Current research interest of my laboratory is to understand the molecular mechanism of cytochrome P450 gene regulation in Drosophila.
Cytochrome P450 monooxygenases or CYPs form one of the largest family of enzymes that are found in almost all living organisms including humans. These enzymes catalyze many endogenous metabolic reactions involved in the biosynthesis of steroid hormones, pigments, prostaglandin, and etc. Besides these endogenous functions, CYPs are also involved in the metabolism and detoxification of innumerable xenobiotic (foreign) compounds that we consume every day. In other words, CYPs provide a chemical defense against various toxic compounds. Insects use this particular property of these enzymes to develop resistance against various insecticides. The resistant insects cause huge economic loss worldwide by destroying plants and various agricultural products, and by carrying germs for malaria and other fatal diseases affecting humans and farm animals. Although several billion pounds of insecticides are used every year, controlling resistant insects still remains a major challenge for agricultural industries.
Analysis of variety of insect species suggests that CYPs play a major role in conferring insecticide resistance. It has also been observed that the resistant insects show much higher level of expression of multiple CYP genes compared to the susceptible ones. We demonstrated by using whole genome microarray analysis and real-time PCR that in DDT resistant strain of Drosophila Cyp6a2, Cyp6a8, Cyp6g1, Cyp6w1 and Cyp12d1 genes are significantly overexpressed relative to the susceptible strains. It is not known whether overexpression of all these genes makes a strain super-resistant to DDT. However, it has been demonstrated that low level of DDT resistance is conferred by Cyp6g1 gene. In addition, Cyp6g1, Cyp6a2 and Cyp12d1 genes can metabolize DDT in heterologous expression system. It has also been shown that DDT selection pressure in a laboratory strain of Drosophila selects an overexpressing allele of the Cyp6a8 gene. However, very little is known about the mechanism of overexpression and the regulation of CYP genes in Drosophila and other insects.
To better understand these regulatory mechanisms, my laboratory has been using Drosophila as a model insect and Cyp6a8 as a prototype gene. To identify the cis-regulatory DNA sequences and transregulatory factors involved in the expression of Cyp6a8 gene, we have been using caffeine and phenobarbital as chemical tools. We demonstrated by using transgenic reporter technology, microarray technology and transient transfection assay of tissue culture cells that a large number of genes including Cyp6a8 are highly induced by these two chemicals. We have shown that the cis-regulatory elements needed for caffeine and phenobarbital induction are located in the first 200-bp upstream DNA. Our current objectives are to fine-map the regulatory sequences for caffeine and phenobarbital induction by site-directed mutagenesis, and identify the transregulatory factors that interact with the cis-regulatory sequences using 3-D gel electrophoresis and mass spectrometry. Our results may help understand the regulation of CYP genes in economically and agriculturally important insects.
- B.S-University of Calcutta, India.
- M.S-University of Calcutta, India.
- Ph.D-University of Nebraska, Lincoln
R. Morra, S. Kuruganti, V. Lam, J. C. Lucchesi and R. Ganguly (2010) Functional analysis of the cis-acting elements responsible for the induction of the Cyp6a8 and Cyp6g1 genes of Drosophila melanogaster by DDT, phenobarbital and caffeine. Insect Molecular Biology 19, 121–130
Bhaskara, S., Chandrasekharan, M. B., and Ganguly, R (2008) Caffeine induction of Cyp6a2 and Cyp6a8 genes of Drosophila melanogaster is modulated by cAMP and D-JUN protein levels. Gene 415, 49-59
Kuruganti, S., Lam, V., Zhou, X., Bennett, G., Pittendrigh, B. R. and Ganguly, R (2007) High expression of Cyp6g1, a cytochrome P450 gene, does not necessarily confer DDT resistance in Drosophila melanogaster. Gene 388:43-53.
Bhaskara, S., Dean, E. D., Lam. V and Ganguly, R. (2006) Induction of two cytochrome P450 genes, Cyp6a2 and Cyp6a8, of Drosophila melanogaster by caffeine in adult flies and in cell culture. Gene 377:56-64.
Boake CRB, McDona K, Maitra S and Ganguly R (2003) Forty years of solitude: life-history divergence and behavioral isolation between laboratory lines of Drosophila melanogaster. J. Evol. Biol. 16:83-90.
Maitra S, Price C and Ganguly R (2002) Cyp6a8 of Drosophila melanogaster: Gene structure, and sequence and functional analysis of the upstream DNA. Insect Biochem. Mol. Biol. 32: 859-870.
Maitra S, Dombrowski SM, Basu M, Raustol O, Waters LC and Ganguly R (2000) Factors on the third chromosome affect the level of Cyp6a2 and Cyp6a8 expression in Drosophila melanogaster. Gene 248:147-156.
Dombrowski SM, Krishnan R, Witte M, Maitra S, Diesing C, Waters LC and Ganguly R (1998) Constitutive and barbital-induced expression of the Cyp6a2 allele of a high producer strain of CYP6A2 in the genetic background of a low producer strain. Gene 221:69-77.
Maitra S, Dombrowski SM, Waters LC and Ganguly R (1996) Three second chromosome-linked clustered Cyp6 genes show differential constitutive and barbital-inducible expression in DDT-resistant and susceptible strains of Drosophila melanogaster. Gene180:165-171