Isolation, purification, and properties of mouse heavy-chain immunoglobulin mRNAs

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Abstract
A procedure was described for the isolation of highly purified H-chain immunoglobulin [Ig] mRNAs from a variety of mouse plasmacytomas (IgA, IgG and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were essential in obtaining large quantities of undegraded H-chain mRNAs. The individual mRNAs were purified by 2 cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate-sucrose gradient centrifugation and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 .gamma.2b and [mouse plasmacytoma] H2020 .alpha. H-chain mRNAs efficiently directed the synthesis of a predominant product of 55,000 MW, while the synthesis of a 70,000 dalton protein in addition to other lower MW polypeptides were observed with [mouse myeloma] MOPC 3741 .mu. mRNA. All of these proteins were immunoprecipitable with class-specific H-chain antisera, and in the case of the .gamma.2b in vitro products good correspondence in a comparative trypsin-chymotrypsin fingerprint with in vivo labeled .gamma.2b H-chain was observed. The .gamma.2b and .alpha. H-chain mRNAs possessed a chain length of .apprx. 1800 nucleotides and the .mu. mRNA a size of .apprx. 2150 nucleotides when examined under stringent denaturation conditions. The purities of the .alpha., .gamma.2b, and .mu. mRNAs were estimated to be 60-80%, 50-70% and 50-83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. H-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even through the c[complementary]DNA-mRNA hybrids are submitted to stringent S1 nuclease digestion. Allotypic determinants probably represent only a minor fraction of the H-chain constant region sequence in the mouse.