Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene

The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of th e class Alpha GSTs, which we have previously reported to be overexpressed i n anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc(1)) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of se ven exons interrupted by introns of different lengths. Exon 1, with a lengt h of 219 bp, contains only the 5'-untranslated region of the gene. Each exo n-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were cha racterized by a combination of primer extension and rapid amplification of the cDNA ends. Position + 1 was identified 219 bp upstream of the first exo n-intron splicing junction. The proximal promoter region of the rat GSTA3 s ubunit gene does not contain typical TATA or CAAT boxes. A computer-based s earch for potential transcription-factor binding sites revealed the existen ce of a number of motifs such as anti-oxidant-responsive element, ras-respo nse element, activator protein-1, nuclear factor-kappa B, cAMP-response-ele ment-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTAS subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-r esistant cells known to have transcriptional activation of this gene by pre vious studies. The structure of the gene, with a large intron upstream of t he translation-initiation site, may explain why the isolation of this promo ter has been so elusive. This information will provide the opportunity to e xamine the involvement of the rat GSTAS subunit gene in drug resistance and carcinogenesis.