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講義
 

講義

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    講義 講義 Presentation Transcript

    • References: Science 240, 1439-1443 (1988). Methods in Enzymology 194, 3-77 (1991). Science 274, 546-567 (1996). Yeast molecular biology-yeast vectors, expression of proteins in Yeast
      • Saccharomyces cerevisiae (budding yeast, bakers yeast)
      • Schizosaccharomyces pombe (fission yeast, brewers yeast)
      Two commonly used yeast in molecular genetics:
    • S. cerevisiae
      • non-pathogenic, edible
      • contain all the advantage of bacterial genetics
      • a monocellular eukaryotic cell with essentially all the organelles
      • a genetically manipulable life cycle
      • well established molecular biology tools
      • well studied biochemical pathway
      • the sequences of S. cerevisiae genome had been determined
      What ‘s special about yeast:
    • Nomenclature in yeast
      • YFG1 : locus or dominant allele (mostly wild type), capital, italic
      • yfg1-119 : a specific recessive mutant of YFG1 , -119 is the name of allele
      • yfg1::LEU2 : YFG1 is integrated by LEU2
      • yfg1  1 : a deletion mutant of YFG1
      • Yfg1p: gene product of YFG1 , a protein
    • Genome of diploid Saccharomyce cerevisiae cell Characteristic Chromosomes 2-mm plasmid Mitochondiral Relative amount (%) 85 5 10 Number of copies 2 x 16 60-100 ~50 (8-130) Size (kbp) 14,000 6.318 70-76 Mutants All kinds none Cyt a.a 3 , b Yeast genome
    • Yeast life cycle
    • Separation of spore products by tetrad dissection
    • Sporulation and tetrad dissection
    • Tetrad dissection
    • Analysis of spore products Complement medium Selection medium
    • Yeast Molecular Genetics vectors cloning Making mutants Gene expression
    • Yeast vectors Plasmids Yeast artificial chromosome Origin of replication Selection markers Yeast strains
      • Need an autonomous replication sequence ( ARS ) for plasmid to replicate.
      • CEN : contain a chromosomal centromere, YCp (yeast centromeric plasmid)
      • 2  m : YEp (yeast episomal plasmid)
      • origin-less : YIp (yeast integrating plasmid) cannot replicate in yeast, integrate into yeast chromosome
      Plasmids Origin of replication: Origin copy number stability (%) # ARS 1-5 ARS -CEN 1-2 90-99 ARS -2  m 10-40 80-95 origin-less 1 100 # stability of plasmid is determined as the percentage of plasmid bearing colonies after overnight culture (~10 cell divisions) in the absence of selection.
    • Plasmids Nutrition dependence: uracil ( URA3 ), adenine ( ADE2 , ADE3 ), leucine ( LEU2 ), tryptophan ( TRP1 ), lysine ( LYS2 ) Selection marker:
      • Diploid vs. halploid
      • Mating type: a or 
      • Genotype: yeast strains should have genotypes that can accommodate plasmids with various selection markers.
      • MATa ade2-1 lys2-1 his3-  200 leu2-  1 trp1-  63 ura3-52
      Strain
    • A typical yeast plasmid
      • High cloning capacity, ~300 kbp.
      • centromere, telomere, selection markers.
      • Linear plasmids (YLp)
      Yeast artificial chromosome (YAC)
    • Yeast cloning vectors
    • URA3 : The gene product of URA3 (orotidine-5’-phosphate decarboxylase) converts 5-FOA (5-fluoroorotic acid) to a toxic product that kills the URA3 cells. LYS2 : The LYS2 gene encodes a-aminoadipate reductase, an enzyme required for lysine biosynthesis. Yeast cells with wild-type LYS2 activity will not grow on media containing  - aminoadipate (  -AA) as a primary nitrogen source. CAN1 : The CAN1 gene encodes an arginine permease. In the absence of arginine, canavanine (arginine analog) is readily incorporated into proteins with lethal consequences; therefore, CAN1 cells are sensitive to canavanine. CYH2 : The CYH2 gene encodes the L29 protein of the yeast ribosome. Cycloheximide blocks translation elongation by interacting with L29. Yeast negative selection systems:
    • Transformation in yeast Li-acetate method Up to 2.2 x 10 7 transformants/  g DNA; simple, easy, and cheap. Spheroplast method ~1-5 x 10 4 transformants/  g DNA; need to digest yeast cells with zymolyase, technically difficult and time consuming Electroporation
      • Transformation efficiency can be ~4 x 10 5 transformants /  g DNA.
      • Need a gene pulser, usually expensive.
    • Unlike the well established plasmid purification methods in E. coli , no easy plasmid purification method is developed in yeast. Plasmids are purified along with chromosomal DNA. Most yeast plasmids are “ shuttle vectors ”, i. e., can propagate in both yeast and E. coli . To recover yeast plasmid DNA, total yeast DNA is purified and transformed into E. coli . Yeast plasmid DNA is then isolated from E. coli . Isolation of yeast DNA Budding yeast has thick walls, to break the cell walls, two methods are used:
      • Mechanical force: use glass beads to break the cell walls.
      • Enzymatic digestion: zymolyase or glusulase are used to digest apart the cell walls.
      • Cloning by mail
      • Complementation of recessive alleles
      • Cloning dominant alleles
      • High-copy suppression
      • Isolating regulated promoters
      • Isolating specific genes from other organisms
      • Yeast genomic and cDNA libraries
      Cloning in yeast
    •  
    • Cloning by complementating a temperature sensitive mutant
      • Plasmid insert size (kbp) selection marker origin reference
        • YRp7 5-20 TRP1 no 1
      • YEp13 5-7 LEU2 2  m
      • YEp24 7-10 URA3 2  m 2
      • YCp50 10-20 URA3 CEN4 3
      • pRS314 6-8 TRP1 CEN6
      • pRS424 6-8 TRP1 2  m
      • pRS425 6-8 LEU2 2  m
      • YEPFAT10 6-8 TRP1 leu2-d 2  m
      • pMAC561 cDNA TRP1 2  m 4
        • pRS316GAL cDNA URA3 CEN 5
        • _____________________________________________________
        • Nasmyth & Reed PNAS 77, 2119-2123, 1980.
      • Carlson and Botstein Cell 28, 145-154, 1982.
      • Rose et al. Gene 60, 237-243, 1987.
      • McKnight & McConaughy PNAS 80, 4412-4416, 1983.
      • Liu et al. Genetics 132, 665-673, 1992
      Yeast genomic and cDNA libraries
    • Gene expression in yeast Copy number Promoter Protease problem
    • Copy number
      • alternate the copy number of DNA alternate the expression of genes.
      • Plasmid copy number: cryptic allele of leu2-d promoter increases the plasmid copy number up to several hundred copies per cell.
      • Ty transposition vector insert semi-randomly into yeast genome
    • Promoter
      • Constitutive: ADH1 (alcohol dehydrogenase I) and PGK (3-phosphoglycerate kinase), produce about 1% each of total yeast mRNA.
      • Inducible: GAL1 , GAL10 (repressed by glucose, induced by galactose), PHO5 (induced by inorganic phosphate), upon induction the level of gene expression increase from 10-30 folds.
    • Protease problem
      • Growth stage
      • Protease deficient strain: there are protease-deficient mutants available that can be used for gene expression purposes. For example: BJ2168 ( MATa leu2 trp1 ura3-52 prb1-1122 prc1-407 pep4-3 prc1-407 gal2 )
      Yeast contains a large number of proteases that are located in various compartments of the cell.
      • Chemicals: ethylmethane sulfonate (EMS), N -methyl- N’ -nitro- N -nitrosoguanidine (MNNG), produce transitions at G-C sites.
      • UV: usually occur in runs of pyrimidines and include both transitions and transversions. Frame-shift mutations are also observed.
      Making mutants Classical mutagenesis techniques: The highest proportion of mutants per treated cell is usually found at doses giving 10 to 50% survival. Gene targeting: In the absence of ARS sequences, DNA transformed into yeast cells integrated into the genome exclusively by homologous recombination.
    • Gene targeting scheme:
    • Gene targeting scheme:
    •