MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation Of Wing See Lam Candidate for the Degree: Doctor of Philosophy Dr. Christopher A. Makaroff, Director Dr. Ann E. Hagerman, Reader Dr. Gary A. Lorigan, Reader Dr. Michael W. Crowder, Reader Dr. David G. Pennock, Graduate School Representative ABSTRACT THE ISOLATION AND CHARACTERIZATION OF ARABIDOPSIS AtSMC1 AND AtSMC3 By Wing See Lam Structural maintenance of chromosome (SMC) proteins are conserved in yeast and all animals examined. Six SMC proteins have been identified and their roles divided into three functional groups. The condensins (SMC2 and SMC4) regulate chromosome condensation and organize long linear chromosomes into compact structures during cell division. The cohesins (SMC1 and SMC3) are responsible for sister chromatid cohesion during DNA replication and the proper segregation of chromosomes. Lastly, DNA recombination and repair complexes (SMC5 and SMC6) participate in postreplicative and recombinational repair of DNA lesions and double-strand breaks. All the complexes have the common theme that they are involved in chromosome folding and dynamics. Cohesin complexes contain SMC1, SMC3, SCC1, and SCC3. Sister chromatid cohesion is generally conserved between all eukaryotes, but there are also species- specific differences in cohesin behavior. For example, cohesin complexes are present in the cytoplasm of animal cells and on spindle poles at the metaphase-anaphase transition in human cells but not in yeast. We have characterized the Arabidopsis AtSMC1 and AtSMC3 genes to investigate cohesin proteins in plants. We found AtSMC1 and AtSMC3 transcripts were present in all tissues. Using antibodies we generated to AtSMC3, we found AtSMC3 was present in the cytoplasm as well as the nucleus. AtSMC3 co-localized with the chromosomes during prophase of mitosis and meiosis. However, we found AtSMC3 associated with the spindle from metaphase to telophase, which is different from what has been observed in both yeast and animal cells. Finally, although Arabidopsis T-DNA insertion mutants for SMC1 and SMC3 have been identified and result in embryo lethality, the mutations did not affect male or female fertility. We proposed that the gametophytes were able to utilize stored SMC1 and SMC3 RNA or proteins to allow development into embryogenesis. In order to investigate this hypothesis, Lat52 AtSMC3 RNAi Arabidopsis lines were generated and analyzed. Three transgenic lines appeared partially sterile due to abnormal formation of pollen grains. These results indicate that AtSMC3 proteins are in fact required during gametophyte development. THE ISOLATION AND CHARACTERIZATION OF ARABIDOPSIS AtSMC1 AND AtSMC3 A DISSERTATION Submitted to the Faculty of Miami University in partial Fulfillment of the requirements For the degree of Doctor of Philosophy Department of Chemistry and Biochemistry By Wing See Lam Miami University Oxford, OH 2004 Dissertation Director: Dr. Christopher A. Makaroff TABLE OF CONTENTS Page I. Introduction 1 II. Characterization of Arabidopsis thaliana SMC: Evidence That AtSMC3 May Have Additional Roles Beyond Chromosome Cohesion A. Summary 32 B. Keywords 33 C. Introduction 34 D. Materials and Methods 40 E. Results 47 F. Discussion 85 G. Acknowledgements 91 H. References 92 III. Real-Time PCR Studies for AtSMC1 and AtSMC3 A. Introduction 100 B. Materials and Methods 105 C. Results 108 D. Discussion 115 E. References 120 ii IV. Preliminary Characterization of Lat52 AtSMC3 RNAi Plants A. Introduction 125 B. Materials and Methods 127 C. Results 131 D. Discussion 153 E. References 157 V. Concluding Remarks 162 iii LIST OF FIGURES Page 1.1. Structural organization of SMC proteins 5 1.2. SMC dimer configuration 7 1.3. Cohesin core complex configuration 11 1.4. Current models for mitotic cohesin function in S. cerevisiae and human 15 1.5. Current models for meiotic cohesin function in S. cerevisiae and rat at meiosis I 19 2.1. Schematic representation of the AtSMC1 and AtSMC3 genes 49 2.2A. Amino acid sequence alignment of SMC1 in Arabidopsis (Ara), human, yeast, and Drosophila 51 2.2B. Amino acid sequence alignment of SMC3 in Arabidopsis (Ara), human, yeast, and Drosophila 53 2.3. Schematic representation of the AtSMC1 genes locus 56 2.4. Transcript studies for AtSMC1, AtSMC1-3’UTR, AtSMC3, PTPG and Actin8 59 2.5. Control studies for SMC1 antibodies in Arabidopsis proteins 62 2.6. AtSMC3 protein levels 65 iv 2.7. Immunolocalization of AtSMC3 proteins in Arabidopsis suspension cells fixed with 4% paraformaldehyde 68 2.8. Western blot analysis of AtSMC3 eluted fractions by the series of extractions of Arabidopsis suspension cells shown in Figure 2.6 70 2.9. Immunolocalization of AtSMC3 proteins in Arabidopsis somatic cells 74 2.10A. AtSMC3 proteins localized with DNA in wild-type Arabidopsis microsporocytes 76 2.10B. AtSMC3 proteins localized with spindle in wild-type Arabidopsis microsporocytes 79 2.11. Immunolocalization of AtSMC3 (A) and SYN1 (B) proteins in wild-type Arabidopsis microsporocytes at pachytene 81 2.12. Immunolocalization of AtSMC3 protein from meiotic interphase to telophase I in microsporocytes of syn1 mutant plants 84 3.1. Schematic representation of the AtSMC1 genes locus 103 3.2. Real-time PCR standard curves for the AtSMC1, AtSMC3, PTPG, and Actin8 genes 111 4.1. AtSMC1 and AtSMC3 RNAi construct maps 133 4.2. Southern blot analysis of Lat52 AtSMC3 RNAi lines 138 4.3. Lat52 AtSMC3 RNAi transgenic plants show reduced seed set 147 v 4.4. Lat52 AtSMC3 RNAi transgenic plants show reduced pollen levels relative to wild-type plants 149 4.5. Phenotypes in pollen sacs and pollen grains in some Lat52 AtSMC3 RNAi transgenic plants 152 vi LIST OF TABLES Page 2.1. Oligonucleotide sequences used in this study 41 3.1. Oligonucleotide sequences used in this study 106 3.2. Analyze of real-time PCR 113 4.1. Oligonucleotide sequences used in this study 128 4.2. Analysis of siliques from Lat52 AtSMC3 RNAi T1 plants 136 4.3. PCR analysis for Lat52 AtSMC3 RNAi plants 140 4.4. Segregation ratios of Lat52 AtSMC3 RNAi plants 141 4.5. Phenotypic analysis of Lat52 AtSMC3 RNAi plants 143 4.6. Phenotypic analysis of Lat52 AtSMC3 RNAi plants 144 vii DEDICATION This dissertation is dedicated to my mother, Yee Fei Chu, whose determination of giving me and my siblings the best that she can provide and much more, loves us unconditionally, and helps us to become who we are and what we will be my sister, Wing Man Lam, who gives me the confidence and support to pursue my education my brother, Shiu Fung Lam, whose accomplishment of becoming the Hong Kong Finest my baby brother, Chun Fung Lam, who is going to be The best Chef in the Universe my host family, Dr. Ron and Cheryl Hale, who love me, teach me and help me throughout the toughest years I had and will have I love you all always viii Chapter I INTRODUCTION Mitosis and Meiosis Genomic stability in all organisms depends on faithful chromosome inheritance during meiosis and mitosis. The processes of mitosis and meiosis are highly conserved in all eukaryotic organisms. During early mitotic prophase the replicated chromosomes condense. Then the nuclear envelope breaks down as the condensed sister chromatids move toward the metaphase plate during late prophase. At metaphase, microtubules from each of the mitotic spindle poles attach to the kinetochores of pairs of sister chromatids at the metaphase plate. In yeast and animals, metaphase is monitored by checkpoints to ensure the proper attachment of the chromosomes to the spindle. If alterations are detected, anaphase will be delayed (Millband et al., 2002). At anaphase, the protease separase cleaves a subunit of the cohesin complex on the sister chromatids that allows the sister chromatids to be pulled to opposite poles of the spindle (Nasmyth, 2002). In telophase, the chromosomes start to decondense. During cytokinesis the cytoplasm divides, and two new daughter cells are formed. In meiosis, two nuclear divisions follow a single round of DNA replication, which is often referred to as S phase. Sister chromatid cohesion is established in premeiotic S phase. During leptotene of prophase I, the chromosomes begin to reorganize starting as a mass of entangled threads. Homologous chromosomes begin to seek each other and pair during leptotene, zygotene, and pachytene. During pachytene, the synaptonemal complex, a proteinaceous complex that links the two homologous chromosomes, forms 1 and recombination takes place. The inability to execute these processes correctly can result in catastrophic chromosome segregation defects as is observed in mutants defective in Spo11, a protein required to initiate recombination (Lin and Smith, 1994; Dernburg et al., 1998; McKim and Hayashi-Hagihara, 1998; Grelon et al., 2001). As the cell reaches diplonema and diakinesis, the chromosomes desynapse and take on a highly condensed, compact structure. At metaphase I, the condensed chromosomes align at the metaphase plate, and the homologous chromosome pairs interact with spindle fibers preparing for segregation. At anaphase I, spindle fibers attached to the kinetochores contract, pulling the homologous chromosomes away from each other. Each chromosome still has two sister chromatids at this point. At telophase I, chromosomes reach the opposite poles of the cell. In general, two nuclear envelopes surround the separated groups of chromosomes at meiotic interphase II; however, this varies from species to species. At prophase II, the condensed sister chromatids, which are still connected at their centromeres, move toward the metaphase plate. At metaphase II, the spindle fibers attach such that one sister chromatid kinetochore will be pulled to one pole, while the other will move to the opposite pole. Similar to mitosis, the protease separase cleaves a subunit of the cohesin complex on the sister chromatids. After the cohesion between sister chromatids is lost the separated chromatids are pulled toward their respective poles. In telophase II, the nuclear envelope forms around the four groups of chromosomes, which begin to decondense, and four new haploid daughter cells are formed after cytokinesis (Ross et al., 1996; Petronczki et al., 2003).