RESUMO
We report here the isolation and nucleotide sequence of genomic clones encoding the chloroplast enzyme sedoheptulose-1,7-bisphosphatase (SBPase) from Arabidopsis thaliana. The coding region of this gene contains eight exons (72-76 bp) and seven introns (75-91 bp) and encodes a polypeptide of 393 amino acids. Unusually, the 5' non-coding region contains two additional AUG codons upstream of the translation initiation codon. A comparison of the deduced Arabidopsis and wheat SBPase polypeptide sequences reveals 78.6%, identity. Expression studies showed that the level of SBPase mRNA in Arabidopsis and wheat is regulated in a light-dependent manner and is also influenced by the developmental stage of the leaf. Although the Arabidopsis SBPase gene is present in a single copy, two hybridizing transcripts were detected in some tissues, suggesting the presence of alternate transcription start sites in the upstream region.
Assuntos
Arabidopsis/genética , Cloroplastos/enzimologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Monoéster Fosfórico Hidrolases/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/efeitos da radiação , Sequência de Bases , Dosagem de Genes , Biblioteca Genômica , Luz , Dados de Sequência Molecular , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fosfatos Açúcares/metabolismo , Distribuição Tecidual , Triticum/enzimologia , Triticum/genética , Triticum/efeitos da radiaçãoRESUMO
The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose-1,7-bisphosphatase [Sed(1,7)P2ase], was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1,7)P2ase gene has eight exons (72-507 bp) and seven introns (85-626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1,7)P2ase with those of fructose-1,6-bisphosphatase [Fru(1,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(1,6)P2ase are highly conserved in the Sed(1,7)P2ase molecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1,7)P2ase and chloroplast Fru(1,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1,6)P2ase is not found in the Sed(1,7)P2ase enzyme.
Assuntos
Cloroplastos/enzimologia , DNA/genética , Frutose-Bifosfatase/genética , Monoéster Fosfórico Hidrolases/genética , Triticum/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Northern Blotting , Éxons , Frutose-Bifosfatase/metabolismo , Íntrons , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Poli A/genética , RNA/genética , RNA Mensageiro , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
We have characterised a DNA-binding interaction common to the upstream sequences of the wheat fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) genes. The recognition site for this sequence-specific binding activity, designated wheat FBPase factor (WF-1), is located within 125 bp of the transcription start site of each gene. Within these regions there are no sequence motifs similar to those shown to be important for light-regulated expression in other species. The binding activity was not detected in wheat root nuclear extracts, or in pea leaf extracts. There was a higher level of binding activity in light-grown than in dark-grown wheat leaves. The level was also found to decline when light-grown plants were given an extended dark treatment, but could be reinduced by light. Utilising the gradient of developmental maturity which exists within the wheat leaf it was found that WF-1 activity increases during leaf development.