IDENTIFICATION OF THE RAT XANTHINE DEHYDROGENASE OXIDASE PROMOTER

Inflammation and ischemia - reperfusion tissue injury are important pa thophysiologic processes with a wide spectrum of clinical presentation s; the enzyme xanthine dehydrogenase/oxidase (XDH/XO) is thought to pl ay a key role in ischemia-reperfusion injury. Recent studies have show n the transcriptional regulation of XDH/XO by cytokines (Dupont et al. , 1992, J. Clin. Invest. 89, 197 - 202). In the present study, the 5' structure of the XDH/XO gene and characterization of its promoter are undertaken providing an initial step to further elucidate the regulato ry mechanism(s) of this enzyme. XDH/XO cDNA from rat bone marrow macro phage has been isolated and used to screen a rat genomic library in or der to identify and characterize the promoter of the XDH/XO gene. By S outhern analysis, XDH/XO was found to be a single copy gene in the rat genome. Primer extension, RNase protection, and anchor-PCR studies in dicate the presence of multiple start sites within a 65 bp window loca ted some 20 - 85 bp upstream of the translation initiator (ATG). Funct ional studies of the sequences up to 116 nt upstream of the translatio nal start site, which encompasses the several transcriptional start si tes, indicate that this region is sufficient to drive the expression o f a luciferase reporter gene and is presumed to represent the promoter . Neither a TATA box nor a CC-rich region are present in close proximi ty to any of the transcriptional start sites; however, sequences with homology to known initiator elements are found within this 116 bp frag ment. Several possible regulatory elements, including a NF-IL6 motif, are also located upstream of the transcriptional start site. This stud y represents the first description of the XDH/XO promoter from a verte brate system.