Oct 11, 2011
Isolation and characterization dominant mutant alleles
Find a mutant
Fast way is to mutagenize your haploid yeast stock
To reduce # of mutants examined you can perform selection instead of
screening if possible.
Determine the number of genes mutated. Perform complementation test-the
mutation is compared to other mutations that are similar in phenotype to determine
whether these mutations are the alleles of the same gene.
Complementation test- is a genetic test to determine if two recessive mutations
are alleles of the same gene. Mutations that are alleles of the same gene fail to
complement.
"complementation" means that you complemented a mutant organism with a gene that
now made it wild-type. In short, you cannot perform complementation test with a
dominant allele.
Between mutants that do not complement you should ask: are they mutants in the same
gene? Dominant mutation will not be complemented.
Classify dominance vs. recessive. Most mutations are recessive and cause a loss or
a reduction in gene’s function, but some are dominant. These include: overproduction of the normal gene product, dominant gain of function, or even
prevention of normal product functioning, dominant negative.
To classify dominant versus recessive: mate your haploids to WT haploid and see
whether the diploids now have WT phenotype.
How can you put a dominant mutation into one of your complementation groups?
Dissect tetrads that you got after the complementation test.
If one of the tetrads are all PDs—your dominant mutation belongs to that particular
complementation group (exception: if genes are extremely close to each other—it’s a
bummer).
If you see WTs among tetrads – dominant mutation does not belong to that
complementation group.
To isolate a dominant allele—you need to map it.
Mapping- means to find the genomic location of a gene.
How do you map a dominant allele?
First: you want to know on which chromosome your mutation is:
Cross your haploid mutant to 16 different strains, where each strain will carry
multiple markers on one of the 16 chromosomes.
If a marker and allele are far apart or on different chromosomes—expect PD:NPD:T
(1:1:≤4)
If one of the markers is very close to your dominant mutant allele, it will rarely
segregate together with it and all of the progeny will be PD.
Once you mapped your gene—you need to clone it. Once you cloned that gene, you
can:
Distinguish between 3 types of dominant mutations:
Dominant gain of function- a dominant allele, because it is 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-type condition. Haploinsufficiency is an exception, elimination of a function of one allele results in a
mutant phenotype; in which a loss of function condition causes a dominant
phenotype
To distinguish between dominant gain of function and dominant negative
of the same gene:
alleles
You need to have/know how a null or complete loss of function mutant for that same
locus looks like
- Dominant gain of function looks like a null