Programmed sequences of gene expression are ubiquitous in biology. None is completely understood, and, in particular, very little is known about mechanisms by which genes are sequentially shut off. This project will analyze sequential gene shut-off during infection of Bacillus subtilis by bacteriophage SPO1. In the initial experiments, two categories of genes will be studied, the host rRNA genes, which are not shut off during infection, and the SPO1 early genes, which are completely shut off by a few minutes after infection. First the two types of promoters will be tested to confirm that the difference in shut-off behavior of the genes resides in their promoters. Promoters from representatives of each category will be isolated and integrated into the host chromosome, each at the same site so each controls transcription of the same reporter gene. Cells containing the different cloned promoters will be infected by wild-type SPO1, and the time and extent of shut-off of each promoter will be measured by pulse-labeling the reporter gene transcripts. A possible role of the unusual base, hmUra, will be explored by cloning certain promoters in the SPO1 genome, and assaying them in the same way. Then, the sites within the promoters that cause some to be shut off, while others are not, will be identified. Chimeras will be formed between promoters with different regulatory specificities, and their regulation will be assayed in the same way as above. Where appropriate, deletion analysis will be used to confirm the conclusions drawn from analysis of chimeras. Once these initial experiments are completed, similar experiments will be performed with representatives of other categories of genes, which are turned off at different times during infection, and which therefore respond to different shut-off mechanisms. However, it is not anticipated that these additional experiments can be completed within this project period. When a virus infects a bacterial cell, a complex series of changes in gene expression occurs. Some, but not all, of the host cell's genes are turned off. The virus'es own genes are turned on in a very strictly controlled order; deviation from this order results in partial or complete failure of the infection process. As part of this strictly controlled series of events, particular viral genes are also turned off at closely specified times. It is emerging from some of Dr. Stewart's prior studies that close control of turn-off of certain viral genes is just as critical for the success of the infection as is control of the time of turn-on of other viral genes. Over the last 30 years a great deal has come to be know about mechanisms of specific gene turn-on, but as yet virtually nothing is known about mechanisms of specific gene turn-off. This is a proposal to elucidate some of these important but as yet obscure mechanisms.
