The first objective of the proposed research is to define the function of a novel retinal guanyl nucleotide binding protein recently identified in bovine retinas. A cDNA clone encoding this protein was selected from a retinal cDNA library using probes that were designed to detect the alpha subunit of transducin, the photoreceptor G protein. This cDNA clone, referred to as T-alpha-II, encodes a protein that is 80% homologous to the transducin alpha subunit. T-alpha-II mRNA is expressed only in the retina, but the location and function of T-alpha-II product are unknown. Retinal mRNA corresponding to this clone is to be localized by in situ hybridization to bovine retina sections. Peptides are to be synthesized that correspond to amino acid sequences specifically found in the novel alpha subunit. Antibodies are to be raised against these peptides and used to localize the novel alpha subunit by immunocytochemical analysis of retina sections. These antibodies are to be used to purify the novel alpha subunit and the properties of the purified protein are to be compared with properties of the transducin alpha subunit. The second objective of the proposed research is to define structure-function relationships within the alpha subunit. A mammalian cell expression system is to be used to make proteins that have been altered by oligonucleotide directed site-specific mutagenesis. The normal and altered proteins are to be purified from cell extracts by affinity to specific antibodies or by conventional methods and then analysed. Sequences that are thought to be involved with GTP binding and hydrolysis as well as with protein-protein interactions are to be examined by this method.
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