This proposal is divided into two parts: a) the structural organization of the genetic region that encodes the polypeptides of aspartate carbamoyl transferase (ATCase) and b) the regulation of the transcriptional expression of these loci. It is necessary to elucidate the general structural organization before a complete understanding of the genetic regulation can be described. Current status: We have cloned the genetic regions encoding the ATCase polypeptides and identified a 1400 base pair fragment containing both cistrons, designated pyrBr and pyrBc (specifying the regulatory and catalytic polypeptides respectively). Furthermore, we have removed pyrBr by restriction endonuclease deletion while maintaining the controlled expression of pyrBc. Research objectives: 1) Develop a genetic fine structure map defining the limits of the regulatory and catalytic cistrons by restriction endonuclease analysis and transposon mutagenesis. 2) Determine the nature of the mRNA, mono or dicistronic, by immunoprecipitation of in vitro translational products. 3) Determine the nucleotide sequences of selected regions of pyrB (ultimately the entire region) by the Sanger technique. 4) Establish the nature of the physiological expression of pyrBr and pyrBc relative to one another by enzymatic assay and message production in response to manipulated endogenous pool concentrations. 5) Analyze the trans expression of pyrBr and pyrBc cistrons simultaneously present in the same cell (e.g. c plus r minus on pBR322 and c minus r plus on pSC101 and vice versa in a complete pyrB minus background). 6) Select additional regulatory variants displaying anomalous expression of the pyrB cistrons by utilizing our recently constructed op(pyrB):lacZ fusion (Casadaban technique). It has not escaped our notice that the overall regulation of pyrBr and pyrBc has not been defined. The expression of pyrBr seems to be dependent on the pyrBc gene product whose expression is dependent on some hitherto unidentified regulatory system. Ultimately, we anticipate that these studies will lead to a clear definition of the structure and expression of the pyrB cistrons encoding ATCase in E. coli.