PCB3103C Exam 2 (take home) Name___________________________

Summer 2015

Answer all ten questions (10 points each). You can use any references you want but the answer must be your own
and unique. Regarding citing references, if in doubt just cite it.

1. Describe the mechanism by which heterochromatin can spread, once it has been established in one
region of the chromosome.

3. Your friend is working in a lab to study how yeast cells adapt to growth on different carbon sources. He grew
half of his cells in the presence of glucose and the other half in the presence of galactose. Then he harvested the
cells and isolated their DNA with a gentle procedure that leaves nucleosomes and some higher-order chromatin
structure intact. He treated the DNA briefly with a low concentration of M-nuclease, a special enzyme that easily
degrades protein-free stretches of DNA. After removing all the proteins, he separated the resulting DNA on the
basis of length. Finally, he used a procedure to visualize only those DNA fragments from a region near a particular
gene called Sweetie or another gene called Salty. The separated DNA fragments are shown in the Figure. Each
vertical column, called a lane, is from a different sample. DNA spots near the top of the figure represent DNA
molecules that are longer than those near the bottom. Darker spots contain more DNA than fainter spots. The
lanes are as follows:

1. “marker” containing known DNA fragments of indicated lengths

Figure Q5-70

A. The lowest spot (as observed in lanes 2, 4, and 5) has a length of about 150 nucleotides. Can you
propose what it is and how it arose?
B. What are the spots representing longer lengths of DNA? Why is there a ladder of spots?
C. Notice the faint spots and extensive smearing in lane 3, suggesting the DNA could be cut almost
anywhere near the Sweetie gene after growth of the cells in galactose. This was not observed in the
other lanes. What probably happened to the DNA to change the pattern between lanes 2 and 3?
 D. What kinds of enzymes might have been involved in changing the chromatin structure between
lanes 2 and 3?
E. Do you think that gene expression of Sweetie is higher, lower, or the same in galactose compared
to glucose? What about Salty?

3. You prepare bacterial cell extracts by lysing the cells and removing insoluble debris via centrifugation. These
extracts provide the proteins required for DNA replication. Your DNA template is a small, double-stranded circular
piece of DNA (a plasmid) with a single origin of replication and a single replication termination site. The
termination site is on the opposite side of the plasmid from the origin.

A. In addition to the extracts and the plasmid DNA, are there any additional materials you should add to this
in vitro replication system? Explain your answer.

B. Which of the following statements is true with respect to this in vitro replication system?
(a) There will be only one leading strand and one lagging strand produced using this template.
(b) The leading and lagging strands compose one half of each newly synthesized DNA strand.
(c) The DNA replication machinery can assemble at multiple places on this plasmid.
(d) One daughter DNA molecule will be slightly shorter than the other.

You decide to use different bacterial strains (each having one protein of the replication machinery mutated) in
order to examine the role of individual proteins in the normal process of DNA replication.

C. What part of the DNA replication process would be most directly affected if a strain of bacteria lacking
primase were used to make the cell extracts?
(a) initiation of DNA synthesis
(b) Okazaki fragment synthesis
(c) leading-strand elongation
(d) lagging-strand completion

D. What part of the DNA replication process would be most directly affected if a strain of bacteria lacking the
exonuclease activity of DNA polymerase were used to make the cell extracts?
(a) initiation of DNA synthesis
(b) Okazaki fragment synthesis
(c) leading-strand elongation
(d) lagging-strand completion

E. What part of the DNA replication process would be most directly affected if a strain of bacteria lacking
helicase were used to make the cell extracts?
(a) initiation of DNA synthesis
(b) Okazaki fragment synthesis
(c) leading-strand elongation
(d) lagging-strand completion
F. What part of the DNA replication process would be most directly affected if a strain of bacteria lacking
single-strand binding protein were used to make the cell extracts?
(a) initiation of DNA synthesis
(b) Okazaki fragment synthesis
(c) leading-strand elongation
(d) lagging-strand completion

G. What part of the DNA replication process would be most directly affected if a strain of bacteria lacking
DNA ligase were used to make the cell extracts?
(a) initiation of DNA synthesis
(b) Okazaki fragment synthesis
(c) leading-strand elongation
(d) lagging-strand completion

4. Indicate whether the following statements are true or false. If a statement is false, explain why it is false.

A. When DNA is being replicated inside a cell, local heating occurs, allowing the two strands to
separate.
B. DNA replication origins are typically rich in G-C base pairs.
C. Meselson and Stahl ruled out the dispersive model for DNA replication.
D. DNA replication is a bidirectional process that is initiated at multiple locations along
chromosomes in eukaryotic cells.

5. Why is the old dogma “one gene—one protein” not always true for eukaryotic genes?

6.

After treating cells with a mutagen, you isolate two mutants. One carries alanine and the other carries
methionine at a site in the protein that normally contains valine. After treating these two mutants again
with mutagen, you isolate mutants from each that now carry threonine at the site of the original valine
(see Figure below). Assuming that all mutations caused by the mutagen are due to single nucleotide
changes, deduce the codons that are used for valine, alanine, methionine, and threonine at the affected
site.
7. This Figure shows an mRNA molecule.

A. Match the labels given in the list below with the label lines in the Figure.

(a) ribosome-binding site_______

B. Is the mRNA shown prokaryotic or eukaryotic? Explain your answer.

8. The yeast GAL4 gene encodes a transcriptional regulator that can bind DNA upstream of genes required for the
metabolism of the sugar galactose and turn them on. Gal4 has a DNA-binding domain and an activation domain.
The DNA-binding domain allows it to bind to the appropriate sites in the promoters of the galactose metabolism
genes. The activation domain attracts histone-modifying enzymes and also binds to a component of the RNA
polymerase II enzyme complex, attracting it to the promoter so that the regulated genes can be turned on when
Gal4 is also bound to the DNA. When Gal4 is expressed normally, the genes can be maximally activated. You decide
to try to produce more of the galactose metabolism genes by overexpressing the Gal4 protein at levels fiftyfold
greater than normal. You conduct experiments to show that you are overexpressing the Gal4 protein and that it is
properly localized in the nucleus of the yeast cells. To your surprise, you find that too much Gal4 causes the
galactose genes to be transcribed only at a low level. What is the most likely explanation for your findings?
9. The expression of the BRF1 gene in mice is normally quite low, but mutations in a gene called BRF2
lead to increased expression of BRF1. You have a hunch that nucleosomes are involved in the regulation
of BRF1 expression and so you investigate the position of nucleosomes over the TATA box of BRF1 in
normal mice and in mice that lack either the BRF2 protein (BRF2–) or part of histone H4 (HHF–) (histone
H4 is encoded by the HHF gene). Table Q8-37 summarizes your results. A normal functional gene is
indicated by a plus sign (+).

Table Q8-37

Which of the following conclusions cannot be drawn from your data? Explain your answer.
(a) BRF2 is required for the repression of BRF1.
(b) BRF2 is required for the specific pattern of nucleosome positions over the BRF1 upstream region.
(c) The specific pattern of nucleosome positioning over the BRF1 upstream region is required for
BRF1 repression.
(d) The part of histone H4 missing in HHF– mice is not required for the formation of nucleosomes.

10. You have a piece of DNA that includes the following sequence:
5′-TATGGCATTCGATCCGGATAGCAT-3′
3′-ATACCGTAAGCTAGGCCTATCGTA-5′

A. Which of the following RNA molecules could be transcribed from this piece of DNA?

(a) 5′-UAUGGCAUUCGAUCCGGAUAGCAU-3′
(b) 5′-AUACCGUAAGCUAGGCCUAUCGUA-3′
(c) 5′-UACGAUAGGCCUAGCUUACGGUAU-3′
(d) None of the above

B. If this is a bacterial gene draw me the asymmetric promoter region, and show me which way
transcription is proceeding.