Pentatricopeptide repeat proteins (PPRs) are one of the largest newly identified protein families. PPR is characterized by the presence of a tandemly repeated 35 amino acids motif. It is extremely abundant in plants, and found in many organisms including mammals. PPRs are said to play essential roles in mitochondria, probably via binding to organelle transcripts. A number of recent reports characterizing PPR proteins indicate that PPR are important regulatory proteins that play essential roles in polycistronic RNA processing, RNA stability, and translation of special transcripts [2, 64]. To date, there are 23 PPRs identified in T.brucei by Mingler et al. [2] and 28 distinct PPRs detected by Pusnik et al. [1] by using different bioinformatics approaches. T.brucei encodes far more PPRs than the other non-plant organisms such as mammals. Trypanosomes undergoes a complex life cycle and thus it need to adapt to the changes of environment where it resides. T.brucei undergoes different morphological forms, metabolic changes, and alterations in gene expression during the changes of environment between insect and mammalian hosts. Thus, T.brucei serves as a good model to study the function of PPRs. We utilized tetracycline (tet)-regulated RNA interference (RNAi) to study the function of two T.brucei PPR proteins, TbPPR6 and TbPPR7. Northern blot analysis shows that TbPPR6 (Tb11.01.7930) was down-regulated by 49% while TbPPR7 (Tb927.3.4550) showed a decrease level of 23% in tetracycline induction RNAi cells. Knock-down of TbPPR6 caused a slow growth phenotype, while TbPPR7 knock-down caused only a minor slow growth phenotype, suggesting that TbPPR6 and TbPPR7 are essential for optimal growth of T.brucei . Poisoned primer extension assays demonstrated that depletion of TbPPR6 and TbPPR7 to the levels described above did not affect the mRNAs analyzed. However, there may be a modest effect on COI stability, particularly after depletion of TbPPR7. To characterize the RNA binding properties of TbPPR4 (Tb10.70.5780), recombinant TbPPR4 full length (PTbPR4-FL), and two of its truncated versions (TbPPR4-1, TbPPR4-0) were expressed and purified. Homopolymer binding assays show that TbPPR4-FL preferentially binds poly-(G) agarose beads over the other homopolymers. We further characterized the TbPPR4-RNA interaction by changing the salt concentration (NaCl) of the binding buffer. Increasing salt concentration in the binding buffer decreases the binding ability of TbPPR4-FL to poly-(G) agarose beads, indicating that electrostatic interactions play a prominent role in the binding of TbPPR4-FL. Interactions such as hydrogen bonding and hydrophobic interactions may also involve in the interactions of TbPPR4-FL with poly-(G). Titration experiments of TbPPR4-FL, TbPPR4-1, and TbPPR4-0 show that TbPPR4-FL binds significantly better than TbPPR4-1 and TbPPR4-0 while TbPPR4-1 binds better than TbPR4-0 to poly-(G). Thus, PPRs with increasing numbers of PPR motifs have higher affinity to RNA. Our studies add to the growing literature that defines PPR motifs as RNA binding motifs, and suggest that TbPPR6 and TbPPR7 maybe play a role in mitochondrial RNA processing and/or stability.

The protozoan parasite and causative agent of human and animal African trypanosomiasis, Trypanosoma brucei, is a leading cause of morbidity and mortality among epidemic rural regions of sub-Saharan Africa. T. brucei is transmitted to the bloodstream of the mammalian host by the tsetse fly, where it resides extracellularly and evades immune detection by a mechanism called antigenic variation. Due to the antigenic nature of the parasite, the prospect for vaccine development is grim. Instead, disease treatment relies solely on chemotherapeutic strategies that target the unique and exploitable biology of the trypanosome. Such unique aspects of T. brucei biology include polycistronic transcription, pre-mRNA trans -splicing, and kinetoplastid RNA editing. The developmental regulation of trypanosome gene expression relies on coordinated post-transcriptional events in which RNA binding proteins play a leading role. With this in mind, our focus is directed towards a family of protein arginine methyltransferases (PRMTs), which are implicated in a variety of post-transcriptional events and hypothesized to play a regulatory role in trypanosome gene expression. Protein arginine methylation is a post-translational modification that modulates the function of a variety nucleic acid binding proteins, impacting transcriptional and post-transcriptional gene expression. These modifications are catalyzed by a family of PRMTs described in mammals and yeast for which five homologs have been identified in the T. brucei genome. The initial characterization of TbPRMT1 and TbPRMT5 has been explored, the latter of which is described in Chapters II and III of this thesis. TbPRMT1 is a type I PRMT and catalyzes asymmetric dimethylarginine modifications, while TbPRMT5 is a type II PRMT and catalyzes symmetric dimethylarginine modifications. Both TbPRMT1 and TbPRMT5 are constitutively expressed in bloodstream and procyclic form trypanosomes, localize to the cytoplasm, and are not essential for growth. Both enzymes methylate a variety of substrates in vitro, including the mitochondrial regulatory protein, RBP16. In addition, TbPRMT5 was shown to associate in vivo with a kinetoplastid-specific nucleotidyltransferase and an ATP-dependent RNA helicase, the latter of which is an in vitro TbPRMT5 substrate. In vivo analysis of higher-order TbPRMT5-TAP-containing complexes indicates that TbPRMT5 associates with two predominant protein complexes with molecular weights of approximately 250 and 700 kDa. The latter of these complexes is unstable and does not withstand glycerol gradient fractionation. While TbPRMT1 and TbPRMT5 do not play a global role in trans -splicing or decay of nuclear encoded RNAs, they both play a role in mitochondrial gene expression. In Chapter III of this dissertation, I show that disruption of TbPRMT5 in procyclic form trypanosomes by RNA interference results in the destabilization of never edited COI and ND4 and both edited and unedited apocytochrome b (CYb) and COII mitochondrial RNAs (Chapter III). In addition, TbPRMT5 disruption resulted in a modest but significant increase in the steady-abundance of mitochondrial-encoded guide RNAs (gRNAs), suggesting that TbPRMT5 plays a role in gRNA turnover. Furthermore, I demonstrate that TbPRMT5 potentially modulates the function of at least four mitochondrial methylproteins. Several in vivo hypomethylation defects were observed in mitochondrial lysates of TbPRMT5-disrupted cells, and TbPRMT5 appears to affect the expression and/or mitochondrial localization of one putative in vivo substrate. The characterization of TbPRMT5 substrates, as well as the remaining T. brucei PRMTs, is currently being explored. In summary, I provide evidence that TbPRMT5-catalyzed arginine methylation plays important regulatory roles in T. brucei mitochondrial gene expression, and its association with RNA modifying enzymes suggests that it likely impacts additional specific RNA metabolic events in the nucleus and/or cytoplasm.

This detailed volume presents a wide range of techniques for plant mitochondrial analysis, ranging from tried-and-tested work horse techniques to the latest innovations. Within its pages, it explores subjects such as affinity-based isolation of mitochondria with magnetic beads, mitochondrial quality assessment protocols, measurement of uptake and release of specific metabolites, mitochondrial protein identification and visualization, as well as gene splicing and editing, and much more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Plant Mitochondria: Methods and Protocols provides a highly useful set of methodologies for the plant mitochondrial community to help discover more interesting aspects of plant mitochondria in the years to come.

We have taught plant molecular biology and biotechnology at the undergraduate and graduate level for over 20 years. In the past few decades, the field of plant organelle molecular biology and biotechnology has made immense strides. From the green revolution to golden rice, plant organelles have revolutionized agriculture. Given the exponential growth in research, the problem of finding appropriate textbooks for courses in plant biotechnology and molecular biology has become a major challenge. After years of handing out photocopies of various journal articles and reviews scattered through out the print and electronic media, a serendipitous meeting occurred at the 2002 IATPC World Congress held in Orlando, Florida. After my talk and evaluating several posters presented by investigators from my laboratory, Dr. Jacco Flipsen, Publishing Manager of Kluwer Publishers asked me whether I would consider editing a book on Plant Organelles. I accepted this challenge, after months of deliberations, primarily because I was unsuccessful in finding a text book in this area for many years. I signed the contract with Kluwer in March 2003 with a promise to deliver a camera-ready textbook on July 1, 2004. Given the short deadline and the complexity of the task, I quickly realized this task would need a co-editor. Dr. Christine Chase was the first scientist who came to my mind because of her expertise in plant mitochondria, and she readily agreed to work with me on this book.

This much-needed book is the first definitive volume on Euglena in twenty-fire years, offering information on its atypical biochemistry, cell and molecular biology, and potential biotechnology applications. This volume gathers together contributions from well-known experts, who in many cases played major roles in elucidating the phenomenon discussed. Presented in three parts, the first section of this comprehensive book describes novel biochemical pathways which in some instances have an atypical subcellular localization. The second section details atypical cellular mechanisms of organelle protein import, organelle nuclear genome interdependence, gene regulation and expression that provides insights into the evolutionary origins of eukaryotic cells. The final section discusses how biotechnologists have capitalized on the novel cellular and biochemical features of Euglena to produce value added products. Euglena: Biochemistry, Cell and Molecular Biology will provide essential reading for cell and molecular biologists with interests in evolution, novel biochemical pathways, organelle biogenesis and algal biotechnology. Readers will come away from this volume with a full understanding of the complexities of the Euglena as well as new realizations regarding the diversity of cellular processes yet to be discovered.

The Genetics and Genomics of the Brassicaceae provides a review of this important family (commonly termed the mustard family, or Cruciferae). The family contains several cultivated species, including radish, rocket, watercress, wasabi and horseradish, in addition to the vegetable and oil crops of the Brassica genus. There are numerous further species with great potential for exploitation in 21st century agriculture, particularly as sources of bioactive chemicals. These opportunities are reviewed, in the context of the Brassicaceae in agriculture. More detailed descriptions are provided of the genetics of the cultivated Brassica crops, including both the species producing most of the brassica vegetable crops (B. rapa and B. oleracea) and the principal species producing oilseed crops (B. napus and B. juncea). The Brassicaceae also include important “model” plant species. Most prominent is Arabidopsis thaliana, the first plant species to have its genome sequenced. Natural genetic variation is reviewed for A. thaliana, as are the genetics of the closely related A. lyrata and of the genus Capsella. Self incompatibility is widespread in the Brassicaceae, and this subject is reviewed. Interest arising from both the commercial value of crop species of the Brassicaceae and the importance of Arabidopsis thaliana as a model species, has led to the development of numerous resources to support research. These are reviewed, including germplasm and genomic library resources, and resources for reverse genetics, metabolomics, bioinformatics and transformation. Molecular studies of the genomes of species of the Brassicaceae revealed extensive genome duplication, indicative of multiple polyploidy events during evolution. In some species, such as Brassica napus, there is evidence of multiple rounds of polyploidy during its relatively recent evolution, thus the Brassicaceae represent an excellent model system for the study of the impacts of polyploidy and the subsequent process of diploidisation, whereby the genome stabilises. Sequence-level characterization of the genomes of Arabidopsis thaliana and Brassica rapa are presented, along with summaries of comparative studies conducted at both linkage map and sequence level, and analysis of the structural and functional evolution of resynthesised polyploids, along with a description of the phylogeny and karyotype evolution of the Brassicaceae. Finally, some perspectives of the editors are presented. These focus upon the Brassicaceae species as models for studying genome evolution following polyploidy, the impact of advances in genome sequencing technology, prospects for future transcriptome analysis and upcoming model systems.