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Analysis of Immunoglobulin Genes: DNA/RNA Hybridization with Immunoglobulin -Chain mRNA and Isolation and Translation of Hybridized RNA¹

From the Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195

Abstract

Immunoglobulin -chain mRNA was hybridized with DNA in order to assess the -gene frequency. -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 -chain precursor molecules in cell-free protein synthesis and essentially no other proteins. MOPC-41 -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/2 450 to 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.

It was further investigated whether the rapid hybrids contained translatable -mRNA or were due to impurities in the RNA preparations. -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. It was found that approximately equal quantities of translatable - and globin-mRNA were hybridized maximally 1.7%). The results do not support the hypothesis that -mRNA is a transcript of both repetitive and unique DNA sequences.

Footnotes

¹ This work was supported by Grants AI-10685 and DE-02600 from the National Institutes of Health, Bethesda, Maryland.