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

Abstract

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...

Structure of human glutathione S-transferase class Mu genes

“…The subunits of mammalian GSTs fall into three classes, Alpha, Mu and Pi, according to their primary structures (Mannervik et al, 1985;Pickett, 1987), and their gene structure is conserved between rat (Telakowski-Hopkins et al, 1986) and mouse (Daniel et al, 1987) for Alpha class genes and between rat (Okuda et al, 1987) and human (Cowell et al, 1988;Morrow et al, 1989) for the Pi class genes. For Mu class genes, three rat sequences have been compared and gene conversion (i.e.…”

Cloning, sequencing and characterization of the human Alpha glutathione S-transferase gene corresponding to the cDNA clone pGTH2

“…4). It is interesting that the 5' region of the human GST gene presented here shows no striking similarity in the analysed 5' region to the corresponding regions of the rat [11] and mouse [12,27] Alpha GST genes, although the NFl and the half ATTAAC motifs are also located at the 5' region of the mouse gene [27]. The SPI site was also found in the human GST Pi gene [14].…”

“…1 and 2). Like its homologue in rat [11] and mouse [12] the human GST Alpha gene contains seven exons (El-E7) interrupted by six introns. Furthermore, clone cosGTH2 contains additional regions corresponding to exons 3 and 4 (E3+ and E4+) ( Figs.…”

“…3. Table 1 shows the length of the exons (E) and introns of the isolated human Alpha gene compared with the Ya gene of rat [11]. Upstream region of cosGTH2 Fig.…”

“…Therefore cosGTH2 represents the second Alpha GST isoenzyme a2 (or ay) which is the less basic subunit. It is of interest to note that in an enzymological study [26] of the human kidney enzymes, there was no difference in substrate specificity, but the Vmax with l-chloro-2, 4-dinitrobenzene was about double in al compared with a2 With regard to the Ya rat gene [11], there are differences in the length of exons 1, 2 and 7, which are 43, 109 and 234 bp in the rat respectively (see Table 1). …”