Forward Genetics
What is forward genetics?
• Genetic screens designed to find genes that affect a trait of interest.
PHENOTYPE
GENE
• Random mutagenesis
• Screen for interesting phenotypes
• Track down the gene(s) responsible
• Traits can include morphology, physiology, behavior
Reverse genetics?
• Genetic screens designed to identify phenotypes association with disruption of a particular gene or DNA
sequence of interest.
GENE
PHENOTYPE
Forward Genetics
Retinotectal projections in zebrafish
Wild type
Wild type
belladonna
never mind
astray
Baier et al., Kalstrom et al., and Trowe et al., 1996, Development
Saturation mutagenesis
• Large scale genetic screens that aim to create a mutation in every
gene required for a trait.
• How do you know when you get there? When new mutations
represent a second or multiple hit in a previously identified gene.
• The saturation point defines the
set of genes required
INDIVIDUALLY for a particular
trait.
• Genes whose function are
redundantly provided by
another loci will be missed.
Mutagens
Radiation
+
+/-
-
Strong mutations –
deletions, inversions,
translocations
May disrupt multiple
genes
Can be difficult to clone
Chemicals
Insertions
+
Full spectrum of
mutations, usually small
+
+
-
Randomly distributed
+
Difficult to clone
Full spectrum of
mutations
Mutation is tagged, and
easy to clone
+ Often reversible
- Nonrandom distribution
Genetic Screens
Screen for dominant
mutations (F1 screen)
Screen for recessive
mutations (F3 screen)
* ¼ of families will
produce m/m offspring!
Genetic Screens
Classification of Mutants: Complementation
?
If you have two mutants that have the same phenotype, how
to test if they are mutations in the same or different genes?
?
• Complement: provide the function of
• Complementation group: a set of alleles that fail to complement
one another
• Complementation testing
reduces a large number of
mutations to a smaller set of
loci.
Hartl and Jones, 1998
•Non-allelic noncomplementation can occur
if mutations affect genes in
the same pathway
Classification of Mutants: Complementation
X
X
WT phenotype
Complementation
Mutations are in different genes
Mutant phenotype
Non-Complementation
Mutations are in the same gene
Classification of Mutants: The ‘Morphs’
Loss of Function alleles
• Amorph
• null allele, no WT activity.
• genetic definition m/m = m/Df
• Hypomorph
• partial l-o-f, reduced WT activity.
• Usually recessive, however can be dominant if it is
haploinsufficient (eg mutations on the X in males)
• genetic definition m/m < m/Df
Classification of Mutants: The ‘Morphs’
Gain of function alleles
• Hypermorph
• extra WT function (usually dominant)
• Examples include constitutive promoters, constitutively active
proteins
• genetic definition: m/m> m/+ > m/Df
• Neomorph
• novel function (usually dominant)
• Examples include chimeric proteins due to translocations
• genetic definition: additional alleles (+ or Df) don’t affect the
phenotype. So m/+ = m/Df
Classification of Mutants: The ‘Morphs’
Gain of function alleles
• Antimorph
• dominant negative
• protein product antagonizes (poisons) the WT protein.
• Occurs with proteins that function as multimers.
B-galactosidase functions as a tetramer
Wild Type
Mutant
Molecular Characterization of Mutants
• Chromosomal Mapping (recombination mapping)
• Molecular identification
•
•
•
•
Positional cloning
Rescue with candidate genes
Identification of altered transcriptional profiles
Cloning based for insertions