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Zeeb Road, Ann Arbor, Ml 48106 Copyright 1986 by Ralph, David Allen All Rights Reserved PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark ■/ . 1. Glossy photographs or pages______ 2. Colored illustrations, paper or print _______ 3. Photographs with dark background i / 4. Illustrations are poor copy_______ 5. Pages with black marks, not original copy _______ 6. Print shows through as there is text on both sides of page_______ 7. Indistinct, broken or small print on several pages _______ 8. Print exceeds margin requirements_______ 9. Tightly bound copy with print lost in spine________ 10. Computer printout pages with indistinct print _______ 11. Page(s)____________lacking when material received, and not available from school or author. 12. Page(s) seem to be missing in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curling and wrinkled pages ______ 15. Dissertation contains pages with print at a slant, filmed as received 16. Other University Microfilms International EVOLUTION OF CYTOPLASMIC GENOMES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Phylosophy in the Graduate School of the Ohio State University By David Allen Ralph, B.S., M.S. ft « « * » The Ohio State University 1986 Dissertation Committee: Approved by P. S. Perlman P. A. Fuerst Advisor S. Falkenthal Molecular, Cellular and Developmental Biology Program Copyright by David Allen Ralph 1986 This dissertation is dedicated to Peggy McDaniel without whose help I could not have completed this work. 11 ACKNOWLEDGEMENTS I would like to express my deepest thanks to John Briggs, Karl Joplin, Jason Tash, Makis Skoulakis, Elio Vanin, Kirk Mecklenburg, Chip Pretzman, Yasoko Rikihisha, and Phil Perlman for assisting and supporting me during my years in graduate school. iii VITA Augest 2, 1954 Born - Dallas Texas 1976...... B.S., Ohio State University 1979 M.S., Ohio State University 1981-1985.... Intern, Ohio Department of Health PUBLICATIONS Streett, D.A., David Ralph, and.Fred Hihk. 1980. Replication of Nosema algerae in three insect cell lines. J. Protozool. 27:113-117. Hinlc,W.F., D.A. Ralph, and K.H. Joplin. 1985. Metabolism and characterization of Insect cell lines, in Comprehensive Insect Physiology Biochemistry and Pharmacology, eds. G.A. Kerkut and L.I. Gilbert. Pergamon Press. New York. Pretzman Jr. Charles I., David Ralph, Lynn Mishler, and Joyce Bodine. 1985. Rapid separation of IgM from whole serum using spun column chromatography. J. Immunological Methods. 63:301-307. Pretzman Jr., Charles I., Yasoko Rikihisha, and David Ralph. 1986. Serological diagnosis of Plutomic horse fever. Clinical Microbiology (submitted) PUBLICATIONS IN PREPARATIONS Ralph, David and Phil Perlman. Structure of the Cox I gene in ten species of yeast Ralph, David and Phil Perlman. A newly observed intron in the Cox I gene of Saccharomyces capensis has an unusual structure. Ralph, David, Charles I. Pretzman Jr., Karl Poetter, Scott Gordon, Jon Clark, Paul Fuerst, and Phil Perlman. Molecular differenciation of Rickettsia. FIELDS OF STUDY Major Field: Molecular Biology (Dr. Phil Perlman-advisor) Minor Field: Molecular biology and in vitro cultivation of parasitic organisms. (Dr. Paul Fuerst, Dr.Fred Hink, and Dr. Yasoko Rikihisha) V TABLE OF CONTENTS DEDICATION.................................................... ii ACKNOWLEDGMENTS............................................. iii VITA........................................................... iv TABLE OF CONTENTS............................................vi LIST OF TABLES...................................... ix LIST OF FIGURES...................................... X CHAPTER I: INTRODUCTION.....................................1 I.A. Preface...................................................1 I.B. Introduction to organelle introns................... 5 I.B.1. Why choose Saceharomyces sp. as a model system to study mitochondrial functions?..........................5 I.B.2. Types of mutants which affect mitochondrial function........................... 8 I.B.3. The organization of yeast and mammalian mitochondrial genomes................ 13 I.B.4. Yeast mitochondrial genes contain introns.... 18 I.B.5. Other fungal mitochondrial genomes contain introns................................... 23 I.B.6. Translation products of fungal mitochondrial introns......... 35 I.B.7. Other translation products of fungal mitochondrial introns: Maturases.................40 I.B.8. Cis-acting sequences required for introns related to cob 14...................... 50 I.B.9. Cis -acting mutants in other introns in the S_j_ cerevisiae mitochondrial genome............................. 54 I.B.10. Computer modelling RNA secondary structures of yeast mitochondrial introns. ........................................ 56 I.B.11. Amino acid homologies between intronic ORF proteins of yeast........................... 58 I.B.12. Autocatalytic and catalytic activities of group I introns...............................61 I.B.13. Oxi I5g, a group II intron, is also autocatalytic......................... 70 I.B.14. Nuclear pre-mRNA splicing: Similarities with group II mitochondrial introns.......... 72 I.B.15. All fungal mitochondrial introns belong to either class I or class II.......... 76 I.B.16. Introns in organelle genes of plants......... 79 I.B.16.a. The cytochrome oxidase subunit II gene in the mitochondrial genomes vi of flowering plants......................... 79 I.B.16.b. Introns in chloroplast tRNA genes........81 I.B.16.c. Introns in chloroplast genes other than tRNA genes...................... 85 I.B.17* A class I intron in the thymidylate synthase gene of the bacteriophage T4........89 I.B.18. Possible class I intron in an archaebacterial 23S rRNA gene....... 90 I.B.19* Short regions of high G+C content in the mitochondrial genome of S. cerevlslae .................................. 91 I.B.20. Conclusion................ 92 I.C. Taxonomy and physiology of Rickettsia.............95 I.C.1. Taxonomy of the family Rickettsiaceae ........95 I.C.2. Typhus group rickettsia..........................98 I.C.3* The Rocky Mountain Spotted Fever group.......105 I.C.4. Scrub typhus...................................... 120 I.C.5. Antigens shared between different groups within the genus Rickettsia..........125 I.C.6. Rickettsial physiology.......................... 127 I.C.7. The genus Rochalimaea ............'............. 132 I.C.8. Rickettsia are a good model for early mitochondrial evolution.................131* CHAPTER II: METHODS...................................... 138 II.A. Strains used......................................138 II.B. Media and buffers used..........................139 II.C. Purification of mitochondrial DNA............ 140 II.D. Tissue culture, growth of rickettsia and purification of rickettsial DNA...........141 II.E. Separation of restriction endonuclease derived fragments of DNA on agarose gels and band isolation............................... 146 II.E. DNA blot hybridization..........................147 II.F. Labeling of DNA probes..........................148 II.G. Construction of recombinant clones........... 149 II.H. Transformation of bacteria..................... 151 II.I. Bal-31 deletions.................................154 II.J. Screening colorless M13 plaques for inserts........................................155 U.K. Isolation of single stranded M13 DNA......... 155 II.L. DNA sequencing of M13 derived ssDNA.......... 157 II.M. The Gimenez hemolymph stain....................158 II.N. The microimmunofluorescence (micro IF) test.. 159 CHAPTER III: