MCB 421 Third Exam

December 8, 2004

1) (25 pts) When wild type E. coli strains that are lysogens for wild type
lambda are grown for many generations in liquid medium, the medium
5
contains about 10 lambda phage, all of which form clear plaques on a
wild type host strain that is a non-lysogen. Explain;

a) Why are all of the plaques made by these phage clear? If this is due
to mutation, in which gene (or genes) does the mutation lie and why?

The phage are λcI (loss of function) mutants that result from
spontaneous mutagenesis. The loss of cI function results in induction
and give the phage in the culture medium (= true result)

Although cII and cIII mutants also give clear plaques, the rare
lysogens these mutants form have normal stability so inactivating these
genes in a lysogen would have no effect.

b) Suppose that the phage all gave turbid plaques rather than clear
plaques. Give an hypothesis to explain the turbid plaques. What
phage lambda mutant could prove (or disprove) this hypothesis?

Turbid plaques would say that cI function had been inactivated

temporarily, but that the cI gene is functional. Hence these are most
probably host mutants that result in clipping of cI repressor (such as a
mutant RecA that does not need ss DNA to become RecA*). If so a λ
Ind- mutant (makes a cI that cannot be clipped) would block phage
release into the medium.
 MCB 421 Third Exam
December 8, 2004

1c) The receptor on the outer membrane that lambda phage binds to during
adsorption is part of the system that transports the disaccharide, maltose, into
E. coli. You find 50-fold fewer phage in the medium when you grow the
lysogen in the presence of maltose than when you grow without an added
sugar. If you grow the lysogen with glucose you obtain 100-fold more
phage than when grown without an added sugar. So phage yields are
glucose>>>no sugar>>>maltose. Maltose is composed of two glucose
molecules. Given a molecular explanation of these data

Maltose addition results in more receptors since maltose induces

synthesis of the receptor. The released phage bind to these receptors
and inject their DNA into a lysogen resulting in loss of the plaque
forming ability of that phage. Glucose addition obviously represses
synthesis of the receptor. Since maltose is two glucose units, it is of no
advantage for E. coli to transport and metabolize maltose so the crp-
CAP regulatory pathway will shut down receptor synthesis and the
phage will not be lost by adsorption to lysogenic cells. The medium with
no sugar gives a basal level of receptors (neither induced or repressed)
so intermediate phage loss results (= true result).
 MCB 421 Third Exam
December 8, 2004

2a) (15 pts) Describe how you would tell if two yeast mutants that have the
same phenotype are mutant in the same or in different genes.

The two strains would have to have opposite mating types a or α. Mix
the two strains (or cross streak) on a plate on which the mutants cannot
grow and incubate. If growth occurs when the strains are mixed (and
thus mate to form diploids) then the mutations are in different genes. If
no such growth is seen they are in the same gene. In the second case you
would need to prove that diploids had formed. This could be done by
looking a morphological or biochemical markers specific to diploid cells,
but is most easily done by having other markers in the strain, say his3 in
one and ura3 in the other and showing growth on medium lacking
histidine and uracil.

b) Describe how you would detect a yeast strain that carries a dominant
mutation.

First the strain would not complement any other mutant having the
same phenotype when mated as above. Hence, it would be classified as
having a mutation in each of the genes involved (a most unlikely
possibility). Final proof would be to mate it with a wild type strain and
see no growth of the proven diploid.

c) Describe how you would tell if the phenotype of a yeast mutant strain is
due to a single mutation or two unlinked mutations.

Mate the mutant strain with the wild type parental strain of opposite
mating type to form a diploid. Purify the diploid and put it on the poor
sporulation medium. Pick out ascospores containing four spores and
dissect about 20 into individual spores. Test for growth of the spores on
a plate on which the mutants cannot grow and incubate. If two spores
from each ascus grow and two spores do not grow, then it is a single
mutation. Any other result means that there is another mutation. The
only exception would be one or two gene conversions.
 MCB 421 Third Exam
December 8, 2004

3) (20 pts) You have isolated an unusual trp- (tryptophan-requiring) strain of

E. coli. Introduction of a wild type trpEDCBA operon on an F’ into this
strain fails to allow growth in the absence of tryptophan. However,
introduction of a wild type trpR gene on an F’ allows very slow growth in
the absence of tryptophan (growth is normal in the presence of tryptophan).
If the F’ carries one of several trpR missense mutants rather than the wild
type trpR gene, normal growth in the absence of tryptophan is seen. In
contrast if F’ carrying various trpR nonsense mutants are used there is no
growth in the absence of tryptophan. Give a molecular explanation to
explain the unusual trp- (tryptophan-requiring) strain and all of the other data
given.

This is a dominant-negative mutation in trpR, the repressor of

trpEDCBA. (the trpR gene is located far from trpEDCB on the
chromosome). This mutation could encode a TrpR that does not
require tryptophan to bind the trpEDCBA operator. TrpR is a dimeric
protein so mixing with wild type subunits can partially “detoxify” the
dominant TrpR, but better is a full length, but inactive TrpR that has
no DNA binding ability (the missense mutants). Nonsense mutant
proteins will not work because even if they are not degraded since they
are too short to form the stable dimers needed to neutralize the
dominant negative TrpR.
 MCB 421 Third Exam
December 8, 2004

4) (20 pts) You have isolated a temperature-sensitive mutant of E. coli that

grows well at 30 oC, but cannot grow at 42 oC . When shifted to 42 oC the
strain stops growth and the cellular chromosomal DNA is fragmented. If
you grow phage lambda on this strain at 37oC growth you get some phage,
but these phage cannot form plaques on a wild type host strain, but do form
plaques on either hsdR or hsdS strains. In what gene does the temperature-
sensitive mutation map and why is the chromosomal DNA fragmented at 42
o
C?

This must be a temperature-sensitive mutation in hsdM, the

modification (methylation) enzyme of the host restriction-modification
system. At high temperature the E. coli DNA is not modified and gets
restricted (chopped into fragments) and any phage grown under these
conditions are not modified and so are restricted when plated on a wild
type strain. However, if the phage are plated on an hsdR strain then
plaques will form since there is no restriction activity. On an hsdS
strain plaques will form since the restriction activity requires both
HsdR and HsdS (the specificity subunit).
 MCB 421 Third Exam
December 8, 2004

5) (20 pts) You grow a phage lambda lysogen and transform the strain with a
suicide plasmid that carries Tn5 and select for kanamycin-resistant colonies.
You then induce the lysogen and most of the cells lyse. However a few cells
do not lyse and when you recover these cells and lyse them artificially you
get mutant phage that transduce a wild type E. coli strain to kanamycin-
resistance. However, when you plate the phage on a non-lysogenic wild
type strain you get no plaques. Look at the lambda map on the next page
and give some genes in which the mutation carried by the mutant phage
might be found. Also, explain how phages can be made despite the mutation
and the characteristics of Tn5 insertions into bacterial chromosomal operons.

The defect in these mutants is in the lysis process so this would be a Tn5
insertion into the S or R genes (see map - actually there are two R
genes). The reason that phage are formed is that (unlike what happens
in bacterial genes) there is no polarity on downstream genes. This is
because of the anti-termination activity of Q that reads through the Tn5
transcription terminators. The mutants cannot form plaques because
the infected cells cannot lyse and release phage to infect and lyse the
neighboring cells.