The Drosophila genome is thought to have five recA like proteins: Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3. In Drosophila Rad51/SpnA, XRCC3/SpnB, and Rad51 C/SpnD participate in homologous recombination repair. The function of DmRad51 D and DmXRCC2 are unknown. The goal of this project was to elucidate the function ofXRCC2 in Drosophila. RNA interference allowed us to knockdown the function XRCC2 and its possible binding partner Rad51D. It was seen the knocking down the function of either XRCC2 or Rad51D does not affect the viability of the fly. However, drug treatment data does not allow us to make any conclusions about how the knockdown ofXRCC2 affects the viability of the fly. RNA in-situ hybridization shows highly intricate and complex branching patterns for XRCC2, which resembles the embryonic tracheal system. Lastly, XRCC2 was purified to generate an antibody made to recognize the XRCC2 protein will help localize the XRCC2 protein in future studies as well as determine protein-protein interactions with XRCC2.

No description available.

The recent call to reconstruct a detailed picture of the tree of life for all organisms has forever changed the field of molecular phylogenetics. Sequencing technology has improved to the point that scientics can now routinely sequence complete plastid/mitochondrial genomes and thus, vast amounts of data can be used to reconstruct phylogenies. These data are accumulating in DNA sequence repositories, such as GenBank, where everyone can benefit from the vast growth of information. The trend of generating genomic-region rich datasets has far outpaced the expasion of datasets by sampling a broader array of taxa. We show here that expanding a dataset both by increasing genomic regions and species sampled using GenBank data, despite the inherent missing DNA that comes with GenBank data, can provide a robust phylogeny for the plant order Caryophyllales (angiosperms). We also investigate the utility of trnK intron in phylogeny reconstruction at relativley deep evolutionary history (the caryophyllid order) by comparing it with rapidly evolving matK. We show that trnK intron is comparable to matK in terms of the proportion of variable sites, parsimony informative sites, the distribution of those sites among rate classes, and phylogenetic informativness across the history of the order. This is especailly useful since trnK intron is often sequenced concurrently with matK which saves on time and resources by increasing the phylogenetic utility of a single genomic region (rapidly evolving matK/trnK). Finally, we show that the inclusion of RNA edited sites in datasets for phylogeny reconstruction did not appear to impact resolution or support in the Gnetales indicating that edited sites in such low proportions do not need to be a consideration when building datasets. We also propose an alternate start codon for matK in Ephedra based on the presense of a 38 base pair indel in several species that otherwise result in pre-mature stop codons, and present 20 RNA edited sites in two Zamiaceae and three Pinaceae species. / Ph. D.

No description available.

Slo2, a large conductance potassium channel in the nervous system is important for regulating neuronal function and excitability. Mutations in the gene that encodes the Slo2 channel are associated with neurological disorders, including epilepsy and intellectual disabilities in humans. However, much remains unknown about the genes and proteins that regulate Slo2 channel activity in the physiological system. This study investigates regulation of SLO-2, a homologue of mammalian Slo2 in C. elegans, by thoc-7 in an RNA editing-dependent pathway. Prior research has shown that adr-1, the gene important for RNA editing, promotes SLO-2 function by RNA editing of scyl-1 that encodes a regulator of SLO-2. To gain a better understanding of the regulation of SLO-2, this study employed a forward genetic approach to screen for mutants with a specific phenotype. Through SNP mapping and whole genome sequencing, we identified the gene thoc-7, which is predicted to be involved in mRNA export from nucleus, from the isolated mutants. The identification was further confirmed by CRISPR/Cas9-mediated gene knock-out, which showed a similar phenotype to the mutant strain. Results of electrophysiological recordings suggest that thoc-7 likely contributes to SLO-2 function in a common pathway with scyl-1. A reporter gene revealed strong expression of thoc-7 in most of the cells of C. elegans, particularly muscular and digestive system. Translational fusion with GFP showed the primary localization of the THOC-7 protein in cytoplasm, with some weak expression in the nucleus. RT-qPCR analysis suggests that thoc-7 regulates scyl-1 by through a post-transcriptional mechanism, possibly involving the transport of mRNA from cytoplasm to nucleus. This study highlights thoc-7 as a potential key regulator recruited by adr-1 to control SLO-2 via scyl-1 expression.

Bacterial transcriptomes are highly complex, comprising not only protein-coding RNAs and translation-related non-coding RNAs, but also non-coding RNAs that function as regulators of gene expression. The post-transcriptional modification of RNA sequences by RNA editing enzymes, which has recently been shown to affect diverse RNA substrates in several bacteria, can magnify this complexity further still. However, little is known about RNA editing and non-coding RNAs in Streptomyces venezuelae, a model organism for studying complex bacterial development and specialized metabolism. This thesis investigates RNA editing and non-coding regulatory RNAs in S. venezuelae using RNA sequencing data from wild type and mutant strains at various stages of development and under several laboratory-controlled conditions. We identified hundreds of adenosine-to-inosine editing events throughout the transcriptome and predicted the potential impact of the edits occurring in protein-coding RNAs. The potential role of the adenosine deaminase enzyme TadA in facilitating these RNA editing events is also considered. Additionally, we detected thousands of transcripts that are expressed from unannotated regions of the S. venezuelae genome, many of which we predict are non-coding RNAs. Furthermore, we highlight our efforts to characterize a highly expressed putative non-coding RNA that exhibits considerable sequence conservation in other streptomycetes. This work provides new insights into the transcriptomic complexity of S. venezuelae and expands our understanding of RNA-based regulation in bacteria. / Thesis / Master of Science (MSc) / All living things have DNA, which contains the instructions for maintaining life in the form of genes. These genes are copied into RNAs, and some of these RNA molecules are used to make proteins, which are the building blocks and machinery of cells. However, not all RNAs make proteins; some act as regulators, controlling which genes and proteins are active. Additionally, some proteins edit the instructions contained by RNA molecules after they are made, adding another layer of complexity to how cells regulate their activities. This thesis investigates these processes in Streptomyces venezuelae, a soil-dwelling bacterium known for its complex development and metabolism. We found hundreds of cases where RNA molecules are edited, potentially affecting their functions in the cell, and discovered thousands of non-protein-coding RNAs that may regulate genes or proteins. Our findings expand our understanding of how Streptomyces bacteria manage their complex genetic activities at the RNA level.

The transcriptome of the mammalian brain is extensively modified by adenosine to inosine (A-to-I) nucleotide conversion by two adenosine deaminases (ADAR1 and ADAR2). As adenosine and inosine have different base pairing properties, A-to-I RNA editing shapes the functional output of both coding and non-coding RNAs (ncRNAs) in the brain. The aim of this thesis was to identify editing events in small regulatory ncRNAs (miRNAs) and to determine their temporal and spatial editing status in the developing and adult mouse brain. To do this, we initially analyzed the editing status of miRNAs from different developmental time points of the mouse brain. We detected novel miRNA substrates subjected to A-to-I editing and found a general increase in miRNA editing during brain development, implicating a more stringent control of miRNAs as the brain matures. Most of the edited miRNAs were found to be transcribed as a single long consecutive transcript from a large gene cluster. However, maturation from this primary miRNA (pri-miRNA) transcript into functional forms of miRNAs is regulated individually, and might be influenced by the ADAR proteins in an editing independent matter. We also found that edited miRNAs were highly expressed at the synapse, implicating a role as local regulators of synaptic translation. We further show that the increase in editing during development is explained by a gradual accumulation of the ADAR enzymes in the nucleus. Specifically for ADAR2, we found a developmentally increasing interaction with two factors, importin-α4 and Pin1, that facilitate nuclear localization of the editing enzyme. We have also found that selectively edited stem loops often are flanked by other long stem loop structures that induce editing in cis. This may explain why multiple pri-miRNAs are edited within the same cluster. In conclusion, this thesis has significantly increased the understanding of the dynamics of both editing substrates and enzymes in the developing and mature brain. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

The trypanosomatid protists belonging to Order Kinetoplastida are some of the most successful parasites ever known to mankind. Their extreme physiological diversity and adaptability to different environmental conditions and host systems make them some of the most widespread parasites, causing deadly diseases in humans and other vertebrates. This project focuses on their unique mitochondrion, called the kinetoplast, and more specifically involves the characterization of a part of their mitochondrial DNA (also called kinetoplast DNA or kDNA), the maxicircles, which are functional homologs of eukaryotic mitochondrial DNA in the kinetoplastid protists. We have sequenced and characterized the maxicircle genomes of 20 new trypanosomatids and compared them with 8 previously published maxicircle genomes of other trypanosomatids. Transcripts of ~13 of the 20 total genes in these maxicircles undergo post-transcriptional modifications involving the insertion and deletion of U residues at precise sites, to yield the final, fully-edited, translatable mRNA. We have deciphered the diverse patterns and extents of RNA editing of each edited gene in the maxicircle of each organism, and inferred the sequences of the putative fully edited mitochondrial transcripts and proteins. Using a binary value - based strategy (1/0), we quantified the RNA editing in all these trypanosomatids and estimated the evolution of RNA editing in the group. Additionally, we conducted phylogenetic analyses using a subset of unedited maxicircle genes to predict the relationships between the various trypanosomatids in this project, and compared them to the previously published nuclear gene-based phylogenies. For convenience of analysis, the 28 total trypanosomatids in this work were divided into two groups: the first group consisting of the endosymbiont-bearing and related insect trypanosomatids, which constitute the first half of the project, and the second group consisting of trypanosomatids of the Trypanosoma genus, including T. cruzi-related and unrelated parasites, constituting the latter half of the project. In summary, most of the trypanosomatid maxicircles showed a syntenic panel of 20 protein-coding genes (excluding any guide RNA genes), beginning with the mitochondrial ribosomal genes and ending with the gene encoding NADH dehydrogenase-5. Although some genes were partially or completely absent in the maxcircles of some species, the remaining genes were completely syntenic. The total number of genes edited and their editing patterns varied considerably among the first group of insect trypanosomatids, but were remarkably similar in the second group of the Trypanosoma genus. On a broad scale, the mitochondrial phylogeny reflects the nuclear phylogeny for these trypanosomatids, except within the T. cruzi population. Similarly, RNA editing appears to have evolved in parallel with the nuclear genes, although subtle differences are again noticeable within the T. cruzi family.

The fern genus Pteridium comprises a number of closely related species distributed throughout the world. Collectively they are called bracken ferns and have historically been treated as a single species, Pteridium aquilinum. Bracken is notorious as a toxic weed that colonizes open fields and poisons livestock. Bracken is also easily cultured and has become one of the most intensively studied ferns. Bracken has been used as a model system for the study of the fern life cycle, fern gametophyte development, the pheromonal mechanism of sex determination, toxicology, invasion ecology, and climate change. This dissertation places bracken within a global evolutionary perspective and establishes bracken as an emerging system for evolutionary genomics in ferns. Bracken samples from around the world were examined for chloroplast DNA variation to infer historical phylogenetic and biogeographic evolutionary events. New high-throughput DNA sequencing technologies and bioinformatic approaches were used to determine the complete chloroplast genome sequence in bracken, to identify novel RNA editing sites in chloroplast transcripts, and to identify gene sequences that are expressed in the gametophyte stage of the fern life cycle. These data represent an important genomic resource in ferns and were examined within a functional and evolutionary perspective. Several novel approaches and analyses were developed in the course of this research. Results presented in this dissertation provide novel insights into fern biology and land plant evolution.

RNA editing had been considered as a rare exception to the central dogma of molecular biology in which the mRNA truthfully carries genetic code from nucleus to the ribosome for translation. However, researches in the last five years have revealed numerous, widespread RNA A-to-I editing sites in human genome. Although the effects of these editing events require further study, this finding strongly suggests RNA editing occurs frequently, and affects large number of genes. By selectively modifying a few sequences of a gene, RNA editing allows a cell to produce a population of proteins with different properties from a single gene. The major question of this thesis study is whether such editing event is actually dynamically regulated when the cellular physiological processes have to be adjusted in response to changing environment. A previous study screening for Drosophila mutants defected in hypoxia and heat tolerance discovered a hypnos-2 mutant strain which was later found to be defective in dADAR, the drosophila gene encoding the A-to-I editing enzyme, supporting the hypothesis that cells/organisms response to stressful environment by dADAR-mediated RNA editing. Two directions are used to approach how Drosophila uses A-to-I editing to adapt ¡§heat¡¨ environment stress. First, whether the expression pattern of dADAR changes after heat shock was investegated. The result showed the dADAR gene exon 7 self-editing frequency was decreased by heat shock, thus possibly enhances dADAR activity after heat shock processing. Moreover it is worth noting that the isoform without -1 exon transcript were obviously up-regulated, and transcript with -1 sequence is relatively down-regulated. On the other hand, no significant changes in the dADAR mRNA expression levels and in the degrees of two dADAR promoters activity were observed. Second, the changes of editing frequency of 30 known A-to-I editing sites were investigated. Generally the editing frequency of majority editing sites changed after heat shock. Therefore, the dADAR activity, the dADAR gene transcript expression alternations, and A-to-I editing frequency of dADAR target genes did change after heat shock, supporting the notion that change of RNA editing pattern is a mechanism for organism to adapt to drastic environmental change. However, how the edited protein isoforms contribute to heat resistance requires further investigation.