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Role of Mitochondrial Beta-Oxidation in Ethanol Response: a Candidate Gene Study Using Caenorhabditis Elegans

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Role of Mitochondrial Beta-Oxidation in Ethanol Response: a Candidate Gene Study Using Caenorhabditis Elegans Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2017 Role of mitochondrial beta-oxidation in ethanol response: A candidate gene study using Caenorhabditis elegans Harini Pallikarana Tirumala Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Behavioral Neurobiology Commons, Molecular Genetics Commons, and the Pharmacology Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/4993 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Role of mitochondrial beta-oxidation in ethanol response: A candidate gene study using Caenorhabditis elegans A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Human Genetics at Virginia Commonwealth University. By Harini Pallikarana Tirumala Master of Science in DNA Profiling from University of Central Lancashire, UK 2012 Directed by Jill C. Bettinger, Ph.D., Associate Professor of Pharmacology and Toxicology Virginia Commonwealth University Richmond, Virginia June, 2017 ACKNOWLEDGEMENTS I would like to thank my thesis advisor, Dr. Jill Bettinger for helping me grow as a scientist and for encouraging me to think out of the box. She has been immensely supportive during the course of my research project and has been a great mentor to me. I would like to thank my committee members, Dr. Andrew Davies, Dr. Laura Mathies and Dr. Rita Shiang. Their valuable inputs during committee meetings have helped me formulate and shape this project better. I would also like to thank all the members of the Davies-Bettinger lab, particularly Gina Blackwell, who has been a pillar of support from my first day in the lab and has patiently answered the umpteen questions I had. I would like to extend thanks to all the Human Genetics students, particularly Saranya Regupathy, Megan Hept and Javeria Aijaz for their support and confidence in me. I would also like to thank all my family members and friends for their support during these two years. To my parents who always believed in me and gave me the confidence to pursue my dreams. A final thank you, to my husband, Swaminathan Balasubramanian, for listening to me talk endlessly about my worms and, for his constant love and support. ii TABLE OF CONTENTS List of Tables ………………………………………………………………………vi List of Figures………………………………………………………………………viii List of Abbreviations and Acronyms.……………………………………………..xii Clarification of Contributions……………………………………………………...xvi Abstract…………………………………………………………………………….xvii 1. Introduction……………………………………………………………………….1 1.1 Alcoholism – National and Global Impact………………………………..1 1.2 Genetics of alcoholism…………………………………………………...2 1.3 Neuronal response to alcohol……………………………………………4 1.3.1 Using C. elegans to study the molecular basis of neuronal response to alcohol………………………………………………….6 1.4 Background research for current project…………………………………9 1.5 Mitochondrial Beta-Oxidation…………………………………………….15 1.5.1 Mitochondrial Beta-oxidation in C. elegans……………………..17 1.5.1.1 Acyl CoA Synth(et)ases (ACS).…………………………..20 1.5.1.2 Carnitine Palmitoyl Transferases (CPT).………………..21 1.5.1.3 Acyl CoA dehydrogenases (ACAD/ACDH).……………..22 1.5.1.4 Enoyl CoA hydratase (ECH), 3-hydroxy acyl CoA dehydrogenase (HADH) and 3-keto acyl CoA thiolase (KAT).………………………………………………………..24 1.6 Goal of Present Study…………………………………………………….25 1.7 Specific Aims………………………………………………………………26 2. Materials and Methods………………………………………………………...27 2.1. Selection of candidate genes……………………………………………27 2.2. Maintaining strains……………………………………………………….28 iii 2.3. Chunking…………………………………………………………………...29 2.4. Making OP50………………………………………………………………29 2.5. Seeding plates with OP50………………………………………………..29 2.6. C. elegans strains…………………………………………………………30 2.7. DNA Isolation………………………………………………………………30 2.7.1. DNA Isolation for PCR…………………………………………….31 2.7.2. DNA Isolation for Single Worm PCR…………………………….31 2.8. Polymerase Chain Reaction (PCR)……………………………………..32 2.8.1. Primers for PCR……………………………………………………32 2.8.2. PCR Setup………………………………………………………….32 2.8.3. Temperature gradient PCR……………………………………….33 2.8.4. Single worm PCR (SWPCR)……………………………………...34 2.9. Restriction digestion……………………………………………………...34 2.10. Agarose Gel Electrophoresis……………………………………..35 2.11. DNA sequencing…………………………………………………...36 2.12. RNA interference (RNAi) …………………………………………37 2.13. Backcrossing mutant strains……………………………………...40 2.14. Behavioral assays on ethanol…………………………………….44 3. Results and Discussion………………………………………………………..48 3.1. Candidate genes in mitochondrial beta-oxidation…………………….48 3.2. Ethanol response phenotypes of candidate genes in mitochondrial beta-oxidation……………………………………………………………...53 3.3. Ethanol affects mitochondrial beta-oxidation…………………………..62 3.4. Acyl CoA Synthases………………………………………………………65 3.5. Carnitine Palmitoyl Transferases………………………………………..74 iv 3.6. Acyl CoA dehydrogenases……………………………………………….76 3.7. Enoyl CoA Hydratases……………………………………………………78 3.8. Hydroxy acyl CoA dehydrogenases…………………………………….80 3.9. Keto-acyl CoA Thiolases…………………………………………………82 3.10. Conclusion………………………………………………………….84 4. List of references……………………………………………………………….86 Appendices………………………………………………………………………..100 Appendix I………………………………………………………………..........100 Appendix II……………………………………………………………………..119 Appendix III…………………………………………………………………….125 Vita…………………………………………………………………………………164 v LIST OF TABLES Table 1. Step-by-step comparison of mitochondrial beta-oxidation in humans and C. elegans Table 2. List of C. elegans strains used for the project Table 3. PCR Reaction components Table 4. Standard PCR conditions Table 5. PCR reaction components for SWPCR Table 6. Restriction digestion reaction mix components Table 7. List of all genes reviewed Table 8. Reasons for exclusion of genes Table 9. Ethanol response phenotype of 2x backcrossed loss of function (lf) mutants in the mitochondrial beta-oxidation pathway in C. elegans Table 10. Ethanol response phenotype of 6x backcrossed lf mutants and RNAi knockdown strains in mitochondrial beta-oxidation pathway in C. elegans Table 11. Human and Mouse orthologs of acs genes tested for ethanol responses Table 12. Concentration of DNA template and primer used for Purified PCR products in Sanger sequencing sample preparation Table 13. Concentration of DNA template and primer used for Plasmid DNA template in Sanger sequencing sample preparation Table 14. Primers for PCR of mutant C. elegans strains obtained from Caenorhabditis Genetics Center website. Table 15. Primers for PCR of mutant C. elegans strains designed on NCBI Primer-BLAST vi Table 16. Restriction enzymes and digest conditions used for snipSNP genotype detection Table 17. RNAi plasmid DNA concentrations quantified using NanoDrop Table 18. 100% Ethanol volumes for various plate weights for specific final concentrations of ethanol (in mM). Table 19. Average basal speeds of 2x-backcrossed mutants at 0mM ethanol concentration at two different time points (10 minutes and 30 minutes after being transferred to the assay plate) Table 20. Average basal speeds of 6x-backcrossed mutants at 0mM ethanol concentration at two different time points (10 minutes and 30 minutes after being transferred to the assay plate) Table 21. Average basal speeds of RNAi-mediated knockdown worms at 0mM ethanol concentration at two different time points (10 minutes and 30 minutes after being transferred to the assay plate) vii LIST OF FIGURES Figure 1. acs-2 modifies AFT and does not alter ethanol metabolism Figure 2. Mitochondrial β-oxidation may influence acute ethanol behaviors Figure 3. Mitochondrial beta-oxidation of fatty acids Figure 4. Metabolic reactions of acyl-CoAs Figure 5. Genotypes and ratios of outcrossed progeny in the 2x outcross of mutants of genes on the autosomes (Chromosomes I-V) Figure 6. Genotypes and ratios of outcrossed progeny in the 2x outcross of mutants of genes on the X-Chromosome Figure 7. Genotypes of F1 and F2 progeny in the 2x backcross of mutants of balanced mutant strain: acs-4(ok2872) III/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III) Figure 8. Mitochondrial beta-oxidation genes in C. elegans and humans Figure 9. Mitochondrial beta-oxidation of fatty acids showing genes tested at each step of the pathway and their ethanol response phenotypes Figure 10. Inhibitors suggest possible products of acyl-CoA produced by ACS isoforms Figure 11. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed acs-3 lf mutant Figure 12. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed acs-4 lf mutant Figure 13. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed acs-5 lf mutant viii Figure 14. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed acs-13 lf mutant Figure 15. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed acs-17 lf mutant Figure 16. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed acs-22 lf mutant Figure 17. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed cpt-3 lf mutant Figure 18. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed cpt-4 lf mutant Figure 19. Ethanol sensitivity and acute functional tolerance (AFT) of 2x backcrossed cpt-6 lf mutant Figure 20. Ethanol sensitivity and acute functional tolerance
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