INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text direct^ from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter Ace, indnle others may be from axy type o f 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 photogrq>hs, print bleedthrough, substandard margins, and improper alignment can adversety afifect 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, b%innmg at the upper left-hand comer and continuing from left to right in equal sections with small overl^s. Each original is also photographed in one exposure and is included in reduced form at the back o f the book. Photogrtq)hs included in the original manuscript have been reproduced xerographically in this copy, ifigher quality 6” x 9” black and white photographic prints are available fiar any photographs or illustrations appearing in this copy fi)r an additional charge. Contact UMI directly to order. UMI A Bell f t Howell Infonnation Company 300 North Zeeb Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE SEQUENCE AND ANALYSIS OF APPROXIMATELY 0.5 MEGA-BASEPAIR OF THE DIGEORGE SYNDROME CRITICAL REGION IN HUMAN CHROMOSOME 22 BAND QII AND A SYNTENIC MOUSE BAC A Dissertation SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the degree of Doctor of Philosophy By FENG CHEN Norman, Oklahoma 1997 UMI Number: 9808400 UMI Microform 9808400 Copyright 1997, 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 SEQUENCE AND ANALYSIS OF APPROXIMATELY OJ MEGA-B ASEPAIR OF THE DIGEORGE SYNDROME CRITICAL REGION IN HUMAN CHROMOSOME 22 BAND QI I AND A SYNTENIC MOUSE BAC A Dissertation APPROVED FOR THE DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY © Copyright by FENG CHEN 1997 All Rights Reserved Acknowledgements I would like to express my sincere gratitude to my wife Jing Li, and my parents for their support and love. I also would like to thank my brother and sisters for their encouragement. I would like to thank all the teachers I had in the way of my growth for everything they taught me. A very special acknowledgment goes to my major professor. Dr. Bruce A. Roe, for his guidance, knowledge, and support. I also extend my appreciation to the other members of my graduate advisory committee, Drs. Leroy Blank, Phil E. Klebba, David R. McCarthy, and Arm West. A big thanks goes to everyone in Dr. Roe's Lab, both past and present, for their positive support, mutual help, team effort, and inspiring discussion; Mueed Ahmad, Cathy Anadu, Dennis Burian, Linda Cantu, Lingzhi Chu, Sandy Clifton, Arlena Coulberson, Judy Crabtree, Chesca Craig, Angela Dorman, Whimey Elkins, Jennifer Gray, Glenda Hall, Karen Hartman, Jennifer Hausner, Andrew Homing, Naiqing Hu, Emily Huang, Axin Hua, Nora Ivanoff, Steve Kenton, Akbar Khan, Doris Kupfer, Hongshing Lai, Lisa Lane, Hioleong Lao, Michele Lasley, Sharon Lewis, Shaoping Lin, Eda Malaj, Anthony McDaniel, Sean Meadows, Fares Najar, Nientung Ma, Yichen Ma, Thuan Nguyen, Huaqin Pan, Murli Rao, Adonis Reece, Qun Ren, Ging Sobhraksha, Lin Song, Rhys Strasia, Will Tankersley, Steve Toth, Yonathan Tilahun, Jeffery Viken, Ha Vu, YingPing Wang, Yungfang Wang, Zhili Wang, Rusty Wayt, Heather Wright, Ziyun Yao, Sola Yu, Xiling Yuan, Min Zhan, Guozhong Zhang, and Hua Zhu. I would like to especially thank Drs. Nientung Ma, Steve Toth, and Zhili Wang for their help on the project. I thank our collaborators, Drs. Marcia L. Budarf and Beverly S. Emanuel from the Children’s Hospital of Philadelphia, PA and member of their research groups for IV providing mapped cosmids and B ACs and their collaboration. This work was supported by grants &om the National Institute of Health, the Oklahoma Center for the Advancement of Science and Technology and the Oklahoma Department of Commerce Center of Excellence in Molecular Medicine Program. Hnancial supports to the author provided by the Department of Chemistry and Biochemistry and the Graduate College of the University of Oklahoma are deeply appreciated. Table of Contents List of Tables IX List of Figures X List of Abbreviations xii Abstract xvii Chapter I: Introduction I I. I DiGeorge syndrome I 1.1.1 History and nomenclature 1 1.1.2 Description and clinical features 2 1.1.3 Genetics of DiGeorge syndrome 3 1.1.4 Sequencing analysis 13 1.2 DNA 16 1.2.1 Inheritance and DNA 16 1.2.2 DNA components 20 1.2.3 DNA structure 22 1.2.4 DNA and genetic information 27 1.2.5 DNA packaging and chromosome 31 1.3 Large scale DNA sequencing 33 1.3.1 History of DNA sequencing 33 1.3.2 Improvement of DNA sequencing 35 1.3.3 Large scale DNA sequencing strategy 39 Chapter II: Methods and Materials 49 2.1 Random shotgun sequencing strategy 49 2.2 Material preparation 54 2.2.1 Large scale DNA isolation 54 VI 2.2.2 Mini-prep isolation of subcloned DNA 57 2.3 Random shotgun DNA subclone library construction 60 2.3.1 DNA shearing 61 2.3.2 End-repair and phosphorylation 65 2.3.3 DNA size selection 66 2.3.4 DNA ligation and transformation 67 2.4 Dideoxynucleotide DNA sequencing 70 2.4.1 AmpliTaq catalyzed fluorescent labeled primer cycle sequencing 71 2.4.2 Sequenase catalyzed fluorescent dye terminator sequencing 72 2.4.3 AmpliTaq catalyzed fluorescent labeled terminator cycle sequencing 73 2.4.4 KlenTaq-TR (Taquenase) catalyzed fluorescent labeled terminator cycle sequencing 74 2.4.5 AmpliTaq-FS catalyzed fluorescent labeled terminator cycle sequencing 74 2.4.6 Unincorporated dye terminator removal by Sephadex G-50 spin coliunn 75 2.4.7 Sequencing gel preparation, sample loading and electrophoresis on ABI377 76 2.5 Shotgun sequence data editing and assembling 77 2.6 Final sequence data analysis 80 2.7 Gap closure 82 2.7.1 Oligonucleotide primer synthesis and PCR 84 Chapter HI: Results and Discussion 87 3.1 Summary 87 3.2 Comparison of 36 kb of genomic sequences fiom two individuals 90 VII 3.3 Analysis of contig I 92 3.3.1 Qathrin heavy chain-Uke gene 94 3.3.2 Ribosomal protein L34 pseudogene 105 3.3.3 Repetitive elements in contig 1 107 3.4 Analysis of contig 2 108 3.4.1 Contig 2.1 108 3.4.1.1 Human HIRA gene 110 3.4.1.2 Nucleotide composition of the 5' region of the HIRA gene 125 3.4.1.3 Repetitive elements in the HIRA gene region 128 3.4.2 contig 2.2 129 3.4.2.1 Human ubiquitin fusion-degradation protein gene 129 3.4.2.2 A putative gene in the distal region of contig 2.2 144 3.4.2.3 Repetitive elements in contig 2.2 159 3.5 Analysis of contig 3 160 3.5.1 Major similarities found in database by Powerblast in contig 3 160 3.5.2 Repetitive elements in contig 3 171 3.6 Analysis of contig 4 172 3.6.1 TlOgene 172 3.6.2 Repetitive elements in contig 4 182 3.7 Analysis of a mouse BAC containing the mouse HIRA gene 183 3.7.1 Mouse HIRA gene 185 3.7.2 Comparison of the human and mouse HIRA gene 196 Chapter IV: Conclusion 206 Chapter V: Literature Cited 216 V lll List of Tables 1. Summary of cosmids sequenced 88 2. Splice sites of CLTCL comparing with the consensus sequence 104 3. Interspersed repetitive elements in contig I 107 4. Position and size of exons and size of introns of human HIRA gene in contig 2.1 111 5. Exon/intron boundary of HIRA gene 112 6. Interspersed repetitive elements in contig 2.1 128 7. Size and boundary of exons and introns of human UFDIL gene 140 8. Comparison of the exon/intron boundary of putative gene and the consensus sequence 146 9. Possible transmembrane helices in putative gene 156 10. Preferences of the orientation of helices in putative gene 158 11. Suggested model for transmembrane topology of putative gene 158 12. Interspersed repetitive elements in contig 2.2 159 13. Interspersed repetitive elements in contig 3 171 14. Position and size of exons and size of introns of human T 10 gene in contig 4 179 15. Exon/intron boundary of human TlOgene 179 16. Interspersed repetitive elements in contig 4 183 17. Position and size of exons and size of introns of mouse HIRA gene 186 18. Exon/intron boundary of mouse HIRA gene 187 19. Sizes comparison of introns of human and mouse HIRA gene 200 20. List of genes in the DiGeorge syndrome critical region 212 IX List of Figures 1. Map ofDlGeorge syndrome critical region 8 2. Map of cosmids, fosmid, BACs and PAC in EKj CR 15 3. Deoxynucleotide structures 21 4. H-bonds in B-form DNA 24 5. Comparison of A-form, B-form, and Z-form DNA structures 26 6. Central dogma of molecular biology 28 7. Illustration of chromosome in metaphase 32 8. Random shotgun DNA sequencing strategy 51 9.