title: 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.
abstract: The levels of one or more cytochrome P450 (CYP) enzymes and the respective mRNAs are found to be higher in insecticide-resistant insects than in susceptible insects. To understand better how insects regulate the levels of CYPs, we examined the expression of the Cyp6a2 gene in various strains of Drosophila melanogaster. We also took a transgenic approach to understand the molecular mechanisms that are involved in strain variation of Cyp6a2 expression. RNA blot analysis showed that the constitutive expression of Cyp6a2 varies from strain to strain; the level of CYP6A2 mRNA is barely detectable in the underproducer ry506 strain, whereas it is very high in the overproducer 91-R and MHIII-D23 strains. 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 strains does not appear to have any role on the steady-state CYP6A2 mRNA level. We also found that the Cyp6a2 gene is inducible by barbital in 91-R, ry506 as well as 91-C, which carries an LTR insertion. To examine the genetic background of the underproducer ry506 strain with respect to Cyp6a2 expression, we transformed the ry506 strain with the Cyp6a2 allele of the overproducer 91-R strain (Cyp6a2-91 R) and measured the constitutive and barbital-induced expression of the Cyp6a2-91 R transgene in the transformed flies. The Cyp6a2-91 R transgene carrying 129 bp of DNA upstream of the ATG codon did not show any constitutive or barbital-induced expression in the ry506 host genome. However, transgenes with 1331 and 985 bp upstream DNA showed similar levels of constitutive expression that were higher than that of the endogenous Cyp6a2 gene of the ry506 host strain, but lower than the expression of the 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 sequence analysis revealed that in both 91-R and ry506, the upstream DNA between +1 and -985 bp contains a distal and a proximal group of three potential barbie boxes, i.e. cis-elements that are thought to be involved in barbiturate-mediated induction of CYP genes. Except for four bases located near the distal cluster of barbie boxes and two other bases, the base sequence of the upstream DNA is identical in ry506 and 91-R strains. These results suggest that the underproducer ry506 strain has the trans-regulatory factors to support constitutive and induced expression of the Cyp6a2-91 R allele carrying DNA between -129 and -1331 bp regions. Possible reasons for low constitutive expression of the endogenous Cyp6a2 gene and moderate level of expression of the Cyp6a2-91 R allele in the ry506 genetic background are discussed.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville 37996, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Dombrowski
authors.forename: R
authors.initials: R
authors.lastname: Krishnan
authors.forename: M
authors.initials: M
authors.lastname: Witte
authors.forename: S
authors.initials: S
authors.lastname: Maitra
authors.forename: C
authors.initials: C
authors.lastname: Diesing
authors.forename: L C
authors.initials: LC
authors.lastname: Waters
authors.forename: R
authors.initials: R
authors.lastname: Ganguly
mesh_terms: D000483:Alleles; D000818:Animals; D001462:Barbital / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072472:Cytochrome P450 Family 6; D004247:DNA / Q000737:chemistry / Q000235:genetics; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D017344:Genes, Insect / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
chemical_list: D029721:Drosophila Proteins; D001462:Barbital; D012313:RNA; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; C109499:Cyp6a2 protein, Drosophila; D000072472:Cytochrome P450 Family 6