Extraction and translation of collagen mRNA from fetal calf skin.

Abstract

RNA was extracted from fetal calf skin by two different procedures, using phenol or guanidine hydrochloride. Poly(A)-rich RNA was separated by oligo(dT)-cellulose affinity chromatography and was further fractionated by sucrose density gradient centrifugation. When translated in an optimized wheat germ extract cell-free system, unfractionated guanidine-hydrochloride-extracted poly(A)-rich RNA directed the synthesis of two collagenase-sensitive protein bands, while phenol-extracted poly(A)-rich RNA with a sedimentation coefficient higher than 25 S was the only fraction to direct the same synthesis. On the basis of their electrophoretic mobility on a sodium dodecylsulfate/urea/polyacrylamide gel, these proteins were identified with procollagen alpha 1(I) and procollagen alpha 2. Inhibition of translation by phenol-extracted poly(A)-rich RNA with a sedimentation coefficient lower than 25 S was also observed. Guanidine-hydrochloride-extracted poly(A)-rich RNA from fetal skin directed the synthesis of three distinct collagenase-sensitive proteins in the micrococcal-nuclease-digested rabbit reticulocyte cell-free system; these seemed to correspond to procollagen alpha 1(I), procollagen alpha 2 and procollagen alpha 1 (III).