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Synthesis, molecular cloning, and restriction analysis of DNA complementary to vitamin D-dependent calcium-binding

C Desplan, M Thomasset, M Moukhtar

    The Journal of Biological Chemistry
    |March 10, 1983
    PubMed

    Insights

    Researchers purified messenger RNA (mRNA) for rat intestinal calcium-binding protein. They created DNA clones of this mRNA, aiding future studies on vitamin D-dependent calcium absorption.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Nutritional Science

    Background:

    • Vitamin D is crucial for calcium absorption in the intestine.
    • Vitamin D-dependent calcium-binding protein (CaBP) plays a key role in this process.
    • Understanding the genetic regulation of CaBP is essential for studying calcium homeostasis.

    Purpose of the Study:

    • To isolate and clone the messenger RNA (mRNA) encoding rat intestinal calcium-binding protein.
    • To develop molecular tools for further investigation of vitamin D-regulated gene expression.
    • To facilitate research into the molecular mechanisms of vitamin D-mediated calcium transport.

    Main Methods:

    • Partial purification of mRNA for rat intestinal calcium-binding protein from rat duodenum.
    • Synthesis of double-stranded DNA (cDNA) from purified mRNA.
    • Cloning of cDNA into the pBR322 plasmid using oligo(dG-dC) tailing.
    • Selection of recombinant clones via differential colony hybridization.
    • Verification of clones using hybrid-arrest and hybrid-selection assays.
    • Analysis of cloned DNA inserts using endonuclease restriction mapping.

    Main Results:

    • Successfully created recombinant DNA clones containing sequences complementary to vitamin D-dependent calcium-binding protein mRNA.
    • Identified four distinct clones with identical endonuclease restriction maps.
    • Determined insert sizes ranging from 250 to 380 base pairs for the selected clones.

    Conclusions:

    • The study successfully generated molecular clones representing the rat intestinal calcium-binding protein mRNA.
    • These clones serve as valuable tools for studying the regulation and function of this vitamin D-induced protein.
    • Provides a foundation for future research into the molecular basis of vitamin D-dependent calcium absorption.

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