University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2006 Studies on Cytochrome P450 genes in Drosophila melanogaster: Relationship between over expression and DDT resistance, and xenobiotic induction Srilalitha Kuruganti University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Kuruganti, Srilalitha, "Studies on Cytochrome P450 genes in Drosophila melanogaster: Relationship between over expression and DDT resistance, and xenobiotic induction. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1976 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Srilalitha Kuruganti entitled "Studies on Cytochrome P450 genes in Drosophila melanogaster: Relationship between over expression and DDT resistance, and xenobiotic induction." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Biochemistry and Cellular and Molecular Biology. Ranjan Ganguly, Major Professor We have read this dissertation and recommend its acceptance: Bruce McKee, John Koontz, Albrecht von Arnim, Sundar Venkatachalam Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Srilalitha Kuruganti entitled “Studies on Cytochrome P450 genes in Drosophila melanogaster: Relationship between over expression and DDT resistance, and xenobiotic induction.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Biochemistry, Cellular and Molecular Biology. Ranjan Ganguly _____________________________ Major Professor We have read this thesis and recommend its acceptance: Bruce McKee ___________________ John Koontz ___________________ Albrecht von Arnim ___________________ Sundar Venkatachalam ___________________ Accepted for the Council: Linda Painter ________________________ Interim Dean of Graduate Studies (Original signatures are on file with official student records) Studies on Cytochrome P450 genes in Drosophila melanogaster: Relationship between over expression and DDT resistance, and xenobiotic induction A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Srilalitha Kuruganti December 2006 To my father, Kuruganti Prabhakar Sastry, without whose constant love, encouragement and motivation, this would have been impossible ii ACKNOWLEDGEMENTS It was a great pleasure working in Dr. Ranjan Ganguly’s lab. Dr.Ganguly’s constant attention and support have helped me a lot towards my scientific development. He is a great advisor helping students in academics as well as non-academics. He is always available and provided excellent guidance, trouble shooting in all endeavors from painful microinjections to cloning to setting up numerous genetic crosses. I would like to sincerely thank him for all his advice and help. I would also like to thank Dr. Nivedita Ganguly for being so warm and friendly during all my visits to their house. I would like to thank my committee members, Dr. Bruce McKee, Dr. John Koontz, Dr. Von Arnim and Dr. Sundar Venkatachalam for their encouragement and critical evaluation of my research. My sincere thanks to Dr. Jae Park and Dr. Gyunghee Lee for friendly advice regarding microinjection and also for providing balancer stocks for various host strains that made my research easier. I would like to thank Dr. Liz Howell, Dr. Barry Bruce, Dr. Cynthia Peterson, Dr. Ranjan Ganguly and Dr. Bruce McKee for their excellent teaching that improved my scientific knowledge. I would like to thank Dr. Sharon Thomas for kindly advice during stressful times and for reading my prelim grants critically. I want to thank Dr. Arnold Saxton from the Animal Sciences department for helping me with probit and microarray data analysis in SAS. I would like to thank Sally Chopra, my friend for being there during the happy and difficult times. I can never forget the chats with her in the department. I would like to thank Srividya Bhaskara, my friend for constant support and for maintaining a friendly atmosphere in the lab. I would like to thank Vita Lam for helping with endless DDT iii bioassays and Chandrashis Bhowmick for his help in the lab. I want to thank Charity Olson for helping with the PCR amplification of recombinant lines. My sincere thanks to all the undergraduates, Kim Crowder, Lauren, Maggie, Neha for making fly food that made life much easier. I want to thank Julia Gouffon and Pradeep Chimakurthy for patiently guiding me through the microarray software and data analysis. I would like to thank BCMB department for providing this excellent opportunity and for all the resources. I would like to specially thank my mother, Swarajya lakshmi for sending me for studies to US. It was a difficult decision and I would not have been here without her courage and support. I would like to extend my sincere thanks and gratitude to my husband, PD Kumar for guiding me and helping me through all the aspects. Being a Ph.D student himself, he always understood what I was going through and encouraged me towards completing my goal. I would like to thank my brother, Teja for being there for me all the time. Without him, this would have been impossible. I would like to thank my sister and my best friend, Srivalli for sharing my happiness and sorrows all the time. I would like to thank all my family members for their love and support. I would like to thank all my friends in Knoxville especially Sampath and Sankar for making my Knoxville stay more fun. I would like to thank my friends, Malathi, Harini, Satya and Priya for making me laugh all the time, even during my bad times and also for helping with my thesis formatting and corrections. I will take these memories with me wherever I go in the future. iv ABSTRACT Cytochrome P450 monooxygenases (CYPs), are involved in the metabolism of a diverse group of endogenous and xenobiotic compounds. In insects, CYPs are involved in conferring resistance against insecticides. In Drosophila, the expression of Cyp6a2, Cyp6a8, Cyp6g1, Cyp6w1 and Cyp12d1 is higher in the resistant compared to susceptible strains. Recent study by Daborn et al (2002, Science, 297, 2253-2256) showed that Cyp6g1 alone can confer resistance phenotype. The aim of my first objective was to re- examine this claim and second to examine the effect of common xenobiotic compounds on the transcriptome of Drosophila. In first objective, six strains of Drosophila were examined for DDT resistance and Cyp6g1 expression. Results showed that some of the highly susceptible strains showed high level of Cyp6g1 expression and Accord element in the Cyp6g1 upstream DNA. When Cyp6g1 allele of the resistant 91-R strain was substituted with that of the susceptible 91-C strain via recombination, the resulting three recombinant lines retained high level of resistance like the 91-R strain, but showed very low Cyp6g1 expression. This suggests that there is a correlation between overexpression of Cyp6g1 and the presence of Accord transposable element but not DDT resistance. In the second part of the first objective, I directly examined the role of the Cyp6a2 and Cyp6g1 in DDT resistance. Germ line transformation in susceptible strain showed that there was a two-fold increase in DDT resistance (LD50) in transformed flies showing v two-fold higher expression of GAL4/UAS driven CYP6A2 or CYP6G1 cDNA. A cumulative increase (4-fold) in DDT resistance was observed when both cDNAs were overexpressed in the same fly. Results suggest that the expression of multiple Cyp genes may be needed to confer a high level of DDT resistance. In the second objective, microarray was used to examine the transcripts induced by caffeine and phenobarbital. Results showed that genes involved in detoxification, carbohydrate metabolism, signal transduction and Cyp genes are induced by caffeine and phenobarbital. These are the same group of genes overexpressed in the resistant 91-R and recombinant strains. These studies shed light on the molecular basis of induction of Cyp genes and insecticide resistance. vi COMPREHENSIVE ABSTRACT Cytochrome P450 monooxygenases or CYPs, a superfamily of enzymes present in all organisms, are involved in the metabolism of a diverse group of endogenous and xenobiotic (foreign) compounds. In insects, CYPs are involved in conferring metabolic resistance against various insecticides. In many insects, over expression of one or more CYP gene is found in the resistant strain. In Drosophila, the level of Cyp6a2, Cyp6a8, Cyp6g1, Cyp6w1 and Cyp12d1 expression is much higher in the resistant strains than in the susceptible ones. However, it is not known how many of these genes are actually involved in resistance, although