exm1_ans

exm1_ans - MIT Department of Biology 7.28, Spring 2005 -...

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______________________________ MIT Department of Biology 7.28, Spring 2005 - Molecular Biology 7.28 Spring 2005 Name Exam One Question 1 (28 Points). Your lab is studying a novel thermophilic eukaryote called S. mokin that has an optimum growth temperature of 75°C. Surprisingly, it has a circular genome and your student has decided to identify its origin(s) of replication. Another student has found that some S. mokin cells contain a 4 kb plasmid that confers resistance to Chromium. Based on this finding, your student wants to use a plasmid rescue assay to identify the genomic origin in S. mokin . As a first step, they use 2D gel analysis to locate the origin of replication in the plasmid and obtain the following results. Bam HI Probe 1 Probe 2 4000/0 1000 2000 3000 Sal I Probe 3 Xho I Sma I Eco R1 Hind III Eco RI and Sma I/Probe 1 Bam HI and Sma I/Probe 2 Increasing Shape Increasing Size Increasing Size Increasing Shape Increasing Size Increasing Shape Increasing Size Increasing Shape and Bam HI /Probe 3 Hind III and Sal I/Probe 3 Eco RI 1A (6 Points). Based on their data, what two enzymes do you suggest that your student use to cut the plasmid and genomic DNA for the plasmid rescue assay? Explain your reasoning. 1
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7.28 Spring 2005 Exam One Name ______________________________ The bubble to y arc gels are the only gels that give you useful information in this experiment. They show that the origin is between EcoR1 and SmaI, and HindIII and SalI. As long as there is only one origin in this plasmid (as was stated in the question), the origin must be in the sequences that overlap these two regions, namely between EcoRI and HindIII. Some people wanted to take out the whole region between SmaI and SalI (they received half credit), but in this sort of experiment, the less cut out of the plasmid the better and the data clearly shows where the origin is and is not. At the conclusion of your plasmid rescue assay you identify three non- overlapping DNA fragments that act as replicators. You are surprised since most archaebacterial genomes have only a single origin of replication. 1B (6 points). You want to know if there are multiple origins in the S. mokin genome. You take advantage of a DNA microarray that was developed by another lab to study gene expression. However, before you will buy the DNA microarrays, you insist that your students identify a conditional initiation mutant. Why do you need this mutant to perform the microarray analysis of DNA replication? Microarray analysis of DNA replication requires that you are able to arrest cells prior to the initiation of DNA replication and then release them from this arrest. This allows the investigator to: 1) have a population of unreplicated cells that can be used as an unreplicated control DNA sample and 2) obtain cells that are going through DNA replication synchronously. Partial credit was given for either of these two answers and full credit was given if both answers were included. Because of the thermophilic nature of the
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This note was uploaded on 11/11/2011 for the course BIO 7.344 taught by Professor Bobsauer during the Spring '08 term at MIT.

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exm1_ans - MIT Department of Biology 7.28, Spring 2005 -...

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