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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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Flow cytometric  analysis and sorting of B cell developmental stages from Balb/c  bone marrow. (A) Triple staining for B220, cIgμ, and CD2  (left dot plot), B220, CD2, and  sIgM (middle dot plot), and B220,  sIgM, and sIgD (right dot plot). In  each case, the data are gated on  lymphocytes by forward and side  scatter and on B220+ cells by fluorescent staining. Numbered  boxes identify the defined B cell  subpopulations (see Table 1): 1 =  pro-B cells; 2 = early pre-B  cells; 3 = late pre-B cells; 4 =  immature B cells; 5 = mature B  cells. (B) Analysis of B cell populations numbered as in A) after  sorting. The staining criteria used  for sorting and the purity of each  sorted subpopulation are indicated in each panel. The data are  ungated.
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Figure 1: Flow cytometric analysis and sorting of B cell developmental stages from Balb/c bone marrow. (A) Triple staining for B220, cIgμ, and CD2 (left dot plot), B220, CD2, and sIgM (middle dot plot), and B220, sIgM, and sIgD (right dot plot). In each case, the data are gated on lymphocytes by forward and side scatter and on B220+ cells by fluorescent staining. Numbered boxes identify the defined B cell subpopulations (see Table 1): 1 = pro-B cells; 2 = early pre-B cells; 3 = late pre-B cells; 4 = immature B cells; 5 = mature B cells. (B) Analysis of B cell populations numbered as in A) after sorting. The staining criteria used for sorting and the purity of each sorted subpopulation are indicated in each panel. The data are ungated.

Mentions: Despite the wealth of known regulated surface antigens on developing B cells (9, 39), the only markers of unambiguous functional significance with regard to V(D)J recombination are the products of the Ig genes themselves. Accordingly, we have divided the B cell lineage into pro-B cells, which do not express functional Igμ, pre-B cells, which express Igμ in their cytoplasm (cIgμ) but not on their surface, immature B cells, which express Igμ and IgL (IgM complex) on their surface, but little or no δ isotype heavy chains (IgD), and mature B cells, which express both IgM and high levels of IgD on their surface. The pre-B cell population was further subdivided based on the expression of the CD2 surface antigen into early CD2− and late CD2+ pre-B cells. This marker was chosen because it is developmentally regulated to appear at a point intermediate between cIgμ and sIgM (5) (Fig. 1 A), and its acquisition correlates well with the cell cycle behavior of pre-B cells (see below). These populations were identified by staining bone marrow cells from adult Balb/c mice with various combinations of antibodies to the B220, cIgμ, CD2, sIgM, and sIgD antigens and analyzing them by 3-color flow cytometry (Fig. 1 A and Table 1).


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)

Flow cytometric  analysis and sorting of B cell developmental stages from Balb/c  bone marrow. (A) Triple staining for B220, cIgμ, and CD2  (left dot plot), B220, CD2, and  sIgM (middle dot plot), and B220,  sIgM, and sIgD (right dot plot). In  each case, the data are gated on  lymphocytes by forward and side  scatter and on B220+ cells by fluorescent staining. Numbered  boxes identify the defined B cell  subpopulations (see Table 1): 1 =  pro-B cells; 2 = early pre-B  cells; 3 = late pre-B cells; 4 =  immature B cells; 5 = mature B  cells. (B) Analysis of B cell populations numbered as in A) after  sorting. The staining criteria used  for sorting and the purity of each  sorted subpopulation are indicated in each panel. The data are  ungated.
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Related In: Results  -  Collection

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Figure 1: Flow cytometric analysis and sorting of B cell developmental stages from Balb/c bone marrow. (A) Triple staining for B220, cIgμ, and CD2 (left dot plot), B220, CD2, and sIgM (middle dot plot), and B220, sIgM, and sIgD (right dot plot). In each case, the data are gated on lymphocytes by forward and side scatter and on B220+ cells by fluorescent staining. Numbered boxes identify the defined B cell subpopulations (see Table 1): 1 = pro-B cells; 2 = early pre-B cells; 3 = late pre-B cells; 4 = immature B cells; 5 = mature B cells. (B) Analysis of B cell populations numbered as in A) after sorting. The staining criteria used for sorting and the purity of each sorted subpopulation are indicated in each panel. The data are ungated.
Mentions: Despite the wealth of known regulated surface antigens on developing B cells (9, 39), the only markers of unambiguous functional significance with regard to V(D)J recombination are the products of the Ig genes themselves. Accordingly, we have divided the B cell lineage into pro-B cells, which do not express functional Igμ, pre-B cells, which express Igμ in their cytoplasm (cIgμ) but not on their surface, immature B cells, which express Igμ and IgL (IgM complex) on their surface, but little or no δ isotype heavy chains (IgD), and mature B cells, which express both IgM and high levels of IgD on their surface. The pre-B cell population was further subdivided based on the expression of the CD2 surface antigen into early CD2− and late CD2+ pre-B cells. This marker was chosen because it is developmentally regulated to appear at a point intermediate between cIgμ and sIgM (5) (Fig. 1 A), and its acquisition correlates well with the cell cycle behavior of pre-B cells (see below). These populations were identified by staining bone marrow cells from adult Balb/c mice with various combinations of antibodies to the B220, cIgμ, CD2, sIgM, and sIgD antigens and analyzing them by 3-color flow cytometry (Fig. 1 A and Table 1).

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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