BL414 Genetics
Problem Set 4 Answer sheet
I. This question assumes that the revertants are caused by an exact reversal of the original
mutation. It is also possible that a mutation at another site within the same gene or in
another gene could suppress the original mutation – in such a case it would be
impossible to tell what the original mutation was without doing molecular analysis at
the DNA sequencing level. However if we assume that the revertants are actually
reversing the original mutation we would get the following answers: Mutation 1 is
reverted by the mutagen that causes transitions, so mutation 1 must have been a
transition. Consistent with this conclusion is the fact that the UV light can also revert
the mutation and the intercalating agent proflavin does not cause reversion. Mutation 2
is reverted by proflavin and therefore must be either an insertion or a deletion of a base.
The other two mutagens do not revert mutation 2. Mutation 3 is not reverted by any of
these mutagenic agents. It is therefore not a single-base substitution, a single-base
insertion, or a single-base deletion. Mutation 3 could be a deletion of several bases or
an inversion. Mutation 4 is reverted only by UV light so it is a single-base change, but
not a transition, since EMS did not revert the mutation. Mutation 4 must be a
transversion.
b. The colonies on the plates could arise by spontaneous reversion of the
mutation. Spontaneous reversion should occur with lower frequency than mutageninduced reversion. The important control here is to spread each mutant culture without
any mutagen treatment onto selective media to assess the level of spontaneous
reversion. A dose response curve would be a good way to quantitatively analyze the
mutagenic properties of each mutagen and also normalize the contribution from
spontaneous mutations.
II.
Again we have to make some assumptions to answer this question – the question
wants us to assume that the normal tissue is from a patient with one defective p53 gene,
and in the tumor tissue, both copies of the p53 gene are defective. By looking at the
DNA blot, we try to deduce what the mutations in p53 were. It is also possible that a
patient could have two normal copies of p53 and get mutations in both copies to cause
cancerous tissue, but that is not the case in this problem. The wildtype p53 region, as
seen in the “wild-type” individual, has three hybridizing bands. Because p53 is a tumor
suppressor, it is recessive at the cellular level and both copies must be defective in the
tumor cells. No observable changes are apparent in patient 1, so this patient must have
inherited a small mutation in p53 and in the tumor cells the second copy would also
contain a small mutation thereby inactivating both copies of p53 in the tumor. In patient
2, a small mutation must have been inherited. In the tumor, the whole region containing
p53 was deleted (thereby removing the second copy of the gene) as seen by the loss of
restriction fragments. (If this DNA blot consisted of cDNA which is amplifying the
mRNA, the tumor lane could indicate a defect in the promoter region of p53). In patient
3, a deletion mutation (smaller than that in the tumor cells of individual 2), as seen in
the altered restriction pattern, was inherited. In the tumor, the second copy of the gene
was similarly deleted (probably by homologous recombination causing gene conversion
since the pattern is the same for both copies).