No description available.

Miniature Inverted-repeat Transposable Elements (MITEs) are a type of transposable element (TE) that lacks coding capacity. It has been established that in rice that certain Stowaway MITEs are mobilized by transposases from the Tc1/Mariner superfamily of TEs. To retrieve all Tc1/Mariner TEs from the genome, bioinformatic approaches were performed. A total of 295 Tc1/Mariner TEs that encoded a full or partial transposase were recorded which 100 were newly described. Sequence alignment, and identification of the catalytic motif placed these transposases into eight groups. A functional link was established by comparing the terminal sequences of the Stowaway-like MITEs to the termini of the terminal sequences of Tc1/Mariner TEs. A yeast excision assay was used to experimentally test these functional links. Majority of the Stowaway-like MITE and transposase combinations tested did not indicate a functional link. However, a possible functional link was observed between the AATp3-13 transposase and AAStow-5 Stowaway-like MITEs.

Miniature Inverted-repeat Transposable Elements (MITEs) are a type of transposable element (TE) that lacks coding capacity. It has been established that in rice that certain Stowaway MITEs are mobilized by transposases from the Tc1/Mariner superfamily of TEs. To retrieve all Tc1/Mariner TEs from the genome, bioinformatic approaches were performed. A total of 295 Tc1/Mariner TEs that encoded a full or partial transposase were recorded which 100 were newly described. Sequence alignment, and identification of the catalytic motif placed these transposases into eight groups. A functional link was established by comparing the terminal sequences of the Stowaway-like MITEs to the termini of the terminal sequences of Tc1/Mariner TEs. A yeast excision assay was used to experimentally test these functional links. Majority of the Stowaway-like MITE and transposase combinations tested did not indicate a functional link. However, a possible functional link was observed between the AATp3-13 transposase and AAStow-5 Stowaway-like MITEs.

Indiana University-Purdue University Indianapolis (IUPUI) / SETMAR is a chimeric protein that originates from the fusion of a SET domain to the mariner Hsmar1 transposase. This fusion event occurred approximately 50 million years ago, after the split of an anthropoid primate ancestor from the prosimians. Thus, SETMAR is only expressed in anthropoid primates, such as humans, apes, and New World monkeys. Evolutionary sequence analyses have revealed that the DNA-binding domain, one of the two functional domains in the Hsmar1 transposase, has been subjected to a strong purifying selection. Consistent with these analyses, SETMAR retains robust binding specificity to its ancestral terminal inverted repeat (TIR) DNA. In the human genome, this TIR sequence is dispersed in over 1500 perfect or nearly perfect sites. Given that many DNA-binding domains of transcriptional regulators are derived from transposases, we hypothesized that SETMAR may play a role in gene regulation. In this thesis, we determined the crystal structures of the DNA-binding domain bound to both its ancestral TIR DNA and a variant TIR DNA sequence at 2.37 and 3.07 Å, respectively. Overall, the DNA-binding domain contains two helix-turn-helix (HTH) motifs linked by two AT-hook motifs and dimerizes through its HTH1 motif. In both complexes, minor groove interactions with the AT-hook motifs are similar, and major groove interactions with HTH1 involve a single residue. However, four residues from HTH2 participate in nucleobase-specific interactions with the TIR and only two with the variant DNA sequence. Despite these differences in nucleobase-specific interactions, the DNA-binding affinities of SETMAR to TIR or variant TIR differ by less than two-fold. From cell-based studies, we found that SETMAR represses firefly luciferase gene expression while the DNA-binding deficient mutant does not. A chromatin immunoprecipitation assay further confirms that SETMAR binds the TIR sequence in cells. Collectively, our studies suggest that SETMAR functions in gene regulation.

Dispatched 3 (Disp3), a thyroid hormone-regulated gene, is studied extensively in our laboratory. Phenotype of cells with overexpressed Disp3 and its expression pattern make it a perfect candidate for a molecular link between thyroid hormone action and cholesterol homeostasis in the brain. Moreover, we hypothesize that it might play a role in certain neurodegenerative disorders and brain tumours. This thesis is aimed at the process of regulation of this gene via thyroid hormone receptor (TR), specifically identification of responsive elements of the thyroid hormone receptor that are necessary for the regulation. Also, we searched for elements recognized by liver X receptor (LXR), as LXR binds to the same arrangement of repeats as TR and there are a number of genes regulated by both of them. We combined in silico analysis of the Disp3 locus with reporter luciferase assays. A cluster of six elements identified around the first exon with two of them being conserved among human and mice draw our attention. In order to analyze this sequence in more detail, reporter vectors of various truncations of 3 kb region around exon 1 were constructed and tested in reporter assays. Reporter assays did not reveal any substantial element activated by TR or LXR; on the other hand, region containing repressor element(s)...