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 free, while others may be from any 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 photographs, print bleedthrough, substandard margins, and improper alignment can adversely afreet 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. UMI A Bell & Howell Information Company 300 North Zed) Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 REGULATIONS OF UREIDE BIOSYNTHESIS GENES IN TROPICAL LEGUME ROOT NODULES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Jong Heon Kim, B.S., M.S. ***** The Ohio State University 1996 Dissertation Committee: Approved by Dr. Desh Pal S. Verma Dr. L. Mark Lagrimini Dr. Richard T. Sayre J^sor Dr. Randall L. Scholl Molecular, Cellul^ and Developmental Biology Program UMI Number: 9710593 UMI Microform 9710593 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 To my wife, Sou Hyun, and parents 11 If I ~ ACKNOWLEDGEMENTS I express sincere appreciation to Dr. Desh Pal S. Verma for his guidance and advice throughout this research. I would like to thank the other members of my advisoty committee, Drs. L. Mark Lagrimini, Richard T. Sayre and Randall L. Scholl, for their suggestions and comments on this study. Gratitude is expressed to members of our lab for their fidendship and encouragement I offer sincere thanks for my wife. Sou Hyun, and my parents for their love, faith and standing with me through the long period of my endeavors. To my children, Cassidy and Lauren, I thank you for understanding my frequent absences during this study. I ll VTTA March 9,1960 Bom - Busan, Korea 1983 B.S., Korea University, Seoul, Korea 1985 M.S., Korea University, Seoul, Korea 1/1988 - 5/1989 Graduate Assistant, University of Oklahoma, Norman, Oklahoma 9/1989 - Present Graduate Associate, The Ohio State University, Columbus, Ohio PUBLICATIONS Verma, D.P.S., Kim, JJH. and Wu, T. 1996. Assimilation of reduced nitrogen in tropical legume nodules: Regulation of de novo purine biosynthesis and peroxisome proliferation. 2nd European Nitrogen Fixation Conference. Poznan, Poland. Kim, JJL and Verma, D J*.S. 1996. Induction and characterization of glutamine phosphoribosylpyrophosphate amidotransferase gene promoter from soybean. Plant Physiol. 111(2)S:101. Kim, J.H., Delaimey, A.J. and Verma, D.P.S. 1995. Control of de novo pmine biosynthesis genes in ureide-producing legumes: Induction of glutamine phosphoribosylpyrophosphate amidotransferase gene and characterization of its cDNA from soybean and Vigna. Plant J. 7(1): 77-86. Kim, J.H., Hmnphreys, J.M. and Verma, DJ*.S. 1995. Regulation of ureide biosynthesis genes in soybean root nodules. Plant Physiol. 108(2)5:72. Kim, J.H., Humphreys, J.M. and Verma, DJ’.S. 1995. Regulation of ureide biosynthesis genes in tropical legume root nodules. The 21st Annual ICSABER Society Graduate Research Forum., Columbus, OH. i V Chapman, K.A., Delauney, A.J., Kim, JJI. and Verma, D.P.S. 1994. Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and S-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia. Plant Mol. Biol. 24:389-395. Kim, J.H., Delauney, A.J., Chapman, KA. and Verma, D.P.S. 1993. Regulation of de novo purine biosynthesis in ureide-producing legumes. Plant Physiol. 1023:26. Chapman, K.A., Delauney, A.J., Kim, J.H. and Verma, D P S. 1993. De novo purine biosynthesis in ureide-producing legumes: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs &om Vigna aconitifolia. Mid-Atlantic Plant Molecular Biology Society Aimual Meeting. Newark, DW. Kim, JJL, Lim, B.S., Lee, S.Y. and Chun, M. 1985. Frequency improvement of protoplast fusion in coryneform bacteria. Korean J. Microbiol. 23(3): 190-196. Chun, M., Lim, B.S., Lee, S.Y., Kim, J.H. and Kyung, K.C. 1985. Studies on breeding of L-lysine producing microorganisms by cell fusion. In: Thesis Col. of Agr. and Forest, Korea Univ., Seoul, Korea. 25: 27-33. Kim, JJL, Lim, B.S., Lee, S.Y. and Chun, M. 1985. Studies on the improvement of protoplast fusion frequency in coryneform bacteria. Annual Meeting of the Korean Society of Microbiologists. Seoul, Korea. FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology TABLE OF CONTENTS ACKNOWLEDGEMENTS.............................................................................. iü VITA.................................................................................................................... iv LIST OF TABLES........................................................................................... vi LIST OF FIGURES....................................................................................... vü CHAPTER PAGE I. TROPICAL LEGUME-RHIZOBIA SYMBIOSIS..................... 1 Introduction ................................................................................... 1 Proposed Research .................................................................... 36 II. INDUCnON OF GLUTAMINE PHOSPHORIBOSYL­ PYROPHOSPHATE AMIDOTRANSFERASE GENE AND CHARACTERIZATION OF ITS cDNA FROM SOYBEANS................................................................................. 38 A bstract......................................................................................... 38 Introduction .................................................................................. 39 Materials and Methods ............................................................ 41 R esults........................................................................................... 44 Discussion ..................................................................................... 52 m . REGULATION OF UREIDE BIOSYNTHESIS GENES 60 A bstract......................................................................................... 60 Introduction ................................................................................... 61 Materials and Methods ............................................................ 63 R esults........................................................................................... 66 Discussion ..................................................................................... 73 VI IV. INDUCTION OF PRAT GENE IN TRANSGENIC PLANTS........................................................................................ 78 A bstract........................................................................................ 78 Introduction ................................................................................. 79 Materials and Methods ............................................................ 80 R esults.......................................................................................... 84 D iscussion .................................................................................... 89 V. SUMMARY AND CONCLUSIONS...................................... 93 LIST OF REFERENCES............................................................................. 97 Vll UST OF TABLES TABLE PAGE 1, Rhizobium-^\zni associations ............................................................3 2. De novo purine-synthesis pathway genes and their identification in different organisms .............................................23 Vlll LISTOFHGURES FIGURES PAGE 1. Early interactions and signal exchange(s) between legume plants and rhizobia leading to endocytosis and establishment of the symbiotic association ..................................................... 5 2. The ureide biosynthesis pathway in determinate nodules 11 3. Proposed pathway and subcellular organization of the reactions involved in amide synthesis and ureide biogenesis in ureide- exporting legume nodules ......................................................... 16 4. Pathway of de novo purine biosynthesis and its regulation 22 5. Comparison of putative operators for deoP2, purM, and purL with galOE. Gal 0/, lac and purF operators ...................... 27 6 . General structure of GalR-LacI family repressors ....................... 28 7. Comparison of cDNA and deduced amino acid sequences of soybean (Glycine max, pGFl) and mothbean (Vigna aconitifolia, pVF5) glutamine PRPP