Biology Reference
In-Depth Information
FIGURE 22.2
Sequencing chromatogram from a diploid strain heterozygous for
mutant
tubulin. Double peak (arrow) indicates the presence of both wild-type and mutant alleles.
tub2-E421K
Note : We recommend sequencing the full gene of interest to exclude the
possibility of additional mutations in the final strain.
22.3 ANALYSIS OF TETRAD VIABILITY
Linking genetic alterations to phenotypic changes through tetrad dissection and sub-
sequent analysis of spores is fundamental for determining the functional conse-
quences of specific mutations. In response to nitrogen starvation and in the
presence of a nonfermentable carbon source, diploid yeast cells sporulate. During
this process, meiotic segregation gives rise to four haploid daughter cells or spores.
Once spores have formed, the intact anucleate mother cell collapses around them
forming a mature tetrahedral ascus ( Fig. 22.3 A). Microdissection and germination
of the spores from a single tetrad yields four haploid strains where a given genetic
locus originating from the pair of homologous parent chromosomes is represented in
two of the four strains.
Mutations that disrupt the function of essential genes, such as TUB1 and TUB2 ,
typically exhibit recessive lethality and produce a characteristic pattern of two viable
and two inviable spores (2
). The surviving spores contain the wild-type version
of the gene, which is reflected by the phenotype. Alternatively, viability of all four
spores is indicative of less harmful mutations and allows analysis of the resultant
haploids that contain only the mutated tubulin. In certain cases, spore viability
may digress from the classic “all or nothing” 2
þ
/2
þ
hypothesis. With regard to tu-
bulin mutations, low viability can result from severe disruption of meiotic chromo-
some segregation, which is a microtubule-dependent process. However, the survival
of none, one, or three spores can also indicate the presence of independent genomic
variations that
/2
influence spore viability,
for example, suppressor mutations.
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