Alpha-amylase synthesis in Bacillus subtilis 168 is activated at the end of exponential growth, is apparently not inducible, and is repressed by more-readily-metabolized carbon sources, such as glucose. The mechanisms of Alpha amylase regulation are currently unknown. We find that the activation and the repression of Alpha-amylase synthesis are distinct, genetically separable events. We have determined that a cloned copy of the B. subtilis 168 Alpha amylase gene contains the flanking DNA necessary for the regulation of its expression. We have localized the regulatory DNA sequences to the 5' end of the cloned Alpha-amylase gene. We propose experiments to further characterize at the molecular level the activation and repression of Alpha-amylase synthesis, and to define the flanking DNA sequences involved in Alpha-amylase regulation. Specifically, we propose: 1. Quantitation of Alpha-amylase mRNA levels, 2. Determination of the start site(s) of transcription for the Alpha-amylase gene, 3. Characterization of in vivo-generated cis-regulatory mutations, 4. Characterization of in vitro-generated regulatory mutations. 5. Isolation & localization of mutations which act in trans. For the latter, we propose a novel technique for the isolation of mutations in genes encoding factors which act upon the flanking regulatory DNA to modulate Alpha-amylase expression. Elucidation of the mechanism of Alpha-amylase regulation may provide insights into the regulatory mechanism governing the expression of other early postexponential genes in B. subtilis.
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