Mutant hunts - introduction

Question 1

What is a mutant hunt?

Answer 1

An experimental strategy to identify an organism with a mutant phenotype of interest

Question 2

What are the 4 motivations for doing a mutant hunt?

Answer 2

1. Identify genes required for a specific biological process
2. Obtain multiple alleles for a gene of interest
3. To obtain mutational tools for structure-function analysis
4. To isolate mutations in a gene so far identified only by molecular methods

Question 3

What is the first step in a mutant hunt?

Answer 3

Mutagenize the gametes of the males

Question 4

Why do we want to mutagenize males?

Answer 4

More sperm than eggs, so more targets to potentially have a mutation

Question 5

Why do the chromosomes of the non-mutagenized females have genetic markers?

Answer 5

So we can track which chromosomes in the progeny have been mutagenized and could potentially carry a mutation and which ones haven’t

Question 6

Which F1 progeny would we select from the cross between the mutagenized males and the molecular marker phenotypes?

Answer 6

Select the F1 that have the phenotypes of the molecular markers

Question 7

What do we do once we select the F1 progeny with the molecular marker phenotypes?

Answer 7

Randomly cross them with each other to see if we get the mutant phenotype, since most mutations are recessive

Question 8

What are two complications for a mutant hunt?

Answer 8

Multiple mutations: background mutations can interfere with what we are interested in
Recombination: the mutation might end up on the homolog with the selectable marker

Question 9

How do we stop multiple mutations from complicating a mutant hunt?

Answer 9

Backcrossing

Question 10

What is backcrossing?

Answer 10

Cross the mutant of interest with the parent that has the molecular markers multiple times to replace the genome of the mutant and get rid of background mutations

Question 11

How do we make sure we still keep the mutation we want while backcrossing?

Answer 11

Cross the backcrossed progeny and make sure we still can get homozygosity for the mutant allele

Question 12

What is the goal of backcrossing?

Answer 12

Replace the entire genome so that we only have the one mutation

Question 13

How do we stop recombination from complicating a mutant hunt?

Answer 13

Balancer chromosomes

Question 14

Why would we want to obtain mutational tools for structure-function analysis?

Answer 14

Can do site-specific mutagenesis to change very specific amino acids to determine their functions

Question 15

Why would we want to isolate mutations in genes identified only with molecular methods?

Answer 15

We need to mutate genes identified by homology to determine what they do. Is a reverse genetics approach