A human gene coding for a membrane-associated nucleic acid-binding protein.

Studies to clone a cell-surface DNA-binding protein involved in the binding and internalization of extracellular DNA have led to the isolation of a gene for a membrane-associated nucleic acid-binding protein (MNAB). The full-length cDNA is 4.3 kilobases with an open reading frame of 3576 base pairs encoding a protein of approximately 130 kDa (GenBank accession numbers and ). The MNAB gene is on human chromosome 9 with wide expression in normal tissues and tumor cells. A C3HC4 RING finger and a CCCH zinc finger have been identified in the amino-terminal half of the protein. MNAB bound DNA (K(D) approximately 4 nm) and mutagenesis of a single conserved amino acid in the zinc finger reduced DNA binding by 50%. A potential transmembrane domain exists near the carboxyl terminus. Antibodies against the amino-terminal half of the protein immunoprecipitated a protein of molecular mass approximately 150 kDa and reacted with cell surfaces. The MNAB protein is membrane-associated and primarily localized to the perinuclear space, probably to the endoplasmic reticulum or trans-Golgi network. Characterization of the MNAB protein as a cell-surface DNA-binding protein, critical in binding and internalization of extracellular DNA, awaits confirmation of its localization to cell surfaces.

Constitutive expression of steel factor gene by human stromal cells.

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.