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.

We have isolated and characterized a complete structural gene encoding a rat liver glutathione Stransferase (glutathione transferase; EC 2.5.1.18) Ya subunit. The gene spans =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).The glutathione S-transferases (glutathione transferase; EC 2.5.1.18) are a family of enzymes that catalyze the conjugation of glutathione to a variety of electrophilic ligands. In addition, the transferases bind heme and bilirubin as well as various exogenous hydrophobic compounds with high affinity (1-3). The enzymes are comprised of binary combinations of at least seven major subunits, Ya, Ya, Ybl, Yb2, Yc, Yn, and Yp, which are electrophoretically distinguishable on one-dimensional NaDodSO4/polyacrylamide gels (4-7). Peptide mapping experiments and carboxyl-terminal sequence analysis have indicated that Ya and Yc subunits are comprised of a mixture of at least two microheterogeneous polypeptides (8-10).Our laboratory has reported the construction of Ya, Yc, Ybl, and Yb2 cDNA clones (11)(12)(13)(14). We have used these clones in RNA blot hybridization and nuclear run-off assays to demonstrate that the rat liver glutathione S-transferase genes are transcriptionally activated by phenobarbital and 3-methylcholanthrene (11,14,15). DNA sequence analysis of the four cDNA clones indicates that the Ya and Yc genes are members of the same gene family, whereas the Yb1 and Yb2 genes are members of a second glutathione S-transferase gene family. Genomic blots using the Ya and Yc cDNAs as probes suggest the presence of at least five to seven Ya and/or Yc genes in the rat (16).In the present study, we have isolated a complete structural gene encoding a Ya subunit of the rat liver glutathione S-transferases. The Ya structural gene spans ""11 kilobases (kb) and is comprised of seven exons separated by six introns. Nucleotide sequence analysis of the exons of the glutathione S-transferase structural gene indicates that the sequence is equivalent to the Ya cDNA clone, pGTB38, which has been isolated and characterized by our laboratory (11). (23). MATERIALS AND METHODS RESULTSIsolation of a Rat Glutathione S-Transferase Structural Gene. In a previous study from our laboratory, we reported Abbreviations: bp, base pair(s); k...