We have identified a related group of genes which specify chloramphenicol-inducible, chloramphenicol acetyltransferase in strains of Bacillus pumilus. Among several such cat genes cloned into B. subtilis, cat-86 was selected as the indicator gene for the construction of a B. subtilis promoter-cloning plasmid, pPL603. The cloned B. pumilus DNA in pPL603 has been sequenced and the regulatory signals necessary to chloramphenicol-inducible gene expression have been localized to a 234 bp region of the DNA. This 234 bp region spans the cat-86 ribosome binding site and the first 29 codons of cat-86. Several lines of evidence suggest that the chloramphenicol-inducibility of cat-86 expression may result from post-transcriptional control or a coupled transcriptional and post-transcriptional control system: inducibility seems independent of the promoter used to activate the gene; cat-86 mRNA is present in cells grown in the presence or absence of chloramphenicol but at different levels when the gene is activated by a weak promoter; sequences spanning the cat-86 ribosome binding site region in DNA should result in the sequestering of the ribosome binding site in RNA through the formation of a stem-loop configuration. This proposal describes experiments to analyze the regulation of cat-86. Two approaches will be used to introduce mutations into the plasmid which cause constitutive expression of cat-86: a random approach and a site directed method. The minimum region of the 234 bp fragment which is responsible for chloramphenical-inducibility will be identified by exonuclease digestion. Host gene products (possibly ribosomal proteins) may contribute to the regulation, and host mutations will be sought that alter inducibility. Attempts will be made to achieve in vitro induction of cat-86 using a coupled transcription-translation system. Analysis of the transcription of cat-86 in vivo, by hybridization, and in vitro using purified RNA polymerase will be performed on derivative plasmids in which cat-86 is activated by a strong, phage promoter, or by a """"""""natural"""""""" cat promoter.
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