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Yeast molecular biology-yeast vectors, expression of proteins in Yeast Reference: Science 240, 1439-1443 (1988). Methods in Enzymology 194, 3-77 (1991). Science 274, 546-567 (1996). Two commonly used yeast in molecular genetics: • Saccharomyces cerevisiae (budding yeast, bakers yeast) • Schizosaccharomyces pombe (fission yeast, brewers yeast) What ‘s special about yeast: • 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 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 • yfg1D1: a deletion mutant of YFG1 • Yfg1p: gene product of YFG1, a protein Yeast genome Genome of diploid Saccharomyce cerevisiae cell Characteristic Relative amount (%) Number of copies Size (kbp) Mutants Chromosomes 2-mm plasmid Mitochondiral 85 5 10 2 x 16 60-100 ~50 (8-130) 14,000 6.318 70-76 All kinds none Cyt a.a3, b Yeast life cycle Sporulation and tetrad dissection Tetrad dissection Analysis of spore products Complement medium Selection medium Yeast Molecular Genetics vectors cloning Gene expression Making mutants Yeast vectors Plasmids Origin of replication Selection markers Yeast strains Yeast artificial chromosome Plasmids Origin of replication: • Need an autonomous replication sequence (ARS) for plasmid to replicate. • CEN: contain a chromosomal centromere, YCp (yeast centromeric plasmid) • 2 mm: YEp (yeast episomal plasmid) • origin-less: YIp (yeast integrating plasmid) cannot replicate in yeast, integrate into yeast chromosome Origin ARS ARS -CEN ARS -2 mm origin-less copy number 1-2 10-40 1 stability (%)# 1-5 90-99 80-95 100 # stability of plasmid is determined as the percentage of plasmid bearing colonies after overnight culture (~10 cell divisions) in the absence of selection. Plasmids Selection marker: Nutrition dependence: uracil (URA3), adenine (ADE2, ADE3), leucine (LEU2), tryptophan (TRP1), lysine (LYS2) Strain • Diploid vs. halploid • Mating type: a or a • Genotype: yeast strains should have genotypes that can accommodate plasmids with various selection markers. MATa ade2-1 lys2-1 his3-D200 leu2-D1 trp1-D63 ura3-52 Yeast negative selection systems: 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 a-aminoadipate (a-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. A typical yeast plasmid Yeast artificial chromosome (YAC) • High cloning capacity, ~300 kbp. • centromere, telomere, selection markers. • Linear plasmids (YLp) Transformation in yeast Li-acetate method Up to 2.2 x 107 transformants/mg DNA; simple, easy, and cheap. Spheroplast method ~1-5 x 104 transformants/mg DNA; need to digest yeast cells with zymolyase, technically difficult and time consuming Electroporation • Transformation efficiency can be ~4 x 105 transformants /mg DNA. • Need a gene pulser, usually expensive. 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. 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. Cloning in yeast • 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 林敬哲 副教授 國立陽明大學 生物藥學研究所 臺北市石牌 立農街二段 155 號 電話: (2) 2826-7258 傳真: (2) 2820-0067 E-mail: [email protected] Yeast genomic and cDNA libraries Plasmid insert size (kbp) selection marker origin reference YRp7 5-20 TRP1 no 1 YEp13 5-7 LEU2 2 mm YEp24 7-10 URA3 2 mm 2 YCp50 10-20 URA3 CEN4 3 pRS314 6-8 TRP1 CEN6 pRS424 6-8 TRP1 2 mm pRS425 6-8 LEU2 2 mm YEPFAT10 6-8 TRP1 leu2-d 2 mm pMAC561 cDNA TRP1 2 mm 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 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 (3phosphoglycerate 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 Yeast contains a large number of proteases that are located in various compartments of the cell. • 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) Making mutants Classical mutagenesis techniques: The highest proportion of mutants per treated cell is usually found at doses giving 10 to 50% survival. • Chemicals: ethylmethane sulfonate (EMS), N-methyl-N’-nitroN-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. Gene targeting: In the absence of ARS sequences, DNA transformed into yeast cells integrated into the genome exclusively by homologous recombination. Gene targeting scheme: 林敬哲 副教授 國立陽明大學 生物藥學研究所 臺北市石牌 立農街二段 155 號 電話: (2) 2826-7258 傳真: (2) 2820-0067 E-mail: [email protected]