Synthesis in vitro of keratin polypeptides directed by mRNA isolated from newborn and adult mouse epidermis.
A method is described which allows the isolation of undegraded total cellular RNA from living epidermis in a one-step procedure. Epidermal tissue is homogenized in 4 M guanidinium thiocyanate/2% sarkosyl buffer and centrifuged through a 5.7 M CsCl cushion. Under appropriate density conditions in the homogenate, large amounts of RNA are sedimented free of DNA and protein to the bottom of the tubes. A prerequisite for the applicability of this method is the preparation of pure epidermis under conditions which efficiently inactivate endogenous RNases. Oligo(dT) affinity chromatography of total RNA yields 3-4% of polyadenylated RNA. Translation of poly(A)-rich fractions from both newborn and adult mouse epidermis in a cell-free reticulocyte system leads to the synthesis of proteins with electrophoretic mobilities identical to those of native keratin polypeptides of the corresponding tissue. Similar to native keratin polypeptides, these proteins are highly insoluble and can selectively be enriched by high-salt extraction of the translation assays. The CNBr cleavage pattern of these purified proteins is identical to that of native keratins. There is no evidence for the synthesis of precursors of these proteins.
D000375:Aging; D000818:Animals; D000831:Animals, Newborn; D002474:Cell-Free System; D007633:Keratins / Q000096:biosynthesis*; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012867:Skin / Q000254:growth & development / Q000378:metabolism*
D012333:RNA, Messenger; D011061:Poly A; D007633:Keratins