Functional analysis of linker-scan mutants spanning the-376-308,-244, and-238 polymorphic sites of the TNF-alpha promoter

Tumor necrosis factor alpha (TNT-alpha) promoter polymorphisms have been li nked to a large number of diseases but studies examining. the possible dire ct functional effects of these polymorphisms have been contradictory. Previ ous studies compared TNF-alpha promoter constructs containing single nucleo tide changes. We have now made a series of mutant constructs in which regio ns of the TNF-alpha promoter containing suspected functional single nucleot ide polymorphisms, including - 376, - 308, - 244 and - 238, were replaced b y a 10 bp linker scan sequence. These constructs were transiently transfect ed into the T cell line Jurkat, the B cell line Raji, and the monocytic cel l line U937, and tested for basal and induced transcriptional activity. Mut ant constructs covering both the - 308 and - 376 polymorphisms showed no si gnificant differences in either basal or induced transcriptional activity. Constructs covering the - 244/ - 238 region showed a small increase in basa l activity in the U937 cell line. These results indicate (i) that the - 308 and - 376 regions are of no functional relevance for TNF-alpha promoter tr anscription, and (ii) that the - 244/ - 238 region does not influence trans cription in some cell lines but may have some role in transcription in othe rs. (C) 2001 Academic Press.