References: Science 240, 1439-1443 (1988). Methods in Enzymology 194, 3-77 (1991). Science 274, 546-567 (1996). Yeast mole...
<ul><li>Saccharomyces cerevisiae  (budding yeast, bakers yeast) </li></ul><ul><li>Schizosaccharomyces pombe  (fission yeas...
S. cerevisiae
<ul><li>non-pathogenic, edible </li></ul><ul><li>contain all the advantage of bacterial genetics </li></ul><ul><li>a monoc...
Nomenclature in yeast <ul><li>YFG1 : locus or dominant allele (mostly wild type), capital, italic </li></ul><ul><li>yfg1-1...
Genome of diploid  Saccharomyce cerevisiae  cell  Characteristic Chromosomes 2-mm plasmid Mitochondiral Relative amount (%...
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
<ul><li>Need an autonomous replication sequence ( ARS ) for plasmid to replicate. </li></ul><ul><li>CEN : contain a chromo...
Plasmids Nutrition dependence: uracil ( URA3 ), adenine ( ADE2 ,  ADE3 ), leucine ( LEU2 ), tryptophan ( TRP1 ), lysine ( ...
A typical yeast plasmid
<ul><li>High cloning capacity, ~300 kbp. </li></ul><ul><li>centromere, telomere, selection markers. </li></ul><ul><li>Line...
Yeast cloning vectors
URA3 : The gene product of  URA3  (orotidine-5’-phosphate decarboxylase) converts 5-FOA (5-fluoroorotic acid) to a toxic p...
Transformation in yeast Li-acetate method Up to 2.2 x 10 7  transformants/  g DNA; simple, easy, and cheap. Spheroplast m...
Unlike the well established plasmid purification methods in  E. coli , no easy plasmid purification method is developed in...
<ul><li>Cloning by mail </li></ul><ul><li>Complementation of recessive alleles </li></ul><ul><li>Cloning dominant alleles ...
 
Cloning by complementating a temperature sensitive mutant
<ul><li>Plasmid insert size (kbp) selection marker origin reference </li></ul><ul><ul><li>YRp7 5-20  TRP1 no 1 </li></ul><...
Gene expression in yeast Copy number Promoter Protease problem
Copy number <ul><li>alternate the copy number of DNA alternate the expression of genes. </li></ul><ul><li>Plasmid copy num...
Promoter <ul><li>Constitutive:  ADH1  (alcohol dehydrogenase I) and  PGK  (3-phosphoglycerate kinase), produce about 1% ea...
Protease problem <ul><li>Growth stage </li></ul><ul><li>Protease deficient strain: there are protease-deficient mutants av...
<ul><li>Chemicals: ethylmethane sulfonate (EMS),  N -methyl- N’ -nitro- N -nitrosoguanidine (MNNG), produce transitions at...
Gene targeting scheme:
Gene targeting scheme:
 
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講義

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講義

  1. 1. 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
  2. 2. <ul><li>Saccharomyces cerevisiae (budding yeast, bakers yeast) </li></ul><ul><li>Schizosaccharomyces pombe (fission yeast, brewers yeast) </li></ul>Two commonly used yeast in molecular genetics:
  3. 3. S. cerevisiae
  4. 4. <ul><li>non-pathogenic, edible </li></ul><ul><li>contain all the advantage of bacterial genetics </li></ul><ul><li>a monocellular eukaryotic cell with essentially all the organelles </li></ul><ul><li>a genetically manipulable life cycle </li></ul><ul><li>well established molecular biology tools </li></ul><ul><li>well studied biochemical pathway </li></ul><ul><li>the sequences of S. cerevisiae genome had been determined </li></ul>What ‘s special about yeast:
  5. 5. Nomenclature in yeast <ul><li>YFG1 : locus or dominant allele (mostly wild type), capital, italic </li></ul><ul><li>yfg1-119 : a specific recessive mutant of YFG1 , -119 is the name of allele </li></ul><ul><li>yfg1::LEU2 : YFG1 is integrated by LEU2 </li></ul><ul><li>yfg1  1 : a deletion mutant of YFG1 </li></ul><ul><li>Yfg1p: gene product of YFG1 , a protein </li></ul>
  6. 6. 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
  7. 7. Yeast life cycle
  8. 8. Separation of spore products by tetrad dissection
  9. 9. Sporulation and tetrad dissection
  10. 10. Tetrad dissection
  11. 11. Analysis of spore products Complement medium Selection medium
  12. 12. Yeast Molecular Genetics vectors cloning Making mutants Gene expression
  13. 13. Yeast vectors Plasmids Yeast artificial chromosome Origin of replication Selection markers Yeast strains
  14. 14. <ul><li>Need an autonomous replication sequence ( ARS ) for plasmid to replicate. </li></ul><ul><li>CEN : contain a chromosomal centromere, YCp (yeast centromeric plasmid) </li></ul><ul><li>2  m : YEp (yeast episomal plasmid) </li></ul><ul><li>origin-less : YIp (yeast integrating plasmid) cannot replicate in yeast, integrate into yeast chromosome </li></ul>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.
  15. 15. Plasmids Nutrition dependence: uracil ( URA3 ), adenine ( ADE2 , ADE3 ), leucine ( LEU2 ), tryptophan ( TRP1 ), lysine ( LYS2 ) Selection marker: <ul><li>Diploid vs. halploid </li></ul><ul><li>Mating type: a or  </li></ul><ul><li>Genotype: yeast strains should have genotypes that can accommodate plasmids with various selection markers. </li></ul><ul><li>MATa ade2-1 lys2-1 his3-  200 leu2-  1 trp1-  63 ura3-52 </li></ul>Strain
  16. 16. A typical yeast plasmid
  17. 17. <ul><li>High cloning capacity, ~300 kbp. </li></ul><ul><li>centromere, telomere, selection markers. </li></ul><ul><li>Linear plasmids (YLp) </li></ul>Yeast artificial chromosome (YAC)
  18. 18. Yeast cloning vectors
  19. 19. 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:
  20. 20. 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 <ul><li>Transformation efficiency can be ~4 x 10 5 transformants /  g DNA. </li></ul><ul><li>Need a gene pulser, usually expensive. </li></ul>
  21. 21. 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: <ul><li>Mechanical force: use glass beads to break the cell walls. </li></ul><ul><li>Enzymatic digestion: zymolyase or glusulase are used to digest apart the cell walls. </li></ul>
  22. 22. <ul><li>Cloning by mail </li></ul><ul><li>Complementation of recessive alleles </li></ul><ul><li>Cloning dominant alleles </li></ul><ul><li>High-copy suppression </li></ul><ul><li>Isolating regulated promoters </li></ul><ul><li>Isolating specific genes from other organisms </li></ul><ul><li>Yeast genomic and cDNA libraries </li></ul>Cloning in yeast
  23. 24. Cloning by complementating a temperature sensitive mutant
  24. 25. <ul><li>Plasmid insert size (kbp) selection marker origin reference </li></ul><ul><ul><li>YRp7 5-20 TRP1 no 1 </li></ul></ul><ul><li>YEp13 5-7 LEU2 2  m </li></ul><ul><li>YEp24 7-10 URA3 2  m 2 </li></ul><ul><li>YCp50 10-20 URA3 CEN4 3 </li></ul><ul><li>pRS314 6-8 TRP1 CEN6 </li></ul><ul><li>pRS424 6-8 TRP1 2  m </li></ul><ul><li>pRS425 6-8 LEU2 2  m </li></ul><ul><li>YEPFAT10 6-8 TRP1 leu2-d 2  m </li></ul><ul><li>pMAC561 cDNA TRP1 2  m 4 </li></ul><ul><ul><li>pRS316GAL cDNA URA3 CEN 5 </li></ul></ul><ul><ul><li>_____________________________________________________ </li></ul></ul><ul><ul><li>Nasmyth & Reed PNAS 77, 2119-2123, 1980. </li></ul></ul><ul><li> Carlson and Botstein Cell 28, 145-154, 1982. </li></ul><ul><li>Rose et al. Gene 60, 237-243, 1987. </li></ul><ul><li>McKnight & McConaughy PNAS 80, 4412-4416, 1983. </li></ul><ul><li>Liu et al. Genetics 132, 665-673, 1992 </li></ul>Yeast genomic and cDNA libraries
  25. 26. Gene expression in yeast Copy number Promoter Protease problem
  26. 27. Copy number <ul><li>alternate the copy number of DNA alternate the expression of genes. </li></ul><ul><li>Plasmid copy number: cryptic allele of leu2-d promoter increases the plasmid copy number up to several hundred copies per cell. </li></ul><ul><li>Ty transposition vector insert semi-randomly into yeast genome </li></ul>
  27. 28. Promoter <ul><li>Constitutive: ADH1 (alcohol dehydrogenase I) and PGK (3-phosphoglycerate kinase), produce about 1% each of total yeast mRNA. </li></ul><ul><li>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. </li></ul>
  28. 29. Protease problem <ul><li>Growth stage </li></ul><ul><li>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 ) </li></ul>Yeast contains a large number of proteases that are located in various compartments of the cell.
  29. 30. <ul><li>Chemicals: ethylmethane sulfonate (EMS), N -methyl- N’ -nitro- N -nitrosoguanidine (MNNG), produce transitions at G-C sites. </li></ul><ul><li>UV: usually occur in runs of pyrimidines and include both transitions and transversions. Frame-shift mutations are also observed. </li></ul>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.
  30. 31. Gene targeting scheme:
  31. 32. Gene targeting scheme:
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