Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2007 Isolation and characterization of two closely related beta-carbonic anhydrases of Chlamydomonas reinhardtii Ruby Agno Ynalvez Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Ynalvez, Ruby Agno, "Isolation and characterization of two closely related beta-carbonic anhydrases of Chlamydomonas reinhardtii" (2007). LSU Doctoral Dissertations. 778. https://digitalcommons.lsu.edu/gradschool_dissertations/778 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 Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. ISOLATION AND CHARACTERIZATION OF TWO CLOSELY RELATED BETA- CARBONIC ANHYDRASES OF CHLAMYDOMONAS REINHARDTII 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 Biological Sciences by Ruby A. Ynalvez B.S., University of the Philippines Los Baños, 1989 M.S., University of the Philippines Los Baños, 1997 August 2007 ACKNOWLEDGMENTS I would like to express my utmost gratitude to my major professor, Dr. James V. Moroney, who mentored me throughout my graduate studies, and who has been so patient and encouraging. I am also grateful for the support and guidance of Drs. Sue G. Bartlett, John C. Larkin and David J. Longstreth all of whom served as members of my graduate committee. I also wish to thank Dr. Bin Chen for his time being a part of my graduate committee. I would like to thank the financial support of the Department of Biological Sciences, the Louisiana Economic Development Award, and the LSU Graduate School. My sincerest gratitude goes to Ying Xiao for the electron microscopy work for the immunolocalization of CAH7 and CAH8. My appreciation goes to the members of the Moroney Laboratory especially to Kristy Brumfield, Steve Pollock, Mautusi Mitra and Catherine Mason for all the kind assistance and warm friendship they have provided. I am equally grateful to Ayana Ward and Krishen Cunnusamy for assisting with the experiments and being hardworking undergraduates. I thank Monica for patiently checking the references. I also like to thank Bingbing, Brahtati, Dev, Megan, Michelle, and Lauren who made my stay in the laboratory more tolerable. I am likewise indebted to all my friends at LSU for all their kindness and for providing me with fond memories of graduate school in the U.S. I would like to thank my husband, Marcus Antonius, for his unconditional love; to my children, Leslie and Boon, who are my constant inspiration; and to Ina, for all her prayers. Most importantly, I thank the Almighty God for His guidance. ii TABLE OF CONTENTS ACKNOWLEDGMENTS…………………………………………………………………… ii LIST OF TABLES………………………………………………………………………...… v LIST OF FIGURES……………………………………………………………………….…. vi ABSTRACT………………………………………………………………………………….. x CHAPTER 1: INTRODUCTION....…………………………………………………….…... 1 CHAPTER 2: REVIEW OF LITERATURE….………………………………………..….... 3 Carbon Dioxide Concentrating Mechanism..……………………………………...… 3 The Carbon Dioxide Concentrating Mechanism in C. reinhardtii…………………... 8 Carbonic Anhydrases……………………………………………………………..…. 19 The C. reinhardtii Carbonic Anhydrases.………………………………………...…. 27 CHAPTER 3: MATERIALS AND METHODS…………………………………………..... 32 Cell Cultures….………….……………………………………………………….….. 32 DNA Preparation, Sequencing and Homology Analysis……………………………. 32 cDNA Library Preparation……..……………………………………………………. 34 Amplification of CAH7 Genomic DNA from the Cosmid Library….…….............… 35 Total RNA Isolation….…………………………………………………………..….. 36 5’RLM-RACE (RNA Ligase Mediated Rapid Amplification of cDNA Ends)……… 37 PCR Protocols….……….…………………………………………….……………... 37 Construction of Overexpression Constructs….……………………..……………..... 39 Overexpression and Purification of MBP Fusion Proteins ….……………................ 43 Generation of Polyclonal CAH7 and CAH8 Primary Antibodies….……………...... 44 Carbonic Anhydrase Assays and Other Biochemical Experiments….…………...…. 46 Immunolocalization Studies Using Electron Microscopy …………………………... 47 Generation of RNAi Mutants …………………………………………………..….... 48 Other Methods ………………………………………………………………………. 49 CHAPTER 4: IDENTIFICATION AND CHARACTERIZATION OF CHLAMYDOMONAS REINHARDTII CHLOROPLAST β-CA CAH7..……………. 51 Introduction.………...………………………………………….………….………… 51 Results.………………....………………………………………………….………… 53 Discussion………………………………………………………..………….……..... 86 CHAPTER 5: IDENTIFICATION AND CHARACTERIZATION OF CHLAMYDOMONAS REINHARDTII PERIPLASMIC β-CARBONIC ANHYDRASE CAH8……………………………………………………………….. 92 Introduction……………………………………………………………………..…..... 92 Results………………………………………...…………………………………........ 94 Discussion…………………………………...……………...………………….…..… 120 iii CHAPTER 6: ISOLATION AND CHARACTERIZATION OF AN INSERTIONAL MUTANT OF CHLAMYDOMONAS REINHARDTII …………………....…….…… 129 Introduction………………………………………………………………….………. 129 Results..………………………………...…………………………….………….….... 131 Discussion………………………………………………………………………....…. 160 CHAPTER 7: CONCLUSIONS…………………………………………………...……........ 168 REFERENCES…………………………………………………………………………......... 174 APPENDIX A: CAH 3 AND CAH6 RNAI CONSTRUCTS………………………………... 191 APPENDIX B: LIST OF PRIMERS………………………………………………………… 198 VITA ………………………………………………………………………………..……..… 200 iv LIST OF TABLES 4.1 The Purification of MBP-CAH7 Fusion Protein from 1L of E. coli Culture….….......... 69 4.2 Comparison of the Expression Levels of the Different C. reinhardtii Carbonic Anhydrases Based on Ct Valuesa from Real-time PCR Using Sbyr Green……………... 72 4.3 Intracellular Localization of CAH7 Using Wild Type C. reinhardtii ………..…..……. 77 4.4 The Inhibition Constants of the Bovine CAII, C. reinhardtii CAH6 and CAH7.…...…. 80 5.1 The Purification of MBP-CAH8 Fusion Protein from 1L of E. coli Culture…..………. 107 5.2 Intracellular Localization of CAH8 Using Wild Type C. reinhardtii Cells..…...……… 114 5.3 The Inhibition Constants of the Bovine CAII, C. reinhardtii CAH6 and CAH8............ 119 6.1 Linkage Analysis of Several Insertional Mutants……..……..……………………….… 132 6.2 Identification of Genes Disrupted by pSP124S……...……………………………..…... 133 6.3 The Amino Acid Composition of CIA7……………………………………………….. 145 7.1 A Summary of Carbonic Anhydrases Known to Date in C. reinhardtii………………... 169 7.2 The Comparison of Some Characteristic Properties of the Recombinant CAH7 and CAH8 Activity…………………………………………………………………………. 170 v LIST OF FIGURES 2.1 Model of the Carbon Dioxide Concentrating Mechanism of Chlamydomonas reinhardtii........................................................................................................................ 11 3.1 Diagram Showing Some Features of the pMal-c2x Expression Vector….………..…… 40 3.2 Partial Sequence of the Polylinker Cloning Site of pMal-c2x……………....….............. 42 3.3 A Schematic Diagram to Show the Cloning and Amylose Column Purification of the Recombinant Fusion Protein……………………………………................................... 45 4.1 CAH7 in Scaffold 116 of JGI Chlamydomonas Genome Browser Version 2…..…….... 54 4.2 Exon-Intron Structure of CAH7........................................................................................ 54 4.3 The Genomic Sequence of CAH7..................................................................................... 55 4.4 The cDNA Sequence of CAH7......................................................................................... 56 4.5 The Amino Acid Sequence of CAH7............................................................................... 58 4.6 Amino Acid Sequence Alignment of CAH7 with other β-CAs........................................ 59 4.7 Multiple Sequence Alignment of C. reinhardtii CAH7 with other C. reinhardtii β CAs 60 4.8 Secondary Structure Prediction of CAH7…………….………………………….…….. 62 4.9 PCR Amplification of the 1.5 kb Insert in Ampicillin Resistant Bacterial Colonies Transformed with PQ80EL-CAH7……………..……………..……..…..………..…… 64 4.10 Overexpression of CAH7 in PQ80EL ………….…..…………………….……….…... 64 4.11 6XHis-CAH7 Fractions ……………………………………………………..……..….. 65 4.12 Overexpression of CAH7 pMal-c2x ……………………………..…………..…….….. 66 4.13 Purification of MBP-CAH7……………………………………..……………………... 67 4.14 The Qualitative Expression Analysis of CAH7……………..……..…………………... 70 4.15 The Quantitative Expression Analysis of CAH7………………………………............. 71 4.16 The Comparison of the Expression Levels of C. reinhardtii Carbonic Anhydrases Based on Ct values …………………………….…………………………………….... 73 4.17 The Relative Log Fold Difference in the Amount of Message of the Different C. reinhardtii Carbonic Anhydrases under Low CO2 Relative to High CO2……………... 74 vi 4.18 Time Course Analysis of the C. reinhardtii Carbonic Anhydrases…...…….…………. 75 4.19 Immunogold Labeling of C. reinhardtii WT137 Cells Probed with CAH7 Antibody... 78 4.20 Immunogold Labeling of C. reinhardtii WT137 Cells Probed with CAH7 Pre- immune Serum...........................................................………………………………….. 79 4.21 The Thermostability of CAH7 Activity........................................................................... 81 4.22 Effect of the Reducing Agent, β-mercaptoethanol on the Activity of the Recombinant MBP-CAH7……………………..………………………………………………….….