Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley

Abstract

A 1420 bp genomic fragment (lambda-hor1-17) encompassing a Hor-1 gene encoding a C-hordein polypeptide is presented. The deduced amino acid sequence is 261 residues long. It comprises a 20 amino acid signal peptide, unique NH2- and COOH-terminal regions and a coding region comprised of pentapeptide (PQQPY) and octapeptide (PQQPFPQQ) repeat motifs. The 431 bp of 5' non-coding region contains a 'TATA box' at -105, a 'CACA box' (-181 to -201) and a -300 prolamin element. In the 3' non-coding region there are two putative polyadenylation signals located 88 and 142 bp downstream of the stop codon. The structure of lambda-hor1-17 is compared with that of another gene (lambda-hor1-14) encoding a C-hordein polypeptide, which contains an amber codon interrupting the ORF. A functional assay in which the 5' non-coding regions of the two genes were fused to the beta-glucuronidase (GUS) gene demonstrated that both genes were transcriptionally active and that circa 430 bp of the C-hordein promoters were sufficient to drive the expression of the GUS gene in developing barley endosperms. It also demonstrated that both promoters had transcriptional efficiencies comparable with that of the 35S CaMV promoter. The in vitro translation of the coding region of lambda-hor1-14 in the wheat germ system showed that the premature stop codon could be partially suppressed. The suppression was also demonstrated in a transient expression assay in vivo using isolated barley endosperms.

Barley BLZ2, a Seed-specific bZIP Protein That Interacts with BLZ1 in Vivo and Activates Transcription from the GCN4-like motif of B-hordein Promoters in Barley Endosperm

“…The EB of the promoter of a Hor-2 gene is made of two cis-motifs, PB and GLM, that are well conserved in the promoters of prolamin genes of the Pooideae grasses, such are those encoding B-and C-hordeins in barley (22,36,37), ␣/␤ gliadins, and LMW-glutenins in wheat (38,39) and -secalins in rye (40). Our results show that BLZ2 binds in vitro in a sequence-specific manner to the GLM within the EB of a Hor-2 gene promoter and that mutations altering the sequence of the GLM disrupt the interaction.…”

Plant viruses as model systems for the study of non-canonical translation mechanisms in higher plants

“…Evidence for readthrough was obtained both in vitro by translation in the wheatgerm system and in vivo by transient expression of reporter gene fusions in barley endosperm tissue after particle gun bombardment. Although the authors demonstrated that the promoter of this particular gene is even more active than that of a control wild-type Chordein gene, the absence of both full-length or truncated proteins after in vitro translation of barley endosperm mRNA showed that they represent a minor component of the storage proteins in barley endosperm (Entwistle et al, 1991). The sequencing of genes belonging to a maize c~-zein subfamily 4 (SF4) gene cluster provided evidence that four of the genes designated type 2 (T2) contained one or two early in-frame stop codons (Liu & Rubenstein, 1993).…”

“…Stop codon suppression has also been implicated in the expression of plant genes such as the synthesis of storage proteins in barley (C-hordeins; Entwistle et al, 1991) and maize (c~-zeins; Liu & Rubenstein, 1993). The C-hordein ORF in the genomic clone 2-horl-14 is interrupted by an amber stop codon within the coding sequence for the octapeptide repeats.…”

From Analysis of Mutants to Genetic Engineering

“…Cloning and sequencing of structural genes for B-, C-, and γ-hordein polypeptides have corroborated that the Hor 1, Hor 2, and Hor F loci each consist of a family of closely linked structural genes for these proteins, with each gene having its own promoter (7,10,18,24,62,63,70). Some of these genes contain stop codons, but transient transformation experiments revealed that expression of such a gene in the endosperm can take place by amber codon suppression (19).…”