Association of allotypic specificities of group a with allotypic specificities A11 and A12 in rabbit immunoglobulin

(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...

Section: Ctgaggtaggttaga----cag-ggtt-gtctg---tagcttgagcttaccttaggn---mentioning

confidence: 99%

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

Obata¹,

Kataoka²,

Nakai³

et al. 1981

“…Genetic studies using allotype markers have shown that only one of the alleles of immunoglobulin genes is expressed in activated B lymphocytes (31)(32)(33). Genetic information for the VH and CH which are genetically linked on one parental chromosome are coordinately expressed, which is referred to as cis expression (34)(35)(36). Apparently, the allelic deletion model postulates allelic exclusion and cis expression by nature.…”

mentioning

confidence: 99%

Organization of immunoglobulin heavy chain genes and allelic deletion model.

Honjo¹,

Kataoka²

1978

265

We have assessed the number of times the gene sequence encoding constant regions of mouse immunoglobulin heavy chains gamma1, gamma2a, and gamma3 are represented in the mouse genome by hybridization kinetic analysis. All three genes are present at one copy each per haploid genome in normal tissues and myelomas producing IgM or IgG3. IgG1-producing myelomas, however, contain 1 copy each of the gamma1 and gamma2a genes and 0.5 copy of the gamma3 gene per haploid genome. IgG2b-producing myelomas contain 1 copy of the gamma2a gene and 0.5 copy each of the gamma1 and gamma3 genes per haploid genome. IgG2a-producing myelomas contain 1 copy of the gamma2a gene and 0.5 copy each of the gamma1 and gamma3 genes per haploid genome. In myelomas producing IgA, all three gamma genes are represented 0.5 times per haploid genome. In order to account for the results we propose an allelic deletion model: (i) The specific deletion of heavy chain constant region genes accompanies the recombination of a variable region gene to a constant region gene. (ii) The portion of the chromosome that resides between two joining sequences is excised out of the chromosome. (iii) The recombination occurs on one of the alleles. Based on this model we also propose that heavy chain genes are arranged on one chromosome in the following order; variable region genes, unknown spacer sequence, mu, gamma3, gamma1, gamma2b, gamma2a, and alpha.

“…V and C genes are genetically linked and expressed as a cis combination (1)(2)(3). In a given lymphocyte, immunoglobulin genes on one allele are exclusively expressed (4)(5)(6).…”

mentioning

confidence: 99%

Cloning immunoglobulin gamma 2b chain gene of mouse: characterization and partial sequence determination.

Kataoka¹,

Yamawaki-Kataoka²,

Yamagishi³

et al. 1979

DNA from newborn mice was digested with restriction endonuclease EcoRI, and a 6.6-kilobase fragment encoding immunoglobulin 'y2b chain mRNA derived from MPC 11 myeloma was enriched about 100-fold by RPC-5 column chromatography and agarose gel electrophoresis. The 6.6-kilobase fragment was Immunoglobulin heavy chain genes consist of a family of variable region (V) genes and several constant region (C) genes. V and C genes are genetically linked and expressed as a cis combination (1-3). In a given lymphocyte, immunoglobulin genes on one allele are exclusively expressed (4-6). Recently, we have shown that specific C genes are deleted from one allele in mouse myelomas depending on which C genes are expressed (7,8). We have proposed an allelic deletion model as the mechanism of the V-C gene recombination that results in the expression of the specific immunoglobulin genes (7,8 two or more neighboring genes. For this purpose it is essential to clone two putative adjacent y chain genes. In this paper we will describe cloning of the -y2b chain C gene, which is proposed to be adjacent to the y1 chain gene, and its structure. EXPERIMENTAL PROCEDURES Partial Purification of DNA Fragment. High molecular weight DNA was extracted from whole newborn mice (killed within 24 hr after birth). DNA was digested with EcoRI, extracted with phenol, and precipitated with ethanol. Forty milligrams of EcoRI-digested DNA was chromatographed on an RPC-5 column (0.8 X 90 cm) as described (10,12). Fractions that hybridized with the y2b chain cDNA by in situ hybridization were pooled and concentrated by ethanol precipitation. The DNA obtained was applied to a 1% agarose gel (7 X 15 X 4 cm) and fractionated as described (13). Fractions were assayed by in situ hybridization with nick-translated y2b chain [32P]cDNA cloned in a plasmid (pG2b-4).Preparation and Screening of Recombinant Phage. Bacteriophage XgtWES-XB (9) was used as the EK2 vector and propagated in ED8656 (14). Cloning experiments were carried out in a P2 facility (15, 16). Bacterial strains NS428 [N205 (X Aamll b2 red 3 cI857 Sam7)I (17) and Xdg8O5 [W3350 (Xdgal8O5 cl857 Sam7)] were used for in vitro packaging.Partially purified 6.6-kilobase (kb) mouse DNA (2 jig) was ligated with 5.1 ,ug of XgtWES outer fragments in a 50-,ul reaction mixture with 0.6 unit of T4 ligase as described (18). The recombinant DNA was packed into coat proteins in vitro and the recombinant phages were plated by the method of Blattner et al. (19). Twenty sets of about 2000 plaques were directly transferred from LB broth agar plates to Millipore filters as described (20). The Millipore filters were hybridized to 32p-labeled Hha I fragment of pG2b-4 and autoradiographed. Recombinant phage DNA was prepared as described (18).In Situ Hybridization. DNA, fixed on a Millipore filter (21), was hybridized to an appropriate probe as described by Jeffrey and Flavell (22) except that 1 M NaCl/50 mM Tris-HCI, pH 7.4/10 mM EDTA replaced 0.9 M NaCl/0.09 M sodium citrate.The probes used were [32P]cDNA (specific ac...