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.
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 [alpha-32P]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 [35S]GTP gamma S to the recombinant DCARF1 protein was detected only in the presence of millimolar levels of both MgCl2 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).
Kiyosue
T
T
Laboratory of Plant Molecular Biology, Institute of Physical and Chemical Research RIKEN, Ibaraki, Japan.
Shinozaki
K
K
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018552:Daucus carota / Q000196:embryology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution
D009711:Nucleotides; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors