Tumor necrosis factor alpha and beta (TNF-alpha and TNF-beta) bind surface receptors on a variety of cell types to mediate a wide range of immunological responses, inflammatory reactions, and anti-tumor effects. A cDNA clone encoding an integral membrane protein of 461 amino acids was isolated from a human lung fibroblast library by direct expression screening with radiolabeled TNF-alpha. The encoded receptor was also able to bind TNF-beta. The predicted cysteine-rich extracellular domain has extensive sequence similarity with five proteins, including nerve growth factor receptor and a transcriptionally active open reading frame from Shope fibroma virus, and thus defines a family of receptors.

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We report the isolation from a placental library, of two cDNAs that can encode high affinity receptors for granulocyte colony-stimulating factor (G-CSF) when expressed in COS-7 cells. The cDNAs are predicted to encode integral membrane proteins of 759 and 812 amino acids in length. The predicted extracellular and membrane spanning sequences of the two clones are identical, as are the first 96 amino acids of their respective cytoplasmic regions. Different COOH termini of 34 or 87 residues are predicted for the two cDNAs, due apparently to alternate splicing. The receptor with the longer cytoplasmic domain is the closest human homologue of the murine G-CSF receptor recently described by Fukunaga et al. (Fukunaga, R., E. Ishizaka-Ikeda, Y. Seto, and S. Nagata. 1990. Cell. 61:341). A hybridization probe derived from the placental G-CSF receptor cDNA detects a approximately 3-kb transcript in RNAs isolated from placenta and a number of lymphoid and myeloid cells. The extracellular region of the G-CSF receptors is composed of four distinct types of structural domains, previously recognized in other cell surface proteins. In addition to the two domains of the HP receptor family-defining region (Patthy, L. 1990. Cell. 61:13) it incorporates one NH2-terminal Ig-like domain, and three additional repeats of fibronectin type III-like domains. The presence of both an NH2-terminal Ig-like domain and multiple membrane-proximal FN3-like domains suggests that the G-CSF receptor may be derived from an ancestral NCAM-like molecule and that the G-CSF receptor may function in some adhesion or recognition events at the cell surface in addition to the binding of G-CSF.

We have located a positive, cis-acting DNA sequence element within the 5' flanking DNA of the c-myc gene (-125 base pairs). This DNA sequence element has a large purine-pyrimidine strand asymmetry and can assume the H-DNA conformation. A factor with the properties of a ribonucleoprotein (RNP) interacts with this DNA region. The interaction of the c-myc DNA sequence element and the RNP involves an RNase H-sensitive mechanism and, therefore, may involve an RNA-DNA hybrid. In addition, a protein factor(s) binds to this DNA sequence element. DNA footprinting and mutant oligonucleotide binding/competition assays implicate a punctate, poly(G-C) recognition/binding sequence for the RNP factor, whereas the major protein factor requires two ACCCT sequence motifs for maximal binding. These results suggest that RNP and protein factors act as positive transcriptional regulators of the c-myc gene, perhaps by altering DNA topology.Several genes have been identified that regulate growth and differentiation. One such gene is the protooncogene c-myc. Enforced expression of c-myc in cultured mouse erythroleukemia cells disrupts differentiation and allows continued growth (1-3). When the c-myc gene is linked to the immunoglobulin A enhancer, and made a transgene in mice, a pre-B cell hyperplasia results. A large percentage of these transgenics progress to a B-cell lymphoma (4, 5). c-myc antisense oligonucleotides inhibit cellular growth and induce differentiation in the human promyelocytic leukemia cell line HL-60 (6). These results demonstrate a direct role for c-myc in the regulation of cellular growth and an indirect role in cellular differentiation.Sequence-specific DNA binding proteins regulate eukaryotic transcription (7)(8)(9)(10)(11)(12). Although several protein factors have been shown to bind to the c-myc gene, the binding of these factors has not shown a correlation with the activity of the c-myc gene (13-15).We, and others, have observed non-B DNA structures in the 5' flanking region of the c-myc gene (ref. 16; T.L.D. and A.J.K., unpublished results). One of the single-strand nuclease-sensitive sites in the c-myc gene maps near and may correspond to a DNase I-hypersensitive site termed III1 (17). DNase I sensitivity at the I1I1 site disappears when cells become committed to terminal differentiation, a time when c-myc transcription is turned off (18). These data imply that the II1 nuclease-hypersensitive site is a cis-acting regulatory element of the c-myc gene. Therefore, we examined the role of this DNA region in c-myc expression. MATERIALS AND METHODSc-myc Fusion Gene Constructs, Gene Transfection, and Chloramphenicol Acetyltransferase (CAT) Assays. Deletions were made in a subclone of the 866-base-pair (bp) Pvu II fragment containing exon I and 353 bp of 5' flanking DNA.This subclone was digested with Sma I, and deletions were created with BAL-31 exonuclease and were sequenced by the dideoxy method (19). CAT constructs were transfected into HeLa cells as described (20). CAT assays were performed as described...

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Clones encoding the type 1 (p80) and type 2 (p60) forms of the murine receptors for tumor necrosis factor (TNF) were isolated by cross-hybridization using probes derived from the cloned human TNF receptors. Each of the murine receptors shows strong sequence homology to the corresponding human receptor (-65% amino acid identity) throughout the molecule but only modest homology, limited to ligand-binding domains, between themselves. The ligand-binding characteristics of the recombinant murine receptors mirror those of the human homologs: both receptor types bind TNF-a and -0 with multiple affinity classes, and the ligands cross-compete.Analysis of the murine transcripts encoding these receptors revealed the presence of RNAs for one or both forms of the receptors in all cells examined. It was also demonstrated that for both types of human TNF receptor, variably sized transcripts are observed in different cells. The murine cDNAs were further used to determine the chromosomal locations of the TNF receptor genes. They are not linked, in contrast to the ligands, and map to chromosomes 4 (type 1) and 6 (type 2).