Download Genetics 314 – Spring 2004

Name: __________________________
Genetics 314 – Spring 2004
First Exam – 100 points
1. You are asked to determine whether three unknown DNA samples are from a
virus, prokaryotic organism or a eukaryotic organism. You decide to produce Cot
curves for the three samples and get the following results:
B
1.0
C/Co 0.5
C
A
0.0
Cot
Based on the Cot curve information what type of organism is represented by each
sample and briefly explain your reasoning.
Sample A.
Virus or phage DNA based on having the smallest amount of total genomic
DNA made up of unique sequences of DNA.
Sample B.
Prokaryotic or bacterial DNA based on having the next largest amount of
total genomic DNA made up of primarily unique sequences of DNA.
Sample C.
Eukaryotic DNA based on the presence of highly repetitive and moderately
repetitive DNA sequences plus having the largest amount of total genomic
DNA and the largest amount of unique sequences based on the length of time
needed to get reannealing of the DNA strands.
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Name: __________________________
2. The Mars rovers make a surprising discovery, not only did they find evidence of
water they also found evidence of life on Mars! Surprisingly Mars life also had
DNA but the DNA replicated differently. It was found to replicate in a
conservative manner. How does this differ from life here on earth and how could
you demonstrate the difference? (you can use diagrams if you wish)
To determine method of replication you would need to label the old and new
strands in such a way as to tell if the DNA molecules or strands stay intact
after replication.
For conservative you would observe the old strands still together after
replication and the new strands together.
For semi-conservative you would observe that the covalent bonds between
the nucleotides of a strand stay intact but the hydrogen bonds between the
strands are broken resulting in DNA having one old strand and one new
strand after replication
3. You are asked to determine the amino acid sequence for one of the Mars DNA
samples. You are given the following DNA sequence but are told that the
sequence is complementary to the template DNA strand for the polypeptide.
5’ ATG GGG ACC CCT AGA TTT TGC AAA CCA AGG TGA 3’
Assuming the genetic code is truly universal:
a. What is the template DNA sequence?
3’TAC CCC TGG GGA TCT AAA ACG TTT GGT TCC ACT 5’
b. What is the mRNA sequence?
5’ AUG GGG ACC CCU AGA UUU UGC AAA CCA AGG UGA 3’
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Name: __________________________
c. What is the polypeptide sequence coded for by this DNA sequence?
met gly thr pro arg phe cys lys pro arg stop
d. You observe that you have two of the same amino acids in the polypeptide but
no duplication in the DNA or mRNA sequence. How is this possible?
Due to the degeneracy of the genetic code where more than one codon codes
for an amino acid.
4. You would like to produce a large supply of this protein for study so you want to
have the gene be transcribed and translated.
a) You decide to put the gene into bacteria. What other DNA sequences
would you need to add to your DNA fragment to insure expression of the
gene in bacteria? Diagram your ‘complete’ gene and briefly explain what
the purpose of each sequence.
promoter seq.
leader seq.
gene seq.
termination seq.
promoter sequence – needed to provide a binding site for RNA polymerase to
start transcription
leader sequence – needed as a binding site for the small sub-unit of the
ribosome to start translation
termination sequence – needed for the termination of transcription
b) NASA decides it only wants its DNA in eukaryotic organisms. What
would you need to change in your ‘complete’ gene to get expression in a
eukaryote? Briefly explain your reasons.
You would need to change the promoter and termination sequence for
transcription and the leader sequence for translation. Enzymes in eukaryotes
recognize different base sequences than the enzymes needed for transcription
and translation in prokaryotes.
5. You have been so successful with your first Mars sample NASA gives you a
complete sample from the first organism found on Mars. You isolate the DNA
polymerase from the sample and discover that it does not need an open 3’ end to
start replication. How would this change how DNA replicates?
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Name: __________________________
You would not need to have primase or RNA nucleotides to start replication.
You would still get Okazaki fragments due to the anti-parallel nature of DNA
and the fact that DNA polymerase can only synthesize DNA in one direction.
In addition, if you are dealing with a eukaryotic system you would need to add
telomerase to insure that the ends of any linear DNA were properly replicated.
6. You decide to replicate the Mars DNA so you buy a DNA replication kit. It
supposedly carried all the components for replication. In replicating the DNA
you discover a couple of problems:
a) The DNA sequences are not complete; on one strand there appear to be
fragments instead of one continuous strand. What is the problem and how
would you fix it?
The synthesized pieces of the lagging strand are not being connected (so the
Okazaki fragments are remaining as fragments) indicating that ligase is missing.
The solution then is to add ligase.
b) If you had the unique Mars DNA polymerase (from problem 5) would this
problem have occurred? Briefly explain your reasoning.
Yes, DNA polymerase does not have the ability to connect the fragments so even
without needing an open 3’ end the enzyme could not make the necessary
connection and ligase would still need to be added.
7. You next try to transcribe your replicated Martian DNA so you buy a
transcription kit. You appear to get only random lengths of mRNA that do not
appear to code for anything.
a) What is causing the production of random lengths of mRNA and how
would you solve the problem?
If random lengths of mRNA are being produced it means the RNA polymerase is
not binding in the correct location. This indicates that the proper sigma factor is
not present or the sigma factor is completely missing. The sigma factor is
necessary to properly orientate the core enzyme of the RNA polymerase in the
proper location (in the promoter region). Solution, add the sigma factor that
corresponds to the sequence in the promoter region.
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Name: __________________________
b) If you solve the first problem you start getting transcripts of the same
length but they all appear to be too long. What could be causing the
production of extremely long transcripts and how could it be repaired?
The problem is either with the termination sequence for transcription being
missing or having the wrong sequence or that the termination for transcription
of this gene is rho dependent and rho is missing. Depending on the problem the
solution would be to either change/add the proper termination sequence or add
the missing rho factor.
8. You now translate your mRNAs even though they are too long. Would you
expect to get a functional polypeptide? Briefly explain your answer?
Yes, if the stop codon is in the correct location translation will function normally
and produce the proper length polypeptide even if the transcript is too long.
9. You get tired of working on extraterrestrial life so you decide to work on
eukaryotic genes. You isolate two genes and put them into a prokaryote. For the
first sample you discover you get a non-functional protein but for the second
sample you do get a functional protein.
a) Why would translation of the first gene produce a non-functional protein?
There are introns present that the prokaryote can not process out. This results
in the transcript having unnecessary bases resulting in a polypeptide with
undesired amino acids resulting in a non-functional protein.
b) What is different with the second gene?
This is a case of a eukaryotic gene that does not have any introns. So when the
gene is transcribed and translated in a prokaryote the message has no extra
bases resulting in a polypeptide that does hot have any extra amino acids.
Extra credit
PCR (polymerase chain reaction) is a method to rapidly replicate small pieces of DNA
using a single enzyme. Please explain how this is possible and what is replacing the
other enzymes needed for normal DNA replication.
Heat is used to separate the DNA strands replacing the functions of helicase, gyrase,
topoisomerase and the single-strand binding proteins. Primers are then added so
primase is not needed. The DNA being replicated is small so no fragments are
produced so ligase is not needed. All that is needed is a heat tolerant DNA
polymerase.
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Name: __________________________
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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