ANALYSIS OF IMMUNOGLOBULIN GENES - DNA-RNA HYBRIDIZATION WITH IMMUNOGLOBULIN KAPPA-CHAIN MESSENGER-RNA AND ISOLATION AND TRANSLATION OF HYBRIDIZED RNA

  • 1 January 1976
    • journal article
    • research article
    • Vol. 117  (1) , 259-268
Abstract
Immunoglobulin .kappa.-chain mRNA was hybridized with DNA to assess the .kappa.-gene frequency. .kappa.-mRNA was purified from membrane-bound ribosomes of mouse myeloma MOPC-41 by poly (U) chromatography and isolation of a 13S RNA by successive sucrose density gradient centrifugations. The RNA coded for .kappa.-chain precursor molecules in cell-free protein synthesis and essentially no other proteins. MOPC-41 .kappa.-mRNA hybridized with MOPC-41, MPC-11 and Krebs DNA with the same kinetics: the majority of the hybrids was formed with rare or unique DNA sequences (Cot/2 [1/2 nucleotide concentration] 450-900), a small portion with highly repetitive sequences (Cot/2 5-6). The slow hybrids were well matched and the rapid hybrids were mismatched by about 4%, regardless of the DNA used. Whether the rapid hybrids contained translatable .kappa.-mRNA or were due to impurities in the RNA preparations. .kappa.-mRNA and globin-mRNA (as an internal standard for a unique transcript) were hybridized with DNA to Cot 20 or 48, the hybridized and unhybridized RNA were isolated by hydroxyopatite-urea chromatography and, after removal of the DNA, translated in a cell-free system. The cell-free products were analyzed by SDS-polyacrylamide gel electrophoresis and immunoprecipitation. Approximately equal quantities of translatable .kappa.- and globin-mRNA were hybridized maximally 1.7%. .kappa.-mRNA may not be a transcript of both repetitive and unique DNA sequences.