We have identified a region in the 5' flanking sequence of the glutathione S-transferase (RX:glutathione Rtransferase, EC 2.5.1.18) Ya subunit gene that contains a unique xenobiotic-responsive element (XRE). The regulatory region spans nucleotides -722 to -682 of the 5' flanking sequence and is responsible for part of the basal level as well as inducible expression of the Ya subunit gene by planar aromatic compounds such as P-naphthoflavone ((3-NF) and 3-methylcholanthrene. The DNA sequence of this region (J3-NF-responsive element) is distinct from the DNA sequence ofthe XRE found in the cytochrome P-450 IAl gene. In addition to the region containing the fi-NF-responsive element, two other regulatory regions of the Ya subunit gene have been identified. One region spans nucleotides -867 to -857 and has a DNA sequence with identity to the hepatocyte nuclear factor 1 recognition motif found in several liver-specific genes. The second region spans nucleotides -908 to -899 and contains a DNA sequence with identity to the XRE found in the cytochrome P450 IA1 gene. The XRE sequence also contributes to part of the responsiveness of the Ya subunit gene to planar aromatic compounds. Our data suggest that regulation of gene expression by planar aromatic compounds can be mediated by a DNA sequence that is distinct from the XRE sequence.The glutathione S-transferases (RX:glutathione R-transferase, EC 2.5.1.18) represent a family of proteins that catalyze the conjugation of glutathione to a variety of electrophiles. In addition, they bind with high affinity a number of exogenous hydrophobic compounds, such as heme, bilirubin, dexamethasone, and polycyclic aromatic hydrocarbons (1-7). The cytosolic forms are heterodimers or homodimers and are composed of at least seven subunits (8)(9)(10)(11)(12).Previous work from our laboratory has included the construction and characterization of cDNA clones complementary to the rat liver Ya, Yb1, Yb2, One region, which is required for basal level expression, has a DNA sequence with identity to the hepatocyte nuclear factor 1 (HNF1) recognition sequence (20, 21). The second region has a DNA sequence with identity to the xenobioticresponsive element (XRE) core sequence found in the planar aromatic hydrocarbon-responsive cytochrome P450 IA1 gene (22, 23). MATERIALS AND METHODSTissue Culture and Cell Lines. Human hepatoma cells, HepG2, were maintained in Eagle's minimum essential medium (Hazleton Laboratories, Lenexa, KS) with nonessential amino acids, sodium pyruvate, 1o (vol/vol) fetal bovine serum, penicillin (10 units/ml), and streptomycin (10 units/ ml) at 370C in humidified 7% C02/93% air. The class II variant mouse cell line, obtained from James Whitlock (Stanford University), was maintained in minimal essential medium (a-MEM) with L-glutamine (Hazleton Laboratories), 10%6 fetal bovine serum, penicillin (10 units/ml), and streptomycin (10 units/ml) at 370C in humidified 7% C02/93% air.Construction of Deletion Mutants of the 5' Flanking Sequence of the Ya Subunit Gene. The initia...

Excitatory amino acids (EAAs) are important neurotransmitters in the vertebrate central nervous system. Electrophysiological and ligand-binding studies indicate that at least three different receptor subtypes for EAAs exist--N-methyl-D-aspartate, kainate and quisqualate receptor subtypes--on the basis of the preferred agonist of the receptors. We recently purified a kainate-binding protein (KBP) from frog (Rana pipiens berlandieri) brain by domoic acid (a high-affinity kainate analogue) affinity chromatography, and showed that the kainate-binding activity was associated with a protein of relative molecular mass 48,000 (Mr 48 K). The pharmacological properties and the anatomical distribution of KBP were consistent with those of a kainate receptor-ionophore complex. We have now isolated a complementary DNA encoding KBP of Mr 48 K. The deduced amino-acid sequence of the KBP has similar hydrophobic profiles to those found in other ligand-gated ion channel subunits, and shows some amino-acid sequence similarities to the corresponding regions of brain nicotinic acetylcholine receptor subunits. Localization of the KBP messenger RNAs by in situ hybridization histochemistry is compatible with the results of immunohistochemistry and receptor autoradiography studies. COS-7 cells transfected with the cDNA encoding the KBP show high-affinity kainate-binding activity with pharmacological properties similar to those of the biochemically purified KBP. These results provide the first molecular characterization of an EAA-binding site and raise the possibility that the KBP cDNA encodes a ligand-binding subunit of a kainate receptor-ionophore complex.

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 the second element is required for inducible expression of the Ya gene by planar aromatic compounds such as P-naph- The rat liver glutathione S-transferases (RX:glutathione Rtransferase, EC 2.5.1.18) are a family of proteins that catalyze the conjugation of glutathione to various electrophilic ligands (1-8). In addition, they bind heme, bilirubin, steroids, and polycyclic aromatic hydrocarbons with high affinity (1-8). Nucleotide and amino acid sequence analysis of cDNA clones and purified proteins indicate a minimum of four glutathione S-transferase gene families in the rat (9)(10)(11)(12)(13)(14)(15)(16)(17) (FAO-H4IIEC3) and the mouse cell line Hepa 1clc7 were obtained from Oliver Hankinson (University of California, Los Angeles). The mouse class I and class II variant cells were obtained from James P. Whitlock, Jr. (Stanford University, Palo Alto, CA). These cell lines were grown on minimal essential medium (a-MEM), L-glutamine, 10% (vol/vol) fetal bovine serum, penicillin at 10 units/ml, and streptomycin at 10 units/ml at 37TC in humidified 7% C02/93% air. In the induction studies the mouse hepatoma cell lines were treated with 15 AM j8-naphthoflavone, whereas the rat cell lines were treated with 50 ,uM /3-naphthoflavone. These concentrations were chosen from dose-response curves and gave maximum induction with maximum cell viability.Human hepatoma cells, HepG2, were grown in Eagle's minimum essential medium, nonessential amino acids, sodium pyruvate, 10% (vol/vol) fetal bovine serum, penicillin at 10 units/ml, and streptomycin at 10 units/ml at 37°C in humidified 7% C02/93% air. In the induction studies the human hepatoma cell line was treated with 50 AM f3-naphthoflavone.

We have cloned and sequenced a cDNA (rBa2) encoding an a2 subunit of the voltage-sensitive L-type calcium channel (dihydropyridine receptor) of rat brain. The cDNA (3823 base pairs) encodes a protein of 1091 amino acids with a Mr of 123,822. The deduced amino acid sequence of rB-a2 cDNA is highly similar (95% amino acid identity) to that of rabbit skeletal muscle a2 subunit. The rB-a2 protein is distinct from the previously cloned skeletal muscle a2-subunit protein [Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H., Leung, A. T., Campbell, K. P., McKenna, E., Koch, W., Hui, A., Schwartz, A. & Harpold, M. M. (1988) Science 241, 1661-1664] because it contains an insertion of 7 amino acid residues and a deletion of a 19-amino acid segment between putative transmembrane domains 1 and 2. We show that the rB-ai2 and skeletal muscle a2-subunit transcripts are the variants produced by alternative splicing of the primary transcript and that they are differentially expressed in brain and skeletal muscle, respectively.The slowly inactivating voltage-sensitive L-type Ca2' channel activated by strong depolarization is sensitive to organic Ca2+ channel blockers such as 1,4-dihydropyridines (DHP), phenylalkylamines, and benzothiazepines. The biochemical and molecular properties of the DHP-sensitive L-type Ca2+ channel have been well characterized from skeletal muscle and heart. Purified skeletal muscle Ca2+ channel is an oligomeric protein composed of two high-molecular polypeptide subunits (al and a2) and three smaller subunits (J3, y, and 8) (reviewed in refs. 1 and 2). Molecular cloning studies have elucidated the primary structure of the DHP-sensitive Ca2+ channel al subunit from both skeletal muscle (3) and cardiac muscle (4). The cloned skeletal and cardiac muscle al subunits express functionally active, DHP-sensitive Ca2' channels when transfected into fibroblasts or microinjected into myotubes (5, 6). Thus, the al subunit appears to have the structural features required for Ca2+ channel function and contains the binding sites for Ca2' antagonists. Subsequent cloning ofcDNAs encoding the a2, p, and y subunits ofrabbit skeletal muscle (7-10) has allowed electrophysiologic characterization of coexpressed cloned Ca2+ channel subunits and also has demonstrated specific functional roles for these subunits to modulate Ca2+ channel function. While the al subunit mRNA alone produced Ca2+ currents in Xenopus oocytes, coinjection of mRNAs for skeletal muscle a2 and ,3 subunits together with that for the al subunit produced very high levels of expression (11-13).Although MATERIALS AND METHODS Isolation of Rat Brain Ca21 Channel a2-Subunit (rB-a2) cDNAs, Subcloning, and DNA Sequencing Analysis. Approximately 6 x 105 recombinant plaques from a rat hippocampus cDNA library in Lambda Zap II (a gift from J. Boulter of the Salk Institute, La Jolla, CA) were screened with the oligonucleotide probes whose sequences were derived from rabbit skeletal muscle DHP-sensitive Ca2+ channel a2-subunit cDNA (7). Two prob...