Genetic Analyses of Mutants- Exam II

Micro 4130 Exam 2

Forward/classical genetics

Starting with the phenotype and looking for the causative gene

Reverse/modern genetics

Starting with the gene and looking for the phenotype

What you can learn from studying mutants

Gene regulation, identifying members of pathways, function of RNAs and proteins, important domains in RNAs and proteins

Neutral mutation

Has no effect on the function of the protein

Loss of function

Can be either total loss of function or partial, but the gene/protein has lessened activity. Ex total loss could be that the substrate doesn’t bind anymore, while partial loss could be that the substrate just binds less efficiently.

Gain of function

Increased activity or new activity. Ex: enzymes can now use another substrate or activators become constitutively active. Gain of new function can mean loss of the original function (evolution of paralogs)

Complementation test

Allows you to determine whether the mutant allele is recessive or dominant with respect to the WT strain. Start with a mutant cell, and add the wt allele back in to create a merodiploid strain. Behavior of the merodiploid cell tells you whether the mutant allele is dominant or recessive

If merodiploid cell acts like WT

This means that the mutant allele iss recessive

If the merodiploid cell still acts like the mutant

This means the mutant allele is dominant

Working in cis

The elements you are considering work together within the gene. For example promoters, regulator binding sites, and SD

Complementation tests with factors that work in cis

You need to include the area changed by the mutation along with all the downstream elements that might be affected by it when creating the merodiploid strain. For example, if your mutation is in the promoter region you need to include both the WT promoter and the WT gene when making the merodiploid because just the promoter will tell you nothing.

Dominance/Recessiveness of Gain of function mutations

Gain of function mutations are typically dominant

Dominance/Recessiveness of Loss of Function Mutations

Generally loss of function mutations are recessive, with the exception of Dominant-negative alleles

Dominant-negative allele

A loss of function allele (typically total) that is dominant over the wild type allele. The mutant factor interferes with the function of the wild type factor, telling you that they are physically interacting in the cell. Ex: in a homomeric complex where the mutant nonfunctional subunit kills the function of the complex- the mutant interacting with the WT shuts down WT function.