Topic 3 - Mutant Hunts

Strategy for Mutant Hunt/Collecting Mutants

1. Defining your biological question/process of interest (ex: ethanol tolerance/sensitivity)

2. Identify an appropriate organism in which to address your question (ex; using fruitflies due to skill set/available tools)

3. Devise your mutagenesis scheme (based on the type of alterations to be generated)

Mutant Hunt - Organism

Drosophila - 2n = 8

-Autosomal chromosomes are 2,3,4

-1st chromosomal pair is the sex pair (X/Y)

Semi colons between homologues mean they independently assort

X/Y ; 2/2 ; 3/3 ; 4/4

Mutant Hunt - Inducing Mutations

Using chemical mutagen (EMS)

-We expect point mutations (likely SBS)

-Increases our chance of generating a wider array of Muller’s morphs

-Mutagenizing males since they produce far more meiotic products than females, therefore more targets for our mutagenic event

Mutant Hunt - Meiocytes

Mutagenesis is random

-Each meiocyte is independently influenced

-Creating chances in meiocyte DNA will transmit changes to next generation

-Hope that atleast 1 sperm has mutation in the GOI

Mutant Hunt - Parental Generation

Males are mated with wildtype females (only wildtype gametes)

Mutant Hunt - F1 Generation

Each F1 fly is unique (different genotype due to different sperm-egg fertilization events)

-F1 flies are heterozygous with mutant alleles, but won’t display mutant phenotypes unless mutation is dominant (rare)

-Flies are treated as individuals since they’re different from each other

Mutant Hunt - F2 Generation

Male flies placed into separate vials, then mated with a single wildtype female (F2 generation)

-Each vial contains multiple flies of both sexes with the same mutation

-Still heterozygotes → wildtype phenotypically

Mutant Hunt - F3 Generation

Flies in each vial randomly mate to produce an F3 generation

-Screen F3 progeny for phenotype of interest

Issues in a Mutant Hunt

1. Flies selected at various times in the mutagenesis scheme may not have a chromosome that was present in the originally mutagenized parent

2. Recombination can “move” a mutation between homologues, during each meiosis in a mutagenesis scheme

3. Mutants may possess multiple mutations, thus complicating the phenotype-genotype correlation

Dealing with Unwanted Mutations

Backcross - Mutants are repeatedly crossed to wildtype individuals to replace regions of the genome containing unwanted mutations

-used to remove unwanted mutations in an effort to get to a state where we have one mutation only

Balancer Chromosome

Used to suppress recombination and aid selecting individuals with a mutation

-Carries a dominant allele, allowing for easy recognition of the presence of a balancer

Often carry a recessive lethal allele (not for Sex Chromsome) → allele present in two copies = death

-Would never be able to have males with balancer chromosome on the X chromosome due to recessive allele

-Prevents homozygosity for the balancer

Able to distinguish between individuals with one or two balancers

Balancer Chromosomes - Inversions

Balancer chromosomes carry inversions to prevent recombination

-Structural inhibition due to inverted sequences on the balancer

-Inviable recombinant products if recombination does occur

-Many of the original balancers generated via X-ray treatment

Balancer chromosomes thereby eliminate recombination

Balancer Names

FM - First multiple, balancer for chromosome one (X) → dominant allele for bar eyes

SM - Second multiple, balancer for chromosome two → dominant allele for curly wings

TM - Third multiple, balancer for chromosome three → dominant allele for leg antenna

MK - balancer for chromosome three → dominant allele for stubble bristles