You mutated the DNA polymerae I gene so that it is temperature sensitive and the

Question

You mutated the DNA polymerae I gene so that it is temperature sensitive and therefore inactivated with temperatures higher than 42 degrees celsius. You grew E. coli in 3H medium for 24 hours at 30 degrees C, which labeled the DNA. Then you palced the cells in non-labeled medium, split them into two tubes and incubated one tube at each of the following temperatures : 30 degrees C and 45 degrees C for 2 hours. After incubation you collected an denatures all the DNA, and pun the DNA in a sucrose gradient. What you observe in the gradient tubes at each temperature? Explain the differences overseved at the two temperatures.

Explanation / Answer

DNA polymerase I is required for the replacement of RNA primers with DNA on the lagging strand.

So, the tube incubated at restrictive temperature can not form a continuous strand by joining Okazaki fragments on the lagging strand.

So, we would observe small fragments on the gradient tube.

Tu=he tube grown at permissive temperature can complete DNA replication properly. So, we would observe a single crisp band.

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