Genomic organization, chromosomal localization, and expression of the murine thromboxane synthase gene (Tbxas1)

Thromboxane synthase (TS) is a membrane-bound cytochrome P450 enzyme catalyzing the synthesis of TxA(2), a potent modulator of vascular smooth muscle contraction and platelet aggregation. TS plays an important role in hemostasis and may be intimately involved in the etiology of cardiovascular, renal, and immune diseases. Restriction enzyme mapping, subcloning, and DNA sequencing analysis of recombinant phage lambda and 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 encoded by 13 exons ranging in size from 53 (exon III) to 315 bp (exon IX). Genomic Southern analysis and fluorescence in situ hybridization suggested that the gene is a single-copy gene, located on chromosome 6 near the midpoint between the centromere and the Ig kappa gene. An alternatively spliced variant of the Tbxas1 transcript, lacking the exon XII-encoded sequence, has been detected in normal mouse tissues. Ribonuclease protection and 8'-RACE assays identified at least five major transcription start sites clustered within 31 bp of the Tbxas1 promoter, The 5'-most start site is not preceded by a TATA box, suggesting transcription can be initiated in a TATA-independent manner. Transfection analyses indicated that the expression of Tbxasl is controlled by a short (70-bp) positive regulatory sequence and several upstream repressive elements. Mutational studies further demonstrated that NP-E2/AP-1 and 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 genetic contribution of TS in normal physiology as well as in disease states. (C) 1997 Academic Press.