Glutathione S-transferase Ya subunit gene: identification of regulatory elements required for basal level and inducible expression.

The function of the 5'-flanking region of a rat glutathione S-transferase Ya subunit structural gene has been examined in homologous and heterologous cells. By using the 5'-flanking region of the Ya subunit gene fused to the structural gene encoding chloramphenicol acetyltransferase, we have identified two cis-acting regulatory elements in the upstream region of the Ya gene. One element is required for maximum basal level expression in homologous cells, whereas maximum basal level expression in homologous cells, whereas the second element is required for inducible expression of the Ya gene by planar aromatic compounds such as beta-naphthoflavone.

Sequence analysis and regulation of rat liver glutathione S-transferase mRNAs.

We have utilized polysomal immunoadsorption techniques to purify the rat liver glutathione S-transferase mRNAs. Using the purified mRNAs as template, cDNA clones complementary to the Ya, Yb1, and Yc mRNAs have been constructed. The cDNA clones have been utilized in RNA blot hybridization and nuclear run-off assays to demonstrate that the Ya and Yb mRNAs are elevated 8 and 5-fold, respectively by phenobarbital; whereas the Yc mRNA is elevated only 2.

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.

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.

Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit.

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S.

Rat liver glutathione S-transferases. Complete nucleotide sequence of a glutathione S-transferase mRNA and the regulation of the Ya, Yb, and Yc mRNAs by 3-methylcholanthrene and phenobarbital.

With the use of cDNA probes reverse transcribed from purified glutathione S-transferase mRNA templates, four cDNA clones complementary to transferase mRNAs have been identified and characterized. Two clones, pGTB38 and pGTB34, have cDNA inserts of approximately 950 and 900 base pairs, respectively, and hybridize to a mRNA(s) whose size is approximately 980 nucleotides. In hybrid-select translation experiments, pGTB38 and pGTB34 select mRNAs specific for the Ya and Yc subunits of rat liver glutathione S-transferases.

Regulation of glutathione S-transferase mRNAs by phenobarbital and 3-methylcholanthrene: analysis using cDNA probes.

Using antibody directed against glutathione S-transferase B (YaYc heterodimer) and glutathione S-transferases A and C (Yb homodimers), we have purified the corresponding mRNAs by polysome immunoprecipitation. These mRNAs have been utilized to synthesize high specific activity cDNA probes which have been used to screen a cDNA library constructed from RNA fractions enriched in the various transferase mRNAs. Two clones which have been identified, pGTB38 and pGTA/C36, are complementary to the Ya/Yc mRNAs and Yb mRNAs, respectively.

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA.

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing.

Isolation and characterization of monoclonal antibodies against rat liver epoxide hydrolase.

We have established hybridoma cell lines which secrete monoclonal antibody to rat liver microsomal epoxide hydrolase. All of the monoclonal antibodies formed against epoxide hydrolase are mouse immunoglobulin subclass IgG1. Utilizing double immunodiffusion analysis, we have found that the monoclonal antibodies bind to and precipitate epoxide hydrolase in solubilized rat liver microsomes.