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MCB 421 HOMEWORK #4
FALL 2011
Page 1 of 2
Due Sept 27th
You need to only answer questions 1-3.
1. You have obtained five phage T4 plaque morphology mutants that form tiny plaques on E. coli whereas
the wild-type phage forms large plaques. The mutants are designated r1, r2, r3, r4, and r5. In order to try to
determine the type of mutation present in the r gene of each phage, you perform a reversion analysis of the
mutants and measure the frequency that you get wild-type plaques (Don’t worry about how this was done).
The results are shown in the Table below.
Mutagen X is an unknown mutagenic compound.
(+) means that revertants are frequent
(-) means revertants are very rare
(+/-) means that the frequency is intermediate
Mutagen
Mutant
r1
r2
r3
r4
r5
5BU
+
+
+
-
EMS
+/+/+
-
NG
+/+/+
-
ICR191
+
-
X
+
+
-
HA
+
-
a. What is the likely type of mutation (ie. missense, nonsense, frameshift, insertion, or deletion) in each
mutant phage? Why?
b. Can any specific predictions be made about the base changes made by any of the mutagens?
c. Which mutagens would most likely produce revertants that are TS or CS? How could such revertants
occur?
2. Mutations in genes that repair DNA damage often result in a “mutator” phenotype. Such mutator strains
accumulate mutations at a much higher frequency than that observed in wild-type cells.
a. Some investigators do not like to use mutator strains to isolate mutations in genes within the bacterial
chromosome. Instead they use mutator strains to isolate mutations in phage chromosomes or in plasmids.
Suggest a reason for the preference. If you did use a mutator strain, what would be a high priority for the
next step in your project? (You may not know how to do what should be done, but what would you propose
to do?)
b. You grow phage lambda lytically on a wild-type strain and on a mutD strain. You then check the phage
particles from each infection for phage mutants that affect the morphology of the phage plaques on normal
bacteria. Which host strain would give you the highest yield of mutants? Why?
c. Often, investigators grow multiple independent cultures inoculated from independent colonies of mutator
strains for mutagenesis. Why?
End of Questions Due Sept 27th. The rest are to ponder before the exam.
3. You need to isolate a temperature sensitive (TS) tryptophan auxotroph. Such a mutant would be able to
synthesize the amino acid tryptophan at 30 C but unable to synthesize tryptophan at 42C.
a. What mutation type would most likely give rise to this TS mutant?
MCB 421 HOMEWORK #4
FALL 2011
Page 2 of 2
Due Sept 27th
b. Design a protocol (starting with a mutagen) that would allow you to isolate such a mutant. Be sure to use
an ENRICHMENT, to give the COMPOSITION OF THE MEDIA at each step and give the
TEMPERATURE you would use at each step. (Hint: This is very similar to the example discussed in class
except that the temperature of the steps needs to be specified).
4. Strain FC40 is unable to grow on lactose as a sole carbon source (Lac -). A Luria-Delbruck fluctuation
test was done by plating 108 cells from 10 independent cultures (#1-10) onto minimal lactose plates, and at
the same time plating 108 cells from a single culture (#11) onto 10 minimal lactose plates. After a long
incubation, the following results were obtained.
Independent Cultures
(Culture #)
1
2
3
4
5
6
7
8
9
10
Number of
Colonies
22
18
19
24
20
23
21
22
21
17
Culture 11
(Plate #)
1
2
3
4
5
6
7
8
9
10
Number of
Colonies
17
24
23
26
20
22
21
24
20
19
Based upon these results, would you conclude that the mutation to Lac + is random or adaptive? (Briefly
explain your answer.)
5. Strains with null mutations in the leuA, leuB or leuC genes are leucine auxotrophs. You decide to isolate
o
revertants of a leuA mutant at 30 C.
a. How would you isolate the revertants? What medium would you use? Is this a selection or a screen?.
b. You isolate a leuB revertant and find that the reversion is a mutation at a second site in the leuB gene. In
addition, the revertant is temperature sensitive for leucine synthesis. How would you demonstrate that the
revertant is temperature sensitive for leucine synthesis?
c. What is the most likely explanation for the temperature sensitive phenotype?
d. It is possible to isolate leucine auxotrophs that do not have mutations in the leuA, B, or C genes. Some
have mutations in the leuS gene, which codes for the leucyl-tRNA synthetase protein. The enzyme is not
required for leucine biosynthesis but is responsible for amino acylation of leu-tRNA for protein synthesis.
What type of mutation would the leuS mutant likely contain? Would you expect the mutation to be a null
mutation?
e. You isolate a revertant of the leuS mutant and find that the mutation maps at a site adjacent to the leuS
gene. What is the likely mechanism for the revertant phenotype?