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

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Abstract

A 1420 bp genomic fragment (λ-horl-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′ noncoding region there are two putative polyadenylation signals located 88 and 142 bp downstream of the stop codon.

The structure of λ-hor1-17 is compared with that of another gene (λ-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 β-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 λ-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.

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Abbreviations

b:

base

CaMV:

cauliflower mosaic virus

GUS:

β-glucuronidase

HMW:

high molecular weight

kb:

kilobase pair

LMW:

low molecular weight

Mabs:

monoclonal antibodies

NOS:

nopaline synthase

nt:

nucleotide

ORF:

open reading frame

PCR:

polymerase chain reaction

TMV:

tobacco mosaic virus

Ψ:

pseudouridine

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Authors and Affiliations

  1. Department of Physiology, Carlsberg Laboratory, Gl. Carlsbergvej 10, DK-2500, Valby, Copenhagen, Denmark

    Entwistle Joycelyn, Søren Knudsen, Martin Müller & Verena Cameron-Mills

  2. Carlsberg Research Laboratory, Gl. Carlsbergvej 10, DK-2500, Valby, Copenhagen, Denmark

    Entwistle Joycelyn, Søren Knudsen, Martin Müller & Verena Cameron-Mills

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  1. Entwistle Joycelyn
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Entwistle, J., Knudsen, S., Müller, M. et al. Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley. Plant Mol Biol 17, 1217–1231 (1991). https://doi.org/10.1007/BF00028737

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