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

Mutant Hunt for Chromosome-Specific Mutation

Setup mutant hunt to identify genes on chromosome 3

Mutation of Flies and Balancer Stock

Balancer Stock - Flies contain balancer chromosome + non-balancer homolog with a dominant marker

-After mutating male flies, cross with balancer stock of female flies

F1 Generation

Select male F1 flies with Balancer chromosome phenotype

-Genotype of F1 males of interest would be x/y ; +/y ; */TM6b, (balancer)

Crossing F1 Flies

Cross F1 flies with females of balancer stock

F2 Generation

Select F2 males/females with balancer chromosome phenotype, but not the non-balancer homolog phenotype

-Female flies provide balancer chromosome, male flies provide mutated chromosome

Crossing of F2 Generation

Cross F2 male/female flies together

F3 Generation

Contain males/females that are homozygous with mutation and flies with balancer chromosome phenotype

-F3 flies that DO NOT have balancer chromosome phenotype are selected (flies of interest)

-Flies WITH balancer chromosome become the “Balanced Stock”

-No flies in F3 generation with no balancer chromosome phenotype indicates mutation is recessively lethal

Obtaining Additional Mutant Alleles

After recovering an initial mutant allele, we want to recover additional mutant alleles

-Requires having a second balancer chromosome that is distinguishable from the first one used

-Goal is to isolate additional viable mutant alleles of our original mutation event

F1 Generation

-After mutagenizing males, cross with balancer stock females w/ non-balancer homolog with dominant marker

-Balancer chromosome should be different that the original one used for the first mutant allele

-Select F1 males w/ balancer-chromosome phenotype

F2 Generation

Cross F1 males with mutant balancer stock females

Selection and Screening

Screening - Need to test individually for those that have mutation of interest

-Vast majority of mutant hunt uses screening, takes lots of time/resources

Selection - Only those that have mutation of interest survive

-Ex; 5-FOA kills ura+ prototrophs

Saturation

When we are “confident” we have enough mutants (one representative mutant allele for every gene involved in our biological process of interest)

-Considered to be at saturation when m >= 5

-f(0) = e^-m

F(0) = Fraction of genes NOT in the collection

m = mean number of alleles recovered for genes already identified (alles/genes)

Phenomena Impacting Interpretation of Mutant Hunts

1. Incomplete penetrance

2. Variable expressivity

3. Pleiotropy (alleles participate in multiple phenotypes)

4. Lethal Mutation

5. Lack of Phenotype

Lack of Phenotype

1. Eukaryotes have multi-gene families; mutation in 1 gene is compensated by other genes of the family

2. Genetic networks compensate for knock-out of one pathway via rerouting to other pathways

3. Mutant phenotypes may only emerge during stress/non-standard lab conditions

4. Mutants may have a small/not noticeable phenotype (requires careful analysis)

5. Some genes have functions only required every few generations