Doctor of Philosophy
Molecular Biology and Genetics
Gregory A. Buck
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.
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Available for download on Sunday, November 22, 2020