title: Induction of two cytochrome P450 genes, Cyp6a2 and Cyp6a8, of Drosophila melanogaster by caffeine in adult flies and in cell culture.
abstract: To examine whether caffeine, the most widely used xenobiotic compound, would induce insect cytochrome P450 or CYP gene expression, upstream DNA fragments of Cyp6a2 (0.12, 0.26, 0.52 and 0.98-kb) and Cyp6a8 (0.06, 0.1, 0.2, 0.5 and 0.8-kb) genes of Drosophila melanogaster were individually fused to the firefly luciferase (luc) reporter gene. Promoter activities of these constructs were examined in Drosophila SL-2 cells using luciferase assays. Activity of 0.2- and 0.8-kb upstream DNA of Cyp6a8 was also measured in transgenic female flies. When these flies were treated with 2 mM pure caffeine or Vivarin caffeine, both DNA fragments showed a 4-5-fold induction of promoter activity. Endogenous Cyp6a8 and Cyp6a2 genes in these flies also showed caffeine-induced expression. In addition, both 0.2- and 0.8-kb DNAs showed differential basal and caffeine-induced activity in head, ovaries, gut, cuticle plus fat body and malpighian tubules. However, in all tissues 0.8-kb DNA always showed higher basal and caffeine-induced activities compared to the 0.2-kb DNA, suggesting that the additional DNA present in the 0.8-kb fragment has sequences that enhance both activities. In SL-2 cells, all reporter constructs of each Cyp6 gene showed significantly higher basal activity than the empty vector. Sequences that boost basal activity are located in -265/-129 and -983/-522 DNA of Cyp6a2, and -199/-109 and -491/-199 DNA of Cyp6a8 genes. While the 0.12- and 0.1-kb upstream DNAs of Cyp6a2 and Cyp6a8 genes respectively did not show caffeine-inducibility in SL-2 cells, the longest upstream DNA of each gene gave the highest level of induction. Caffeine-responsive sequences are not clustered at one place; they appear to be dispersed in -983/-126 and -761/-109 regions of Cyp6a2 and Cyp6a8 genes which also contain many binding sites for activator protein 1 (AP1) and cyclic AMP response element binding protein (CRE-BP). Significance of these binding sites in caffeine-inducibility has been discussed.
authors.affiliation: Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, 37996, USA.
authors.forename: Srividya
authors.initials: S
authors.lastname: Bhaskara
authors.forename: Erika Danielle
authors.initials: ED
authors.lastname: Dean
authors.forename: Vita
authors.initials: V
authors.lastname: Lam
authors.forename: Ranjan
authors.initials: R
authors.lastname: Ganguly
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002110:Caffeine / Q000494:pharmacology*; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072472:Cytochrome P450 Family 6; D017931:DNA Primers / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics*; D004331:Drosophila melanogaster / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D017344:Genes, Insect / Q000187:drug effects*; D017930:Genes, Reporter; D006899:Mixed Function Oxygenases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic
chemical_list: D017931:DNA Primers; D029721:Drosophila Proteins; D002110:Caffeine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C495067:CYP6A8 protein, Drosophila; C109499:Cyp6a2 protein, Drosophila; D000072472:Cytochrome P450 Family 6