RAG-1 and RAG-2 are not sufficient to direct all phases of immunoglobulin gene rearrangement in pre-B-cell lines.

To probe the factors controlling immunoglobulin heavy-chain gene rearrangement, we analyzed Abelson virus-transformed pre-B-cell lines that fail to undergo VH-to-DJH joining at an appreciable frequency. Despite this feature, some of these cell lines (rechi) rearrange an extrachromosomal recombination substrate at levels normal for transformed pre-B cells. Others (reclo) rearrange these substrates at levels characteristic of nonlymphoid hematopoietic cells. The DJH rearrangements from a representative rechi cell line were aberrant, suggesting that these cells probably fail to complete heavy-chain gene assembly because some of the necessary cis-acting signals are missing. In contrast, both DJH rearrangements from a reclo cell line appeared normal in structure, indicating that trans-acting factors necessary for recombination might be missing. Introduction of the RAG-1 and RAG-2 genes, genes encoding two such factors, failed to confer a rechi phenotype to these cells. However, fusion of the reclo cells to a rechi cell line generated a high frequency of rechi hybrids. In addition, most of the hybrids rearranged the endogenous kappa light-chain locus. Neither the rechi phenotype nor kappa-chain rearrangement correlated with levels of RAG-1 and RAG-2 expression in all of the hybrids. Thus, both gene transfer and cell fusion experiments indicate that RAG-1 and RAG-2 are not sufficient to activate immunoglobulin gene recombination in at least some pre-B-cell lines. In addition, the fusion experiments suggest that two gene products in addition to RAG-1 and RAG-2 may be required for kappa-gene rearrangement.

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Immunoglobulin gene rearrangements and expression in diffuse histiocytic lymphomas reveal cellular lineage, molecular defects, and sites of chromosomal translocation

Blood ◽

10.1182/blood.v67.2.391.391 ◽

1986 ◽

Vol 67 (2) ◽

pp. 391-397 ◽

Cited By ~ 18

Author(s):

KA Siminovitch ◽

JP Jensen ◽

AL Epstein ◽

SJ Korsmeyer

Keyword(s):

B Cell ◽

Cell Line ◽

Heavy Chain ◽

Light Chain ◽

Cell Lineage ◽

Chromosomal Translocation ◽

Chain Gene ◽

Gene Rearrangements ◽

Immunoglobulin Gene ◽

Light Chain Gene

Abstract We have examined the immunoglobulin gene configurations in cell lines from eight patients with diffuse histiocytic lymphoma in order to establish the cellular lineage and stage of differentiation of these lymphomas. The presence of heavy and light chain gene rearrangements as well as heavy chain class switching in seven cells placed these tumors within the B cell lineage. In contrast, one cell (SU-DHL-1), which lacks B cell-restricted surface antigens, retained germline heavy and light chain loci, indicating that it may represent a true histiocyte or uncommitted cell. Truncated RNAs for both the heavy and light chain immunoglobulins were responsible for the lack of surface immunoglobulin in the SU-DHL-2 cell line. Another cell line (SU-DHL-6), which possesses a t(14;18)(q32;q21) translocation, demonstrated an unexpected recombination within its heavy chain gene locus that may be the interchromosomal breakpoint.

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Stimulation of kappa light-chain gene rearrangement by the immunoglobulin mu heavy chain in a pre-B-cell line

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5679-5690.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5679-5690

Author(s):

A M Shapiro ◽

M S Schlissel ◽

D Baltimore ◽

A L DeFranco

Keyword(s):

B Cell ◽

Cell Lines ◽

Heavy Chain ◽

Gene Rearrangement ◽

Germ Line ◽

Gene Rearrangements ◽

Matched Pairs ◽

Immunoglobulin Gene ◽

Kappa Light Chain ◽

Immunoglobulin Mu

B-lymphocyte development exhibits a characteristic order of immunoglobulin gene rearrangements. Previous work has led to the hypothesis that expression of the immunoglobulin mu heavy chain induces rearrangement activity at the kappa light-chain locus. To examine this issue in more detail, we isolated five matched pairs of mu- and endogenously rearranged mu+ cell lines from the Abelson murine leukemia virus-transformed pro-B-cell line K.40. In four of the five mu+ cell lines, substantial expression of mu protein on the cell surface was observed, and this correlated with an enhanced frequency of kappa immunoglobulin gene rearrangement compared with that in the matched mu- cell lines. This increased kappa gene rearrangement frequency was not due to a general increase in the amount of V(D)J recombinase activity in the mu+ cells. Consistently, introduction of a functionally rearranged mu gene into one of the mu- pre-B-cell lines resulted in a fivefold increase in kappa gene rearrangements. In three of the four clonally matched pairs with increased kappa gene rearrangements, the increase in rearrangement frequency was not accompanied by a significant increase in germ line transcripts from the C kappa locus. However, in the fourth pair, K.40D, we observed an increase in germ line transcription of the kappa locus after expression of mu protein encoded by either an endogenously rearranged or a transfected functional heavy-chain allele. In these cells, the amount of the germ line C kappa transcript correlated with the measured frequency of rearranged kappa genes. These results support a regulated model of B-cell development in which mu protein expression in some way targets the V(D)J recombinase to the kappa gene locus.

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Heavy chain immunoglobulin gene rearrangement in acute nonlymphocytic leukemia

Blood ◽

10.1182/blood.v63.5.1023.bloodjournal6351023 ◽

1984 ◽

Vol 63 (5) ◽

pp. 1023-1027

Author(s):

U Rovigatti ◽

J Mirro ◽

G Kitchingman ◽

G Dahl ◽

J Ochs ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Heavy Chain ◽

Gene Rearrangement ◽

Leukemia Cell ◽

Leukemic Cell ◽

Immunoglobulin Gene ◽

Acute Nonlymphocytic Leukemia ◽

Lymphoid Leukemia ◽

Immunoglobulin Gene Rearrangement

Samples of leukemic cell DNA from 14 children with acute nonlymphocytic leukemia (ANLL) and 4 human myeloid leukemia cell lines were analyzed for rearrangement in the heavy chain region of the immunoglobulin gene. The diagnosis of ANLL was confirmed in all patients by morphological, cytochemical, and immunologic studies. By restriction endonuclease digestion and hybridization with cloned heavy chain immunoglobulin gene probes for the constant (Cmu) and joining (JH) regions, the DNA of 2 patients and 1 cell line (ML-1) was found to contain rearrangements. The DNA from the remaining 12 patients and 3 cell lines was not rearranged (germline configuration). Both patients with apparent immunoglobulin gene rearrangement achieved complete remission on therapy for ANLL. Immunoglobulin gene rearrangement in phenotypically defined ANLL suggests (1) that such changes may not be limited to lymphoid leukemia of B cell lineage, or (2) that, in some patients, the leukemic transforming event may involve stem cells capable of both B cell and myeloid differentiation.

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Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains.

Journal of Experimental Medicine ◽

10.1084/jem.178.1.139 ◽

1993 ◽

Vol 178 (1) ◽

pp. 139-149 ◽

Cited By ~ 45

Author(s):

M E Pauza ◽

J A Rehmann ◽

T W LeBien

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Surface ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Gene Rearrangement ◽

Immunoglobulin Gene ◽

Human B Cells ◽

Sds Page

Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow cytometry and were clonal by Southern blotting. Surprisingly, two of the mu/lambda-expressing cell lines contained both kappa alleles in germline configuration, and synthesis/expression of conventional lambda L chains was directly proven by immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in one of them. Thus, human fetal bone marrow B lineage cells harbor the capacity to make functional lambda L chain gene rearrangements without rearranging or deleting either kappa allele. A third unusual cell line, designated 30.30, was observed to coexpress cell surface kappa and lambda L chains associated with mu H chains. The 30.30 cell line had a diploid karyotype, a single H chain rearrangement, both kappa alleles rearranged, and a single lambda rearrangement. Immunoprecipitation/SDS-PAGE confirmed that 30.30 cells synthesized and expressed kappa and lambda L chains. Multiparameter flow cytometry was used to demonstrate the existence of kappa+/lambda+ cells in fetal bone marrow and fetal spleen at frequencies of 2-3% of the total surface Ig+ B cell population. The flow cytometry data was confirmed by two-color immunofluorescence microscopy. The existence of normal human B cells expressing cell surface kappa and lambda refutes the widely accepted concept that expression of a single L chain isotype is immutable. The kappa+/lambda+ cells may represent transients undergoing L chain isotype switching.

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