PURIFICATION OF MESSENGER RNAS ENCODING FOR NEUROTROPIC FACTORS IN RAT BRAIN

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It has been shown that brain injury induces the appearance of neurotrophic activity at the lesion site. The neurotrophic activity can be assayed in vitro on sympathetic and parasympathic neurons in culture. Because of the potential importance of this and other neurotrophic and neurite promoting factors for much of the work at the Neuropsychiatry Branch, we decided to determine if we could use techniques from molecular biology to get a better understanding of their function. The first steps at the project have consisted in the development of the assays for testing for neurotrophic activity and their production. Different techniques were used for identification of lesioned brain-specific mRNAs. Messenger RNAs were prepared from the walls of the wound cavity of lesioned rat brains and from equivalent areas of controls. They were fractionnated by sucrose density gradient centrifugation. Each fraction was translated into proteins in Xenopus cocytes and the products tested for neurotrophic activity in the neuron cultures. The fraction of mRNA from lesioned brains responsible for the neurotrophic activity was used as a template for cDNA synthesis. The cDNA was hybridized with mRNA from controls to subtract the sequences common to the two preparations.