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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 I 75-4121 I STUDIES ON THE DNA CONTENT OF A PLASTID MUTANT IN GOSSYPIUM HIRSUTUM (L.) Item Type text; Dissertation-Reproduction (electronic) Authors Kestler, Daniel Paul, 1948- 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 05/10/2021 13:29:13 Link to Item http://hdl.handle.net/10150/288307 INFORMATION TO USERS This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. 5. PLEASE NOTE: Some pages may have indistinct print. Filmed as received. Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 I 75-4121 I KESTLER, Daniel Paxil, 1948- STUDIES ON THE DNA CONTENT OF A PLASTID MUTANT IN GOSSYPIUM HIRStTIUM L.. The University of Arizona, Ph.D., 1974 Chemistry, analytical Xerox University Microfilms, Ann Arbor, Michigan 48106 THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. STUDIES ON THE DNA CONTENT OF A PLASTID MUTANT IN GOSSYPIUM HIRSUTUM L. by Daniel Paul Kestler A Dissertation Submitted to the Faculty of the COMMITTEE ON GENETICS CGRADUATE) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 19 7 4 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE I hereby recommend that this dissertation prepared under my direction by Daniel Paul Kestler . , STUDIES ON THE DNA CONTENT OF A PLASTID MUTANT entitled IN GOSSYPIUM HIRSUTUM L. be accepted as fulfilling the dissertation requirement of the degree of Doctor of Philosophy k- ?JU& )issertation Director Date 7 After inspection of the final copy of the dissertation, the following members of the Final Examination Committee concur in its approval and recommend its acceptance: dJU 7y fi>P. Ee^w-jU, f W This approval and acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination. 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 allow­ able without special permission, provided that accurate ac­ knowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manu­ script 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 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. SIGNED: DEDICATION This manuscript and work which it represents is dedicated to the memories of John Paul Kestler and Rose Ann MacDonald Kestler. iii ACKNOWLEDGMENTS The author wishes to express his gratitude: to Dr. Frank Katterman for many helpful suggestions during the course of the research and the preparation of this manuscript. to Dr. John Endrizzi for use of mutant cotton seeds and greenhouse facilities. to Dr. Kaoru Matsuda and Dr. William Bemis for advice in the preparation of this manuscript. to Dr. Wayne Ferris for use of the electron micro­ scope and laboratory facilities. to Mr. Glenn Lipman for preparation of the electron micrographs of leaf sections and DNA. iv TABLE OF CONTENTS Page LIST OF TABLES vii LIST OF ILLUSTRATIONS viii ABSTRACT xi INTRODUCTION . , 1 LITERATURE REVIEW 3 Light Microscopic Evidence of Nucleic Acids in Chloroplasts 3 Evidence of Nucleic Acids in Chloro­ plasts from Electron Microscopy ....... 4 Radioisotopic and Autoradiographic Evidence of Nucleic Acids in Chloro­ plasts 5 Evidence of the Existence of Chloroplast Nucleic Acids from Nucleic Acid Bio­ chemistry 6 Mitochondrial DNA of Higher Plants 22 Uniparental Inheritance in Higher Plants . 22 MATERIALS AND METHODS 29 Isolation of Chloroplasts 29 Light Microscopic Examination of Purified Chloroplast Preparations 32 Isolation of Nuclei 33 Isolation of Chloroplast DNA 33 Isolation of Nuclear DNA 36 Preparation of Denatured and Denatured- Renatured Nuclear and Chloroplast DNA .... 37 Preparation of DNA for CsCl Analytical Buoyant Density Centrifugation . 37 DNA Preparation for Electron Micrographs . 39 Preparation of Leaf-Sections for Electron Microscopy 39 v vi TABLE OF CONTENTS (Continued) Page RESULTS 41 Analysis of Cotton Chloroplastic and Nuclear DNA in Mutant and Wild Type Tissue 41 Summary of Results from Buoyant Density Studies of Cotton Nuclear and Chloro- plast DNA 62 Chloroplast Preparations Examined by Electron Microscopy 63 Electron Microscopic Study of Isolated Chloroplast DNA 73 DISCUSSION 75 SUMMARY 86 APPENDIX A: EVIDENCE THAT THE CHLOROPLAST MUTANT OF COTTON IS A TRUE CYTOPLASMIC MUTANT 88 LITERATURE CITED , 91 LIST OF TABLES Table Page 1. Base composition (moles percent) of ribosomal RNA from some higher plants 13 2. Chloroplast and nuclear DNA buoyant density components of selected higher plants 16 3. List of higher plants with uniparental- maternal transmission of their plastids .... 24 4. Cotton chloroplast and nuclear DNA buoyant density data 45 5. Percentages of cotton chloroplast DNA from CsCl buoyant density analysis .... 50 6. Cross pollinations between variegated plants and normal green plants 89 7. Typical segregation ratios observed in the F^ generation from individual crosses of variegated-A mutant plants X normal green plants 90 vii LIST OF ILLUSTRATIONS Figure Page 1. Green, yellow-green mosaic, and white- green mosaic leaves from Gossypium hirsutum L 30 2. Typical absorption profile of purified white tissue cotton chloroplast DNA 43 3. Typical absorption profile of purified green tissue cotton chloroplast DNA 44 4. Microdensitometer tracing of cotton chloroplast DNA from green leaf tissue .... 46 5. Microdensitometer tracing of cotton chloroplast DNA from yellow leaf tissue and yellow cotyledon tissue 47 6. Microdensitometer tracings of cotton chloroplast DNA from white leaf tissue 48 7. Comparisons of microdensitometer tracings of analytical centrifugations of chloroplast DNA from white, yellow and green tissue 52 8. Microdensitometer tracings of cotton nuclear DNA from green leaf tissue and white leaf tissue 53 9. Microdensitometer tracings of cotton chloroplast DNA from green tissue in comparison to cotton nuclear DNA from green tissue 54 10. Microdensitometer tracings of cotton chloroplast DNA from white tissue in comparison to cotton nuclear DNA from white tissue . , . 55 viii ix LIST OF ILLUSTRATIONS (Continued) Figure Page 11. Microdensitometer tracings of cotton chloroplast DNA from yellow leaf tissue with a slight amount of nuclear con­ tamination in comparison to cotton nuclear DNA from green leaf tissue 56 12. Microdensitometer tracings of heat de­ natured and heat denatured-renatured nuclear DNA . , . 57 13. Microdensitometer tracings of heat de­ natured and heat denatured-renatured chloroplast DNA from green tissue 58 14. Microdensitometer tracings of chloro­ plast DNA which had heat denatured- renatured chloroplast DNA from white tissue 59 15. Microdensitometer tracings comparing heat denatured and heat denatured- renatured chloroplast and nuclear DNA from green leaf tissue 61 16. Electron micrograph of green leaf tissue chloroplast 64 17. Electron micrograph of green leaf chloroplasts 65 18. Electron micrograph of chloroplasts of green leaf tissue 66 19. Electron micrograph of yellow leaf tissue chloroplast
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