RESUMEN
The NucleoLink surface is a physically modified, thermostable, optically clear resin. It allows the covalent binding of 5'-phosphorylated oligonucleotides. Target DNA amplification by polymerase chain reaction (PCR) is accomplished by asymmetric amplification on the covalently immobilized primer that develops into immobilized amplicons. A DNA fragment of bovine leukemia virus is used as a model system for the detection of immobilized amplicons by ELISA-like techniques. Covalently bound oligonucleotides are also utilized as capture probe in the hybridization-based signal amplification for detection of an infectious organism.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Reacción en Cadena de la Polimerasa/métodos , ADN Viral/análisis , Virus de la Leucemia Bovina/genética , Resinas de PlantasRESUMEN
The ability of scavengers of hydroxyl radical (OH radical) to modulate the photosensitized relaxation (induction of the first single-strand break) of supercoiled plasmid DNA with UVA photoactivated 4'-aminomethyl-4,5',8-trimethylpsoralen was examined by comparing the dose reduction factor (DRF: the ratio of fluence required to induce the same degree of relaxation in the absence to the presence of OH radical scavengers). The addition of mannitol, azide, acetate, or formate at concentrations inversely proportional to the value of the rate constants for the scavenging of OH radicals partially attenuated the supercoiled DNA relaxation. The degrees of protection afforded by the four scavengers in the presence of AMT photoactivated by either 334 nm or 365 nm monochromatic photons were similar, giving an average DRF of about 0.25 in all cases. Given the diverse chemical nature of the scavengers and their wide range of concentrations utilized, these findings are evidence for the involvement of a Type I photosensitization in the induction of DNA single-strand breaks by photoactivated AMT.
Asunto(s)
ADN Superhelicoidal/efectos de los fármacos , ADN Superhelicoidal/efectos de la radiación , Depuradores de Radicales Libres/farmacología , Radical Hidroxilo/química , Fármacos Fotosensibilizantes/farmacología , Trioxsaleno/análogos & derivados , Acetatos/farmacología , Ácido Acético , Azidas/farmacología , ADN Superhelicoidal/química , Formiatos/farmacología , Radical Hidroxilo/efectos de la radiación , Manitol/farmacología , Plásmidos/efectos de los fármacos , Plásmidos/efectos de la radiación , Azida Sódica , Trioxsaleno/farmacología , Rayos UltravioletaRESUMEN
An action spectrum for the relaxation of supercoiled plasmid DNA (induction of the first single-strand break) by photoactivated 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) has been determined using monochromatic UV photons from 254 to 405 nm. The spectrum of AMT-induced plasmid DNA relaxation fits closely with the absorbance spectrum of AMT in the spectral region between 313 nm and 405 nm but deviates at wavelengths shorter than 313 nm. This assay also reveals that the psoralen photosensitization reaction with DNA also produces piperidine-labile sites. Addition of mannitol and azide partially quenches the supercoil relaxation reaction, evidence for a role of Type II photosensitization pathway.
Asunto(s)
ADN Superhelicoidal/efectos de los fármacos , ADN Superhelicoidal/efectos de la radiación , Trioxsaleno/análogos & derivados , Rayos Ultravioleta , Reactivos de Enlaces Cruzados/farmacología , Escherichia coli/genética , Genes myc/efectos de los fármacos , Genes myc/efectos de la radiación , Plásmidos/efectos de los fármacos , Plásmidos/efectos de la radiación , Radiación , Trioxsaleno/farmacologíaRESUMEN
vhs1 is a mutant of herpes simplex virus type 1 that is defective in the virion host shutoff function responsible for the degradation of cellular mRNAs and the concomitant shutoff of host protein synthesis. In this study, the effect of the vhs1 mutation on the metabolism of viral mRNAs was examined by measuring the half-lives and patterns of accumulation of 10 different viral mRNAs representing all kinetic classes. The vhs1 mutation had the effect of dramatically lengthening the cytoplasmic half-lives of all 10 mRNAs. In wild-type virus infections, the 10 mRNAs had similar half-lives, suggesting that little, if any, target mRNA selectivity was exhibited by the vhs function. The vhs1 mutation caused overaccumulation of a number of mRNAs. The effect was most dramatic for the alpha (immediate-early) mRNA for ICP27 and the beta (early) mRNAs encoding thymidine kinase, ICP8, and DNA polymerase. Whereas in wild-type infections these mRNAs increased to peak levels and subsequently declined in abundance, in vhs1 infections they continued to accumulate until late times. A significant but less dramatic overaccumulation was observed for several beta-gamma (delayed-early) and gamma (late) mRNAs. The results suggest that the vhs protein plays an important role in determining the half-lives of viral mRNAs belonging to all kinetic classes and in so doing is important in the normal downregulation at late times of alpha and beta gene expression.
Asunto(s)
Regulación de la Expresión Génica , Genes Virales , ARN Mensajero/genética , ARN Viral/genética , Simplexvirus/genética , Animales , Dactinomicina/farmacología , Células HeLa , Factores de Tiempo , Células VeroRESUMEN
vhs1 is a herpes simplex virus type 1 mutant defective in the shutoff of both host and alpha polypeptide synthesis. In cycloheximide reversal experiments, alpha mRNAs were significantly more stable in vhs1-infected cells than in cells infected with wild-type virus, whether assayed by in vitro translation or Northern blotting.