Physical interaction and functional synergy between glucocorticoid receptor and Ets2 proteins for transcription activation of the rat cytochrome P-450c27 promoter.

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a "weak" Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.

Cloning, characterisation and bacterial expression of full length cDNA for the mouse liver microsomal glutathione S-transferase.

We have isolated a cDNA encoding full length microsomal glutathione S-transferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using primers designed from published cDNA sequence of rat MGST with the addition of 5' Nde-1 and 3' HindIII sites, and cloned into bacterial expression vector pSP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver which has sequence homology with that of rat and human liver MGST1. Recombinant GST cDNA (Genbank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring catalytic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and Western blot analysis. We have demonstrated the cloning and expression of full length cDNA for MGST from mouse liver and have characterised the functionally active product as MGST protein. These results should facilitate studies on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemicals.

Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP-dependent phosphorylation at ser128.

We have investigated mechanisms of mitochondrial targeting of the phenobarbital-inducible hepatic mitochondrial P450MT4, which cross-reacts with antibody to microsomal P4502B1. Results show that P4502B1 and P450MT4 have identical primary sequence but different levels of phosphorylation and secondary structure. We demonstrate that P4502B1 contains a chimeric mitochondrial and endoplasmic reticulum (ER) targeting signal at its N-terminus. Inducers of cAMP and protein kinase A-mediated phosphorylation of P4502B1 at Ser128 activate the signal for mitochondrial targeting and modulate its mitochondrial or ER destination. S128A mutation inhibits in vitro mitochondrial transport and also in vivo mitochondrial targeting in COS cells. A fragment of P4502B1 containing the N-terminal signal and the phosphorylation site could drive the transport of dihydrofolate reductase (DHFR) into mitochondria. Ser128 phosphorylation reduced the affinity of 2B1 protein for binding to SRP, but increased the affinity of the 2B1-DHFR fusion protein for binding to yeast mitochondrial translocase proteins, TOM40 and TIM44, and matrix Hsp70. We describe a novel regulatory mechanism by which cAMP modulates the targeting of a protein to two distinct organelles.

Down-regulation of the rat hepatic sterol 27-hydroxylase gene by bile acids in transfected primary hepatocytes: possible role of hepatic nuclear factor 1alpha.

In vitro and in vivo studies have shown that the sterol 27-hydroxylase (CYP27) gene is transcriptionally repressed by hydrophobic bile acids. The molecular mechanism(s) of repression of CYP27 by bile acids is unknown. To identify the bile acid responsive element (BARE) and transcription factor(s) that mediate the repression of CYP27 by bile acids, constructs of the CYP27 5'-flanking DNA were linked to either the CAT or luciferase reporter gene and transiently transfected into primary rat hepatocytes. Taurocholate (TCA), taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) significantly reduced CAT activities of the -840/+23, -329/+23, and -195/+23 mCAT constructs. A -76/+23 construct showed no regulation by bile acids. When a DNA fragment (-110/-86) from this region was cloned in front of an SV 40 promoter it showed down-regulation by TDCA. 'Super'-electrophoretic mobility shift assays (EMSA) indicated that both HNF1alpha and C/EBP bind to the -110 to -86 bp DNA fragment. Recombinant rat HNF1alpha and C/EBPalpha competitively bound to this DNA fragment. 'Super'-EMSA showed that TDCA addition to hepatocytes in culture decreased HNF1alpha, but not C/EBP, binding to the -110/-86 bp DNA fragment. A four base pair substitution mutation (-103 to -99) in this sequence eliminated TCA and TDCA regulation of the (-840/+23) construct. The substitution mutation also eliminated (>95%) HNF1alpha, but not C/EBP, binding to this DNA fragment. We conclude that bile acids repress CYP27 transcription through a putative BARE located between -110 and -86 bp of the CYP27 promoter. The data suggest that bile acids repress CYP27 transcriptional activity by decreasing HNF1alpha binding to the CYP27 promoter.

Constitutive and inducible cytochromes P450 in rat lung mitochondria: xenobiotic induction, relative abundance, and catalytic properties.

The presence of xenobiotic-inducible CYP1A1, 2B1/2, and 3A1/2 in rat lung mitochondria was investigated using mitochondrial preparations of defined purity. The mitochondrial P450 content in uninduced lung was 1.5-fold higher compared to microsomes. Administration of BNF induced the P450 contents by twofold in both mitochondrial and microsomal membrane fractions. BNF treatment induced EROD activity to about 40-fold in the microsomal fraction and 25-fold in the mitochondrial fraction. The microsomal induction was observed at 4 days of BNF treatment, while the mitochondrial induction required 10 days of treatment. Consistent with the activity profile, Western blot analysis showed the presence of CYP1A1 antibody reactive protein only in lung mitochondria from BNF-treated rats. BNF administration also caused a 50 to 80% reduction in the CYP2B1/2-associated PROD and BROD activities and CYP3A1/2-associated ERND activity in both mitochondria and microsomes. There was also a parallel reduction in the antibody reactive CYP2B1/2 and 3A1/2 proteins in both of these membrane fractions. Administration of DEX for 4 days induced mitochondrial and microsomal ERND activity by 1. 7- and 2.5-fold, respectively. Mitochondrial EROD activity was inhibited by antibodies to P450MT2, as well as Adx, but not by antibody against P450 reductase, indicating the mitochondrial localization of CYP1A1. Protease protection and alkaline extraction experiments indicated that CYP1A1 associated with lung mitochondria is localized inside the inner membrane and exists as a membrane extrinsic protein. In summary, this is probably the first report of inducible P450s in rat lung mitochondria, and our results suggest a possible functional role for these mitochondrial enzymes in xenobiotic metabolism.

Physiological role of the N-terminal processed P4501A1 targeted to mitochondria in erythromycin metabolism and reversal of erythromycin-mediated inhibition of mitochondrial protein synthesis.

Recently, we showed that the major species of beta-naphthoflavone-inducible rat liver mitochondrial P450MT2 consists of N-terminal truncated microsomal P4501A1 (+33/1A1) and that the truncated enzyme exhibits different substrate specificity as compared with intact P4501A1. The results of the present study show that P450MT2 targeted to COS cell mitochondria by transient transfection of P4501A1 cDNA is localized inside the mitochondrial inner membrane in a membrane-extrinsic orientation. Co-expression with wild type P4501A1 and adrenodoxin (Adx) cDNAs resulted in 5-7-fold higher erythromycin N-demethylation (ERND) in the mitochondrial fraction but minimal changes in the microsomal fraction of transfected cells. Erythromycin, a potent inhibitor of bacterial and mitochondrial protein synthesis, caused 8-12-fold higher accumulation of CYP1A1 mRNA, preferential accumulation of P450MT2, and 5-6-fold higher ERND activity in the mitochondrial compartment of rat C6 glioma cells. Consistent with the increased mitochondrial ERND activity, co-expression with P4501A1 and Adx in COS cells rendered complete protection against erythromycin-mediated mitochondrial translation inhibition. Mutations that specifically affect the mitochondrial targeting of P4501A1 also abolished protection against mitochondrial translation inhibition. These results for the first time suggest a physiological function for the xenobiotic inducible cytochrome P4501A1 against drug-mediated mitochondrial toxicity.

Preferential effects of nicotine and 4-(N-methyl-N-nitrosamine)-1-(3-pyridyl)-1-butanone on mitochondrial glutathione S-transferase A4-4 induction and increased oxidative stress in the rat brain.

We have investigated the in vivo effects of the tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on antioxidant defense systems in the mitochondrial, microsomal, and cytosolic compartments of rat brain, lung, and liver. Nicotine induced maximum oxidative stress in brain mitochondria, as seen from a 1.9-fold (P < 0.001) increase in thiobarbituric acid-reactive substance (TBARS) and a 2-fold (P < 0.001) increase in glutathione S-transferase (GST) A4-4 (also referred to as rGST 8-8) activities. These changes were accompanied by a 25-40% increase in reactive oxygen species and a 20-30% decrease in alcohol dehydrogenase activities. The 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-induced oxidative damage was apparent in the microsomal fraction of brain, lung, and liver, and it also increased 4-hydroxynonenal specific GST A4-4 activity in the brain and lung mitochondrial matrix fraction. The levels of microsomal thiobarbituric acid reactive substance, cytochrome P4502E1 activity, and reactive oxygen species were also increased significantly (P < 0.001) in all tissues. Both of these toxins induced the level of GST A4-4 mRNA in the brain, while they caused a marked reduction in the liver GST A4-4 mRNA pool. Additionally, the brain mitochondrial matrix showed a markedly higher level of 4-hydroxynonenal specific GST activity and mGST A4-4 antibody-reactive protein than did the cytosolic fraction. In conclusion, the present study provides evidence for the occurrence of GST A4-4 enzyme activity in mammalian mitochondria, in addition to demonstrating that both mitochondria and microsomes are intracellular targets for nicotine- and NNK-induced organ toxicity.

Structural organization and transcription regulation of nuclear genes encoding the mammalian cytochrome c oxidase complex.

Cytochrome c Oxidase (COX) is the terminal component of the bacterial as well as the mitochondrial respiratory chain complex that catalyzes the conversion of redox energy to ATP. In eukaryotes, the oligomeric enzyme is bound to mitochondrial innermembrane with subunits ranging from 7 to 13. Thus, its biosynthesis involves a coordinate interplay between nuclear and mitochondrial genomes. The largest subunits, I, II, and III, which represent the catalytic core of the enzyme, are encoded by the mitochondrial DNA and are synthesized within the mitochondria. The rest of the smaller subunits implicated in the regulatory function are encoded on the nuclear DNA and imported into mitochondria following their synthesis in the cytosol. Some of the nuclear coded subunits are expressed in tissue and developmental specific isologs. The ubiquitous subunits IV, Va, Vb, VIb, VIc, VIIb, VIIc, and VIII (L) are detected in all the tissues, although the mRNA levels for the individual subunits vary in different tissues. The tissue specific isologs VIa (H), VIIa (H), and VIII (H) are exclusive to heart and skeletal muscle. cDNA sequence analysis of nuclear coded subunits reveals 60 to 90% conservation among species both at the amino acid and nucleotide level, with the exception of subunit VIII, which exhibits 40 to 80% interspecies homology. Functional genes for COX subunits IV, Vb, VIa 'L' & 'H', VIIa 'L' & 'H', VIIc and VIII (H) from different mammalian species and their 5' flanking putative promoter regions have been sequenced and extensively characterized. The size of the genes range from 2 to 10 kb in length. Although the number of introns and exons are identical between different species for a given gene, the size varies across the species. A majority of COX genes investigated, with the exception of muscle-specific COXVIII(H) gene, lack the canonical 'TATAA' sequence and contain GC-rich sequences at the immediate upstream region of transcription start site(s). In this respect, the promoter structure of COX genes resemble those of many house-keeping genes. The ubiquitous COX genes show extensive 5' heterogeneity with multiple transcription initiation sites that bind to both general as well as specialized transcription factors such as YY1 and GABP (NRF2/ets). The transcription activity of the promoter in most of the ubiquitous genes is regulated by factors binding to the 5' upstream Sp1, NRF1, GABP (NRF2), and YY1 sites. Additionally, the murine COXVb promoter contains a negative regulatory region that encompasses the binding motifs with partial or full consensus to YY1, GTG, CArG, and ets. Interestingly, the muscle-specific COX genes contain a number of striated muscle-specific regulatory motifs such as E box, CArG, and MEF2 at the proximal promoter regions. While the regulation of COXVIa (H) gene involves factors binding to both MEF2 and E box in a skeletal muscle-specific fashion, the COXVIII (H) gene is regulated by factors binding to two tandomly duplicated E boxes in both skeletal and cardiac myocytes. The cardiac-specific factor has been suggested to be a novel bHLH protein. Mammalian COX genes provide a valuable system to study mechanisms of coordinated regulation of nuclear and mitochondrial genes. The presence of conserved sequence motifs common to several of the nuclear genes, which encode mitochondrial proteins, suggest a possible regulatory function by common physiological factors like heme/O2/carbon source. Thus, a well-orchestrated regulatory control and cross talks between the nuclear and mitochondrial genomes in response to changes in the mitochondrial metabolic conditions are key factors in the overall regulation of mitochondrial biogenesis.

Differential response of cytosolic, microsomal, and mitochondrial glutathione S-transferases to xenobiotic inducers.

Subcellular levels of different isoenzymes of glutathione S-transferases (GSTs) and their catalytic activities in rat liver, lung and brain tissues were compared following treatment with phenobarbital (PB), -naphthoflavone (BNF) and dexamethasone (DEX). The constitutive expression of á and mu classes of GSTs, but not the GST , was maximum in the liver cytosol as compared to other tissues. Cytosolic GST activity using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate was 2-4 fold higher than that in the microsomal and mitochondrial fractions. Glutathione peroxidase activity with cumene hydroperoxide as a substrate was also highest in the rat liver cytosol. PB and BNF treatments markedly induced the amount of GST proteins in all the tissues studied with the maximum induction in the cytosol after 4 days of PB and 10 days of BNF treatments, respectively. The longer duration of treatments had a suppressive effect on the GST activity, particularly in the mitochondrial and microsomal fractions. DEX treatment, on the other hand, only marginally induced the cytosolic GST, while the mitochondrial GST and the membrane bound microsomal GST activities were mostly decreased. Northern blot analysis also showed an increase in the GST-á mRNA level indicating a possible upregulation of the GST gene expression by the xenobiotic agent. Differences between the subcellular GSTs were studied by the in vitro addition of N-ethylmaleimide (NEM), a selective activator of the microsomal GST. The cytosolic GST activity, both in livers of uninduced and PB-treated, was inhibited to about 50% of the control levels by NEM. The mitochondrial activity, on the other hand, was significantly activated by the addition of NEM, similar to that reported for the microsomal GST. These results suggest selectivity in the effects of different xenobiotics on the expression and catalytic activity of GST isoenzymes from different subcellular compartments of tissues. More importantly, these observations are also relevant in studies on xenobiotic induced organ-specific toxicity and carcinogenicity.

Interaction of adrenodoxin with P4501A1 and its truncated form P450MT2 through different domains: differential modulation of enzyme activities.

Recently we showed that the beta-naphthoflavone-inducible liver mitochondrial P450MT2 consists of two N-terminal truncated forms of the microsomal P4501A1, termed P450MT2a (+5/1A1) and MT2b (+33/1A1) [Addya et al. (1997) J. Cell Biol. 139, 589-599]. In the present study, we demonstrate that intact P4501A1 and the major mitochondrial form, P450MT2b (routinely referred to as P450MT2), show distinct substrate specificities and preference for different electron transport proteins. Enzyme reconstitution and spectral studies show that the wild-type adrenodoxin (Adx), but not the mutant Adx, binds to P450MT2 in a functionally productive manner (Kd = 0.6 microM) and induces a characteristic high-spin state. Adx binding to intact P4501A1 or +5/1A1 is less efficient as seen from spectral shift patterns (Kd = 1.8-2.0 microM) and reconstitution of enzyme activity. Use of Adx--Sepharose affinity matrix yielded < 90% pure P450MT2 (specific activity: 13.5 nmol/mg of protein) starting from a partially purified fraction of 10-15% purity, further demonstrating the specificity of P450MT2 and Adx interaction. Chemical cross-linking studies show that the bovine Adx forms heteroduplexes with both P450MT2 and intact P4501A1, though at different efficiencies. Our results show that Adx interacts with P450MT2 through its C-terminal acidic domain 2, while interaction with intact P4501A1 likely involves the N-terminal acidic domain 1. These results point to an interesting possibility that different electron transfer proteins may differently modulate the enzyme activity. Our results also demonstrate for the first time as to how a different mode of Adx interaction differently modulates the substrate specificities of the two P450 forms.

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2.

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

Localization of multiple forms of inducible cytochromes P450 in rat liver mitochondria: immunological characteristics and patterns of xenobiotic substrate metabolism.

Hepatic mitochondria contain inducible cytochromes P450 that cross-react with antibodies to P4501A1/2 and 2B1/2. In the present study, we present evidence for the occurrence of additional P450 forms in rat liver mitochondria that cross-react with antibodies to microsomal P4503A1/2 and 2E1. Protease protection and also immunoelectron microscopy studies were carried out to support the mitochondrial location of the immunoreactive P450s. The solubility of immunoreactive proteins in 0.1 M Na2CO3 suggests that the mitochondrial P450 forms tested are not membrane-integral proteins. The mitochondrial-associated P450 forms are capable of metabolizing resorufin derivatives, erythromycin, and p-nitrophenol in an adrenodoxin- and adrenodoxin reductase-supported system. Treatment of rats with phenobarbital (PB) resulted in the induction of mitochondrial pentoxyresorufin O-deethylase (PROD), benzoxyresorufin O-deethylase (BROD), and erythromycin N-demethylase (ERND) activities by 17-, 23-, and 2-fold, respectively. These activities were inhibited by 33 to 64% by antibodies to P4502B1/2 and P4503A1/2. The induction of the above monooxygenase activities correlated with the levels of mitochondrial proteins cross-reacting with antibodies to P4502B1/2 and P4503A1/2 in PB-treated livers. Similarly, administration of beta-naphthoflavone (BNF) resulted in a marked elevation of O-deethylation of ethoxy-, benzoxy-, and methoxyresorufins and a 2-fold increase in ERND activity. Immunoblot and immunoinhibition experiments using P4501A1/2, P4502B1/2, P4503A1/2, and P4502E1 antibodies revealed the presence of P450 forms closely related to the microsomal inducible forms. Results of immunoinhibition studies, using antibodies to adrenodoxin and reconstitution of enzyme activity with purified P450 forms, suggested a role for the mitochondrial P450 in the metabolism of xenobiotic substrates. The purified mitochondrial P450s also exhibited overlapping substrate specificities for resorufin derivatives and erythromycin.

Regulation of murine cytochrome oxidase Vb gene expression in different tissues and during myogenesis. Role of a YY-1 factor-binding negative enhancer.

The mouse cytochrome oxidase (COX) Vb promoter contains three sequence motifs with partial or full consensus for YY-1 and GTG factor binding and a CArG box, located between positions -480 and -390. Individually, all three motifs stimulated transcription of the TKCAT promoter, and bound distinctly different proteins from the liver and differentiated C2C12 nuclear extracts. Collectively, these motifs, together with the downstream flanking sequence, -378 to -320, suppressed the transcription activity of heterologous promoters, thymidine kinase-chloramphenicol acetyltransferase (TKCAT) and COXIV/CAT. The transcription activities of both TKCAT and COXIV/CAT constructs were induced 3-4-fold during induced myogenesis of C2C12 cells. The downstream CArG-like motif binds transcription factor YY-1, while the upstream YY-1-like motif binds to a yet unidentified factor. Co-expression with intact YY-1, but not that lacking the DNA binding domain suppressed the transcriptional activity. Mutations targeted to the CArG-like motif abolished the suppressive effect of the negative enhancer and the inducibility of the promoter during myogenic differentiation. Our results suggest that the activity of the negative enhancer may determine the level of expression of the COX Vb gene in different tissues.

The role of an E box binding basic helix loop helix protein in the cardiac muscle-specific expression of the rat cytochrome oxidase subunit VIII gene.

We have characterized the rat gene for muscle-specific cytochrome oxidase VIII (COX VIII(H)) and mapped the distal promoter region responsible for transcription activation in C2C12 skeletal myocytes and H9C2 cardiomyocytes. In both cell types, the promoter elements responding to the induced differentiation of myocytes map to two E boxes, designated as E1 and E2 boxes with a core sequence of CAGCTG. Gel mobility shift analysis showed that both E1 and E2 box motifs form complexes with nuclear extracts from H9C2 cardiomyocytes that were supershifted with monoclonal antibody to E2A but not with antibody to myo-D. Extracts from induced and uninduced H9C2 cardiomyocytes yielded different gel mobility patterns and also different E2A antibody supershifts suggesting a difference in the DNA-bound protein complexes cross-reacting with the E2A antibody. Transcriptional activity of the promoter construct containing intact E boxes was inhibited by coexpression with Id in differentiated H9C2 cardiomyocytes. Our results show the involvement of an E box binding basic helix loop helix protein in the cardiac muscle-specific regulation of the COX VIII(H) promoter.

Localization of a transcription promoter within the second exon of the cytochrome P-450c27/25 gene for the expression of the major species of two-kilobase mRNA.

The rat P-450c27/25 (CYP27) gene is expressed as two distinctly sized mRNAs of 2 and 2.3 kb (kilobase). The 2 kb mRNA is the predominant form in the liver with negligible 2.3 kb species. Rat kidney and hepatoma, on the other hand, contain significant levels of the 2.3 kb species. Rat CYP27 gene contains 11 exons of 80-415 nucleotides that are separated by 10 introns of 83 bases to approximately 10 kb. S1 nuclease protection and primer extension analyses using liver RNA showed a prominent 5' terminus 86 nucleotides downstream from the start of exon 2. This site, designated as +1, is the start site for the 2 kb mRNA. 5' RACE analysis of rat kidney and hepatoma RNAs showed the presence of a 5' extended mRNA with a sequence complementary to the Spi2 mRNA. A cryptic TATA box (TTTAAA) is located 24 nucleotides upstream of the 2 kb mRNA transcription initiation site at +1. A 106 bp DNA fragment (sequence -83 to +23) that houses the putative TATA motif forms three differently migrating complexes with nuclear extract from the murine 3T3 cells. DNAse I footprinting and competition with synthetic DNA showed that complex A represents the bound Sp1 factor and complexes B and C are due to unknown factors binding to the -83 to -71 and -20 to -12 sequences, respectively. In vivo transcription analysis using -840/+23 DNA and its 5' deletions cloned in a CAT reporter plasmid suggests that the basal promoter elements are located within sequence -45 to +23 of the gene. Finally, in vitro transcription analysis in HeLa cell nuclear extract showed that intact TTTAAA motif and complex C-forming sequence from this region are essential for transcription initiation at the +1 position of the promoter. Our results demonstrate that the 2 kb mRNA is transcribed as an independent transcript driven by an immediate upstream promoter located within exon 2.

Identification of a stable RNA encoded by the H-strand of the mouse mitochondrial D-loop region and a conserved sequence motif immediately upstream of its polyadenylation site.

By using a combination of Northern blot hybridization with strand-specific DNA probes, S1 nuclease protection, and sequencing of oligo-dT-primed cDNA clones, we have identified a 0.8 kb poly(A)-containing RNA encoded by the H-strand of the mouse mitochondrial D-loop region. The 5' end of the RNA maps to nucleotide 15417, a region complementary to the start of tRNA(Pro) gene and the 3' polyadenylated end maps to nucleotide 16295 of the genome, immediately upstream of tRNA(Phe) gene. The H-strand D-loop region encoded transcripts of similar size are also detected in other vertebrate systems. In the mouse, rat, and human systems, the 3' ends of the D-loop encoded RNA are preceded by conserved sequences AAUAAA, AAUUAA, or AACUAA, that resemble the polyadenylation signal. The steady-state level of the RNA is generally low in dividing or in vitro cultured cells, and markedly higher in differentiated tissues like liver, kidney, heart, and brain. Furthermore, an over 10-fold increase in the level of this RNA is observed during the induced differentiation of C2C12 mouse myoblast cells into myotubes. These results suggest that the D-loop H-strand encoded RNA may have yet unknown biological functions. A 20 base pair DNA sequence from the 3' terminal region containing the conserved sequence motif binds to a protein from the mitochondrial extracts in a sequence-specific manner. The binding specificity of this protein is distinctly different from the previously characterized H-strand DNA termination sequence in the D-loop or the H-strand transcription terminator immediately downstream of the 16S rRNA gene. Thus, we have characterized a novel poly(A)-containing RNA encoded by the H-strand of the mitochondrial D-loop region and also identified the putative ultimate termination site for the H-strand transcription.

Purification and characterization of a hepatic mitochondrial glutathione S-transferase exhibiting immunochemical relationship to the alpha-class of cytosolic isoenzymes.

Hepatic mitochondria from different mammalian species contain varying levels of glutathione S-transferase (GST) activities. More than 70% of the activity detectable in the mouse liver mitochondria is associated with the soluble matrix. The mouse mitochondrial matrix GST was purified using a combination of (NH4)2SO4 fractionation, Sephadex gel filtration and affinity chromatography on glutathione (GSH) conjugated Sepharose. The purified GST comigrates with the mouse cytosolic MI (or alpha form), and exhibits an apparent molecular mass of 25 kD on sodium dodecyl sulfate-polyacrylamide gels. Polyclonal antibody to the purified mitochondrial GST cross-reacted with the similarly migrating cytosolic MI GST, suggesting extensive immunochemical relatedness between these two forms. As previously demonstrated for the cytosolic alpha form, the mitochondrial GST catalyzes aflatoxin B1-GSH conjugation (6.3 nmol/mg protein/min) and exhibits peroxidase activity (6.7 mumol/mg protein/min). The putative mitochondrial GST only in intact mitochondria, but not in sonic disrupted mitochondria, is resistant to proteolytic digestion with trypsin, demonstrating its intramitochondrial location. Isoelectric focusing on the flat bed polyacrylamide gel system resolves the mitochondrial GST into two distinct components with apparent pI of 9.9 and 9.7, both of which cross-react with polyclonal antibody to the mitochondrial GST. Under the identical conditions, the most cationic form of cytosolic GST cross-reacting intensely with the antibody resolves as a single component with an apparent pI of 9.4. Thus the mitochondrial GST resembles the alpha family of isoenzymes, though it appears to represent independent molecular species different from the cytosolic forms.