iGenetics 3rd Edition by Peter Russell

Exercise 8

Key experiments indicating that the genetic code was a triplet code came from the work of Crick and his colleagues with proflavin-induced rII mutants in T4 phage. Answer the following questions to explore the reasoning behind Crick's experiments.
a. What types of DNA changes does proflavin induce? What are the effects of these mutations if they occur within a gene?
b. Suppose you expose T4 phage to proflavin, and infect the phage into E. coli. What type of E. coli would you infect the phage into to select for rII mutants? How would you know if you had recovered an rII mutant?
c. Suppose you isolate two proflavin-induced rII mutations at exactly the same site in the rII gene. Mutation rII X is caused by the insertion of one base pair (a mutation), while mutation rII Y is caused by the deletion of one base pair (a mutation). How would you select for revertants of these mutations?
d. Suppose you isolate five revertants of rII X. Using a diagram, explain whether all of them are likely to affect the same DNA base pair.
e. A colleague in your lab analyzes your revertants, and tells you that none of them result from the deletion of the base pair that was inserted in the rII X mutation. Does this mean that all of the revertants are double mutants? If so, explain how a double mutant can have a phenotype.
f. Your colleague uses recombination to separate the nucleotide changes induced in your revertants from the chromosome with the original rII X mutation, and gives you five phage, each of which has only the DNA change introduced by the reversion event. Will these phage show an rII phenotype, that is, are these phage rII mutants? If they are, what type of mutations are present in them, how would you select for revertants, and what type of additional mutation in a revertant would lead to an phenotype?
g. Your colleague uses recombination to combine the rII Y mutation with each of the five mutations that led to reversion of the rII X mutation. Explain whether the five double mutants she gives you will have an phenotype. If not, and you treat the double mutants with proflavin and select for revertants, what type of mutation would lead to an phenotype? Use diagrams in your answers.
h. Use diagrams to explain which of your answers in part (g) require the genetic code to be a triplet code. For example, could you recover proflavin-induced revertants in part (g) if the genetic code were not a triplet code?