Analysis of Tc1/mariner Transposable Element Protein Localization in Saccharomyces Cerevisiae and Genome-wide de novo Classification of Miniature Inverted-Repeat Transposable Elements in Brassica rap

Transposable elements (TEs) are segments of mobile DNA that have accumulated in eukaryotic genomes at significant levels, while constantly evolving with host systems. Autonomous transposable elements are of particular interest, as they have been implicated in restructuring genomes, and epigenetically regulating specific genes, and taking on novel functions. Osmar14 (Oryza sativa Mariner-like element) is a transposable element found in rice. Its transposase (Tpase) Osmar14A can mobilize its non-autonomous cognate Osmar14NAS, as well as cross mobilize a Stowaway MITE, Ost-35 at a high frequency. The observed transposition frequency of Ost-35 by Osmar14A is an area of interest, specifically the structural components of Osmar14A that allow for this increased transposition. Nuclear localization signals (NLS) and nuclear export signals (NES) are hypothesized as playing a role in Tpase activity by facilitating protein cellular localization. It was determined in a yeast transposition assay that Osmar14A-NLS mutants were unable to mobilize Ost-35, with a transposition frequency of zero. Furthermore, Osmar14A-NES mutants exhibited decreased transposition frequency, implicating a potential inhibitory effect of increased Tpase protein within the nucleus. A protein localization system using pAG415GAL-cerulean-ccdB based vectors were designed and constructed, which could potentially be employed to determine whether there is an accumulation of Osmar14A-NES protein within the nucleus when compared to Osmar14A wild-type protein.;Along with the studies of transposases produced by autonomous transposable elements, classification of non-autonomous elements is also essential as they provide evidence of previous transposase element activity In Brassica rapa, MITEs comprise a significant portion of the genome, and accordingly have led to diversification and evolution of the genome. Using MITE digger, and MITE analysis Kit, 85 novel B. rapa MITE families were uncovered in the B. rapa genome. These MITEs were further grouped into Tc1/mariner, PIF/Harbinger, CACTA, hAT, MULE and PiggyBac super-families according to sequence length, terminal inverted repeats and target site duplications. Of the B. rapa Stowaway MITEs, 836 share sequence similarity with Brassica Olocerea Stowaway mite BraSto. CACTA, hAT, and PiggyBac MITEs have been shown to have most recently diverged. Furthermore, it has been shown that Stowaway MITEs inserted at the upstream region of a B. rapa MADS DNA domain binding transcription factor, perhaps playing a regulatory function. Gene association was also noted with Tourist, hAT and CACTA-like elements, commonly with regulatory regions of genes. The recent divergence of MITEs suggests that they may be recently active within the Brassica rapa genome. Classification of MITEs in B. rapa can aid in the development of genetic engineering tools.