GENETIC MECHANISMS AFFECTING IMMUNOGLOBULIN GENE EXPRESSION

Abstract

Immunoglobulin genes are regulated by a complex set of mechanisms which cause major DNA rearrangements and create antigen binding diversity. Characterization of Ig variants which express structurally altered heavy chains should help define the spectrum of recombination and repair mechanisms which impinge upon these genes. To examine these questions, I have characterized M 311 and A68.13.9, independent variants of the mouse myeloma cell line, MPC 11, which produce short immunoglobulin heavy chains of 40,000MW compared with the parental H chain of 55,000MW.;Biochemical analysis of the short heavy chains produced by M 311 and A68.13.9, was carried out. Automated sequential degradation of the variant C-terminal CNBr fragment showed it to be identical to a corresponding segment of parental heavy chain CNBr fragment with the exception of a lysine to asparagine substitution at the C-terminal residue. This defined the loss of MW as a C-terminal deletion comprising the C(,H)3 domain. It also enabled prediction of the parental DNA sequence in this region and the genetic mechanism which generated the variant, a -2 frameshift followed by premature termination. This hypothesis is substantiated by Southern analysis of genomic DNA and Northern blot analysis of the mRNA which show that the parental MPC 11 and short variants are identical in these respects.;In addition, M 311 and A68.13.9 express an aberrant assembly pathway leading to the secretion of both H(,2)L(,2) and HL molecules. Variant Ig H chains radiolabeled in the presence of Tunicamycin, a glycosylation inhibitor, were shown to give at least 2 bands when analysed by gel electrophoresis. This suggested that differential glycosylation plays a role in the specific segregation of these H chains into Ig molecules.;Many of the variants isolated from MPC 11 produce (gamma)(,2b)-(gamma)(,2a) hybrid heavy chains or have undergone a complete (gamma)(,2b) to (gamma)(,2a) heavy chain class switch in vitro. Generalized recombination is a phenomenon dependent on recognition of homologous DNA and may be responsible for these types of variants. Chi, a promoter of generalized recombination in lambda phage, is present in immunoglobulin genes. Consideration of the frequency at which Chi occurs in bulk mouse DNA, Ig genes and other unique eukaryotic sequences and the positions at which Chi occurs in Ig genes suggests that homologous recombination may be applicable to the C(,H) class switch.