Genetics Assignment: Analyzing IOB Mutation Reversion Rates

[shanwill]

Homework Statement

You decide to revert an iob- mutant to wild type (IOB+) using different mutagens and find the following reversion rates for each mutagen tested:
2-amino purine - 98%
5-bromouracil - 95%
Nitrous acid - 94%
Proflavin - 2%
Spontaneous - 4%

1. What is the most likely molecular basis of the original iob- mutation?
2. What does the reversion test data suggest about the most prominent mechanism of spontaneous mutations in this organism?
3. You decide to isolate deletion mutations in the IOB gene. Suggest a genetic screen that would enrich for deletion mutations in this gene over other types of loss-of-function mutations.
4. Suggest two genetic tests that would show you have indeed identified iob deletion mutants.

Homework Equations

The Attempt at a Solution

1. I assumed that since bromouracil, nitrous acid, and aminopurine all cause transition mutations, the original mutation is likely a transition - but i wasn't sure if that's what they meant by molecular basis?
2. Since I'm assuming the original mutation was a transition, and the rate of spontaneous revertants is very low, I was thinking I'd suggest that the most prominent mechanism of mutations in this organism is not transition mutations - but again i wasn't sure if i was answering what the question was asking
3. I'm at a loss here - any ideas?! *** I was thinking possibly using a mutagen that is known to cause deletions (but I couldn't seem to find any) or one that causes both deletions and insertions and then screening for insertions by the methods described in 4, but I couldn't seem to find a mutagen like that either. HEEEELP!
4. If I could answer number 3, then i was thinking i could use PCR analysis or comparative genomic hybridization, does that make sense?

Your opinions would be much appreciated!

 

[nate808]

1. The most likely molecular basis of the original iob- mutation is a transition mutation, specifically a point mutation in the IOB gene.
2. The reversion test data suggests that the most prominent mechanism of spontaneous mutations in this organism is not transition mutations, as the rate of spontaneous reversion is very low compared to the other mutagens tested.
3. One possible genetic screen to enrich for deletion mutations in the IOB gene could be to use a mutagen that specifically targets and induces deletions, such as CRISPR-Cas9. This would increase the likelihood of identifying deletion mutations in the IOB gene over other types of mutations.
4. Two genetic tests that could confirm the identification of iob deletion mutants could be PCR analysis and sequencing of the IOB gene in the mutants, as well as a complementation test where the iob deletion mutants are crossed with a known IOB- mutant strain to see if the phenotype is restored.