Download 1 A. You have the following piece of genomic DNA with the two

Seroudeʼs lectures
MBIO218 Tutorial
2009
_______________________________________________________________________________________
A. You have the following piece of genomic DNA with the two strands and the location of
various restriction enzyme recognition sequences indicated:
( HindIII: A↓AGCTT
EcoRI: G↓AATTC
KpnI: GGTAC↓C )
You perform the following steps:
i.
Digest the DNA with EcoRI
ii.
Add DNA polymerase and radioactive deoxyribonucleotides (αP32)
iii.
Heat the reaction at 100˚C for 10min
iv.
Slowly cool down the reaction
v.
Digest with KpnI
vi.
Separate fragments by electrophoresis
vii.
Purify the 380bp fragment
viii.
Use the purified fragment as a Northern-blot probe
1. If after step ii, you separate the fragment and perform an autoradiography, draw the
expected result
2. If after step vi, you perform an autoradiography, draw the expected result
3. Why is necessary to perform step iii? (hint: what are enzymatic activities of the DNA
polymerase)
4. Why is it necessary to perform step iv? (hint: What are the properties of restriction
enzymes)
5. The northern-blot does not show any bands (blank). What do you conclude?
6. You repeat the same steps, but this time you use HindIII in the step v and purify the
300bp fragment. This time, the northern-blot reveals a 480bp band. What can you
conclude?
7. How can you modify the protocol in order to reach the same conclusion in 5 steps
instead of 8?
Additional questions for problem A. (optional)
You digest the DNA with EcoRI, HindIII or KpnI (single digest) and purify respectively the
450bp (EcoRI), 400bp (HindIII) or 460bp (KpnI) fragment. Each fragment is incubated
(separately, one at a time) by a cell extract in presence of radioactive ribonucleotides
(αP32). After incubation, you load the reaction on an electrophoresis gel and perform
autoradiography.
8. What is the purpose of such experiment?
The EcoRI, HindIII and KpnI fragments yield respectively a 300bp (EcoRI), 150bp (HindIII)
and 380bp (KpnI) band.
9. What can you conclude?
10. Name an alternative strategy that uses the reverse transcriptase enzyme and that can
be used to confirm this conclusion?
1
Seroudeʼs lectures
MBIO218 Tutorial
2009
_______________________________________________________________________________________
Here is the complete nucleotide sequence of this DNA (top strand, 5ʼ-3ʼ):
10
20
30
40
TTCTCATGTT TGACAGCTTA TCATCGATAT GCTTTAATGC
60
70
80
90
AGCTTTTAAA TTGCTAACGC AGTCAGGCAC CGTGTATGAA
110
120
130
GCGCTCATCG TCATCCTCGG CACCGTCACC
160
170
180
AATTCTTATG CCGGTACTGC CGGGCCTCTT
Kpn1
210
220
230
ACAGCATCGC CAGTCGGTAC CGCGTGCTGC
260
270
280
CAATTTCTAT GCGCACCCGT TCTCGGAGCA
310
320
330
CCGCCCAGTC CTGCTCGCTT CGCTACTTGG
360
370
380
TCATGGCGAC CACACCCGTC CTGTGGATCC
140
CTGGATGCTG
190
GCGGGATATC
240
TAGCGCTATA
290
CTGTCCGACC
340
AGCCACTATC
390
TCTACGCCGG
410
420
430
440
GCCGGCATCA CCGGCGCCAC AGGTGCGGTT GCTGGCGCCT
460
470
480
490
AGCTTCCGAT GGGGAAGATC GGGCTCGCCA CTTCGGGCTC
510
520
530
540
GTTTCGGCGT GGGTATGGTG GCAGGCCCCG TGGCCGGGGG
Hind3
50
GGTAGTTTAA
100
ATCTAACAAT
EcoR1
150
TAGGCATAGG
200
GTCCATTCCG
250
TGCGTTGATG
300
GCTTTGGCCG
350
GACTACGCGA
400
ACGCATCGTG
Hind3
450
ATATCGCCGA
500
ATGAGCGCTT
550
ACTGTTGGGC
EcoR1
600
TCAACGGCCG
650
AAGGGAGAGC
560
570
580
590
GCCATCTCCT TGCATGCACC ATTCCTTGCG GCGGCGGTGC
610
620
630
640
AATTCTACTA CTGGGCTGCT TCCTAATGCA GGAGTCGCAT
Kpn1
660
670
680
690
700
GTCGACCGAT GCCCTTGAGA GCCTTGGTAC CAGTCAGCTC CTTCCGGTGG
710
720
730
740
750
GCGCGGGGCA TGACTATCGT CGCCGCACTT ATGACTGTCT TCTTTATCAT
760
770
780
GCAACTCGTA GGACAGGTGC CGGCAGCGCT
11. Give the 5ʼ-3ʼ sequence of the primer you would use for this alternative strategy.
12. Name another alternative strategy that can be used to confirm this conclusion?
13. Describe the steps you would use to perform this second alternative strategy?
14. Taking into account the conclusions you have made up to this point, examine the
provided sequence. What can you conclude about the nature of the gene that is under
study with this DNA?
15. How could you modify the experiment in step 8 to gain further information about the
nature of this gene?
2
Seroudeʼs lectures
MBIO218 Tutorial
2009
_______________________________________________________________________________________
B. The following diagram shows the analysis of RNA polymerase activity of the fractions
obtained by chromatography:
1. Draw this diagram if the analysis of the fractions is done in presence of 0.1µg/ml of αamanitin.
2. Draw this diagram if the analysis of the fractions is done in presence of 0.1mg/ml of αamanitin.
3. What modifications would you do in order to demonstrate that RNA PolI is solely
responsible for the transcription of the gene encoding the large rRNA precursor?
Relevant lecture information (provided at the exam):
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Seroudeʼs lectures
MBIO218 Tutorial
2009
_______________________________________________________________________________________
C. Following epitope tagging and immunoprecipitation, the different
subunits of the RNA Pol II can be revealed (right figure)
The same experiment is repeated but after the immunoprecipitation,
the proteins are fractionated by chromatography according to their
size. The fraction containing proteins ranging between 80 and
800KDa is analyzed by electrophoresis and the following result is
obtained:
1. Interpret the result.
2. Propose a genetic experiment(s) to demonstrate that the lower band is Rpb2
3. Propose a biochemical experiment(s) to demonstrate that the lower band is Rpb2
4. Propose experiment(s) to confirm your interpretation.
Additional problems (optional)
D. Propose an experiment that would allow you to demonstrate that the RNA polymerase
synthetize RNA in the 5ʼ to 3ʼ direction.
E. Propose an experiment that would allow you to demonstrate that TFIIH phosphorylate
the CTD of Rpb1 subunit of the RNA Pol II.
F.
In the lectures, we examined an experiment to determine by which mechanism
enhancers stimulate transcription. Based on your knowledge of gene regulation and how it
involves interactions between trans-acting regulators and cis-regulatory sequences,
propose an alternative strategy you could use to demonstrate that the tracking model is not
correct.
G. You should now be familiar with many enzymes that are used in molecular biology to
manipulate (modification, digestion, synthesis, degradation..) DNA and RNA. Using this
knowledge, propose an experiment to demonstrate that the 5ʼ of RNA is modified (capping)
in Eukaryotes.
H. Why is it not possible to determine the 3ʼ end of a transcript with the primer extension
technique?
4
Seroudeʼs lectures
MBIO218 Tutorial
2009
_______________________________________________________________________________________
I. You study a neurodegenerative disease caused by a viral infection of the brain. You
isolated a DNA fragment that contains an enhancer for a viral gene necessary for the
pathogenic effects of the virus (without this gene, the virus is harmless). You prepare cell
extracts from neurons, glial cells, blood cells and lung cells. Each extract is incubated with
the DNA fragment and the results analyzed with a band shift assay. The following results
are obtained:
1
2
3
4
5
1: no extract added
2: lung extract
3: blood extract
4: neuron extract
5: glial extract
1. What can you conclude?
2. Each extract is incubated with the DNA fragment and the results analyzed with an S1
nuclease assay. The following results are obtained:
1: no extract added, no S1 added
2: no extract added
3: lung extract
4: blood extract
5: neuron extract
6: glial extract
3. What are your conclusions about the enhancer under study?
4. Based on those two results, give an explanation why this virus does not cause any
pathology in the blood?
5. Based on those two results, give an explanation why this virus does not cause any
pathology in the lung?
6. Describe an experiment to prove or disprove the explanation provided in 5.
5