Question

1. In class we discussed temperature sensitive mutants that are functional at lower temperatures but non-functional at higher temperatures (e.g. above 42 "C). Although less common, there are also examples of cold sensitive mutants that are functional at higher temperatures but non functional at lower temperatures (eg. below 28 °C). Instead of performing a normal pulse-chase experiment, we can combine the pulse-chase with a temperature shift. The experiment is performed by first growing cells at a permissive temperature (30-35 °C) in the pres cold dNTPs and then shifting the cells to a higher temperature (45 "C) and adding radioactive dNTPs. The chase is then performed at a lower temperature (25 °C) with cold dNTPs. I will call this a high temp pulse/low temp chase. Note that temperature sensitive mutations will be non- functional during the pulse and cold sensitive mutations will be non-functional during the chase What results would you expect if you performed a high temp pulse/low temp chase experiment with a temperature sensitive mutation in the gene for DNA Polymerase 1 and cold sensitive mutation in the gene for DNA ligase. The tube on the left is after the pulse and the tube on the right is after the chase. Using the following illustrations of a replication fork immediately after the pulse (top) and then after the chase (bottom), draw the newly synthesized DNA with an arrow at the end (m ) to indicate the direction of synthesis and use cross-hatching (XXXXXX) to indicate radioactive nucleotides. Use a squiggly line to indicate RNA (M ). For any radioactive fragment, indicate where it is in the appropriate tube above. Label the 5 and 3 ends of all fragments. (10 points) DNA Polymerase I non-functional in in pulse DNA ligase non-functional pulse