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

Baier et al., Kalstrom et al., and Trowe et al., 1996, Development

Retinotectal projections in zebrafish

Wild type

belladonna astrayWild type never mind

Saturation mutagenesis

• Large scale genetic screens that aim to create a mutation in everygene 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 Chemicals Insertions

+ Strong mutations – deletions, inversions, translocations

+/- May disrupt multiple genes

- Can be difficult to clone

+ 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

• Complement: provide the function of• Complementation group: a set of alleles that fail to complement one another

If you have two mutants that have the same phenotype, how to test if they are mutations in the same or different genes?? ?

• Complementation testing reduces a large number of mutations to a smaller set of loci.

•Non-allelic non-complementation can occur if mutations affect genes in the same pathway

Hartl and Jones, 1998

X

WT phenotype

ComplementationMutations are in different genes

X

Mutant phenotype

Non-ComplementationMutations are in the same gene

Classification of Mutants: Complementation

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

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.

Classification of Mutants: The ‘Morphs’

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