The chief objective of this project is to investigate the molecular mechanism of genetic control of the histidine operon of Salmonella typhimurium. The major control over expression of this operon is at the level of transcription, utilizing an attenuator mechanism. I propose to DNA sequence a substantial number of the 150 available mutations in the target genetic control signals, with the application of M13 cloning, classical recombination, and the dideoxy method of DNA sequencing. I will also investigate the in vitro transcription behavior of the mutations. I will apply the footprinting method to determine the binding sequences recognized by RNA polymerase at the promoter and while dynamically interacting with the terminator of the attenuator. I propose to determine the DNA sequence of the entire histidine operon, while developing an improved technical strategy for this purpose, so that 8 kb can be sequenced accurately in two to four man-months. I will then determine several mutational base changes in the genes of the operon, beginning with those used by the Ames test, which uses this same DNA to test for possible carcinogens among environmental chemicals.
