Name: ______________________________
Genetics 314 – Spring 2006
Exam 2 – 100 points
1. You have been hired by the University of Idaho’s Department of Environmental
Safety to determine why the sewage treatment plant’s bacterial fermentation tanks
are not working properly. You suspect illegal chemicals are being dumped down
the drains in the molecular biology labs.
a) You check one lab and find that several of their chemicals are listed as mutagens,
some are listed as causing base change mutations and other chemicals are listed as
causing frameshift mutations. What is the difference between the two mutagens
and which one would have the greater potential to cause deleterious mutations in
bacteria if it was poured down the drain? Briefly explain your answer.
A mutagen that causes a change in a single base will only affect the codon where
the change has taken place. This may have a major effect if the codon is a start
codon, a codon for a critical amino acid or the change is in a promoter region
and affects how a regulatory protein can bind. It can also have little or no effect
if the base that is changed is the third base in a codon so the amino acid does not
change (redundancy in the code) or if the codon codes for a non-critical amino
acid. A frameshift mutation is a deletion or addition of bases in a DNA
sequence. By adding 1 or 2 bases the entire reading frame for the gene
downstream from the mutation will be changed, potentially changing all the
codons and therefore all the amino acids downstream of the mutation. This
would have a greater chance to result in a non-functional gene product. Because
of the potential for multiple amino acid changes with a deletion or addition of a
base, frameshift mutations have a greater potential to cause deleterious
mutations.
b) You accuse one lab of causing mutations in the sewage treatment plant’s bacteria
but they claim that since the fermentation tank did not have a cover that U.V. light
is the cause of the mutations.
1) Why would they claim that the U.V. light was the cause of the mutations? Briefly
explain your answer.
Ultra violet light can cause adjacent thymines to form a covalent bond producing
a thymine dimer. The presence of the dimer can cause problems during DNA
replication resulting in errors in replication.
2) What information do you know about bacteria that would make you doubt their
claim?
Bacteria have two repair mechanisms for U.V. light damage. There is a light
activated system that uses the enzyme photolyase and energy from light to break
the thymine dimers. There is a second excision and repair mechanism that does
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Name: ______________________________
not use light energy and involves two steps. First the region of the DNA strand
that has the thymine dimer is removed and then the remaining strand is used as
a template for DNA polymerase I to synthesis new DNA to replace the excised
piece and ligase comes in to connect the last phosphodiester bond.
c) You go into a genetic engineering lab and find that a graduate student was
pouring a chemical down the drain that is listed as a base analog to adenine.
Could this also potentially be the source of your bacterial mutation problem?
Briefly explain your answer.
Yes, a base analog is a chemical that mimics a base during replication (such as
adenine) but in a subsequent cycle of replication will pair to a base other than
thymine resulting in a base change mutation.
2. The people in Bacteriology are so impressed with your work that they hire you to
run their bacteria fermentation tanks that are being used to produce genetically
engineered proteins that require a high level of the amino acid histidine. The
protein is produced by the reactions catalyzed by three enzymes that are coded for
by three genes. They would like the bacteria to produce a relatively constant level
of the protein and ask you how you would arrange the genes for simultaneous
regulation and what type of regulatory system they should include on their gene
construct.
a) You first recommend they use an operon system for regulation of expression of
the three genes. What is an operon system of regulation?
An operon system of regulation in bacteria is where the genes coding for the
enzymes in a specific biochemical pathway are placed in a series (one right after
the other) and are controlled by a single promoter region directly upstream of
the first gene. This insures all the genes needed for a biochemical pathway are
turned on or off at the same time.
b) What type of prokaryotic regulatory system would you choose to obtain a
relatively constant level of protein production?
A repressible system
c) Describe how your system would work to control expression of your protein.
A repressible system would allow for a constant level of expression with a
minimum of external control by using the end-product as a co-repressor of the
operon. In such a system there is a repressor protein present but it is inactive
unless there is an adequate level of the co-repressor present in the system. When
production of the end-product gets too high then the co-repressor binds with the
repressor making it active so transcription is shut down. As the level of end-
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Name: ______________________________
product drops the co-repressor becomes less available inactivating the repressor
protein resulting in the restarting of transcription.
3. A friend suggests that you could increase production by putting the gene construct
into a eukaryotic system such as yeast and add a secondary level of regulation for
gene expression, specifically attenuation.
a) Describe how attenuation works to reduce expression of a gene.
Attenuation is a level of secondary gene regulation where the relationship of the
RNA polymerase to the ribosome on the newly synthesized mRNA will dictate if
the RNA polymerase continues or stops transcription before the gene sequences
are transcribed. This control system is based on regions of complimentarity in
the mRNA that allows for dsRNA loops to form depending on the location of the
ribosome on the mRNA. If there is adequate end-product the ribosome proceeds
at its normal speed and a loop similar to a transcription termination loop is
formed halting transcription. If the level of end-product is low then the
ribosome’s pace is slowed allowing for a different loop to form preventing
formation of a termination loop allowing transcription to continue.
b) Would this work in a eukaryotic system as your friend suggests? Briefly explain
your answer.
No, for attenuation to work you must be able to have simultaneous transcription
and translation which only occurs in prokaryotes. In eukaryotes there is a
nuclear membrane separating transcription from translation so RNA
polymerase and ribosomes can not interact directly.
4. You are asked officially to switch to working in a eukaryotic system because they
discover that some of the genes they are working on carry introns. Your first
comment is that an operon would not work. Describe a system that would allow
you to express all three genes simultaneously without an operon.
Since operons are not available in eukaryotes the way to get the genes
responsible for the enzymes in a biochemical pathway to turn on at the same
time is to make sure all the genes have the same enhancer sequences and
promoter sequence upstream of each gene. In this way when the activator
proteins that correspond to the enhancer sequences are present, all the genes will
be ‘turned on’ at the same time.
5. Your system in the eukaryotic organism appears to be working fine but you start
to notice a decrease in production of the protein by the bacteria. You discover
that the genes are being transcribed but not translated. Describe a posttranscriptional form of regulation that could prevent expression of a gene in a
eukaryotic cell.
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Name: ______________________________
A post-transcription form of regulation that could prevent expression of a gene
in a eukaryotic cell could involve destruction of the transcript or delay in
modification of the transcript preventing its movement from the nucleus to the
cytoplasm. Destruction of the transcript could be due to si (small interfering)
RNA that in association with an endonuclease could identify and cut up specific
RNA sequences. A second problem could be in the placement of the cap and
poly A tail on the transcript during the mRNA processing. If the cap and tail
are not added the mRNA probably can not get out of the nucleus and or would
be degraded rapidly. A third possibility is that the poly A tail is added but it is
short and the mRNA is broken down before it can be translated.
6. You are walking by the virology lab and hear the sounds of champagne bottles
being opened. You ask why and they say they have just developed a method to
stop replication of virus in a cell without impacting the cells ability to grow and
replicate. You ask what type of RNA virus they are working on and they ask why
you suspect they are working on an RNA virus.
a) Why would you assume they are working on an RNA virus?
Because DNA virus utilize the host enzymes for replication and if any of these
enzymes were targeted for preventing virus replication the host cell would also
be affected. With RNA virus, non-host enzymes must be supplied by the virus
for replication. If these enzymes (replicase or reverse transcriptase) were
targeted, virus replication could be reduced without affecting the growth and
development of the host cells.
b) Briefly describe how such a system might work.
If a protein could be designed that would either bind to replicase or reverse
transcriptase, or bind to the RNA that codes for these enzymes preventing them
from being translated the virus could not produce the intermediary product
(minus RNA strand or DNA template) it needs to successfully replicate. This
would prevent additional copies of the virus from being produced, halting its
spread.
7. You see people leaving the adjacent virology lab in a hurry and you ask the
graduate student left to clean up the lab why. The student says that some of the
lab animals were accidentally infected by two strains of flu virus. What could be
the problem with infecting an animal with two strains of flu virus at the same
time? Briefly explain your answer.
If simultaneous infection of an animal by different strains of a virus occurs, it is
possible for a recombinant virus to be produced that is more virulent than either
of the two original strains. Recombination can occur by direct recombination
between complimentary regions of viral RNA, copy-choice errors during
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Name: ______________________________
replication of the viral RNA, and simple packaging errors when the pieces of
RNA are put in the viral protein capsule.
8. Returning to your lab by the recombinant DNA lab you are asked for advice on
getting a new gene they have developed into a bacteria for expression.
a) You ask first to see what type of expression vector plasmid they propose to use.
What would you need in your expression vector to get expression of your gene in
bacteria and to be able to select bacteria that carry the selection vector?
For an expression vector to work you need a promoter, leader and termination
sequence for proper expression of the gene, a series of restriction endonuclease
cut sites between the leader and termination sequences to allow insertion of you
DNA sequence, and an antibiotic resistance gene on the vector to allow for
selection of transformed cells.
b) How would you propose to get the expression vector plasmid into the bacteria.
Briefly explain your answer.
The easiest method to get the vector into the bacteria is by transformation. Here
the expression vector is placed in a solution with competent cells and the
bacterial cells take up the DNA from the environment.
A second method could be transduction of viral capsules is available to transfer
the DNA into the bacterial. This method works by placing the DNa in the viral
capsule and then allowing the virus to infect the bacteria, injecting the
expression vector carrying the gene into the bacteria.
9. You are studying the ability of bacteria to transfer genes by conjugation. You
have four Hfr strains and produce the following set of data:
strain
first gene out
A 444
B 333
C 555
D 777
trp
lac
bio
glu
bio
ser
trp
ser
last gene out
met
glu
his
lac
gal
amp
kam
gal
lac
kam
amp
met
Draw a circular gene map using this data showing the order of the genes, and the
insertion and orientation of the F plasmids in these Hfr strains.
trp
his
his
trp
trp
bio
met
gal
lac
lac
ser
glu
lac
lac
ser
ser
glu
glu
bio
bio
met
met
gal
gal
amp
amp
kam
kam
amp
kam
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Name: ______________________________
10. You are studying transduction and discover only two genes, bio and gal, are being
observed in the bacterial recombinants. What is causing the recombination of
only these two genes? Briefly explain your answer.
You are seeing specialized or specific transduction occurring. This is where a
temperate phage has a single insertion site in a bacterial chromosome and this
site is between the two genes bio and gal. After insertion, when the phage
attempts to disassociate from the bacterial chromosome it does not disassociate
properly and one of the two genes is taken by accident and part of the viral
genome is left behind. When a phage carrying either gene infects another
bacteria it will transfer the gene and since the phage is incomplete it will not
cause the death of the infected cell.
Extra credit:
Briefly describe how DNA fingerprinting works.
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