Cellular selection leads to age-dependent and reversible down-regulation of transgenic immunoglobulin light chain genes

following correction should be noted. On page 2475, the second line of the left column that reads ". . . were reconstituted with aly͞aly BM cells after lethal irradiation" should read ". . . were reconstituted with aly͞ϩ BM cells after lethal irradiation."Immunology. In the article "Modifying the sequence of an immunoglobulin V-gene alters the resulting pattern of hypermutation," by Beatriz Goyenechea and César Milstein, which appeared in number 24, November 26, 1996, of Proc. Natl. Acad. Sci. USA (93, 13979-13984), the following revision should be noted: The formula at the bottom of the first column of page 13980 was misprinted. The correct formula is:FIG. 6. Three-dimensional structure of rQR1 in complex with FAD and NADP ϩ (12). The C-terminal 43 amino acids (232-274) are shown in red (Left), and have been truncated at the arrow (Right). Different colors of the backbone indicate the two different subunits. It can be seen that the truncated portion of QR2 is critical for binding of the pyrophosphate moiety of the NAD(P)H cofactor. Note that the contact regions between the enzyme and FAD are presumably preserved in QR2 (Right), consistent with the tight binding of FAD. The structural comparison between rQR1 and hQR2 is appropriate since the homology between rQR1 and hQR1 is very high (85% identity). CorrectionsProc. Natl. Acad. Sci. USA 94 (1997) ABSTRACT Affinity maturation of antibodies requires localized hypermutation and antigen selection. Hypermutation is particularly active in certain regions (notably the CDRs of light and heavy chains) due to the local accumulation of hot spots. We have now analyzed the role of individual nucleotides in the origin of hot spots and show that mutability is largely defined by the nucleotide sequence. We compared the mutability profile of wild-type and modified transgenes that contain silent mutations in the CDR1 segment. We found a new hot spot created at the third base of Ser-31 when its wild-type AGT codon was substituted by AGC. Two major hot spots associated with this AGC vanished when Ser-31 was encoded by the synonymous TCA. In addition to these, which were the most prominent changes, there were compensatory alterations in mutability of residues not directly related to the introduced silent mutations, so that the average hypermutation remained constant. Thus, mutations arising early in the immune response, even silent ones, could affect the mutability of critical residues and alter the pattern of affinity maturation. When analyzing hybridomas, we detected such alterations, but they seemed to better correlate with changes in average rather than local mutation rates. Overall, this paper shows how evolution could have optimized the mutability of individual residues to minimize deleterious mutations. Thus, the optimal strategy for affinity maturation may involve the incorporation of multiple point mutations before antigen selection of the relevant cells.

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“…Founder animals were identified by expression of rat chain in serum, as revealed by ELISA (35), and by Southern blot…”

Section: Methodsmentioning

confidence: 99%

Modifying the sequence of an immunoglobulin V-gene alters the resulting pattern of hypermutation

Goyenechea

Milstein

1996

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

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“…In certain experiments, BAL-17 cells were stimulated with 100 ng/ml TPA or TPA plus 400 ng/ml ionomycin (Sigma). A single-cell suspension of whole spleens was made as described previously (Pettersson et al, 1989). Resting lymphocyte cells were prepared using Percoll density gradient separation (Arulampalam et al, 1994).…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

IgM receptor-mediated transactivation of the IgH 3′ enhancer couples a novel Elf-1-AP-1 protein complex to the developmental control of enhancer function.

1995

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“…1) is composed of a rearranged mouse V, gene linked to a rat C, [17]. The V, is the germ-line V,-0x1 sequence [ 181 rearranged to J,5 [ 191, and the C, gene downstream of the XbaI site is derived from the LOU allotype rat C, [20].…”

Section: The Xox Transgene Construct and Transgenic Micementioning

confidence: 99%

“…Transgene x chains were detected by an ELISA using the mouse anti-rat x mAb MARK-1 as previously described [17]. Antibodies specific to phOx were assayed by an ELISA in which serial dilutions were incubated in wells of a microtiter plate coated with OxloBSA, and blocked with 1% FSA.…”

Section: Elisa and Determination Of H Chain Subclassmentioning

confidence: 99%

“…Cytoplasmic immunofluorescence of permeabilized cells attached to slides was performed as previously described [17]. Mouse x chain was detected using FITC-conjugated OX20 (Serotec), and transgenic x chain was detected using biotinylated sheep anti-rat antiserum in the presence of 5% normal mouse serum, followed by streptavidin conjugated to TRITC (Serotec).…”

Section: Immunofluorescencementioning

confidence: 99%

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Lack of somatic mutation in a χ light chain transgene

et al. 1990

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Analysis of mice transgenic for immunoglobulin genes should allow definition of the cis-acting DNA sequences required to target somatic mutation to antibody V genes. We have looked for mutations in a chimeric kappa transgene encoding a V region specific for the hapten 2-phenyloxazolone (phOx) linked to a rat C kappa gene. Two independent lines of transgenic mice were hyperimmunized with phOx and splenic hybridomas established. In B cells that had been selected by antigen and which used mouse anti-phOx genes, the endogenous sequences were found to be mutated whereas the transgene remained unchanged. These results suggest either that (a) if the transgene is a "passenger" gene expressed at a low level, transgene mutation is a rare event, or that (b) sequences far from the kappa coding region are necessary to direct somatic mutation.

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Cellular selection leads to age-dependent and reversible down-regulation of transgenic immunoglobulin light chain genes

Cited by 33 publications

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Modifying the sequence of an immunoglobulin V-gene alters the resulting pattern of hypermutation

Modifying the sequence of an immunoglobulin V-gene alters the resulting pattern of hypermutation

IgM receptor-mediated transactivation of the IgH 3′ enhancer couples a novel Elf-1-AP-1 protein complex to the developmental control of enhancer function.

Lack of somatic mutation in a χ light chain transgene

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