THE MOLECULAR-CLONING AND CHARACTERIZATION OF BM1P1 AND BM1P2 PROTEINS, PUTATIVE POSITIVE TRANSCRIPTION FACTORS INVOLVED IN BARBITURATE-MEDIATED INDUCTION OF THE GENES ENCODING CYTOCHROME P450(BM-1) OF BACILLUS-MEGATERIUM

Analysis of a 1,3-kilobase segment of 5'-flanking DNA from the barbitu rate-inducible P450(BM-1) gene (CYP106) of Bacillus megaterium reveale d two open reading frames. One, BMIP1, encodes 98 amino acids and is l ocated 267 base pairs upstream from the sequence encoding cytochrome P 450(BM-1) but in the opposite orientation, The second, B2M1P2 (88 amin o acids), is 892 base pairs upstream from the P450(BM-1) coding sequen ce and in the same coding strand. The expression of BM1P1 and BM1P2 wa s strongly stimulated in cells grown in the presence of pentobarbital, and the BM1P1 gene product exerted positive control on expression of P450(BM-1), When a 177-base pair fragment encompassing the overlapping promoter regions of the P450(BM-1) and BM1P1 genes was used as a prob e in DNA binding assays, the BM1P1 and BM1P2 gene products and Bm3R1 ( the repressor protein regulating the barbiturate-mediated expression o f P450(BM-3)) could bind individually, but the addition of BM1P1 or BM 1P2 to a binding mixture containing Bm3R1 completely prevented the app earance of a Bm3R1 binding band, When a 208 base pair fragment contain ing a Barbie box sequence and located upstream of the 177-base pair fr agment was used as a probe, only a Bm3R1 binding band was detected, Al though neither BM1P1 and BM1P2 appeared to bind to this 208-base pair fragment, their presence strongly inhibited the binding of Bm3R1 to th e same probe, The evidence suggests that BM1P1 and BM1P2 may, in part, act as positive regulatory proteins involved in the expression of the P450(BM-1) gene by interfering with the binding of the repressor prot ein, Bm3R1, to the regulatory regions of P450(BM-1).