Appukuttan, Binoy; (1997) Molecular genetics of mammalian blue cone pigment genes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Since the isolation of the human cone opsin genes 10 years ago, the advance of molecular biology techniques has played a significant role in increasing our knowledge on the genetics of visual pigment genes from a wide variety of vertebrate and invertebrate species. However, only two non-primate mammalian blue cone pigment (BCP) genes have been characterised to date, and the amino acid residues which are responsible for maintaining the short wavelength sensitivity of this particular class of pigment (the S group) have yet to be determined. Which cis-acting elements confer photoreceptor or cone-specific gene expression. The identification of cone and rod-specific cis-acting elements will not only increase our understanding of photoreceptor-specific gene expression but also contribute to gene therapy applications which target inherited retinal disorders. The BCP gene from the domestic pig (Sus scrofa) was sequenced and characterised, and analysis of the deduced amino acid sequence showed that this opsin is a new member of the S group of visual pigments. Comparison of the S group of opsins with other vertebrate visual pigments resulted in the identification of amino acids which are specific to this family of opsins. Amino acids at 16 positions were identified as putative spectral tuning sites which may contribute to maintaining the short λmax observed in the S group of opsins. 1194 bp, 949 bp, 573 bp and 570 bp of the 5' flanking regions from the BCP genes of human, porcine, talapoin monkey and capuchin monkey were sequenced, respectively. A comparison of 5' flanking BCP gene sequences, between species, revealed several evolutionary conserved motifs, one of which is the photoreceptor conserved element (PCE1). The cells of a retinoblastoma cell line, Weri-Rb1, were shown to express cone- specific transcripts by RT-PCR. It was determined that nuclear proteins isolated from the Weri-Rb1 cells could be used for electrophoretic band shift assay (EBSA) experiments on fragments of the 5' flanking region of the human BCP gene. Competitive EBSA, using short double-stranded oligonucleotides designed to sequences within the human BCP gene 5' flanking region, showed that the specific regions within the BCP proximal promoter to which proteins were binding could be identified. The yeast, Pichia pastoris was tested as an alternative to mammalian cells for the in vitro expression of opsins. The human BCP gene was found to be associated with two Alu repeats, one is situated within intron 3, and the other is between 1194 and 916 bp upstream, of the gene.

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