INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. 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. A Bell & Howell InformationCompany 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313.'761-4700 800/521-0600 THE H-2S/D REGION OF THE MOUSE MAJOR HISTOCOMPATIBILITY COMPLEX: COSMID CLONING, NOVEL GENE IDENTIFICATION, AND TISSUE EXPRESSION STUDIES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Dennis Keith Lanning, B.S. ***** The Ohio State University 1995 Dissertation Committee: Arthur Burghes Approved by William P. Lafuse \WdJU*^ Deborah Parris Advisor Department of Medical Bruce Zwilling Microbiology and Immunology UMI Number: 9526051 UMI Microform 9526051 Copyright 1995, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 My Parents ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor, Dr. William P. Lafuse, for his guidance and friendship throughout the course of this research. I would also like to thank the other members of my advisory committee, Drs. Arthur Burghes, Deborah Parris, and Bruce Zwilling, for their enthusiasm, support, and advice. My heartfelt appreciation to Heidi Sahlbom for supporting my return to graduate school and standing by me despite the many miles between us. Finally, my deepest thanks to my parents and family for their unwavering support and encouragement. VITA June 16, 1955 ...............................Bom - Grand Rapids, Michigan 1977 ................................................ B.S., Biology, Michigan State University, E. Lansing, MI 1 9 8 1 ............................................. Continuing Education, Calvin College, Grand Rapids, MI. 1984 - 1988 ................................. Continuing Education, Grand Valley State University, Allendale, MI. 1988 - p re s e n t .......................... Graduate Research Associate, Department of Medical Microbiology and Immunology, The Ohio State University, Columbus, OH. PUBLICATIONS Lafuse, W .P., Lanning, D., Spies, T., and David, C.S. (1992). PFGE mapping and RFLP analysis of the S/D region of the mouse H-2 complex. Immunogenetics 36: 110-116. FIELDS OF STUDY Major field: Medical Microbiology and Immunology Studies in Molecular Immunology - Dr. William P. Lafuse TABLE OF CONTENTS DEDICATION....................................................................................................... ii ACKNOWLEDGEMENTS.................................................................................... iii VITA ................................................................................................................................... iv LIST OF FIGURES.................................................................................................. vii LIST OF PLATES....................................................................................................viii LIST OF TABLES.................................................................................................. x CHAPTER I - INTRODUCTION......................................................................... 1 CHAPTER H - MATERIALS AND METHODS................................................ 54 Cell Lines .............................. - ............................................................. 54 DNA P ro b es ............................................................................................... 55 RNA Isolation ............................................................................................ 55 Long-Range Restriction Mapping ........................................................... 56 Cosmid Library Screening ...................................................................... 58 Exon Trapping .......................................................... 63 CHAPTER m - LONG-RANGE RESTRICTION MAPPING OF THE H-2S/D REGION................................................ 70 CHAPTER IV - COSMID CLONING OF THE H-2S/D REGION................. 77 CHAPTER V - EXON TRAPPING STUDIES....................• 102 CHAPTER VI - TISSUE EXPRESSION STUDIES ..........................................157 v CHAPTER VII - DISCUSSION LIST OF REFERENCES LIST OF TABLES TABLE PAGE 1. Summary of restriction fragment sizes ...........................................................73 2. Recognition sequences and cleavage sites of the restriction enzymes used in cosmid clone restriction m apping ......................................79 3. Genomic fragments isolated by exon trapping ........................................... 156 vii LIST OF FIGURES FIGURES PAGE 1. Pulsed field gels hybridized with cDNA probes to the Tnfa, B f BAT3, and BATS genes ................................................... 72 2. Long-range restriction map of the S/D region of the BALB/c mouse H-2 com plex .............................................................. 75 3. Cosmid clone U6.1 restriction map ......................... ............................... 89 4. Cosmid clone U6.1 restriction map with the positions of the Bat4 and Bat5 loci indicated ......................................................... 92 5. Composite restriction map of the telomeric segment of theH-2S/D region showing overlapping cosmid clones and the positions of the Bat2, Bat3, Bat4, BatS, Bf, and Tnfa lo c i ................................................................................................ 94 6. Composite restriction map of the centromeric segment of the H-2S/D region showing overlapping cosmid clones and the position of the Bat6 lo c u s ........................................................... 99 7. Cosmid clone Cl.l restriction map showing the map position of genomic fragment C. 1.350(2) ............................................. 117 8. Sequence homology between genomic fragment C. 1.350(2) and the humanBA T3 coding sequence ........................................................... 119 9. Cosmid clone Cl.l restriction map showing the map position of genomic fragment C. 1.440(1) .............................................................. 122 10. Cosmid clone Cl.l restriction map showing the map position of genomic fragment C.3.220(1) .............................................................. 125 viii 11. Cosmid clone B5.2 restriction map showing map position of genomic fragment B.5.800(1) .................................................................... 131 12. Cosmid clone U6.1 restriction map showing the map position of genomic fragment U6.1 280(2) ........................................................... 137 13. Sequence homology between genomic fragment U6.1 280(2) and the mouse casein kinase U 0 subunit (CA7//3) coding sequence 138 14. Cosmid clone E9.1 restriction map showing the map position of genomic fragment E. 1.550(2) .............................................................. 143 15. Cosmid clone E9.1 restriction map showing the map position of genomic fragment E. 1.280(2) .............................................................. 146 16. Cosmid clone E9.1 restriction map showing the map position of genomic fragment E.3.230(2) .............................................................. 148 17. Cosmid clone E9.1 restriction map showing the map position of genomic fragment E. 1.230(2) .............................................................. 151 18. Cosmid clone E9.1 restriction map showing the map position of genomic fragment E.4.270(2) ................................................................154 ix LIST OF PLATES PLATES PAGE I. Cosmid clone U6.1 restriction mapping g e l ......................................... 81 n . Cosmid clone U 6.1 restriction mapping blot hybridized with 32P-end-labeled T3 oligonucleotide p r o b e .................................. 84 HI. Cosmid clone U6.1 restriction mapping blot hybridized with 32P-end-labeled T7 oligonucleotide p ro b e .................................. 85 IV. Cosmid clone U6.1 Kpnl partial digest blot hybridized with 32P-end-labeled T3 oligonucleotide p r o b e ......................... 87 V. Cosmid clone U6.1 restriction mapping blot hybridized with 32P-labeled human BAT4 cDNA probe .......................................