Deciphering Pleiotropic Effects: A molecular characterization of the foraging gene in Drosophila melanogaster. by Aaron Munro Allen A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Cell and Systems Biology University of Toronto © Copyright by Aaron Munro Allen 2016 Deciphering Pleiotropic Effects: A molecular characterization of the foraging gene in Drosophila melanogaster. Aaron Munro Allen Doctor of Philosophy Cell and Systems Biology University of Toronto 2016 Abstract Pleiotropy is defined as the manifold effects of a gene at the phenotypic level. Understanding the mechanisms of manifold gene action has important implications for many fields of biology ranging from evolution and to medicine. The foraging gene, in Drosophila melanogaster, has long been a pivotal example of a single gene with natural variants that affect feeding-related phenotypes. One possible mode of action for foraging’s pleiotropy is through independent regulation of its gene products. Characterization of the foraging gene revealed 4 distinct promoters that produce 21 transcripts, and 9 ORFs. A foraging null mutant of the locus was generated using Ends-out gene targetting. foraging null mutants had reduced foraging behaviour, reduced food intake behaviour, and increased lipid levels. A recombineered full genomic rescue of the gene rescued the effects of the null mutation. By comparing the effects of the null mutant with those of the natural variants, I showed that these feeding-related phenotypes were differentially regulated. A promoter manipulation strategy identified diverse, and non-overlapping expression patterns associated with the 4 foraging promoters. Expression ii was seen in the nervous and gastric systems of the larva and adult fly, as well as the reproductive systems of adult fly. This expression suggests potential new roles for the foraging gene in the larva and adult fly. Characterizing the regulation of foraging's gene products will further our understanding of its role in behaviour, and shed light on the evolutionary origins of natural variants of the foraging. Not only will this study further our understanding of this gene's conserved function across taxa, but it will also help elucidate the role for differential transcriptional regulation in achieving multiple functions of a gene. This could further serve as fodder for investigations intro the roles of neo-functionalization versus escape from adaptive constraint. iii Acknowledgements I’d like to thank Prof. Marla Sokolowski for her supervision, guidance and support throughout this work. I have learned many valuable lessons and have had many unique experiences throughout my time in the lab. There was always something new to learn with such a diverse group. Prof. Joel Levine for the many technical discussions and frankness throughout the degree, his out of the box thinking and for instigating the collaboration with Prof. Stephen Goodwin at the University of Oxford. Prof. Tim Westwood for his insightful comments and discussion on transcriptional regulation, and for keeping in touch with multiple interesting and relevant studies and speakers. Prof. Mariana Wolfner, and Prof. Henry Krause for making time to give their feedback and examination of this thesis. To Dr. Scott Douglas for teaching me many of the basics of molecular biology, including many now defunct techniques. Prof. Jean-Christophe Billeter for multiple discussion earlier in my degree that were pivotal in the overall design of the this project, and for introducing me to the bioinformatic software package Geneious, which single handedly saved a minimum of two years of work. Dr. Amsale Belay for teaching me immunohistochemistry and many valuable discussion on the molecular biology of the foraging gene. Dr. Tony So for many earlier morning coffee discussions, giving me yet another perspective into the voodoo of molecular biology. Prof. Stephen Goodwin who graciously collaborated on the HR and recombineering projects, and for allowing me to conduct some of the early work on these projects for 4 months in his laboratory at the University of Oxford. Dr. Megan Neville for her advice, instruction and discussions that helped guide the HR and recombineering project while in the Goodwin Lab. Yet another invaluable insight into the voodoo of molecular biology. To the other members of the Goodwin lab for making it such a welcoming environment. To many Sokolowski lab members over the years for the many science and non-science discussions. To Bryon Hughson for many for plotting and scheming on how we’re going to fix the world, and for educating me in areas of metabolism. The new kids, Ina Anreiter, and Oscar iv Vasquez for their shenanigans and many useful discussions into topics of chromatin modifications and biochemistry, respectively. Dr. Jeff Dason for many useful discussion with a grounded and pragmatic points of view on science. To the many graduate students through the years. Thomas Braukmann for providing a valuable outside perspective, and for always being a willing participant in procrastination and tomfoolery. Kevin Judge, Marion Andrews, Matt Janicki, Amy Wong, Becky Rooke, Aaron LeBlanc, Mark McDougall, Laura Junker, Audrey Reid, and the whole UTM Friday Beers crew for always providing interesting and entertaining discussions. To the Earth Science Centre community of graduate students on the St. George campus who made our lab’s move to downtown such an easy transition. My mother, Michele Robinson, and my father, David Allen, for their genes and the environmental conditions to cultivate them. For instilling a sense of curiosity, wonder, and skepticism in interrogating the world around me. For giving me a grounded work ethic and true value of a dollar. Dorothy and Russell Merrifield, my second set of parents, for even further instilling the a solid work ethic. For trying to keep my smart aleck responses in check; it always made for a good challenge. My brothers, Patrick, Kyle and Sean who helped me cut my pedantic and semantic teeth from a very young age. For our countless silly, ridiculous, and Monty Python inspired academic discussion that frequently go all through the night. To Dr. Kyla Ercit for her support and companionship throughout the degree. You helped me retain the last remnants of my sanity. For always listening when I need to vent. For distracting me when I need distracting. For providing an invaluable outside perspective on my work; making sure I am explaining myself in an interpretable way. v Table of Contents Abstract.........................................................................................................................................ii Acknowledgements......................................................................................................................iv Table of Contents.........................................................................................................................vi List of Tables................................................................................................................................xi List of Figures.............................................................................................................................xii List of Appendices......................................................................................................................xiv List of Initialisms and Acronyms...............................................................................................xv Chapter 1:......................................................................................................................................1 General Introduction..................................................................................................................1 Pleiotropy..............................................................................................................................2 Origins..............................................................................................................................2 “One gene, one enzyme”..................................................................................................3 Introducing molecular biology.........................................................................................3 Philosophical utility..........................................................................................................4 Transcriptional regulation.....................................................................................................5 Chromatin remodeling......................................................................................................5 Insulators act to delineate regulatory domains.................................................................6 Transcription factor binding and prediction.....................................................................6 The long reach of the enhancer.........................................................................................7 Promoter structure............................................................................................................7 Promoter-proximal pausing..............................................................................................8 Alternative promoters and splicing...................................................................................9 The model organism Drosophila melanogaster....................................................................9 Advances in tools for