The Regulation of Gene Expression During the Aerobic Germination of Mucor Racemosus Sporangiospores
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Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1983 The Regulation of Gene Expression During the Aerobic Germination of Mucor Racemosus Sporangiospores. John Edgar Linz Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Linz, John Edgar, "The Regulation of Gene Expression During the Aerobic Germination of Mucor Racemosus Sporangiospores." (1983). LSU Historical Dissertations and Theses. 3930. https://digitalcommons.lsu.edu/gradschool_disstheses/3930 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. 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University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8409586 Linz, John Edgar THE REGULATION OF GENE EXPRESSION DURING THE AEROBIC GERMINATION OF MUCOR RACEMOSUS SPORANGIOSPORES The Louisiana State University and Agricultural and Mechanical Ph.D. Col. 1983 University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106 Copyright 1984 by Linz, John Edgar All Rights Reserved PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages.S' 2. Colored illustrations, paper or print______ 3. Photographs with dark background 4. Illustrations are poor copy_______ 5. P ages with black marks, not original copy_______ 6. Print shows through as there is text on both sides of page______ 7. Indistinct, broken or small print on several pages^ 8. Print exceeds margin requirements______ 9. Tightly bound copy with print lost in spine_______ 10. Computer printout pages with indistinct print_______ 11. P a g e(s) _____________ lacking when material received, and not available from school or author. 12. Page(s) _____________ seem to be missing in numbering only as text follows. 13. Two pages numbered_____________. Text follows. 14. Curling and wrinkled p a g e_______ s 15. Other____________________________________________________________________________ _ University Microfilms International THE REGULATION OF GENE EXPRESSION DURING THE AEROBIC GERMINATION OF MUCOR RACEMOSUS SPORANGIOSPORES A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Microbiology by John E . Linz B.A., Albion College, 1976 M.S., University of Illinois, 1979 December 1983 © 1984 JOHN EDGAR LINZ All Rights Reserved Dedication I would like to dedicate this work to my parents, Henry and Beatrice Linz, who provided me with support and encourage ment in my pursuit of a career in academics. Acknowledgement I would like to thank Dr. Michael Orlowski for all of the advice, support, and friendship he has extended to me over the past few years. He has helped to increase my understanding of Microbiology through our conversations and has been a great inspi ration and source of motivation in the laboratory. I would also like to thank the members of my graduate com mittee: Dr. A. D. Larson, Dr. H. D. Braymer, Dr. D. F. Day, Dr. S. Chang, and Dr. V. R. Srinivasan for their timely advice and constructive criticisms of my work. Special thanks go to Dr. H. D. Braymer for performing amino acid analysis; to Dr. Rod Nelson for expert photography work; to Karol Katayama for invaluable advice in cell free protein synthesis; to Mary Jo Brooks for typing this dissertation; and to the students and faculty of LSU for making my stay a pleasant learning experience. Finally, I would like to thank my wife, Susan, and daughter, Sarah, for their love, understanding and support in my work. Table of Contents Page Dedication..................................................... ii Acknowledgement...................... iii Table of C o n t e n t s ............................................. iv List of T a b l e s ............................ vi List of F i g u r e s ........................ vii Abstract....................................................... ix Introduction ................................................... 1 Literature Review ............................................. Introduction ............................................... 4 Fungal Spore Germination ................................... 5 Yeast-to-Hypha Morphogenesis in Mucor ...................... 12 Other Fungal Cell Differentiation Systems .................. 16 Materials and Methods Organism and Cultivation ................................... 19 Spore P r o d u c t i o n ........................................... 19 Germination................................................. 20 Preparation of L-[35s]Methionine from [35s]Sulfate ........ 21 Determination of the Kinetics of RNA and Protein Synthesis . 22 Measurement of Free Intracellular Amino Acid and Nucleotide Pools ......................................... 23 Density Gradient Analysis of Ribosomes .................... 25 RNA Purification........................................... 27 Density Gradient Analysis of RNA ........................... 28 Oligo dT Cellulose Fractionation of R N A .................... 31 Prelabeling/Pulse Labeling of Sporangiospore Proteins .... 32 Cell-Free Protein Synthesizing System ...................... 32 Polyacrylamide Gel Electrophoresis ......................... 35 Results Incorporation of L-[^C]Leucine into Protein during Sporangiospore Germination ............................... 43 Ribosome Analysis ........ 44 iv Table of Contents (Cont.) Page o 32 Incorporation of [JH]Uracil and [ PjPhosphate into RNA during Sporangiospore Germination .................... 45 Analyses of RNA Synthesized during Sporangiospore Germination............................................... 46 Analyses of RNA from Dormant Sporangiospores .............. 47 Analyses of 3[n Vivo Pulse-Labeled Proteins from Germinating Sporangiospores ............................... 48 Analyses of Functional mRNA Levels in Dormant and Germinating Sporangiospores ............................... 53 Discussion..................................................... 59 Literature Cited ............................................... 71 Appendix........................................................ 79 V i t a .............................................................. 130 v List of Tables Table Page 1 Total Amino Acid Pool and L-Leucine Pool in Germinating Sporangiospores of a Leucine Auxotroph of racemosus 1216 B .................... 80 2 The Effect of Sporangiospore Harvest Conditions on the Percentage of Ribosomes Associated with m R N A ........................................... 81 32 3 Equilibration of [ P]Phosphate into Nucleotide Pools during Germination of Sporangiospores of M. racemosus..................................... 82 32 4 Sucrose Density Gradient Analysis of P-Labeled RNA Purified from Germinating Sporangiospores of M. racemosus..................................... 83 5 Specific Activity of Proteins Pulse-Labeled with L-[35s]Methionine during Germination of M. racemosus Sporangiospores ........................... 84 6 Size of Amino Acid Pools in Germinating Sporangio spores of M^_ racemosus 1216 B ....................... 85 7 Specific Activity of L-[^S]Methionine in