MOUSE-TUMOR NECROSIS FACTOR-RECEPTOR TYPE-I - GENOMIC STRUCTURE, POLYMORPHISM, AND IDENTIFICATION OF REGULATORY REGIONS

The mouse tumor necrosis factor receptor (TNFR)-I gene was cloned, sequenced, and characterized. The nucleotide sequence analysis shows that the TNFR-I is composed of 10 exons and nine introns. The first intron includes two simple dinucleotide repeat sequences, (GA), and (TC)9(TG)19. The (TC)9(TG)19 tandem repeat was found to be polymorphic in its length among various mouse strains. The nucleotide sequence of the 1076 bp 5' flanking region of the TNFR-I was also determined. Various possible regulatory sequences were identified in the 5' flanking region of the TNFR-1 gene. For functional analysis, the 5' flanking region of the TNFR-I gene was isolated, ligated upstream of the luciferase reporter gene, and transiently transfected into L929, Hela, and a T cell hybridoma cell line. The results show that the isolated 5' flanking region has functional promoter activity and is responsible for constitutive expression of the TNFR-I gene. A series of truncated promoter constructs were generated and studied in a transient transfection system. Analysis of transient expression in L929 cells shows that the regions -1076/-939, -615/-425, and -425/-198 include positive regulatory elements, while the region -939/-615 may contain negative cis-acting elements for the constitutive expression of the TNFR-I. The shortest construct containing 198 bp of the 5' flanking region still has significant promoter activity, suggesting that the two GC-rich elements in this region may play an important role in the constitutive expression of the TNFR-I gene.