Abstract
Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.
Highlights
Encoding a polypeptide of M, = 29,545
The sequence of the DB58 Previous studies on the two subunit types of the seed lectin lectin exhibits 84%homology to the D . biflorus seed indicated that both subunits arisferom differential proteolytic lectin at the amino acid level, suggesting that these processing of a single polypeptide precursor [16, 17].The lectins are encoded by differentially expressed genes marked similarities between the subunits of DB58 suggested and may have evolvedto carry out tissue-specific func- that the subunittypes of this lectin may arise from tions
The unamplified library was plated on replicate subunits of DB58 indicate extensivehomology with subunits I and I1of the D. biflorus seedlectin
Summary
Isolation of DB58 cDNA and in Vitro Translationof DB58 construction. All steps were identical to the originparlocedure, except mRNA-Data previously obtained on the structuresof both for the use of T 4 DNA ligase instead of Escherichia coli DNA ligase in the plasmid circularization and repair steps. The filterswere washed for 1 h in several changesof 2 isolated from stems and leaves of D.biflorus identifies an mRNA of approximately 1200 nucleotides (Fig. 2). This mRNA is in the correscitze range for encoding either subunit of DB58. Amounts of these two lectin mRNAs can be discriminated using the seed lectin cDNA probe, pDolSL5 (Fig. 2) These resultsindicatethatpDolSL5iscapable of identifying a homologous but nonidentical mRNA in stems and leaves of. Determine whether it represented DB58 mRNA, pDowlSasL5 used to isolate cDNA clones from a D.biflorus stem and leaf cDNA library. The cDNA library was constructedinthe vector pSV7186 using mRNA isolated from the stems and leaves of 3-week-old D.biflorus plants.
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