What mechanisms do differentiating cells use to activate particular sets of genes in the proper sequence to ensure an orderly progression from one cell type to another? Possible answers to this question will be sought by examining the control machanisms that activate genes at late stages of endospore formation in the bacterium Bacillus subtilis. During sporulation this organism undergoes a sequential alteration in morphology and physiology that may be regulated, at least during the early stages, by modification of the cell's RNA polymerase. The regulatory factors that activate genes transcribed at late stages of spore formation as well as the timing mechanism that permits these factors to function at this particular stage of development are unknown. We will investigate whether further RNA polymerase modifications could play a role in late gene transcription by isolating this enzyme at several times during the final half of endospore formation to examine its subunit composition and in vitro transcriptional specificity. In addition, RNA's that are uniquely synthesized during these periods will be detected. DNA sequences that are complementary to these RNA's will be cloned and used as templates for in vitro transcription. The transcribing enzymes will be either partially purified RNA polymerases that were isolated during """"""""late sporulation"""""""" or, if these enzymes fail to transcribe the cloned sequences, then crude protein extracts prepared from cells that had progressed to this late stage. The factors responsible for """"""""late transcription"""""""" would be identified and purified using their activity in the in vitro transcription assay as a guide. The putative regulatory factors would then be analyzed to determine how they are able to perform their function only at a particular stage in the developmental program. Monoclonal antibodies will be prepared against anticipated novel RNA polymerase subunits and/or other late transcription factors for use as immunological probes to determine when these factors are present in cells as well as conditions that influence their synthesis. The antibodies would also be used in the development of an antibody-based rapid purification system for these proteins. Polyclonal antibody and oligonucleotide probes will be prepared and used to clone the structural genes that code for the late transcription factors. Once cloned, these genes could then be analyzed by gene fusion and in vitro transcription techniques.
