Order N u m b e r 9201787 Manipulation of the chloroplast genome Ye, Jingsong, Ph.D. The Ohio State University, 1991 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 MANIPULATION OF THE CHLOROPLAST GENOME DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Jingsong Ye, B.S., M.S. ***** The Ohio State University 1991 Dissertation Committee: Approved by R. Sayre V . Raghavan F. Sack Advisor Department of Plant Biology D. Bisaro To my Father and Mother To my wife Hua and my daughter Qian-qian • * 11 ACKNOWLEDGMENTS I would like to express my sincere appreciation and gratitude to all those involved in the completion of this dissertation: Dr. Richard T. Sayre, my advisor, for his continually valuable advice and guidance during the investigation and preparation of the dissertation. His gentle nature, kindness and personal concerns greatly helped me complete the study and adapt to the life in the U.S.A. Dr. V. Raghavan, Dr. F. Sack, Dr. D. Bisaro, Dr. T. Sims and Dr. R. Hangarter for their advice and suggestions concerning my Ph.D. project and my career. Ms. Susan Ketchner, Ms. Robin Roffey, Ms. Elizabeth Wrobel-Boerner, Mr. John Okuley and Mr. Reed Clark for their guidance and assistance in laboratory work and for their kindness which made my life at The Ohio State University enjoyable and memorable. Finally, my wife, Yan Hua and my daughter, Qian-qian, for their endless love, encouragement and sacrifice during my study for Ph.D. VITA May 4, 1957 Born in Nanjing, P.R. China 1982 B.S., Wuhan University, Hubei, P.R. China 1986 M.S. , University of Illinois at Urbana-Champaign 1986-Present Graduate Student Teaching Assistant, Ohio State Plant Biology Program, Columbus, Ohio PUBLICATIONS Ye J, Hauptmann RM, Smith AG, Widholm JM (1987) Selection of a Nicotiana plumbaainifolia universal hybridizer and in intergeneric somatic hybrid formation. Mol Gen Genet 208:474-480 Ye, J, Sayre RT (1990) Reduction of chloroplast DNA content in Solanum nigrum suspension cells by treatment with chloroplast DNA synthesis inhibitors. Plant Physiology 94:1477-1483 FIELDS OF STUDY Major Field: Plant Biology Studies in development of a universal somatic hybrid selection system for plant protoplast fusion under Dr. Jack M. Widholm Studies in manipulation of chloroplast genome of Solanum nigrum and Chlamydomonas in vivo under Dr. Richard T. Sayre TABLE 07 CONTENTS ACKNOWLEDGMENTS ......................................... iii VITA ..................................................... iv LIST OF TABLES.......................................... viii LIST OF FIGURES......................................... ix ABBREVIATIONS ........................................... xi CHAPTER PAGE I. INTRODUCTION ......................................... 1 1. 1 INTRODUCTION......................................1 1.2 REGULATION of CHLOROPLAST DNA SYNTHESIS .......... 2 1.2.1 Chloroplast Genome ........................ 2 1.2.2 Plastid DNA Copy Number.................... 4 1.2.3 Chloroplast DNA Replication .............. 7 1.2.4 Inhibitors of Chloroplast DNA Replication . 9 1.3 CHLOROPLAST TRANSFORMATION ...................... 11 1.3.1 Introduction of DNA into the Nucleus 12 1.3.2 Introduction of Foreign Plastid ......... 13 1.3.3 Autonomously Replicating Plasmids ...... 14 1.3.4 Introduction of Foreign Genes into Chloroplasts.............................. 15 1.3.5 Problems and Prospects.................... 18 1.4 TRANSFORMATION OF CHLAMYDOMONAS CHLOROPLASTS _____ 19 1.4.1 Chiamydomonas is a Unique Transformation System.................................... 19 1.4.2 Delivery of DNA to Chloroplasts.......... 20 1.4.3 Selectable Marker for Chloroplast Transformation ........................... 25 1.5 RECA PROTEIN..................................... 28 1.5.1 General Characteristic .................. 28 1.5.2 Role in General Recombination.............32 1.5.3 Role in DNA Re p a i r ........................ 40 1.5.4 RecA Protein Analogues....................44 v II.REDUCTION OF CHLOROPLAST DNA CONTENT IN SOLANUM NIGRUM SUSPENSION CELLS BY TREATMENT WITH CHLOROPLAST DNA SYNTHESIS INHIBITORS ................ 46 2.1 INTRODUCTION..................................... 46 2.2 MATERIALS AND METHODS............................ 48 2.2.1 Plant Material and Growth Conditions ... 48 2.2.2 Isolation of Protoplasts..................48 2.2.3 Treatment of Suspension Cells with Chloroplast DNA Synthesis Inhibitors ... 49 2.2.4 Analyses of Chloroplast and Nuclear DNA Content................................... 50 2.2.5 Quantification of Total Nucleic Acids by Fluorescent DNA Binding D y e s ..............52 2.3 RESULTS.......................................... 53 2.3.1 Effects of DNA Synthesis Inhibitors on Cell Growth and Viability................. 53 2.3.2 Effects of DNA Synthesis Inhibitors on Nuclear and Chloroplast DNA Content ..... 57 2.4 DISCUSSION....................................... 66 III. CHLOROPLAST TRANSFORMATION AND MUTAGENESIS ....... 71 3.1 INTRODUCTION..................................... 71 3.2 MATERIALS AND METHODS............................ 74 3.2.1 Plasmids and Chlamvdomonas Strains ...... 74 3.2.2 Reduction of Chloroplast DNA Content in Chlamvdomonas ............................ 75 3.2.3 Ch1amydomonas Transformation..............76 3.2.4 Regeneration of Plants from Suspension Cells and Leaves.......................... 77 3.2.5 Transformation of Solanum niorum Cells ... 77 3.2.6 Southern Hybridization Analyses ......... 78 3.2.7 Preparation of Oligonucleotide Probe .... 79 3.2.8 DNA Hybridizations........................ 80 3.2.9 Chloroplast Mutagenesis .................. 81 3.3 RESULTS ...................................... 81 3.3.1 Regulation of Chloroplast DNA Content in Chlamvdomonas.......................... 81 3.3.2 Transformation Frequency in Chlamvdomonas 84 3.3.3 Regeneration of Plants from Suspension and Leaf Cells ........................... 85 3.3.4 Chloroplast Transformation and Mutagenesis............................... 87 3.4 DISCUSSION....................................... 95 IV. CHLOROPLAST RECA-LIKE PROTEIN IN CHLAMYDOMONAS ___ 104 4.1 INTRODUCTION.................................... 104 4.2 MATERIALS AND METHODS........................... 106 Vi 4.2.1 Chlamvdomonas Strain and Plasmid ....... 106 4.2.2 Insolation of Chloroplast Proteins and Western Blotting a..**................... 10*7 4.2.3 Treatment of Chlamvdomonas Cells with Novobiocin............................... 108 4.2.4 Labeling Plasmid DNA with Tritiated Nucleotides.............................. 109 4.2.5 DNA Renaturation As s a y ................... 110 4.2.6 Purification of RecA-like Protein ...... Ill 4.2.7 Preparation of pX13 s s D N A ................ 112 4.2.8 Preparation of [MP]-dsDNA............. 113 4.2.9 Assay of Three-stranded D N A .............. 114 4.3 RESULTS......................................... 115 4.3.1 Identification of "RecA-like" Protein in the Chloroplast of Chiamydomonas ....... 115 4.3.2 Renaturation of D N A ..................... 120 4.3.3 Determination of Optimal Conditions for Chloroplast Protein Catalyzed DNA Renaturation Reaction...................123 4.3.4 Partial Purification of RecA-like Protein................................. 140 4.3.5 Formation of Three-Stranded D N A ........ 14 6 4.4 DISCUSSION...................................... 155 V. SUMMARY............................................. 161 5.1 REDUCTION cpDNA CONTENT BY TREATMENT WITH cpDNA SYNTHESIS INHIBITORS ..................... 161 5.2 TRANSFORMATION OF S. NIGRUM PLASTIDS AND MUTAGENIC EFFECTS OF CpDNA SYNTHESIS INHIBITORS.......................................163 5.3 REGULATION OF cpDNA CONTENT AND TRANSFORMATION FREQUENCY IN CHLAMYDOMONAS ..................... 164 5.4 RecA-LIKE PROTEIN IN CHLAMYDOMONAS CHLOROPLASTS ................................... 165 5.5 PROSPECTS....................................... 168 REFERENCES............................................... 170 vii LIST OF TABLES TABLE 1. Effects of DNA Synthesis Inhibitors on the DNA Content and Fresh Weight of £. nigrum Suspension Cells (8 day Culture) ................................ 58 2. Effects of DNA Synthesis Inhibitors on the DNA Content and Fresh Weight of £. nigrum Suspension Cells (2X4 day culture) ............................ 59 3. Quantification of Total DNA in Protoplasts Labeled with the Fluorescent DNA Binding Dye, DAPI ............63 4. Effects of Chloroplast DNA Synthesis Inhibitors on DNA Content of Protoplasts Isolated from 2 X 4 day Suspension Cultures of S. nigrum as Determined by Dot Blot Hybridizations..............................65 5. Effects of cpDNA Synthesis Inhibitors on DNA Content of Chlamydomonas as Determined by Dot Blot Hybridizations ...................................... 83 6. Increase of the Transformation Frequency for Chlamydomonas Chloroplast psbA Deletion Mutant ....... 86 7. Effect of BAP and IAA on Green Shoot Formation from Callus...............................................88 8. Mutagenic Effect of Novobiocin on Chloroplast DNA ... 96 9. Ammonium Sulfate Fractionation .................. 141 10. Cellulose Phosphate Purification.. ................ 142 11. DEAE Sephadex Purification......................... 143 LIST or FIGURES FIGURE 1. Schematic of a Microprojectile Delivery System .... 21 2. Reactions Promoted by recA Protein..................34 3. Reaction Pathway for the Transfer of a DNA Strand from a Linear Duplex to a Circular s s D N A ........... 39 4. Model of the SOS Regulatory System...............