Transcript Document

Chapter 12: Alternative approaches to mutational dissection
Fig. 16-1
Types of mutational analysis
• 1. “Classical” “forward genetics” approach to
understanding gene function:
– Collect mutations.
– Select those that affect the biological process of
interest.
– Study the mutant phenotype to discern the role of genes
in the process
– Clone the gene and carry out molecular analysis
• 2. “Post-genomics” “reverse genetics” approach:
– Start with the cloned/sequences gene of unknown
function
– Create mutants of the gene
– Study the mutant phenotype to discern the biological
role of the gene
Selecting general mutagenic agents
Genetic screening versus selection
Genetic screen: produce and sort through many
non-mutant individuals to find the rare desired mutation
Genetic selection: only the desired mutation survives
Fig. 16-4
Genetic screens can be carried out for a wide
variety of biological functions (phenotypes):
•
•
•
•
biochemical mutations
morphological mutations
lethal mutations
conditional mutations
(restrictive/permissive conditions)
• behavioral mutations
• secondary screens:
• modifier mutations
• gene expression mutations (using “reporters”)
Forward selection criteria: testing for auxotrophy
Fig. 16-6
Forward selection criteria: testing for phototaxis
Fig. 16-7
Forward selection criteria: cell cycle progression
Aspergillus nidulans
Fig. 16-10
Forward selection criteria: developmental morphology
Danio rerio
Fig. 16-12
Screen strategy: survey haploids for mutant phenotypes
Fig. 16-13
Genetic screen strategies
• Haploid screen
• Diploid screen for dominant mutations (“F1 screen”)
• Diploid screen for recessive mutations (“F2 screen”)
• Diploid screen for recessive mutations – specific locus screen
• “Special tricks” screens
Enhancer trap screen to identify tissue-specific enhancers
Fig. 16-14
Reverse genetics
Knowing the sequence of a gene permits
experiments to determine its function by
directed mutation or phenocopy analysis
• Targeted gene knockout
Knowing a gene sequence, it can become
a target for knockout or replacement
Fig. 16-15
Reverse genetics
Knowing the sequence of a gene permits
experiments to determine its function by
directed mutation or phenocopy analysis
• Targeted gene knockout
• Site-directed mutagenesis
•
Knowing a gene sequence, it can become
a target of in vitro mutagenesis
Fig. 16-16
Knowing a gene sequence, it can become
a target of in vitro mutagenesis
Fig. 16-16
Reverse genetics
Knowing the sequence of a gene permits
experiments to determine its function by
directed mutation or phenocopy analysis
• Targeted gene knockout
• Site-directed mutagenesis
• Produce phenocopies with antisense RNA
Knowing a gene sequence, it can become
a target for RNA-interference experiments
dsRNA induces cellular complexes
that degrade dsRNA
Fig. 16-19
Knowing a gene sequence, it can become
a target for RNA-interference experiments
Can induce RNA-specific degradation by
deliberately introducing dsRNA into cells
Look for phenotypes in RNAi-treated cells/organisms
Fig. 16-18
Fig. 16-21
Understanding the functional basis of dominant mutations
Fig. 16-22
Understanding the functional basis of dominant mutations
Fig. 16-22
Understanding the functional basis of dominant mutations
Fig. 16-22
Understanding the functional basis of dominant mutations
Fig. 16-22
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