RESUMEN
During NASA's Apollo missions, inhalation of dust particles from lunar regolith was identified as a potential occupational hazard for astronauts. These fine particles adhered tightly to spacesuits and were unavoidably brought into the living areas of the spacecraft. Apollo astronauts reported that exposure to the dust caused intense respiratory and ocular irritation. This problem is a potential challenge for the Artemis Program, which aims to return humans to the Moon for extended stays in this decade. Since lunar dust is "weathered" by space radiation, solar wind, and the incessant bombardment of micrometeorites, we investigated whether treatment of lunar regolith simulants to mimic space weathering enhanced their toxicity. Two such simulants were employed in this research, Lunar Mare Simulant-1 (LMS-1), and Lunar Highlands Simulant-1 (LHS-1), which were added to cultures of human lung epithelial cells (A549) to simulate lung exposure to the dusts. In addition to pulverization, previously shown to increase dust toxicity sharply, the simulants were exposed to hydrogen gas at high temperature as a proxy for solar wind exposure. This treatment further increased the toxicity of both simulants, as measured by the disruption of mitochondrial function, and damage to DNA both in mitochondria and in the nucleus. By testing the effects of supplementing the cells with an antioxidant (N-acetylcysteine), we showed that a substantial component of this toxicity arises from free radicals. It remains to be determined to what extent the radicals arise from the dust itself, as opposed to their active generation by inflammatory processes in the treated cells.
RESUMEN
Protein iron-sulfur (FeS) centers have recently been implicated in the regulation of gene expression. In the redox-sensing SoxR protein, the oxidation state of [2Fe-2S] centers controls its activity as a transcription activator independent of DNA-binding ability. Thus, FeS centers allosterically link cellular oxidative stress to the expression of defense genes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteínas Bacterianas/genética , Regulación de la Expresión Génica/genética , Óxido Nítrico/metabolismo , Oxidación-Reducción , Regiones Promotoras Genéticas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
New insights into the workings of the repair enzymes that police the genome for damage to DNA come from the recently determined structures of two uracil-DNA glycosylases.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , N-Glicosil Hidrolasas/metabolismo , Humanos , Conformación Proteica , Uracilo/metabolismo , Uracil-ADN Glicosidasa , Proteínas Virales/metabolismoRESUMEN
The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells. Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells. The rate of spontaneous mutation in apn1 mutant S. cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired.
Asunto(s)
Daño del ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Mutación , Saccharomyces cerevisiae/enzimología , Alquilantes/farmacología , Alquilación , Centrifugación por Gradiente de Densidad , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido) , Genes Fúngicos , Peróxido de Hidrógeno/farmacología , Metilmetanosulfonato/farmacología , Oxidación-Reducción , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genéticaRESUMEN
Adenine-DNA glycosylase MutY of Escherichia coli catalyzes the cleavage of adenine when mismatched with 7,8-dihydro-8-oxoguanine (GO), an oxidatively damaged base. The biological outcome is the prevention of C/G-->A/T transversions. The molecular mechanism of base excision repair (BER) of A/GO in mammals is not well understood. In this study we report stimulation of mammalian adenine-DNA glycosylase activity by apurinic/apyrimidinic (AP) endonuclease using murine homolog of MutY (Myh) and human AP endonuclease (Ape1), which shares 94% amino acid identity with its murine homolog Apex. After removal of adenine by the Myh glycosylase activity, intact AP DNA remains due to lack of an efficient Myh AP lyase activity. The study of wild-type Ape1 and its catalytic mutant H309N demonstrates that Ape1 catalytic activity is required for formation of cleaved AP DNA. It also appears that Ape1 stimulates Myh glycosylase activity by increasing formation of the Myh-DNA complex. This stimulation is independent of the catalytic activity of Ape1. Consequently, Ape1 preserves the Myh preference for A/GO over A/G and improves overall glycosylase efficiency. Our study suggests that protein-protein interactions may occur in vivo to achieve efficient BER of A/GO.
Asunto(s)
Liasas de Carbono-Oxígeno/metabolismo , Reparación del ADN , Proteínas de Escherichia coli , Guanina/análogos & derivados , N-Glicosil Hidrolasas/genética , Secuencia de Aminoácidos , Animales , Ácido Apurínico/metabolismo , Disparidad de Par Base/genética , Clonación Molecular , ADN/genética , ADN/metabolismo , Daño del ADN , ADN Glicosilasas , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Electroforesis en Gel de Poliacrilamida , Guanina/metabolismo , Ratones , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/metabolismo , Unión Proteica , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Oxidative stress exerted by superoxide-generating (redox-cycling) agents such as paraquat triggers the soxRS regulon of Escherichia coli. In this system, SoxR protein is the redox-sensitive activator of the soxS gene, the product of which then activates the approximately 10 promoters of this regulon. We found that 4-nitroquinoline-N-oxide (4NQO) is a powerful inducer of soxS, > 10-fold more potent than paraquat. The transcriptional induction of the soxS gene by 4NQO was tightly dependent on a functional soxR gene and on the presence of molecular oxygen, as found previously for several well characterized redox-cycling agents. Two 4NQO-related compounds were also shown to induce soxS:4-nitropyridine-N-oxide, with an efficiency only slightly less than 4NQO, and 4-hydroxyaminoquinoline-N-oxide, at approximately 50-fold lower potency than 4NQO. E. coli strains that are hypersensitive to oxidative stress (owing to deficiency in either superoxide dismutases or oxidative DNA repair enzymes) were hypersensitive to killing by 4NQO. Thus, considerable oxidative stress is induced in cells by 4NQO, which might contribute to the carcinogenic potency of this compound.
Asunto(s)
4-Nitroquinolina-1-Óxido/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas de Escherichia coli , Transactivadores , Factores de Transcripción/biosíntesis , 4-Nitroquinolina-1-Óxido/análogos & derivados , Proteínas Bacterianas/genética , Escherichia coli/genética , Peróxido de Hidrógeno/metabolismo , Metosulfato de Metilfenazonio/farmacología , Oxidación-Reducción/efectos de los fármacos , Superóxido Dismutasa/farmacología , Superóxidos/metabolismo , Factores de Transcripción/genéticaRESUMEN
NO is a biologically generated free radical that serves diverse roles in mammalian cell signaling and immune-mediated cell killing. Because mammalian cells might be exposed to varying levels of NO, we tested for possible defense genes and proteins induced upon treatment of cells with sublethal fluxes of pure NO. Two-dimensional gel analysis was performed for human embryonic lung fibroblasts (IMR-90) exposed for 90 min to pure NO at approximately 280 nM/s, which revealed the reproducible induction of at least 12 proteins. Among these, a prominent polypeptide had Mr approximately 32,000, similar to the well-known oxidative stress protein heme oxygenase-1 (HO-1). Northern blot analysis of IMR-90 and HeLa cells demonstrated the NO-mediated induction of HO-1 mRNA up to 70-fold over the levels in untreated cells. HO-1 induction depended on the NO dose and subsequent expression time and was maximal 3-5 h after a 1-h exposure to NO at a constant flux of approximately 280 nM/s. The mRNA encoding a tyrosine/threonine phosphatase (CL100/MKP-1) was also NO inducible (approximately 20 fold), whereas there was no increase in expression of the mRNA encoding manganese-containing superoxide dismutase. Induction of HO-1 mRNA was independent of the guanylate cyclase signaling pathway; addition of the analogue 8-bromo-cyclic GMP did not induce the HO-1 transcript, and the soluble guanylate cyclase inhibitor LY-83583 did not block HO-1 induction by NO in IMR-90 cells. Luciferase reporter constructs containing up to 4.7 kb of DNA upstream of the HO-1 transcription start site showed < or = 2.5-fold induction in IMR-90 or HeLa cells exposed to NO. However, HO-1 mRNA was dramatically stabilized after exposure of IMR-90 cells to NO. Even a transient NO exposure produced elevated levels of HO-1 protein for > or = 10 h, whereas continuous low-level NO treatment (35 nM/s) maintained elevated HO-1 mRNA expression for > or = 8 h. These results reveal a complex mammalian response to NO that involves a new level of posttranscriptional control in response to this radical.
Asunto(s)
Proteínas de Ciclo Celular , Hemo Oxigenasa (Desciclizante)/biosíntesis , Proteínas Inmediatas-Precoces/biosíntesis , Óxido Nítrico/farmacología , Fosfoproteínas Fosfatasas , Biosíntesis de Proteínas , Proteínas Tirosina Fosfatasas/biosíntesis , Relación Dosis-Respuesta a Droga , Fosfatasa 1 de Especificidad Dual , Electroforesis en Gel Bidimensional , Inducción Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Guanilato Ciclasa/metabolismo , Células HeLa/efectos de los fármacos , Células HeLa/enzimología , Hemo-Oxigenasa 1 , Humanos , Pulmón/citología , Proteínas de la Membrana , Proteína Fosfatasa 1 , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismoRESUMEN
The 19,000 Mr C-terminal domain of the Escherichia coli ada gene product that contains O6-methylguanine-DNA methyltransferase DNA repair activity has been crystallized in a low-salt environment. The crystals, which diffract to 2.3 A (1 A = 0.1 nm), are suitable for detailed structural studies. The space group is P21 with unit cell dimensions a = 46.3 A, b = 45.8 A, c = 46.9 A and beta = 113.3 degrees.
Asunto(s)
Escherichia coli/enzimología , Metiltransferasas , Cristalización , O(6)-Metilguanina-ADN MetiltransferasaRESUMEN
Two redox-responsive transcription regulators have been well defined in Escherichia coli and serve as paradigms of redox-operated genetic switches. SoxR contains iron-sulfur centers that activate the protein when they are one-electron oxidized, or nitrosylated by nitric oxide. OxyR contains a pair of redox-active cysteine residues that activate the protein when they are oxidized to form a disulfide bond.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Bacterianas/química , Biotecnología , Disulfuros/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Radicales Libres/metabolismo , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico/metabolismo , Oxidación-Reducción , Proteínas Represoras/química , Superóxidos/metabolismo , Factores de Transcripción/química , Activación TranscripcionalRESUMEN
The soxRS regulon of Escherichia coli coordinates the induction of at least twelve genes in response to superoxide or nitric oxide. This review describes recent progress in understanding the signal transduction and transcriptional control mechanisms that activate the soxRS regulon, and some aspects of the physiological functions of this system. The SoxS protein represents a growing family of transcription activators that stimulate genes for resistance to oxidative stress and antibiotics. SoxR is an unusual transcription factor whose activity in vitro can be switched off by the removal of [2Fe-2S] centers, and activated by their reinsertion. The activated form of SoxR remodels the structure of the soxS promoter to activate transcription. When the soxRS system is activated, bacteria gain resistance to oxidants, antibiotics and immune cells that generate nitric oxide. The latter features could increase the success (virulence) of some bacterial infections.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo/genética , Regulón , Transactivadores , Factores de Transcripción/genética , Transducción de Señal/genéticaRESUMEN
Nitric oxide (NO) is a free radical produced actively by mammalian cells, including neurons. Low levels of NO can function in intercellular signaling, but high levels are cytotoxic. This cytotoxic potential suggests that cells at risk for NO damage, such as neurons, might have NO resistance mechanisms to prevent cell death, and adaptive resistance to NO-releasing compounds has been reported for some non-neuronal cell types. Here we show that immortalized mouse motor neurons (NSC34 cells) respond to sub-lethal fluxes of pure NO by activating adaptive resistance mechanisms that counteract cytotoxic NO exposure. This adaptive NO resistance is reversible and is paralleled by the induction of the oxidative stress enzyme heme oxygenase 1 (HO-1). An inhibitor of both HO-1 and heme-dependent guanylate cyclase (tin-protoporphyrin IX) greatly sensitized NO-pretreated NSC34 cells to the NO challenge. However, readdition of cyclic GMP (in the form of the 8-bromo derivative) restored rather little resistance, and a more selective guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxaline-1-one (at 10 microM), did not have the sensitizing effect. Therefore, the inducible HO-1 pathway contributes substantially to adaptive NO resistance, while cyclic GMP seems to play at most a small role. A similar adaptive resistance to NO was observed in primary rat spinal chord motor neurons. The activation of NO resistance in motor neurons may counteract age- or disease-related neurodegeneration.
Asunto(s)
Neuronas Motoras/citología , Óxido Nítrico/farmacología , Médula Espinal/citología , Animales , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Resistencia a Medicamentos , Embrión de Mamíferos , Cinética , Ratones , Neuronas Motoras/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/fisiología , Fármacos Neuroprotectores , Estrés Oxidativo , Ratas , Ratas Long-EvansRESUMEN
Genetic responses to oxidative stress are triggered by excessive levels of agents such as superoxide. The soxRS regulon of Escherichia coli includes at least a dozen oxidative-stress and antibiotic-resistance genes that are activated by the SoxS protein, the synthesis of which is controlled by the redox-sensing SoxR protein. SoxR is a homodimer of 17 kDa subunits, each of which contains a [2Fe-2S] cluster. Transcriptional activation by SoxR is controlled by the oxidation state of these metal centres. In the absence of oxidative stress, the [2Fe-2S] centres are in the reduced form and the protein is inactive, although it still binds the soxS promoter. Agents that generate superoxide in the cell (e.g. paraquat) cause rapid oxidation of the metal centres, which triggers the transcriptional activity of SoxR; removal of the oxidative stress is followed by rapid re-reduction of the [2Fe-2S] centres. This facile mechanism links oxidation state to control of protein activity and may be used widely to allow cells to respond to oxidative stress.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Hierro-Azufre/metabolismo , Estrés Oxidativo , Transcripción Genética , Regulación Alostérica , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Oxidación-Reducción , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Two different DNA repair enzymes are induced in E. coli by methylating agents: a methyltransferase acting on 0(6)-methylguanine residues, and a DNA glycosylate for N-methylated purines. The properties of the homogeneous methyltransferase are described.
Asunto(s)
Alquilantes/farmacología , ADN Glicosilasas , Reparación del ADN , Escherichia coli/enzimología , Metiltransferasas/metabolismo , N-Glicosil Hidrolasas/metabolismo , Metilación , Peso Molecular , O(6)-Metilguanina-ADN MetiltransferasaRESUMEN
DNA strand breaks with terminal 3'-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4'-carbon of DNA deoxyribose groups. Included among these agents are gamma-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin. However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks. Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents. Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from gamma-irradiated DNA, although there appeared to be a small resistant subpopulation. The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes. These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.
Asunto(s)
Antibacterianos/toxicidad , Antibióticos Antineoplásicos/toxicidad , Liasas de Carbono-Oxígeno/metabolismo , Daño del ADN , ADN/metabolismo , Proteínas de Escherichia coli , Exodesoxirribonucleasas/metabolismo , Glicolatos , Cinostatina/toxicidad , Aminoglicósidos , Animales , Bovinos , ADN/efectos de los fármacos , ADN/efectos de la radiación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Escherichia coli/enzimología , Rayos gamma , Humanos , CinéticaRESUMEN
Abasic (AP) sites in DNA arise from spontaneous reactions or the action of DNA glycosylases and represent a loss of genetic information. The AP sites can be mutagenic or cytotoxic, and their repair is initiated by class II AP endonucleases, which incise immediately 5' to AP sites. The main enzyme of S. cerevisiae. Apn1, provides cellular resistance to oxidants (e.g., H2O2) or alkylating agents, and limits the spontaneous mutation rate. AP endonucleases from other species can replace Apn1 function in yeast to different extents. We studied the main human enzyme, Ape, with respect to its incision specificity in vitro and the expression of the APE gene in vivo. The results suggest that Ape evolved to act preferentially on AP sites compared to deoxyribose fragments located at oxidative strand breaks and that the incision modes of Ape and Apn1 may be fundamentally different. We also defined the functional APE promoter, and showed that APE expression is transiently downregulated during the regeneration of epidermis after wounding. This latter effect may lead to a window of vulnerability for DNA damage and perhaps mutagenesis during the healing of epidermal and other wounds. Such unexpected effects on the expression of DNA repair enzymes need to be taken into account in analyzing the susceptibility of different tissues to carcinogens.
Asunto(s)
Daño del ADN , Reparación del ADN , Liasas/fisiología , Cicatrización de Heridas , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Humanos , Saccharomyces cerevisiae/genéticaRESUMEN
Apurinic/apyrimidinic endonucleases initiate repair at sites of base loss produced by many carcinogens and maintain genetic stability by repairing abasic sites produced spontaneously. We examined whether expression of the main apurinic endonuclease of human cells, encoded by the APE gene, might be decreased in malignant cells and thus potentiate an elevated mutation rate. Northern blotting and in situ hybridisation were used to quantitate the level of APE mRNA in various normal tissues and in 24 different brain tumors. There were no differences in APE expression among the normal tissues, or between malignant astrocytomas and meningiomas and the surrounding normal brain tissues. This suggests that diminished expression of the apurinic endonuclease does not underly the induction of these cancers, or explain clinical variations in presentation and response to treatment.
RESUMEN
Gene regulation mechanisms have evolved allowing cells to finetune the level of "endogenous" oxidative stress and to cope with increased free radicals from external sources. Levels of H2O2 are tightly controlled in E. coli by OxyR, which is activated by H2O2 to increase scavenging activities and limit H2O2 generation by the respiratory chain. Sub-micromolar levels of H2O2 are maintained in mammalian tissues, though the regulatory systems that govern this control are unknown. Excess superoxide triggers the soxRS system in E. coli, which is controlled by the oxidant-sensitive iron-sulfur centers of the SoxR protein. Nitric oxide activates SoxR by a different modification of the iron-sulfur centers. The soxRS regulon mobilizes diverse functions to scavenge free radicals and repair oxidative damage in macromolecules, and other mechanisms that exclude many environmental agents from the cell. Mammalian cells also sense and respond to sub-toxic levels of nitric oxide, activating expression of heme oxygenase 1 through stabilization of its mRNA. These inductions give rise to adaptive resistance to nitric oxide in neuronal and other cell types.
Asunto(s)
Homeostasis/fisiología , Animales , Proteínas Bacterianas/genética , Escherichia coli/metabolismo , Radicales Libres , Humanos , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico/fisiología , Salmonella typhimurium/metabolismoAsunto(s)
Chlamydomonas reinhardtii/metabolismo , Proteínas de Unión al ADN , Escherichia coli/metabolismo , Estrés Oxidativo , Oxidorreductasas , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos , Animales , Chlamydomonas reinhardtii/genética , Cisteína/metabolismo , Disulfuros/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli , Regulación de la Expresión Génica , Glutarredoxinas , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Modelos Biológicos , Oxidación-Reducción , Proteína Disulfuro Isomerasas/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Compuestos de Sulfhidrilo/metabolismo , Tiorredoxinas/metabolismoRESUMEN
We investigated the minimal promoter of APEX, which encodes mouse apurinic DNA repair endonuclease. A 1.85-kb fragment with APEX upstream sequences and approximately 290 bp of the transcribed region linked to a chloramphenicol acetyltransferase (CAT) reporter gene was assayed by transient transfection in NIH-3T3 cells. The minimal APEX promoter was comprised of approximately 190 bp of upstream and approximately 170 bp of transcribed DNA (exon 1 and most of intron 1). This approximately 360-bp region contains two CCAAT boxes and other consensus protein binding sites, but no TATA box. Deletion of the 5'-most CCAAT box decreased activity approximately 5-fold. The second CCAAT box (situated in exon 1) may play an independent role in APEX expression. Transcription start sites have been identified downstream of the second CCAAT box, and DNase I footprinting demonstrated NIH-3T3 nuclear proteins binding this region, including an Spl site located between the CCAAT boxes. Electrophoretic mobility-shift assays indicated binding by purified Sp1. Mouse proteins did not bind three myc-like (USF) sites in the APEX promoter, in contrast to the APE promoter. The APEX and APE promoter had similar activity in Hela cells, but in mouse cells, the murine promoter had approximately 5-fold higher activity than did the human promoter. Both the APEX and APE promoters exhibited bidirectional activity in their cognate cells.
Asunto(s)
Liasas de Carbono-Oxígeno , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Complejos Multienzimáticos/genética , Proteínas Nucleares/genética , Células 3T3 , Animales , Secuencia de Bases , Células Cultivadas , Mapeo Cromosómico , Clonación Molecular , Huella de ADN , Reparación del ADN , ADN Complementario/análisis , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Desoxirribonucleasa I/metabolismo , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Análisis de Secuencia de ADN , Transcripción Genética , TransfecciónRESUMEN
The metallothionein-III gene (MT-IIA) is a major member of the human MT gene family. Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that bind and detoxify heavy metals. At least two different MT-IIA polymorphisms have been identified in humans, one or both of which may affect susceptibility to metal toxicity. The purpose of this study was to investigate whether these different genotypes affect the inducibility of MT-IIA mRNA in human lymphocytes treated with zinc (Zn), the major known inducer of MT-IIA in vitro. Fresh lymphocytes obtained from 16 healthy volunteers, aged 23-38 yr, were genotyped for the MT-IIA gene and tested for expression. A 43.5-bp HindIII-Taql fragment of the MT-IIA promoter was used to probe for the two known polymorphisms (a 7.8-kb vs. a 5.3-kb fragmnent, and a 1.7-kb vs. a 1.6-kb fragment). The allele frequencies of the 16 subjects were 14%, for 5.3-kb allele and 19% for 1.6-kb allele. In Northern blotting experiments, MT-II mRNA levels were induced over a wide range of Zn concentrations during 2-h exposures; specifcally, levels increased by 9- to 115-fold with exposure to 100 microM ZnCl, and by 16- to 311-fold with exposure to 200 microM ZnCl2. However, no significant differences in MT-IIA inducibility were found between the 7.8/5.3-kb allele pair (n = 4) and the 7.8/7.8-kb allele pair (n = 12) or between the 1.7/1.6-kb allele pair (n = 5) and the 1.7/1.7-kb allele pair (n = 11). Thus. MT-IIA is strongly inducible by Zn in human lymphocytes, but individual variations exceed those that can be attributed to the known promoter-region polymorphisms.