References:

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Yeast molecular biology-yeast vectors, expression
of proteins in Yeast
References Science 240, 1439-1443
(1988). Methods in Enzymology 194, 3-77
(1991). Science 274, 546-567 (1996).
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Two commonly used yeast in molecular genetics

• Saccharomyces cerevisiae (budding yeast, bakers
  yeast)
• Schizosaccharomyces pombe (fission yeast, brewers
  yeast)

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S. cerevisiae
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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

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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
• yfg1LEU2 YFG1 is integrated by LEU2
• yfg1D1 a deletion mutant of YFG1
• Yfg1p gene product of YFG1, a protein

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Yeast genome
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.a3, b
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Yeast life cycle
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Separation of spore products by tetrad dissection
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Sporulation and tetrad dissection
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Tetrad dissection
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Analysis of spore products
Complement medium
Selection medium
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Yeast Molecular Genetics
vectors
cloning
Gene expression
Making mutants
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Yeast vectors
Plasmids
Origin of replication Selection markers Yeast
strains
Yeast artificial chromosome
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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 copy number stability () ARS 1-5 ARS
-CEN 1-2 90-99 ARS -2 mm 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.
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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

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A typical yeast plasmid
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Yeast artificial chromosome (YAC)

• High cloning capacity, 300 kbp.
• centromere, telomere, selection markers.
• Linear plasmids (YLp)

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Yeast cloning vectors
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Yeast negative selection systems
URA3 The gene product of URA3 (orotidine-5-phosp
hate 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.
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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.

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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.
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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

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Cloning by complementating a temperature
sensitive mutant
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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 C
EN6 pRS424 6-8 TRP1 2 mm pRS425 6-8 LEU2 2
mm YEPFAT10 6-8 TRP1 leu2-d 2 mm pMAC561 cDNA TR
P1 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
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Gene expression in yeast
Copy number
Promoter
Protease problem
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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

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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.

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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)

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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-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.

Gene targeting In the absence of ARS sequences,
DNA transformed into yeast cells integrated into
the genome exclusively by homologous
recombination.
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Gene targeting scheme
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Gene targeting scheme
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