Gene, 132 (1993) 227-236 Q 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l Il9/93/$06.00 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH GENE 07249 227 Cloning and sequence analysis of the rat tumor necrosis factor-encoding genes (Astrocytes; sequence comparison; transcriptional regulation; nuclear factor-K@ Jongbum Kwon, 11 Yup Chung and Etty N. Benveniste Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Received by J.A. Engler: 12 February 1993; Accepted: 9 April 1993; Received at publishers: 6 May 1993 SUMMARY We have isolated an approximately 22-kb TNF locus (encoding tumor necrosis factor) from a rat genomic library and sequenced the 7105bp fragment that comprises the TNF- cr and TNF- fi genes, including their flanking sequences. The two genes are tandemly arranged with TNF-/? 5’ to TNF- a and separated by approximately 1.1 kb of intergenic space, and each gene consists of four exons and three introns, similar to those of the other species examined thus far. Comparison analysis showed that the rat TNF have high sequence homology with the mouse TNF (TNF-cr, 86.5%; TNF-P, 89.3%) and relatively low homology with the human, rabbit, and porcine TNF. The upstream sequence of rat TNF- ct contains a number of sequence motifs implicated in the expression and regulation of eukaryotic genes, including binding sites for the transcription factors Sp- 1, Ap-2, IFN. 1 and NF-KB. The possible significance of potential regulatory sequence elements found in the rat TNF- ct in the context of transcriptional regulatory mechanisms is discussed. INTRODUCTION Tumor necrosis factor-a (TNF-a) and TNF-l3 (or lym- photoxin) are two structurally related pleiotropic cytok- ines with overlapping biological functions, including an involvement in inflammatory processes (Paul and Ruddle, 1988; Beutler and Cerami, 1989; Vilcek and Lee, 1991). Although both cytokines share many immunologi- Correspondence to: Dr. E.N. Benveniste, Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Tel. (I-205) 934-7667; Fax (I-205) 975-6748. Abbreviations: aa, amino acid(s); bp, base pair(s); CAT, chloramphenicol acetyltransferase; cat, gene encoding CAT; cDNA, DNA complemen- tary to mRNA; CNS, central nervous system; CTF, CCAAT-binding transcription factor; GCG, Genetics Computer Group (Madison, WI, USA); IFN-7, interferon-gamma; IL-l, interleukin-1; kb, kilobase or 1000 bp; LPS, hpopolysaccharide; MHC, major histocompatibility complex; MS, multiple sclerosis; NF-kB, nuclear factor kappa B; nt, nucleotide(s); ohgo, oligodeoxyribonucleotide; PCR, polymerase chain reaction; PKC, protein kinase C; TNF, tumor necrosis factor: TNF, gene encoding TNF; TPA, 12-0-tetradecanoylphorbol-13-acetate; tsp, transcription start point(s); UTR, untranslated region. cal functions and bind to the same cell-surface receptor (Aggarwal et al., 1985), they are produced by different cell types: TNF-a is produced primarily by activated macro- phages, whereas TNF-P is mainly a product of activated T-lymphoid cells. In addition to their beneficial roles as immunostimulants and as factors with cytotoxic or cyto- static activity against tumor cell lines (Old, 1985), they also mediate the deleterious effects of endotoxin septic shock (Cerami and Beutler, 1988) and the cachexia associ- ated with cancer (Oliff et al., 1987). The genes encoding TNF-a and TNF-P for human, mouse, rabbit, and porcine have been cloned and sequenced (Nedwin et al., 1985; Ito et al., 1986; Semon et al., 1987; Shirai et al., 1988; Shakhov et al., 1990b; Kuhnert et al., 1991). In all species examined, the two genes are arranged in a tandem orientation with TNF- B 5’ to the TNF- a and separated by approximately 1 kb of intergenic space; each gene consists of four exons and three introns. The human and mouse TNF have been shown to be closely linked and located adjacent to the MHC complex (Muller et al., 1987; Spies et al., 1989).