Combinatorial regulation of the murine RAG-2 promoter by Sp1 and distinct lymphocyte-specific transcription factors.

The recombination activation genes, RAG-1 and RAG-2, encode the critical components of the recombinase complex responsible for the generation of functional antigen receptor genes. In order to gain an insight into the transcription factors and cis-acting elements that regulate the lymphocyte-specific expression of RAG-2, the promoter-region of this gene was isolated and characterized. This analysis demonstrated that a relatively small promoter fragment could confer lymphocyte-restricted expression to a reporter construct. Our work and that of others subsequently revealed that RAG-2 promoter expression is positively regulated by BSAP (PAX-5) and c-Myb transcription factors in B- and T-lineage cells, respectively. Although BSAP and c-Myb were deemed necessary for lymphocyte-specific expression, our analysis also uncovered a G-rich region at the 5'-end of the core promoter that was essential for full activity in lymphocyte cell lines. Site-directed mutagenesis revealed that a GA-box within the G-rich region was required for full promoter activity and subsequent DNA binding assays demonstrated that this element was specifically recognized by Sp1. Apart from showing that Sp1 interacts within the RAG-2 promoter, we also demonstrate that the Sp1-binding site is necessary for the high-level activation of this promoter.

Bc3h1 myogenic cells produce an infectious ecotropic murine leukemia virus.

cDNAs representing an endogenous C-type ecotropic murine leukemia virus were isolated from a cDNA library constructed to represent mRNAs present in BC3H1 myogenic cells but not in C2C12 myogenic cells. RNA blot hybridization analysis using the cDNA inserts as probes revealed that BC3H1 cells produce MuLV-related transcirpts of at least three different size classes. A polymerase chain reaction enhanced assay for reverse transcriptase activity revealed the presence of reverse transcriptase in a viral pellet from medium conditioned by BC3H1 cells. A fungizone enhanced assay for syncitium formation provided further evidence of ecotropic retroviral particle production. Exposure of 3T3 cells to medium conditioned by BC3H1 cells, using conditions that facilitate infection, resulted in infection of the 3T3 cells, as confirmed by the syncitium formation assay. We conclude that BC3H1 cells produce an infectious ecotropic murine leukemia virus. Whether or not this feature of BC3H1 cells contributes to their inability to express some muscle-specific genes or to carry out myotube formation is unknown. Investigators will want to take into account that BC3H1 cells are virus producers when planning experiments that involve coculture of BC3H1 with other cell types, BC3H1 conditioned medium, retrovirally mediated transfection into BC3H1 cells, or study of the mCAT-1 amino acid transporter (the viral receptor) in BC3H1 cells. BC3H1 cells and the virus they produce may be of interest to those studying retroviral genomes and products and their effects.

Further characterization of BC3H1 myogenic cells reveals lack of p53 activity and underexpression of several p53 regulated and extracellular matrix-associated gene products.

To catalog factors that may contribute to the completion of myogenesis, we have been looking for molecular differences between BC3H1 and C2C12 cells. Cells of the BC3H1 tumor line, though myogenic, are nonfusing, and withdraw from the cell cycle only reversibly, whereas cells of the C2C12 line fuse, differentiate terminally, and express several muscle-specific gene products that BC3H1 cells do not. Relative to C2C12 cells, BC3H1 cells underaccumulated cyclin-dependent kinase inhibitor p21 and underaccumulated transcripts for p21, GADD45, CDO, decorin, osteopontin, H19, fibronectin, and thrombospondin-1 (tsp-1). Levels of accumulation of H19, tsp-1, and larger isoforms of fibronectin messenger ribonucleic acid (mRNA) were found to increase in response to expression of myogenic regulatory factors as shown by their accumulation in differentiated myogenically converted 10T1/2 cells but not in 10T1/2 fibroblasts. BC3H1s accumulated a temperature-insensitive, geldanamycin-sensitive, misfolded form of p53 incapable of transactivating a p53 responsive reporter, consistent with underexpression of p21, GADD45, and tsp-1. BC3H1 and C2C12 cells were similar with respect to upregulation of p27 protein, downregulation of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein, upregulation of retinoblastoma (Rb) mRNA, and nuclear localization of hypophosphorylated Rb. Cells of both lines expressed the muscle-specific 1b isoform of MEF2D. Although nonfusing in the short term, after more than 18 d in differentiation medium, some cultures of BC3H1 cells formed viable multinucleated cells in which the nuclei did not reinitiate synthesis of DNA in response to serum. Our findings suggest participation of tsp-1 and specific isoforms of fibronectin in myogenesis and suggest additional avenues of research in myogenesis and oncogenesis.

Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis.

We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the approximately 160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.