Vision is the most precious of our senses, and its appearance in the animal kingdom was almost certainly a major driving force in the radiation of species that occurred around 550 million years ago. Our research addresses both of these issues, firstly by way of understanding the basic cause and potential treatment of inherited retinal diseases, and secondly by investigating the origin and evolution of proteins involved in the visual process. This process is initiated by the capture of photons by the rod and cone visual pigments that are present in the photoreceptors of the retina, and culminates via the phototransduction cascade in a change in membrane potential. In this way, light is converted into an electrical signal. In order to understand the evolution of this process in vertebrates, our studies include ancient groups such as the lampreys, hagfishes, and chimaeras that appeared at the base of the vertebrate radiation, together with more recent divergences among the bony fishes, reptiles, and mammals. Inherited retinal disease is a major cause of blindness. In many cases, the causative mutations are found in these same processes of phototransduction. Therefore, our aim is to identify such mutations and to determine the mechanism of mutant gene action, a necessary precursor to the development of a management strategy or treatment for the disorder.
The key to understanding the structure-function relationship of genes and their role in pathological disease lies at the level of RNA and protein. However, one of the difficulties in achieving this goal is the inability to obtain fresh tissue for RNA or protein work, particularly where tissue biopsy is very invasive (as in the eye or brain) or where the animals are highly endangered. In many cases, only genomic DNA (gDNA) is available to scientific researchers. As an example, we recently obtained the full-length sequences of the visual pigment (opsin) genes in the duck-billed platypus genome. However, to complete the analysis we needed to determine the spectral properties of the pigments. The platypus is an endangered and highly protected species so we were unable to obtain an eye for direct measurement of the pigments, or for the isolation of the spliced opsin transcripts. Nonetheless, by using our SPLICE technique we were able to produce full-length coding sequences with all intronic regions removed, which were subsequently used to express the pigments in vitro. This relatively simple but efficient technique results in the generation of a DNA fragment containing the coding information of any gene from the gDNA of any species for subsequent use by downstream procedures for the assay of gene function.
See “SPLICE: A technique for generating in vitro spliced coding sequences from genomic DNA” on page 785.