Cellular, intracellular, and developmental expression patterns of murine SWAP-70.

SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.

Cloning and characterization of mammalian SMC1 and SMC3 genes and proteins, components of the DNA recombination complexes RC-1.

Members of the evolutionary conserved Structural Maintenance of Chromosomes (SMC) protein family are involved in chromosome condensation and gene dosage compensation with the SMC2 and SMC4 subtypes, and sister chromatid cohesion with the SMC1 and SMC3 subtypes. The bovine recombination protein complex RC-1, which catalyzes DNA transfer reactions, contains two heterodimeric SMC polypeptides, the genes of which have now been cloned, sequenced, and classified as bovine (b)SMC1 and bSMC3. Both proteins display all the characteristic features of the SMC family. FISH analysis localized the mouse SMC3 gene to chromosome 19D2-D3. Mono- and polyclonal antibodies specific for either subtype detected high levels of protein expression in lymphoid tissues, lung, testis and ovary. No change in levels of bSMC1 and bSMC3 proteins occurred after X-ray or UV-light irradiation of various cell lines or primary cells, and the amounts of individual proteins and the heterodimer are roughly constant throughout the cell cycle. Immunofluorescence of mouse cells detected the SMC1 protein in foci associated with the chromatin. These foci dissolve and the SMC protein dissociates from the chromatin during M phase.

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.

Expression of DNA-dependent protein kinase holoenzyme upon induction of lymphocyte differentiation and V(D)J recombination.

Murine preB lymphocytes grow in tissue culture in the presence of stromal cells and interleukin 7 (IL-7), and can be induced to differentiate to surface-immunoglobulin-positive B cells in vitro by withdrawal of IL-7. Upon differentiation, proliferation ceases, and upregulation of Rag-1 and Rag-2 expression, and induction of V(D)J immunoglobulin-gene rearrangements occur. DNA-dependent protein kinase (DNA-PK) is required for effective V(D)J recombination and repair of DNA double-strand breaks. The holoenzyme comprises a catalytic subunit (DNA-PKcs) and the Ku heterodimer (Ku70/Ku80). We have analyzed expression of Ku70, Ku80 and DNA-PKcs upon induction of differentiation in preB cells derived from wild-type, severe combined immunodeficiency (SCID) and Rag-2-/- mice. Protein levels of Ku80 and Ku70 moderately decrease after induction in all three cell types. A distinct polypeptide that crossreacts with anti-Ku Ig appears in the cytoplasm of wild-type and Rag-2-/- cells, but not of SCID cells. In mouse preB cells, Ku70 and Ku80 are present in the nuclei and cytoplasm before and after onset of differentiation. In vivo, Ku70 is predominantly expressed in V(D)J-recombination-active, early-preB and CD4-/CD8- thymocyte cell populations. Upon differentiation, protein levels of DNA-PKcs are unaltered. DNA-PK activity, which is not detectable in SCID cells, increases in wild-type and Rag-2-/- cells more than twofold shortly after induction of differentiation, then falls back to about 50% of starting levels.

SMC proteins constitute two subunits of the mammalian recombination complex RC-1.

Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC-1. N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities.

Role of environmental antigen in the development of IgG+ cells in the bursa of fabricius.

IgG+ cells were detected in the bursa of Fabricius after hatching by immunofluorescence staining of single cells and immunohistologic studies using mAb anti-Ig gamma-heavy chain. The frequency of IgG+ cells in the bursa increased rapidly immediately after hatching. In histologic studies, most of the IgG+ cells were found in the medullary areas of the bursal follicle. Isolation of the bursa from the gut by bursal duct ligation before hatching suppressed the development of IgG+ cells in the bursa after hatching. In addition, administration of Ags into the bursal lumen just before bursal duct ligation caused a significant increase of IgG+ cells in the bursa compared with the isolated bursa. However, parenteral administration of Ags had no effect on the frequency of IgG+ cells in the isolated and normal bursa. These results suggest that Ags in the bursa are a prerequisite for the development of IgG+ cells in the bursa. Although the majority of IgG+ bursal cells were IgM+ IgG+ double-positive cells, sorted Bu1+ bursal cells, which contained 99.9% of IgG+ cells but not plasma cells, synthesized de novo IgM but little or no IgG. Therefore, it is likely that surface IgG on the bursal cells is not produced locally, but is trapped by IgM+ bursal cells. We speculate that Ags derived from the bursal lumen form immune complexes within the bursa, which are subsequently trapped on the surface of IgM+ bursal cells mediated either by Fc-receptors, C3 receptors, or surface Ig.

Stimulation of defective DNA transfer activity in recombination deficient SCID cell extracts by a 72-kDa protein from wild-type thymocytes.

The SCID (Severe Combined Immune Deficiency) mutation causes two DNA recombination deficiencies: an aberrant joining of V(D)J immunoglobulin gene elements and a failure to perform efficient repair of DNA double-strand breaks. A recently established cell-free assay for DNA transfer (DTA) was applied to study nuclear extracts from normal and SCID-derived cells. The recombination deficiency was reflected in the cell-free system: SCID lymphocyte and fibroblast extracts showed reduced levels of DTA activity on a variety of DNA substrates. Analysis of nuclear extracts prepared from wild-type thymocytes and B cells representing different stages in lymphocyte ontogeny revealed the highest activities at the most immature stages. With progression of development, DTA activity decreased. Corresponding to their early developmental arrest, V(D)J rearrangement-incompetent RAG-2-/- lymphocyte extracts show high DTA activity. In contrast, extracts from SCID early lymphocytes express very low DNA transfer activity. Induction of V(D)J rearrangement in vivo in a normal preB cell line lead to a co-induction of the cell-free recombination activity. This indicates a development stage specificity of cell-free DNA recombination, which temporally parallels V(D)J recombination. A protein could be purified to near-homogeneity from wild-type thymocytes which stimulates the recombination activity specifically in SCID thymocyte and proB cell extracts. This protein, SRSP (SCID Recombination Stimulatory Protein), migrates as a single band of approximately 72 kDa in SDS-polyacrylamide gel electrophoresis.

Characterization of the putative avian CD2 homologue.

We describe two mouse mAb recognizing the putative chicken homologue of mammalian CD2 Ag and provide evidence for both structural and functional conservation between the avian and mammalian CD2 molecules. The antibodies were T cell-specific and immunoprecipitated a single diffuse band of Mr 40,000 from lysates of surface-labeled chicken thymocytes and peripheral T cells. Removal of N-linked carbohydrate with endo-beta-N-acetylglucosaminidase F revealed the core protein size of Mr 25,000. A rabbit antiserum raised against a synthetic peptide (CD2-300), composed of 18 amino acid residues of the conserved cytoplasmic domain of human, mouse, and rat CD2, precipitated an Ag similar to chicken CD2. Sequential precipitation with CD2-300 antiserum indicated the conservation of an avian and mammalian CD2 epitope. CD2 expression on thymocytes starts at day 11 of embryonic development, and, during subsequent development, thymic gamma delta cells are all CD2+, whereas most peripheral gamma delta-T cells lack CD2. Functional conservation between the chicken and mammalian CD2 molecules was demonstrated by the induction of DNA synthesis in chicken thymocytes and peripheral T cells with the combination of anti-CD2 mAb and PMA.