Induction of two cytochrome P450 genes, Cyp6a2 and Cyp6a8, of Drosophila melanogaster by caffeine in adult flies and in cell culture.
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.
Bhaskara
Srividya
S
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, 37996, USA.
Dean
Erika Danielle
ED
Lam
Vita
V
Ganguly
Ranjan
R
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
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