Doctoral Dissertations

Date of Award

5-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Ranjan Ganguly

Committee Members

Mary Ann Handel, Bruce McKee, David Brian, Larry Waters

Abstract

Metabolic detoxification of insecticides is a major factor that causes resistance in insects. Cytochrome P450s (CYPs) play an important role in the process as these detoxifying enzymes are expressed at high levels in resistant insects. However, not much is known about the mechanisms responsible for the high level of CYP gene expression in resistant insects. Such knowledge is important in the understanding and development of 'biorational' strategies to control insecticide resistance. This study was initiated to gain an understanding about the mechanisms of CYP gene regulation using Drosophila as a model organism. Two new clustered CYP genes (Cyp6a8 and Cyp6a9) were isolated from Drosophila, of these Cyp6a8 was found to be highly expressed in insecticide resistant strains. Hence, this gene was used as a prototype to study insect CYP gene regulation. Analysis of cw-acting sequences of Cyp6a8, showed the presence of basal promoter elements within 0.2-kb of the upstream DNA. This region also had sequences that could confer the barbiturate inducibility and sexual dimorphism shown by Cyp6a8, to a heterologous luciferase reporter gene. However, analysis of the upstream region did not reveal sequences that could be responsible for the overexpression of Cyp6a8 in resistant strains. Chromosome substitution studies, on the other hand, showed that the 3rd chromosome from wild type, susceptible, CYP-underproducer strains carry negative traws-regulatory loci (locus) that downregulate the 2nd chromosomal Cyp6a8 allele from resistant, CYP-overproducer strains. A similar effect was also observed for Cyp6a2, another 2nd chromosomal gene that shows overexpression in resistant strains, implying that the regulatory effects are global, affecting more than one CYP gene. The 2nd chromosome was found to influence the phenobarbital response of Cyp6a8 and Cyp6a2 in the same way, implying that common regulatory controls exist that influence both constitutive and inducible expression of at least two CYP genes. The CYP-regulatory mechanisms indicated in this study were found to be similar to the ones shown for the housefly, implying a conservation of such mechanisms between insect species. This study also generated several molecular reagents that can be used to further dissect the mechanisms of CYP gene regulation in insects.

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