Publications

the role of rna polymerase sigma subunit in promoter-independent initiation of transcription.	in bacteria, initiation of transcription depends on the rna polymerase sigma subunit, which brings catalytically proficient rna polymerase core to promoters by binding to specific dna elements located upstream of the transcription start point. here, we study sigma-dependent synthesis of a transcript that is used to prime replication of the single-stranded genome of bacteriophage m13. we show that, in this system, sigma plays no role in dna recognition, which is accomplished solely through rna po ...	2004	15070729
identification of the xpg region that causes the onset of cockayne syndrome by using xpg mutant mice generated by the cdna-mediated knock-in method.	in addition to xeroderma pigmentosum (xp), mutations in the human xpg gene cause early onset of cockayne syndrome (cs) in some patients (xpg/cs). the cs-causing mutations in such patients all produce truncated xpg proteins. to test the hypothesis that the cs phenotype, with characteristics such as growth retardation and a short life span in xpg/cs patients, results from c-terminal truncations, we constructed mutants with c-terminal truncations in mouse xpg (xpg) (from residue d811 to the stop co ...	2004	15082767
functional interaction between tfiib and the rpb2 subunit of rna polymerase ii: implications for the mechanism of transcription initiation.	the general transcription factor tfiib is required for accurate initiation, although the mechanism by which rna polymerase ii (rnap ii) identifies initiation sites is not well understood. here we describe results from genetic and biochemical analyses of an altered form of yeast tfiib containing an arginine-78 --> cysteine (r78c) replacement in the "b-finger" domain. tfiib r78c shifts start site selection downstream of normal and confers a cold-sensitive growth defect (csm(-)). suppression of the ...	2004	15082791
structure and mechanism of the rna polymerase ii transcription machinery.	advances in structure determination of the bacterial and eukaryotic transcription machinery have led to a marked increase in the understanding of the mechanism of transcription. models for the specific assembly of the rna polymerase ii transcription machinery at a promoter, conformational changes that occur during initiation of transcription, and the mechanism of initiation are discussed in light of recent developments.	2004	15114340
poliovirus rna-dependent rna polymerase (3dpol): kinetic, thermodynamic, and structural analysis of ribonucleotide selection.	we have performed a kinetic and thermodynamic analysis of 3d(pol) derivatives containing substitutions in the ribose-binding pocket with atp analogues containing correct and incorrect sugar configurations. we find that asp-238, a residue in structural motif a that is conserved in all rna-dependent rna polymerases, is a key determinant of polymerase fidelity. alterations in the position of the asp-238 side chain destabilize the catalytically competent 3d(pol)-primer/template-ntp complex and reduc ...	2004	15122880
distinctive protein signatures provide molecular markers and evidence for the monophyletic nature of the deinococcus-thermus phylum.	the deinococcus-thermus group of species is currently recognized as a distinct phylum solely on the basis of their branching in 16s rrna trees. no unique biochemical or molecular characteristics that can distinguish this group from all other bacteria are known at present. in this work, we describe eight conserved indels (viz., inserts or deletions) in seven widely distributed proteins that are distinctive characteristics of the deinococcus-thermus phylum but are not found in any other group of b ...	2004	15126471
a new method for detection of pfmdr1 mutations in plasmodium falciparum dna using real-time pcr.	surveillance for drug-resistant plasmodium falciparum should be a component of malaria control programmes. real-time pcr methods for the detection of parasite single-nucleotide polymorphisms (snps) and gene amplification could be useful survellance tools.	2004	15132750
predicting transmembrane beta-barrels in proteomes.	very few methods address the problem of predicting beta-barrel membrane proteins directly from sequence. one reason is that only very few high-resolution structures for transmembrane beta-barrel (tmb) proteins have been determined thus far. here we introduced the design, statistics and results of a novel profile-based hidden markov model for the prediction and discrimination of tmbs. the method carefully attempts to avoid over-fitting the sparse experimental data. while our model training and sc ...	2004	15141026
topology of the substrate-binding site of a lys49-phospholipase a2 influences ca2+-independent membrane-damaging activity.	bthtx-i (bothropstoxin-i) is a myotoxic lys49-pla2 (phospholipase a2 with lys49) isolated from bothrops jararacussu venom, which damages liposome membranes by a ca2+-independent mechanism. the highly conserved phe5/ala102/phe106 motif in the hydrophobic substrate-binding site of the asp49-pla2s is substituted by leu5/val102/leu106 in the lys49-pla2s. the leu5/val102/leu106 triad in bthtx-i was sequentially mutated via all single- and double-mutant combinations to the phe5/ala102/phe106 mutant. a ...	2004	15147240
crystal structure of the deinococcus radiodurans single-stranded dna-binding protein suggests a mechanism for coping with dna damage.	single-stranded dna (ssdna)-binding (ssb) proteins are uniformly required to bind and protect single-stranded intermediates in dna metabolic pathways. all bacterial and eukaryotic ssb proteins studied to date oligomerize to assemble four copies of a conserved domain, called an oligonucleotide/oligosaccharide-binding (ob) fold, that cooperate in nonspecific ssdna binding. the vast majority of bacterial ssb family members function as homotetramers, with each monomer contributing a single ob fold. ...	2004	15159541
cloning, sequencing, and characterization of a heat- and alkali-stable type i pullulanase from anaerobranca gottschalkii.	the gene encoding a type i pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium anaerobranca gottschalkii. in addition, the homologous gene was isolated from a gene library of anaerobranca horikoshii and sequenced. the proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria fervidobacterium pennivorans and thermotoga maritima. the pullulanase gene from a. gottschalkii (en ...	2004	15184138
protein tolerance to random amino acid change.	mutagenesis of protein-encoding sequences occurs ubiquitously; it enables evolution, accumulates during aging, and is associated with disease. many biotechnological methods exploit random mutations to evolve novel proteins. to quantitate protein tolerance to random change, it is vital to understand the probability that a random amino acid replacement will lead to a protein's functional inactivation. we define this probability as the "x factor." here, we develop a broadly applicable approach to c ...	2004	15197260
efficient and error-free replication past a minor-groove dna adduct by the sequential action of human dna polymerases iota and kappa.	dna polymerase iota (poliota) is a member of the y family of dna polymerases, which promote replication through dna lesions. the role of poliota in lesion bypass, however, has remained unclear. poliota is highly unusual in that it incorporates nucleotides opposite different template bases with very different efficiencies and fidelities. since interactions of dna polymerases with the dna minor groove provide for the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposit ...	2004	15199127
antibacterial peptide microcin j25 inhibits transcription by binding within and obstructing the rna polymerase secondary channel.	the antibacterial peptide microcin j25 (mccj25) inhibits transcription by bacterial rna polymerase (rnap). biochemical results indicate that inhibition of transcription occurs at the level of ntp uptake or ntp binding by rnap. genetic results indicate that inhibition of transcription requires an extensive determinant, comprising more than 50 amino acid residues, within the rnap secondary channel (also known as the "ntp-uptake channel" or "pore"). biophysical results indicate that inhibition of t ...	2004	15200952
evidence for transcriptional and posttranscriptional alterations of the sodium/iodide symporter expression in hypofunctioning benign and malignant thyroid tumors.	the uptake of iodide by epithelial thyroid cells requires the expression of a specific transporter, the na(+)/i(-) symporter, nis. benign and malignant thyroid tumors of epithelial origin show a decrease up to a loss of iodide uptake activity. previous studies of the human nis (hnis) gene expression in these tumors, based on the amplification of transcripts and/or immunohistochemical detection of the protein, have yielded divergent data; hnis expression was found either increased or decreased. t ...	2004	15215159
bacillus subtilis ydih is a direct negative regulator of the cydabcd operon.	during aerobic respiration, bacillus subtilis utilizes three terminal oxidases, cytochromes aa3, caa3, and bd. cytochrome bd is encoded by the cydabcd operon. we report here the first identification of a regulator for the cydabcd operon, ydih. while working with deltaresde mutant strains, we identified colonies which contained suppressor mutations (cmp) which bypassed the requirement for resd for all phenotypes not associated with cytochrome aa3 or caa3. mapping identified a class of tn10 insert ...	2004	15231791
expression and functional activity of ppargamma in pancreatic beta cells.	rosiglitazone is an agonist of peroxisome proliferator activated receptor-gamma (ppargamma) and ameliorates insulin resistance in type ii diabetes. in addition, it may also promote increased pancreatic beta-cell viability, although it is not known whether this effect is mediated by a direct action on the beta cell. we have investigated this possibility. semiquantitative real-time reverse transcription-polymerase chain reaction analysis (taqman) revealed that freshly isolated rat islets and the c ...	2004	15237101
reverse gyrase has heat-protective dna chaperone activity independent of supercoiling.	hyperthermophilic organisms must protect their constituent macromolecules from heat-induced degradation. a general mechanism for thermoprotection of dna in active cells is unknown. we show that reverse gyrase, the only protein that is both specific and common to all hyperthermophiles, reduces the rate of double-stranded dna breakage approximately 8-fold at 90 degrees c. this activity does not require atp hydrolysis and is independent of the positive supercoiling activity of the enzyme. reverse g ...	2004	15247343
the strong efficiency of the escherichia coli gapa p1 promoter depends on a complex combination of functional determinants.	the escherichia coli multi-promoter region of the gapa gene ensures a high level of gapdh (glyceraldehyde-3-phosphate dehydrogenase) production under various growth conditions. in the exponential phase of growth, gapa mrnas are mainly initiated at the highly efficient gapa p1 promoter. in the present study, by using site-directed mutagenesis and chemical probing of the rpo (open complex) formed by esigma70 (holoenzyme associated with sigma70) rnap (rna polymerase) at promoter gapa p1, we show th ...	2004	15250823
substrate specificity of helicobacter pylori histone-like hu protein is determined by insufficient stabilization of dna flexure points.	the histone-like hu protein is ubiquitous in the eubacteria. a role for escherichia coli hu in compaction of the bacterial genome has been reported, along with regulatory roles in dna replication, transposition, repair and transcription. we show here that hu from the human pathogen helicobacter pylori, which has been implicated in the development of ulcers and gastric cancer, exhibits enhanced thermal stability and distinct dna substrate specificity. thermal denaturation of hpyhu (h. pylori hu) ...	2004	15255779
construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag.	the thermophilic inorganic pyrophosphatase (pyr) from thermus thermophilus has been produced in escherichia coli fused to the c terminus of the choline-binding tag (chb tag) derived from the choline-binding domain (chbd) of pneumococcal lyta autolysin. the chimeric chbd-pyr protein retains its thermostable activity and can be purified in a single step by deae-cellulose affinity chromatography. pyr can be further released from the chbd by thrombin, using the specific protease recognition site inc ...	2004	15294797
use of 16s ribosomal dna pcr and denaturing gradient gel electrophoresis for analysis of the microfloras of healing and nonhealing chronic venous leg ulcers.	the bacterial microfloras of 8 healing and 10 nonhealing chronic venous leg ulcers were compared by using a combination of cultural analysis and denaturing gradient gel electrophoresis (dgge) of pcr-amplified 16s rrna gene products. cultural analysis of the microflora revealed that the majority of both wound types carried the aerobes staphylococcus and pseudomonas spp. (89 and 80%, respectively). sequencing of 16s ribosomal dnas selected on the basis of dgge profiling allowed the identification ...	2004	15297496
amino acid contacts between sigma 70 domain 4 and the transcription activators rhas and rhar.	the rhas and rhar proteins are transcription activators that respond to the availability of l-rhamnose and activate transcription of the operons in the escherichia coli l-rhamnose catabolic regulon. rhar activates transcription of rhasr, and rhas activates transcription of the operon that encodes the l-rhamnose catabolic enzymes, rhabad, as well as the operon that encodes the l-rhamnose transport protein, rhat. rhas is 30% identical to rhar at the amino acid level, and both are members of the ar ...	2004	15342598
dependence of dna polymerase replication rate on external forces: a model based on molecular dynamics simulations.	molecular dynamics simulations are presented for a thermus aquaticus (taq) dna polymerase i complex (consisting of the protein, the primer-template dna strands, and the incoming nucleotide) subjected to external forces. the results obtained with a force applied to the dna template strand provide insights into the effect of the tension on the activity of the enzyme. at forces below 30 pn a local model based on the parameters determined from the simulations, including the restricted motion of the ...	2004	15345530
reverse transcriptase activity innate to dna polymerase i and dna topoisomerase i proteins of streptomyces telomere complex.	replication of streptomyces linear chromosomes and plasmids proceeds bidirectionally from a central origin, leaving recessed 5' termini that are extended by a telomere binding complex. this complex contains both a telomere-protecting terminal protein (tpg) and a telomere-associated protein that interacts with tpg and the dna ends of linear streptomyces replicons. by using histidine-tagged telomere-associated protein (tap) as a scaffold, we identified dna polymerase (pola) and topoisomerase i (to ...	2004	15353591
mechanism of association and reciprocal activation of two gtpases.	the signal recognition particle (srp) mediates the cotranslational targeting of nascent proteins to the eukaryotic endoplasmic reticulum membrane or the bacterial plasma membrane. during this process, two gtpases, one in srp and one in the srp receptor (named ffh and ftsy in bacteria, respectively), form a complex in which both proteins reciprocally activate the gtpase reaction of one another. here, we explore by site-directed mutagenesis the role of 45 conserved surface residues in the ffh-ftsy ...	2004	15383838
reverse transcriptase at bacterial telomeres.		2004	15454610
the dna primase of sulfolobus solfataricus is activated by substrates containing a thymine-rich bubble and has a 3'-terminal nucleotidyl-transferase activity.	dna primases are responsible for the synthesis of the short rna primers that are used by the replicative dna polymerases to initiate dna synthesis on the leading- and lagging-strand at the replication fork. in this study, we report the purification and biochemical characterization of a dna primase (sso dna primase) from the thermoacidophilic crenarchaeon sulfolobus solfataricus. the sso dna primase is a heterodimer composed of two subunits of 36 kda (small subunit) and 38 kda (large subunit), wh ...	2004	15459292
regulated communication between the upstream face of rna polymerase and the beta' subunit jaw domain.	we used bacteriophage t7-encoded transcription inhibitor gene protein 2 (gp2) as a probe to study the contribution of the escherichia coli rna polymerase (rnap) beta' subunit jaw domain--the site of gp2 binding--to activator and atp hydrolysis-dependent open complex formation by the sigma(54)-rnap. we show that, unlike sigma(70)-dependent transcription, activated transcription by sigma(54)-rnap is resistant to gp2. in contrast, activator and atp hydrolysis-independent transcription by sigma(54)- ...	2004	15470503
reorganisation of an rna polymerase-promoter dna complex for dna melting.	sigma factors, the key regulatory components of the bacterial rna polymerase (rnap), direct promoter dna binding and dna melting. the sigma(54)-rnap forms promoter complexes in which dna melting is only triggered by an activator and atp hydrolysis-driven reorganisation of an initial sigma(54)-rnap-promoter complex. we report that an initial bacterial rnap-dna complex can be reorganised by an activator to form an intermediate transcription initiation complex where full dna melting has not yet occ ...	2004	15470504
asymmetric atp binding and hydrolysis activity of the thermus aquaticus muts dimer is key to modulation of its interactions with mismatched dna.	prokaryotic muts and eukaryotic msh proteins recognize base pair mismatches and insertions or deletions in dna and initiate mismatch repair. these proteins function as dimers (and perhaps higher order oligomers) and possess an atpase activity that is essential for dna repair. previous studies of escherichia coli muts and eukaryotic msh2-msh6 proteins have revealed asymmetry within the dimer with respect to both dna binding and atpase activities. we have found the thermus aquaticus muts protein a ...	2004	15476405
the a2453-c2499 wobble base pair in escherichia coli 23s ribosomal rna is responsible for ph sensitivity of the peptidyltransferase active site conformation.	peptide bond formation, catalyzed by the ribosomal peptidyltransferase, has long been known to be sensitive to monovalent cation concentrations and ph. more recently, we and others have shown that residue a2451 in the peptidyltransferase center of the escherichia coli 50s ribosomal subunit changes conformation in response to alterations in ph, depending on ionic conditions and temperature. two wobble pairs, a2453-c2499 and a2450-c2063, have been proposed as potential candidates to convey ph-depe ...	2004	15479786
distinct roles for the saccharomyces cerevisiae mismatch repair proteins in heteroduplex rejection, mismatch repair and nonhomologous tail removal.	the saccharomyces cerevisiae mismatch repair (mmr) protein msh6 and the sgs1 helicase were recently shown to play similarly important roles in preventing recombination between divergent dna sequences in a single-strand annealing (ssa) assay. in contrast, mmr factors such as mlh1p, pms1p, and exo1p were shown to not be required or to play only minimal roles. in this study we tested mutations that disrupt sgs1p helicase activity, msh2p-msh6p mismatch recognition, and atp binding and hydrolysis act ...	2005	15489516
high-efficiency bypass of dna damage by human dna polymerase q.	endogenous dna damage arises frequently, particularly apurinic (ap) sites. these must be dealt with by cells in order to avoid genotoxic effects. dna polymerase theta; is a newly identified enzyme encoded by the human polq gene. we find that polq has an exceptional ability to bypass an ap site, inserting a with 22% of the efficiency of a normal template, and continuing extension as avidly as with a normally paired base. polq preferentially incorporates a opposite an ap site and strongly disfavor ...	2004	15496986
measurement of relative copy number of cdkn2a/arf and cdkn2b in bladder cancer by real-time quantitative pcr and multiplex ligation-dependent probe amplification.	many tumors have large homozygous deletions of the cdkn2a locus (encoding p14(arf) and p16) and of cdkn2b (p15). our aim was to determine which gene is the major target in bladder cancer. we used quantitative real-time pcr (rtq-pcr) to determine copy number of p15, of p14(arf) exon 1beta, and p16 exon 2 in 22 tumor cell lines and 83 bladder tumors, some of which had been assessed previously by duplex pcr. titration experiments showed that homozygous deletion could be detected in the presence of ...	2004	15507675
cold sensitivity of thermophilic and mesophilic rna polymerases.	rna polymerase from mesophilic deinococcus radiodurans displays the same cold sensitivity of promoter opening as rna polymerase from the closely related thermophilic thermus aquaticus. this suggests that, contrary to the accepted view, cold sensitivity of promoter opening by thermophilic rna polymerases may not be a consequence of their thermostability.	2004	15516599
crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with t7 dna polymerase reveal mechanisms of mutagenesis.	the carcinogen 2-acetylaminofluorene forms two major dna adducts: n-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dg-aaf) and its deacetylated derivative, n-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dg-af). although the dg-aaf and dg-af adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on dna replication. dg-aaf poses a strong block to dna synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of ...	2004	15528277
distance-restrained docking of rifampicin and rifamycin sv to rna polymerase using systematic fret measurements: developing benchmarks of model quality and reliability.	we are developing distance-restrained docking strategies for modeling macromolecular complexes that combine available high-resolution structures of the components and intercomponent distance restraints derived from systematic fluorescence resonance energy transfer (fret) measurements. in this article, we consider the problem of docking small-molecule ligands within macromolecular complexes. using simulated fret data, we have generated a series of benchmarks that permit estimation of model accura ...	2004	15542547
distance-restrained docking of rifampicin and rifamycin sv to rna polymerase using systematic fret measurements: developing benchmarks of model quality and reliability.	we are developing distance-restrained docking strategies for modeling macromolecular complexes that combine available high-resolution structures of the components and intercomponent distance restraints derived from systematic fluorescence resonance energy transfer (fret) measurements. in this article, we consider the problem of docking small-molecule ligands within macromolecular complexes. using simulated fret data, we have generated a series of benchmarks that permit estimation of model accura ...	2004	15542547
unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry.	among the methods used to unravel protein interaction surfaces, chemical cross-linking followed by identification of the cross-linked peptides by mass spectrometry has proven especially useful in dynamic and complex systems. during the signal recognition particle (srp)-dependent targeting of proteins to the bacterial plasma membrane, the specific interaction between ffh (the protein component of srp) and ftsy (the srp receptor) is known to be essential for the efficiency and fidelity of this pro ...	2004	15546976
nucleotide specificity of hiv-1 reverse transcriptases with amino acid substitutions affecting ala-114.	ala-114, together with asp-113, tyr-115 and gln-151, form the pocket that accommodates the 3'-oh of the incoming dntp in the hiv-1 rt (reverse transcriptase). four mutant rts having serine, glycine, threonine or valine instead of ala-114 were obtained by site-directed mutagenesis. while mutants a114s and a114g retained significant dna polymerase activity, a114t and a114v showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent k(m) values for dntp. disc ...	2005	15548134
protein-mediated error correction for de novo dna synthesis.	the availability of inexpensive, on demand synthetic dna has enabled numerous powerful applications in biotechnology, in turn driving considerable present interest in the de novo synthesis of increasingly longer dna constructs. the synthesis of dna from oligonucleotides into products even as large as small viral genomes has been accomplished. despite such achievements, the costs and time required to generate such long constructs has, to date, precluded gene-length (and longer) dna synthesis from ...	2004	15561997
genetic bases of the rifampin resistance phenotype in brucella spp.	rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens. its bactericidal activity is due to its ability to bind to the beta subunit of the dna-dependent rna polymerase encoded by the rpob gene. mutations of the rpob gene have been characterized in rifampin-resistant (rif(r)) strains of escherichia coli and mycobacterium tuberculosis. the genetic bases of rif(r) in brucella spp. are still unknown. in the present study, the nucleotide sequences of the rpob ge ...	2004	15583262
low-fidelity pyrococcus furiosus dna polymerase mutants useful in error-prone pcr.	random mutagenesis constitutes an important approach for identifying critical regions of proteins, studying structure-function relations and developing novel proteins with desired properties. perhaps, the most popular method is the error-prone pcr, in which mistakes are introduced into a gene, and hence a protein, during dna polymerase-catalysed amplification cycles. unfortunately, the relatively high fidelities of the thermostable dna polymerases commonly used for pcr result in too few mistakes ...	2004	15601989
selenocysteine trna-specific elongation factor selb is a structural chimaera of elongation and initiation factors.	in all three kingdoms of life, selb is a specialized translation elongation factor responsible for the cotranslational incorporation of selenocysteine into proteins by recoding of a uga stop codon in the presence of a downstream mrna hairpin loop. here, we present the x-ray structures of selb from the archaeon methanococcus maripaludis in the apo-, gdp- and gppnhp-bound form and use mutational analysis to investigate the role of individual amino acids in its aminoacyl-binding pocket. all three s ...	2004	15616587
selenocysteine trna-specific elongation factor selb is a structural chimaera of elongation and initiation factors.	in all three kingdoms of life, selb is a specialized translation elongation factor responsible for the cotranslational incorporation of selenocysteine into proteins by recoding of a uga stop codon in the presence of a downstream mrna hairpin loop. here, we present the x-ray structures of selb from the archaeon methanococcus maripaludis in the apo-, gdp- and gppnhp-bound form and use mutational analysis to investigate the role of individual amino acids in its aminoacyl-binding pocket. all three s ...	2004	15616587
the effects of upstream dna on open complex formation by escherichia coli rna polymerase.	binding of activators to upstream dna sequences regulates transcription initiation by affecting the stability of the initial rna polymerase (rnap)-promoter complex and/or the rate of subsequent conformational changes required to form the open complex (rp(o)). here we observe that the presence of nonspecific upstream dna profoundly affects an early step in formation of the transcription bubble. kinetic studies with the lambdap(r) promoter and escherichia coli rnap reveal that the presence of dna ...	2004	15626761
the effects of upstream dna on open complex formation by escherichia coli rna polymerase.	binding of activators to upstream dna sequences regulates transcription initiation by affecting the stability of the initial rna polymerase (rnap)-promoter complex and/or the rate of subsequent conformational changes required to form the open complex (rp(o)). here we observe that the presence of nonspecific upstream dna profoundly affects an early step in formation of the transcription bubble. kinetic studies with the lambdap(r) promoter and escherichia coli rnap reveal that the presence of dna ...	2004	15626761
presence of beta-lactamase gene tem-1 dna sequence in commercial taq dna polymerase.		2005	15635039
non-cross-linking gold nanoparticle aggregation as a detection method for single-base substitutions.	aggregation of dna-modified gold nanoparticles in a non-cross-linking configuration has extraordinary selectivity against terminal mismatch of the surface-bound duplex. in this paper, we demonstrate the utility of this selectivity for detection of single-base substitutions. the samples were prepared through standard protocols: dna extraction, pcr amplification and single-base primer extension. oligonucleotide-modified nanoparticles correctly responded to the unpurified products from the primer e ...	2005	15640441
use of a restriction enzyme-digested pcr product as substrate for helicase assays.	dna helicases play essential roles in many cellular processes. the currently available techniques to generate substrates for helicase assays are fairly complicated and need some expertise not available in all laboratories. here, a pcr-based method to generate a substrate for a helicase assay is described, and its application for several archaeal, bacterial and viral enzymes is demonstrated.	2005	15653629
mutants with temperature-sensitive defects in the escherichia coli mismatch repair system: sensitivity to mispairs generated in vivo.	we have used direct selections to generate large numbers of mutants of escherichia coli defective in the mismatch repair system and have screened these to identify mutants with temperature-sensitive defects. we detected and sequenced mutations that give rise to temperature-sensitive muts, mutl, and muth proteins. one mutation, muts60, results in almost normal levels of spontaneous mutations at 37 degrees c but above this temperature gives rise to higher and higher levels of mutations, reaching t ...	2005	15659661
identification and characterization of genes involved in the downstream degradation pathway of gamma-hexachlorocyclohexane in sphingomonas paucimobilis ut26.	sphingomonas paucimobilis ut26 utilizes gamma-hexachlorocyclohexane (gamma-hch) as a sole source of carbon and energy. in our previous study, we cloned and characterized genes that are involved in the conversion of gamma-hch to maleylacetate (ma) via chlorohydroquinone (chq) in ut26. in this study, we identified and characterized an ma reductase gene, designated linf, that is essential for the utilization of gamma-hch in ut26. a gene named lineb, whose deduced product showed significant identity ...	2005	15659662
substrate requirements for regulated intramembrane proteolysis of bacillus subtilis pro-sigmak.	during sporulation of bacillus subtilis, pro-sigmak is activated by regulated intramembrane proteolysis (rip) in response to a signal from the forespore. rip of pro-sigmak removes its prosequence (amino acids 1 to 20), releasing sigmak from the outer forespore membrane into the mother cell cytoplasm, in a reaction catalyzed by spoivfb, a metalloprotease in the s2p family of intramembrane-cleaving proteases. the requirements for pro-sigmak to serve as a substrate for rip were investigated by prod ...	2005	15659674
low copy number dna template can render polymerase chain reaction error prone in a sequence-dependent manner.	paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. we used dna isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-dna glycosylase (ung), encoding the ung catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (pcr) artifacts. we demonstrate that low dna template input i ...	2005	15681472
rna polymerase mutants defective in the initiation of transcription-coupled dna repair.	the bacterial mfd protein is a transcription-repair coupling factor that performs two key functions during transcription-coupled dna repair. the first is to remove rna polymerase (rnap) complexes that have been stalled by a dna lesion from the site of damage, and the second is to mediate the recruitment of dna repair proteins. mfd also displaces transcription complexes that have been stalled by protein roadblocks, and catalyses the reactivation of transcription complexes that have become 'backtr ...	2005	15687384
structural, functional, and genetic analysis of sorangicin inhibition of bacterial rna polymerase.	a combined structural, functional, and genetic approach was used to investigate inhibition of bacterial rna polymerase (rnap) by sorangicin (sor), a macrolide polyether antibiotic. sor lacks chemical and structural similarity to the ansamycin rifampicin (rif), an rnap inhibitor widely used to treat tuberculosis. nevertheless, structural analysis revealed sor binds in the same rnap beta subunit pocket as rif, with almost complete overlap of rnap binding determinants, and functional analysis revea ...	2005	15692574
pseudomonas aeruginosa mutl protein functions in escherichia coli.	escherichia coli muts, mutl and muth proteins act sequentially in the mmrs (mismatch repair system). muth directs the repair system to the newly synthesized strand due to its transient lack of dam (dna-adenine methylase) methylation. although pseudomonas aeruginosa does not have the corresponding e. coli muth and dam homologues, and consequently the mmrs seems to work differently, we show that the mutl gene from p. aeruginosa is capable of complementing a mutl-deficient strain of e. coli. mutl f ...	2005	15709980
rifampin-resistant rna polymerase mutants of chlamydia trachomatis remain susceptible to the ansamycin rifalazil.	stable, homotypic mutants of chlamydia trachomatis for which mics of rifampin and rifalazil are elevated were isolated by serial passage at sub-mic concentrations of these compounds. an alternative approach, in which chlamydia cells were incubated and subsequently passaged three times, all in the presence of the selective agent at concentrations above the mic, appeared to be a more effective means of selecting for mutants. in every instance where an elevation in the mic occurred, one or more mut ...	2005	15728912
separation of mutation avoidance and antirecombination functions in an escherichia coli muts mutant.	dna mismatch repair in escherichia coli has been shown to be involved in two distinct processes: mutation avoidance, which removes potential mutations arising as replication errors, and antirecombination which prevents recombination between related, but not identical (homeologous), dna sequences. we show that cells with the mutsdelta800 mutation (which removes the c-terminal 53 amino acids of muts) on a multicopy plasmid are proficient for mutation avoidance. in interspecies genetic crosses, how ...	2005	15731339
chemical engineering of the peptidyl transferase center reveals an important role of the 2'-hydroxyl group of a2451.	the main enzymatic reaction of the large ribosomal subunit is peptide bond formation. ribosome crystallography showed that a2451 of 23s rrna makes the closest approach to the attacking amino group of aminoacyl-trna. mutations of a2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleoside ...	2005	15767286
single-stranded dna-binding protein of deinococcus radiodurans: a biophysical characterization.	the highly conserved bacterial single-stranded dna-binding (ssb) proteins play an important role in dna replication, repair and recombination and are essential for the survival of the cell. they are functional as tetramers, in which four ob(oligonucleotide/oligosaccharide binding)-folds act as dna-binding domains. the protomer of the ssb protein from the extremely radiation-resistant organism deinococcus radiodurans (drassb) has twice the size of the other bacterial ssb proteins and contains two ...	2005	15781492
the thermophilic, homohexameric aminopeptidase of borrelia burgdorferi is a member of the m29 family of metallopeptidases.	proteases are implicated in several aspects of the physiology of microorganisms, as well as in host-pathogen interactions. aminopeptidases are also emerging as novel drug targets in infectious agents. in this study, we have characterized an aminopeptidase from the spirochete borrelia burgdorferi, the causative agent of lyme disease. the aminopeptidolytic activity was identified in cell extracts from b. burgdorferi by using the substrate leucine-7-amido-4-methylcoumarin. a protein displaying this ...	2005	15784569
different rifampin sensitivities of escherichia coli and mycobacterium tuberculosis rna polymerases are not explained by the difference in the beta-subunit rifampin regions i and ii.	mycobacterium tuberculosis rna polymerase is 1,000-fold more sensitive to rifampin than escherichia coli rna polymerase. chimeric e. coli rna polymerase in which the beta-subunit segment encompassing rifampin regions i and ii (amino acids [aa] 463 through 590) was replaced with the corresponding region from m. tuberculosis (aa 382 through 509) did not show an increased sensitivity to the antibiotic. thus, the difference in amino acid sequence between the rifampin regions i and ii of the two spec ...	2005	15793146
a ring-like nucleoid is not necessary for radioresistance in the deinococcaceae.	transmission electron microscopy images of deinococcus radiodurans r1 suggest that the nucleoid of this species exists as a "ring-like" body, and have led to speculation that this structure contributes to the radioresistance of the species. since extreme radioresistance is characteristic of six other species of deinococcus, we have attempted to correlate nucleoid morphology and radioresistance by determining whether the genomic dna of each of these species exhibit similar structures.	2005	15799787
correcting errors in synthetic dna through consensus shuffling.	although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1-3 random errors/kb of dna. here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic dna. in this method, errors are revealed as mismatches by re-hybridization of the population. the dna is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding prote ...	2005	15800206
perspectives on biotechnological applications of archaea.	many archaea colonize extreme environments. they include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. this review summarizes current knowledge about the biotechnolog ...	2002	15803645
cross-resistance of escherichia coli rna polymerases conferring rifampin resistance to different antibiotics.	in this study we further defined the rifampin-binding sites in escherichia coli rna polymerase (rnap) and determined the relationship between rifampin-binding sites and the binding sites of other antibiotics, including two rifamycin derivatives, rifabutin and rifapentine, and streptolydigin and sorangicin a, which are unrelated to rifampin, using a purified in vitro system. we found that there is almost a complete correlation between resistance to rifampin (rif(r)) and reduced rifampin binding t ...	2005	15805525
nonbridging phosphate oxygens in 16s rrna important for 30s subunit assembly and association with the 50s ribosomal subunit.	ribosomes are composed of rna and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. detailed information about the structure of the ribosome has come from the recent x-ray crystal structures of the ribosome and the ribosomal subunits. however, the molecular interactions between the rrnas and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. here we describe a modification-interference ...	2005	15811917
helicobacter pylori dnab helicase can bypass escherichia coli dnac function in vivo.	in escherichia coli, dnac is essential for loading dnab helicase at oric (the origin of chromosomal dna replication). the question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnac in their genomes. previously, we have reported the characterization of hpdnab (helicobacter pylori dnab) both in vitro and in vivo. interestingly, h. pylori does not have a dnac homologue. using two different e. coli dnac (ecdnac) temperature-sensi ...	2005	15836434
genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.	pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (dsg3) and dsg1. mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. using antibody phage display, we have isolated repertoires of human anti-dsg mabs as single-chain variable-region fragments (scfvs) from a patient with active mucocutaneous pemphigus vulgaris. scfv mabs demonstrated bin ...	2005	15841178
surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions.	surface plasmon field-enhanced fluorescence spectroscopy (spfs) utilizes the evanescent electromagnetic field of a surface plasmon to excite chromophors in close proximity to the surface. while conventional surface plasmon resonance spectroscopy allows the observation of surface reactions by means of refractive index changes, spfs additionally provides a channel for the read-out of fluorescence changes. thus, the detection limit for low mass compounds, whose adsorption is only accompanied by sma ...	2005	15849312
piecemaker: selection of dna fragments for selector-guided multiplex amplification.	we describe piecemaker, a software tool for the design of applications of selector probes-oligonucleotide probes that direct circularization of target nucleic acid molecules. such probes can be combined in parallel to circularize a selection of fragments from restriction digested total genomic dna. these fragments can then be amplified in a single pcr using a common primer pair, yielding substrates for subsequent analyses, such as parallel genotyping or sequencing. however, designing multiplex s ...	2005	15860769
role of muts and mutl genes in hypermutability and recombination in staphylococcus aureus.	the mutator phenotype has been linked in several bacterial genera to a defect in the methyl-mismatch repair system, in which the major components are muts and mutl. this system is involved both in mismatch repair and in prevention of recombination between homeologous fragments in escherichia coli and has been shown to play an important role in the adaptation of bacterial populations in changing and stressful environments. in this report we describe the molecular analysis of the muts and mutl gen ...	2005	15866932
structural and functional divergence of muts2 from bacterial muts1 and eukaryotic msh4-msh5 homologs.	muts homologs, identified in nearly all bacteria and eukaryotes, include the bacterial proteins muts1 and muts2 and the eukaryotic muts homologs 1 to 7, and they often are involved in recognition and repair of mismatched bases and small insertion/deletions, thereby limiting illegitimate recombination and spontaneous mutation. to explore the relationship of muts2 to other muts homologs, we examined conserved protein domains. fundamental differences in structure between muts2 and other muts homolo ...	2005	15866941
distribution of genes for synthesis of trehalose and mannosylglycerate in thermus spp. and direct correlation of these genes with halotolerance.	in this study we correlate the presence of genes leading to the synthesis of trehalose and mannosylglycerate (mg) in 17 strains of the genus thermus with the ability of the strains to grow and accumulate these compatible solutes in a defined medium containing nacl. the two sets of genes, namely, otsa/otsb for the synthesis of trehalose and mpgs/mpgp for the synthesis of mg, were necessary for the growth of thermus thermophilus in a defined medium containing up to 6% nacl. strains lacking a compl ...	2005	15870334
comparison of standard exponential and linear techniques to amplify small cdna samples for microarrays.	the need to perform microarray experiments with small amounts of tissue has led to the development of several protocols for amplifying the target transcripts. the use of different amplification protocols could affect the comparability of microarray experiments.	2005	15871737
localization of spermine binding sites in 23s rrna by photoaffinity labeling: parsing the spermine contribution to ribosomal 50s subunit functions.	polyamine binding to 23s rrna was investigated, using a photoaffinity labeling approach. this was based on the covalent binding of a photoreactive analog of spermine, n1-azidobenzamidino (aba)-spermine, to escherichia coli ribosomes or naked 23s rrna under mild irradiation conditions. the cross-linking sites of aba-spermine in 23s rrna were determined by rnase h digestion and primer-extension analysis. domains i, ii, iv and v in naked 23s rrna were identified as discrete regions of preferred cro ...	2005	15897324
new mutations and horizontal transfer of rpob among rifampin-resistant streptococcus pneumoniae from four spanish hospitals.	a total of 103 (0.7%) of 14,236 streptococcus pneumoniae isolates collected in four spanish hospitals from 1989 to 2003 were resistant to rifampin (mics, 4 to 512 microg/ml). only sixty-one (59.2%) of these isolates were available for molecular characterization. resistance was mostly related to human immunodeficiency virus (hiv) infection in adult patients and to conjunctivitis in children. thirty-six different pulsed-field gel electrophoresis patterns were identified among resistant isolates, f ...	2005	15917517
heptameric (l12)6/l10 rather than canonical pentameric complexes are found by tandem ms of intact ribosomes from thermophilic bacteria.	ribosomes are universal translators of the genetic code into protein and represent macromolecular structures that are asymmetric, often heterogeneous, and contain dynamic regions. these properties pose considerable challenges for modern-day structural biology. despite these obstacles, high-resolution x-ray structures of the 30s and 50s subunits have revealed the rna architecture and its interactions with proteins for ribosomes from thermus thermophilus, deinococcus radiodurans, and haloarcula ma ...	2005	15923259
aminoacylation and conformational properties of yeast mitochondrial trna mutants with respiratory deficiency.	we report the identification and characterization of eight yeast mitochondrial trna mutants, located in mitochondrial trna(gln), trna(arg2), trna(ile), trna(his), and trna(cys), the respiratory phenotypes of which exhibit various degrees of deficiency. the mutations consist in single-base substitutions, insertions, or deletions, and are distributed all over the trna sequence and structure. to identify the features responsible for the defective phenotypes, we analyzed the effect of the different ...	2005	15923375
domain rearrangement of srp protein ffh upon binding 4.5s rna and the srp receptor ftsy.	the signal recognition particle (srp) mediates membrane targeting of translating ribosomes displaying a signal-anchor sequence. in escherichia coli, srp consists of 4.5s rna and a protein, ffh, that recognizes the signal peptide emerging from the ribosome and the srp receptor at the membrane, ftsy. in the present work, we studied the interactions between the ng and m domains in ffh and their rearrangements upon complex formation with 4.5s rna and/or ftsy. in free ffh, the ng and m domains are fa ...	2005	15923378
sequence determination of nucleic acids containing 5-methylisocytosine and isoguanine: identification and insight into polymerase replication of the non-natural nucleobases.	nucleobase analogs 5-methylisocytosine ((me)isoc) and isoguanine (isog) form a non-natural base pair in duplex nucleic acids with base pairing specificity orthogonal to the natural nucleobase pairs. sequencing reactions were conducted with oligodeoxyribonucleotides (odns) containing d(me)isoc and disog using modified pyrosequencing and dye terminator methods. modified dye terminator sequencing was generally useful for the sequence identification of odns containing the non-natural nucleobases. th ...	2005	15933210
the haloferax volcanii ftsy homolog is critical for haloarchaeal growth but does not require the a domain.	the targeting of many sec substrates to the membrane-associated translocation pore requires the cytoplasmic signal recognition particle (srp). in eukarya and bacteria it has been shown that membrane docking of the srp-substrate complex occurs via the universally conserved srp receptor (sralpha/beta and ftsy, respectively). while much has been learned about the archaeal srp in recent years, few studies have examined archaeal sralpha/ftsy homologs. in the present study the ftsy homolog of halofera ...	2005	15937164
the bacterial helicase-primase interaction: a common structural/functional module.	the lack of a high-resolution structure for the bacterial helicase-primase complex and the fragmented structural information for the individual proteins have been hindering our detailed understanding of this crucial binary protein interaction. two new structures for the helicase-interacting domain of the bacterial primases from escherichia coli and bacillus stearothermophilus have recently been solved and both revealed a unique and surprising structural similarity to the amino-terminal domain of ...	2005	15939015
gene transfer and genome plasticity in thermotoga maritima, a model hyperthermophilic species.	the genome sequence of the hyperthermophilic bacterium thermotoga maritima msb8 presents evidence for lateral gene transfer events between bacterial and archaeal species. to estimate the extent of genomic diversity across the order thermotogales, a comparative genomic hybridization study was initiated to compare nine thermotoga strains to the sequenced t. maritima msb8. many differences could be associated with substrate utilization patterns, which are most likely a reflection of the environment ...	2005	15995209
cloning and expression of islandisin, a new thermostable subtilisin from fervidobacterium islandicum, in escherichia coli.	a gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium fervidobacterium islandicum (dsmz 5733) was cloned and actively expressed in escherichia coli. the gene was identified by pcr using degenerated primers based on conserved regions around two of the three catalytic residues (asp, his, and ser) of subtilisin-like serine protease-encoding genes. using inverse pcr regions flanking the catalytic residues, the gene could be cloned. sequencing re ...	2005	16000809
ethanol feeding enhances age-related deterioration of the rat hepatic mitochondrion.	chronic ethanol feeding damages the hepatic mitochondrion by increasing mitochondrial dna (mtdna) oxidation, lowering mtdna yields and impairing mitochondrial respiration. these effects are also seen during aging. by employing a 21-day chronic feeding regimen, we investigated the effects of ethanol consumption on mtdna content and mitochondrial respiration in 2-, 12-, and 24-mo-old male rats. aging resulted in decreased mtdna content, increased mtdna damage (as indicated by inhibition of taq pol ...	2005	16020655
origin and evolution of the archaeo-eukaryotic primase superfamily and related palm-domain proteins: structural insights and new members.	we report an in-depth computational study of the protein sequences and structures of the superfamily of archaeo-eukaryotic primases (aeps). this analysis greatly expands the range of diversity of the aeps and reveals the unique active site shared by all members of this superfamily. in particular, it is shown that eukaryotic nucleo-cytoplasmic large dna viruses, including poxviruses, asfarviruses, iridoviruses, phycodnaviruses and the mimivirus, encode aeps of a distinct family, which also includ ...	2005	16027112
uracil content of 16s rrna of thermophilic and psychrophilic prokaryotes correlates inversely with their optimal growth temperatures.	we report here the finding of a highly significant inverse correlation of the uracil content of 16s rrna and the optimum growth temperature (t(opt)) of cultured thermophilic and psychrophilic prokaryotes. this correlation was significantly different from the weaker correlations between the contents of other nucleotides and t(opt). analysis of the 16s rrna secondary structure regions revealed a fall in the a:u base-pair content in step with the increase in t(opt) that was much steeper than that o ...	2005	16030352
molecular mimicry: quantitative methods to study structural similarity between protein and rna.	with rapidly increasing availability of three-dimensional structures, one major challenge for the post-genome era is to infer the functions of biological molecules based on their structural similarity. while quantitative studies of structural similarity between the same type of biological molecules (e.g., protein vs. protein) have been carried out intensively, the comparable study of structural similarity between different types of biological molecules (e.g., protein vs. rna) remains unexplored. ...	2005	16043503
polyphosphate kinase regulates error-prone replication by dna polymerase iv in escherichia coli.	the ppk gene encodes polyphosphate kinase (ppk), an enzyme that catalyses the polymerization of inorganic phosphate into long chains of polyphosphate (polyp). an insertion mutation in ppk causes a decrease in adaptive mutation in escherichia coli strain fc40. adaptive mutation in fc40 mostly results from error-prone dna polymerase iv (pol iv), encoded by dinb; most of the antimutagenic phenotype of the ppk mutant disappears in a dinb mutant strain. in addition, the ppk mutant causes a decrease i ...	2005	16045619
the involvement of the aspartate triad of the active center in all catalytic activities of multisubunit rna polymerase.	three conserved aspartate residues in the largest subunit of multisubunit rna polymerases (rnaps) coordinate two mg2+ ions involved in the catalysis of phosphodiester bond synthesis. a structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these mg2+ ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent rna. here, we check these pred ...	2005	16049026
evidence for a watson-crick hydrogen bonding requirement in dna synthesis by human dna polymerase kappa.	the efficiency and fidelity of nucleotide incorporation by high-fidelity replicative dna polymerases (pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural dna bases but which lack the ability to engage in watson-crick (w-c) hydrogen bonding. dna synthesis by poleta, a low-fidelity polymerase able to replicate through ...	2005	16055723
nucleotide exchange and excision technology (next) dna shuffling: a robust method for dna fragmentation and directed evolution.	dna shuffling is widely used for optimizing complex properties contained within dna and proteins. demonstrated here is the amplification of a gene library by pcr using uridine triphosphate (dutp) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. the incorporated uracil bases were excised using uracil-dna-glycosylase and the dna backbone subsequently cleaved with piperidine. these end-point reactions required no adjustments. polyacrylamide ...	2005	16061932
determination of protein-dna binding constants and specificities from statistical analyses of single molecules: muts-dna interactions.	atomic force microscopy (afm) is a powerful technique for examining the conformations of protein-dna complexes and determining the stoichiometries and affinities of protein-protein complexes. we extend the capabilities of afm to the determination of protein-dna binding constants and specificities. the distribution of positions of the protein on the dna fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. the fractional occupancies of the ...	2005	16061937
evolution and molecular phylogeny of listeria monocytogenes isolated from human and animal listeriosis cases and foods.	to probe the evolution and phylogeny of listeria monocytogenes from defined host species and environments, l. monocytogenes isolates from human (n = 60) and animal (n = 30) listeriosis cases and food samples (n = 30) were randomly selected from a larger collection of isolates (n = 354) obtained in new york state between 1999 and 2001. partial sequencing of four housekeeping genes (gap, prs, purm, and ribc), one stress response gene (sigb), and two virulence genes (acta and inla) revealed between ...	2005	16077098
dna sequence-based subtyping and evolutionary analysis of selected salmonella enterica serotypes.	while serotyping and phage typing have been used widely to characterize salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining salmonella transmission, ecology, and evolution. a set of 25 salmonella enterica isolates, representing five clinically relevant serotypes (serotypes agona, heidelberg, schwarzengrund, typhimurium, and typhimurium var. copenhagen) was initially used to develop a multilocus sequence typing (mlst) scheme for salmon ...	2005	16081897
multiplex pcr: use of heat-stable thermus thermophilus reca protein to minimize non-specific pcr products.	in this paper we report that the inclusion of heat-resistant reca protein from a thermophilic bacteria, thermus thermophilus, and its cofactor (atp) in pcr effectively eliminates non-specific pcr products. the effect of reca protein, which catalyzes pairing between homologous dna molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in pcr (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). in ...	2005	16087733
fidelity of dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis.	we report the crystal structures of a translesion dna polymerase, dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. these structures suggest two mechanisms by which dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. first, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. by replacing mg2+ with mn2+, w ...	2005	16107880