ISOLATION AND CHARACTERIZATION OF THE PROMOTER OF THE HUMAN 5'-NUCLEOTIDASE (CD73)-ENCODING GENE

Ecto-5'-nucleotidase (NT, CD73) is a purine salvage-pathway enzyme loc ated on the surface of various cell types, including subsets of human lymphocytes and certain leukemias and lymphomas. In addition to purine salvage, NT has proposed roles in lymphocyte maturation and activatio n, and its expression has been associated with the resistance of some tumor cell lines to chemotherapeutic agents. To better understand the regulation of NT gene expression in normal lymphocyte development and the elevated expression seen in some drug-resistant tumor cell lines, we isolated NT genomic clones containing the promoter region. The geno mic DNA upstream from the NT start codon is high in G+C content, with one cAMP-responsive element and five consensus Sp-1 binding sites, but no TATAA box. RNase protection assays identified a cluster of potenti al transcription start points (tsp). One tsp, at -63 bp relative to th e start codon, was confirmed as authentic by 5'-RACE (rapid amplificat ion of cDNA ends) cloning. Transient transfection experiments utilizin g luc as a reporter gene have demonstrated that a 155-bp NT genomic DN A segment inclusive of the tsp functions as a promoter in both NT+ (WI -L2 and MG) and NT- (Jurkat, Hela and Raji) cell lines. The addition o f 5'-flanking sequences extending as far as -1.9 kb did not confer cel l-type-specific expression to the core promoter, However, nuclear run- on analysis of nascent NT transcripts suggested that differential tran scription initiation is at least partially responsible for the regulat ion of NT expression. Thus, additional information. is necessary, eith er at the chromatin level, or within elements outside of the promoter region, to direct tissue-specific expression of NT.