GENOMIC ORGANIZATION, CHROMOSOMAL LOCALIZATION, AND EXPRESSION OF THEMURINE THROMBOXANE SYNTHASE GENE (TBXAS1)

Thromboxane synthase (TS) is a membrane-bound cytochrome P450 enzyme c atalyzing the synthesis of TxA(2), a potent modulator of vascular smoo th muscle contraction and platelet aggregation. TS plays an important role in hemostasis and may be intimately involved in the etiology of c ardiovascular, renal, and immune diseases. Restriction enzyme mapping, subcloning, and DNA sequencing analysis of recombinant phage lambda a nd P1 clones revealed that exons encoding the 1.9-kb mouse TS mRNA are dispersed over >150 kb genomic DNA. Determination of the intron-exon splicing junctions established that the mouse TS gene (Tbxas1) is enco ded by 13 exons ranging in size from 53 (exon III) to 315 bp (exon IX) . Genomic Southern analysis and fluorescence in situ hybridization sug gested that the gene is a single-copy gene, located on chromosome 6 ne ar the midpoint between the centromere and the Ig kappa gene. An alter natively spliced variant of the Tbxas1 transcript, lacking the exon XI I-encoded sequence, has been detected in normal mouse tissues. Ribonuc lease protection and 8'-RACE assays identified at least five major tra nscription start sites clustered within 31 bp of the Tbxas1 promoter, The 5'-most start site is not preceded by a TATA box, suggesting trans cription can be initiated in a TATA-independent manner. Transfection a nalyses indicated that the expression of Tbxasl is controlled by a sho rt (70-bp) positive regulatory sequence and several upstream repressiv e elements. Mutational studies further demonstrated that NP-E2/AP-1 an d Sp1 exerted activating and repressive, respectively, effects on the promoter. These studies provide the genetic tools and information for TS research in mice, which should expedite understanding of the geneti c contribution of TS in normal physiology as well as in disease states . (C) 1997 Academic Press.