Ginkgo 11S seed storage protein family mRNA: unusual Asn-Asn linkage as post-translational cleavage site.
By reducing the amount of ginkgo water-soluble polysaccharides, which occupy about 35% of the wet seed mass and interfere with the extraction of RNA, cDNA-quality mRNA was obtained from developing seeds of Ginkgo biloba. Based on the NH2-terminal 17-amino acid sequence and an internal 12-amino acid sequence derived from the basic subunit of ginnacin, 11S-seed storage protein family of ginkgo, two degenerate oligonucleotide primers were synthesized and used for polymerase chain reaction (PCR). The resulting PCR product was used for screening the above endosperm cDNA library, and a plaque carrying the 1614 bp cDNA insert, which contained the entire coding region for a precursor of ginnacin was isolated. This is the first reported cloning of cDNA from ginkgo seeds. The deduced primary sequence is composed of a signal peptide segment (25 amino acid residues) and an acidic subunit (248 residues) followed by a basic subunit (187 residues). It was also found that the post-translational cleavage site in the ginnacin precursor is the Asn-Asn rather than the Asn-Gly bond found in a variety of the major subunit precursors in 11S seed protein family known to date. We showed that a purified soybean extract and an extract of ginkgo seeds can specifically hydrolyze -Asn248-Asn249- but not -Asn249-Val250-, in the heptapeptide Gly-Asn248-Asn-Val-Glu-Glu-Leu that corresponds to the ginnacin cleavage region.
Arahira
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M
Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan.
Fukazawa
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C
D000595:Amino Acid Sequence; D001216:Asparagine; D001483:Base Sequence; D017931:DNA Primers; D015723:Gene Library; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis*; D010944:Plants / Q000201:enzymology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology
D017931:DNA Primers; D009842:Oligopeptides; D010940:Plant Proteins; D012333:RNA, Messenger; C088977:ginnacin protein, Ginkgo biloba; D001216:Asparagine