Isolation and Translation of Hordein Messenger RNA from Wild Type and Mutant Endosperms in Barley

Nucleotide sequences of four cDNA clones coding for the carboxy-terminal portion of at least two different B1 hordein polypeptides are presented. The open reading frame in the nucleotide sequence of the the largest clone (pc hor2-4, 720 nucleotides) translates into the 181 carboxy-terminal amino acids of a polypeptide chain showing close homology to the previously determined primary structure of B1 hordein peptides. Of the 74 amino acid residues which can be compared 61 proved to be identical. The second cDNA clone (pc hor2-3, 257 nucleotides) encodes the 54 carboxy-terminal amino acids of a different B1 hordein polypeptide, which is revealed by 21 nucleotide substitutions resulting in 9 amino acid changes. Messenger RNA has been isolated from developing barley endosperms by sucrose gradient sedimentation, Sepharose 4B gel filtration and preparative gel electrophoresis. Hordein messenger RNA was found to be a major constituent of the total messenger RNA population of the endosperm cell. Polyadenylated hordein messenger RNA sedimented at 11S in sucrose gradients and electrophoretic analysis reveals the presence of at least three RNA species with apparent molecular weights of 0.45, 0.36 and 0.30 megadaltons. The 11S messenger RNA was translated in vitro into hordein precursor polypeptides which are 2–4 kilodaltons larger than the native hordein polypeptides. The endosperm cell of mutant No. 1508 contained twice as much RNA as the wild type endosperm cell but the same amount of polyadenylated 11S RNA. The template activity of the latter was 10% of that found for the 11S hordein messenger RNA from the wild type and was limited to translation into one hordein precursor polypeptide.