Medical University of South Carolina MEDICA MUSC Theses and Dissertations 2011 Cell Cycle Regulation in the Florida Red Tide Dinoflagellate, Karenia brevis Stephanie Alexandra Brunelle Medical University of South Carolina Follow this and additional works at: https://medica-musc.researchcommons.org/theses Recommended Citation Brunelle, Stephanie Alexandra, "Cell Cycle Regulation in the Florida Red Tide Dinoflagellate, Karenia brevis" (2011). MUSC Theses and Dissertations. 176. https://medica-musc.researchcommons.org/theses/176 This Dissertation is brought to you for free and open access by MEDICA. It has been accepted for inclusion in MUSC Theses and Dissertations by an authorized administrator of MEDICA. For more information, please contact [email protected]. Cell Cycle Regulation in the Florida Red Tide Dinoflagellate, Karenia brevis By Stephanie Alexandra Brunelle A dissertation submitted to the faculty of the Medical University of South Carolina in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies. Department of Molecular and Cellular Biology and Pathobiology 2011 Approved by: Chairman, Advisory Committee TABLE OF CONTENTS ACKNOWLEDGMENTS .............................................................................................. i LIST OF FIGURES .............................................................................. ii LIST OF TABLES .............................................................................................. v LIST OF ABBREVIATIONS ................................................................... vi ABSTRACT ....................................................................................... vii CHAPTER 1: Introduction ................................................................................... l CHAPTER 2: Global protein and translation patterns in Karenia brevis over the diel cycle ................................................................................................. 13 Introduction ......................................................................................................... 14 Methods ............................................................................................................... 20 Results ................................................................................................................. 29 Discussion ............................................................................................................ 36 CHAPTER 3: Characterization and regulation ofS-phase genes and proteins in K. brevis Introduction ............................................................................... 65 Methods .................................................................................... 70 Results ..................................................................................... 79 Discussion .................................................................................. 88 CHAPTER 4: S-phase Expression of PCNA: Possible Post-translational Modifications, CDK Dependence, and its Potential Utility as a Biomarker of Growth Status in Karenia brevis Red Tides ............................................................................... 118 Introduction ........................................................................... 119 Methods ................................................................................ 122 Results ...............................................' ................................... 130 Discussion .............................................................................. 139 CHAPTER 5: Conclusions, Significance, and Future Directions ........................ 179 REFERENCES ................................................................................. 191 APPENDIX 1 ..................................................................................210 APPENDIX 2 .................................................................................. 217 ACKNOWLEDGMENTS I would like to extend a heart felt thank you to my mentor, Dr. Fran Van Dolah. Throughout this endeavor, she provided wonderful support and training. I would also like to thank my committee members who provided advice and shared knowledge that made this project successful: Dr. Michael Janech, Dr. Lauren Ball, Dr. David Kurtz and Dr. Paul McDermott. The members of the Van Dolah laboratory were immensely helpful and were not only wonderful colleagues but dear friends as well: Jeanine Morey, Emily Monroe, Jillian Johnson, Mackenzie Zippay and Peter Feltman. I would also like to thank the members of the Marine Biotoxins Program including Tod Leighfield and the Marine Biomedicine and Environmental Sciences students. Lastly, I need to thank my family, Alyce and Roger Brunelle who provided the love and encouragement I required to complete this PhD program. 1 LIST OF FIGURES CHAPTER 1 FIGURE 1: Tree of life ............................................................................. 12 CHAPTER 2 FIGURE 1: Polysome profile of L. polyedrum over circadian time .......................... 47 FIGURE 2: Polysome profile of K. brevis cultures during the light and dark phase ...... 48 FIGURE 3: Flow cytometry over the K. brevis cell cycle: polysome experiment. ........ 49 FIGURE 4: Analysis of translational activity over the cell cycle/diel cycle of K. brevis: polysome fractionation ............................................................................ 50 FIGURE 5: Two-dimensional gels of K. brevis proteins: extraction optimization ........ 52 FIGURE 6: Flow cytometry over the K. brevis cell cycle: 20 study ....................... 53 FIGURE 7: 20 gels of K. brevis proteins over the cell cycle ................................. 54 FIGURE 8: Flow cytometry over the K. brevis cell cycle: OIGE study .................... 55 FIGURE 9: 20 gels: resolved proteins labeled with Cy2, Cy3 and Cy5 .................. 56 FIGURE 10: Significantly changing proteins as determined by OIGE in K. brevis ...... 57 FIGURE 11: Scans of experimental gels: resolved proteins stained with SYPRO Ruby ................................................................................................ 60 FIGURE 12: Trends of significantly changing proteins in K. brevis ........................61 FIGURE 13: 20E gels of K. brevis nuclear proteins ..........................................62 CHAPTER 3 FIGURE 1: Multiple Alignments of the amino acid sequences of S-phase proteins ...... 95 FIGURE 2: Phylogenetic tree ofPCNA ...................................................... 102 FIGURE 3: Three-dimensional protein models of K. brevis PCNA ...................... 104 11 FIGURE 4: Spliced leader sequences on S-phase transcripts .............................. 105 FIGURE 5: Flow cytometry over the cell cycle .............................................. 106 FIGURE 6: qPCR analysis of S-phase transcripts over the cell cycle in K. brevis ...... l 07 FIGURE 7: Western blot analysis ofS-pha~e over the cell cycle in K. brevis ........... .108 FIGURE 8: Peptide competition assay of anti-K. brevis PCNA ........................... 109 FIGURE 9: Immunolocalization of PCNA ................................................... 110 FIGURE 10: Flow cytometry of the cell population after an extended dark phase ...... 112 CHAPTER 4 FIGURE 1: Multiple alignments of the SUMO and ubiquitin polypeptides .............. 157 FIGURE 2: K. brevis PCNA amino acid sequence: analysis of potential sumoylation and ubiquitin sites ...................................................................................... 158 FIGURE 3: The ubiquitin and SUMO proteins in K. brevis ................................ .159 FIGURE 4: Immunoprecipitation of K. brevis PCNA ....................................... 160 FIGURE 5: Immunoprecipitation of K. brevis PCNA, western blot with anti-SUMO antibody ..................................................................................................................... 161 FIGURE 6: Growth curve and flow cytometry data of K. brevis in 125mL flasks ..... 162 FIGURE 7: Western blot analysis ofPCNA over the K. brevis growth curve .......... 163 FIGURE 8: Western blot analysis ofPCNA in log and stationary phase cultures ..... .164 FIGURE 9: Pattern of ubi quit in at ion in K. brevis stationary phase cultures ............ 166 FIGURE 10: Full length nucleotide sequence of a cyclin-dependent kinase in K. brevis .............................................................................................. 167 FIGURE 11: Amino acid sequence of full length K. brevis cyclin-dependent kinase .. 168 FIGURE 12: Partial alignment of K. brevis CDKs: cyclin binding motif. .............. 169 111 FIGURE 13: Cyclin-dependent kinase inhibitors (CDKIs), olomoucine and fascaplysin ...................................................................................... 170 FIGURE 14: SYTOX staining of K. brevis ................................................ .171 FIGURE 15: PCNA protein abundance throughout S-phase ............................ 172 FIGURE 16: PCNA protein abundance at LDT12 after inhibition with CDKIs ..... .173 FIGURE 17: Linear correlation of % S-phase and PCNA protein abundance ........ 174 IV LIST OF TABLES CHAPTER 2 Table 1: Proteins found to significantly change over the diel cycle using DIGE .......... 63 Table 2: Nuclear protein identifications using tandem MS (LTQ) ............................ 64 CHAPTER 3 Table 1: Primers used for qPCR and SL - gene specific PCR .............................. 114 Table 2: Peptides used