Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors.
Poly(A)-rich RNA was isolated from developing soybean seeds (Glycine max (L.) Merr.) and fractionated on linear log sucrose gradients. Two major fractions sedimenting at 18 S and 20 S were separated and then purified by further sucrose gradient fractionation. Both fractions were active as messengers when added to a rabbit reticulocyte lysate protein synthesis system. The 18 S fraction caused proteins migrating primarily to the 60,000-dalton region of a sodium dodecyl sulfate gel to be produced, while translation of the 20 S fraction preferentially directed the synthesis of polypeptides similar in size to the alpha and alpha' subunits of beta-conglycinin. Evidence that many of the 60,000-dalton polypeptides were related to glycinin and the high molecular weight 20 S translation products were related to beta-conglycinin was obtained by immunoprecipitation using monospecific antibodies against glycinin and beta-conglycinin, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated products revealed that the glycinin precursor region contained at least three different size components and that the family of glycinin precursors had larger apparent molecular weight (58,000-63,000) than the disulfide-linked complexes between acidic and basic glycinin subunits (57,000). Unlike the disulfide-linked glycinin complexes which were cleaved by disulfide reduction, glycinin precursors were insensitive to reducing agents. The alpha and alpha' subunits synthesized in vitro also had slightly larger apparent molecular weights than purified alpha and alpha' standards.
D000818:Animals; D052179:Antigens, Plant; D005916:Globulins / Q000235:genetics*; D008970:Molecular Weight; D014674:Plant Proteins, Dietary / Q000235:genetics*; D011061:Poly A / Q000235:genetics; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D055314:Seed Storage Proteins; D012639:Seeds / Q000378:metabolism*; D030262:Soybean Proteins; D013025:Glycine max
D052179:Antigens, Plant; D005916:Globulins; D014674:Plant Proteins, Dietary; D011498:Protein Precursors; D012333:RNA, Messenger; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max; D011061:Poly A; D012313:RNA; C011474:glycinin