?(Fig.7C).7C). PKC cleavage product in vitro, and caspase inhibitors prevent the generation of this product during apoptosis in transfected cell lines. Inducible overexpression of either the full-length or truncated form of PKC results in cell cycle arrest at the G1/S transition. These results suggest that the expression and proteolytic activation of PKC play an important role in the regulation of cell division and cell death during early B-cell development. B-cell development is usually characterized by the ordered assembly of immunoglobulin (Ig) heavy- and light-chain genes from their component gene segments by a site-specific DNA rearrangement reaction known as V(D)J recombination (61). This reaction is usually regulated such that heavy-chain genes assemble before light-chain genes, and an individual B cell expresses only one functional gene of each type (allelic exclusion [44, 46]). Heavy-chain protein is usually expressed around the surfaces of developing AM679 pre-B cells along AM679 with surrogate light chains and the transmission transduction molecules Ig and Ig in a complex known as the pre-B-cell receptor (pre-BCR). The pre-BCR is usually a critical regulator of development, responsible for the activation of Ig-light-chain locus rearrangement and the inactivation of allelic heavy-chain locus rearrangement (35, 49, 52). Mutational inactivation of any of the components of the pre-BCR prospects to developmental arrest at a distinct stage of B-cell development (13, 23, 24). Developing pro-B cells which fail to assemble the pre-BCR undergo apoptosis, whereas cells expressing the pre-BCR increase expression of the anti-apoptotic Bcl-xL gene and survive for an extended period (10). Furthermore, the ongoing expression of surface Ig is essential for B-cell viability (26). Due to the sum of these processes, the great majority of developing B cells fail to survive. In addition to regulating gene rearrangement and cell survival, the pre-BCR signals specific alterations in the transcription of several developmentally regulated genes, including those encoding Bcl-x, TdT, and 5, and the germline light chain locus (10, 27, 58). In order to more fully define the set of genes regulated by expression of the pre-BCR, we isolated developmentally arrested pro-B cells from RAG1-deficient mice, pre-B cells from RAG1-deficient/-transgenic mice (58) and mature B cells from wild-type spleen, and used RNA from these cells to perform representational difference analysis (RDA [19, 29]). This approach led to the isolation of a large set of cDNA fragments whose expression was either positively or negatively regulated by expression of the pre-BCR. Strikingly, many of the genes encode proteins involved in apoptosis. We statement here around the regulated expression and posttranslational modification of one of these genes, encoding protein kinase C (PKC), and present evidence suggesting that PKC may be involved in the regulation of programmed cell death by the pre-BCR. MATERIALS AND METHODS Purification of CD19+ B cells. B cells were purified from your bone marrow of RAG1-deficient and RAG1-deficient/-transgenic AM679 mice (58) and from your spleen of wild-type mice by using biotinylated monoclonal rat anti-mouse CD19 antibody (25) and streptavidin paramagnetic beads AM679 (MiniMacs system; Milltenyi Biotech) as previously explained (54). In some experiments, less-mature bone marrow B-lineage cells were purified by the depletion of secretory IgM-positive (sIgM+) cells by using a monoclonal rat anti-mouse IgM antibody, yielding a mixed populace of pro-B and pre-B cells, followed by selection with biotinylated anti-CD19 antibody. In addition, wild-type pro-B and pre-B cells were processed in a fluorescence-activated cell sorter (FACS) with anti-CD19, -CD43, and -IgM antibodies. The purity of selected populations was assessed by circulation cytometry by using biotinylated monoclonal rat anti-mouse CD19, fluorescein isothiocyanate-conjugated monoclonal rat anti-mouse CD43 (17), and phycoerythrin-conjugated goat anti-mouse IgM antiserum (Southern Biotech). RDA process. Cells were pelleted and poly(A)+ RNA was directly purified with the Micro-FastTrack mRNA Isolation Kit (Invitrogen). Poly(A)+ RNA was converted to double-stranded cDNA by using the cDNA Synthesis System (Gibco BRL) according to the manufacturers instructions. cDNA (2 g) was then digested with has been shown to induce apoptosis in cell extracts (30), NADH-ubiquinone oxidoreductase is usually a potent generator of reactive oxygen species (47), and inhibitors of F1-ATPase have been shown AM679 to induce apoptosis in the WEHI 231 B cell collection CYSLTR2 (39). Galectin 9 is usually a recently recognized member of a family of proteins which have been shown to stimulate superoxide production (62) and to induce apoptosis of T cells (42, 43). A similar set of genes was also recognized in a screen for transcripts induced by p53 expression before the onset of apoptosis (45). TABLE 1 Subtracted?genesa Pre-B-cell-specific genes ?Group I ??Effector cell protease receptor 1??Stathmin??Calpactin ??Topoisomerase II ??Inositol triphosphate receptor 1??Ki-67 ??LPS-inducible gene ??Cyclin-dependent kinase ??Rag 2 ?Group II ??Mi-2 ??Y-box transcription factor ??ZNF 131 ??TIS 21??ALL-1 ??SAM synthetase ??p78 kinase ??Sox-4 ??Tax-1 ??21-kDa polypeptide ??Cytochrome tetracycline repressor fused to.