eg. Biosynthetic pathway in Neurospora:

• wild-type = prototrophic (makes all 20 amino acids)

• mutant = auxotrophic (lacks one amino acid because enzyme in pathway missing)

• supplement medium with missing amino acid --> rescues growth

• supplement medium with different precursors --> reveals enzymatic step affected by mutation

• mutations disrupting different enzymatic steps "complement" each other's function in heterozygous diploid --> rescues growth

• mutations disrupting same gene (therefore same enzyme/same step in pathway) "fail to complement" --> no growth without supplement = allelic

• select for rare wild-type revertant of auxotrophic mutant allele

• revertants RARE except in case of transposon insertion

(1) intragenic reversion = changes back to original nt.

(2) extragenic reversion = suppressor gene

Genetic screen in diploid requires extra steps to see homozygous recessive phenotype

• only dominant or recessive X-linked mutations show phenotype in F1 generation

• special techniques in zebrafish to produce haploid F1

• "balancer" chromosomes in Drosophila to establish pure-breeding lines --> analyze F2 generation for mutant phenotype

• somatic mutant clones to analyze homozygous mutant phenotype in sector of body

"Reverse Genetics" = start with gene sequence --> make mutant to determine function

(1) Targeted gene knock-out

• homologous recombination used to insert defective clone of gene into endogenous gene

• transgenic technology for introducing cloned DNA into organism's germline --> transmitted to progeny

(2) RNA interference

• double-stranded RNA triggers "silencing" of homologous mRNA in cell

• "phenocopy" of loss of function mutation because endogenous transcript degraded