MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation Of Brian Junior Henson Candidate for the Degree: Doctor of Philosophy Advisor ______________________ (Susan R. Barnum) Advisor ______________________ (Linda E. Watson) Reader ______________________ (David A. Francko) Reader ______________________ (John Z. Kiss) Grad School Representative ______________________ (Luis A. Actis) Abstract EVOLUTION, VARIATION, AND EXCISION OF DEVELOPMENTALLY REGULATED DNA ELEMENTS IN THE HETEROCYSTOUS CYANOBACTERIA by Brian Junior Henson In some cyanobacteria, heterocyst differentiation is accompanied by developmentally regulated DNA rearrangements that occur within the nifD, fdxN, and hupL genes, referred to as the nifD, fdxN, and hupL elements. These elements are excised from the genome by site-specific recombination during the latter stages of heterocyst differentiation. In this dissertation, two major questions are addressed: 1) what is the evolutionary history of the nifD and hupL elements and 2) how is the nifD element excised? To answer the first question, full length nifD and hupL element sequences were characterized and compared; and xisA and xisC sequences (which encode the recombinases that excise the nifD and hupL elements, respectively) were phylogenetically analyzed. Results indicated extensive structural and compositional variation within the nifD and hupL elements. The data suggests that the nifD and hupL elements are of viral origin and that they have variable patterns of evolution in the cyanobacteria. To answer the second question, a recombination system was devised where the ability of XisA to excise or recombine variants of the nifD element (substrate plasmids) was tested. Using PCR directed mutagenesis, specific nucleotides within the flanking regions of the nifD element were altered and the effects on recombination determined. Results indicate that nucleotides within and outside of the direct repeats are involved in excision, and that not all nucleotides within the direct repeats are required. In certain nucleotide positions, the presence of a purine versus a pyrimidine greatly affected recombination. Although excision was inhibited when certain nucleotides were mutated, PCR analyses revealed that excision occurred at a low level. The data also indicate that the site of excision occurs within the direct repeats. The results presented here suggest that the elements may be variable in size, composition, and excision. EVOLUTION, VARIATION, AND EXCISION OF DEVELOPMENTALLY REGULATED DNA REARRANGEMENTS IN THE HETEROCYSTOUS CYANOBACTERIA A Dissertation Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Botany by Brian Junior Henson Miami University Oxford, Ohio 2005 Major Advisors: Susan R. Barnum Ph.D. and Linda E. Watson Ph.D. TABLE OF CONTENTS PAGE LIST OF TABLES.................................................................................................................... iii LIST OF FIGURES....................................................................................................................iv DEDICATION............................................................................................................................v ACKNOWLEDGEMENTS........................................................................................................vi INTRODUCTION ......................................................................................................................1 REFERENCES..................................................................................................................9 CHAPTER 1 .............................................................................................................................14 ABSTRACT....................................................................................................................15 INTRODUCTION...........................................................................................................16 MATERIALS AND METHODS.....................................................................................17 RESULTS .......................................................................................................................18 DISCUSSION .................................................................................................................19 REFERENCES................................................................................................................23 CHAPTER 2 .............................................................................................................................26 ABSTRACT....................................................................................................................27 INTRODUCTION...........................................................................................................28 MATERIALS AND METHODS.....................................................................................31 RESULTS .......................................................................................................................34 DISCUSSION .................................................................................................................38 REFERENCES................................................................................................................57 CHAPTER 3 .............................................................................................................................65 ABSTRACT....................................................................................................................66 INTRODUCTION...........................................................................................................67 MATERIALS AND METHODS.....................................................................................70 RESULTS .......................................................................................................................76 DISCUSSION .................................................................................................................80 REFERENCES..............................................................................................................113 SUMMARY ...........................................................................................................................118 ii LIST OF TABLES TABLES PAGE CHAPTER I 1. Similarities within conserved regions of the nifD element...........................................21 CHAPTER II 1. Prevalence of the nifD element ...................................................................................48 2. Prevalence of the hupL element .................................................................................49 3. G+C content of the elements.......................................................................................50 CHAPTER III 1. Primer table ................................................................................................................83 2. Results of recombination assay ...................................................................................89 iii LIST OF FIGURES FIGURE PAGE INTRODUCTION 1. NifD element .................................................................................................................7 2. HupL element................................................................................................................8 CHAPTER I 1. Similarity between conserved regions of the nifD elements.........................................22 CHAPTER II 1. Comparison of nifD elements......................................................................................51 2. Comparison of Cylindrospermum PCC 7417 and N. punctiforme nifD elements..........52 3. Comparison of hupL elements ....................................................................................53 4. Combined xisA and xisC phylogeny............................................................................54 5. XisA phylogeny ..........................................................................................................55 6. XisC phylogeny ..........................................................................................................56 CHAPTER III 1. NifD element...............................................................................................................91 2. Map of pDK1 and pSUB1...........................................................................................92 3. Map of pSUB9B and its derivatives ............................................................................93 4. pRE-18 .......................................................................................................................94 5. pDK1..........................................................................................................................94 6. pSUB1........................................................................................................................95 7. pSUB2........................................................................................................................95 8. pSUB9B .....................................................................................................................96 9. nifD and nifK proximal regions in pSUB9B ................................................................96 10 pSUB16Z, 10..............................................................................................................97