MURINE MYELOMA IMMUNOGLOBULIN HEAVY-CHAIN MESSENGER-RNA - ISOLATION, PARTIAL-PURIFICATION, AND CHARACTERIZATION OF GAMMA-1, GAMMA-2A, GAMMA-2B, GAMMA-3, MU- AND ALPHA HEAVY-CHAIN MESSENGER-RNAS

Seven mouse immunoglobulin heavy-chain mRNAs have been partially purified from several myeloma tumors. Each of the individual, partially purified γ1, γ2a, γ2b, γ3, or α heavy-chain mRNA migrated as a 17S species on sucrose velocity sedimentation gradients. However, these heavy-chain mRNAs migrated slightly slower than 18S ribosomal RNA, by use of denaturing acrylamide or agarose gel analyses. Each different heavy-chain mRNA has a molecular weight of 750 000, equivalent to 2200 nucleotides. The partially purified μ. heavy-chain mRNA migrated as a 20S species on sucrose gradients and ran significantly slower than 18S rRNA on denaturing formamide polyacrylamide gels, with a molecular weight equal to 950 000, or 2800 nucleotides. This RNA fraction also contained a small and distinctly separate 18S RNA moiety—not coding for detectable μ heavy chain. The 17S or 20S mRNA fractions coded for the cell-free syntheses of several detectable proteins in the expected size range of the various authentic heavy chains. In all cases, only one size class of cell-free synthesized protein was serologically reactive with the respective monospecific antiserum. These immunoprecipitated proteins accounted for as much as two-thirds of the cell-free synthesized, [35S]methionine-labeled product. All in vitro generated γ heavy chains have an apparent molecular weight of 52 000, as determined by denaturing sodium dodecyl sulfate slab gel analyses. This compares with an apparent molecular weight of 57 000 for the authentically secreted γ, heavy chain of MOPC-31C and 52 000 for each of the other authentically secreted γ1, γ2a, γ2b, and γ, heavy chains studied here, as determined in the same slab gel. The α heavy chain 17S mRNA fraction coded for the in vitro synthesis of only one major serologically reactive protein component with an estimated molecular weight of 56 000. A minor one (<5%) also was detected with an estimated molecular weight of 48 000. These compare with an estimated molecular weight of 54 000 for the in vivo authentically secreted a heavy chain. The 20S μ heavy-chain mRNA fraction coded for the synthesis of only-one major serologically reactive protein component with an estimated molecular weight of 64 000. This is significantlysmaller than the in vivo authentically secreted homologous μ heavy chain, estimated at 70 000 daltons. A small amount (<5%) of the immunoprecipitated product appeared to have nearly the same molecular weight as the in vivo μ heavy chain. Both these IgM and IgA, from MOPC-104E and J 558, respectively, have the same naturally occurring idiotype of all immunized normal BALB/c mice, suggesting very similar heavy-chain variable regions. Tryptic peptide analyses revealed significant homologies between the authentically secreted heavy chains and their respective cell-free synthesized, immunoprecipitated products, verifying the identity of that particular heavy-chain mRNA. Kinetic complexity analyses between the mRNA and their respective complementary DNA revealed RNA fractions which were from 40 to 75% homogeneous, depending upon the particular isotype of heavy-chain mRNA studied. Models for the mature cytoplasmic form of all murine γ, α, and μ heavy-chain mRNA are presented. Only about 60% of the nucleotide sequence of any subclass of RNA is directly responsible for encoding the structural protein. This means as few as 600-700 nucleotides are unaccounted for in both location and function for all murine γ and α heavy-chain isotypes, while a minimum of 800 nucleotides of the μ heavy-chain isotype fit this situation. Therefore, any cDNA probe used to measure any parameters of the constant region of any γ or α heavy-chain gene must be significantly longer than 700 bases to ensure that at least part of that heavy-chain constant region coding segment is included. Similarly, the minimum length of cDNA to be used as probe for the constant region of the μ heavy-chain gene must be significantly larger than 800 bases. © 1979, American Chemical Society. All rights reserved.