11.

In an effort to determine the location of an operator site for a negatively regulated gene, you
have made a series of deletions within the regulatory region. The extent of each deletion is
shown by the line underneath the sequence, and the resulting expression from the operon
(i = inducible; c = constitutive; − = no expression) is also indicated.

a.What can you conclude from these data about the location of the operator site?
In the first option, a sequence deletion results in an induced operon, which indicates that
structural genes are expressed when a catabolite is present. In the absence of a catabolite
repressor, structural gene transcription is inhibited. Therefore, the operator sequence is still
present in the sequence in the first option, even though a sequence deletion does not result in its
deletion.
Because the operator sequence is absent, there is no place for a repressor to bind and control
expression. As a result, the polymerase binds to the promoter and begins transcription of the
structural genes. From the Third and Fifth options, the deletion results in constitutive expression,
which means the structural genes are continuously expressing their products. From this, we
concluded that the deletion leads to deletion of the operator sequence. Consequently, these
regions include the operator sequence.
b. Why do you think deletions 2 and 4 show no expression of the gene?

The deletion in the second and fourth alternatives causes no expression, which indicates that no
structural gene product is present. Here, we can assume two things: 1) that in the absence of
catabolite, the repressor will not have bound to the operator, and 2) that even in the presence of
catabolite, there will be no expression. Based on these assumptions, we conclude that the
deletion in this region includes the Promoter region, because in its absence, there will be no
place for RNA polymerase to bind, which will result in no transcription of structural genes and no
expression.

2. Six strains of E. coli(mutants 1–6) that had one of the following mutations (i–vi) affecting the
lacoperon were isolated.
i. deletion of lacY
ii. oc mutation
iii. missense mutation in lacZ
iv. inversion of the lac operon (but not an inversion of the lacI gene)
v. superrepressor mutation
vi. inversion of lacZ, Y, and A but not lacI, P, o
a. Which of these mutations would prevent the strain from utilizing lactose?
Stains i,iii,iv,v would not utilize lactose.
b.The entire lac operon (including the lacI gene and its promoter) from each of the six E. coli
strains was cloned into a plasmid vector containing an ampicillin resistance gene. Each
recombinant plasmid was transformed into each of the six strains to create partial diploids. In
analysis of these strains, mutant 1 was found to carry a deletion of lacY, so this strain corresponds
to mutation i in the list above. Which of the other types of mutations would be expected to
complement mutant 1 in these partial diploids so as to allow lactose utilization?
ii, iii, vi mutation would complement mutation 1
c.In part (b), each strain was plated on ampicillin media in which lactose was the only carbon
source. (Ampicillin was included to ensure maintenance of the plasmid.) Growth of the
transformants is scored below (a + sign indicates growth, a − sign means no growth). Synthesis of
β-galactosidase and permease are both required for growth on this medium. Results of this
merodiploid analysis are shown here. Which mutant bacterial strain (1– 6) contained each of the
alterations (i–vi) listed previously?