STUDIES ON THE SYNTHESIS OF 12S STORAGE GLOBULIN IN OAT SEEDS
Abstract
The experiments reported in this thesis were undertaken to describe the molecular features of oat seed globulin synthesis and in so doing account for the high levels of globulin present in oat seeds. Since oat seed 12S globulin had been only partially described, the initial experiments were to further characterize the protein. Globulin was isolated from mature seeds, purified to a single 12S protein, and the polypeptides present were analyzed by two-dimensional gel electrophoresis. The two types of polypeptides were resolved by ion-exchange chromatography. Subunits containing the two disulfide-linked polypeptides were identified. The synthesis of oat 12S globulin as precursors was determined by in vitro translation of poly(A) RNA isolated from developing oat seeds. Translation products were immunoprecipitated with antisera raised against oat globulin and visualized by gel electrophoresis and fluorography. In vivo labeling of newly synthesized polypeptides by incubating oat groats in radioactive solution showed the major polypeptides synthesized corresponded in size to oat globulin precursors and were immunoprecipitable with anti-globulin sera. Oat seed poly(A) RNA was sized on sucrose gradients and fractions active in synthesizing globulin precursors (('(TURN))18S) were identified. These RNA fractions were used to synthesize double-stranded cDNAs which were cloned into bacterial plasmids. Globulin clones were identified with synthetic oligonucleotides whose sequences were derived from oat globulin amino acid sequences. The cDNA clones were characterized by restriction enzyme mapping and hybridization analysis. The nucleotide sequence of the longest clone was determined. The number of genes encoding oat globulin cDNA polypeptides was estimated by hybridization of an oat globulin cDNA clone with oat genomic DNA digested with Eco R1, separated by gel electrophoresis, and transferred to nitrocellulose. A family of eight to twelve genes was found to encode the 12S oat globulins.
Degree
Ph.D.
Subject Area
Molecular biology
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