## True Reversion

The pathway of proline biosynthesis is shown below:

A strain with a missense mutation in the proA gene is unable to grow on minimal medium without proline. You can isolate Pro+ revertants by selecting for prototrophs. What medium would you use to select for Pro+ prototrophs?

ANSWER: Minimal medium with glucose as a carbon source without proline.

A Luria-Delbruck fluctuation test was done to determine the reversion rate of a histidine auxotroph. Twenty tubes of rich medium were each inoculated with a few auxotrophic cells and the cultures grown to 108 cells/ml. A 0.1 ml sample of each culture was plated on minimal medium to detect His+ revertants. The results are shown below.

Culture# Revertants
10
21
35
410
5100
61
70
80
93
1030
111
120
134
141
155
16300
1710
180
191
205

Calculate the reversion rate of the his mutants in reversion events/cell division.

ANSWER: 5 out of 20 tubes contained no revertants ("mutants" for our purposes). So, you can use the Poisson equation to measure the reversion rate in the same way you use it to calculate the mutation rate. Thus:

h= -ln [5/20] = 1.386

a = h / N where N= 108 cells / ml x 0.1 ml = 107 cells therefore approximately 107 cell divisions

Reversion rate = 1.386 / 107 = 1.386 x 10-7 reversions / cell division

Why are mutant reversion studies more sensitive than forward mutation studies for the detection of mutagenic properties of chemicals? What are the limitations of determining mutagenic specificities by reversion studies?

Reversion studies are often quite sensitive because they provide a simple, genetic selection for mutants in a specific target gene (for example, in the Ames test the selection is for His+). The problem with reversion studies is that any particular type of mutation will only be reverted by certain classes of mutations. Therefore, it is necessary to test for reversion of several different types of mutant alleles.

Reversion analysis of five his mutants was done by treating 108 mutant cells with a mutagen then plating the cells on minimal medium without histidine. The results are shown below. Based on the reversion analysis, what is the most likely type of the mutation in each of the his mutants?

MUTANTMUTAGEN*
NONEEMSICR
1++++-
2++++-
3++++-
4--+
5---
*Legend: - = no colonies; + = a few colonies; +++ = 100-500 colonies

ANSWER: Mutants #1-3 are probably base substitution mutations because they are stimulated to revert by EMS which causes base substitutions. Mutant #4 is probably a frameshift mutation because it is stimulated to revert by ICR, an intercalating agent which causes frameshift mutations. Mutant #5 may be a deletion or insertion mutation because it reverts very rarely and is not stimulated to refert by either EMS or ICR.

When grown on MacConkey-Lactose plates, Lac+ cells from red colonies and Lac- cells form white colonies. How could you screen for strains with a mutator phenotype using MacConkey-Lactose plates? [Hint: you would want to start with a Lac- mutant. Why?]

ANSWER: "Mutator phenotype" indicates that the desired mutant strains exhibit an enhanced spontaneous rate of mutagenesis due to lesions in one or more of the DNA repair genes (ones that you should know about are: mutD which results in loss of proof-reading and mutSLH which results in loss of mismatch repair). [The key to this question is thinking about the phenotype of the Lac- colonies -- Lac- colonies will appear white on the MacConkey lactose medium thus allowing rare red sections within a colony (papillae or sectors) to be easily identified. So what are these red sections and how do they get there? The lac- mutation can revert to Lac at a low frequency because the mutant allele (point mutation) can be repaired by a mutation in a specific base pair to restore the wild-type gene amino acid sequence. Any Lac+ revertants will appear produce offspring that make this part of the colony red on MacConkey medium. However, normally such reversion events are so rare that most colonies would not show red papilli after overnight growth. However, if a strain exhibits a high level of spontaneous mutation (e.g. a mutator phenotype), it will accumulate multiple mutations during the growth of a colony. If a reversion occurs early in the life of the colony, the daughter cells within the colony will be numerous and form a sector, if the reversion occurs later, fewer daughter cells will arise before the colony stops growing and resulting in small red spots around the edge of the colony called papilli. This idea is analogous to the "jackpot" concept that underlies the Luria-Delbruck fluctuation test.]

The reversion frequency of four different Salmonella typhimurium his mutants was assayed as shown below.

MutantNumber of Revertants /108 Cells
ICR-191EMSSpontaneousQ
his-10000
his-21926182022
his-31785918367
his-48996

1. What is the most likely type of mutation present in each of the mutants?

• his-1 is probably a deletion because it doesn't revert.
• his-2 is probably a frameshift mutation because reversion is increased by the intercalating agent ICR-191.
• his-3 is probably a base substitution mutation because reversion is increased by the alkylating agent EMS
• his-4 is probably an insertion mutation because reverts at a low frequency, but the frequency of reversion is not stimulated by mutagens.

2. Is compound Q a mutagen? If so, what can you say about its specificity?

ANSWER: Q seems to be a mutagen because it causes a dramatic increase in the reversion frequency of his-3. (Although not as powerful of a mutagen as EMS.) Because it increases the reversion frequency of his-3 which is likely to be a base substitution mutation (as described above) but does not increase the reversion frequency of his-2 which is likely to be a frameshift mutation, Q probably causes base substitution mutations.