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Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9427817 Analysis of the in vivo role of myosin regulatory light chain phosphory lat ion Tohtong, Rutaiwan, Ph.D. The Ohio State University, 1994 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 ANALYSIS OF THE IN VIVO ROLE OF MYOSIN REGULATORY LIGHT CHAIN PHOSPHORYLATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Rutaiwan Tohtong, B.Sc. ***** The Ohio State University 1994 Dissertation Commitee: Approved by Berl Oakley Amanda Simcox Caroline Breitenberger Richard Swenson Department of Molecular Grenetics To my parents ACKNOWLEDGEMENTS I would like to thank my parents for their constant moral support through out these years. I thank Dr. Berl Oakley and Dr. Amanda Simcox for their guidance over the last three years. I am especially grateful to Dr. Simcox for being both a friend and an adviser. I would like to thank my committee members for their valuable advice and time. To all my friends in Molecular Genetics, thank you for having made these years a good memory. And lastly, I would like to express my gratitude to Dr. Scott Falkenthal who helped initiate this project. VITA April 18, 1962 Bom - Bangkok, Thailand 1982 ................ B.Sc. Microbiology, Chulalongkom University, Bangkok, Thailand 1986-present Graduate Research and Teaching Associate Department of Molecular Genetics The Ohio State University Columbus, Ohio PUBLICATIONS Tohtong, R., M. Graham, R. Schaaf, J. Hurley, A. Simcox, and D. Maughan. Analysis of the Role of Myosin Regulatory Light Chain (MRLC) Phosphorylation in Drosophila melanogaster. 1993. Biophys. J. 64(2): 8a. Yamashita, H., R. Tohtong, A. Simcox. J. Vigoreaux, J. Haeberle, C. Hyatt, S. Brown, and D. Maughan. 1994. Assessment of the Role of Myosin Regulatory Light Chain (MRLC) Phosphorylation by In Vivo Mutagenesis in Drosophila. Biophys. J. 66(2): A123. FIELDS OF STUDY Major Field: Molecular Genetics iv TABLE OF CONTENTS ACKNOWLEDGEMENTS............................................................... iii VITA................................................................................................... iv LIST OF TABLES............................................................................. xi LIST OF FIGURES.............................................................................. xii CHAPTER I. INTRODUCTION ........................................................... 1 Introduction ................................................................................ 1 Muscle structure...................................................................... 2 Skeletal muscle.................................................................... 2 Smooth muscle .................................................................... 8 Cardiac................................................................................... 9 Composition of myofilaments ................................................. 9 The thick filaments. ........................................................... 9 Myosin heavy chain ...................................................... 10 Myosin regulatory light chain .................................... 11 Myosin essential light chain ........................................ 15 The hinge region ................................................................... 16 The thin filaments ................................................................ 17 Actin .................................................................................... 17 Tropomyosin ...................................................................... 18 The troponin complex ....................................................... 18 Other accessory proteins ..................................................... 19 a-actinin ............................................................................ 19 Titin ..................................................................................... 19 Muscle contraction ..................................................................... 20 The sliding filament model of muscle contraction 20 v Regulation of muscle contraction ............................................ 21 Thin filament control ........................................................... 21 Thick filament control .......................................................... 22 Regulation by phosphorylation of myosin light chain-2 ........................................................... 23 Drosophila musculature.......................................................... 24 Supercontractile muscle ..................................................... 24 Tubular muscle ..................................................................... 27 Fibrillar muscle ..................................................................... 28 Muscle proteins of Drosophila................................................ 29 Myosin heavy chain ............................................................ 29 Myosin light chain 2 ........................................................... 30 Myosin alkali light chain .................................................... 34 Actin ....................................................................................... 34 Tropomyosin ............................................................ 35 Troponin ................................................................................ 36 Param yosin .................................................. 36 Alpha actinin and other Z disc associated proteins ...................................................... 37 Objectives .................................................................................. 39 II. MATERIALS AND METHODS............................................... 41 Fly stocks and culture conditions ........................................ 41 Construction of P element transformation vector and generation of transformants by germline transformation ................................................... 44 Mobilization of the P[Mlc2S66 67A] insert to multiple chromosome sites ......................................... 47 Rescue of the dominant flightless phenotype and the recessive lethality of Mlc2 null mutation ................ 48 Flight test ................................................................................... 53 vi Muscle protein preparation .................................................... 53 Protein electrophoresis ............................................................ 54 In vivo labeling of muscle proteins ........................................ 55 Expression of Drosophila MLC-2 fusion protein in E. coll................................................................. 56 Purification of the Drosophila MLC-2 fusion protein ....................................................................... 56 Production of MLC-2 specific antibodies and Western Hybridization ......................................................... 59 Peptide sequencing.................................................................... 60 Electron microscopy.. ................................................................ 60 Preparation of genomic DNA from adult Drosophila............................................................................. 61 PCR amplification of the genomic DNA for cloning of the MLCK gene ................................................. 62 Isolation of Drosophila MLCK cDNA clones .......................... 63 Southern Hybridization ........................................................... 64 DNA sequencing ........................................................................ 64 Total RNA preparation ......................... 64 Synthesis of RNA probes for RNAase protection assays ................................................................. 65 RNAase protection assays ....................................................... 65 III. MULTIPLE PHOSPHORYLATED VARIANTS OF DROSOPHILA MYOSIN LIGHT CHAIN 2 PROTEIN ACCUMULATE M VIVO...............................................................