Search

Plasmodium knowlesi is a zoonotic malaria parasite of Southeastern macaque monkeys that causes zoonotic malaria in humans. P. knowlesi is also an experimental model for malaria, and information on P. knowlesi largely stem from experimental lines first isolated >four decades ago, rather than contemporary isolates causing human infections. The experimental lines are laboratory-restricted and have become relatively genetically stagnant and free from the selection pressure that would naturally occur in nature. Within the P. knowlesi genome exist the Schizont Infected Cell Agglutination variant antigen (SICAvar) and Plasmodium knowlesi interspersed repeat (kir) multigene families significant, which are of biological and scientific interest. To provide context using contemporary clinical isolates, this project aimed to generate high-quality genome sequences using long-sequencing from clinical 'wild-type' samples from infected patients. This includes generating new information on variant multigene families in P. knowlesi genomes generated from clinical patient whole blood. The work presented here details a method to deplete leucocytes in thawed P. knowlesi-infected patient whole blood samples to generate parasite-enriched DNA for whole-genome sequencing, resulting in >95% human DNA reduction. The extracted DNA was sequenced with long-read sequencing technology to create de novo whole-genome assemblies. From these, two isolate genomes representing the two dimorphic clusters of P. knowlesi in clinical samples were analysed. The generated genomes are highly syntenic to the published reference genome, sharing >4500 orthologous clusters with the PKNH reference genome. However, the number of SICAvar and kir genes present in the dataset deviated from the published reference genomes of P. knowlesi. The successful generation and construction of these patient genomes aid further interrogation of the contemporary P. knowlesi genome, with a focus on the constituent genes present in comparison to the experimental line.

Species-specific expansions of gene duplicates foster adaptation, genetic innovation, and phenotypic diversification. While it is recognized that events during their early evolutionary history are important for determining if a gene duplicate will be retained, lost, or become nonfunctional, models of gene family evolution have mostly been built from studies of relatively ancient gene families. Therefore, how genes overcome the immediate consequences of duplication, i.e., dosage increase, and accumulate the molecular diversity required for novel functions, while also being impacted by molecular mechanisms such as gene conversion, and evolutionary forces such as genetic drift and selection along the path to fixation, remains largely uncharacterized.My goal was to characterize the functional evolution of a young tandem gene expansion found only in Drosophila melanogaster: Sperm-specific dynein intermediate chain (Sdic). I aimed to accurately reconstruct the Sdic region at the structural and sequence level while obtaining accurate information about sequence diversity among the Sdic paralogs in different strains from different geographical origins (Chapters 1 & 2); investigate the extent of Sdic copy number variation (CNV) (Chapter 2) while examining the relationship between Sdic copy number and total Sdic expression (Chapters 2 & 3); and probe the divergence of different expression attributes among Sdic paralogs within and between strains while gauging the impact of cis and trans regulatory variation (Chapters 1 & 3). Through my research, I established the correct structure of the Sdic region in the D. melanogaster reference genome using raw long read sequences and showed the Sdic paralogs exhibit variable expression in both abundance and breadth using qRT-PCR and RNA-seq. I generated a precise portrait of Sdic copy number variation using reference-quality genome annotations, qPCR, and read-depth methods. Only one Sdic paralog is fixed across populations and there is no evidence of pseudogenization among paralogs. While artificially doubling copy number within the same genomic background increased male expression over two-fold, I observed no correlation between copy number and total Sdic expression across natural populations, suggesting differential regulatory modifiers likely play key roles in shaping Sdic expression. Further, I used RNA-seq to quantify Sdic expression in testes from populations with Sdic CNV, as well as testis, heads, and accessory glands from males with identical genomes except for different Y chromosomes. In testis, I found clear evidence of variable expression among Sdic paralogs and a positive correlation between Sdic CNV and expression. The Y chromosome seems to impact total expression of Sdic in accessory glands but not testes or heads. My dissertation represents a rare interpopulation characterization of a species-specific multigene family at the sequence, structural, and functional levels. Sdic epitomizes how quickly a tandem multigene family can functionally diversify at both the coding and regulatory levels, even in the face of gene conversion. Beyond maintaining a minimally optimal expression level, the presence of Sdic duplicates appears to act as a catalyst for generating protein and regulatory diversity, showcasing a possible evolutionary path that novel gene functions can follow toward long-term consolidation within eukaryotic genomes.