Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-1-carboxylate synthase in tomato.
1-Aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14), the key enzyme in ethylene biosynthesis, was purified 5000-fold from induced tomato pericarp. ACC synthase activity was unambiguously correlated with a 45-kDa protein by two independent methods. Peptide sequences were obtained both from the N terminus after electroblotting and from tryptic peptides separated by reversed-phase chromatography. Mixed oligonucleotide probes were used to screen a lambda gt11 library prepared from RNA of induced pericarp tissue. Putative ACC synthase clones were isolated with a frequency of 0.01%. One of these contained a 1.9-kilobase insert with a single open reading frame encoding a polypeptide of 55 kDa. A second, partial cDNA clone was found that differed from the first one in 18% of its bases. Genomic Southern blotting suggests possible tandem organization of the two genes in tomato. The entire coding region was expressed in Escherichia coli and the denatured recombinant polypeptide was used to raise polyclonal antibodies. The antibody preparation both immunoinhibits and immunoprecipitates ACC synthase activity from an enriched tomato extract, confirming the identity of the clone. Northern blot analysis demonstrates that the ACC synthase messenger accumulation is coordinated with fruit ripening.
Laboratorium voor Genetica, Rijksuniversiteit Ghent, Belgium.
D
D
Van der Straeten
L
L
Van Wiemeersch
H M
HM
Goodman
M
M
Van Montagu
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008190:Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010449:Peptide Mapping; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014357:Trypsin
D007527:Isoenzymes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004247:DNA; D014357:Trypsin; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase