#3: Answer due week of Oct. 12, 2009. #3A. An artificial plasmid is constructed. It is double-stranded circular DNA with one origin of replication— oriC (the E. coli chromosomal origin). This plasmid replicates with exactly the same requirements and by exactly the same mechanism as the E. coli chromosome. The plasmid has a unit length that we’ll define as 1 . It takes about 50 Okazaki fragments to make a whole circle. You start with a bacterial culture in which cells are actively dividing and the plasmid is actively replicating. Each plasmid in the culture is at a different stage in the replication process. a. You do a pulse-label experiment with radioactive thymidine, extract the DNA, and analyze it on an alkaline sucrose gradient, which displays single stranded DNA. Diagram what you expect to see. [Your diagram should show the distribution of radioactivity in ss DNA in relation to the expected position in the gradient of ss DNA of unit length (1) . This is tricky, so be very careful about deducing the lengths of ss DNA observed.]
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