Construction and selection of bacterial clones hybridized with the light-chain immunoglobulin mRNA

S. M. Deyev, A. V. Karlyshev, O. L. Polyanovsky

    Research output: Contribution to journalArticle

    1 Citation (Scopus)

    Abstract

    The copy-DNA was synthesized on the mRNA fraction which was isolated from MOPC 21 mice myeloma by reverse transcription. This copy was completed with another chain without adding the exogenous primer, with the aid of the Klenov fragment of DNA polymerase I. After treatment of the double-stranded pin DNA with endonuclease S1, the poly (dA) sequences were built up using terminal deoxynucleotidyl transferase. The building up of the poly(dT) sequences at the DNA 3'-termini of pBR322 plasmid previously treated with BamHI was performed in a similar way. The average length of connecting polynucleotide sequences was 50 nucleotides. After annealing and transformation of the cells with Escherichia coli hybrid plasmids, selection of clones was made for Ap(R) Tc(S) phenotype. Further, we applied a stepwise selection of clones by means of increasing the specificity: colony hybridization, quantitative hybridization of the excess of plasmid DNA with ( 32P)-cDNA and hybridization of plasmid DNAs with mRNA units which were transferred from electrophoregrams to the diazo-papers. As a result, bacterial clones were selected which contained gene fragments showing the ability to hybridize with the RNA fraction characterized by mRNA mobility of the light-chain immunoglobulin G.

    Original languageEnglish
    Pages (from-to)880-887
    Number of pages8
    JournalGenetika
    Volume18
    Issue number6
    Publication statusPublished - 1982

    Fingerprint

    Immunoglobulin Light Chains
    Plasmids
    Clone Cells
    Messenger RNA
    DNA
    Poly T
    Polynucleotides
    DNA Polymerase I
    DNA Nucleotidylexotransferase
    Deoxyribonuclease I
    Reverse Transcription
    Nucleotides
    Complementary DNA
    Immunoglobulin G
    RNA
    Escherichia coli
    Phenotype
    Genes

    ASJC Scopus subject areas

    • Genetics

    Cite this

    Construction and selection of bacterial clones hybridized with the light-chain immunoglobulin mRNA. / Deyev, S. M.; Karlyshev, A. V.; Polyanovsky, O. L.

    In: Genetika, Vol. 18, No. 6, 1982, p. 880-887.

    Research output: Contribution to journalArticle

    Deyev, S. M. ; Karlyshev, A. V. ; Polyanovsky, O. L. / Construction and selection of bacterial clones hybridized with the light-chain immunoglobulin mRNA. In: Genetika. 1982 ; Vol. 18, No. 6. pp. 880-887.
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