Cloning of a Carrot cDNA for a Member of the Family of ADP-ribosylation Factors(ARFs) and Characterization of the Binding of Nucleotides by its Product after Expression in E. coli : GROWTH AND DEVELOPMENT : GENE STRUCTURE AND EXPRESSION

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A cDNA clone, designated DcArf1, was isolated from a cDNA library prepared from embryogenic cell clusters of Daucus carota and characterized. The cDNA (883 bp) contained an open reading frame that encoded a protein of 181 amino acid residues with significant homology to the ADP-ribosylation factors (ARFs) of animals and yeast. The DcArf1-related transcripts were detected at a nearly constant level during somatic embryogenesis. The DcArf1-encoded protein was expressed as a fusion protein in E. coli and was shown to have dithiothreitol-independent [a-^<32>P]GTP-binding activity. This binding was completely prevented by a 100-fold molar excess of unlabeled GTP or GDP, while GMP, ANP, TNP, CNP and UNP had no effect on the binding. The binding of [^<35>S]GTPγS to the recombinant DCARF1 protein was detected only in the presence of millimolar levels of both MgCl_2 and dimyristoylphosphatidylcholine (DMPC). The amino acid sequence and the GTP-binding characteristics of the DCARF1 protein suggest that the DcArf1 cDNA encodes an ARF of carrot rather than a protein that only resembles such a factor, ADP-ribosylation factor-like protein (ARL).
日本植物生理学会の論文