Download Genetics 314 - Spring 2005

Name: _________________________________
Genetics 314 - Spring 2005
Exam 1 – 100 points
1. You are given three samples of DNA from three different organisms: a virus, a
bacteria and a pygmy owl. You are asked to identify the three samples just by
heating and cooling the DNA.
a) What information would heating the DNA tell you and would this help in
identifying the source organism for each sample?
By heating the DNA you could tell what temperature it would denature which is
related to the amount of G – C pairs and A – T pairs in the DNA samples. Because
of the greater number of hydrogen bonds shared between G – C pairs, DNA with a
high proportion of G-C pairs will require a higher temperature to denature than
DNA rich in A – T pairs. While this information may be useful, it will not give you
any information that could be used to identify the source organism of each sample.
b) What information could you gain by cooling the heated DNA and how would this
information help in determining the source of each organism?
The information you will get from cooling the DNA will be the size of the genome of
the organism (number of unique sequences/genes) and the presence of highly
repetitive and moderately repetitive sequences in the sample. This information
could be used to determine the source organism for each sample because virus have
a small genome size compared to bacteria so these two can be differentiated by the
rate of renaturation (virus faster than bacteria) and pygmy owl could be
differentiated from the virus and bacteria by the presence of repetitive sequences in
the eukaryotic (pygmy owl) and by the time it takes for the whole sample to
renature due to the much larger genome size and greater number of unique
sequences found in eukaryotic DNA samples.
c) Draw a set of Cot curves for how you would expect the three samples to behave,
labeling which curve represented the virus, the bacteria and the pygmy owl.
pygmy owl
virus
bacteria
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Name: _________________________________
2. DNA is said to be replicated in a semi-conservative manner. What is meant by
the term semi-conservative replication and how was this determined?
Semi-conservative replication of DNA means that the hydrogen bonds are broken
during replication allowing the strands to separate but the phosphor-diester bonds
remain intact the each individual strand of the original DNA is conserved. This
results in the DNA after replication consisting of one new strand and one old strand
of DNA.
This was determined by using a radioisotope of nitrogen (N15) to label the
nucleotides in the DNA prior to replication then allowing replication to occur with
non-labeled nucleotides (N14) and look at the density of the DNA after successive
rounds of replication. A single band consisting of a mix of N14 and N15 DNA was
expected after the first replication and two bands one just N14 and the other a mix
of N14/N15 after the second replication with semi-conservative replication. This is
what was observed in the experiment, confirming DNA replicated in a semiconservative manner.
3. You are given the following sense strand of DNA that codes for a simple protein
that could be added to food to control obesity.
3’ TAC CCC ACG GGC TTT GCA AAA TCG GCC AGC ACT 5’
a) What would be the mRNA sequence coded for by this DNA sequence? (For full
credit label the 3’ and 5’ ends of the mRNA)
5’ AUG GGG UGC CCG AAA CGU UUU AGC CGG UCG UGA 3’
b) What would be the amino acid sequence coded for by your mRNA sequence?
MET GLY CYS PRO LYS ARG PHE SER ARG SER STOP
c) A friend says the following sequence codes for the same polypeptide but it does
not have the exact base sequence of your DNA.
3’ TAC CCA ACA GGT TTC GCA AAG TCG GCT AGC ACT 5’
Is your friend correct and if so how is that possible?
5’ AUG GGU UGU CCA AAG CGU UUC AGC CGA UCG UGA
MET GLY CYS PRO LYS ARG PHE SER ARG SER STOP
Yes, because of the redundancy of the cod where more than one codon can code
for the same amino acid. Note the deviation is usually in the third base of the
codon.
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Name: _________________________________
4. You would like to have the obesity-preventing protein from question 3 mass
produced so you want to insert it into an organism to get the DNA sequence
transcribed and translated.
a) What other DNA sequences would you need to add to your DNA sequence to
insure proper transcription and translation in a prokaryotic organism? For full
credit indicate the purpose of each sequence.
promoter
sequence
leader sequence
gene sequence
termination sequence
promoter sequence – binding site for RNA polymerase for the initiation of
transcription
leader sequence – binding site for the small subunit of the ribosome for the initiation
of translation
termination sequence – sequence that signals the end of transcription
b) If you decided to move your gene from the prokaryotic organism to a eukaryotic
organism would you need to change the additional DNA sequences you added to
get expression in the prokaryotic organism? Briefly explain your answer.
Yes, the promoter sequence, leader sequence and termination sequence are different
between prokaryotic and eukaryotic organisms so they would need to be changed
for the enzymes involved in transcription and translation to be able to recognize the
initiation or termination sequences for transcription and the sequence needed for
binding of the small ribosomal subunit in translation.
5. You have isolated a gene from an eukaryotic organism and get it expressed in a
prokaryotic organism and discover you do not get a functional protein. If you go
back to the eukaryotic organism and isolate the mRNA for this gene from the
cytoplasm and then create a DNA sequence from the mRNA sequence, the
synthesized gene will produce a functional protein when inserted in a prokaryote.
Based on the information given answer the following questions:
a) Why would the gene not produce the desired protein when it was placed in the
prokaryotic organism the first time?
The gene has introns that need to be processed out of the mRNA for the transcript
to code for a functional protein. Because prokaryotes do not have introns they do
not have the necessary systems needed to remove the introns before translation
occurred.
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Name: _________________________________
b) Why would synthesizing a gene based on the mRNA base sequence produce the
desired protein?
If the DNA sequence of the gene is based on the mRNA isolated from the cytoplasm
of a eukaryotic cell the introns will not be presence in the sequence because mRNA
in the cytoplasm has been processed so no introns would be present in the sequence.
With no introns present the protein product produced by the prokaryote would be
functional.
6. Being frustrated by the difference you find in gene expression using eukaryotic
and prokaryotic cells you decide to take control by working with the gene in vitro.
You buy a DNA replication kit from a USA company but notice the kit was really
produced in Eastern Europe.
a) You add your gene to the ingredients in the kit and discover that you get no
replication of your DNA. Upon further inspection you find that the DNA had
opened up but no DNA synthesis was occurring. What could be causing the
problem and how could it be fixed.
If the DNA strand has opened it means that helicase, topoisomerase and the single
strand binding proteins are present and functioning but the DNA polymerases do
not have an open 3’ end to initiate synthesis of DNA. The 3’ end is provided by
RNA primers that are synthesized by the enzyme primase using RNA nucleotides.
The absence of the primer indicates either primase is missing or the RNA
nucleotides are missing.
Solution, add RNA nucleotides and primase to produce RNA primers.
b) You think you solve the problem and start the reaction again. This time you get
DNA synthesis but it is much slower than expected. Now what is the problem
and how could it be fixed?
If this is based off of a prokaryotic model then the problem is that the DNA
polymerase in the kit is DNA polymerase I not DNA polymerase III. The two
enzymes differ in their rate of synthesis with DNA polymerase III being much faster
that DNA polymerase I.
Solution, add DNA polymerase III.
7. Now that you have your gene properly replicated you turn to doing in vitro
transcription. You go to another American company for your transcription kit but
discover it is now made in China.
a) Undaunted, you combine the ingredients in your kit but discover that the
transcripts that are produced are of variable lengths. What could be missing from
your kit and how could you fix the problem?
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Name: _________________________________
Again using a prokaryotic system as a model, the problem is that the RNA
polymerase is not attaching at the promoter site but instead is randomly attaching
on the DNA. This would be due to the lack of a sigma factor. The role of the sigma
factor is to orientate the RNA polymerase (core enzyme) in the proper location on
the gene on the promoter,
Solution, add the correct sigma factor.
b) Starting to get frustrated, you decide to read the instructions. You read one
warning saying that the kit will only work for genes with rho independent
termination. What is meant by this and why would it possibly be a concern?
The only potential problem here is if you were using a termination sequence for
your gene that required the rho factor (protein) to facilitate termination of
transcription. The end result would be extra long transcripts.
Solution, add the rho factor or make sure the termination sequence of your gene is a
rho independent termination sequence.
8. You have now produced the transcripts you want, so you now are ready to attempt
in vitro translation. You find a kit that is actually made in America but discover
that the company to save money only included 45 of the possible 61 tRNAs in the
kit. The disclaimer in the kit says this should not cause problem with translation.
a) How could the company claim that its cost saving measures would not affect your
ability to translate your gene?
There is a redundancy in the code where more that one codon codes for a specific
amino acid. Combine this with the wobble theory where the third base in the codon
is not that necessary for matching the t-RNA anti-codon with a specific codon and
you could have one anti-codon work for several codons meaning not all the t-RNA’s
would be necessary. There would need to be at least one t-RNA for each amino acid.
b) You try the kit and you get no translation. You discover you never see a complete
ribosome attached to your DNA. You call the company and claim that their costsaving measures caused the problem. They say the problem is with your DNA
sequence. What is the basis of your argument and what could the company think
is wrong with your DNA sequence?
If the small ribosome is attaching but the complete ribosome is not being assembled
you could be missing the special methionine needed for initiation or missing the tRNA associated with methionine. If either are missing the second step of ribosome
assembly can not occur so no complete ribosomes are assembled and translation can
not begin. A second possibility is if the initiation factors for translation that
facilitate assembly of the ribosome are missing then no ribosomes would be
assembled and no translation would occur.
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Name: _________________________________
If the DNA lacked a start codon AUG the second step of ribosome assembly would
not occur again resulting in the lack of complete ribosomes and no translation.
RNA Codons
UUU
UUC
UUA
UUG
phe
phe
leu
leu
UCU
UCC
UCA
UCG
ser
ser
ser
ser
UAU
UAC
UAA
UAG
tyr
tyr
stop
stop
UGU cys
UGC cys
UGA stop
UGG trp
CUU
CUC
CUA
CUG
leu
leu
leu
leu
CCU
CCC
CCA
CCG
pro
pro
pro
pro
CAU
CAC
CAA
CAG
his
his
gln
gln
CGU
CGC
CGA
CGG
arg
arg
arg
arg
AUU
AUC
AUA
AUG
ile
ile
ile
met
ACU
ACC
ACA
ACG
thr
thr
thr
thr
AAU
AAC
AAA
AAG
asn
asn
lys
lys
AGU
AGC
AGA
AGG
ser
ser
arg
arg
GUU
GUC
GUA
GUG
val
val
val
val
GCU
GCC
GCA
GCG
ala
ala
ala
ala
GAU
GAC
GAA
GAG
asp
asp
glu
glu
GGU
GGC
GGA
GGG
gly
gly
gly
gly
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