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Mutagenesis and Genetic
Screens
General pathway for
mutational dissection
of a biological process
“Forward Genetics”
Fig. 12-39
From phenotype to gene
• Once an interesting
mutant is found and chromosome
characterized, we
want to find the gene
in which the mutant
occurred
• Positional cloning
– First use genetic
mapping
– Then use
chromosome walking
contig
candidate genes mutation
Candidate-gene approach
• If the mutated gene is
localized to a
sequenced region of
the chromosome,
then look for genes
that could be involved
in the process under
study
• Last step: confirm
gene identification
– Rescue of phenotype
– Mutations in same
gene in different
alleles
Insertional mutagenesis
• Alternative to chromosome walking
– To reduce time and effort required to identify
mutant gene
• Insert piece of DNA that disrupts genes
– Inserts randomly in chromosomes
• Make collection of individuals
– Each with insertion in different place
• Screen collection for phenotypes
• Use inserted DNA to identify mutated gene
General pathway for
mutational dissection
of a biological process
“Forward Genetics”
Fig. 12-39
Isolated 148 temperature-sensitive cell division cycle (cdc) mutants out of
1500 total ts mutants
Complementation analysis: 32 complementation groups (genes/cistrons)
Mapped 14 genes (no apparent clustering)
Phenotypic characterizations:
•Variety of cell cycle points disrupted (can dissect stages)
•Mutations of single gene blocked in very similar point (same
morphology indicates action at a single point))
•Temperature shift immediately blocks some mutants; block delayed 1-3
cell cycles for others (suggests temperature effects are on protein
function versus protein synthesis)
•Mutants block cell cycle in haploid and diploid cells
2 cdc
2 marker
2 cdc & marker
2 wild-type
1 cdc
1 marker
1 cdc & marker
1 wild-type
pp. 46-51, 1974