HIGH ORDER STRUCTURE IN THE BABESIA BOVIS LOCUS OF ACTIVE Ves TRANSCRIPTION By YINGLING HUANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1 © 2009 Yingling Huang 2 To my husband, Wenqiang Tian, and my dear daughter, Alina Hanning Tian 3 ACKNOWLEDGMENTS I sincerely thank my major mentor Dr. David Allred, who provides me with extensive support which is crucial to the completion of my work throughout my doctoral study. During this work, I benefited a lot from numerous in-depth and inspiring scientific discussions between Dr. Allred and myself. With his great encouragement and assistance, I became more confident and independent as a grown researcher needs to be. I also gratefully thank my supervisory committee (Dr. John Dame, Dr. Anthony Barbet, Dr. David Ostrov and Dr. Jorg Bungert) who put forward many helpful suggestions to guide my research in this project. I would like to give many thanks to Dr. Basima Al-Khedery, who offered me important help in teaching me southern blotting procedures. Additionally, I want to sincerely thank Ms. Xuping Xiao and other labmates such as Xinyi Wang, Anne Bouchut and Alexia Berg and so on for providing me technical assistance and extensive laboratory support. Finally, I would also like to thank my husband and my dear daughter for their love and support, which was the strongest drive for me to complete this work. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 LIST OF ABBREVIATIONS ........................................................................................................10 ABSTRACT ...................................................................................................................................12 INTRODUCTION .........................................................................................................................14 B. bovis Life Cycle and Pathogenesis .....................................................................................14 Antigenic Variation in B.bovis ...............................................................................................15 VESA1 Protein and C9.1 ves1α/β LAT ..................................................................................16 Inverted Repeats and Their Biological Importance ................................................................18 DNA Two Dimensional Gel Electrophoresis and Its Application ..........................................20 Chromatin Structure and Gene Transcription .........................................................................21 Nucleosomal Organization on Genes ..............................................................................22 Nucleases and Their Applications in Studying Chromatin Structure ..............................24 Hypothesis ..............................................................................................................................26 HIGH ORDER DNA STRUCTURE withIN BABESIA BOVIS LOCUS OF ACTIVE ves TRANSCRIPTION .................................................................................................................27 Abstract ...................................................................................................................................27 Introduction .............................................................................................................................27 Methods and Materials ...........................................................................................................29 Parasite Culture ...............................................................................................................29 Rupture of Infected Red Blood Cells (IRBCs) by Saponin Lysis ...................................29 Construction of Artificial DNA Cruciform Molecule .....................................................30 In vitro DNA Thermal Remodeling Assay ......................................................................30 DNA 2-D Gel Electrophoresis System ............................................................................31 DNA Electron Microscopy ..............................................................................................33 Detection of In vivo LAT-Associated DNA Structure with Structure-Protective Extraction .....................................................................................................................34 Detection of In vivo LAT-Associated DNA Structure with Psoralen Crosslinking ........35 Southern Blot Analysis ....................................................................................................36 Results .....................................................................................................................................38 Establishment of a DNA 2-D Gel System to Detect DNAs with Higher-order Structure .......................................................................................................................38 Construction of DNA cruciform control molecule ..................................................38 Migration of thermally-remodeled cruciform molecule ...........................................38 DNA electron microscopy with constructured cruciform ........................................40 5 Analysis of In vitro Structural Potential of Ves Genes ....................................................41 Migration of thermally remodeled ves gene fragments ............................................42 DNA electron microscopy with structured ves fragments........................................43 Detection of In vivo DNA Structure Associated with Ves Genes ...................................43 Detection of In vivo DNA Structure without DNA Crosslinking ....................................44 Detection of In vivo DNA Structure with DNA crosslinking ..........................................49 Discussion ...............................................................................................................................51 Establishment of 2-D Gel System to Detect Higher-order DNA Structure in Chromatin ....................................................................................................................51 In vitro Structure Study of Ves Genes .............................................................................55 Detection of In vivo LAT-Associated DNA Strucutre ....................................................57 CHROMATIN STRUCTURAL CHARACTERISTICS AMONG B. BOVIS ves GENES ..........89 Abstract ...................................................................................................................................89 Introduction .............................................................................................................................89 Methods and Materials ...........................................................................................................91 Parasite Culture ...............................................................................................................91 Isolation of Parasite Nuclei .............................................................................................91 Nuclease Digestion of Intact Nuclei ................................................................................91 Micococcal nuclease digestion .................................................................................91 DNase I digestion .....................................................................................................92 S1 nuclease digestion ...............................................................................................92 Mung bean nuclease digestion .................................................................................92 Southern blot analysis ..............................................................................................93 Results .....................................................................................................................................93 MNase Mapping ..............................................................................................................94 DNase I Digestion ...........................................................................................................97 Discussion ...............................................................................................................................99 Nucleosomal Positioning Pattern among Ves Genes .....................................................100 Nuclease Sensitivity Analysis .......................................................................................102 SUMMARY .................................................................................................................................123 PUTATIVE HISTONES AND HISTONE MODIFIING ENZYMES IN BABESIA BOVIS ...130 COMPARISON OF HISTONE H3 N-TERMINI BETWEEN BABESIA BOVIS AND OTHER ORGANISMS ........................................................................................................131 THE VALIDATION OF COMMERIAL ANTIBOIDIES