Single Cell Gel Electrophoresis for the Detection of Genomic Ribonucleotides.

Single cell gel electrophoresis or comet assay enables the quantification of DNA damage such as single-strand or double-strand breaks on a single cell level. Here, we describe a variant of this method for the detection of ribonucleotides embedded in genomic DNA. Briefly, cells are embedded in agarose on a microscopic slide, lysed under high salt and alkaline conditions and then subjected to in situ treatment with E. coli RNase HII which nicks 5' to a ribonucleotide within the context of a DNA duplex thereby converting genomic ribonucleotides into strand breaks. After unwinding of genomic DNA using a highly alkaline buffer, electrophoresis under mild alkaline conditions is performed resulting in formation of comets due to migration of fragmented DNA toward the anode. Following SYBR Gold staining comets can be visualized by fluorescence microscopy. In this setting, the length and the intensity of comets formed reflect the level of genomic ribonucleotides present in a given cell.

NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils.

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors, patients with vasculitis, and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1, a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor.