Nucleotide sequence and endosperm-specific expression of the structural gene for the toxin alpha-hordothionin in barley (Hordeum vulgare L.).
A barley genomic library, obtained by cloning in the vector lambda EMBL-4, was screened with a cDNA probe encoding the alpha-hordothionin toxin. A positive clone, designated lambda TH1, was selected for further characterization. The coding and flanking regions of the alpha-hordothionin gene (Hth-1) were sequenced. Hth-1 has two introns of 420 and 91 nucleotides (nt), respectively. The promoter region has the following main features: one TATA box; three CATC boxes; an enhancer-like sequence, starting at nt position -282 from the first ATG codon, which is homologous to sequences appearing at similar positions in other endosperm genes; two versions of an 18-nt sequence that is more highly repeated in structural domains of several prolamin genes; two extensive regions close to the first ATG codon that are homologous to a sequence located much further upstream in the B-hordein promoter. The transcription start point was determined at nt positions -46 to -47, both by the S1 nuclease-protection and by the primer-extension assays. A maximum of 2-4 copies of the Hth-1 gene per haploid genome was determined by Southern-blot hybridization. Expression of the Hth-1 gene was detected during the cell proliferation stage of endosperm development (maximum at 13-16 days after pollinization) and was not detected in either etiolated or green coleoptiles.
Rodríguez-Palenzuela
P
P
Departamento de Bioquimica, Escuela Técnica Superior de Ingenieros Agrónomos, Madrid, Spain.
Pintor-Toro
J A
JA
Carbonero
P
P
García-Olmedo
F
F
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA; D002523:Edible Grain / Q000235:genetics*; D004720:Endonucleases; D005796:Genes; D001467:Hordeum / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic
D023181:Antimicrobial Cationic Peptides; D003062:Codon; D010940:Plant Proteins; C036092:hordothionin protein, Hordeum vulgare; D004247:DNA; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases