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.
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
Isolating regulated promoters
Isolating specific genes from other organisms
Yeast genomic and cDNA libraries
Cloning in yeast
Cloning by complementating a temperature sensitive mutant
Gene expression in yeast Copy number Promoter Protease problem
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
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 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.