Name: _____Answer Key___________ 1 Recitation Section or TA: 7.013 Exam Two -- 2007 Exam starts at 11:05 am and ends at 11:55 am. There are 10 pages including this cover page. Please write your name on each page. Only writing on the FRONT of every page will be graded. (You may use the backs, but only as scratch paper.) Questions that call for short answers should be limited to 15 words or fewer. Topic 1 10 pts________ Topic 2 24 pts________ Topic 3 8 pts________ Topic 4 12 pts________ Topic 5 14 pts________ Topic 6 20 pts________ Topic 7 12 pts________ TOTAL out of 100_______Name: _____Answer Key___________ 2Topic 1. Vasopressin is a small peptide secreted by the hypothalamus (part of the brain) into the bloodstream. Vasopressin has the following amino acid structure. FY CQ NGC P RFY CQ NGC P R The DNA sequence and the mature transcript from the human gene that encodes vasopressin are depicted on Page 9 of this exam. Based on the information provided here and on Page 9, answer the following questions. 1a. (2 pts.) How many exons does this gene have? 3 Name two processes that modify the primary vasopressin transcript to form the mature vasopressin transcript. 1b. (2 pts.) intron splicing 1c. (2 pts.) polyadenylation (also: 5’ capping) Name one post-translational modification that must be occurring if the primary translation product from the gene on Page 9 forms the vasopressin depicted above. 1d. (4 pts.) Proteolytic processing (cleavage of the primary polypeptide) or Formation of disulfide bond between C (cysteine) residuesName: _____Answer Key___________ 3Topic 2. Binding of brain-derived vasopressin to receptors on the surfaces of cells lining the kidneys will activate a signal transduction cascade as depicted here. Vasopressin (V) signal transduction in kidney cells. Heavy arrows indicate activation of an enzyme, pathway or process. Dotted arrows indicate chemical pathways, processes or movements. Abbreviations: AC = adenylylcyclase; PD = phosphodiesterase; PKA, P1, P2 and P3 = individual enzymes; RNAP = RNA polymerase; AQP2 = aquaporin 2a. (2 pts.)Is the vasopressin/receptor interaction an example of an autocrine, endocrine, paracrine, or amacrine pathway? endocrine 2b. (2 pts.) What name would best describe the enzyme that metabolizes GTP (indicated by the small circles labeled α, β, γ?) G-protein or GTPase 2c. (4 pts.) What is the advantage of having multiple enzymes in a protein kinase cascade? Signal amplification 2d. (4 pts.) Of all the labeled proteins in the figure, which would best be described as a possible transcriptional activator? P3 2e. (2 pts.) Only AQP2 tetramers (not monomers) in the cytoplasm form active water channels. The assembly of AQP2 tetramers is a regulated process. Would a chemical that inhibits the phosphorylation of P1 inhibit, enhance or have no effect on the expression of new AQP2 monomers? inhibit 2f. (2 pts.) Would a chemical that inhibits the phosphorylation of P1 inhibit, enhance or have no effect on the assembly of AQP2 tetramers from previously synthesized monomers? have no effect 2g. (4 pts.) Of the enzymes and proteins depicted in the above diagram, which one acts to suppress (or weaken) the vasopressin response? Explain in 15 words or fewer. PD weakens the vasopressin response by lowering the concentration of (metabolizing) cAMP in the cell 2h. (4 pts.) Diabetes insipidus is a disorder in which the hypothalamus fails to produce vasopressin. Based on the diagram above, what would be the most obvious symptom in someone who suffers from diabetes insipidus? Explain in 15 words or fewer. Frequent or excessive urination; excessive thirst/dehydration VβγαACGTPGDPATPPKAP1P2P3ATPADPATPADPATPADPRNAPAQP2 GeneNucleusAQP2AQP2AQP2AQP2AQP2AQP2AQP2AQP2AQP2AQP2AQP2H2OH2OH2OH2OAQP2Excretion(in urine)Re-absorptionof H2OVVVVPDAMPcAMP(from brain)VβγαACGTPGDPATPPKAP1P2P3ATPADPATPADPATPADPRNAPAQP2 GeneNucleusAQP2AQP2AQP2AQP2AQP2AQP2AQP2AQP2AQP2AQP2AQP2H2OH2OH2OH2OAQP2Excretion(in urine)Re-absorptionof H2OVVVVPDAMPcAMP(from brain)Name: _____Answer Key___________ 4 Topic 3. In order to develop a treatment for patients who do not make sufficient vasopression, you set out to use cultured E. coli to produce recombinant vasopressin. To begin, you will use PCR to amplify a portion of the vasopressin gene (underlined sequences on page 9). 3a. (4 pts.) Using the vasopressin gene sequence as presented on Page 9 of this exam, design two primers, each 10 bases long, that could be used to precisely amplify only the underlined portion of the gene (recall that your primers will become part of the resulting PCR product). Label the 5’ and 3’ ends of your primer sequences. Primer 1 sequence: 5’-CTCGGCCTAC-3’ Primer 2 sequence: 5’-CTGTCTCAGC-3’ 3b. (4 pts.) To express the recombinant vasopressin in E. coli, select the most appropriate promoter from the following list. Explain your selection (15 words or fewer). • The promoter from the uromodulin gene, which directs kidney-specific transcription • The promoter from the beta-actin gene, which is expressed in nearly every mammalian cell type • A constitutive promoter from the lambda phage, a virus that infects bacterial cells • The original promoter from the human vasopressin gene Of these promoters, only the lambda phage promoter will be active in the E. coli (bacterial) cells that you are using to produce the recombinant vasopressinName: _____Answer Key___________ 5Topic 4. You now have the vasopressin PCR product cloned in a plasmid vector. A portion of the expected plasmid sequence is shown below. 5’-CTCGGGCAGTTCTGGAAGTAGCACGCGGAGGAGAAGGCCA-3’ 3’-GAGCCCGTCAAGACCTTCATCGTGCGCCTCCTCTTCCGGT-5’ In order to verify that your PCR-product does not contain any errors (misincorporated bases) you choose to determine its sequence. You obtain the following primer: 5’-CAGTTCT-3’ You carry out a Sanger sequencing reaction using DNA polymerase, high levels of dATP, dCTP, dGTP, dTTP, low levels of labeled-ddATP, the primer, the DNA template (depicted above), some magnesium, and a buffer to control pH. After the reaction has gone to completion, you separate the reaction products on a sequencing gel and detect the labeled DNA. 4a. (12 pts.) Use the diagram to the right to indicate where the labeled reaction products would
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