A gene coding for tomato fruit beta-galactosidase II is expressed during fruit ripening. Cloning, characterization, and expression pattern.
beta-Galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes characterized by their ability to hydrolyze terminal, nonreducing beta-D-galactosyl residues from beta-D-galactosides. Several beta-galactosidases, sometimes referred to as exo-galactanases, have been purified from plants and shown to possess in vitro activity against extracted cell wall material via the release of galactose from wall polymers containing beta(1-->4)-D-galactan. Although beta-galactosidase II, a protein present in tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan, has been purified, cloning of the corresponding gene has been elusive. We report here the cloning of a cDNA, pTombetagal 4 (accession no. AF020390), corresponding to beta-galactosidase II, and show that its corresponding gene is expressed during fruit ripening. Northern-blot analysis revealed that the beta-galactosidase II gene transcript was detectable at the breaker stage of ripeness, maximum at the turning stage, and present at decreasing levels during the later stages of normal tomato fruit ripening. At the turning stage of ripeness, the transcript was present in all fruit tissues and was highest in the outermost tissues (including the peel). Confirmation that pTombetagal 4 codes for beta-galactosidase II was derived from matching protein and deduced amino acid sequences. Furthermore, analysis of the deduced amino acid sequence of pTombetagal 4 suggested a high probability for secretion based on the presence of a hydrophobic leader sequence, a leader-sequence cleavage site, and three possible N-glycosylation sites. The predicted molecular mass and isoelectric point of the pTombetagal 4-encoded mature protein were similar to those reported for the purified beta-galactosidase II protein from tomato fruit.
Smith
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Horticultural Crops Quality Laboratory, Plant Sciences Institute, Agricultural Research Service, United States Department of Agriculture, Building 002, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350, USA.
Starrett
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Gross
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017343:Genes, Plant; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D001616:beta-Galactosidase