Molecular Genetics Problem Set 5

1. You have identified a set of human genes that are expressed at much higher levels when
the cells are exposed to the hormone cortisol. Based on what you learned in this class, you
hypothesize that the same cortisol-responsive transcriptional activator regulates all of these
genes.

Which of the following statements below should be true if your hypothesis is correct?

a.) The cortisol-responsive genes must all be in an operon.

b.) All of the cortisol-responsive genes share a DNA sequence in their regulatory regions
that binds the cortisol-responsive transcriptional activator.
c.) All of the cortisol-responsive genes must have exactly the same set of activators binding
to their regulator regions (enhancers and promoters).

Statement B
2. This question is about a protein that is found in liver and kidney cells in mice called
Livkid. You are trying to understand the gene regulation of Livkid, so you first look for a
cell that doesn't have that protein. You are able to find one: skin cells do not express Livkid.
To understand how gene regulators interact with the promoter of Livkid, you clone a
fragment just upstream of the Livkid gene, which contains the promoter. You then clone
that piece of DNA upstream of the gene for green fluorescent protein (GFP), and insert this
entire piece of recombinant DNA into mice. You find GFP is expressed the same way that
Livkid is: you see GFP glowing in the liver and kidney cells but not in skin cells. After more
experiments, you demonstrate that there are three regions in the promoter that contribute
to this expression pattern labeled A, B, and C in the figure below.

Assume that a single and unique transcription factor binds each site such that:
o protein X binds site A
o protein Y binds site B
o protein Z binds site C.

You want to determine which region is responsible for tissue-specific expression, and create
mutations in the promoter to determine the function of each of these regions. In the figure
below, if a site is missing, it means that it is mutated such that it can not bind its
corresponding transcription factor.

A. Which of the following proteins is most likely to function as a repressor?

(a) factor X (b) factor Y (c) factor Z (d) none of the above
Factor Z

B Which of the following protein(s) are likely to act as gene activators?

(a) factors X and Y (b) factors X and Z (c) factors Y and Z (d) factor X only
Factors X and Y
C In what tissue is factor Z normally present and bound to the DNA?
Skin
a) kidney (b) liver (c) skin (d) none of the above
 3. You
have
performed
a screen
for
mutants
that you
believe
may affect
the
regulation
of your
favorite
gene in
your
favorite
eukaryotic organism. You have exhaustively characterized these mutants in a number of
experiments:
• You have run RNA from each mutant on a gel to separate molecules by size, and
hybridized this gel with a probe for your favorite gene. (Note: larger molecules will be
towards the top of the gel.)
• You have performed in situ hybridization on each mutant at an early developmental
stage, using a probe for your favorite gene. In situ hybridization is like a Northern Blot,
except instead of hybridizing your probe to RNA that has been extracted, you hybridize
your probe directly to a slice of tissue and detect the RNA in situ.
(Your organism is round at this stage; the probe is blue.)

• You have performed an in vitro experiment to assay the enzymatic activity of the protein
encoded by your favorite gene from each mutant.
• You have measured the level of mRNA from your favorite gene expressed in the mutant
organisms.
The results of your industry are displayed below. For each mutant, propose a reasonable
mutational event which may account for the way the function of your favorite gene is
disrupted.
Mutation 1: This mutation could be caused by a frameshift mutation.
Mutation 2: This mutation could be caused by a frameshift mutation.
Mutation 3: This mutation could be caused by a duplication mutation.
Mutation 4: This mutation could be caused by a point deletion mutation.
Mutation 5: This mutation could be caused by a complete deletion mutation.
Mutation 6: This mutation could be caused by a frameshift mutation.

4. Yeast are great models for genetic analysis. Mutant screens are performed in haploid cells,
so you don’t need to worry about whether your mutation is dominant or recessive. Reporter
gene constructs can be introduced on a circular piece of DNA called a plasmid, which is
maintained in the cell but does not integrate into the genome. You can read more about yeast
genetics in the textbook.
Mating two haploid cells results in a diploid, allowing you to assess allele interactions.
Diploids sporulate to generate 4 haploid cells in a “tetrad”, much like meiosis generates 4
haploid gametes.
You have discovered a gene in yeast that is involved in repairing damaged DNA. Mutations
in this gene make yeast more sensitive to DNA-damaging agents such as UV radiation. You
designate your new gene Rad66. To study the regulation of Rad66, you fuse the cis
regulatory region upstream of the Rad66 open reading frame to the LacZ coding sequence.
You then place this hybrid gene (designated Prad66–LacZ, the P stands for “promoter”) on
a yeast plasmid. As hoped, yeast cells carrying the Prad66–LacZ plasmid do not express ß-
galactosidase activity unless exposed to UV light, showing that the hybrid gene is regulated
in the same way that the Rad66 gene is normally regulated.
(a) You next identify a mutant that you call Reg1–, which causes expression of the Prad66–
LacZ reporter gene construct, regardless of whether or not the cells have been exposed to
UV light. By mating a Reg1– haploid mutant strain to a wild-type haploid strain, you find
that the resulting diploid only expresses the reporter gene in the presence of UV light.
Classify the Reg1– mutation in terms of its basic genetic properties (constitutive vs
uninducible, dominant vs recessive). Explain the rationale behind your conclusions. Based
on these properties, make a proposal for the wild-type regulatory function of the Reg1
gene.
Recessive and uninducible; we know this because the diploid containing it only expresses
the reporter gene in the presence of UV light despite expressing the reporter gene
regardless of UV light presence.

(b) Give a model for the regulatory pathway for Rad66 that is consistent with the data
presented in this problem so far. In the model you diagram, include the wild-type Rad66
and Reg1 genes. Also be sure to include in your model a role for UV radiation.

(c) Next, you isolate a mutant that you call Reg2–, which will not express the Prad66–LacZ
reporter, even after cells have been exposed to UV radiation. Mating a Reg2– haploid
mutant strain to a wild-type haploid strain gives a diploid that can expresses the reporter
only in the presence of UV radiation. Given all of the available data on the Reg1– and
Reg2– mutants, diagram two potential pathways that could explain the roles of the Reg1
and Reg2 gene products and UV radiation in the regulation of Rad66.
 (d) You cross a haploid strain that carries the Reg1– mutation to a haploid strain that
carries the Reg2– mutation. You then induce sporulation of the resulting diploid. You
analyze only two tetrads, and they both show the same pattern of expression of Prad66–
LacZ in response to UV light: one spore shows normal induction in response to UV
radiation, two spores express the reporter even in the absence of UV radiation, and one
does not express the reporter. What is the phenotype of a Reg1– Reg2– double mutant
haploid strain?

(e) Select the model from part (c) that is consistent with the double mutant data above.

Next you evaluate the cis regulatory sequences necessary for expression of the Prad66–
LacZ reporter gene construct. The figure below shows the effect of six different 50 basepair
long deletions (#1 - #6) in the regulatory region on the amount of ß- galactosidase activity
expressed by the reporter gene.

(f) In light of the experiments from parts (a) – (e), propose a specific function for any cis
acting segment that has a clear regulatory role as defined by these deletion constructs.