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Changes in locus-specific V(D)J recombinase activity induced by immunoglobulin gene products during B cell development.

Constantinescu A, Schlissel MS - J. Exp. Med. (1997)

Bottom Line: This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus.We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele.Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an E mu-myc transgene.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an E mu-myc transgene.

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Proliferative behavior of B cells at different stages of  development. (A) DNA staining  of cells at different stages in the B  cell lineage in wild-type Balb/c  mice. Bone marrow cells were  stained for two developmental  markers using FITC- and PEconjugated antibodies and counterstained with 7-AAD to determine their DNA content. The  histograms represent data gated  on the indicated combination of  antigenic markers. The percentage of cells with DNA content  greater than G1 is indicated. (B)  Southern blot of RT-PCR analysis of cyclin D1 gene expression  in sorted B cell lineage cells.  Lane 1, pro-B and early pre-B  cells (B220+ CD2−); lane 2, late  pre-B cells (B220+CD2+sIgM−);  lane 3, immature and mature B  cells (B220+sIgM+); lanes 4–8,  serial dilution of cDNA from  3T3 cells in exponential growth  phase. The primers used in this  amplification reaction span an  intron, eliminating the possibility  of a false signal from contaminating genomic DNA. Transcripts  amplified by the H2 primers  come from a non-polymorphic  region of several MHC class I  molecules and are expressed at  constant levels. (C) DNA staining of cells at different stages in  the B cell lineage in Eμ-myc  transgenic mice. Staining and  data acquisition were done as in A.
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Figure 2: Proliferative behavior of B cells at different stages of development. (A) DNA staining of cells at different stages in the B cell lineage in wild-type Balb/c mice. Bone marrow cells were stained for two developmental markers using FITC- and PEconjugated antibodies and counterstained with 7-AAD to determine their DNA content. The histograms represent data gated on the indicated combination of antigenic markers. The percentage of cells with DNA content greater than G1 is indicated. (B) Southern blot of RT-PCR analysis of cyclin D1 gene expression in sorted B cell lineage cells. Lane 1, pro-B and early pre-B cells (B220+ CD2−); lane 2, late pre-B cells (B220+CD2+sIgM−); lane 3, immature and mature B cells (B220+sIgM+); lanes 4–8, serial dilution of cDNA from 3T3 cells in exponential growth phase. The primers used in this amplification reaction span an intron, eliminating the possibility of a false signal from contaminating genomic DNA. Transcripts amplified by the H2 primers come from a non-polymorphic region of several MHC class I molecules and are expressed at constant levels. (C) DNA staining of cells at different stages in the B cell lineage in Eμ-myc transgenic mice. Staining and data acquisition were done as in A.

Mentions: To assess the proliferative status of cells at each developmental stage, the DNA content of these cells was determined by flow cytometry. This was done using the DNAbinding dye 7-aminoactinomycin D (7-AAD) in conjunction with staining for two antigenic markers to identify the subpopulation of interest (B220+cIgμ− for pro-B cells, cIgμ+ CD2− for early pre-B cells, CD2+sIgM− for late pre-B cells, sIgM+sIgD− for immature B cells, and sIgM+sIgD+ for mature B cells). This analysis revealed that early pre-B cells had the highest fraction of cells with DNA content greater than G1 (Fig. 2 A). This population presumably comprises cells undergoing a proliferative burst in response to preBCR signaling. In contrast, late pre-B cells, as well as immature and mature B cells, appeared to be almost entirely quiescent, with very few cells in the S or G2/M phases of the cell cycle (Fig. 2 A).


Changes in locus-specific V(D)J recombinase activity induced by immunoglobulin gene products during B cell development.

Constantinescu A, Schlissel MS - J. Exp. Med. (1997)

Proliferative behavior of B cells at different stages of  development. (A) DNA staining  of cells at different stages in the B  cell lineage in wild-type Balb/c  mice. Bone marrow cells were  stained for two developmental  markers using FITC- and PEconjugated antibodies and counterstained with 7-AAD to determine their DNA content. The  histograms represent data gated  on the indicated combination of  antigenic markers. The percentage of cells with DNA content  greater than G1 is indicated. (B)  Southern blot of RT-PCR analysis of cyclin D1 gene expression  in sorted B cell lineage cells.  Lane 1, pro-B and early pre-B  cells (B220+ CD2−); lane 2, late  pre-B cells (B220+CD2+sIgM−);  lane 3, immature and mature B  cells (B220+sIgM+); lanes 4–8,  serial dilution of cDNA from  3T3 cells in exponential growth  phase. The primers used in this  amplification reaction span an  intron, eliminating the possibility  of a false signal from contaminating genomic DNA. Transcripts  amplified by the H2 primers  come from a non-polymorphic  region of several MHC class I  molecules and are expressed at  constant levels. (C) DNA staining of cells at different stages in  the B cell lineage in Eμ-myc  transgenic mice. Staining and  data acquisition were done as in A.
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Figure 2: Proliferative behavior of B cells at different stages of development. (A) DNA staining of cells at different stages in the B cell lineage in wild-type Balb/c mice. Bone marrow cells were stained for two developmental markers using FITC- and PEconjugated antibodies and counterstained with 7-AAD to determine their DNA content. The histograms represent data gated on the indicated combination of antigenic markers. The percentage of cells with DNA content greater than G1 is indicated. (B) Southern blot of RT-PCR analysis of cyclin D1 gene expression in sorted B cell lineage cells. Lane 1, pro-B and early pre-B cells (B220+ CD2−); lane 2, late pre-B cells (B220+CD2+sIgM−); lane 3, immature and mature B cells (B220+sIgM+); lanes 4–8, serial dilution of cDNA from 3T3 cells in exponential growth phase. The primers used in this amplification reaction span an intron, eliminating the possibility of a false signal from contaminating genomic DNA. Transcripts amplified by the H2 primers come from a non-polymorphic region of several MHC class I molecules and are expressed at constant levels. (C) DNA staining of cells at different stages in the B cell lineage in Eμ-myc transgenic mice. Staining and data acquisition were done as in A.
Mentions: To assess the proliferative status of cells at each developmental stage, the DNA content of these cells was determined by flow cytometry. This was done using the DNAbinding dye 7-aminoactinomycin D (7-AAD) in conjunction with staining for two antigenic markers to identify the subpopulation of interest (B220+cIgμ− for pro-B cells, cIgμ+ CD2− for early pre-B cells, CD2+sIgM− for late pre-B cells, sIgM+sIgD− for immature B cells, and sIgM+sIgD+ for mature B cells). This analysis revealed that early pre-B cells had the highest fraction of cells with DNA content greater than G1 (Fig. 2 A). This population presumably comprises cells undergoing a proliferative burst in response to preBCR signaling. In contrast, late pre-B cells, as well as immature and mature B cells, appeared to be almost entirely quiescent, with very few cells in the S or G2/M phases of the cell cycle (Fig. 2 A).

Bottom Line: This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus.We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele.Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an E mu-myc transgene.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an E mu-myc transgene.

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