Two alpha heavy chain disease proteins with different genomic deletions demonstrate that nonexpressed alpha heavy chain genes contain methylated bases.

Abstract

Two independently arising a heavy chain mutants have been found to synthesize heavy chains with CH, deletions ofapproximately equal extent. Both were isolated from heavy chain-producing variants of the mouse myeloma W3129 and demonstrate that it is possible to arrive at the heavy chain disease phenotype by the pathway H + L -e H -+ AH. Analysis of genomic DNA by digestion with restriction endonucleases followed by molecular hybridization showed that one mutant (A37) had a deletion of approximately 0.2 kilobase and the second mutant (A15) had a deletion of approximately 0.5 kilobase. Mouse myeloma cells contain several a chain alleles but only one is expressed; the presence of the deletion in A37 and A15 made it possible to identify the restriction fragments from the expressed allele. Analysis of the fragments produced after cleavage with an isoschizomeric pair of restriction enzymes, Map I and Hpa H, indicated that, in the W3129 cell line and its variants, the unexpressed a alleles contain methylated bases. The influence of methylation on gene expression remains to be elucidated.

Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products.

“…The cell line A15.8, which synthesizes and secretes an alpha heavy chain with a deletion in CH1, was cloned in soft agarose to isolate myeloma cells deficient in their secretion of immunoglobulin. A15.8 was isolated in two steps from the myeloma W3129 (20). The interrelationship of the mutants is shown in Table 1.…”

Methylation and rearrangement of mouse intracisternal a particle genes in development, aging, and myeloma.

“…DNA methylation is correlated with transcriptional silence of several eucaryotic genes (12,17,29,34,39,42), including immunoglobulin genes (8), ovalbumin genes (22), human globin genes (40), thymidine kinase genes (6), and genes of endogenous retroviruses (1,12,28,39). Methylation appears to regulate gene transcription via controls over DNA-protein interactions in chromatin (11,43).…”

Isolation and characterization of a variant of mouse plasmacytoma J558 synthesizing a 110,000-dalton immunoglobulin heavy chain and of secondary variants synthesizing either a 55,000-dalton or an 80,000-dalton immunoglobulin heavy chain: possible implications.

“…Intact alpha chains are secreted associated with L chains, but are not secreted if they do not have noncovalently associated L chains. However, alpha chains which contain deletions of CH, can be secreted in the absence of L-chain synthesis (11).…”

“…7B, lanes 1 and 5). In addition, a minor band of approximately 11 kb was seen after hybridization with the 3' probe, and analysis of clone 2.10.6 DNA after digestion with enzymes which cut within the EcoRI fragments suggests that some rearrangement or sequence changes may have occurred within these fragments in clone 2.10.6 (data not shown). It should be noted that the J558 cell line appears to have multiple copies of alpha-chain genes; the pattern after EcoRI digestion and hybridization with the 0.8-kb probes suggests a minimum of three alpha-chain genes in J558.…”

“…None of these appears to go away in the 6.5.2 mutant; instead a new band appears and a second band increases in intensity, suggestive of its being present in increased amounts. (2,3,6,11,32) and mutants synthesizing H chains with an alteration in subclass (17,33,38). However, to date only one mutant has been reported which synthesizes an H chain of increased length (27); the reported mutant, derived from the _Y2b myeloma MPC-11, synthesized an H chain of 75,000 daltons.…”