Date of Award
Master of Science
Biochemistry and Cellular and Molecular Biology
Mariano Labrador, Bruce D. McKee
Cytochrome P450 enzymes (CYPs), found in almost all organisms, are involved in endobiotic metabolism and detoxification of xenobiotic compounds, such as drugs, pollutants, and insecticides. In insects, CYPs play a major role in conferring resistance to various insecticides including DDT. In Drosophila and other insects, DDT-resistant strains exhibit increased expression of multiple P450 genes; however, the mechanism of overexpression is unknown. Since many CYP genes including Cyp6a8 of Drosophila are induced by caffeine and other xenobiotics, these chemicals are used as tools to understand the regulation of these genes. Previously it was shown that the 0.8-kb (-1/-732) and 0.2-kb (-1/-170) upstream DNA of Cyp6a8 of the DDT-resistant 91-R strain support caffeine, DDT, and Phenobarbital induction in adult flies and S2 cells, the 0.2-kb DNA has many transcriptionally important sequence motifs. In the present investigation, site-directed mutagenesis was performed on the putative TATA box and CREB/AP-1 motifs located at the -97/-101, -57/-61, -43/-47, and -6/-10 regions of the 0.2- and 0.8 DNAs to determine their cis-regulatory role in caffeine and PB induction in S2 cells using luciferase reporter system. Results showed that all four deletions in 0.2- and 0.8-kb DNA decreased both basal and caffeine-induced activities, but maximum effect was seen with the -57/-61 deletion. Second, the TATA mutations greatly decreased basal activity, but they did not decrease caffeine-inducibility as much as the -57/-61 mutations. Third, the effects of other three deletions on basal activities were not as pronounced in the 0.8-kb environment as were seen in the 0.2-kb environment. Taken together these results suggest that of all four putative CREB/AP1 sites the one located at -57/-61 region is most important for both basal and caffeine-induced activities. The results also suggest that the additional 600 bases upstream of -1/-170 have distal elements that interact with the proximal promoter in the 0.2-kb DNA and boost basal transcription. A model suggesting interactions of all cis elements with the basal promoter for basal and induced transcription has been proposed.
Hill, Olivia Nichole, "Functional analysis of the Cyp6a8 gene promoter of Drosophila melanogaster for caffeine- and Phenobarbital-inducibility by site-directed mutagenesis. " Master's Thesis, University of Tennessee, 2011.