Characterization of DNA Mutations Caused by MNNG Using a Shuttle Vector System.

Abstract

This project was designed to be a background reference when examining the mutations caused by nitrosamine-derivatives, direct-acting carcinogens, that cause lung cancer. Because the nitrosamine derivatives react via a similar mechanism as MNNG, it serves as a useful reference. The alkylating agent MNNG is a known carcinogen for many organisms and is a likely carcinogen for humans. We are interested in the mutations that it causes in human cells, but the mutation will be expressed in a bacterial host strain. Treating the plasmid, pSP189, with MNNG will induce damages in the DNA located approximately every thousand base pairs. pSP189 is a shuttle vector which contains a mammalian origin of replication in addition to a bacterial origin of replication. This specific vector is also unique due to its eight base pair signature sequence, which allows an entire population of plasmid to be scanned at once. Once treated, the plasmid is transfected into a human cell line. After replication has occurred, the plasmid is then recovered as low molecular weight DNA and transformed into the host E.Coli strain MBM7070. The colonies undergo blue-white selection; colonies containing a mutation in the target supF gene will appear white. These colonies? mutations are then analyzed for frequency, mutation location, and mutation type.