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      Molecular cloning and expression of the fas ligand, a novel member of the tumor necrosis factor family

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      Cell

      Elsevier BV

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          Abstract

          The Fas antigen (Fas) belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family, and it mediates apoptosis. Using a soluble form of mouse Fas, prepared by fusion with human immunoglobulin Fc, Fas ligand was detected on the cell surface of a cytotoxic T cell hybridoma, PC60-d10S. A cell population that highly expresses Fas ligand was sorted using a fluorescence-activated cell sorter, and its cDNA was isolated from the sorted cells by expression cloning. The amino acid sequence indicated that Fas ligand is a type II transmembrane protein that belongs to the TNF family. The recombinant Fas ligand expressed in COS cells induced apoptosis in Fas-expressing target cells. Northern hybridization revealed that Fas ligand is expressed in activated splenocytes and thymocytes, consistent with its involvement in T cell-mediated cytotoxicity and in several nonlymphoid tissues, such as testis.

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          Most cited references 33

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          Single step method of RNA isolation by acid guanidium thiocyanate phenol chloroform extraction. Anal

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            High efficiency transformation of E. coli by high voltage electroporation.

            E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.
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              Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis.

              Fas antigen is a cell-surface protein that mediates apoptosis. It is expressed in various tissues including the thymus and has structural homology with a number of cell-surface receptors, including tumour necrosis factor receptor and nerve growth factor receptor. Mice carrying the lymphoproliferation (lpr) mutation have defects in the Fas antigen gene. The lpr mice develop lymphadenopathy and suffer from a systemic lupus erythematosus-like autoimmune disease, indicating an important role for Fas antigen in the negative selection of autoreactive T cells in the thymus.
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                Author and article information

                Journal
                Cell
                Cell
                Elsevier BV
                00928674
                December 1993
                December 1993
                : 75
                : 6
                : 1169-1178
                Article
                10.1016/0092-8674(93)90326-L
                7505205
                © 1993

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