Structure of a rearranged gamma 1 chain gene and its implication to immunoglobulin class-switch mechanism.

Abstract

(S,, region) indicates that the heavy chain class switch may not be mediated by stepwise linear deletion along the order of the heavy chain constant-region genes (5'-,j-y3-y1-y2b-y2a-a-3'). We propose a sister-chromatid exchange model that explains class switch-associated deletion of heavy chain genes by unequal crossing-over events between sister chromatids.Immunoglobulins are divided into five classes, IgM, IgG, IgA, IgD, and IgE, which are defined by their heavy (H) chain constituents, ,u, y, a, 8, and e, respectively. Each gene locus is itself split into two areas encoding the variable (V) and constant (C) regions (1-5). In the H chain, C region (CH) genes share a family ofV region (VH) genes. During differentiation of a given B lymphocyte, a single VH region gene (VH gene) is first expressed as a part ofthe ,t chain, and at a later stage the expressed H chain switches the C region from u chain to y or a chains without alteration of the VH-region sequence. Recent studies on the expressed H chain genes clearly demonstrate that two distinct types of DNA rearrangements take place during the differentiation of B lymphocytes (6-9). One type, termed V-J recombination, generates the complete VH gene by joining the VH-, D-, and JH-gene segments (8, 10, 11). In CH-region genes (CH genes) only one set ofthe J-region genes is proposed to be present in the 5' flanking region ofthe ,u chain gene (,u gene) (7, 9). A second type of recombination mediates the H chain class switch that associates a particular antigenbinding specificity, the completed V region, with a series of different CH regions. The rearrangement is termed S-S recombination because itjoins two switch (S) regions which are located in the 5' flanking region of each CH gene (7, 9). To switch from A to ychain, the S, region recombines with the SYregion, keeping the VH gene unaffected.It has been shown that the S-S recombination is associated with the deletion ofthe intervening DNA between the VH gene and the CH gene (12)(13)(14)(15)(16)(17)(18). Based on the order of CH-gene deletion in various myeloma cells, we have proposed an H chain gene order of 5'-g-y3-y1-y2b-'y2a-a-3'. Recent cloning experiments have directly demonstrated the order of 5'-yl-,y2b--y2a-E-a-3' (19,20).In this paper we report a detailed structure of a rearranged (expressed) yl chain gene (yl gene) cloned from a myeloma MC 101 cell. Partial nucleotide sequence determination indicates that the rearranged yl gene contains a small S. segment between the SM and S,, segments. The unique structure ofthis yl gene implicates the molecular mechanism for the class-switch (S-S) recombination. EXPERIMENTAL PROCEDURESCloning. Charon 4A (21) phage library of partial EcoRI digests of MC101 DNA was screened by using yl chain-cDNA clone pGl-6 (22) as a probe as described (23). Germline V genes were cloned from mouse embryo DNA library (provided by P. Leder, National Institutes of Health) with a V portion of the expressed yl gene (Igyl-704) as a probe. Cloning and nucleotide sequence determination of these...

Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch.

“…The rearrangement is accomplished by a recombination event between segments of DNA (called S,u and Sa regions) located between two and six kilobases upstream of the C, and Ca genes. This recombination event between S regions is similar to that observed in the vast majority of heavy-chain switches in other immunoglobulin-producing cells (3,12,14,17).…”

“…The unusual composition of switch regions might also cause DNA synthesis to be error prone. Errorprone DNA synthesis in the switch would explain discrepancies previously observed between recombined and germ line switch regions (14,24). Nucleotides not accounted for by either the donor or recipient switch region at switch recombination sites (25) might be explained by non-template-directed DNA synthesis.…”

“…DISCUSSION We assume that molecular clones with identical switch recombination sites are derived from a single switch recombination event. No two independent switch sites have ever been shown to be identical at the sequence level (3,12,14,16,17,24,25). In every case studied to date the two Igh loci within a single cell are found in distinguishable, rearranged restriction enzyme fragments (2,6,8,20,25 9,1989 R. quences.…”

“…For example, the S,u region is composed of the repeat unit GGGGT(GAGCT)n, where n is usually between one and seven (13,17). The Sa region is composed of an 80-base-pair (bp) long repeat which is based on pentamers similar to those found in the S,u region (3,14). Switch recombination events in both the S,u and the Sa regions have been shown to be scattered over most of the respective switch regions (3,10,12,14).…”

Independent immunoglobulin class-switch events occurring in a single myeloma cell line.

“…The experimental materials available for these studies generally do not allow for the detection of subsequent switches among the other (non-,u) classes (only surface immunoglobulin M [IgM]-positive and surface IgA-positive B-cell populations can be obtained in sufficient numbers for analysis). However, the immunoglobulin gene structure in some myelomas suggests that B cells (or plasmacytes) sometimes undergo this type of class switch as well (e.g., ,u to -y2b to e; 27,28).…”