PMID: 2500432Jul 5, 1989Paper

Requirement for a 1-kilobase 5'-flanking sequence for barbiturate-inducible expression of the cytochrome P-450BM-3 gene in Bacillus megaterium.

The Journal of Biological Chemistry
L P WenA J Fulco

Abstract

In a previous publication (Wen, L.-P., and Fulco, A. J. (1987) J. Biol. Chem. 262, 6676-6682), we described the cloning of the gene encoding cytochrome P-450BM-3, a catalytically self-sufficient fatty acid monooxygenase induced by barbiturates in Bacillus megaterium. We have now subcloned a 1.6-kilobase segment of DNA from this cloned gene that includes the barbiturate-responsive regulatory region as well as 88 bases encoding the NH2-terminal portion of cytochrome P-450BM-3. From this, we generated two series of 5' and 3' deletion derivatives and examined their effects on gene expression. When the 1.6-kilobase fragment or the 1.3-kilobase 5'----3' deletion derivative is inserted into Escherichia coli on a vector containing a promoterless chloramphenicol acetyltransferase (CAT) gene with the sequence encoding the NH2-terminal portion of P-450BM-3 placed immediately in front of the CAT gene, CAT activity is constitutive and unaffected by pentobarbital. On the other hand, the basal level of CAT is low in B. megaterium transformed by the same construct but is strongly inducible by pentobarbital. Furthermore, the multicopy plasmid containing this regulatory region causes a dramatic decrease in both the basal and pentobarbital-induce...Continue Reading

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