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UBC Theses and Dissertations

Characterization of IAPLTR1 subclasses and bidirectional promoter activity : "making sense of it all" Little, Natasha W.


Endogenous retroviruses (ERVs) are over five-times more prevalent than gene coding sequences in the mouse and human genomes (5). The long terminal repeats (LTRs) of these elements are promoter-enhancers that can have many regulatory effects on the host genome (3). Intracisternal A-Particles (IAPs) are a highly active, murine-specific class of ERV that is known to have strong LTR-driven promoter activity (10). In a recent study, a sequence divergence-based subclass nomenclature was suggested for several classes of IAPLTR – including IAPLTR1 (59). However, it remained unknown whether these high (H1) and low (L1) divergence subclasses provided any more biologically relevant information than the current class system (with no subclasses). Some, but not all, IAPLTRs can initiate sense and antisense transcripts and have thus been considered bidirectional promoters; however, due to growing interest in their form and function, a set of criteria has recently been established for what defines a bidirectional promoter and bidirectional reporter constructs have been developed. The research presented in this thesis provides a detailed analysis of bidirectional and unidirectional reporter constructs and provides evidence that bidirectional reporter constructs should be used when assaying bidirectional promoters. Using a bidirectional reporter construct, IAPLTR1 was determined to meet the criteria for a bidirectional promoter. The core promoters for IAPLTR1 and putative transcription factor binding sites that were unique to each subclass were identified. Point mutagenesis experiments revealed that functional divergence accompanied the sequence divergence of H1 and L1 subclasses. In fact, 95% of the total promoter activity of an L1 LTR was removed by changing three base-pairs to resemble the same region of an H1 LTR. The reciprocal experiment resulted in the H1 LTR losing 100% of antisense promoter activity, but maintaining 100% of sense promoter activity. These experiments, among others, provide evidence that the subclass system provides more biologically relevant information than the single IAPLTR1 class, and therefore should be adopted as part of IAP nomenclature.

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