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Genetics of S. cerevisiae
Tetrad analysis
Double crossovers
Yeast Plasmids
• Low copy: ars, CEN
-plasmid segregates as an ordinary chromosome
• High-copy (2μ): ars
-useful when for overexpression
Generating a Strain
• One-step gene disruption: transform yeast with a selectable marker
flanked by homology to either side of a gene’s open reading frame
• Nomenclature:
WT gene YFG
null yfgΔ::HIS3
• Original strain his3Δ
• Selection on -Ura followed by PCR
To Make pt Mutation w/o Marker
Nomenclature:
WTgene ADE1
Mutant ade1-1
-Ura
Isolation and characterization
dominant mutant alleles
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Isolate a mutant
Determine the number of genes mutated
Classify dominance vs. recessive
To isolate a dominant allele—you need to map it. If one of
the markers is very close to your dominant mutant allele, it
will rarely segregate together with it and most of the progeny
will be PD.
• Clone your gene
Types of Dominant Mutations
• Dominant gain of function- over-active
• Dominant-negative – a mutation, which results in a gene
product that neutralizes the wild-type gene product.
Antimorph is a mutant with a dominant-negative phenotype.
• Haplo-insufficiency - occurs when a diploid organism only has
a single working copy of a wild-type gene, and the single
functional copy of the gene does not produce enough of a
gene product to bring about a wild-typy condition.
Cloning a recessive mutant
For recessive mutation-transfrorm mutant with a genomic library from WT
What type of plasmid would you use?
Finding more genes in a biological
pathway
• High-copy suppressor screens
-high copy plasmid or GAL promoter on CEN
plasmid
ura3Δ
What would you get
from this screen?
Finding more genes in a biological
pathway
Second site suppressors screens
-mutagenize your mutant and look for WT colonies
Extragenic vs Intragenic?
Synthetic lethal screen
-Genes can be in the same pathway where a mutation
causes partial loss of function in this pathway
-Genes could be in parallel pathways that perform
redundant functions
-Genes can be in parallel pathways that do not have
related functions, but one is required when other is
missing (Ex. checkpoint mutation and DNA repair machinery).
Synthetic Lethal Screen
• Haploid=>Diploid=>Haploid
• Need to be able to select for diploids and then
for haploids.