae was integrated at various sites in the CANl locus by one-step gene disruption method (Rothstein, ). After subcloning of the kb long BamHI-Chin fragment of the CANl gene (see Fig. 1) in pUC18 (pWH), this plasmid was digested with DNase in the presence of manganese. "LEU2" - Beta-isopropylmalate dehydrogenase in Saccharomyces i.e. each node represents all the proteins produced by a single, protein-coding gene locus. Node Color. colored nodes: query proteins and first shell of interactors NRRL Y, S. cerevisiae, Saccharomyces capensis, Saccharomyces cerevisiae, Saccharomyces italicus. the Saccharomyces cerevisiae HIS4 and GALI genes and the E. coli lac2 gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5’-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by. In addition, we evaluated the effect of S. cerevisiae MIG1 protein on this promoter. 2. Materials and methods Strains, transformation and culture conditions. S. cerevisiae strains WA (MATa SUC2 ade can his,15 leu, trp ura) and IMb (MATa his4 leu, sta° STA10) were from our collection.
An integrated GAL1-lacZ fusion provided a useful phenotypic marker for the gal80 − regulatory mutation in Saccharomyces minimal glucose plates containing a β-galactosidase indicator, a GAL80 strain containing the fusion gave white colonies, whereas a gal80 − strain gave blue colonies. This color difference was used to isolate the GAL80 gene from a plasmid . Disruption of the ORF YGLc was also made in (MATa /α ura3‐52/URA3 his3Δ1/HIS3 leu2‐3,/LEU2 trp1‐/TRP1). For routine cultures, S. cerevisiae was grown on glucose complete medium (YPD): 1% yeast extract, 2% peptone and 2% glucose. Glycerol complete medium (YPG) contained 3% (v/v) of glycerol instead of glucose. A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control. In the present study, data from functional analysis of a septin component Cdc11 containing four different epitopes shed light on the usefulness of the tagging vectors for rapid analysis of gene function in S. cerevisiae. 2 Materials and methods Strains and culture condition. Table 1 lists the yeast strains and plasmids used in this study.
DiCarlo, J. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Resea (). Horwitz, A. et al. Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas. Cell Systems 1, (). Jakočiūnas, T., Jensen, M. & Keasling, J. Northern(RNA)analysisshowedthatgcr2andgcrl affectENO] mRNAlevels. The glycolytic pathway is a major metabolic route in Saccharomyces cerevisiae. Most of the S. cerevisiae en- fusion gene for. IFM analysis of nucleolar structures in strains transcribing the GALS rDNA fusion gene on a plasmid (NOYa) or the same fusion gene integrated into chromosome XII at the RDN locus (YJV) by use of Pol II. Nucleolar protein Ssb1p was analyzed by IFM and DNA was stained by DAPI as described in Materials and Methods. that targets integration to the genomic LEU2 locus  and the p77x plasmid that targets integration to the TOK1 locus. p77x was gener-ated from p by replacing the LEU2 targeting regions with bp of TOK1 50 and 30 non-coding sequence, respectively. In both gene replacement constructs, the expression of the GFP-mKir construct.