| structure of the fmet-trna(fmet)-binding domain of b. stearothermophilus initiation factor if2. | the three-dimensional structure of the fmet-trna(fmet) -binding domain of translation initiation factor if2 from bacillus stearothermophilus has been determined by heteronuclear nmr spectroscopy. its structure consists of six antiparallel beta-strands, connected via loops, and forms a closed beta-barrel similar to domain ii of elongation factors ef-tu and ef-g, despite low sequence homology. two structures of the ternary complexes of the ef-tu small middle dotaminoacyl-trna small middle dot gdp ... | 2000 | 10775275 |
| functional interaction between the werner syndrome protein and dna polymerase delta. | werner syndrome (ws) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. the ws gene encodes a 1,432-amino acid polypeptide (wrn) with a central domain homologous to the recq family of dna helicases. purified wrn unwinds dna with 3'-->5' polarity, and also possesses 3'-->5' exonuclease activity. elucidation of the physiologic function(s) of wrn may be aided by the identification of wrn-interacting proteins. we show here that wrn ... | 2000 | 10781066 |
| a novel amidase (half-amidase) for half-amide hydrolysis involved in the bacterial metabolism of cyclic imides. | a novel amidase involved in bacterial cyclic imide metabolism was purified from blastobacter sp. strain a17p-4. the enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. the purified amidase showed h ... | 2000 | 10788365 |
| dna polymerase i is essential for growth of methylobacterium dichloromethanicum dm4 with dichloromethane. | methylobacterium dichloromethanicum dm4 grows with dichloromethane as the unique carbon and energy source by virtue of a single enzyme, dichloromethane dehalogenase-glutathione s-transferase. a mutant of the dichloromethane-degrading strain m. dichloromethanicum dm4, strain dm4-1445, was obtained by mini-tn5 transposon mutagenesis that was no longer able to grow with dichloromethane. dichloromethane dehalogenase activity in this mutant was comparable to that of the wild-type strain. the site of ... | 2000 | 10986246 |
| reduction of fe(iii), cr(vi), u(vi), and tc(vii) by deinococcus radiodurans r1. | deinococcus radiodurans is an exceptionally radiation-resistant microorganism capable of surviving acute exposures to ionizing radiation doses of 15,000 gy and previously described as having a strictly aerobic respiratory metabolism. under strict anaerobic conditions, d. radiodurans r1 reduced fe(iii)-nitrilotriacetic acid coupled to the oxidation of lactate to co(2) and acetate but was unable to link this process to growth. d. radiodurans reduced the humic acid analog anthraquinone-2,6-disulfon ... | 2000 | 10788374 |
| the autographa californica nuclear polyhedrosis virus p143 gene encodes a dna helicase. | the p143 protein of autographa californica nuclear polyhedrosis virus is essential for replication of viral dna. to determine the function of p143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress p143. atpase activity copurified with p143 protein during purification and also during gel filtration at a high salt concentration. the atpase activity did not require the presence of single-stranded dna, but was stimulated fourfold by the additi ... | 2000 | 10799604 |
| listeriolysin o as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen listeria monocytogenes. | listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin o (llo) encoded by hly. llo-negative mutants of l. monocytogenes are avirulent in the mouse model. we have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible pro ... | 2000 | 10816469 |
| production of exopolysaccharide by lactobacillus rhamnosus r and analysis of its enzymatic degradation during prolonged fermentation. | the potential of lactobacillus rhamnosus r for producing exopolysaccharide (eps) when grown on basal minimum medium supplemented with glucose or lactose was investigated. eps production by l. rhamnosus r is partially growth associated and about 500 mg of eps per liter was synthesized with both sugars. the product yield coefficient (y(eps/s)) was 3.15 (0.0315 g of eps [g of lactose](-1)) and 2.88 (0.0288 g of eps [g of glucose](-1)). it was clearly shown that the amount of eps produced declined u ... | 2000 | 10831403 |
| arabidopsis muts homologs-atmsh2, atmsh3, atmsh6, and a novel atmsh7-form three distinct protein heterodimers with different specificities for mismatched dna. | arabidopsis mismatch repair genes predict muts-like proteins remarkably similar to eukaryotic muts homologs-msh2, msh3, and msh6. a novel feature in arabidopsis is the presence of two msh6-like proteins, designated atmsh6 and atmsh7. combinations of arabidopsis atmsh2 with atmsh3, atmsh6, or atmsh7 proteins-products of in vitro transcription and translation-were analyzed for interactions by analytical gel filtration chromatography. the atmsh2 protein formed heterodimers with atmsh3, atmsh6, and ... | 2000 | 10852942 |
| conservation of sigma-core rna polymerase proximity relationships between the enhancer-independent and enhancer-dependent sigma classes. | two distinct classes of rna polymerase sigma factors (sigma) exist in bacteria and are largely unrelated in primary amino acid sequence and their modes of transcription activation. using tethered iron chelate (fe-babe) derivatives of the enhancer-dependent sigma(54), we mapped several sites of proximity to the beta and beta' subunits of the core rna polymerase. remarkably, most sites localized to those previously identified as close to the enhancer-independent sigma(70) and sigma(38). this indic ... | 2000 | 10856247 |
| rapid 5' nuclease (taqman) assay for detection of virulent strains of yersinia enterocolitica. | we have developed a rapid procedure for the detection of virulent yersinia enterocolitica in ground pork by combining a previously described pcr with fluorescent dye technologies. the detection method, known as the fluorogenic 5' nuclease assay (taqman), produces results by measuring the fluorescence produced during pcr amplification, requiring no post-pcr processing. the specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 no ... | 2000 | 10966441 |
| male gender predisposes to development of endotoxic shock in the rat. | after intravenous (i.v.) injection of lipopolysaccharide (lps) macrophages release nitric oxide (no) due to the expression of the inducible no synthase (inos). after lps no is abundantly produced also in the cardiovascular system and may contribute to the development of hypotension and shock. since the immune response, the synthesis of no and the regulation of blood pressure (bp) differ between males and females, in the present study the effect of lps on bp, renal function, the plasma and urinar ... | 2000 | 10869545 |
| influence of sulfide and temperature on species composition and community structure of hot spring microbial mats. | in solfataric fields in southwestern iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80 degrees c that are white or yellow from precipitated sulfur (sulfur mats). in low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90 degrees c, and a chloroflexus mat is formed at 65 to 70 degrees c. we have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one chloroflexus mat (low-sulfide) hot spr ... | 2000 | 10877776 |
| characterization of mselb, a novel mammalian elongation factor for selenoprotein translation. | decoding of uga selenocysteine codons in eubacteria is mediated by the specialized elongation factor selb, which conveys the charged trna(sec) to the a site of the ribosome, through binding to the secis mrna hairpin. in an attempt to isolate the eukaryotic homolog of selb, a database search in this work identified a mouse expressed sequence tag containing the complete cdna encoding a novel protein of 583 amino acids, which we called mselb. several lines of evidence enabled us to establish that m ... | 2000 | 10970870 |
| promoter opening by sigma(54) and sigma(70) rna polymerases: sigma factor-directed alterations in the mechanism and tightness of control. | transcription control at the melting step is not yet understood. here, band shift, cross-linking, and transcription experiments on diverse dna probes were used with two bacterial rna polymerase holoenzymes that differ in how they regulate melting. data indicated that both sigma(54) and sigma(70) holoenzymes assume a default closed form that cannot establish single-strand binding. upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction ... | 2000 | 10970887 |
| pcr bias in ecological analysis: a case study for quantitative taq nuclease assays in analyses of microbial communities. | succession of ecotypes, physiologically diverse strains with negligible rrna sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (c. postius and a. ernst, arch. microbiol. 172:69-75, 1999). in order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. in this study, we examined the perform ... | 2000 | 11055948 |
| quantitation of pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid 'real-time' polymerase chain reaction. | statement of findings: we developed a real-time detection (rtd) polymerase chain reaction (pcr) with rapid thermal cycling to detect and quantify pseudomonas aeruginosa in wound biopsy samples. this method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (cfu)/g tissue or a few copies per reaction. the time from sample collection to result was less than 1h. rtd-pcr has potential for rapid quantitative detection of pathogens in cri ... | 2000 | 11056755 |
| requirement for phe36 for dna binding and mismatch repair by escherichia coli muts protein. | the muts family of dna repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. photocrosslinking and mutational studies previously identified a highly conserved phe residue at the n-terminus of thermus aquaticus muts protein that is critical for mismatch recognition in vitro. here, a mutant escherichia coli muts protein harboring a substitution of ala for the corresponding phe36 residue is assessed for proficiency ... | 2000 | 10982877 |
| uv-induced crosslinks in the 16s rrnas of escherichia coli, bacillus subtilis and thermus aquaticus and their implications for ribosome structure and photochemistry. | sixteen long-range crosslinks are induced in escherichia coli 16s rrna by far-uv irradiation. crosslinking patterns in two other organisms, bacillus subtilis and thermus aquaticus, were investigated to determine if the number and location of crosslinks in e.coli occur because of unusually photoreactive nucleotides at particular locations in the rrna sequence. thirteen long-range crosslinks in b.subtilis and 15 long-range crosslinks in t.aquaticus were detected by gel electrophoresis and 10 cross ... | 2000 | 11000271 |
| a dual-specificity aminoacyl-trna synthetase in the deep-rooted eukaryote giardia lamblia. | cysteinyl-trna (cys-trna) is essential for protein synthesis. in most organisms the enzyme responsible for the formation of cys-trna is cysteinyl-trna synthetase (cysrs). the only known exceptions are the euryarchaea methanococcus jannaschii and methanobacterium thermoautotrophicum, which do not encode a cysrs. deviating from the accepted concept of one aminoacyl-trna synthetase per amino acid, these organisms employ prolyl-trna synthetase as the enzyme that carries out cys-trna formation. to da ... | 2000 | 11078517 |
| application of 5'-nuclease pcr for quantitative detection of listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk. | pcr techniques have significantly improved the detection and identification of bacterial pathogens. countless adaptations and applications have been described, including quantitative pcr and the latest innovation, real-time pcr. in real-time pcr, e.g., the 5'-nuclease chemistry renders the automated and direct detection and quantification of pcr products possible (p. m. holland et al., proc. natl. acad. sci. usa 88:7276-7280, 1991). we present an assay for the quantitative detection of listeria ... | 2000 | 11010869 |
| quantification of fungal dna by using fluorescence resonance energy transfer and the light cycler system. | the light cycler technique combines rapid in vitro amplification of dna in glass capillaries with real-time species determination and quantification of dna load. we have established a quantitative pcr protocol for two clinically important pathogens, candida albicans and aspergillus fumigatus. the sensitivity of the assay was comparable to those of previously described pcr protocols (5 cfu/ml). specific detection of c. albicans and a. fumigatus could be achieved. the assay showed a high reproduci ... | 2000 | 10655350 |
| induction of apoptosis in human mitogen-activated peripheral blood t-lymphocytes by the ether phospholipid et-18-och3: involvement of the fas receptor/ligand system. | 1. activated t-cells constitute a target for treatment of autoimmune diseases. we have found that the antitumour ether phospholipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och3; edelfosine) induced dose- and time-dependent apoptosis in human mitogen-activated peripheral blood t-lymphocytes, but not in resting t-cells. t-lymphocytes were stimulated with phytohemagglutinin and interleukin-2 or with concanavalin a. apoptosis was assessed by dna fragmentation through cell cycle ... | 1999 | 10433487 |
| evidence for horizontal gene transfer in evolution of elongation factor tu in enterococci. | the elongation factor tu, encoded by tuf genes, is a gtp binding protein that plays a central role in protein synthesis. one to three tuf genes per genome are present, depending on the bacterial species. most low-g+c-content gram-positive bacteria carry only one tuf gene. we have designed degenerate pcr primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. the amplified dna fragments were ... | 2000 | 11092850 |
| effects of amplification facilitators on diagnostic pcr in the presence of blood, feces, and meat. | the full potential of diagnostic pcr is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. therefore, different pre-pcr treatments are being used to reduce the effects of pcr inhibitors. the aim of the present study was to investigate the effects of 16 amplification facilitators to enhance dna amplification in the presence of blood, feces, or meat. different concentrations of amplification facilitators and inhibitory samples we ... | 2000 | 11101581 |
| detection of ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic pcr (taqman) assay. | a fluorogenic (taqman) pcr assay was developed to detect ralstonia solanacearum strains. two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (rs) detected all biovars of r. solanacearum, and a second more specific probe (b2) detected only biovar 2a. amplification of the target was measured by the 5' nuclease activity of taq dna polymerase on each probe, resulting in emission of fluorescence. taqman pcr was performed with dna extracted from 42 r. solanacearum and g ... | 2000 | 10877778 |
| contamination and sensitivity issues with a real-time universal 16s rrna pcr. | a set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16s rrna gene was designed for use with the real-time pcr applied biosystems 7700 (taqman) system. during the development of this pcr, problems were noted with the use of this gene as an amplification target. contamination of reagents with bacterial dna was a major problem exacerbated by the highly sensitive nature of the real-time pcr chemistry. this was compounded by the use of a small amplicon of ... | 2000 | 10790092 |
| eosinophil granule-derived major basic protein induces il-8 expression in human intestinal myofibroblasts. | eosinophil infiltration occurs in a variety of allergic and inflammatory diseases. the release of preformed mediators from eosinophils may contribute to inflammatory responses. we investigated the ability of eosinophil-derived major basic protein and eosinophil-derived neurotoxin to stimulate production of il-8 from intestinal myofibroblasts. intestinal myofibroblasts (18-co cells) were incubated with major basic protein, eosinophil-derived neurotoxin, or a synthetic analogue of major basic prot ... | 2000 | 11012615 |
| a 5' nuclease pcr (taqman) high-throughput assay for detection of the meca gene in staphylococci. | in an effort to find a rapid, efficient, and reliable method of screening large numbers of bacterial isolates for specific antimicrobial resistance genes, we compared conventional pcr results to the results generated using the taqman 5' nuclease pcr kit in conjunction with an abi prism 7700 sequence detector for detecting the meca gene in various species of staphylococci. dna was extracted using two techniques. the first used a high-salt extraction method suitable for conventional pcr but result ... | 2000 | 10878035 |
| identification of an outer segment targeting signal in the cooh terminus of rhodopsin using transgenic xenopus laevis. | mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ros). various green fluorescent protein (gfp)/rhodopsin cooh-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic xenopus laevis under the control of the xenopus opsin promoter. the fusio ... | 2000 | 11134067 |
| isolation and expression of lactate dehydrogenase genes from rhizopus oryzae. | rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. in this study i cloned two genes, ldha and ldhb, which code for nad(+)-dependent l-lactate dehydrogenases (ldh) (ec 1.1.1.27), from a lactic acid-producing strain of r. oryzae. these genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. this is the first description of ldh genes in a fungus, and sequence comparisons ... | 2000 | 10831409 |
| detection of human cytomegalovirus dna by real-time quantitative pcr. | a real-time pcr assay was developed to quantify human cytomegalovirus (cmv) dna. this assay was used to demonstrate a higher cmv dna load in plasma of bone marrow transplant patients than in that of blood donors. the cmv load was higher in cmv antigen-positive patients than in antigen-negative patients. | 2000 | 10878073 |
| cloning and sequencing of defective particles derived from the autonomous parvovirus minute virus of mice for the construction of vectors with minimal cis-acting sequences. | the production of wild-type-free stocks of recombinant parvovirus minute virus of mice [mvm(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. we have therefore cloned and sequenced spontaneously occurring defective particles of mvm(p) with very small genomes to identify the minimal cis-acting sequences required for dna amplification and virus production. one of them has lost all capsid ... | 2001 | 11152501 |
| extensive homologous recombination among widely divergent tt viruses. | analyses of a collection of full-length tt virus genomes showed nearly half of them to be recombinant. the results were highly significant and revealed homologous recombination both within and among genotypes, often involving extremely divergent lineages. recombination breakpoints were significantly more common in the noncoding region of the tt virus genome than in the coding region. | 2000 | 10906223 |
| common fold in helix-hairpin-helix proteins. | helix-hairpin-helix (hhh) is a widespread motif involved in non-sequence-specific dna binding. the majority of hhh motifs function as dna-binding modules, however, some of them are used to mediate protein-protein interactions or have acquired enzymatic activity by incorporating catalytic residues (dna glycosylases). from sequence and structural analysis of hhh-containing proteins we conclude that most hhh motifs are integrated as a part of a five-helical domain, termed (hhh)(2) domain here. it t ... | 2000 | 10908318 |
| novel genes coding for lithotrophic sulfur oxidation of paracoccus pantotrophus gb17. | the gene region coding for lithotrophic sulfur oxidation of paracoccus pantotrophus gb17 is located on a 13-kb insert of plasmid peg12. upstream of the previously described six open reading frames (orfs) soxabcdef with a partial sequence of soxa and soxf (c. wodara, f. bardischewsky, and c. g. friedrich, j. bacteriol. 179:5014-5023, 1997), 4,350 bp were sequenced. the sequence completed soxa, and uncovered six new orfs upstream of soxa, designated orf1, orf2, and orf3, and soxxyz. orf1 could enc ... | 2000 | 10940005 |
| reduction of gc --> ta transversion mutation by overexpression of muts in escherichia coli k-12. | overexpression of the muts repair protein significantly decreased the rate of lacz gc --> ta transversion mutation in stationary-phase and exponentially growing bacteria and in muty and mutm mutants, which accumulate mismatches between 8-oxoguanine (8-oxog) and adenine residues in dna. conversely, gc --> ta transversion increased in mutl or muts mutants in stationary phase. in contrast, overexpression of muts did not appreciably reduce lacz at --> cg transversion mutation in a mutt mutant. these ... | 2000 | 10940054 |
| identifying a core rna polymerase surface critical for interactions with a sigma-like specificity factor. | cyclic interactions occurring between a core rna polymerase (rnap) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. in this work we have identified regions of the single-subunit yeast mitochondrial rnap (rpo41p) important for interaction with its sigma-like specificity factor (mtf1p). previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of mtf1p are re ... | 2000 | 10958696 |
| new host-vector system for thermus spp. based on the malate dehydrogenase gene. | a thermus thermophilus hb27 strain was constructed in which the malate dehydrogenase (mdh) gene was deleted. the deltamdh colonies are recognized by a small-colony phenotype. wild-type phenotype is restored by transformation with thermus plasmids or integration vector containing an intact mdh gene. the wild-type phenotype provides a positive selection tool for the introduction of plasmid dna into thermus spp., and because mdh levels can be readily quantified, this host-vector system is a conveni ... | 2001 | 11160114 |
| a hydrogen peroxide-forming nadh oxidase that functions as an alkyl hydroperoxide reductase in amphibacillus xylanus. | the amphibacillus xylanus nadh oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-nadh, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (fad) or the small disulfide-containing salmonella enterica ahpc protein. the enzyme has two disulfide bonds, cys128-cys131 and cys337-cys340, which can act as redox centers in addition to the enzyme-bound fad (k. ohnishi, y. niimura, m. hidaka, h. masaki, h. suzuki, t. uozumi, and t. ni ... | 2000 | 10960086 |
| a positive cis-acting dna element is required for high-level transcription in chlamydia. | the spacer a/t region is a positive cis-acting dna element that was identified in the chlamydia trachomatis rrna promoter region. we have now demonstrated that similar sequences in other chlamydial promoters are important for transcription. substitution of candidate spacer a/t regions in four chlamydial promoters decreased transcription by partially purified c. trachomatis rna polymerase in an in vitro transcription assay. addition of a spacer a/t region to the dnak promoter, which does not cont ... | 2000 | 10960101 |
| rna polymerases from bacillus subtilis and escherichia coli differ in recognition of regulatory signals in vitro. | adaptation of bacterial cells to diverse habitats relies on the ability of rna polymerase to respond to various regulatory signals. some of these signals are conserved throughout evolution, whereas others are species specific. in this study we present a comprehensive comparative analysis of rna polymerases from two distantly related bacterial species, escherichia coli and bacillus subtilis, using a panel of in vitro transcription assays. we found substantial species-specific differences in the a ... | 2000 | 11029421 |
| functional interaction between ssu72 and the rpb2 subunit of rna polymerase ii in saccharomyces cerevisiae. | ssu72 is an essential gene encoding a phylogenetically conserved protein of unknown function that interacts with the general transcription factor tfiib. a recessive ssu72-1 allele was identified as a synthetic enhancer of a tfiib (sua7-1) defect, resulting in a heat-sensitive (ts(-)) phenotype and a dramatic downstream shift in transcription start site selection. here we describe a new allele, ssu72-2, that confers a ts(-) phenotype in a sua7 wild-type background. in an effort to further define ... | 2000 | 11046131 |
| fine structure of e. coli rna polymerase-promoter interactions: alpha subunit binding to the up element minor groove. | the alpha subunit of e. coli rnap plays an important role in the recognition of many promoters by binding to the a+t-rich up element, a dna sequence located upstream of the recognition elements for the sigma subunit, the -35 and -10 hexamers. we examined dna-rnap interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin. our results suggest that alpha interacts with bases in the dna minor groove and with the dna backbon ... | 2001 | 11238372 |
| genome of the extremely radiation-resistant bacterium deinococcus radiodurans viewed from the perspective of comparative genomics. | the bacterium deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, uv radiation, oxidizing agents, and electrophilic mutagens. d. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. these characte ... | 2001 | 11238985 |
| sec-dependent protein export and the involvement of the molecular chaperone secb. | | 2000 | 11048650 |
| a new thermoactive pullulanase from desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in bacillus subtilis. | the gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon desulfurococcus mucosus (apua) was cloned in escherichia coli and sequenced. apua from d. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apua encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kda after signal cleavage. the apua gene wa ... | 2000 | 11053376 |
| development of real-time pcr assays for rapid detection of pfiesteria piscicida and related dinoflagellates. | pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the east coast of north america, particularly in its largest (chesapeake bay in maryland) and second largest (albermarle-pamlico sound in north carolina) estuaries. in response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. h ... | 2000 | 11055905 |
| real-time pcr for quantitative detection of toxoplasma gondii. | the protozoan toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. we report here the development of a real-time pcr-based assay for the detection of t. gondii. oligonucleotide primers and a fluorescence-labeled taqman probe were designed to amplify the t. gondii b1 gene. after 40 pcr cycles, the cycle threshold values (c(t)) indicative of the quantity of the target gene were determine ... | 2000 | 11060078 |
| fecal excretion of a novel human circovirus, tt virus, in healthy children. | the role of tt virus (ttv) as a human pathogen is unclear, as is the mode of ttv transmission. to determine the prevalence of ttv infection and the possible fecal-oral route of transmission, we analyzed fecal specimens from 67 healthy, nontransfused children for ttv dna sequences by heminested pcr, using the ng and t primer sets. the overall prevalence of ttv fecal excretion was 22.4% (15 of 67), with the t primer set (19.4%) being more sensitive than the ng primer set (10.4%). ttv prevalence ba ... | 2000 | 11063506 |
| identification of conserved residues contributing to the activities of adenovirus dna polymerase. | adenovirus codes for a dna polymerase that is a member of the dna polymerase alpha family and uses a protein primer for initiation of dna synthesis. it contains motifs characteristic of a proofreading 3'-5'-exonuclease domain located in the n-terminal region and several polymerase motifs located in the c-terminal region. to determine the role of adenovirus dna polymerase in dna replication, 22 site-directed mutations were introduced into the conserved dna polymerase motifs in the c-terminal regi ... | 2000 | 11090167 |
| use of pulsed-field gel electrophoresis and flagellin gene typing in identifying clonal groups of campylobacter jejuni and campylobacter coli in farm and clinical environments. | although campylobacters have been isolated from a wide range of animal hosts, the association between campylobacters isolated from humans and animals in the farm environment is unclear. we used flagellin gene typing and pulsed-field gel electrophoresis (pfge) to investigate the genetic diversity among isolates from animals (cattle, sheep, and turkey) in farm environments and sporadic cases of campylobacteriosis in the same geographical area. forty-eight combined fla types were seen among the 315 ... | 2001 | 11282587 |
| recombinant thermus aquaticus rna polymerase, a new tool for structure-based analysis of transcription. | the three-dimensional structure of dna-dependent rna polymerase (rnap) from thermophilic thermus aquaticus has recently been determined at 3.3 a resolution. currently, very little is known about t. aquaticus transcription and no genetic system to study t. aquaticus rnap genes is available. to overcome these limitations, we cloned and overexpressed t. aquaticus rnap genes in escherichia coli. overproduced t. aquaticus rnap subunits assembled into functional rnap in vitro and in vivo when coexpres ... | 2001 | 11114902 |
| identification of ccda in paracoccus pantotrophus gb17: disruption of ccda causes complete deficiency in c-type cytochromes. | a transposon tn5-mob insertional mutant of paracoccus pantotrophus gb17, strain tp43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20%. strain tp43 was unable to form functional c-type cytochromes, as determined by difference spectroscopy and heme staining. however, formation of apocytochromes and their transport to the periplasm were not affected, as seen with soxd, a c-type cytochrome associated with ... | 2001 | 11114924 |
| escherichia coli rna polymerase core and holoenzyme structures. | multisubunit rna polymerase is an essential enzyme for regulated gene expression. here we report two escherichia coli rna polymerase structures: an 11.0 a structure of the core rna polymerase and a 9.5 a structure of the sigma(70) holoenzyme. both structures were obtained by cryo-electron microscopy and angular reconstitution. core rna polymerase exists in an open conformation. extensive conformational changes occur between the core and the holoenzyme forms of the rna polymerase, which are large ... | 2000 | 11118218 |
| rapid, quantitative pcr monitoring of growth of clostridium botulinum type e in modified-atmosphere-packaged fish. | a rapid, quantitative pcr assay (taqman assay) which quantifies clostridium botulinum type e by amplifying a 280-bp sequence from the botulinum neurotoxin type e (bont/e) gene is described. with this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during pcr by using the abi prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the c. botulinum type e ... | 2001 | 11133447 |
| mapping the fmet-trna(f)(met) binding site of initiation factor if2. | the interaction between fmet-trna(f)(met) and bacillus stearothermophilus translation initiation factor if2 has been characterized. we demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fmet-3'accaac of the initiator trna and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid c-terminal domain of if2 (if2 c-2). a weak but specific interaction ... | 2000 | 11013225 |
| evaluation of 5' nuclease assay for detection of actinobacillus pleuropneumoniae. | sequence detection by the 5' nuclease taqman assay uses online detection of internal fluorogenic probes in closed pcr tubes. primers and probe were chosen from a part of the omla gene common to all serotypes of actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp. the test was evaluated with 73 lung isolates and 120 tonsil isolates of a. pleuropneumoniae as well as with a collection of reference strains. by using a c(t) value (cycle number in which the fluorescence exceeds the thresh ... | 2001 | 11136780 |
| ribosomal protein l2 is involved in the association of the ribosomal subunits, trna binding to a and p sites and peptidyl transfer. | ribosomal proteins l2, l3 and l4, together with the 23s rna, are the main candidates for catalyzing peptide bond formation on the 50s subunit. that l2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50s particles either lacking l2 or harboring a mutated l2. l2 does not play a dominant role in the assembly of the 50s subunit or in the fixation of the 3'-ends of the trnas at the peptidyl-transferase center. however, it is absolutely required f ... | 2000 | 11013226 |
| isolation of segments of homologous genes with only one conserved amino acid region via pcr. | we present a method which allows the isolation of fragments from genes coding for homologous proteins via pcr when only one block of conserved amino acids is available. sets of degenerated primers are defined by reverse translation of the conserved amino acids such that each set contains not more than 128 different sequences. the second primer binding site is provided by a special cassette that is designed such that it does not allow binding of the second primer prior to being copied by dna synt ... | 2001 | 11139638 |
| novel bifunctional hyperthermostable carboxypeptidase/aminoacylase from pyrococcus horikoshii ot3. | genome sequencing of the thermophilic archaeon pyrococcus horikoshii ot3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of sulfolobus solfataricus and also to that encoding the aminoacylase from bacillus stearothermophilus. the gene from p. horikoshii comprises an open reading frame of 1,164 bp with an atg initiation codon and a tga termination codon, encoding a 43,058-da protein of 387 amino acid residues. however, some of the proposed active-site r ... | 2001 | 11157230 |
| pseudomonas stutzeri nitrite reductase gene abundance in environmental samples measured by real-time pcr. | we used real-time pcr to quantify the denitrifying nitrite reductase gene (nirs), a functional gene of biogeochemical significance. the assay was tested in vitro and applied to environmental samples. the primer-probe set selected was specific for nirs sequences that corresponded approximately to the pseudomonas stutzeri species. the assay was linear from 1 to 10(6) gene copies (r2 = 0.999). variability at low gene concentrations did not allow detection of twofold differences in gene copy number ... | 2001 | 11157241 |
| quantification of human cytomegalovirus dna by real-time pcr. | a quantitative real-time pcr assay was developed to measure human cytomegalovirus (hcmv) dna load in peripheral blood leukocytes (pbls). the hcmv dna load in pbls was normalized by means of the quantification of a cellular gene (albumin). the results of the real-time pcr assay correlated with those of the hcmv pp65-antigenemia assay (p < 0.0001). | 2001 | 11158149 |
| bacterial rna polymerase subunit omega and eukaryotic rna polymerase subunit rpb6 are sequence, structural, and functional homologs and promote rna polymerase assembly. | bacterial dna-dependent rna polymerase (rnap) has subunit composition beta'betaalpha(i)alpha(ii)omega. the role of omega has been unclear. we show that omega is homologous in sequence and structure to rpb6, an essential subunit shared in eukaryotic rnap i, ii, and iii. in escherichia coli, overproduction of omega suppresses the assembly defect caused by substitution of residue 1362 of the largest subunit of rnap, beta'. in yeast, overproduction of rpb6 suppresses the assembly defect caused by th ... | 2001 | 11158566 |
| analysis of escherichia coli strains causing bacteriuria during pregnancy: selection for strains that do not express type 1 fimbriae. | escherichia coli isolates from patients with bacteriuria of pregnancy were compared by pcr with isolates from patients with community-acquired cystitis for the presence of established virulence determinants. the strains from patients with bacteriuria of pregnancy were less likely to carry genes for p-family, s-family, and f1c adhesins, cytotoxic necrotizing factor 1, and aerobactin, but virtually all of the strains carried the genes for type 1 fimbriae. standard mannose-sensitive agglutination o ... | 2001 | 11159970 |
| a ty1 reverse transcriptase active-site aspartate mutation blocks transposition but not polymerization. | reverse transcriptases (rts) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. an invariant triad of aspartates is thought to be required for the catalytic function of rts. we generated rt mutants in the yeast retrotransposon ty1, changing each of these active-site aspartates to asparagine or glutamate. all but one of the mutants lacked detectable polymerase activity. the novel exception, d(211)n, retained near wild-typ ... | 2001 | 11413300 |
| mutagenesis of the peptidyltransferase center of 23s rrna: the invariant u2449 is dispensable. | u2449 is one of many invariant residues in the central loop of domain v of 23s rrna, a region that constitutes part of the peptidyltransferase center of the ribosome. in escherichia coli, this u is post-transcriptionally modified to dihydrouridine (d) and is the only d modification found in e.coli rrnas. to analyze the role of this base and its modification in ribosomal function, all three base substitutions were constructed on a plasmid copy of the rrnb operon and assayed for their ability to s ... | 2001 | 11160893 |
| pcr performance of the b-type dna polymerase from the thermophilic euryarchaeon thermococcus aggregans improved by mutations in the y-gg/a motif. | the effect of mutations in the highly conserved y-gg/a motif of b-type dna polymerases was studied in the dna polymerase from the hyperthermophilic euryarchaeon thermococcus aggregans. this motif plays a critical role in the balance between the synthesis and degradation of the dna chain. five different mutations of the tyrosine at position 387 (tyr387-->phe, tyr387-->trp, tyr387-->his, tyr387-->asn and tyr387-->ser) revealed that an aromatic ring system is crucial for the synthetic activity of t ... | 2000 | 11024170 |
| connection between poly-beta-hydroxybutyrate biosynthesis and growth on c(1) and c(2) compounds in the methylotroph methylobacterium extorquens am1. | several dna regions containing genes involved in poly-beta-hydroxybutyrate (phb) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph methylobacterium extorquens am1. genes involved in phb biosynthesis include those encoding beta-ketothiolase (phaa), nadph-linked acetoacetyl coenzyme a (acetyl-coa) reductase (phab), and phb synthase (phac). phaa and phab are closely link ... | 2001 | 11208803 |
| human papillomavirus dna in plasma of patients with cervical cancer. | human papillomavirus (hpv) is a crucial etiological factor for cervical cancer (cc) development. from a diagnostic view-point, the consistent presence of hpv in cc allows the viral dna to be used as a genetic marker. the aims of this study were to evaluate the presence, physical status and clinical significant of hpv dna in circulation of cc patients. | 2001 | 11244579 |
| the dnab.dnac complex: a structure based on dimers assembled around an occluded channel. | replicative helicases are motor proteins that unwind dna at replication forks. escherichia coli dnab is the best characterized member of this family of enzymes. we present the 26 a resolution three-dimensional structure of the dnab hexamer in complex with its loading partner, dnac, obtained from cryo-electron microscopy. analysis of the volume brings insight into the elaborate way the two proteins interact, and provides a structural basis for control of the symmetry state and inactivation of the ... | 2001 | 11250911 |
| identification of new genes involved in the virulence of listeria monocytogenes by signature-tagged transposon mutagenesis. | listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. we have adapted signature-tagged transposon mutagenesis to l. monocytogenes to identify new genes involved in virulence in the murine model of infection. we used transposon tn1545 carried on the integrative vector pat113. forty-eight tagged transposons were constructed and used to generate banks of l. monocytogenes mutants. pools of 48 mutants were assem ... | 2001 | 11254558 |
| stabilizing interactions in the dimer interface of alpha-subunit in escherichia coli rna polymerase: a graph spectral and point mutation study. | the formation of alpha(2) dimer in escherichia coli core rna polymerase (rnap) is thought to be the first step toward the assembly of the functional enzyme. a large number of evidences indicate that the alpha-subunit dimerizes through its n-terminal domain (ntd). the crystal structures of the alpha-subunit ntd and that of a homologous thermus aquaticus core rnap are known. to identify the stabilizing interactions in the dimer interface of the alpha-ntd of e. coli rnap, we identified side-chain c ... | 2001 | 11266593 |
| hydrogen peroxide-forming nadh oxidase belonging to the peroxiredoxin oxidoreductase family: existence and physiological role in bacteria. | amphibacillus xylanus and sporolactobacillus inulinus nadh oxidases belonging to the peroxiredoxin oxidoreductase family show extremely high peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of the small disulfide redox protein, ahpc (peroxiredoxin). in order to investigate the distribution of this enzyme system in bacteria, 15 bacterial strains were selected from typical aerobic, facultatively anaerobic, and anaerobic bacteria. ahpc-linked alkyl hydroper ... | 2001 | 11274101 |
| regulation of the acetoin catabolic pathway is controlled by sigma l in bacillus subtilis. | bacillus subtilis grown in media containing amino acids or glucose secretes acetate, pyruvate, and large quantities of acetoin into the growth medium. acetoin can be reused by the bacteria during stationary phase when other carbon sources have been depleted. the acoabcl operon encodes the e1alpha, e1beta, e2, and e3 subunits of the acetoin dehydrogenase complex in b. subtilis. expression of this operon is induced by acetoin and repressed by glucose in the growth medium. the acor gene is located ... | 2001 | 11274109 |
| comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases. | the actions of cyclomaltodextrin glucanotransferases (cgtase; ec 2.4.1.19) from alkalophilic bacillus sp. strain a2-5a (a2-5a cgtase), bacillus macerans (bmac cgtase), and bacillus stearothermophilus (bste cgtase) on amylose were investigated. all three enzymes produced large cyclic alpha-1,4-glucans (cycloamyloses) at the early stage of the reaction, but these were subsequently converted into smaller cycloamyloses. however, the rates of this conversion differed among the three enzymes. the prod ... | 2001 | 11282590 |
| method for host-independent detection of generalized transducing bacteriophages in natural habitats. | despite an increasing interest in horizontal gene transfer in bacteria, the role of generalized transduction in this process has not been well investigated yet. certainly one of the reasons is that only a small fraction of general transducing bacteriophages have been characterized, because many bacterial hosts needed for propagation and identification are not culturable or are simply unknown. a method for host-independent detection of transducing bacteriophages was developed. phage-encapsulated ... | 2001 | 11282595 |
| self-reporting pna/dna primers for pcr analysis. | we report a new fluorogenic method for sealed-tube pcr analysis using a quencher-labeled peptide nucleic acid (q-pna) probe. the q-pna hybridizes to a complementary tag sequence located at the 5' end of a 5' fluorophore-labeled oligonucleotide primer, quenching the primer's fluorescence. incorporation of the primer into a doublestranded amplicon causes displacement of the q-pna such that the fluorescence of the sample is a direct indication of the amplicon concentration. the q-pna is able to que ... | 2001 | 11282976 |
| identification of yace (coae) as the structural gene for dephosphocoenzyme a kinase in escherichia coli k-12. | dephosphocoenzyme a (dephospho-coa) kinase catalyzes the final step in coenzyme a biosynthesis, the phosphorylation of the 3'-hydroxy group of the ribose sugar moiety. wild-type dephospho-coa kinase from corynebacterium ammoniagenes was purified to homogeneity and subjected to n-terminal sequence analysis. a blast search identified a gene from escherichia coli previously designated yace encoding a highly homologous protein. amplification of the gene and overexpression yielded recombinant dephosp ... | 2001 | 11292795 |
| the pyrimidine operon pyrrpb-cara from lactococcus lactis. | the four genes pyrr, pyrp, pyrb, and cara were found to constitute an operon in lactococcus lactis subsp. lactis mg1363. the functions of the different genes were established by mutational analysis. the first gene in the operon is the pyrimidine regulatory gene, pyrr, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to ump formation. the second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous ... | 2001 | 11292797 |
| a tandem repeats database for bacterial genomes: application to the genotyping of yersinia pestis and bacillus anthracis. | some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. in the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. the rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. the availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies. | 2001 | 11299044 |
| il-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression. | dendritic cell (dc) differentiation from human cd34(+) hematopoietic progenitor cells (hpcs) can be triggered in vitro by a combination of cytokines consisting of stem cell factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. the immune response regulatory cytokines, il-4 and il-13, promote dc maturation from hpcs, induce monocyte-dc transdifferentiation, and selectively up-regulate 15-lipoxygenase 1 (15-lo-1) in blood monocytes. to gain more insight into cy ... | 2001 | 11320251 |
| replication slippage involves dna polymerase pausing and dissociation. | genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. the slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. we analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded dna template that mimics the lagging strand synthesis. we show that slippage involv ... | 2001 | 11350948 |
| crystal structures of a ddatp-, ddttp-, ddctp, and ddgtp- trapped ternary complex of klentaq1: insights into nucleotide incorporation and selectivity. | the mechanism by which dna polymerase i enzymes function has been the subject of extensive biochemical and structural studies. we previously determined the structure of a ternary complex of the large fragment of dna polymerase i from thermus aquaticus (klentaq1) bound to a primer/template dna and a dideoxycytidine 5'-triphosphate (ddctp). in this report, we present the details of the 2.3-a resolution crystal structures of three additional ternary complexes of klentaq1 bound to a primer/template ... | 2001 | 11369861 |
| molecular detection of antimicrobial resistance. | the determination of antimicrobial susceptibility of a clinical isolate, especially with increasing resistance, is often crucial for the optimal antimicrobial therapy of infected patients. nucleic acid-based assays for the detection of resistance may offer advantages over phenotypic assays. examples are the detection of the methicillin resistance-encoding meca gene in staphylococci, rifampin resistance in mycobacterium tuberculosis, and the spread of resistance determinants across the globe. how ... | 2001 | 11585788 |
| partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (slec) precursor to an active enzyme during germination of clostridium perfringens s40 spores and analysis of a gene cluster involved in the activity. | a spore cortex-lytic enzyme of clostridium perfringens s40 which is encoded by slec is synthesized at an early stage of sporulation as a precursor consisting of four domains. after cleavage of an n-terminal presequence and a c-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an n-terminal prosequence with germination-specific protease (gsp) during germination. the present study was undertaken to characterize gsp. in the presence of 3 ... | 2001 | 11371539 |
| identification of the partitioning site within the repabc-type replicon of the composite paracoccus versutus plasmid ptav1. | the replicator region of composite plasmid ptav1 of paracoccus versutus (included in mini-replicon ptav320) belongs to the family of repabc replicons commonly found in plasmids harbored by agrobacterium and rhizobium spp. the repabc replicons encode three genes clustered in an operon, which are involved in partitioning (repa and repb) and replication (repc). in order to localize the partitioning site of ptav320, the two identified incompatibility determinants of this mini-replicon (inc1, located ... | 2001 | 11591666 |
| purification and characterization of a psychrophilic, calcium-induced, growth-phase-dependent metalloprotease from the fish pathogen flavobacterium psychrophilum. | flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. this bacterium produced an extracellular protease with an estimated molecular mass of 55 kda. this enzyme, designated fpp1 (f. psychrophilum protease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. on the basis of its biochemical characteristics, fpp1 can b ... | 2001 | 11375148 |
| differential effects of replacing escherichia coli ribosomal protein l27 with its homologue from aquifex aeolicus. | the rpma gene, which encodes 50s ribosomal subunit protein l27, was cloned from the extreme thermophile aquifex aeolicus, and the protein was overexpressed and purified. comparison of the a. aeolicus protein with its homologue from escherichia coli by circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas the e. coli protein is unstructured under the same conditions. a mutant of e. coli ... | 2001 | 11673426 |
| application of the 5' fluorogenic exonuclease assay (taqman) for quantitative ribosomal dna and rrna analysis in sediments. | in this study, we report on the development of quantitative pcr and reverse transcriptase pcr assays for the 16s rrna of geobacter spp. and identify key issues related to fluorogenic reporter systems for nucleic acid analyses of sediments. the lower detection limit of each assay was 5 to 50 fg of genomic dna or < or =2 pg of 16s rrna. taqman pcr spectral traces from uncontaminated, amended aquifer sediments were significantly lower (p < 0.0002) than traces for the external standard curve. we als ... | 2001 | 11375195 |
| quantitation of viral dna by real-time pcr applying duplex amplification, internal standardization, and two-color fluorescence detection. | a real-time pcr method was developed to quantitate viral dna that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. applied to human parvovirus b19 dna, the linear range was from 10(2) to at least 5 x 10(6) copies per ml of sample. the coefficient of variation was 0.29 using a run control of 2,876 copies per ml. the method reduces the risk of false-negative results, yields high precision, ... | 2001 | 11375203 |
| detection and quantification of methyl tert-butyl ether-degrading strain pm1 by real-time taqman pcr. | the fuel oxygenate methyl tert-butyl ether (mtbe), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. the bacterial strain pm1 rapidly mineralizes and grows on mtbe in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. we applied the taqman quantitative pcr method to detect and quantify strain pm1 in laboratory and field samples. specific primers and probes were designed for the 16s ribosoma ... | 2001 | 11679339 |
| spontaneous erythromycin resistance mutation in a 23s rrna gene, rrla, of the extreme thermophile thermus thermophilus ib-21. | spontaneous, erythromycin-resistant mutants of thermus thermophilus ib-21 were isolated and found to carry the mutation a2058g in one of two 23s rrna operons. the heterozygosity of these mutants indicates that a2058g confers a dominant or codominant phenotype in this organism. this mutation provides a valuable tool for the genetic manipulation of the 23s rrna genes of thermus. | 2001 | 11418580 |
| novel cell type-specific antiviral mechanism of interferon gamma action in macrophages. | interferon (ifn)-gamma and macrophages (mphi) play key roles in acute, persistent, and latent murine cytomegalovirus (mcmv) infection. ifn-gamma mechanisms were compared in embryonic fibroblasts (mefs) and bone marrow mphi (bmmphi). ifn-gamma inhibited mcmv replication in a signal transducer and activator of transcription (stat)-1alpha-dependent manner much more effectively in bmmphi (approximately 100-fold) than mef (5-10-fold). although initial stat-1alpha activation by ifn-gamma was equivalen ... | 2001 | 11181700 |
| subunit interactions and glutamine utilization by escherichia coli imidazole glycerol phosphate synthase. | a selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. the protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. the heterodimeric escherichia coli imidazole glycerol phosphate (igp) synthase was probed to assess if residues in ... | 2001 | 11208798 |
| development and evaluation of a fluorogenic 5' nuclease assay to detect and differentiate between ebola virus subtypes zaire and sudan. | the ability to rapidly recognize ebola virus infections is critical to quickly limit further spread of the disease. a rapid, sensitive, and specific laboratory diagnostic test is needed to confirm outbreaks of ebola virus infection and to distinguish it from other diseases that can cause similar clinical symptoms. a one-tube reverse transcription-pcr assay for the identification of ebola virus subtype zaire (ebola zaire) and ebola virus subtype sudan (ebola sudan) was developed and evaluated by ... | 2001 | 11682540 |
| respiration capacity of the fermenting bacterium lactococcus lactis and its positive effects on growth and survival. | oxygen is a major determinant of both survival and mortality of aerobic organisms. for the facultative anaerobe lactococcus lactis, oxygen has negative effects on both growth and survival. we show here that oxygen can be beneficial to l. lactis if heme is present during aerated growth. the growth period is extended and long-term survival is markedly improved compared to results obtained under the usual fermentation conditions. we considered that improved growth and survival could be due to the c ... | 2001 | 11443085 |
| analysis of mutations at residues a2451 and g2447 of 23s rrna in the peptidyltransferase active site of the 50s ribosomal subunit. | on the basis of the recent atomic-resolution x-ray structure of the 50s ribosomal subunit, residues a2451 and g2447 of 23s rrna were proposed to participate directly in ribosome-catalyzed peptide bond formation. we have examined the peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. in escherichia coli, pure mutant ribosome populations carrying either the g2447a or g2447c mutations maintained cell viability. in vitro, the g2447a ribosomes s ... | 2001 | 11470897 |
| sequence analysis and molecular characterization of the lactococcus lactis temperate bacteriophage bk5-t. | the lactococcus lactis temperate bacteriophage bk5-t is one of twelve type phages that define l. lactis phage species. this paper describes the nucleotide sequence and analysis of a 21-kbp region of the bk5-t genome and completes the nucleotide sequence of the genome of this phage. the 40,003-nucleotide linear genome encodes 63 open reading frames. sequence runoff experiments showed that the cohesive ends of the bk5-t genome contained a 12-bp 3' single-stranded overhang with the sequence 5'-caca ... | 2001 | 11472933 |
| detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative pcr and sybr green chemistry. | a rapid and highly sensitive real-time pcr detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (ihhnv), a single-stranded dna virus, and white spot virus (wsv), a double-stranded dna (dsdna) virus infecting penaeid shrimp (penaeus sp.), was developed using the geneamp 5700 sequence detection system coupled with sybr green chemistry. the pcr mixture contains a fluorescence dye, sybr green, which upon binding to dsdna exhibits fluorescence enhancement. th ... | 2001 | 11474000 |