Adams, Alexander Nichols, authorMueller, Rachel, advisorHanson, Jeffrey, committee memberHoke, Kim, committee memberSloan, Dan, committee member2024-01-012024-01-012023https://hdl.handle.net/10217/237444Across the tree of life, the correlated traits of genome size and cell size both vary by orders of magnitude, with the increase in genome size being largely attributable to an increase in transposable elements (TEs) throughout the genome. This accumulation of TEs affects many facets of the cell including DNA regulation, organellar scaling, and RNA transcription. This dissertation will explore all 3 of these facets through the lens of genome gigantism and address how these facets differ in large cells in comparison to cells that are more typical in size. The first chapter will discuss methylation of cytosines at genomic CpG dinucleotide sites that silence TEs. TE abundance drives differences in genome size, but TE silencing variation across genomes of different sizes remains largely unexplored. Salamanders include most of the largest C-values — 9 to 120 Gb. We measured CpG methylation levels in salamanders with genomes ranging from 2N = ~58 Gb to 4N = ~116 Gb. We compared these levels to results from endo- and ectothermic vertebrates with more typical genomes. Salamander methylation levels are ~90%, higher than all endotherms. However, salamander methylation does not differ from the other ectotherms, despite a ~100-fold difference in nuclear DNA content. Because methylation affects the nucleotide compositional landscape through 5-methylcytosine deamination to thymine, we quantified salamander CpG dinucleotide levels and compared them to other vertebrates. Salamanders have comparable CpG levels to other ectotherms, and ectotherm levels are higher than endotherms. These data show no shift in global methylation at the base of salamanders, despite a dramatic increase in TE load and genome size. This result is reconcilable with previous studies by considering endothermy and ectothermy, which may be more important drivers of methylation in vertebrates than genome size. The next chapter will look at how an increase in cell size affects organellar structure and abundance. Depending on their shape, organelles can scale in larger cells by increasing volume, length, or number. Scaling may also reflect demands placed on organelles by increased cell size. The 8,653 species of amphibians exhibit diverse cell sizes, providing a powerful system to investigate organellar scaling. Using transmission electron microscopy and stereology, we analyzed three frog and salamander species whose enterocyte cell volumes range from 228 to 10,593 μm3. We show that the nucleus increases in radius (i.e. spherical volume) while the mitochondria increase in total network length; the endoplasmic reticulum and Golgi apparatus, with their complex shapes, are intermediate. Notably, all four organelles increase in volume proportionate to cell volume. This pattern suggests that protein concentrations are the same across amphibian species that differ 50-fold in cell size, and that organellar building blocks are incorporated into more or larger organelles following the same "rules" across cell sizes, despite variation in metabolic and transport demands. This conclusion contradicts results from experimental cell size increases, which produce severe proteome dilution. We hypothesize that salamanders have evolved the biosynthetic capacity to maintain a functional proteome despite a huge cell volume. Finally, the last chapter will be discussing differences in intronic splicing, an important step that pre-mRNA transcripts undergo during processing in the nucleus to become mature mRNAs. Although long thought to occur exclusively in a single step, some introns are now also known to be removed in multiple steps through a process called recursive splicing. This non-canonical form of splicing is hypothesized to aid with intron splicing fidelity, particularly in longer introns. Using West African lungfish (Protopterus annectens; genome size ~40Gb) as a model, we use total RNA-seq data to test the hypothesis that gigantic genomes, which have relatively long introns, have increased levels of recursive splicing compared to genomes of more typical size. Our results reveal levels of recursive splicing at conserved sites similar to those seen in humans, suggesting that genome-wide intronic expansion accompanying evolutionary increase in genome size is not associated with the evolution of high levels of recursive splicing. However, in addition to these results, we also observed patterns of decreasing RNA-seq read depths across entire intron lengths and note that both canonical co-transcriptional splicing and stochastic recursive splicing using many random splice sites could produce this pattern. Thus, we infer canonical co-transcriptional splicing and/or stochastic recursive splicing — but not widespread recursive splicing at conserved sites — manage the removal of long introns.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.genome sizeorganellar sizesalamandersmethylationcell sizerecursive splicingText