scholarly journals Mouse Lymphoid Cell Line Selected To Have High Immunoglobulin Promoter Activity

2002 ◽  
Vol 22 (5) ◽  
pp. 1460-1473 ◽  
Author(s):  
Dean Tantin ◽  
Phillip A. Sharp
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Lymphoid Cell ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Immunoglobulin Heavy Chain ◽  
Parental Line ◽  
Lymphoid Cell Line ◽  
Variable Regions

ABSTRACT Immunoglobulin variable region promoters are predominantly B-cell specific, but the molecular basis for this specificity has not been elucidated. To further understand how B-cell-specific immunoglobulin promoter expression is mediated, the murine lymphoid cell line 2017 was engineered to express the green fluorescent protein under the control of an immunoglobulin heavy chain promoter and selected for high activity using multiple rounds of fluorescence-activated cell sorting. Rare clones with intense and stable immunoglobulin promoter activity were isolated. Transient transfection experiments demonstrated that two different immunoglobulin promoters and two other B-cell-specific promoters have higher activities in the selected cell lines relative to the parental line and to the non-cell-type-specific histone H2B promoter. The increased immunoglobulin activity required nucleotide residues downstream of the transcription initiation site which were also important for maximal activity in B cells and which were conserved in other B-cell-specific promoters. Unlike the unselected cells, the 2017 variants also showed activation of their endogenous immunoglobulin heavy chain variable regions.

scholarly journals Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

1987 ◽  
Vol 7 (1) ◽  
pp. 450-457 ◽  
Author(s):  
E H Brown ◽  
M A Iqbal ◽  
S Stuart ◽  
K S Hatton ◽  
J Valinsky ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Cluster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Dna Content ◽  
Immunoglobulin Heavy Chain ◽  
S Phase ◽  
C Value ◽  
Murine Erythroleukemia

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

scholarly journals Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells.

1994 ◽  
Vol 14 (6) ◽  
pp. 4087-4096 ◽  
Author(s):  
Y Jacobs ◽  
X Q Xin ◽  
K Dorshkind ◽  
C Nelson
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
B Lymphocyte ◽  
Myeloid Cells ◽  
Immunoglobulin Heavy Chain ◽  
Cell Development ◽  
B Cell Development ◽  
Chain Gene ◽  
Enhancer Element

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.

scholarly journals CLASS II-RESTRICTED PRESENTATION OF AN IMMUNOGLOBULIN HEAVY-CHAIN-GENE PRODUCT BY A GENE-TRANSFECTED B-CELL LINE

1994 ◽  
Vol 47 (4) ◽  
pp. 195-210
Author(s):  
Yasushi MATSUURA ◽  
Saburo ONISHI ◽  
Yasutake YAMAMOTO ◽  
Taketoshi TANIGUCHI ◽  
Satoshi OBANA ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Product ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Class Ii ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Immunoglobulin Heavy Chain Gene

Immunoglobulin heavy-chain class switching in a pre-B cell line is accompanied by DNA rearrangement

Nature ◽  
1983 ◽  
Vol 306 (5940) ◽  
pp. 243-246 ◽  
Author(s):  
Peter D. Burrows ◽  
Gabriele B. Beck-Engeser ◽  
Matthias R. Wabl
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Dna Rearrangement ◽  
Class Switching

Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells

1994 ◽  
Vol 14 (6) ◽  
pp. 4087-4096
Author(s):  
Y Jacobs ◽  
X Q Xin ◽  
K Dorshkind ◽  
C Nelson
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
B Lymphocyte ◽  
Myeloid Cells ◽  
Immunoglobulin Heavy Chain ◽  
Cell Development ◽  
B Cell Development ◽  
Chain Gene ◽  
Enhancer Element

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.

scholarly journals Biphenotypic leukemic lymphocyte precursors in CD2+CD19+ acute lymphoblastic leukemia and their putative normal counterparts in human fetal hematopoietic tissues

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 1000-1015 ◽  
Author(s):  
FM Uckun ◽  
A Muraguchi ◽  
JA Ledbetter ◽  
T Kishimoto ◽  
RT O'Brien ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Lymphoblastic Leukemia ◽  
Immunoglobulin Heavy Chain ◽  
Lymphoid Cells ◽  
Constant Region ◽  
Leukemic Lymphocyte

Abstract During detailed immunophenotypic analyses of marrow blasts from 336 acute lymphoblastic leukemia (ALL) patients, a very small percentage of cases reactive with B-cell-directed as well as T-cell-directed monoclonal antibodies (MoAbs) were identified. Five ALL cases were biphenotypic since they coexpressed CD2 (Tp50) and CD19 (Bp95) antigens at the single-cell level. The composite immunophenotype of these biphenotypic ALL cases was [TdT+HLA-ABC+CD2+CD3-CD10+CD13-CD14-CD16- CD19+CD20+ ++-CD21-CD33-CD34+Bgp95-C mu- slg-]. Low-molecular-weight B- cell growth factor (LMW-BCGF), recombinant interleukin-2 (rIL-2), and rIL-3 stimulated the proliferative activity of biphenotypic leukemic lymphocyte precursors without inducing differentiation. In the presence of the phorbol ester TPA, leukemic blasts from two cases differentiated along the B precursor pathway to the [CD2-CD10+CD19+CD20+C mu+slg-] pre- B cell stage. Biphenotypic ALL cases did not share a common configuration and gene rearrangement pattern of the immunoglobulin heavy chain genes or T-cell receptor (TCR) genes. Three cases had rearranged C mu genes but germline TCR genes, one case showed rearrangement of both C mu and TCR genes, and the remaining case had rearranged TCR genes but germline C mu genes. All five patients attained prompt remission after standard induction chemotherapy. Three to four years after initial diagnosis, four patients are now off chemotherapy and remain alive in their first remission. One patient relapsed at 3 years, 7 months, but promptly achieved complete remission after reinduction chemotherapy and remains in second remission off chemotherapy greater than 3 years after her reinduction therapy. With two-color immunofluorescence staining techniques and multiparameter flow cytometric analyses, we identified a small population of CD2+CD19+ lymphoid cells in fetal livers (FLs) and fetal bone marrows (FBMs), which may represent the putative normal counterparts of biphenotypic ALL blasts. A CD2+CD19+ normal biphenotypic lymphoid precursor cell line, designated FL 8.2 CD2+, was established from an FL of 8-weeks of gestational age by Epstein-Barr virus (EBV)-induced blastoid transformation. The composite immunophenotype of FL 8.2 CD2+ cell line was [TdT+HLA-ABC+HLA-DR+ CD2+CD5-CD7-CD10+/-CD13-CD19+CD20-CD21+ CD22+CD33-CD34+/-Bgp95-CDw40+C mu-slgD-slgM-]. FL 8.2 CD2+ cells showed germline patterns of immunoglobulin heavy-chain joining region, heavy- chain constant region, kappa light-chain constant region genes, and TCR beta-chain genes. Cross-linking of CD2 as well as CD19 antigens on FL 8.2 CD2+ cells caused an increase of intracellular ionized calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region.

1997 ◽  
Vol 17 (10) ◽  
pp. 6167-6174 ◽  
Author(s):  
J S Michaelson ◽  
O Ermakova ◽  
B K Birshtein ◽  
N Ashouian ◽  
C Chevillard ◽  
...  
Keyword(s):  
Dna Replication ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Regulatory Region ◽  
Replication Timing ◽  
Immunoglobulin Heavy Chain ◽  
Igh Locus ◽  
Early Replication

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.

Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon

1987 ◽  
Vol 7 (1) ◽  
pp. 450-457
Author(s):  
E H Brown ◽  
M A Iqbal ◽  
S Stuart ◽  
K S Hatton ◽  
J Valinsky ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Cluster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Dna Content ◽  
Immunoglobulin Heavy Chain ◽  
S Phase ◽  
C Value ◽  
Murine Erythroleukemia

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

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