Somatic mutations of immunoglobulin transgenes in cultured B cell lines

Abstract

In order to study the molecular mechanism and regulation of variable (V) region somatic mutation, I developed a cell system for in vitro study. pSV2neo plasmids containing an IgM heavy chain gene with nonsense mutations in either V or C region (Vn/{dollar}\mu{dollar} or Cn/{dollar}\mu{dollar}) were transfected into several plasma cell lines: 2C3, S107, T558L and NSO. The rates of reversion of the nonsense mutations were analyzed using the ELISA spot assay and fluctuation analysis. Mutations in both V and C regions were confirmed by sequencing analysis. This system made it possible to compare the frequencies, rates and types of mutations in V and C regions. In S107, spontaneous point mutations occurred in the V region at a rate of 5 {dollar}\times\ 10\sp{lcub}-5{rcub}{dollar}/bp/gen which was more than 100 fold greater than in other cell lines. This result suggested that S107 is a permissive cell line for somatic mutation and that the {dollar}\mu{dollar} construct used contains enough DNA sequence information for mutation, at least in S107.;In order to use this system to study somatic mutation in more cell lines, the Vn/{dollar}\gamma{dollar}2a construct was created by replacing the C{dollar}\mu{dollar} region of Vn/{dollar}\mu{dollar} construct with the C{dollar}\gamma{dollar}2a region. The mutation rates of these two Vn constructs in several cell lines were compared. Both {dollar}\gamma{dollar}2a and {dollar}\mu{dollar} construct mutated at relatively high rates (5-10 {dollar}\times\ 10\sp{lcub}-5{rcub}{dollar}/bp/gen) in S107, and at low rates (5 {dollar}\times\ 10\sp{lcub}-7{rcub}{dollar}/bp/gen) in J558L. This suggested that these two cell lines were expressing different combinations and/or amounts of the transacting factors involved in somatic mutation. In contrast, {dollar}\gamma{dollar}2a construct mutated its V region at a rate of 10{dollar}\sp{lcub}-3{rcub}{dollar}/bp/gen, while the {dollar}\mu{dollar} construct mutated at a 1000 fold lower rate of 10{dollar}\sp{lcub}-7{rcub}{dollar}/bp/gen in NSO. This suggested that NSO was expressing some different factors than S107 and T558L and that the {dollar}\gamma{dollar}2a and {dollar}\mu{dollar} constructs were differentially regulated by these factors. Subsequent studies showed that the presence of the {dollar}\gamma{dollar}2a construct raises the mutation rate of the {dollar}\mu{dollar} construct in NSO, implying trans-acting interaction between these two constructs.