Structural analysis of a rat liver glutathione S-transferase Ya gene.

We have isolated and characterized a complete structural gene encoding a rat liver glutathione S-transferase (glutathione transferase; EC 2.5.1.18) Ya subunit. The gene spans approximately 11 kilobases and is comprised of seven exons separated by six introns. A sequence similar to the Goldberg-Hogness promoter ("TATA" box), TATTA, is located 32 base pairs upstream from the transcription initiation site. Exons 2 and 4 of the glutathione S-transferase gene encode amino acid sequences of the Ya subunit that are highly conserved in the Yc subunit, whereas exons 3 and 5 encode amino acids that are divergent in the Yc subunit. These data suggest that exons 2 and 4 may encode domains of the Ya subunits that have similar structural or functional properties to the corresponding domains in the Yc subunit (e.g., glutathione binding site), whereas exons 3 and 5 may encode domains of the Ya subunit that have unique structural or functional properties to the corresponding domains in the Yc subunit (e.g., substrate binding site).

Multiplicity of glutathione S-transferase genes in the rat and association with a type 2 Alu repetitive element.

Southern blot analysis of rat genomic DNA using glutathione S-transferase Ya and Yc cDNA probes was employed to estimate the size of the Ya/Yc multigene family. A minimum of five to seven Ya/Yc genes were detected; at least two of these are Yc genes. The presence of multiple genes was further supported by the isolation of three nonoverlapping genomic clones from a rat EcoRI library that hybridized to a Ya cDNA clone, pGTB38. However, not all EcoRI bands seen in genomic blots were represented in the clones, suggesting that not all Ya/Yc genes have been isolated. The organization of a Ya gene in one of these EcoRI genomic clones, lambda GTB38-3, and an overlapping clone, lambda GTB45-1, isolated from a HaeIII library, was investigated with 5' and 3' probes prepared from Ya and Yc cDNA clones. Restriction endonuclease mapping and hybridization studies revealed that the gene spans over 10 kilobases and contains at least three introns. Sequences upstream from the 5' untranslated region of the gene, and within an intron in the 5' coding region, were found to contain sequences homologous to a type 2 Alu repetitive element from the rat growth hormone gene [Page, G.S., Smith, S., & Goodman, H.M. (1981) Nucleic Acids Res. 9, 2087-2104]. The repetitive sequences in lambda GTB38-3 were identified by hybridization to a novel Ya cDNA clone, pGTB45. This cDNA clone was isolated from a cDNA library previously described [Telakowski-Hopkins, C.A., Rodkey, J.A., Bennett, C.D., Lu, A.Y.H., & Pickett, C.B. (1985) J. Biol. Chem. 260, 5820-5825] with nick-translated intron sequences as probes. pGTB45 is virtually identical with pGTR261 [Tu, C.-P.D., Lai, H.-C.J., Li, N.-Q., Weiss, M.J., & Reddy, C.C. (1984) J. Biol. Chem. 259, 9434-9439], except that the 3' untranslated region extends 231 base pairs beyond the polyadenylation signal of pGTR261. This elongated 3' untranslated sequence is unique in that it contains a full-length type 2 Alu repetitive element, which includes two additional, overlapping polyadenylation signals.