Analysis of mutants with more than mutation - Exam II

Micro 4130 - Exam 2

Effects of multiple mutations

Can be additive when they affect independent pathways. Can also be unexpected based on the single phenotypes alone when the mutations are functionally related.

Synthetic genetic interactions

Can be either negative or positive. Occurs when two mutations are functionally related, and the sum of mutations are not just the two different phenotypes together.

Negative Synthetic gene interactions

Negative synergy: two single mutants have a mild phenotype each together, but when both present together result in a very strong phenotype.

Synthetic Lethality

A type of negative synergy where the phenotype of the double mutant is death

Positive synthetic gene interactions

Suppression: where one phenotype/mutation is suppressed by another. The phenotype of a mutant gets suppressed (closer to WT) in the presence of a second mutation. Whatever the second mutation effects is related to what the first mutation effects.

What synthetic genetic interactions can reveal

Redundancy (where loss of one doesn’t matter, but loss of both could be catastrophic). Functional/physical interaction between 2 factors/domains

How to use negative synergy to find redundant genes

Start with one mutation (might reduce function, might not). Mutagenizing until you see loss of the pathway then determining what changed tells you the redundant factor.

How negative synergy can demonstrate functional/physical interactions between domains

Altering 2 different factors then analyzing the amount of a particular function that is happening can tell you that they work together.

What synthetic lethality reveals

Factors altered by mutations are redundant for some essential function OR factors altered by mutations are non-essential members of a complex that performs an essential function in the cell; complex can tolerate loss of one but not two essential factors- individually non-essential.

Extragenic suppressor

In a different location than the first mutation. Ex: substrate changing and now matches a mutated substrate binding site

Intragenic second-site suppressor

In the same location as the first mutation. Ex: substrate binding site mutates 2 separate times and can then fit the original substrate. Another Ex: 1 bp deletion followed by a 1 bp insertion close by

Conformational (interactive) suppression

Restores a physical interaction- usually very specific to a particular mutation/change. Typically allelic specific: each conformational suppressor is typically specific for the particular mutation they suppress, and often don’t work with other changes that affect the same site. Ex: changing substrate binding site to fit changed substrate.

Bypass suppression

Overrides the need for a particular gene. Suppresses the phenotypes caused by many different mutations in one same gene or in the same pathway. Specific for the particular pathway but not for the particular mutation. Ex: Mutating an existing transporter to compensate for another missing transporter.

Informational suppression

Changes how mRNAs are read (mutations in tRNA and genes associated with the ribosome) so that stop codon can now encode for an amino acid. Usually a mutation in a gene coding for a tRNA- the mutation changes the anticodon of the tRNA. The mutation in the gene encoding a tRNA can now recognize aa stop codon (suppress a nonsense mutation). This changes the tRNA’s anticodon, so suppressor strains must have another tRNA that recognizes the wt codon.

E. coli nonsense suppressors

Specific for 1 or 2 stop codons, but will do this in every message. They don’t work 100%, can make the strain sick because the proteins are larger than they should be.

How to find suppressors

Targeted must have a good selection/screen. Random must have a good selection or a high-throughput screen.