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Information to Users 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. 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 addition charge. Contact UMI directly to order. Bell & Howell Information and Leaming 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMI' UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE DEVELOPMENT AND ANALYSIS OF AN EXPRESSED SEQUENCE TAG DATABASE FROM FUSARIUM SPOROTRICHIOIDES A Dissertation SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the degree of Doctor of Philosophy By QUNREN Norman, Oklahoma 2001 UMI Number: 3005138 UMI UMI Microform 3005138 Copyright 2001 by Beil & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 © Copyright by Qun Ren 2001 All rights reserved. DEVELOPMENT AND ANALYSIS OF AN EXPRESSED SEQUENCE TAG DATABASE FROM FUSARIUM SPOROTRICHIOIDES A Dissertation APPROVED FOR THE DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY Acknowledgements I am very grateful to my committee members, Dr. Bruce A. Roe, Dr. Paul F. Cook, Dr. Ann H. West, Dr. George B. Richter-Addo and Dr. John S. Downard for their kind help during my study here. Dr. Cook was particularly helpful throughout my program in providing encouragement and insights. I also wish to thank Dr. Phil E. Klebba for his help when I prepared for my general exam. My special thanks go to Dr. Roe, my major professor, who has always provided constant encouragement, excellent advice, and generous financial support for my study here over the past five years. I also appreciate his patience and challenge to me. I wish to thank everyone in Dr. Roe’s lab for giving me advice, support and help. Thanks especially to Mueed Ahmad, Dennis Burian, Linda Cantu, Feng Chen, Lingzhi Chu, Sandra Clifton, Stéphane Deschamps, Sara Downard, An Do, Trang Do, Angela Dorman, Mounir Elharam, Fang Fang, Ying Fu, Kaylynn Hale, Jennifer Gray, Li Hang, Jennifer Hausner, Ping Hu, Xiaohong Hu, Axin Hua, Honggui Jia, Emily Huang, Steve Kenton, Doris Kupfer, Hongshing Lai, Sean Meadows, Lisa Lane, Victoria Lao, Christopher Lau, Sharon Lewis, Shaoping Lin, Phobe Loh, Eda Malaj, Rose Morales- Diaz, Wendy Martin, Jami Milam, Fares Najar, Thuan Nguyen, Shelly Oommen, Huaqin Pan, Andy Peterson, Yudong Qian, Sulan Qi, Murli Rao, Lin Song, Jing Tian, Runying Tian, Yonathan Tilahun, Qiaoyan Wang, Ying-Ping Wang, Doug White, Rusty Wayt, Zhili Wang, Jim White, Mary Catherine Williams, Dixie Wishnuck, Tammy Womack, Heather Wright, Hui Wu, Limei Yang, Ziyun Yao, Xuling Yuan, Min Zhan, Guozhong Zhang and Hua Zhu. IV I greatly appreciate my parents, Qiyu Ren and Yiying Zhou, my brother Min Ren, and my sister-in-laws Zhaomei Zhang and Lanying Han. Their love and help enabled my husband and I to come to the United States of America to study. Finally, I dedicate this dissertation to my husband, Zhaojie Zhang and my daughter, Renfei Zhang, who gave me constant support, encouragement and love. Table of Contents List of Figures ...............................................................................................................x List of Tables ................................................................................................................xii List of Abbreviations .............................................................................................. xiv Abstract................................................................... ......................................................xviii Chapter I. Introduction ...............................................................................................1 1.1 DNA,RNA and Genes............................................................................................1 1.1.1 DNA.......................................................................................................1 1.1.2 RNA....................................................................................................... 4 1.1.3 Genes.....................................................................................................5 1.1.3.1 Definition of gene ....................................................................5 1.1.3.2 Classification of genes ............................................................ 5 1.1.3.3 Structures of genes .................................................................. 6 1.1.3.4 Pseudogenes ............................................................................ 7 1.2 mRNA...................................................................................................................... 8 1.2.1 Splicing .................................................................................................. 8 1.2.2 5’ cap......................................................................................................8 1.2.3 3’ poly(A) tail ........................................................................................ 10 1.2.4 Mature mRNA...................................................................................... 10 1.3 cDNA library............................................................................................................11 1.3.1 Advantages of constructing an cDNA libray .........................................11 1.3.2 General procedures for construction of cDNA library ......................... 12 1.3.3 The X ZAP system as the vector for cDNA library .............................. 15 1.4.EST and UniGene databases................................................................................. 16 1.4.1 Beginning of large scale EST sequencing .............................................17 1.4.2 Recent advances in large scale EST sequencing .................................. 17 1.4.3 3’EST and 5’EST...................................................................................19 1.4.3.1 5’EST is more gene family specific ........................................19 1.4.3.2 3’ EST is more gene specific .................................................. 22 1.4.4 UniGene database....................................................................................24 1.4.5 Applications of EST and UniGene database .........................................25 1.5 DNA sequencing ....................................................................................................27 1.5.1 Basic methods for DNA sequencing ..................................................... 27 1.5.2 Improvements on Sanger method .......................................................... 28 1.5.3 DNA sequencing instruments .................................................................31 1.5.4 Shortgun strategy for large scale DNA sequencing .............................. 32 1.6 Sequence analysis software and data submission web site ...................................33 1.6.1 Phred and Phrap .......................................................................................33 1.6.2 Cross_match, Dotter, Bestfit, Gap and Sim4 .........................................34 1.6.3 Consed .....................................................................................................34 1.6.4 BLAST.....................................................................................................35 1.6.5 Powerblast ................................................................................................36 1.6.6 Genscan and Xgrail, NNPP, Promoter 2.0 and Repeatmasker ............. 36 1.6.7 GenBank, Entrez, Sequin and dbEST .................................................... 37 1.7 Fusarium sporotrichioides cDNA library..............................................................39 VI 1.8 Inborn gap in the published megabase sequence of the Ig light chain genes .....44 1.9 William Syndrome is associated with deletion of chromosome band 7ql 1.23... 46 Chapter II. Materials and Methods (Part 1)-Sequence and analysis of ESTs... 48 2.1 Construction of cDNA library ................................................................................ 48 2.1.1 Source of cDNA library ...........................................................................48 2.1.2 cDNA synthesis........................................................................................48 2.1.2.1 Synthesis of the first strand cDNA ..........................................48 2.1.2.2 Synthesis
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