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  • 1
    ISSN: 1573-5028
    Keywords: cDNA sequence ; expression library ; in vitro translation ; Kunitz-type proteinase inhibitor ; mRNA accumulation in vivo ; signal peptide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The accumulation of the Kunitz-type chymotrypsin inhibitor WCI-3 in winged bean seeds is controlled developmentally. In vitro translation experiments showed that the WCI-3 mRNA was present in 35- and 40-day-old immature seeds after flowering. The size of the in vitro translation product is about 2 000 Da larger than that of the mature WCI-3 protein. The WCI-3 cDNA clones were isolated from a λgtll cDNA library of 35-day-old immature seeds by immunoscreening. A nearly full-length cDNA clone was obtained containing an open reading frame of 207 amino acid residues. The deduced sequence of the 183 carboxy terminal amino acids coincides precisely with the amino acid sequence determined for purified WCI-3. The amino terminal extension of 24 residues has the characteristics of a signal peptide. Northern hybridization analysis of total poly(A)+ RNA showed that the WCI-3 mRNA is approximately 900 nucleotides long and accumulates in 35- and 40-day-old but not in 30-day-old immature seeds.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-5028
    Keywords: DNA-binding proteins ; high mobility group proteins ; HMG proteins ; Kunitz proteinase inhibitor ; seed storage protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Winged bean Kunitz chymotrypsin inhibitor (WCI) accumulates abundantly in seeds and tuberous roots of winged bean plant. In seeds, the WCI mRNA is observed transiently during seed maturation period. The WCI is encoded by a multigene family and the major WCI (WCI-3) is encoded by two nearly identical genes (WCI-3a and WCI-3b genes), in which nucleotide sequences in the 1.1 kb 5′ flanking regions are about 99% homologous to each other and the transcribed regions are completely identical. Here we report the detection of two types of nuclear proteins which bind to the multiple sites in the 5′ upstream region of the WCI-3a gene. One of the proteins, band 1-forming protein, also bound to cauliflower mosaic virus 35S (CaMV35S) promoter, but another protein, band 3-forming protein, did not. DNaseI footprinting analysis showed that these proteins bound to AT-rich upstream regions in the WCI-3a gene. Addition of poly(dA-dT)-poly(dA-dT) to the binding reaction inhibited the formation of the retarded bands, while poly(dI-dC)-poly(dI-dC) did not. In various organs and throughout seed maturation period, proteins with invariable binding specificities were detected, and these binding proteins met some operational criteria for high-mobility-group (HMG) proteins. These results suggest that leguminous seed AT-binding proteins reported on several seed storage protein genes may be HMG-like proteins which are present ubiquitously in plant organs.
    Type of Medium: Electronic Resource
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