RESUMEN
A new method for the qualitative analysis of adenosine nucleotides (AMP, ADP, and ATP) and synthetic oligonucleotides has been proposed, utilizing a pH- and temperature-responsive polymer of N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and N,N-dimethylaminopropylacrylamide (DMAPAAm) as the stationary phase of HPLC. In the chromatographic system using the copolymer with ionizable groups of modified packing materials, we investigated how to separate adenosine nucleotides and oligonucleotides by temperature. The properties of the surface of the copolymer-grafted stationary phase altered from hydrophilic to hydrophobic and from charged to non-charged due to changes in the temperature and in the pH, respectively. In addition, it is possible to exhibit and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena result from changes in the charge and hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface with the controlling temperature. A pH- and temperature-responsive chromatography would be greatly useful for biopolymer and nucleotide separation and purification.
Asunto(s)
Cromatografía Liquida/métodos , Nucleótidos/aislamiento & purificación , Polímeros/química , Acrilamidas/síntesis química , Acrilamidas/química , Resinas Acrílicas/síntesis química , Resinas Acrílicas/química , Nucleótidos de Adenina/análisis , Nucleótidos de Adenina/aislamiento & purificación , Cromatografía Liquida/instrumentación , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Metacrilatos/síntesis química , Metacrilatos/química , Nucleótidos/análisis , Oligonucleótidos/análisis , Oligonucleótidos/aislamiento & purificación , Polímeros/síntesis química , Ácidos Polimetacrílicos/síntesis química , Ácidos Polimetacrílicos/química , Reproducibilidad de los Resultados , Electricidad Estática , Temperatura , Nucleótidos de Timina/análisis , Nucleótidos de Timina/aislamiento & purificación , Agua/químicaRESUMEN
Bizelesin is the first anticancer drug capable of damaging specific regions of the genome with clusters of its binding sites T(A/T)(4)A. This study characterized the sequence- and region-specificity of a bizelesin analogue, U-78779, designed to interact with mixed A/T-G/C motifs. At the nucleotide level, U-78779 was found to prefer runs of A/Ts interspersed with 1 or 2 G/C pairs, although 25% of the identified sites corresponded to pure AT motifs similar to bizelesin sites. The in silico computational analysis showed that the preferred mixed A/T-G/C motifs distribute uniformly at the genomic level. In contrast, the secondary, pure AT motifs (A/T)(6)A were found densely clustered in the same long islands of AT-rich DNA that bizelesin targets. Mapping the sites and quantitating the frequencies of U-78779 adducts in model AT island and non-AT island naked DNAs demonstrated that clusters of pure AT motifs outcompete isolated mixed A/T-G/C sites in attracting drug binding. Regional preference of U-78779 for AT island domains was verified also in DNA from drug-treated cells. Thus, while the primary sequence preference gives rise to non-region-specific scattered lesions, the clustering of the minor pure AT binding motifs seems to determine region-specificity of U-78779 in the human genome. The closely correlated cytotoxic activities of U-78779 and bizelesin in several cell lines further imply that both drugs may share common cellular targets. This study underscores the significance of the genome factor in a drug's potential for region-specific DNA damage, by showing that it can take precedence over drug binding preferences at the nucleotide level.
Asunto(s)
Antineoplásicos Alquilantes/química , Daño del ADN , ADN de Neoplasias/química , ADN Viral/química , Repeticiones de Dinucleótido/efectos de los fármacos , Genoma Humano , Indoles/química , Urea/análogos & derivados , Urea/química , Nucleótidos de Adenina/aislamiento & purificación , Nucleótidos de Adenina/metabolismo , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/toxicidad , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , Nucleótidos de Citosina/metabolismo , Aductos de ADN/química , Aductos de ADN/metabolismo , ADN de Neoplasias/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , ADN Viral/metabolismo , Duocarmicinas , Nucleótidos de Guanina/metabolismo , Humanos , Indoles/metabolismo , Indoles/toxicidad , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Virus 40 de los Simios/efectos de los fármacos , Virus 40 de los Simios/genética , Nucleótidos de Timina/aislamiento & purificación , Nucleótidos de Timina/metabolismo , Células Tumorales Cultivadas , Urea/metabolismo , Urea/toxicidadRESUMEN
The flexible substrate spectrum of the recombinant enzymes from the biosynthetic pathway of dTDP-beta-L-rhamnose in Salmonella enterica, serovar typhimurium (LT2), was exploited for the chemoenzymatic synthesis of deoxythymidine diphosphate- (dTDP-) activated 2,6-dideoxyhexoses. The enzymatic synthesis strategy yielded dTDP-2-deoxy-alpha-D-glucose and dTDP-2,6-dideoxy-4-keto-alpha-D-glucose (13) in a 40-60 mg scale. The nucleotide deoxysugar 13 was further used for the enzymatic synthesis of dTDP-2,6-dideoxy-beta-L-arabino-hexose (dTDP-beta-L-olivose) (15) in a 30-mg scale. The chemical reduction of 13 gave dTDP-2,6-dideoxy-alpha-D-arabino-hexose (dTDP-alpha-D-olivose) (1) as the main isomer after product isolation in a 10-mg scale. With 13 as an important key intermediate, the in vitro characterization of enzymes involved in the biosynthesis of dTDP-activated 2,6-dideoxy-, 2,3,6-trideoxy-D- and L-hexoses can now be addressed. Most importantly, compounds 1 and 15 are donor substrates for the in vitro characterization of glycosyltransferases involved in the biosynthesis of polyketides and other antibiotic/antitumor drugs. Their synthetic access may contribute to the evaluation of the glycosylation potential of bacterial glycosyltransferases to generate hybrid antibiotics.
Asunto(s)
Antibacterianos/biosíntesis , Desoxiazúcares/biosíntesis , Salmonella enterica/enzimología , Nucleótidos de Timina/biosíntesis , Nucleótidos de Timina/metabolismo , Animales , Antibacterianos/química , Deshidrogenasas de Carbohidratos/metabolismo , Carbohidrato Epimerasas/metabolismo , Bovinos , Cromatografía Líquida de Alta Presión , Desoxiazúcares/química , Desoxiazúcares/aislamiento & purificación , Desoxiazúcares/metabolismo , Hidroliasas/metabolismo , Magnesio/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Nucleotidiltransferasas/metabolismo , Conejos , Especificidad por Sustrato , Nucleótidos de Timina/química , Nucleótidos de Timina/aislamiento & purificaciónRESUMEN
dTDP-6-deoxy-4-keto-D-glucose (1), the common intermediate in the biosyntheses of the manifold deoxysugars, was synthesized on a gram-scale by the combination of sucrose synthase and dTDP-D-glucose 4,6-dehydratase in a fed batch, starting the reaction with dTDP. This process allowed a dTDP conversion with a 100% rate. An easy and efficient three-step purification with anion-exchange chromatography and gel filtration gave 1.1 g of 1 in an overall yield of 73%. This work realizes a first step for an economic access to activated deoxysugars.
Asunto(s)
Glucosa/análogos & derivados , Glucosiltransferasas/metabolismo , Hidroliasas/metabolismo , Sacarosa/metabolismo , Nucleótidos de Timina/química , Nucleótidos de Timina/síntesis química , Nucleótidos de Timina/metabolismo , Biotecnología/métodos , Escherichia coli/genética , Glucosa/síntesis química , Glucosa/aislamiento & purificación , Glucosa/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/aislamiento & purificación , Hidroliasas/genética , Hidroliasas/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimología , Sacarosa/química , Nucleótidos de Timina/aislamiento & purificaciónRESUMEN
OBJECTIVES AND DESIGN: Measurement of phosphorylated zidovudine (ZDV) inside infected cells is more likely to provide satisfactory dose response relationships than serum concentrations. This study provides information on ZDV phosphorylation in HIV-seronegative volunteers (n = 5) and in patients with HIV infection (n = 12). METHODS: Intracellular ZDV phosphate metabolites were measured in peripheral blood mononuclear cells isolated from whole blood by density cushion centrifugation. Cells were washed and extracted overnight with 60% methanol prior to analysis by high performance liquid chromatography. Fractions eluted from the column corresponding to ZDV, ZDV monophosphate (ZDV-MP), ZDV diphosphate (ZDV-DP) and ZDV triphosphate (ZDV-TP) were collected, hydrolysed by acid phosphatase and ZDV levels quantified by radioimmunoassay. RESULTS: The area under the plasma ZDV concentration-time curve (AUC0-6 h) was similar in seronegative volunteers and patients [mean +/- SD, 4.64 +/- 2.50 versus 5.56 +/- 2.67 mumoles/l h; 95% confidence interval (CI), -4.39-2.23; P = 0.646, Mann-Whitney U test]. However, ZDV phosphorylation was greater in patients, with the AUC0-6 h for total phosphate metabolites being 5.91 +/- 3.42 pmoles/10(6) cells h compared with seronegative volunteers (0.66 +/- 0.48 pmoles/10(6) cells h; 95% CI, -8.35 to -2.32; P = 0.0003). The concentration of ZDV-TP was similar in both groups, the increase in total phosphates in patients being due primarily to ZDV-MP. ZDV-MP AUC0-6 h and total ZDV phosphate AUC0-6 h were closely correlated (r2 = 0.94). The relationship between total ZDV phosphate AUC0-6 h and the CD4 count demonstrates that patients with a count < 100 x 10(6)/l have much higher ZDV phosphate levels, predominantly ZDV-MP. CONCLUSION: ZDV is phosphorylated to a greater extent in patients than in healthy volunteers. The increased ZDV-MP in patients with low CD4 counts may explain the well known occurrence of increased ZDV toxicity in patients with more advanced disease. The ability to measure ZDV phosphorylated metabolites (without the administration of radiolabelled nucleoside) represents a significant advance in our understanding of the clinical pharmacology of the drug.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/sangre , Infecciones por VIH/sangre , Seronegatividad para VIH/fisiología , Leucocitos Mononucleares/metabolismo , Zidovudina/metabolismo , Adulto , Biotransformación , Didesoxinucleótidos , Humanos , Persona de Mediana Edad , Fosforilación , Valores de Referencia , Nucleótidos de Timina/sangre , Nucleótidos de Timina/aislamiento & purificación , Zidovudina/análogos & derivados , Zidovudina/sangre , Zidovudina/aislamiento & purificaciónRESUMEN
Most human peripheral blood gamma delta T lymphocytes respond to hitherto unidentified mycobacterial antigens. Four ligands from Mycobacterium tuberculosis strain H37Rv that stimulated proliferation of a major human gamma delta T cell subset were isolated and partially characterized. One of these ligands, TUBag4, is a 5' triphosphorylated thymidine-containing compound, to which the three other stimulatory molecules are structurally related. These findings support the hypothesis that some gamma delta T cells recognize nonpeptidic ligands.
Asunto(s)
Antígenos Bacterianos/inmunología , Activación de Linfocitos , Mycobacterium tuberculosis/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Nucleótidos de Timina/inmunología , Antígenos Bacterianos/química , Antígenos Bacterianos/aislamiento & purificación , Células Cultivadas , Cromatografía por Intercambio Iónico , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Nucleótidos de Timina/análisis , Nucleótidos de Timina/química , Nucleótidos de Timina/aislamiento & purificaciónRESUMEN
In vitro studies of zidovudine (ZDV) phosphorylation may not accurately reflect the in vivo dose-response relationship, which is crucial to determining the relationship between ZDV exposure, efficacy, and toxicity. However, measurement of ZDV phosphorylated anabolites in peripheral blood mononuclear cells (PBMCs) from ZDV-treated human immunodeficiency virus (HIV)-infected patients would be extremely useful in the more appropriate utilization of ZDV in the treatment of HIV infection. We developed a specific and sensitive combined high-pressure liquid chromatography (HPLC)-radioimmunoassay (RIA) procedure for the determination of ZDV, ZDV-monophosphate, ZDV-diphosphate, and ZDV-triphosphate in PBMCs taken from ZDV-treated HIV-infected patients. ZDV and its anabolites were extracted from washed, Ficoll-Paque-isolated PBMCs and then separated by HPLC using a strong anion-exchange column. The anabolites were then hydrolyzed to ZDV with acid phosphatase. ZDV was then measured by using a modified commercially available RIA protocol. Our method was validated by measuring [3H]ZDV anabolites generated in Molt-4 cells radioisotopically and simultaneously by the combined HPLC-RIA procedure. The ZDV determinations correlated well (r2 = 0.97) over the range of 0.037 to 5.2 pmol (10 to 1,400 pg) per assay tube. Furthermore, we defined the stability of ZDV anabolites during ficoll isolation and the recovery after extraction and cleanup. We then measured intracellular parent ZDV and its phosphorylated anabolites in PBMCs from six ZDV-treated HIV-infected patients (PBMCs were taken 2 h after a 300-mg oral dose). The mean concentrations ( +/- standard deviations) of parent and of mono-, di-, and triphosphates were 0.15 +/- 0.08, 1.4 +/-, 0.082 +/- 0.02, and 0.081 +/- 0.03 pmol/10(6) PBMC, respectively (one pmol/10(6) PBMC represents a concentration of approximately 1 microm). Concurrent serum ZDV concentrations were between 1.3 and 7.1 microm. This method should provide a useful tool for evaluating in vivo pharmacokinetics of ZDV anabolites in PBMCs and possibly other cell types, even at the low doses of ZDV currently administered therapeutically.
Asunto(s)
Nucleótidos de Timina/sangre , Zidovudina/análogos & derivados , Zidovudina/sangre , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico , Didesoxinucleótidos , Estabilidad de Medicamentos , Infecciones por VIH/sangre , Infecciones por VIH/tratamiento farmacológico , Humanos , Líquido Intracelular/metabolismo , Leucocitos Mononucleares/química , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Fosforilación , Radioinmunoensayo/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Nucleótidos de Timina/aislamiento & purificación , Nucleótidos de Timina/metabolismo , Tritio , Zidovudina/aislamiento & purificación , Zidovudina/metabolismoRESUMEN
Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) J. Biol. Chem. 265, 6112-6117). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3'-azido-3'-deoxythymidine diphosphate diacylglycerol, 3'-deoxythymidine diphosphate diacylglycerol and 2',3'-dideoxycytidine diphosphate diacylglycerol (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and 1H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.
Asunto(s)
Antivirales/síntesis química , Nucleótidos de Desoxicitosina/síntesis química , Diglicéridos/síntesis química , Nucleótidos de Timina/síntesis química , Antivirales/química , Antivirales/aislamiento & purificación , Antivirales/metabolismo , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxicitosina/aislamiento & purificación , Nucleótidos de Desoxicitosina/metabolismo , Diglicéridos/química , Diglicéridos/aislamiento & purificación , Diglicéridos/metabolismo , VIH/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Espectrofotometría Infrarroja , Nucleótidos de Timina/química , Nucleótidos de Timina/aislamiento & purificación , Nucleótidos de Timina/metabolismoRESUMEN
A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-D-xylo-4-hexulose was isolated after a 1-h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-D-xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier. Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Azúcares de Nucleósido Difosfato , Ramnosa/análogos & derivados , Nucleótidos de Timina/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Carbohidratos , Cromatografía de Gases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Espectrometría de Masas , Datos de Secuencia Molecular , Plásmidos , Ramnosa/biosíntesis , Ramnosa/aislamiento & purificación , Salmonella/genética , Nucleótidos de Timina/aislamiento & purificaciónRESUMEN
The nucleotide sugar precursor of the oleandrose units of the avermectins has been purified from a mutant of Streptomyces avermitilis, which does not synthesize any avermectins but which converts avermectin aglycones to their respective disaccharides. This precursor has been identified as dTDP-oleandrose. The purification was achieved by anion exchange and reverse phase high performance liquid chromatography. The purified nucleotide sugar had an absorption spectra characteristic of thymidine, released dTMP when treated with phosphodiesterase, and possessed an NMR spectrum in which three resonances characteristic of oleandrose were seen in addition to the thymidine signals. The enzyme, avermectin aglycone dTDP-oleandrose glycosyltransferase, which catalyzes the stepwise addition of oleandrose to the avermectin aglycones, has been demonstrated in cell-free extracts and (NH4)2SO4 fractions of cell-free extracts of S. avermitilis. The enzyme is specific for dTDP-oleandrose as the glycosyl donor but utilizes all avermectin aglycones as glycosyl acceptors. The stoichiometry between dTDP-oleandrose consumed in the reaction and oleandrose units transferred to the avermectin mono- and disaccharide was found to be 1:1.
Asunto(s)
Antiprotozoarios , Hexosas/aislamiento & purificación , Ivermectina/análogos & derivados , Streptomyces/metabolismo , Nucleótidos de Timina/aislamiento & purificación , Sistema Libre de Células , Cromatografía Líquida de Alta Presión , Glicosilación , Indicadores y ReactivosRESUMEN
Diastereoisomeric thymidine cyclic (3',5')-methanephosphonates (3a), cyclic (3',5')-phosphoranilidates (3b) and cyclic (3',5')-phosphoranilidothioates (3c) were prepared by treatment of diastereoisomerically pure thymidine 3'-O-[O-(4-nitrophenyl)methanephosphonates] (2a), 3'-O-[O-(4-nitrophenyl)phosphoranilidates] (2b) or 3'-O-[O-(4-nitrophenyl)phosphoranilidothioates] (2c), respectively, with sodium hydroxide in dioxane-water solution.
Asunto(s)
Nucleótidos de Timina/síntesis química , Indicadores y Reactivos , Estereoisomerismo , Nucleótidos de Timina/aislamiento & purificaciónRESUMEN
"In solution" synthesis and separation of diastereoisomers of 5'-O-(4,4'-dimethoxytriphenylmethyl)dithymidyl (3',5') 4,4'-dimethoxytriphenylmethanephosphonate (DMTTDMTT) is described. One of diastereoisomers was successfully crystallized and characterized by means of HPLC.
Asunto(s)
Nucleótidos de Timina/síntesis química , Cromatografía Líquida de Alta Presión , Indicadores y Reactivos , Estereoisomerismo , Nucleótidos de Timina/aislamiento & purificaciónAsunto(s)
Nucleótidos de Desoxiuracil/aislamiento & purificación , Nucleótidos de Timina/aislamiento & purificación , Animales , Cromatografía en Capa Delgada/métodos , Nucleótidos de Desoxiuracil/metabolismo , Técnicas In Vitro , Leucemia L1210/metabolismo , Metotrexato/farmacología , Ratones , Nucleótidos de Timina/metabolismoRESUMEN
Dextran gels of varying porosites (Sephadex G series) were chemically modified so as to contain covalently bound monofunctional mercury. Mercurated Sephadex of the porosity G-25 was then used to fractionate mixtures of mono- and dinucleotides into the constituent components. Separation is based on the affinity of the nitrogen binding sites of the purine and pyrimidine derivatives for organomercurial Hg+. Thus, a mixture composed of the four mononucleotides Cyd-5'-P, Ado-3'-P, Guo-2'(3')-P, dThd-5'-P and of the four dinucleotides Cyd-P-Cyd, Ado-P-Ado, Guo-P-Urd, and Urd-P-Urd could be separated into eight well-resolved fractions by using a combination Tris-bicarbonate/carbonate buffer system of increasing pH as an eluent. Complete separation was also achieved when a mixture of Ado 3:5'-P, Ado 5'-P, Ado-5'-PP, and Ado-5'-PPP was fractionated on mercurated Sephadex G-25. Again, Tris-bicarbonate/carbonate buffer served as an eluent. Lastly, fractionation can also be performed at a constant pH by offering other suitable ligands, for instance Cl-, that will compete with nucleotides for monofunctional Hg+. The fractionation behavior of mercurated Sephadex G-25 can be fully understood on the basis of the complexing properties of monofunctional Hg+. This has been shown by calculating the net retention volume ratios of several nucleotides with the help of the known interaction parameters of corresponding nucleosides with CH3 HgOH and by comparing the predicted ratios with the experimentally measured ones. Finally, the acid-base properties of mercurated Sephadex G-25 as well as its affinity for chloride and iodide ions have been determined. The data agree quite well with those known for CH3 HgOH.
Asunto(s)
Cromatografía en Gel/métodos , Ribonucleótidos/aislamiento & purificación , Nucleótidos de Timina/aislamiento & purificación , Nucleótidos de Adenina/aislamiento & purificación , Sitios de Unión , Nucleótidos de Citosina/aislamiento & purificación , Dextranos/análogos & derivados , Nucleótidos de Guanina/aislamiento & purificación , Concentración de Iones de Hidrógeno , Mercurio , Compuestos de Metilmercurio , Nucleótidos Cíclicos/aislamiento & purificación , Nucleótidos de Uracilo/aislamiento & purificaciónRESUMEN
E. coli DNA-dependent RNA-polymerase binding sites on DNAs of T5, T7, and lambda coliphages have been isolated according to three different methods in order to analyze the binding sites themselves as well as the nearest neighboring regions. It is shown that the binding sites are regions that are rather rich in G+C, are about 14 base pairs long and are located between DNA sequences highly enriched in A+T. The biological implications of this result are discussed.
Asunto(s)
ADN Viral/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleótidos/aislamiento & purificación , Nucleótidos de Adenina/aislamiento & purificación , Secuencia de Bases , Sitios de Unión , Colifagos , Virus ADN/metabolismo , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Poliacrilamida , Etidio/metabolismo , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Nucleótidos/metabolismo , Radioisótopos de Fósforo , Espectrometría de Fluorescencia , Nucleótidos de Timina/aislamiento & purificación , Transcripción GenéticaAsunto(s)
Desoxirribonucleótidos/metabolismo , Adenosina Trifosfato/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Autorradiografía , Cromatografía en Capa Delgada , Nucleótidos de Citosina/aislamiento & purificación , Nucleótidos de Citosina/metabolismo , Desoxirribonucleótidos/aislamiento & purificación , Escherichia coli/metabolismo , Estudios de Evaluación como Asunto , Nucleótidos de Guanina/aislamiento & purificación , Nucleótidos de Guanina/metabolismo , Concentración de Iones de Hidrógeno , Métodos , Oxidación-Reducción , Radioisótopos de Fósforo , Ribosa , Nucleótidos de Timina/aislamiento & purificación , Nucleótidos de Timina/metabolismoAsunto(s)
Cromatografía , Desoxirribonucleótidos/aislamiento & purificación , Oligonucleótidos/aislamiento & purificación , Nucleótidos de Adenina/aislamiento & purificación , Secuencia de Bases , Celulosa , Fenómenos Químicos , Química , Nucleótidos de Citosina , Estabilidad de Medicamentos , Nucleótidos de Guanina , Temperatura , Nucleótidos de Timina/aislamiento & purificación , Nucleótidos de Uracilo/aislamiento & purificaciónRESUMEN
Previous isolation and analysis of E. coli RNA polymerase (EC 2.7.7.6) binding sites on lambda DNA had demonstrated the existence of a sigma-dependent process of recognition of A-T-rich DNA sequences. We have now extended this finding to T5 and T7 DNA and häve provided evidence for the double-strandedness of the isolated binding sites. The possible equation of these sites to the genetically defined promoters is discussed.