Glutathione S-transferase Gene Family Research Articles

Modulation of Glutathione S-Transferase Subunits A2, M1, and P1 Expression by Interleukin-1β in Rat Hepatocytes in Primary Culture

The influence of various cytokines on the expression of glutathione S-transferases (GSTs) was investigated in rat hepatocytes in primary culture. Only treatment of hepatocytes with interleukin-1beta (IL-1) was effective, resulting in a marked decrease in GSTs. Steady-state mRNA levels of rGSTA2 and M1 were strongly down-regulated by IL-1 in a dose-dependent manner after a 24-h exposure while rGSTP1 mRNA level was increased by a 48-h treatment. Similar effects of IL-1 were observed at the protein level. The response to IL-1 appeared to be specific for each subunit within GST gene families. In addition, IL-1 strongly suppressed the induction of rGSTA2 by 3-methylcholanthrene, oltipraz (a synthetic derivative of 1, 2-dithiole-3-thione), and phenobarbital and that of rGSTM1 by oltipraz and phenobarbital, whereas it was ineffective on rGSTP1 induction by these compounds. Using in vitro nuclear run-on transcription assay and Northern blot analysis of alpha-amanitin-treated cells, IL-1-mediated rGSTM1 mRNA decrease was found to result from mRNA destabilization. These results provide the first demonstration that IL-1 regulates some major GST subunits in hepatocytes by a post-transcriptional mechanism.

A glutathione S-transferase gene of the vector mosquito, Anopheles gambiae.

A cDNA (D7-8B) 1456 nucleotides in length was isolated from an adult, female-specific Anopheles gambiae library and identified as a member of the glutathione S-transferase (GST) gene family by virtue of the inferred amino acid sequence. The gene, AgGST2-1, specifies a protein that is 57% identical to the Drosophila melanogaster gene, DmGST-2. The conceptual translation product also shows similarity to the pi family of vertebrate GSTs. Northern analysis reveals multiple and abundant transcription products in larval, pupal and adult RNA preparations. Southern analyses of genomic DNA and hybridizations in situ to polytene chromosomes both suggest that there are multiple members of the GST gene family in Anopheles gambiae.

Molecular cloning of a glutathione S-transferase overproduced in an insecticide-resistant strain of the housefly (Musca domestica)

We report the cloning and sequencing of a glutathione S-transferase (GST) gene from the housefly Musca domestica. A cDNA lambda gt11 library was prepared from the organophosphate insecticide-resistant housefly strain Cornell-R--a variant that has elevated GST activity. The lambda phage GST clone was identified on the basis of its ability to cross-hybridize to a GST DNA probe from Drosophila melanogaster. Based on amino acid homology to other GSTs and expression of GST activity in Escherichia coli, the Musca GST gene (MdGST-1) belongs to the GST gene family. Although organophosphate resistance in Cornell-R is largely due to one of the GSTs, MdGST-1 is probably not the enzyme responsible for resistance. The mutation that controls resistance to organophosphate insecticides in Cornell-R is highly unstable and we isolated spontaneous variants to both insecticide sensitivity and to even higher levels of resistance. This provided us with an isogenic set of three strains. We found that MdGST-1 transcript levels as measured by Northern assays are higher in all three Cornell-R strains relative to the sensitive wild type, but that the sensitive Cornell-R strain has more MdGST-1 transcript than does the highly resistant Cornell-R strain. These data as well as Southern analysis of genomic DNA allow us to conclude: (1) there are multiple GST genes in M. domestica; (2) the natural variant Cornell-R overproduces excess transcript from two and probably more of these genes; and (3) the unstable mutation in Cornell-R influences the levels of multiple GSTs.

Coamplification of μ class glutathione S‐transferase genes and an adenylate deaminase gene in coformycin‐resistant Chinese hamster fibroblasts

In Chinese hamster fibroblasts, we previously detected an expressed gene located near the AMP deaminase gene. This gene was named Y1. Upon selection for resistance to coformycin, an inhibitor of AMP deaminase activity, both genes were amplified in several mutants. We have determined the complete nucleotide sequence of Y1 cDNA and identified the Y1 gene as a mu class glutathione S-transferase gene by comparison with sequences present in a data bank. Accordingly, Y1-amplified mutants express an increased glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene; this activity, as well as the abundance of the corresponding RNA, appears, however, to reach a limit despite further increase in the Y1 gene copy number during successive amplification steps. Southern blot experiments showed that Y1 belongs to a multigene family, all or part of which has been amplified in mutant lines. These data provide a method to amplify and to overexpress the mu class of the glutathione S-transferase gene family on the basis of its linkage with the AMP deaminase gene.

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 2.0-fold. The elevation in glutathione S-transferase mRNAs is due in part to transcriptional activation of the corresponding genes. Nucleotide sequence analysis of the three glutathione S-transferase clones suggest that the Ya and Yc genes represent one rat liver glutathione S-transferase gene family whereas the Yb genes represent a second distinct glutathione S-transferase gene family. The construction of these cDNA clones will allow identification and characterization of the glutathione S-transferase structural genes as well as aid in the identification of regulatory elements that are responsible for transcriptional activation of the genes by xenobiotics.