Molecular characterization of full-length cDNAs for nuclear-encoded chloroplast ribosomal proteins L12, L24 and L27 of Nicotiana tabacum. Item Type text; Dissertation-Reproduction (electronic) Authors Elhag, Gasmalla Abdelwahab. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 08/10/2021 23:00:38 Link to Item http://hdl.handle.net/10150/185581 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. 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MI 48106 MOLECULAR CHARACTERIZATION OF FULL-LENGTH <..DNAs FOR NUCLEAR-ENCODED CHLOROPLAST RIBOSOMAL PROTEINS L12, L24 AND L27 OF Nicotiana tabacum by Gasmalla Abdelwahab Elhag A Dissertation Submitted to the Faculty of the DEPARTMENT OF BIOCHEMISTRY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 1991 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE 2 As members of the Final Examination Committee, we certify that we have read the dissertation prepared by ____~G=a=s~m=a=l~l=a~A~.~E~l~h~a~g~ __________________________ en tit led _....;M:..:..O=-L.-::E::....::C.-::U..::L:..:..A_R_C:..:..HA....:..::RA..::..:...:C..::T..::E:..:..R..::I:.:.ZA:.:.T::..:I::..:O:..:::N.:....-..:O..::.F--=...FU.:.:L:.:.L=--..::L::E:.:.N..::G-=T.:.:H--=-cD::..:N:.:.:A.:.:s:......::F-=O..:..:R~ ______ NUCLEAR-ENCODED CHLOROPLAST RIBOSOMAL PROTEINS L12, L24 AND L27 OF NICOTIANA TABACUM and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of____ D~o~c~t~o~r~o~f~P~h~i~l~o~s~o~p~h~y~ ____________________________________ 17)11/ 7/I Date 7 /1::2 /3: f Hans Date I ? 7)/{pjq; Date • 1 ?-/I2-/]/ Date ; 70' z i f / Date I /' Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. Don. P. Bourque Date ~ I 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNE~~ 4 ACKNOWLEDGEMENTS I would like to express my deep gratitude to Dr. Don Bourque for his consistent advice and constructive criticism and especially for tolerating my sometimes obstinate arguments and slow progress. I am very grateful to Dr. Hans Bohnert for his constant interest and encouraging discussions. I am grateful to the other members of my supervisory committee: Drs. Marty Hewlett, Kaoru Matsuda and David Mount for their guidance and support. I especially wish to thank Dr. Mark Hildebrand for his friendship and encouragement and for being patient in answering my many naive questions. I am grateful to Tom McCreery, Betty Glinsmann-Gibson, Steve Carls and Dan Wall for encouragement, interest and for helping with the many computer alignments and graphics. Gail Hewlett prepared the antibodies used in the initial library screening. Dr. Frank Thomas purified tobacco chloroplast ribosomal protein L12 by HPLC for N­ terminal protein sequencing. The assistance and efforts of Jane Dugas Huff in typing this dissertation is especially acknowledged and appreciated. Finally, I would like to thank my wife, my son and my daughter for their patience and support during the course of this research. 5 TABLE OF CONTENTS LIST OF ILLUSTRATIONS ..................................... 11 LIST OF TABLES . .. 15 ABSTRACT ............................................... 16 CHAPTER 1 ................................................ 18 Introduction/ The Plant Genome ................................. 18 A. Evidence that Higher Plants Contain More Than One Genome ..... 19 1. Characteristics of the Nuclear Genome .................. 20 2. Characteristics of the Chloroplast Genome . .. 25 3. Characteristics of the Mitochondrial Genome . .. 27 4. Relationships Between Nuclear, Chloroplast and Mitochondrial DNA. 30 5. Evidence that Some Chloroplast Proteins are Nuclear Encoded. .. 33 B. Ribosomes. .. 37 1. Algal Ribosomes ................................... 38 a. Euglena Ribosomes . .. 38 b. Chlamydomonas Ribosomes . 42 c. Cyanelle Ribosomal Proteins . 48 2. Plant Ribosomes ................................... 50 a. Cytoplasmic Ribosomes. 50 b. Chloroplast Ribosomes. 51 c. Mitochondrial Ribosomes ........................ 54 C. Organelle and Nuclear Contributions to Chloroplast Ribosomal Protein Complement ..................................... 56 1. Chloroplast Encoded Ribosomal Proteins . .. 56 2. Nuclear Encoded Chloroplast Ribosomal Proteins .......... 68 D. Other Prokaryotic and Eukaryotic Ribosomes. .. 78 1. E. coli Ribosomes .................................. 78 2. Yeast Ribosomes . 85 3. Mammalian Ribosomes ............................... 92 4. Xenopus Ribosomes . 96 5. Drosophila Ribosomes ............................... 101 E. Goals of Dissertation " . .. 107 CHAPTER 2 ................................................ 110 Experimental Procedures ....................................... 110 6 A. General Procedures . .. 110 1. Routine Precautions ................................ 110 2. Ethanol Precipitation of Nucleic Acids ................... 110 3. Phenol Extraction .................................. 111 4. Estimation of Nucleic Acids ........................... 111 5. Concentration of Nucleic Acid Solutions with Butanol ....... 112 6. Trichloroacetic Acid (TCA) Precipitation of Nucleic Acids and Measurement of Incorporated Radioactivity. .. 112 7. Sephadex G-50 Chromatography to Desalt Nucleic Acids 113 8. Removal of Ethidium Bromide from DNA Solutions ........ 114 9. Restriction Endonuclease Cleavage of DNA ............... 114 10. Dephosphorylation of DNA prior to DNA Ligation Reactions .. 115 11. Ligation Conditions . .. 115 12. Storage of Bacterial Strains and Phage Stocks .............. 116 B. Isolation of Tobacco Nucleic Acids ........................... 116 1. Preparation of Piant Material ......................... 116 2. Isolation of Tobacco Genomic DNA .................... 117 3. Isolation of Tobacco Leaf RNA ........................ 118 4. Oligo( dT)-Cellulose Chromatography and Separation of Poly(At mRNA ................................... 120 C. In vitro Translation ...................................... 121 1. Preparation of Wheat Germ Extract .................... 121 2. Preparation of the Protein Biosynthesis Reaction Mix for Wheat Germ III vitro Translation System ................. 121 3. In vitro Translation Reaction .......................... 122 D. cDNA Synthesis . .. 123 1. First Strand Synthesis ............................... 123 2. Second Strand Synthesis ............................. 124 3. cDNA Size Estimation . .. 125 4. cDNA End Filling Reaction . .. 125 5. cDNA Synthesis Using a Commercial Kit ................. 125 6. Methylation of EcoRI Sites ........................... 126 7. Phosphorylation of 5'-ends of EcoRI Linkers and Ligation to cDNA ................