Cell‐Free Translation of Messenger RNAs from Adult and Fetal Human Muscle

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Abstract
Using a procedure of ethanol precipitation in concentrated guanidine .cntdot. HCl solutions followed by chloroform/isoamylic alcohol extraction and washing in 3 M sodium acetate, high MW cellular RNA from human fetal and adult skeletal muscle was isolated. About 500 .mu.g RNA were obtained per g of fetal muscle and 50 .mu.g RNA/g of adult muscle. Both RNA preparations were efficiently translated in a cell-free reticulocyte lysate system and directed synthesis of various polypeptides, one of them of MW 200,000 probably corresponding to myosin heavy chains. Dodecyl sulfate/polyacrylamide gel electrophoretic pattern of polypeptides neosynthesized using either fetal or adult RNA exhibited several differences. Three neosynthesized cytosolic muscle enzymes were purified from the translation mixtures using a micromethod of immunoaffinity chromatography; specificity of the neosynthesized polypeptides purified according to this procedure was checked by immunological competition with the corresponding unlabeled pure muscle enzymes. Successful cell-free translation of RNA from adult skeletal muscle and purification of neosynthesized human enzymes are reported for the first time. These methods, indispensable for further studies on human adult muscle gene expression, could also shed light on the mechanism of some inherited molecular diseases and tumoral or dystrophic processes.