Masters Theses

Date of Award

8-2008

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Ranjan Ganguly

Committee Members

Bruce McKee, Mariano Labrador

Abstract

Cytochrome P450 monooxygenases (CYP), a large family of enzymes, play an important role in insecticide resistance. Compared to the susceptible strains, the resistant strains of Drosophila show higher expression of one or more Cyp genes including Cyp6w1, which maps close to the resistance locus. In the present investigation I examined the Cyp6w1 gene’s role in DDT resistance and inducibility with caffeine, DDT and phenobarbital (PB). Utilizing the Gal4:UAS binary system, I overexpressed CYP6W1 RNA in either a susceptible strain alone or in combination with CYP6A2 or CYP6G1 RNA, which are known to give a low level of DDT resistance. When CYP6W1 was combined with CYP6G1 or CYP6A2, a modest increase in DDT resistance was observed. However, CYP6W1 may not be responsible for this increase because overexpression of CYP6W1 alone did not confer any DDT resistance.

In the second objective, the 0.1-, 0.5- and 0.9-kb upstream DNAs of the Cyp6w1 gene spanning -1/-95, -1/-510 and -1/-937 bp regions were examined for caffeine, DDT and PB inducibility using the firefly luciferase reporter gene system and transient transfection of Drosophila S2 cells. Results showed that basal transcriptional activity of the 0.5- and 0.9-kb DNA was 2-fold greater than the activity of the 0.1-kb DNA, but none of the upstream DNA fragments was induced by DDT. While 0.1-kb DNA did not show much induction with any chemical, 0.5-kb DNA showed about 1.5-fold and 2.0-fold inductions following PB and caffeine treatment, respectively. The 0.9-kb DNA, on the other hand, showed almost a 3-fold induction with caffeine or PB. When cells transfected with 0.9-kb or 0.5-kb DNA were treated with a mixture of caffeine and PB, the level of induction was significantly greater than the induction level observed with each chemical alone. Taken together, these results suggest that DNA between -510 and -937 contains cis-acting sequences that give high levels of caffeine and PB induction. It appears that caffeine and PB act via independent pathways because they show a synergistic effect when combined together.

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