Analysis of immunoglobulin genes: DNA/RNA hybridization with immunoglobulin kappa-chain mRNA and isolation and translation of hybridized RNA.
Immunoglobulin kappa-chain mRNA was hybridized with DNA in order 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/2 450 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 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. It was found that approximately equal quantities of translatable kappa- and globin-mRNA were hybridized maximally 1.7%). The results do not support the hypothesis that kappa-mRNA is a transcript of both repetitive and unique DNA sequences.
D000818:Animals; D004247:DNA / Q000276:immunology*; D006358:Hot Temperature; D007147:Immunoglobulin Light Chains / Q000032:analysis*; D007145:Immunoglobulin kappa-Chains / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012313:RNA / Q000276:immunology* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000276:immunology* / Q000302:isolation & purification
D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D012333:RNA, Messenger; D012313:RNA; D004247:DNA