references: science 240, 1439-1443 (1988). methods in enzymology 194, 3-77 (1991)

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• yfg11: a deletion mutant of YFG1• Yfg1p: gene product of YFG1, a protein

Genome of diploid Saccharomyce cerevisiae cell Characteristic Chromosomes2-mm plasmid MitochondiralRelative amount (%) 85 5 10Number of copies 2 x 16 60-100 ~50 (8-130)Size (kbp) 14,000 6.318 70-76Mutants All kinds none Cyt a.a3, 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 replicationSelection markersYeast 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 1-5ARS -CEN 1-2 90-99ARS -2 m 10-40 80-95origin-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 107 transformants/g DNA; simple, easy, and cheap.

Spheroplast method

~1-5 x 104 transformants/g DNA; need to digest yeast cells with zymolyase, technically difficult and time consuming

Electroporation

• Transformation efficiency can be ~4 x 105 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

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: jjlin@ym.edu.tw

Cloning by complementating a temperature sensitive mutant

Plasmid insert size (kbp) selection marker origin referenceYRp7 5-20 TRP1 no 1

YEp13 5-7 LEU2 2 mYEp24 7-10 URA3 2 m 2YCp50 10-20 URA3 CEN4 3

pRS314 6-8 TRP1 CEN6pRS424 6-8 TRP1 2 mpRS425 6-8 LEU2 2 m

YEPFAT10 6-8 TRP1 leu2-d 2 mpMAC561 cDNA TRP1 2 m 4pRS316GAL 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:

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: jjlin@ym.edu.tw