CONSTITUTIVE AND BARBITAL-INDUCED EXPRESSION OF THE CYP6A2 ALLELE OF A HIGH PRODUCER STRAIN OF CYP6A2 IN THE GENETIC BACKGROUND OF A LOW PRODUCER STRAIN

The levels of one or more cytochrome P450 (CYP) enzymes and the respec tive mRNAs are found to be higher in insecticide-resistant insects tha n in susceptible insects. To understand better how insects regulate th e levels of CYPs, we examined the expression of the Cyp6a2 gene in var ious strains of Drosophila melanogaster. We also took a transgenic app roach to understand the molecular mechanisms that are involved in stra in variation of Cyp6a2 expression. RNA blot analysis showed that the c onstitutive expression of Cyp6a2 varies from strain to strain; the lev el of CYP6A2 mRNA is barely detectable in the underproducer ry(506) st rain, whereas it is very high in the overproducer 91-R and MHIII-D23 s trains. The long terminal repeat (LTR) of mobile element 17.6 that is found in the 3' untranslated region (UTR) of the Cyp6a2 gene of some s trains does not appear to have any role on the steady-state CYP6A2 mRN A level. We also found that the Cyp6a2 gene is inducible by barbital i n 91-R, y(506) as well as 91-C, which carries an LTR insertion. To exa mine the genetic background of the underproducer ry506 Strain with res pect to Cyp6a2 expression, we transformed the ry(506) Strain with the Cyp6a2 allele of the overproducer 91-R strain (Cyp6a2-91R) and measure d the constitutive and barbital-induced expression of the Cyp6a2-91R t ransgene in the transformed flies. The Cyp6a2-91R transgene carrying 1 29 bp of DNA upstream of the ATG codon did not show any constitutive o r barbital-induced expression in the ry(506) host genome. However, tra nsgenes with 1331 and 985 bp upstream DNA showed similar levels of con stitutive expression that were higher than that of the endogenous Cyp6 a2 gene of the ry(506) host strain, but lower than the expression of t he same gene in the 91-R strain. Both these transgenes, with 1331 and 985 bp upstream DNA, also showed induction with 0.1 M barbital. DNA se quence analysis revealed that in both 91-R and ry(506), the upstream D NA between +1 and -985 bp contains a distal and a proximal group of th ree potential barbie boxes, i.e. cis-elements that are thought to be i nvolved in barbiturate-mediated induction of CYP genes. Except for fou r bases located near the distal cluster of barbie boxes and two other bases, the base sequence of the upstream DNA is identical in ry(506) a nd 91-R strains. These results suggest that the underproducer ry(506) Strain has the trans-regulatory factors to support constitutive and in duced expression of the Cyp6a2-91R allele carrying DNA between -129 an d -1331 bp regions. Possible reasons for low constitutive expression o f the endogenous Cyp6a2 gene and moderate level of expression of the C yp6a2-91R allele in the ry(506) genetic background are discussed. (C) 1998 Elsevier Science B.V. All rights reserved.