The translation of partially purified retinal mRNA will be studied in the Xenopus laevis oocyte using electrophysiological intracellular means to reveal 1 to 10 days after mRNA injection functional translation products in the plasma membrane of the oocyte. The study concerns the transfer of photosensitivity to another cell as well as the transfer of the sensitivity to retinal synaptic transmitters. The major source of the mRNA will be the retina of young rats for measuring light-sensitivity transfer while the transfer of retinal synaptic transmitter sensitivity will use the mRNA of young chicken retinas because of their richness in cells of the inner layers. Solubilized retinal RNA will be fractionated by affinity chromatography with oligo-d (thymidine) cellulose to obtain poly-(A) mRNA. The isolated oocytes will be microinjected with 30 nonoliters of 1 mg/ml solution of RNA in water. The crucial question is whether or not a functional receptor complex is assembled in the plasma membrane of the oocyte after the injection of retinal mRNA. This has been shown to occur for the retinal synaptic receptors specifically sensitive to gamma amino butyric acid, kainic acid, glutamate and acetylcholine (nicotinic action). The physiological and pharmacological properties of these and other receptors will be analyzed including their dependence on ions of the external and internal medium (intracellular perfusion) and their modulation by neuropeptides and other compounds. Single ionic channel kinetics will be studied by patch clamping. Using the electrophysiological responses as an assay, attempts will be made to purify the receptor mRNAs by gradient fractionation. Simultaneously, the translation of a functional visual transduction complex will be studied after possible modes of 11-cis retinal application have been evaluated. Rhodopsin translation will be assessed in cell-free lysates and in the oocyte using immunoprecipitation. The localization of the translated opsin within the oocyte will be studied by cell fractionation using membrane extraction procedures as developed for the rhodopsin extraction from isolated outer segments.
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