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Download 1 A. You have the following piece of genomic DNA with the two
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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): 3 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