The genetic basis of antibody production: A single heavy chain variable region gene encodes all molecules bearing the dominant anti‐arsonate idiotype in the strain A mouse

Siekevitz, Miriam; Huang, Shu Ying; Gefter, Malcolm L.

doi:10.1002/eji.1830130207

Cited by 151 publications

(130 citation statements)

References 43 publications

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“…We have examined the CXAL 1.40 cell line for the presence of VHARS (VH36-65), the VHJ558 member (1) expressed in the predominant idiotype response (CRI) to p-phenyl-arsonate in Igh`strains (31) . As previously reported, the VH36-65 probe, VId-130 (32), hybridizes to the VHARS-bearing Eco RI fragment (6.2 kbp) of the Igh`haplotype but does not hybridize to BALB/c (Igh°) genomic DNA.…”

Section: Resultsmentioning

confidence: 99%

The organization of the mouse Igh-V locus. Dispersion, interspersion, and the evolution of VH gene family clusters.

Brodeur¹,

Osman²,

Mackle³

et al. 1988

The Journal of Experimental Medicine

108

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Studies ofIg gene structure and organization during the past decade have illuminated the central mechanisms of antibody diversification: the somatic rearrangement and reassortment of multiple gene segments, junctional flexibility, and point mutation . The inherited set of Ig gene segments provides a diversified genetic basis upon which these dynamic processes operate during ontogeny. Thus, the composition of these germline genes imposes a major influence on the development of the antibody repertoire.The present study examines the germline content and organization of the mouse heavy chain variable region genes (V genes) . We set out to analyze the locus in sufficient detail and resolution to provide the basis for determining the extent of inherited Vgene diversity, the evolution ofthe germline repertoire, and any functional consequences of the physical arrangement of the V gene segments .The mouse Igh locus consists of at least 100-200 V genes (1-3). V gene families, defined by nucleotide sequence relationships, comprise distinct sets of highly related V genes that can be identified by hybridization using prototypic V gene probes . This classification of V gene families (l, 4) has provided a useful framework for the study of germline V gene content, polymorphism, and utilization (1-7). Whereas the general organization of the Igh locus is 5'-V HDJ .-C -3' (8, 9), previous studies of V gene family organization have resulted in partial, relatively low resolution maps lacking consistency between reports (10-12)."Deletion mapping" takes advantage of the fact that V, gene rearrangements result in the deletion of DNA originally separating the rearranged V gene segment and the DJ .-CH region (13). We have constructed a panel of 32 pre-B cell lines, most of which have rearranged V, genes on both chromosomes. Since these cell lines were derived from Fi mice heterozygous at the Igh locus, V, gene deletions can be identified using RFLPs. V, gene analyses of 51 independently rearranged chromosomes are consistent with a single V, gene map order of nine V, gene families. The genomic stability of these cell lines and consistent deletion profiles of all 51 rearranged loci provide a high resolution V gene map that has compelling experimental support.

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Section: Resultsmentioning

confidence: 99%

The organization of the mouse Igh-V locus. Dispersion, interspersion, and the evolution of VH gene family clusters.

Brodeur¹,

Osman²,

Mackle³

et al. 1988

The Journal of Experimental Medicine

108

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“…These antibodies are encoded by a unique combination of heavy (H)-and light (L)-chain variable (V), diversity (D), and joining (J) genes and have different amino acid residues at the VH-DH, DH-JH, and VK-JK junctions (3)(4)(5)(6)(7). Additional differences in the amino acid sequence of the V regions are the result of somatic hypermutation induced by antigen (8)(9)(10)(11).…”

mentioning

confidence: 99%

Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold.

Sharon

1990

Proc. Natl. Acad. Sci. U.S.A.

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Communicated by Alfred Nisonoff, April 9, 1990 (received for review February 22, 1990 ABSTRACTThe basis for the 200-fold difference in affinity between two hybridoma antibodies specific for the hapten p-azophenylarsonate (Ars) that have diversified by somatic hypermutation was examined. Oligonucleotide-directed mutagenesis was used to sequentially convert the nucleotide sequence ofthe lower-affinity antibody into that ofthe higher-affinity one, and the mutant antibodies generated by transfection of hybridoma cells were analyzed for affinity to Ars-tyrosine. The data

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“…1 were determined by oligonucleotide-primed dideoxy nucleotide sequencing of the mRNA (40). The heavy and light chain V regions of 36-65 are identical to those reported (43,44). The VH region ( Fig.…”

Section: Resultsmentioning

confidence: 65%

Identification of mutant monoclonal antibodies with increased antigen binding.

Pollock

French

Gefter

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 x iA'. The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.In the course of the immune response to foreign antigen, antibodies with progressively higher affinities are found in the circulation (1). Recent studies of families of monoclonal antibodies encoded by the same germ-line variable (V) region genes (2-9) have shown that this affinity maturation is largely due to somatic hypermutation (2,10) of the heavy and light chain V regions and subsequent selection, presumably by antigen and accessory cells, for the B cells that are producing the highest-affinity antibodies (11)(12)(13)(14)(15)(16). The rate of V region mutation in vivo has been estimated to be as high as 10-3 per base pair per generation (6, 7). Further, the available data suggest that somatic mutation is limited to the V region and its immediate flanking sequences and does not extend into the constant (C) region of the immunoglobulin gene (10,(16)(17)(18).Because it is difficult to study the molecular mechanisms of somatic hypermutation in vivo, investigators have attempted to identify cell lines undergoing a high rate of V region mutation in tissue culture (19). The S107 mouse myeloma cell line frequently and spontaneously generates V region mutations that result in a loss of antigen binding (20-23). However, heavy chain C region mutations also occur frequently in the S107 cell line (24). The 18-81 pre-Bcell line undergoes a high rate (10-' per base pair per generation) of heavy chain V region mutation in culture (25), whereas C region mutations appear to occur much less frequently (26).Hybridoma cell lines producing monoclonal antibodies have also been examined for V region mutations (19). Bruggermann et al. (27) It seemed possible that the low frequency of V region mutations in hybridoma cell lines was due to the negative detection systems that had been used rather than to a seemingly paradoxical stability of the V region genes. Since it would also be very useful if monoclonal antibodies with increased binding could be obtained from hybridomas making low-affinity antibodies, we have developed a "positive" detection system that will identify even rare subclones that are making monoclonal antibodies with increased binding. This report describes the detection system and the identification of such higher-binding mutants. Further, we show that the mutants with greatly increased antigen binding that we have obtained have wild-type heavy and light chain V regions and altered C regions. MATERIALS AND METHODS

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The genetic basis of antibody production: A single heavy chain variable region gene encodes all molecules bearing the dominant anti‐arsonate idiotype in the strain A mouse

Cited by 151 publications

References 43 publications

The organization of the mouse Igh-V locus. Dispersion, interspersion, and the evolution of VH gene family clusters.

The organization of the mouse Igh-V locus. Dispersion, interspersion, and the evolution of VH gene family clusters.

Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold.

Identification of mutant monoclonal antibodies with increased antigen binding.

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