Biomedical subjects

N Logsdon

Publications and source records attributed to N Logsdon.

8 recordsLinked to original sources

PubMed1998

p53-mediated transcription induces resistance of DNA to UV inactivation.

A possible role of p53-dependent transcription in the induction of DNA repair was explored by transfecting a UV-irradiated chloramphenicol acetyl transferase (CAT) reporter plasmid (pRGC.FOS.CAT), containing a minimal FOS promoter driven by a consensus p53 binding site, into a p53 negative-mouse cell line [(10)1]. When a p53-expressing plasmid (pSV.p53) was cotransfected into these cells, CAT expression levels persisted even after prolonged UV irradiation. In comparison, CAT expression from pSV2.CAT, which lacks a p53-responsive element in its SV40 promoter, dropped off much more precipitously after UV irradiation in the absence or presence of WT p53 expression. A similar sharp drop was observed with three other constructs when the reporter gene was under the control of the ras, beta-actin or fos promoter. Mouse cells (A1-5) that constitutively express a temperature-sensitive mutant (135 AV) of mouse p53 also generated, at 32 degrees C, higher levels of enzyme expressed from UV-irradiated pRGC.FOS.CAT than from UV-irradiated pSV2.CAT. The frequency of cyclobutane pyrimidine dimers in UV-irradiated pRGC.FOS.CAT was determined with T4 endo V, and the probability of having an undamaged CAT coding strand was calculated by the Poisson distribution for various times of UV-irradiation. The observed relative CAT expression levels from irradiated pSV2.CAT and pRGC.FOS.CAT in the absence of p53 were consistent with those numbers. These results show that WT p53-mediated transcription directs a resistance of the transcribed DNA to UV inactivation and reactivates the reporter gene. Furthermore, some single point substitution mutants of p53 that maintain a near normal ability to activate transcription had lost their ability to extend CAT gene expression after UV irradiation. Conversely, other mutants with reduced transcriptional activity retained this ability. This indicates that although resistance to UV inactivation is transcriptionally-dependent, these two activities are genetically distinct. These data, taken together, suggest that the transcription of UV-damaged DNA by a p53-dependent process promotes its repair.

Actins↗

PubMed1996

p53 binds to a novel recognition sequence in the proximal promoter of the rat muscle creatine kinase gene and activates its transcription.

The rat muscle creatine kinase (CKM) gene promoter is unusual since it is one of the few cellular promoters containing a p53 response element which is located proximally (bp -168 to -57) to the transcription start site. We have previously shown that p53wt transactivates transcription in vivo of rat CKM, in CV-1 monkey kidney cells, through this 112 bp promoter-proximal fragment which contains at least five degenerate p53-binding elements. In this report, we employed the gel-shift assay and demonstrated that recombinant, immunoaffinity-purified mouse p53wt binds to this 112 bp CKM sequence and activates the in vitro transcription of the proximal CKM promoter by nuclear extracts from CV-1 cells. Also, a competitor plasmid containing this 112 bp CKM fragment interferred with the in vivo transactivation of CKM by p53. This CKM fragment, when cloned upstream of the rat brain creatine kinase (CKB) promoter, mediated the p53 transactivation of CKB. Analyses of p53wt and a series of missense mutants (altered in conserved region II of p53) showed that binding of p53 to the CKM promoter was required but was not sufficient for transactivation. The results are discussed in relation to the possible role of p53wt in the expression of CKM in cell types which may not express the myogenic transcription factors.

Animals↗

PubMed1995

Construction and expression of a chimeric gene encoding human terminal deoxynucleotidyltransferase and DNA polymerase beta.

A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Pol beta and TdT to investigate the region of Pol beta required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154-212 of TdT was replaced by the corresponding region encoding aa 1-60 of POL beta. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of POL beta represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Pol beta protein possessed significant template-dependent polymerase activity suggests that aa 1-60 of Pol beta are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Pol beta possesses ssDNA-binding activity [Kumar et al., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar et al., Biochemistry 29 (1990b) 7156-7159; Prasad et al., J. Biol. Chem. 268 (1993) 22746-22755].

Amino Acid Sequence↗

PubMed1992

Selective 5' modification of T7 RNA polymerase transcripts.

We have developed two methods for selective 5' modification of RNAs generated by enzymatic synthesis using T7 RNA polymerase. The first method involves a two-step procedure. Transcription reactions are performed under standard conditions except that GTP is replaced by GTP gamma S. Since the polymerase initiates transcription with GTP, every transcript contains a 5' gamma-thiophosphate group which is modified with the thiol-specific reagent of choice (e.g., iodoacetyl dansyl derivative) in the second step. In an alternative method, transcription and modification reactions are carried out in a single step, using a mixture of dansylated GTP and GTP. Under the appropriate conditions, dansylated GTP effectively competes with GTP in the initiation reaction but does not substantially inhibit the elongation reaction. Yields of fluorescent 64-mer RNA ranging from 30 to 70% of the total transcription product have been obtained using these methods in combination with HPLC purification. This approach is amenable to large scale synthesis reactions and can be used to produce a wide variety of 5'-modified RNAs of virtually any size for structural or functional studies.

Base Sequence↗

PubMed1987

The role of oxygen radicals in dye-mediated photodynamic effects in Escherichia coli B.

Photosensitive dyes representative of the thiazines, xanthenes, acridines, and phenazines mediated phototoxicity in Escherichia coli B. The observed phototoxicity was sensitizer-, light-, and oxygen-dependent and is therefore a photodynamic effect. Hydroxyl radical scavengers conferred protection against the photodynamic action of all of the representative dyes. The extent of protection was dependent on the concentration of scavenger and on the in vitro reactivity of the scavenger with the hydroxyl radical. Exogenous superoxide dismutase and catalase partially protected the cells against the dye-mediated phototoxicity, and prior induction of intracellular superoxide dismutase and catalase by growth in glucose minimal medium containing manganese and paraquat substantially protected E. coli B against the photodynamic action of all of the dyes examined. Combinations of protective treatments against the phototoxicity of all four classes of dyes, including superoxide dismutase and catalase preinduction and addition of extracellular superoxide dismutase and catalase or the addition of hydroxyl radical scavengers, provided nearly complete protection against the oxygen-dependent component of dye-mediated lethality. E. coli B grown in glucose minimal medium containing manganese and photosensitive dyes induced manganese superoxide dismutase. The extent of induction was correlated with the dyes' ability to photooxidize NADH in vitro. Thus, oxygen radicals are primarily responsible for the oxygen-dependent toxicity of the photosensitive dyes examined, and one adaptive response of E. coli B to a dye-mediated oxidative threat is to induce superoxide dismutase.

Acridines↗

PubMed1987

Oxygen radicals are generated by dye-mediated intracellular photooxidations: a role for superoxide in photodynamic effects.

Representative thiazines, xanthenes, acridines, and phenazines photosensitized the oxidation of reduced pyridine nucleotides and reduced glutathione when illuminated with low intensity visible light. Photooxidation resulted in oxygen consumption and in superoxide generation, assayed as the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. The major pathway of electron transfer involved dye reduction rather than singlet oxygen-mediated oxidation of the substrate, as demonstrated by the relative insensitivity of the oxidation to inhibition by sodium azide and by the observable bleaching of the dye. Hydrogen peroxide was a stable end product of photooxidation. Photosensitive dyes were photoreduced intracellularly. These dyes were transported across the membranes of Escherichia coli B and stimulated a light- and concentration-dependent increase in the cyanide-insensitive respiration. Dyes reduced intracellularly subsequently diffused out of the cell where they reduced extracellular cytochrome c. The photosensitive dyes examined in this study exhibited a light-dependent bacteriostatic effect on E. coli B grown in nutrient broth, manifested as an increased lag prior to growth. Restoration of growth coincided with increased levels of SOD, and the intracellular level of SOD correlated with the level of illumination, the dye concentration, and the reactivity of the dye to NADH in vitro. The thiazine dye, toluidine blue o, imposed a light- and oxygen-dependent lethality on E. coli B grown in glucose minimal medium. Toxicity was relieved by hydroxyl radical scavengers, and their ability to protect the cells was proportional to their reactivity with the hydroxyl radical. The results indicate that oxygen radicals and related species mediate photodynamic effects in E. coli B.

Coloring Agents↗

PubMed1987

Oxygen radicals mediate cell inactivation by acridine dyes, fluorescein, and lucifer yellow CH.

Acridine dyes, fluorescein and lucifer yellow CH are fluorescent photosensitizers used experimentally to selectively stain and photodynamically destroy eukaryotic cells and subcellular structures. We have determined that the mechanism of light- and oxygen-dependent inactivation of E. coli by these dyes involves oxygen radicals and hydrogen peroxide. All of the dyes oxidized NAD(P)H+ under illumination. Superoxide (O2), detected as the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c, was a major product of the dye sensitized photooxidation. Cationic acridine dyes penetrated the membranes of E. coli and were photoreduced intracellularly. Reduced dyes diffused back into the medium and mediated the reduction of extracellular ferricytochrome c. The anionic dyes fluorescein and lucifer yellow CH were unable to mediate extracellular cytochrome c reduction, indicating that these dyes were impermeable to the E. coli membrane. Acridine dyes, when illuminated, inhibited the growth of E. coli in a rich medium, and induced the synthesis of SOD. Fluorescein and lucifer yellow CH did not inhibit growth or induce SOD synthesis because they were unable to enter the cells. Superoxide (O2) and hydrogen peroxide (H2O2), generated by the enzyme xanthine oxidase were toxic to E. coli B. Inactivation by xanthine oxidase was partially inhibited by exogenous SOD and completely inhibited by exogenous catalase or SOD plus catalase. Similarly, exogenous SOD plus catalase protected against inactivation by acridines and fluorescein-NADH or lucifer yellow CH-NADH mixtures. Prior induction of superoxide dismutase and catalase in E. coli B significantly protected cells against a subsequent challenge by illuminated acridine dyes. SOD and catalases preinduction combined with additions of exogenous SOD and catalase completely protected E. coli B against photodynamic inactivation by acridine yellow. The hydroxyl radical scavengers, dimethyl sulfoxide, sodium benzoate and thiourea, protected E. coli B against photodynamic inactivation by acridine orange. The results implicate O2, H2O2, and the hydroxyl radical (OH) as underlying molecular agents of the phototoxicity mediated by acridine orange, acridine yellow, fluorescein and lucifer yellow CH.

Acridine Orange↗

PubMed1987

Role of oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B.

Photoillumination of tetracycline derivatives with low-intensity (320- to 400-nm) light and visible light generated superoxide, observed as the reduction of ferricytochrome c. The rate of reduction was dependent on the tetracycline concentration and on the derivative being examined, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. Tetracycline-mediated cytochrome c reduction was oxygen dependent and inhibited up to 70% by superoxide dismutase. Illuminated tetracyclines were lethal to Escherichia coli B incubated in a glucose minimal medium containing chloramphenicol. This lethality was light dependent, oxygen dependent, and dependent on the concentration of tetracycline. Kill rates also varied according to the derivative under study, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. The addition of superoxide dismutase and catalase to the incubation medium partially protected E. coli B against the light-dependent lethality. Preinduction of intracellular superoxide dismutase and catalase substantially protected E. coli B against the phototoxicity of tetracyclines. Iron EDTA augmented the phototoxicity of tetracyclines, while diethylenetriaminepentaacetic acid protected against their lethality. Hydroxyl radical scavengers also conferred protection against tetracycline phototoxicity. The extent of protection was in order of the in vitro reactivity of the scavengers with the hydroxyl radical. These results indicate that superoxide, hydrogen peroxide, and the hydroxyl radical are generated by illuminated tetracyclines and are molecular agents of tetracycline phototoxicity in E. coli B.

Catalase↗