Transposable elements account for the almost half of the sequence encoded by mammalian genomes, which become silenced during early embryonic development. This thesis sought to explore the hypothesis of the involvement of the RNAi pathway in the silencing of transposable elements in mammals, predominantly through the identification of transposon-associated RNA of -20-25nt using a gel blotting technique. Initially cell lines of embryonic and tumour origin were analysed. This lead to the identification of several previously unreported transposon-associated RNA ranging from 70-90nt. However, it was not until a more detailed analysis of the embryonic cell lines, with the induction of differentiation in cell culture that several discrete RNA of -20nt were detected for the mouse transposons L1 and B2. The differentiation of embryonic cell lines in culture also serendipitously lead to the detection of several short 55 rRNA of -22-26nt, these were also later detected in several human cell lines of breast cancer origin and healthy breast tissue. Intriguingly the -20-26nt repeat-associated identified were predominantly observed after two-days of differentiation in cell culture in several cell lines and often coincided with an ethidium bromide stainable band of -19nt. The latter may indicate a large proportion of these RNAs. Further analysis of the B2 and 55 rRNA repeat-associated short RNA revealed both to have reduce accumulation in Dicer-null embryonic stem cells, implicating a possible association with a known component of the RNAi pathway. Dicer was also observed to process the longer -50-80nt 55rRNA to -20-26nt in vitro. BLAST was also used to identify possible mRNA targets for the short B2 and 55 rRNA. One of these, the mRNA encoding sialic acid acetylesterase (SIAE) was consistently observed to be reduced with the accumulation of the short RNA using end-point RT-PCR, consistent with targeting through the RNAi or a similar pathway. However, no further links with the RNAi pathway were established, with no targeting detected for the short B2 or 55 rRNA using dual luciferase sensor assays. The repeat-associated RNA identified in this thesis are among the first of their type and further work will be required to establish what relevance they have to the RNAi pathway and transposon regulation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:559864 |
| Date | January 2007 |
| Creators | Docherty, Louise E. |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/16/ |
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