Highly selective isolation of unknown mutations in diverse DNA fragments: toward new multiplex screening in cancer.
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Cancer research would greatly benefit from technologies that allow simultaneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the potential of this method, heteroduplexes with a single A/G mismatch are formed via cross-hybridization of mutant (T-->G) and wild-type DNA-fragment populations. Aldehydes are uniquely introduced at the position of mismatched adenines via the Escherichia coli glycosylase, MutY. Subsequent treatment with a biotinylated hydroxylamine results in highly specific and covalent biotinylation of the site of mismatch. For PCR amplification, synthetic linkers are then ligated to the DNA fragments. Biotinylated DNA is then isolated and PCR amplified. Mutation-containing DNA fragments can subsequently be sequenced to identify type and position of mutation. This method correctly detects a single T-->G transversion introduced into a 7-kb plasmid containing full-length cDNA from the p53 gene. In the presence of a high excess wild-type DNA (1:1000 mutant:normal plasmids) or in the presence of diverse DNA fragment sizes, the DNA fragments containing the mutation are readily detectable and can be isolated and amplified. The present Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current limit of detection of one base substitution in 7 Mb of DNA and increases the limit for unknown mutation scanning by two to three orders of magnitude. Homozygous and heterozygous p53 regions (G-->T, exon 4) from genomic DNA are also examined, and correct identification of mutations is demonstrated. This method should allow large-scale detection of genetic alterations in cancer samples without any assumption as to the genes of interest. (+info)
Magnetic field exposure enhances DNA repair through the induction of DnaK/J synthesis.
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In contrast to the common impression that exposure to a magnetic field of low frequency causes mutations to organisms, we have demonstrated that a magnetic field can actually enhance the efficiency of DNA repair. Using Escherichia coli strain XL-1 Blue as the host and plasmid pUC8 that had been mutagenized by hydroxylamine as the vector for assessment, we found that bacterial transformants that had been exposed to a magnetic field of 50 Hz gave lower percentages of white colonies as compared to transformants that had not been exposed to the magnetic field. This result was indicative that the efficiency of DNA repair had been improved. The improvement was found to be mediated by the induced overproduction of heat shock proteins DnaK/J (Hsp70/40). (+info)
Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene.
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The translocation stage of protein synthesis is a highly conserved process in all cells. Although the components necessary for translocation have been delineated, the mechanism of this activity has not been well defined. Ribosome movement on template mRNA must allow for displacement of tRNA-mRNA complexes from the ribosomal A to P sites and P to E sites, while ensuring rigid maintenance of the correct reading frame. In Escherichia coli, translocation of the ribosome is promoted by elongation factor G (EF-G). To examine the role of EF-G and rRNA in translocation we have characterized mutations in rRNA genes that can suppress a temperature-sensitive (ts) allele of fusA, the gene in E. coli that encodes EF-G. This analysis was performed using the ts E. coli strain PEM100, which contains a point mutation within fusA. The ts phenotype of PEM100 can be suppressed by either of two mutations in the decoding region of the 16S rRNA when present in combination with a mutation at position 2058 in the peptidyltransferase domain of the 23S rRNA. Communication between these ribosomal domains is essential for coordinating the events of the elongation cycle. We propose a model in which EF-G promotes translocation by modulating this communication, thereby increasing the efficiency of this fundamental process. (+info)
Restricted passage of reaction intermediates through the ammonia tunnel of carbamoyl phosphate synthetase.
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The x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel. NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of gamma-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase. (+info)
Blue light's effects on rhodopsin: photoreversal of bleaching in living rat eyes.
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PURPOSE: To determine whether blue light induces photoreversal of rhodopsin bleaching in vivo. METHODS: Eyes of anesthetized albino rats were exposed to either green (550 nm) or deep blue (403 nm) light, and the time course of rhodopsin bleaching was determined. Rhodopsin was isolated from whole retinas by detergent extraction and measured photometrically. To inhibit photoreversal of bleaching, rats were perfused with 70 mM hydroxylamine (NH(2)OH), a known inhibitor of photoreversal. To determine whether blue-absorbing, photoreversible photoproducts were formed, rhodopsin was bleached to near completion with green light and then exposed to blue light. Finally, experimental results were simulated on a computer by means of a simple, three-component model involving a long-lived photoreversible photoproduct. RESULTS: Photoreversal of bleaching in blue light occurs in vivo as evidenced by the following: In the absence of NH(2)OH, bleaching of rhodopsin by blue light was slow and complex. In the presence of NH(2)OH, however, blue light bleached rhodopsin very fast with a simple, pseudo-first-order kinetic. A long-lived bleaching intermediate produced by green light exposure was photoreversed to rhodopsin by exposure to blue light. The three-component computer model, invoking a blue-absorbing, photoreversible, long-lived intermediate accurately described the data. CONCLUSIONS: Because of the instantaneous, nonmetabolic regeneration of rhodopsin by the process of photoreversal of bleaching, blue light exposure permits the absorption of large numbers of photons by rhodopsin and by a photoreversible intermediate of bleaching in vivo. These data may have an important impact on resolving mechanisms of blue light-mediated damage to the retina. (+info)
Transcript analysis of multiple copies of amo (encoding ammonia monooxygenase) and hao (encoding hydroxylamine oxidoreductase) in Nitrosomonas europaea.
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The genes encoding ammonia monooxygenase (amoCAB), hydroxylamine oxidoreductase (hao), and the c-type cytochrome c-554 (hcy) are present in multiple copies in the genome of Nitrosomonas europaea. The upstream regions of the two copies of amoC, the three copies of hao, and one copy of hcy were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for amoA. Putative sigma(70) promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with amoC primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that N. europaea cells may be able to use different promoters in the presence and absence of ammonium. (+info)
A nitric oxide-dopamine link pathway in organum vasculosum laminae terminalis of rat brain exerts control over blood pressure.
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1. Experiments were carried out to explore the possible role played by the nitric oxide (NO) and dopamine (DA) system in the organum vasculosum laminae terminalis (OVLT) of rat brain in arterial pressure regulation. 2. Intracerebroventricular (ICV) administration of NO donors such as hydroxylamine or sodium nitroprusside (SNP) caused an up to 59 mmHg decrease in blood pressure (BP) and a decrease in DA release (measured by nafion coated carbon fibre electrodes in combination with voltammetry) in the OVLT. In contrast, ICV administration of N(G)-nitro-L-arginine methyl ester (L-NAME; a constitutive NO synthase inhibitor) or 7-nitroindazol (a neuronal NO synthase inhibitor) caused an up to 98 mmHg increase in BP and an increase in DA release in the OVLT. 3. Intra-OVLT injection of amphetamine (0.1 - 0.3 mg), SKF 38393 (a DA D(1) receptor agonist; 0.01 - 0.03 mg), or apomorphine (a DA D(2,3) receptor agonist; 0.01 - 0.03 mg) caused an increase in BP. On the other hand, intra-OVLT injection of SCH23390 (a DA D(1) receptor antagonist; 0.005 - 0.020 mg) or haloperidol (0.005 - 0.020 mg) caused a decrease in BP. 4. The pressor effects induced by intra-OVLT administration of L-NAME were attenuated by pretreatment with intra-OVLT injection of haloperidol, SCF23390, or 6-hydroxydopamine. In the contrast, the hydroxylamine-, 8-Br-cGMP- or SNP-induced depressor effects were attenuated by pretreatment with intra-OVLT injection of amphetamine, SKF 38393 or apomorphine. 5. The data suggest that activation of a NO-DA link pathway within the OVLT of rat brain exerts control over blood pressure. (+info)
Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible.
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Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents subsequent repalmitoylation. (+info)