Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5' upstream regions of genes coding for sporamin and beta-amylase from sweet potato.
We isolated a cDNA encoding a DNA-binding protein, SPF1, of sweet potato that binds to the SP8a (ACTGTGTA) and SP8b (TACTATT) sequences present in the 5' upstream regions of three different genes coding for sporamin and beta-amylase of tuberous roots. SPF1 comprises 549 amino acids and is enriched in both basic and acidic residues. The amino acid sequence of SPF1 shows no significant homology to any known protein sequences, suggesting that it may represent a new class of DNA-binding protein. Binding studies with 35S-labeled SPF1, synthesized in vitro, and synthetic DNA fragments indicated that, although SPF1 binds to both the SP8a and SP8b sequences, it binds much more strongly to SP8a than to SP8b. SPF1 bound to the SP8a sequence as a monomer. The DNA-binding domain of SPF1 was localized within the C-terminal half of this protein, and a 162-amino acid fragment of SPF1 (Met310-Arg472) showed DNA-binding activity with no change in target sequence specificity. This fragment contains a region enriched in basic amino acids adjacent to a highly acidic stretch. A sequence which is highly homologous to a 40-amino acid sequence in the basic region of the DNA-binding domain is duplicated in the N-terminal part of SPF1. The gene coding for SPF1 is present in one or a few copies per haploid genome and the SPF1 mRNA was detected in leaves, stems and tuberous roots of the sweet potato, in addition to petioles. The level of SPF1 mRNA in the petioles decreased when leaf-petiole cuttings were treated with sucrose to induce accumulation of sporamin and beta-amylase mRNAs.
Laboratory of Biochemistry, School of Agriculture, Nagoya University, Japan.
S
S
Ishiguro
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K
Nakamura
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D018744:DNA, Plant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D014675:Vegetables / Q000201:enzymology* / Q000235:genetics*; D001614:beta-Amylase / Q000235:genetics*
D018076:DNA, Complementary; D018744:DNA, Plant; D004268:DNA-Binding Proteins; D010940:Plant Proteins; C090109:SPF1 protein, plant; C067142:sporamin protein, Ipomoea batatas; D001614:beta-Amylase