Isolation of a clone which induces expression of the gene encoding the human tumor necrosis factor receptor.
Tumor necrosis factor (TNF) is a cytokine with pleiotropic effects upon cell growth, inflammation and immunologic responsiveness. High-affinity TNF receptors (TNFRs) of 55 and 75 kDa are found in many cell types. Using an Epstein-Barr virus (EBV)-based mammalian expression library, we have isolated a clone from human lymphoblastoid transfectants that induces overexpression of the TNFR-encoding gene (TNFR). Transfectants overproducing the TNFR were isolated by multiple rounds of sorting on a fluorescence-activated cell sorter using fluorescent TNF ligand binding as the selection procedure. Among the sorted transfectants were cells producing approx. 150,000 receptors per cell (Kd of approx. 1 nM). These cells have multiple copies of the TNFR gene present as extrachromosomal plasmids. These cells also overproduced the mRNA for TNFR. Low-Mr EBV episomes were isolated from these overproducing cells and used to transform Escherichia coli. One of the colonies isolated contained a plasmid encoding a portion of the noncoding region of the TNFR gene. Transfection of human lymphoblastoid cells with this DNA gave rise to high-level production of TNFR. Fluorescent TNF bound to these transfectants is fully and specifically displaced by an excess of TNF. The rescued clone contains approx. 10 kb of human genomic DNA including the 3'-untranslated region of TNFR and several Alu sequences; apparently during the selection procedure in human cells, recombination occurred to rescue a portion of the TNFR gene. Transient transfection was used to narrow down the region responsible for TNFR induction to 5.2 kb. The mechanism by which this clone induces TNFR expression has not been determined.
Garret
M
M
Department of Neurosciences, Roche Research Center, Nutley, NJ 07110.
McHendry-Rinde
B
B
Spickofsky
N
N
Margolskee
R F
RF
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics*; D018124:Receptors, Tumor Necrosis Factor; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha