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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 corner 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. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9316190 Heterodimeric deoxyguanosine/deoxyadenosine kinase from Lactobacillus acidophilus R-26: Affinity protein purification, molecular cloning, sequence of the genes, and expression in Escherichia coli Ma, Grace Tak-Yi, Ph.D. The Ohio State University, 1993 Copyright ©1993 by Ma, Grace Tak-Yi. All rights reserved. UMI 300 N. ZeebRd. Ann Arbor, MI 48106 HETERODIMERIC DEOXYGUANOSINE/DEOXYADENOSINE KINASE FROM LACTOBACILLUS ACIDOPHILUS R-26: AFFINITY PROTEIN PURIFICATION, MOLECULAR CLONING, SEQUENCE OF THE GENES, AND EXPRESSION IN E. COLI DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Grace Ma, B.S. * * * « * The Ohio State University 1992 Dissertation Committee: Approved by D.H. Ives G.A. Marzluf C.A. Breitenberger Adviser, M.D. Tsai Biochemistry Program, The Ohio State University Copyright by Grace Tak-Yi Ma 1993 To My Parents and My Husband Sik Kwan ACKNOWLEDGEMENTS Words alone cannot express my thanks to my adviser Professor David H. Ives. His guidance, assistance, understanding, support and patience will always be gratefully appreciated. My special thanks go to Dr. Seiichiro Ikeda for his assistance and friendship during this work. Many thanks also go to all my friends in my laboratory-Dr. Min Young Kim, Dr. Ning Ma, Young Soo Hong, Pamphinol Bunnag, and others, whose help and company made my years in the laboratory enjoyable. I would like to gratefully acknowledge the advice and assistance provided by the faculty members of the Department of Biochemistry, especially Professors George Marzluf and Caroline Breitenberger. To my parents, my everlasting appreciation and love go to them for their unshakable, continuous support and love. Finally, I express my deepest thanks, indebtedness and love to my husband, Sik Kwan, whose love, support, encouragement, comfort and lots of patience made this possible. VITA February 26, 1962 Bom - Hong Kong 1985 Bachelor of Science The Pennsylvania State University University Park, Pennsylvania 1986 - present Graduate Teaching/Research Associate, Department of Biochemistry The Ohio State University PUBLICATION 1. Grace T. Ma and David H. Ives. "Cloning, Sequence and Expression of the Heterodimeric Deoxyadenosine Kinase/Deoxyguanosine Kinase from Lactobacillus acidophilus R26." Manuscript in preparation. 2. Seiichiro Ikeda, Grace T. Ma and David H. Ives. "Control of Competing Ionic and Hydrophobic Forces in Affinity Chromatography of the Deoxyguanosine Kinase/Deoxyadenosine Kinase Complex from Lactobacillus acidophilus R-26." Submitted. 3. Seiichiro Ikeda, Grace T. Ma and David H. Ives. "Heterodimeric Deoxynucleoside Kinases of Lactobacillus acidophilus R26: Assignment of Subunit Function by Direct Photoaffinity Labeling with dNTP Analogs and Comparison of N-Terminal Sequences." Manuscript in preparation. v FIELDS OF STUDY Major Field: Biochemistry Studies in Protein Biochemistry Professor David H. Ives Studies in Molecular Biology Professors David H. Ives, George A. Marzluf, Caroline A- Breitenberger, and Donald H. Dean TABLE OF CONTENTS DEDICATION ............................................................................................ iii ACKNOWLEDGEMENTS ........................................................................... iv VITA................................................................................................................ v LIST OF TABLES.......................................................................................... xi UST OF FIGURES...................................................................................... xii ABBREVIATIONS ...................................................................................... xiv ABSTRACT .............................................................................................. xvii CHAPTER PAGE I. INTRODUCTION............................................................................... 1 A. Deoxynucleoside Kinases From L. acidophilus R-26 .................. 1 1. Overview ............................................................................... 1 2. Previous studies .................................................................... 2 B. Deoxynucleoside Kinases From Other Sources ........................ 17 1. Other Bacterial Deoxynucleoside K inases ........................ 17 2. Mammalian Deoxynucleoside Kinase ............................... 18 3. Viral Thymidine Kinases ................................................... 20 C. Affinity M edia ............................................................................. 21 D. Cloning Vector And Host Cell L in e ......................................... 22 1. Cloning Vector ................................................................... 22 2. Host C e ll............................................................................ 25 II. MATERIALS AND METHODS.................................................... 27 A. Materials................................................................................... 27 B. Methods ................................................................................... 28 1. Anion Exchange HPLC ................................................... 28 2. DEAE Trisacryl M ......................................................... 28 3. Inorganic Phosphate Determination ................................ 29 4. Culturing L. acidophilus R-26 ........................................ 30 5. Extraction of Enzymes ................................................... 31 6. Protein A ssay .................................................................. 31 7. Enzyme Activity Assay ................................................... 32 8. SDS-Polyacrylamide Gel Electrophoresis ....................... 33 a. The Laemmli Buffer System ....................................... 33 b. The pH 7.28 MZE 3328.IV buffer system ................... 34 9. Electroblotting ................................................................ 34 10. N-Terminal Protein Sequence Analysis ......................... 36 11. Chromosomal DNA Preparation .................................... 36 12. Oligonucleotides .............................................................. 37 13. Polymerase Chain Reaction (P C R ) ............................... 38 14. Preparation of ^P-labeled DNA P ro b e ......................... 38 15. Synthesis of Biotinylated DNA Probe by P C R ............... 39 16. Gel Electrophoresis for D N A ........................................ 39 17. Elution of DNA from Agarose G e l ............................... 40 18. Southern Transfer and Hybridization ............................. 41 19. Nonisotopic Nucleic Acid Detection ............................. 41 20. Plasmid DNA Preparation .............................................. 42 21. Competent Cells and Transformation ............................. 42 22. Colony Hybridization ..................................................... 43 23. Genomic Library Screening by Polymerase Chain Reaction ............................................................................ 44 viii 24. DNA Sequencing of Cloned D N A .................................... 44 25. Asymmetrical PCR and DNA Sequencing ....................... 45 26. Extraction and Purification of Enzymes from E.coli clones ................................................................................... 46 III. AFFINITY PURIFICATION OF DEOX Y GU ANOSINE/ DEOXYADENOSINE KINASES FROM L. ACIDOPHILUS 47 A. Construction Of dAp 4 -Sepharose Affinity M edium .................. 47 1. Development ..................................................................... 47 2. Chemical Synthesis and Characterization ........................ 49 B. Purification Of Deoxyguanosine Kinase/Deoxyadenosine Kinase By dAp 4 -Sepharose Affinity Chromatography .............. 58 C. Characterization Of Affinity Purified Deoxyguanosine Kinase/Deoxyadenosine K inase .................................................. 62 1. Enzyme Purity and Subunit Molecular
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