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MIT 7 012 - Problem Set 4

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NAME_____________________________________________________________TA__________________ SEC____7.012 Problem Set 4 FRIDAY October 15, 2004Answers to this problem set must be inserted into the box outsideProblem sets will NOT be accepted late. Solutions will be posted on the webQuestion 1NASA astronauts found a mysterious gene from the H zone of outer space. They neededyour help to know more about the gene, so they cloned it into a plasmid vector and toldyou they would send you the plasmid, that they named pSET (plasmid “Sequence Extra-Terrestrial”). When the package arrived, however, you found two unlabeled tubes.Not sure of what they sent you, you decide to make restriction maps of what’s in eachtube, assuming that there is plasmid DNA in each.You perform digestions with the restriction enzymes BglII (B), NotI (N) and SalI (S)either alone or in combination and obtain the following gel-electrophoresis data.BasepairsMIT Biology Department7.012: Introductory Biology - Fall 2004Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel2a) Draw the restriction maps of the plasmids in the space provided below. Write thedistances between the restriction sites. Also write down the total sizes of the plasmidsin the boxes below, in kilobases.b) Confused, you call NASA and ask them what’s going on and why they sent you twotubes. They do not know why you got two tubes, but they know that the gene of interesthad been cloned into the NotI and SalI restriction sites of the vector. Which of theplasmids has the gene? Circle your answer.Plasmid #1 Plasmid #2c) What is the length of the cloned gene in bases?500d) NASA also told you that the plasmid they used is a bacterial expression vector. Whichone of the following elements should be in the vector? Circle all that apply.centromeremultiple cloning sitesnuclear localization signaltelomereinducible terminatorbacterial origin of replicationselectable markernucloid insertion sitescytosolic targeting sequencesinducible promotertopoisomerase genegene encoding restriction enzyme Plasmid #1 kb Plasmid #2 kb35003500Bam H1Bam H1Sal 1Not 1Sal 110001000250050020003Question 2You rename the gene GOSH for “gene from the outer space H.” For some reason, NASAwants you to study the effect of expressing GOSH in amphibians. To express GOSH inamphibian cells, you have to clone it into an amphibian expression plasmid (pEA) with anamphibian promoter. Below is the map of pEA.a) Since pEA does not contain NotI and SalI sites, you decide to PCR-amplify GOSH withprimers that will replace the SalI and NotI sites with BamHI and XhoI sites. You knowthe sequences of the 5’ and 3’ ends of GOSH as they are in the bacterial plasmid: Start Stop 5’GTCGACatggtcgccatgcga………………………..tgctcgatatcgtaaGCGGCCGC3’ SalI NotIWhich of the following primer pairs would you use to successfully amplify the gosh genewith the desired restriction sites at either end? All primer sequences are written in the5’  3’ direction. Circle all that apply.Pair1: GTCGACATGGTCGCCATGCGA and GCGGCCGCTTACGATATCGAGPair2: TCGCATGGCGACCATGTCGAC and GGATCACTACGAGATCGAGCAPair3: GGATCCATGGTCGCCATGCGA and CTCGAGTTACGATATCGAGCAPair4: TCGCATGGCGACCATCTCGAG and TGCTCGATATCGTAAGGATCCPair5: CTCGAGATGGTCGCCATGCGA and GGATCCTTACGATATCGAGCABoth pair 3 and pair 5 will successfully amplify the GOSH gene with BamHI and XhoI sites at theend; however, only one pair, pair 5, will produce a PCR product that can be correctly inserted intopEA such that the GOSH gene will be expressed from the amphibian promoter.5’GGATCC 3’BamHI recognition sequence5’CTCGAG 3’XhoI recognition sequence5’GCGGCCGC 3’NotI recognition sequence5’GTCGAC 3’SalI recognition sequence4Now you try to express the new construct (pEA-GOSH) in frog (Xenopus) embryos.Hmm… Something seems wrong and the expected peptide is not expressed. Since PCR canintroduce errors, you sequence pEA-GOSH from its 5’ end and obtain the followingsequencing gel:b) What is the sequence in the gel? (in 5’  3’ direction) ATGGTCGCCATAGCGAc) Based on the sequence data, why is GOSH not expressed? Circle all that apply.i) Restriction enzyme recognition site is lost, resulting in an immature, nonfunctional peptide.ii) A nonsense mutation is introduced, introducing an early stop codon.iii) The promoter in the plasmid is lost, so GOSH cannot be expressed.iv) A frameshift mutation is introduced, so the encoded protein has a different primarystructure from Gosh.5Question 3After fixing the mutation, you express pEA-GOSH in frog embryos. The transformedfrogs show abnormal eye-tongue coordination. You call this phenotype Helpless. (See anartist’s depiction below.)To better understand the function of Gosh, you construct two more plasmids. The firstone is pEA-GFP, which will express the green fluorescent protein in amphibians. Thesecond one is pEA-GOSH-GFP, which will express a fusion protein of Gosh and GFP. Youexamine the impact of expressing these plasmids in frogs.How to keep GOSH-expressing frogs alive in the laboratory.Place image like so. (Otherwise they would starve to death.)frogs transformed with the pEA-GOSH construct:frogs transformed with pEA vector only:How to keep GOSH-expressing frogs alive in the laboratory.Place image like so. (Otherwise they would starve to death.)frogs transformed with the pEA-GOSH construct:frogs transformed with pEA vector only:How to keep GOSH-expressing frogs alive in the laboratory.Place image like so. (Otherwise they would starve to death.)frogs transformed with the pEA-GOSH construct:frogs transformed with pEA vector only:How to keep GOSH-expressing frogs alive in the laboratory.Place image like so. (Otherwise they would starve to death.)frogs transformed with the pEA-GOSH construct:frogs transformed with pEA vector only:How to keep GOSH-expressing frogs alive in the laboratory.Place image like so. (Otherwise they would starve to death.)frogs transformed with the pEA-GOSH construct:frogs transformed with pEA vector only:How to keep GOSH-expressing frogs alive in the laboratory.Place image like so. (Otherwise they would starve to death.)frogs transformed with the pEA-GOSH construct:frogs transformed with pEA vector only:How to keep GOSH-expressing frogs alive in the laboratory.Place image like so. (Otherwise they would starve to death.)frogs


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MIT 7 012 - Problem Set 4

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