RESUMEN
Salt concentration governs nucleic acid hybridization according to the Schildkraut-Lifson equation. High concentrations of SDS are used in some common protocols, but the effects of SDS on hybridization stringency have not been reported. We investigated hybridization parameters in solutions containing SDS. With targets immobilized on nylon membranes and PCR- or transcription-generated probes, we report that the 50% dissociation temperature (Tm*) in the absence of SDS was 15 degrees C-17degrees C lower than the calculated Tm. SDS had only modest effects on Tm* [1% (w/v) equating to 8 mM NaCl]. RNA/DNA hybrids were approximately 11 degrees C more stable than DNA/DNA hybrids. Incomplete homology (69%) significantly reduced the Tm* for DNA/DNA hybrids (approximately /4degrees C; 0.45 degrees C/% nonhomology) but far less so for RNA/DNA hybrids (approximately 2.3 degrees C; approximately 0.07 degrees C/% non-homology); incomplete homology also markedly reduced the extent of hybridization. On these nylonfilters, SDS had a major effect on nonspecific binding. Buffers lacking SDS, or with low salt concentration, gave high hybridization backgrounds; buffers containing SDS, or high-salt buffers, gave reproducibly low backgrounds.
Asunto(s)
Sondas de ADN/efectos de los fármacos , Hibridación de Ácido Nucleico/efectos de los fármacos , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia de ADN/métodos , Cloruro de Sodio/farmacología , Datos de Secuencia Molecular , Control de Calidad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Dodecil Sulfato de Sodio/farmacología , Especificidad por Sustrato , TemperaturaRESUMEN
We have previously reported that glucocorticoids markedly increase and anti-glucocorticoids (such as RU-486) block c-fms RNA and protein expression in some breast cancer cell lines, but not in others, and that this increase is the consequence of increased transcription from the first, epithelial cell-specific promoter of the c-fms gene (encoding CSF-1R, macrophage colony-stimulating factor receptor). Employing DNaseI protection and electrophoretic mobility shift assays (EMSA), we now demonstrate that DNA-transcription factor protein complexes are formed on the c-fms first promoter at a composite regulatory element containing overlapping binding sites for AP-1 proteins, bHLH factors, and the glucocorticoid receptor (GR). Competition studies indicate that transcription factor proteins bind the AP-1 site and the GR element (GRE) and both GR and AP-1 proteins are involved in DNA-protein complex formation. The complexes differ in quantity and glucocorticoid inducibility in the different breast cancer cell lines studied depending on whether the promoter responds to glucocorticoid stimulation. Transient transfection of promoter/reporter gene constructs resulted in reduced basal transcription activity of this promoter and lack of glucocorticoid stimulation when the AP-1 site was mutated. We conclude that AP-1 proteins, GR and associated co-factors regulate transcription from the c-fms first promoter and that differences in recruitment of the various components are responsible for cell specific repression and activation of this gene in breast carcinoma cell lines.
Asunto(s)
Dexametasona/farmacología , Regulación Neoplásica de la Expresión Génica , Genes Reguladores/efectos de los fármacos , Genes fms , Glucocorticoides/farmacología , Regiones Promotoras Genéticas/fisiología , Anticuerpos/inmunología , Secuencia de Bases , Unión Competitiva , Neoplasias de la Mama/genética , Secuencia de Consenso , Sondas de ADN/efectos de los fármacos , Sondas de ADN/metabolismo , Desoxirribonucleasa I/análisis , Ensayo de Cambio de Movilidad Electroforética/métodos , Células Epiteliales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Mutación , Proto-Oncogenes Mas , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/fisiología , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N:4-ethyldeoxycytidine (d(4Et)C), which hybridises specifically with natural dG to give a G:(4Et)C base pair with reduced stability compared to the natural G:C base pair. Substitution by d(4Et)C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.
Asunto(s)
Sondas de ADN/química , Sondas de ADN/metabolismo , ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico/métodos , Emparejamiento Base/efectos de los fármacos , Secuencia de Bases , ADN/efectos de los fármacos , ADN/genética , Sondas de ADN/efectos de los fármacos , Sondas de ADN/genética , Desoxirribonucleósidos/química , Desoxirribonucleósidos/genética , Desoxirribonucleósidos/metabolismo , Ingeniería Genética , Sustancias Intercalantes/farmacología , Mutación/genética , Conformación de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/genética , Hibridación de Ácido Nucleico/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Temperatura , TermodinámicaRESUMEN
High mobility group (HMG) proteins 1 and 2 are abundant non-histone chromosomal proteins that bind preferentially DNA that is bent or underwound. Previous studies have shown that these proteins preferentially bind to DNA damaged by the crosslinking agents cis-diammine-dichloro-platinum(II), chromium(III) and UV-C radiation. Here we have studied the binding of HMG-1/2 proteins to a duplex oligonucleotide damaged by benzo(a)pyrene diol epoxide or N-acetoxy-acetylaminofluorene using an electrophoretic mobility shift assay. Both chemicals induce monoadducts that are known to distort DNA structure. The affinities of HMG-1/2 for DNA damaged by benzo[a]pyrene diol epoxide or N-acetoxy-acetylaminofluorene were similar to that for UV-irradiated DNA, which were an order of magnitude higher than for undamaged DNA. In contrast, DNA modified by dimethyl sulfate was not preferentially recognised by HMG-1/2.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/farmacología , Acetoxiacetilaminofluoreno/farmacología , Aductos de ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Alquilantes/farmacología , Animales , Sitios de Unión , Southern Blotting , Carcinógenos/farmacología , Sondas de ADN/química , Sondas de ADN/efectos de los fármacos , Sondas de ADN/metabolismo , Sondas de ADN/efectos de la radiación , Masculino , RatasRESUMEN
The use of digoxigenin-labelled probes was studied for quantitation of HBV-DNA during antiviral drug evaluation. Digoxigenin (dig)-labelled probes were generated either via incorporation of dig-dUTP in a polymerase chain reaction (PCR) or a random priming reaction. Using the PCR-labelled probe (delineating a 523 bp fragment in the core gene of the HBV) as little as 1 pg of immobilized HBV-DNA could be detected following an 8 h exposure of the hybridized membrane. A close correlation (r = 0.95) was found between the amount of HBV-DNA (range 2.5-200 pg) and the signal generated by the probe hybridized to its target DNA. By using a probe that was labelled with digoxigenin via random priming, the minimal quantity of immobilized HBV plasmid DNA that could be detected following an 8 h exposure was 4 pg, whereas a 32P-labelled probe, generated in parallel by random priming, allowed the detection of 16 pg of HBV plasmid DNA following a 4-day exposure. The PCR-generated digoxigenin-labelled probe proved to be useful for antiviral drug evaluation, i.e. to detect HBV-DNA in total cellular DNA from HBV-positive hepatoma cells (HepG2.2.15) that had either been treated with reference antiviral agents or left untreated. The 50% effective concentrations (EC50) that were calculated for inhibition of HBV-DNA production by lamivudine (3TC), penciclovir (PCV), lobucavir (LBV), adefovir (PMEA) and tenofovir (PMPA) were comparable to those reported in the literature. The use of digoxigenin-labelled probes thus appears to be a simple, convenient, rapid, reliable and non-radioactive method for use for anti-HBV screening. In addition, and in contrast to 32P-labelled probes, digoxigenin-labelled probes can be stored for >1 year without loss of specific activity, which makes these probes particularly attractive for large-scale antiviral drug evaluation purposes.
Asunto(s)
Antivirales/farmacología , Sondas de ADN/metabolismo , ADN Viral/análisis , Digoxigenina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Virus de la Hepatitis B/genética , Sondas de ADN/efectos de los fármacos , Nucleótidos de Desoxiuracil/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Humanos , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
The repair proteins XPA, XPC and replication protein A (RPA) have been implicated in the primary recognition of damaged DNA sites during nucleotide excision repair. Detailed structural information on the binding of these proteins to DNA lesions is however lacking. We have studied the binding of human RPA (hRPA) and hRPA-XPA-complexes to model oligonucleo-tides containing a single 1, 3-d(GTG)-cisplatin-modification by photocrosslinking and electrophoretic mobility shift experiments. The 70 kDa subunit of hRPA can be crosslinked with high efficiency to cisplatin-modified DNA probes carrying 5-iodo-2"-deoxyuridin (5-IdU) as crosslinking chromophore. High efficiency crosslinking is dependent on the presence of the DNA lesion and occurs preferentially at its 5"-side. Examination of the crosslinking efficiency in dependence on the position of the 5-IdU chromophore indicates a specific positioning of hRPA with respect to the platination site. When hRPA and XPA are both present mainly hRPA is crosslinked to the DNA. Our mobility shift experiments directly show the formation of a stable ternary complex of hRPA, XPA and the damaged DNA. The affinity of the XPA-hRPA complex to the damaged DNA is increased by more than one order of magnitude as compared to hRPA alone.