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Sudips Revised Thesis Investigation Of The Behavior Of The Gal4 Inhibitor Gal80 Of The GAL Genetic Switch In The Yeast Saccharomyces Cerevisiae Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sudip Goswami, M.S. Graduate Program in Molecular Genetics The Ohio State University 2014 Dissertation Committee Dr. James Hopper, Advisor Dr. Stephen Osmani Dr. Hay-Oak Park Dr. Jian-Qiu Wu ii Copyright by Sudip Goswami 2014 iii ABSTRACT The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to upstream activation sequences or UASGAL sites on GAL gene promoters as a dimer both in the absence and presence of galactose. In the absence of galactose, a Gal80 dimer binds to and masKs the Gal4 activation domain, inhibiting its activity. In the presence of galactose, Gal3 interacts with Gal80 and relieves Gal80’s inhibition of Gal4 activity allowing rapid induction of expression of GAL genes. In the first part of this work (Chapter 2) in-vitro chemical crosslinking coupled with SDS PAGE and native PAGE analysis were employed to show that the presence of Gal3 that can interact with Gal80 impairs Gal80 self association. In addition, live cell spinning disK confocal imaging showed that dissipation of newly discovered Gal80-2mYFP/2GFP clusters in galactose is dependent on Gal3’s ability to interact with Gal80. In the second part (Chapter 3), extensive analysis of Gal80 clusters was carried out which showed that these clusters associate strongly with the GAL1-10-7 locus and this association is dependent on the presence of the UASGAL sites at this locus. Moreover, the formation of Gal80 clusters is dependent on the presence of Gal4. Additionally, it was discovered that Gal4-2GFP/2mYFP maKes intranuclear foci, which unlike Gal80-2mYFP/2GFP clusters do not dissipate in galactose and their formation is not dependent on the presence of Gal80. Similar to Gal80 clusters, these Gal4 foci associate strongly with the GAL1-10-7 locus. These evidences suggest that large molecular assemblies of Gal80 and Gal4 associate with the UASGAL sites of the GAL1-10-7 locus, which raises some interesting possibilities as to how this genetic switch is regulated. ii I dedicate this work to the “Future” iii Acknowledgements: I would liKe to thanK Prof. James Hopper, my thesis advisor, for his continuous guidance and advice. I would also like to thanK my advisory committee members Prof. Stephen Osmani, Prof. Hay-Oak Park and Prof. Jian-Qiu Wu for their critical and important input into my worK. I thanK all my present J. Hopper lab members, Eric Jiang, Alexander DiScenna and Pallavi Chandna for their technical assistance. I also acknowledge the technical assistance I received from former lab members Kathleen Dotts, Emily Baas, Jill Steinbrunner, Caitlin Rigsby, and others. I would also like to thanK former graduate student Dr. Onur Egriboz with whom I collaborated quite a bit and also for his ideas and input to my worK, former post docs Dr. Xiarong Tao and Dr. Fenglei Jiang for their help, and former lab mate Jin Shuo with whom I did my first project in the lab. I also acKnowledge many students and post docs of other labs for their generous help and cooperation. I am also grateful to Eric Jiang of the Jim Hopper lab and Joey Marquadt of the Harold FisK lab for their critical reading of this thesis. iv Vita 1995 ……………………………………………………...B.S. Pharmaceutical Technology Jadavpur University Kolkata, West Bengal, India 2007………………………………………………………M.S. Biotechnology West Bengal University of Technology Kolkata, West Bengal, India 2007-2014……………………………………………..Graduate Associate, Department of Molecular Genetics The Ohio State University Publication Egriboz O, Goswami S, Tao X, Dotts K, Schaeffer C, Pilauri V, Hopper JE. 2013 Self- association of the Gal4 inhibitor protein Gal80 is impaired by Gal3: evidence for a new mechanism in the GAL gene switch Mol Cell Biol. Sep;33(18):3667-7 Field of Study Major Field: Molecular Genetics v Table of contents Abstract ................................................................................................................................ ii Dedication ........................................................................................................................... iii AcKnowledgement ........................................................................................................... iv Vita ......................................................................................................................................... v Table of Contents ............................................................................................................. vi List of Figures .................................................................................................................... xii List of Tables ...................................................................................................................... xv List of Abbreviations ...................................................................................................... xvii Chapter 1 Regulation of Transcription and The GAL genetic switch .............................. 1 1.Introduction ................................................................................................................... 2 1.2. Main Factors In Transcription Regulation ................................................... 3 1.2.1Core promoter elements ...................................................................... 4 1.2.2 Basal Transcription factors ............................................................... 4 vi 1.2.3Enhancers ................................................................................................... 4 1.2.3.1 Action at a distance-DNA looping ................................ 5 1.2.4 Activators ................................................................................................................. 11 1.3 Genetic regulatory network of yeast galactose pathway ........................ 15 1.3.1 Enzymes for galactose utilization .................................................................. 16 1.3.2 Gal4 – the transcriptional activator .............................................................. 17 1.3.3 Gal80 – the transcriptional inhibitor ........................................................... 21 1.3.4 Gal3 – the galactose sensor and signal transducer ................................ 23 1.3.5 The current model of GAL gene switch ....................................................... 23 2. Chapter 2: Interaction of Gal3 with Gal80 impairs Gal80 self-association ................................................................................................................................................. 27 2.1 Abstract ........................................................................................................................ 28 2.2 Introduction ................................................................................................................ 29 2.3 Materials and Methods .......................................................................................... 31 2.3.1 Yeast strains and plasmids ............................................................................... 31 2.3.2 Microscopy .............................................................................................................. 32 2.3.3 Protein expression and purification ............................................................. 33 2.3.4 Cross-linking of Gal80 and Gal3 ..................................................................... 34 vii 2.3.5 Discontinuous blue native protein gel electrophoresis ....................... 34 2.4 Results ........................................................................................................................... 35 2.4.1 Gal80-Gal80 self-association is impaired under conditions supporting Gal3-Gal80 interaction .......................................................................... 35 2.4.2 The nuclei of live cells display clusters of Gal80-2mYFP that dissipate in response to galactose-triggered Gal3-Gal80 interaction ................................................................................................................................................. 37 2.4.3 Super repressor Gal80S-2 forms more stable higher order oligomers on native gel compared to WT Gal80 ................................................................................................................................................. 38 2.5 Discussion .................................................................................................................... 39 3. Chapter 3 Investigation Of The Intranuclear Clusters Of Gal80 And Intranuclear Foci of Gal4 ................................................................................................................................................. 51 3.1 Abstract ........................................................................................................................ 52 3.2 Introduction ................................................................................................................ 53 3.3 Materials and Methods .......................................................................................... 54 3.3.1 Yeast strains, plasmids, media and growth condition .........................
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