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sequence-similar, structure-dissimilar protein pairs in the pdb.it is often assumed that in the protein data bank (pdb), two proteins with similar sequences will also have similar structures. accordingly, it has proved useful to develop subsets of the pdb from which "redundant" structures have been removed, based on a sequence-based criterion for similarity. similarly, when predicting protein structure using homology modeling, if a template structure for modeling a target sequence is selected by sequence alone, this implicitly assumes that all sequence-simil ...200718004789
sequence-similar, structure-dissimilar protein pairs in the pdb.it is often assumed that in the protein data bank (pdb), two proteins with similar sequences will also have similar structures. accordingly, it has proved useful to develop subsets of the pdb from which "redundant" structures have been removed, based on a sequence-based criterion for similarity. similarly, when predicting protein structure using homology modeling, if a template structure for modeling a target sequence is selected by sequence alone, this implicitly assumes that all sequence-simil ...200718004789
mapping of rna polymerase residues that interact with bacteriophage xp10 transcription antitermination factor p7.bacteriophage xp10-encoded transcription factor p7 interacts with host xanthomonas oryzae rna polymerase beta' subunit and prevents both promoter recognition by the rna polymerase holoenzyme and transcription termination by the rna polymerase core. p7 does not bind to and has no effect on rna polymerase from escherichia coli. here, we use a combination of biochemical and genetic methods to map the p7 interaction site to within four beta' amino acid residues at the n terminus of x. oryzae rnap be ...200818021805
mapping of rna polymerase residues that interact with bacteriophage xp10 transcription antitermination factor p7.bacteriophage xp10-encoded transcription factor p7 interacts with host xanthomonas oryzae rna polymerase beta' subunit and prevents both promoter recognition by the rna polymerase holoenzyme and transcription termination by the rna polymerase core. p7 does not bind to and has no effect on rna polymerase from escherichia coli. here, we use a combination of biochemical and genetic methods to map the p7 interaction site to within four beta' amino acid residues at the n terminus of x. oryzae rnap be ...200818021805
regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy.we recently showed an increase in vascular endothelial growth factor (vegf), decrease in angiopoietin-1 (ang-1) and unaltered ang-2 expression by the villous placenta with advancing baboon pregnancy. moreover, placental vegf expression was increased by estrogen in early pregnancy. in the present study, we determined whether placental ang-1 and ang-2 are regulated by estrogen. ang-1 and ang-2 mrna and protein were determined by rt-pcr and immunocytochemistry in the placenta of baboons on day 60 o ...200818022824
the methyltransferase yfgb/rlmn is responsible for modification of adenosine 2503 in 23s rrna.a2503 in 23s rrna of the gram-negative bacterium escherichia coli is located in a functionally important region of the ribosome, at the entrance to the nascent peptide exit tunnel. in e. coli, and likely in other species, this adenosine residue is post-transcriptionally modified to m2a. the enzyme responsible for this modification was previously unknown. we identified e. coli protein yfgb, which belongs to the radical sam enzyme superfamily, as the methyltransferase that modifies a2503 of 23s rr ...200818025251
effect of oxidatively damaged dna on the active site preorganization during nucleotide incorporation in a high fidelity polymerase from bacillus stearothermophilus.we study the effect of the oxidative lesion 8-oxoguanine (8oxog) on the preorganization of the active site for dna replication in the closed (active) state of the bacillus fragment (bf), a klenow analog from bacillus stearothermophilus. our molecular dynamics and free energy simulations of explicitly solvated model ternary complexes of bf bound to correct dctp/incorrect datp opposite guanine (g) and 8oxog bases in dna suggest that the lesion introduces structural and energetic changes at the cat ...200818058909
biophysical analysis of thermus aquaticus single-stranded dna binding protein.due to their involvement in processes such as dna replication, repair, and recombination, bacterial single-stranded dna binding (ssb) proteins are essential for the survival of the bacterial cell. whereas most bacterial ssb proteins form homotetramers in solution, dimeric ssb proteins were recently discovered in the thermus/deinococcus group. in this work we characterize the biophysical properties of the ssb protein from thermus aquaticus (taqssb), which is structurally quite similar to the tetr ...200818065449
biophysical analysis of thermus aquaticus single-stranded dna binding protein.due to their involvement in processes such as dna replication, repair, and recombination, bacterial single-stranded dna binding (ssb) proteins are essential for the survival of the bacterial cell. whereas most bacterial ssb proteins form homotetramers in solution, dimeric ssb proteins were recently discovered in the thermus/deinococcus group. in this work we characterize the biophysical properties of the ssb protein from thermus aquaticus (taqssb), which is structurally quite similar to the tetr ...200818065449
miniprimer pcr, a new lens for viewing the microbial world.molecular methods based on the 16s rrna gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. here we show that a new pcr method using an engineered polymerase and 10-nucleotide "miniprimers" expands the scope of detectable sequences beyond those detected by standard methods using longer primers and taq polymerase. after testing the method in silico to identify divergent ribosomal genes in previously cloned environmental seq ...200818083877
miniprimer pcr, a new lens for viewing the microbial world.molecular methods based on the 16s rrna gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. here we show that a new pcr method using an engineered polymerase and 10-nucleotide "miniprimers" expands the scope of detectable sequences beyond those detected by standard methods using longer primers and taq polymerase. after testing the method in silico to identify divergent ribosomal genes in previously cloned environmental seq ...200818083877
threonine 429 of escherichia coli sigma 70 is a key participant in promoter dna melting by rna polymerase.initiation of transcription is an important target for regulation of gene expression. in bacteria, the formation of a transcription-competent complex between rna polymerase and a promoter involves dna strand separation over a stretch of about 14 base pairs. aromatic and basic residues in conserved region 2.3 of escherichia coli sigma(70) had been found to participate in this process, but it is still unclear which amino acid residues initiate it. here we report an essential role for threonine (t) ...200818155246
threonine 429 of escherichia coli sigma 70 is a key participant in promoter dna melting by rna polymerase.initiation of transcription is an important target for regulation of gene expression. in bacteria, the formation of a transcription-competent complex between rna polymerase and a promoter involves dna strand separation over a stretch of about 14 base pairs. aromatic and basic residues in conserved region 2.3 of escherichia coli sigma(70) had been found to participate in this process, but it is still unclear which amino acid residues initiate it. here we report an essential role for threonine (t) ...200818155246
a polymerase iii-like reinitiation mechanism is operating in regulation of histone expression in archaea.an archaeal histone gene from the hyperthermophile pyrococcus furiosus containing four consecutive putative oligo-dt terminator sequences was used as a model system to investigate termination signals and the mechanism of termination in vitro. the archaeal rna polymerase terminated with high efficiency at the first terminator at 90 degrees c when it contained five to six t residues, at 80 degrees c readthrough was significantly increased. a putative hairpin structure upstream of the first termina ...200818182021
ex vivo induction of mrna in human whole blood as a new platform of drug and dietary supplement development.we introduced a new concept of ex vivo gene expression analysis (mitsuhashi, clin chem 53:148-149, 2007), where drug action was simulated under physiological conditions. this model system was applied to study various fields of drug development.200818183479
the x-ray crystal structure of rna polymerase from archaea.the transcription apparatus in archaea can be described as a simplified version of its eukaryotic rna polymerase (rnap) ii counterpart, comprising an rnapii-like enzyme as well as two general transcription factors, the tata-binding protein (tbp) and the eukaryotic tfiib orthologue tfb. it has been widely understood that precise comparisons of cellular rnap crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of ...200818235446
simultaneous fitting of real-time pcr data with efficiency of amplification modeled as gaussian function of target fluorescence.in real-time pcr, it is necessary to consider the efficiency of amplification (ea) of amplicons in order to determine initial target levels properly. eas can be deduced from standard curves, but these involve extra effort and cost and may yield invalid eas. alternatively, ea can be extracted from individual fluorescence curves. unfortunately, this is not reliable enough.200818267040
high-throughput avian molecular sexing by sybr green-based real-time pcr combined with melting curve analysis.combination of chd (chromo-helicase-dna binding protein)-specific polymerase chain reaction (pcr) with electrophoresis (pcr/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. gender determination often fails when the difference in length between the pcr products of chd-z and chd-w genes is too short to be resolved.200818269737
mechanistic consequences of temperature on dna polymerization catalyzed by a y-family dna polymerase.our previous publication shows that sulfolobus solfataricus dpo4 utilizes an 'induced-fit' mechanism to select correct incoming nucleotides at 37 degrees c. here, we provide a comprehensive report elucidating the kinetic mechanism of a dna polymerase at a reaction temperature higher than 37 degrees c in an attempt to determine the effect of temperature on enzyme fidelity and mechanism. the fidelity of dpo4 did not change considerably with a 30 degrees c increase in reaction temperature, suggesti ...200818276639
beta clamp directs localization of mismatch repair in bacillus subtilis.muts homologs function in several cellular pathways including mismatch repair (mmr), the process by which mismatches introduced during dna replication are corrected. we demonstrate that the c terminus of bacillus subtilis muts is necessary for an interaction with beta clamp. this interaction is required for muts-gfp focus formation in response to mismatches. reciprocally, we show that a mutant of the beta clamp causes elevated mutation frequencies and is reduced for muts-gfp focus formation. mut ...200818280235
lineage-specific amino acid substitutions in region 2 of the rna polymerase sigma subunit affect the temperature of promoter opening.highly conserved amino acid residues in region 2 of the rna polymerase sigma subunit are known to participate in promoter recognition and opening. we demonstrated that nonconserved residues in this region collectively determine lineage-specific differences in the temperature of promoter opening.200818281402
fine structure of the promoter-sigma region 1.2 interaction.we recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the -10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the sigma subunit of escherichia coli rna polymerase (rnap). because rrna promoters contain sequences within the discriminator region that are suboptimal for interaction with sigma1.2, these promoters have the kinetic properties required for regulation by the rnap-binding fact ...200818287032
an intramolecular fret system monitors fingers subdomain opening in klentaq1.a major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating dna base is selected and delivered to the polymerase active site. structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. we previously designed a fret system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. however, this fret system was limited ...200818287276
quantification of cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice.inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. these models frequently rely on cre-mediated recombination of sequences flanked by lox-p sites. to understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process.200818298805
"hot cores" in proteins: comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms.a wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. these include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. it has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. in this work, protein crystal structures from hyper/thermop ...200818312638
identification of cross-linked peptides from large sequence databases.we describe a method to identify cross-linked peptides from complex samples and large protein sequence databases by combining isotopically tagged cross-linkers, chromatographic enrichment, targeted proteomics and a new search engine called xquest. this software reduces the search space by an upstream candidate-peptide search before the recombination step. we showed that xquest can identify cross-linked peptides from a total escherichia coli lysate with an unrestricted database search.200818327264
on helicases and other motor proteins.helicases are molecular machines that utilize energy derived from atp hydrolysis to move along nucleic acids and to separate base-paired nucleotides. the movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. recent structural data for the hexameric e1 helicase of papillomavirus in complex with single-stranded dna and mgadp has provided a detailed atomic and mechanistic picture of its atp-driven dna translocation. the structural and mechanistic features ...200818329872
dynamics of recognition between trna and elongation factor tu.elongation factor tu (ef-tu) binds to all standard aminoacyl transfer rnas (aa-trnas) and transports them to the ribosome while protecting the ester linkage between the trna and its cognate amino acid. we use molecular dynamics simulations to investigate the dynamics of the ef-tu.guanosine 5'-triphosphate.aa-trna(cys) complex and the roles played by mg2+ ions and modified nucleosides on the free energy of protein.rna binding. individual modified nucleosides have pronounced effects on the structu ...200818336835
rifamycin antibiotic resistance by adp-ribosylation: structure and diversity of arr.the rifamycin antibiotic rifampin is important for the treatment of tuberculosis and infections caused by multidrug-resistant staphylococcus aureus. recent iterations of the rifampin core structure have resulted in new drugs and drug candidates for the treatment of a much broader range of infectious diseases. this expanded use of rifamycin antibiotics has the potential to select for increased resistance. one poorly characterized mechanism of resistance is through arr enzymes that catalyze adp-ri ...200818349144
endothelial cells support the growth of prostate tissue in vivo.the contribution of vascular endothelial cells to prostate growth has not been investigated. we examined whether endothelial cells support growth of prostate tissue when co-inoculated with prostate epithelial cells under the renal capsule.200818361413
regulation of bacterial rna polymerase sigma factor activity: a structural perspective.in bacteria, sigma factors are essential for the promoter dna-binding specificity of rna polymerase. the sigma factors themselves are regulated by anti-sigma factors that bind and inhibit their cognate sigma factor, and 'appropriators' that deploy a particular sigma-associated rna polymerase to a specific promoter class. adding to the complexity is the regulation of anti-sigma factors by both anti-anti-sigma factors, which turn on sigma factor activity, and co-anti-sigma factors that act in conc ...200818375176
mismatched dntp incorporation by dna polymerase beta does not proceed via globally different conformational pathways.understanding how dna polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dntp incorporations. little is known about the latter because mismatched complexes do not crystallize readily. in this report, we employed small-angle x-ray scattering (saxs) and structural modeling to probe the conformations of different intermediate states of mammalian dna polymerase beta (pol beta) in its wild-type and an error-prone variant, i260q. our structural results indica ...200818385153
novel computational methods for increasing pcr primer design effectiveness in directed sequencing.polymerase chain reaction (pcr) is used in directed sequencing for the discovery of novel polymorphisms. as the first step in pcr directed sequencing, effective pcr primer design is crucial for obtaining high-quality sequence data for target regions. since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. further ...200818405373
dna mismatch repair: molecular mechanism, cancer, and ageing.dna mismatch repair (mmr) proteins are ubiquitous players in a diverse array of important cellular functions. in its role in post-replication repair, mmr safeguards the genome correcting base mispairs arising as a result of replication errors. loss of mmr results in greatly increased rates of spontaneous mutation in organisms ranging from bacteria to humans. mutations in mmr genes cause hereditary nonpolyposis colorectal cancer, and loss of mmr is associated with a significant fraction of sporad ...200818406444
the rate and spectrum of microsatellite mutation in caenorhabditis elegans and daphnia pulex.the effective use of microsatellite loci as tools for microevolutionary analysis requires knowledge of the factors influencing the rate and pattern of mutation, much of which is derived from indirect inference from population samples. interspecific variation in microsatellite stability also provides a glimpse into aspects of phylogenetic constancy of mutational processes. using long-term series of mutation-accumulation lines, we have obtained direct estimates of the spectrum of microsatellite mu ...200818430937
identification of a new motif required for the 3'-5' exonuclease activity of escherichia coli dna polymerase i (klenow fragment): the rrry motif is necessary for the binding of single-stranded dna substrate and the template strand of the mismatched duplex.the klenow fragment of escherichia coli dna polymerase i houses catalytic centers for both polymerase and 3'-5' exonuclease activities that are separated by about 35 a. upon the incorporation of a mismatched nucleotide, the primer terminus is transferred from the polymerase site to an exonuclease site designed for excision of the mismatched nucleotides. the structural comparison of the binary complexes of dna polymerases in the polymerase and the exonuclease modes, together with a molecular mode ...200818448432
de-orphaning the structural proteome through reciprocal comparison of evolutionarily important structural features.function prediction frequently relies on comparing genes or gene products to search for relevant similarities. because the number of protein structures with unknown function is mushrooming, however, we asked here whether such comparisons could be improved by focusing narrowly on the key functional features of protein structures, as defined by the evolutionary trace (et). therefore a series of algorithms was built to (a) extract local motifs (3d templates) from protein structures based on et rank ...200818461181
correlation between rpob gene mutation in mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of crohn's disease.to investigate overlapping regions of the rpob gene previously involved with rifamycin resistance in m. tuberculosis and seek correlation between rpob mutations in clinical map strains with susceptibility to rif and rfb.200818461657
structures of dna polymerase beta with active-site mismatches suggest a transient abasic site intermediate during misincorporation.we report the crystallographic structures of dna polymerase beta with dg-dampcpp and dc-dampcpp mismatches in the active site. these premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dampcpp, and mn(2+). substituting mn(2+) for mg(2+) significantly decreases the fidelity of dna synthesis. the structures reveal that the enzyme is in a closed conformation like that observed with a matched watson-crick base pair. the incorrect dampcpp binds in a conformation i ...200818471977
characterization of pseudomonas chlororaphis myovirus 201varphi2-1 via genomic sequencing, mass spectrometry, and electron microscopy.pseudomonas chlororaphis phage 201varphi2-1 is a relative of pseudomonas aeruginosa myovirus phikz. phage 201 phi2-1 was examined by complete genomic sequencing (316,674 bp), by a comprehensive mass spectrometry survey of its virion proteins and by electron microscopy. seventy-six proteins, of which at least 69 have homologues in phikz, were identified by mass spectrometry. eight proteins, in addition to the major head, tail sheath and tail tube proteins, are abundant in the virion. electron mic ...200818474389
snp analysis using catacleave probes.catacleave probes are catalytically cleavable fluorescence probes having a chimeric deoxyribonucleic acid (dna)-ribonucleic acid (rna)-dna structure that can be used for real-time detection of single nucleotide polymorphisms (snps), insertions, and deletions. fluorescent donor emission is normally quenched by förster resonance energy transfer (fret). upon binding to the target, if the rna/dna hybrid is correctly base-paired, ribonuclease (rnase) h will cleave the rna moiety and the probe fragmen ...200818484652
a basic/hydrophobic cleft of the t4 activator mota interacts with the c-terminus of e.coli sigma70 to activate middle gene transcription.transcriptional activation often employs a direct interaction between an activator and rna polymerase. for activation of its middle genes, bacteriophage t4 appropriates escherichia coli rna polymerase through the action of two phage-encoded proteins, mota and asia. alone, asia inhibits transcription from a large class of host promoters by structurally remodelling region 4 of sigma(70), the primary specificity subunit of e. coli rna polymerase. mota interacts both with sigma(70) region 4 and with ...200818485078
mitochondrial dna, base excision repair and neurodegeneration.neurodegeneration is a growing public health concern because of the rapid increase in median and maximum life expectancy in the developed world. mitochondrial dysfunction seems to play a critical role in neurodegeneration, likely owing to the high energy demand of the central nervous system and its sole reliance on oxidative metabolism for energy production. loss of mitochondrial function has been clearly demonstrated in several neuropathologies, most notably those associated with age, like alzh ...200818485834
low-fidelity dna synthesis by human dna polymerase theta.human dna polymerase theta (pol or polq) is a proofreading-deficient family a enzyme implicated in translesion synthesis (tls) and perhaps in somatic hypermutation (shm) of immunoglobulin genes. these proposed functions and kinetic studies imply that pol may synthesize dna with low fidelity. here, we show that when copying undamaged dna, pol generates single base errors at rates 10- to more than 100-fold higher than for other family a members. pol adds single nucleotides to homopolymeric runs at ...200818503084
homologous recombination is unlikely to play a major role in influenza b virus evolution.influenza b viruses cause a significant amount of morbidity and mortality. the occurrence of homologous recombination in influenza viruses is controversial. to determine the extent of homologous recombination in influenza b viruses, recombination analyses of 2,650 sequences representing all eight segments of the influenza b viruses were carried out. only four sequences were indentified as putative recombinants, which were verified using phylogenetic methods. however, the mosaics detected here we ...200818505573
advances in bacterial promoter recognition and its control by factors that do not bind dna.early work identified two promoter regions, the -10 and -35 elements, that interact sequence specifically with bacterial rna polymerase (rnap). however, we now know that several additional promoter elements contact rnap and influence transcription initiation. furthermore, our picture of promoter control has evolved beyond one in which regulation results solely from activators and repressors that bind to dna sequences near the rnap binding site: many important transcription factors bind directly ...200818521075
a novel universal real-time pcr system using the attached universal duplex probes for quantitative analysis of nucleic acids.real-time pcr techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time pcr is the high cost of the labeled probe for each target molecule.200818522756
the three princes of serendip: notes on a mysterious phenomenon. 200618523622
a comparative analysis of dmc1 and rad51 nucleoprotein filaments.the eukaryotic reca homologs rad51 and dmc1 are essential for strand exchange between homologous chromosomes during meiosis. all members of the reca family of recombinases polymerize on dna to form helical nucleoprotein filaments, which is the active form of the protein. here we compare the filament structures of the rad51 and dmc1 proteins from both human and budding yeast. previous studies of dmc1 filaments suggested that they might be structurally distinct from filaments of other members of t ...200818535008
the rna polymerase ii trigger loop functions in substrate selection and is directly targeted by alpha-amanitin.structural, biochemical, and genetic studies have led to proposals that a mobile element of multisubunit rna polymerases, the trigger loop (tl), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. here we present evidence that the saccharomyces cerevisiae rna polymerase ii (pol ii) tl participates in substrate selection. amino acid substitutions within the pol ii tl preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by alpha ...200818538653
a proposed role for leishmania major carboxypeptidase in peptide catabolism.leishmaniasis is a tropical disease caused by leishmania, eukaryotic parasites transmitted to humans by sand flies. towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two m32 carboxypeptidases present within the leishmania major (lmacp1) genome. enzymatic studies reveal that like previously studied m32 carboxypeptidases, lmacp1 cleaves substrates with a variety of c-terminal amino acids--the primary except ...200818539138
effects of ph and temperature on the composition of polar lipids in thermoplasma acidophilum ho-62.thermoplasma acidophilum ho-62 was grown at different phs and temperatures, and its polar lipid compositions were determined. although the number of cyclopentane rings in the caldarchaeol moiety increased when t. acidophilum was cultured at high temperature, the number decreased at low phs. glycolipids, phosphoglycolipids, and phospholipids were analyzed by high-performance liquid chromatography with an evaporative light-scattering detector. the amount of caldarchaeol with more than two sugar un ...200818539746
characterization of single and double inactivation strains reveals new physiological roles for group 2 sigma factors in the cyanobacterium synechocystis sp. pcc 6803.cyanobacteria are eubacteria that perform oxygenic photosynthesis like plants. the initiation of transcription, mediated by the rna polymerase holoenzyme, is the main determinant of gene regulation in eubacteria. the sigma factor of the rna polymerase holoenzyme is responsible for the recognition of a promoter sequence. in the cyanobacterium synechocystis sp. pcc 6803, the primary sigma factor, siga, is essential for cell viability. the sigb, sigc, sigd, and sige factors show significant sequenc ...200818539776
molecular characterization of the diversity and distribution of a thermal spring microbial community by using rrna and metabolic genes.the diversity and distribution of a bacterial community from coffee pots hot spring, a thermal spring in yellowstone national park with a temperature range of 39.3 to 74.1 degrees c and ph range of 5.75 to 6.91, were investigated by sequencing cloned pcr products and quantitative pcr (qpcr) of 16s rrna and metabolic genes. the spring was inhabited by three aquificae genera--thermocrinis, hydrogenobaculum, and sulfurihydrogenibium--and members of the alpha-, beta-, and gammaproteobacteria, firmic ...200818539788
high-fidelity dna polymerase enhances the sensitivity of a peptide nucleic acid clamp pcr assay for k-ras mutations.sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level. recently, peptide nucleic acid (pna) clamp real-time pcr has provided a time-sparing and sensitive method for the detection of mutations in the presence of a large excess of wild-type dna. we present the first report that the sensitivity of pna clamp pcr is limited by the low fidelity of taqdna polymerase. replication errors introduced b ...200818556764
rifampin and rifaximin resistance in clinical isolates of clostridium difficile.rifaximin, a poorly absorbed rifamycin derivative, is a promising alternative for the treatment of clostridium difficile infections. resistance to this agent has been reported, but no commercial test for rifaximin resistance exists and the molecular basis of this resistance has not been previously studied in c. difficile. to evaluate whether the rifampin etest would be a suitable substitute for rifaximin susceptibility testing in the clinical setting, we analyzed the in vitro rifaximin susceptib ...200818559647
molecular mechanism underlying differential apoptosis between human melanoma cell lines uacc903 and uacc903(+6) revealed by mitochondria-focused cdna microarrays.human malignant melanoma cell line uacc903 is resistant to apoptosis while chromosome 6-mediated suppressed cell line uacc903(+6) is sensitive. here, we describe identification of differential molecular pathways underlying this difference. using our recently developed mitochondria-focused cdna microarrays, we identified 154 differentially expressed genes including proapoptotic (bak1 [6p21.3], bcap31, bnip1, casp3, casp6, fas, fdx1, fdxr, tnfsf10 and vdac1) and antiapoptotic (bcl2l1, cln3 and mcl ...200818563568
structural modules of rna polymerase required for transcription from promoters containing downstream basal promoter element ggga.we recently described a novel basal bacterial promoter element that is located downstream of the -10 consensus promoter element and is recognized by region 1.2 of the sigma subunit of rna polymerase (rnap). in the case of thermus aquaticus rnap, this element has a consensus sequence ggga and allows transcription initiation in the absence of the -35 element. in contrast, the escherichia coli rnap is unable to initiate transcription from ggga-containing promoters that lack the -35 element. in the ...200818574242
engineering of functional replication protein a homologs based on insights into the evolution of oligonucleotide/oligosaccharide-binding folds.the bacterial single-stranded dna-binding protein (ssb) and the archaeal/eukaryotic functional homolog, replication protein a (rpa), are essential for most aspects of dna metabolism. structural analyses of the architecture of ssb and rpa suggest that they are composed of different combinations of a module called the oligonucleotide/oligosaccharide-binding (ob) fold. members of the domains bacteria and eukarya, in general, contain one type of ssb or rpa. in contrast, organisms in the archaeal dom ...200818586938
physiological and biochemical defects in carboxyl-terminal mutants of mitochondrial dna helicase.mitochondrial dna helicase, also called twinkle, is essential for mtdna maintenance. its helicase domain shares high homology with helicases from superfamily 4. structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to ntp hydrolysis required for translocation and dna unwinding, yet genetic and biochemical information is very limited. here, we evaluate the effects of overexpression in drosophila cell culture of variants carrying a series of deletion ...200818593709
gene 1.7 of bacteriophage t7 confers sensitivity of phage growth to dideoxythymidine.bacteriophage t7 dna polymerase efficiently incorporates dideoxynucleotides into dna, resulting in chain termination. dideoxythymidine (ddt) present in the medium at levels not toxic to escherichia coli inhibits phage t7. we isolated 95 t7 phage mutants that were resistant to ddt. all contained a mutation in t7 gene 1.7, a nonessential gene of unknown function. when gene 1.7 was expressed from a plasmid, t7 phage resistant to ddt still arose; analysis of 36 of these mutants revealed that all had ...200818599435
cellular and molecular effects of nonreciprocal chromosome translocations in saccharomyces cerevisiae.saccharomyces cerevisiae strains harboring a nonreciprocal, bridge-induced translocation (bit) between chromosomes viii and xv exhibited an abnormal phenotype comprising elongated buds and multibudded, unevenly nucleated pseudohyphae. in these cells, we found evidence of molecular effects elicited by the translocation event and specific for its particular genomic location. expression of genes flanking both translocation breakpoints increased up to five times, correlating with an increased rna po ...200818599460
tetraalkylammonium derivatives as real-time pcr enhancers and stabilizers of the qpcr mixtures containing sybr green i.tetraalkylammonium (taa) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (pcr) detected by end-point analysis. in this study, we compared the ability of various taa derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time pcr based on fluorescence detection from intercalating dye sybr green i (sgi). our data i ...200818606615
the last universal common ancestor: emergence, constitution and genetic legacy of an elusive forerunner.since the reclassification of all life forms in three domains (archaea, bacteria, eukarya), the identity of their alleged forerunner (last universal common ancestor or luca) has been the subject of extensive controversies: progenote or already complex organism, prokaryote or protoeukaryote, thermophile or mesophile, product of a protracted progression from simple replicators to complex cells or born in the cradle of "catalytically closed" entities? we present a critical survey of the topic and s ...200818613974
demonstration of a multistep mechanism for assembly of the srp x srp receptor complex: implications for the catalytic role of srp rna.two gtpases in the signal recognition particle (srp) and its receptor (sr) control the delivery of newly synthesized proteins to the endoplasmic reticulum or plasma membrane. during the protein targeting reaction, the 4.5s srp rna accelerates the association between the two gtpases by 400-fold. using fluorescence resonance energy transfer, we demonstrate here that formation of a stable srp x sr complex involves two distinct steps: a fast initial association between srp and sr to form a gtp-indep ...200818617187
distinct double- and single-stranded dna binding of e. coli replicative dna polymerase iii alpha subunit.the alpha subunit of the replicative dna polymerase iii of escherichia coli is the active polymerase of the 10-subunit bacterial replicase. the c-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (ob-fold) domain. in a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded dna, in distinct subdomains of the protein. the portion of the protein that binds to double-stranded dna stabilizes the ...200818652472
mechanism and dynamics of translesion dna synthesis catalyzed by the escherichia coli klenow fragment.translesion dna synthesis represents the ability of a dna polymerase to incorporate and extend beyond damaged dna. in this report, the mechanism and dynamics by which the escherichia coli klenow fragment performs translesion dna synthesis during the misreplication of an abasic site were investigated using a series of natural and non-natural nucleotides. like most other high-fidelity dna polymerases, the klenow fragment follows the "a-rule" of translesion dna synthesis by preferentially incorpora ...200818652487
sequence-specific detection of individual dna polymerase complexes in real time using a nanopore.nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single dna or rna molecules. recently, nanopores have been used to measure the binding of enzymes to their dna substrates. in this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. the force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactio ...200718654412
selective recognition of pyrimidine-pyrimidine dna mismatches by distance-constrained macrocyclic bis-intercalators.binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (esi-ms) and fluorescent intercalator displacement (fid) titrations. the macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas ...200818658249
the proofreading exonuclease subunit epsilon of escherichia coli dna polymerase iii is tethered to the polymerase subunit alpha via a flexible linker.escherichia coli dna polymerase iii holoenzyme is composed of 10 different subunits linked by noncovalent interactions. the polymerase activity resides in the alpha-subunit. the epsilon-subunit, which contains the proofreading exonuclease site within its n-terminal 185 residues, binds to alpha via a segment of 57 additional c-terminal residues, and also to theta, whose function is less well defined. the present study shows that theta greatly enhances the solubility of epsilon during cell-free sy ...200818663010
dual promoters control expression of the bacillus anthracis virulence factor atxa.the atxa virulence regulator of bacillus anthracis is required for toxin and capsule gene expression. atxa is a phosphotransferase system regulatory domain-containing protein whose activity is regulated by phosphorylation/dephosphorylation of conserved histidine residues. here we report that transcription of the atxa gene occurs from two independent promoters, p1 (previously described by dai et al. [z. dai, j. c. sirard, m. mock, and t. m. koehler, mol. microbiol. 16:1171-1181, 1995]) and p2, wh ...200818676674
dysregulated mitochondrial genes and networks with drug targets in postmortem brain of patients with posttraumatic stress disorder (ptsd) revealed by human mitochondria-focused cdna microarrays.posttraumatic stress disorder (ptsd) is associated with decreased activity in the dorsolateral prefrontal cortex (dlpfc), the brain region that regulates working memory and preparation and selection of fear responses. we investigated gene expression profiles in dlpfc brodmann area (ba) 46 of postmortem patients with (n=6) and without ptsd (n=6) using human mitochondria-focused cdna microarrays. our study revealed ptsd-specific expression fingerprints of 800 informative mitochondria-focused genes ...200818690294
insights into the replisome from the structure of a ternary complex of the dna polymerase iii alpha-subunit.the crystal structure of the catalytic alpha-subunit of the dna polymerase iii (pol iiialpha) holoenzyme bound to primer-template dna and an incoming deoxy-nucleoside 5'-triphosphate has been determined at 4.6-a resolution. the polymerase interacts with the sugar-phosphate backbone of the dna across its minor groove, which is made possible by significant movements of the thumb, finger, and beta-binding domains relative to their orientations in the unliganded polymerase structure. additionally, t ...200818691598
heminested reverse-transcriptase polymerase chain reaction (hnrt-pcr) as a tool for rabies virus detection in stored and decomposed samples.the use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. reverse-transcriptase polymerase chain reaction (rt-pcr) has been used in rabies diagnosis with good results, even in decomposed materials. additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology.200818710536
identification of pseudouridine methyltransferase in escherichia coli.in ribosomal rna, modified nucleosides are found in functionally important regions, but their function is obscure. stem-loop 69 of escherichia coli 23s rrna contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and n3 methyl-pseudouridine (m(3)psi) at position 1915. the gene for pseudouridine methyltransferase was previously not known. we identified e. coli protein ybea as the methyltransferase methylating psi1915 in 23s rrna. the e. coli ybea gene deletion strain lacks ...200818755836
biomolecular pleiomorphism probed by spatial interpolation of coarse models.in low resolution structures of biological assemblies one can often observe conformational deviations that require a flexible rearrangement of structural domains fitted at the atomic level. we are evaluating interpolation methods for the flexible alignment of atomic models based on coarse models. spatial interpolation is well established in image-processing and visualization to describe the overall deformation or warping of an object or an image. combined with a coarse representation of the biol ...200818757874
crystal structure of escherichia coli rnk, a new rna polymerase-interacting protein.sequence-based searches identified a new family of genes in proteobacteria, named rnk, which shares high sequence similarity with the c-terminal domains of the gre factors (grea and greb) and the thermus/deinococcus anti-gre factor gfh1. we solved the x-ray crystal structure of escherichia coli regulator of nucleoside kinase (rnk) at 1.9 a resolution using the anomalous signal from the native protein. the rnk structure strikingly resembles those of e. coli grea and greb and thermus gfh1, all of ...200818760284
a fundamental study of the pcr amplification of gc-rich dna templates.a theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient pcr amplification of gc-rich templates. the analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency (eta) of annealing as a function of temperature (t(a)), annealing period (t(a)), and template composition. the optimal efficiency lies in a n ...200818760969
enzymes used in molecular biology: a useful guide.since molecular cloning has become routine laboratory technique, manufacturers offer countless sources of enzymes to generate and manipulate nucleic acids. thus, selecting the appropriate enzyme for a specific task may seem difficult to the novice. this review aims at providing the readers with some cues for understanding the function and specificities of the different sources of polymerases, ligases, nucleases, phosphatases, methylases, and topoisomerases used for molecular cloning. we provide ...200818766469
the role of nucleotide cofactor binding in cooperativity and specificity of muts recognition.mismatch repair (mmr) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. the critical first step in escherichia coli mmr is the specific recognition and binding of muts to a heteroduplex, containing either a mismatch or an insertion/deletion loop of up to four nucleotides. all known muts homologs recognize a similar broad spectrum of substrates. binding and hydrolysis of nucleotide cofactors by the muts-heteroduplex ...200818773911
mechanism of the chemical step for the guanosine triphosphate (gtp) hydrolysis catalyzed by elongation factor tu.elongation factor tu (ef-tu), the protein responsible for delivering aminoacyl-trnas (aa-trnas) to ribosomal a site during translation, belongs to the group of guanosine-nucleotide (gtp/gdp) binding proteins. its active 'on'-state corresponds to the gtp-bound form, while the inactive 'off'-state corresponds to the gdp-bound form. in this work we focus on the chemical step, gtp+h(2)o-->gdp+pi, of the hydrolysis mechanism. we apply molecular modeling tools including molecular dynamics simulations ...200818773979
rifamycins do not function by allosteric modulation of binding of mg2+ to the rna polymerase active center.rifamycin antibacterial agents inhibit bacterial rna polymerase (rnap) by binding to a site adjacent to the rnap active center and preventing synthesis of rna products >2-3 nt in length. recently, artsimovitch et al. [(2005) cell 122:351-363] proposed that rifamycins function by allosteric modulation of binding of mg(2+) to the rnap active center and presented three lines of biochemical evidence consistent with this proposal. here, we show that rifamycins do not affect the affinity of binding of ...200818787125
unique substrate spectrum and pcr application of nanoarchaeum equitans family b dna polymerase.the known archaeal family b dna polymerases are unable to participate in the pcr in the presence of uracil. here, we report on a novel archaeal family b dna polymerase from nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to pcr. n. equitans family b dna polymerase (neq dna polymerase) produced lambda dna fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 x 10(-6)) than taq dna polymerase (11.98 x ...200818791030
hot start pcr with heat-activatable primers: a novel approach for improved pcr performance.the polymerase chain reaction (pcr) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. great improvements to pcr performance have been achieved by the use of hot start activation strategies that aim to prevent dna polymerase extension until more stringent, higher temperatures are reached. herein we present a novel hot start activation approach in pcr where primers con ...200818796527
effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the y-family dna polymerases.y-family dna polymerases carry out translesion synthesis past damaged dna. dna polymerases (pol) eta and iota are usually uniformly distributed through the nucleus but accumulate in replication foci during s phase. dna-damaging treatments result in an increase in s phase cells containing polymerase foci. using photobleaching techniques, we show that poleta is highly mobile in human fibroblasts. even when localized in replication foci, it is only transiently immobilized. although ubiquitination o ...200818799611
the rna-binding domain of bacteriophage p22 n protein is highly mutable, and a single mutation relaxes specificity toward lambda.antitermination in bacteriophage p22, a lambdoid phage, uses the arginine-rich domain of the n protein to recognize boxb rnas in the nut site of two regulated transcripts. using an antitermination reporter system, we screened libraries in which each nonconserved residue in the rna-binding domain of p22 n was randomized. mutants were assayed for the ability to complement n-deficient virus and for antitermination with p22 boxb(left) and boxb(right) reporters. single amino acid substitutions comple ...200818820025
the mycoplasma pneumoniae mpn229 gene encodes a protein that selectively binds single-stranded dna and stimulates recombinase a-mediated dna strand exchange.mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. in spite of this genetic stability, homologous dna recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, p1, of m. pneumoniae. in order to identify the proteins that may be involved in homologous dna recombination in m. pneumoniae, we set out to characterize the mpn229 open reading frame (orf), which bears sequence similarity to the ...200818831760
rapid screening assay for kras mutations by the modified smart amplification process.previously, the smart amplification process version 2 (smap-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. the purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the smap-2 assay. we developed modified smap-2 assays that enabled detection of any change in a single codon using a single assay. rapid smap- ...200818832461
a new family of polymerases related to superfamily a dna polymerases and t7-like dna-dependent rna polymerases.using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain dna viruses, including the recently reported sputnik virus. using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and dna polymerase activity, comparable to the previously reported prim-po ...200818834537
site-specific labeling of t7 dna polymerase with a conformationally sensitive fluorophore and its use in detecting single-nucleotide polymorphisms.like most enzymes, dna polymerases undergo a large conformational change on the binding of a correct nucleotide. to determine how the conformational change contributes to substrate specificity, we labeled the t7 dna polymerase with a conformationally sensitive fluorophore at a position that provides a signal coincident with structural changes following nucleotide binding and distinguishes correct base pairs from incorrect ones by the sign of the fluorescence change. here we describe methods to d ...200818834851
site-specific labeling of t7 dna polymerase with a conformationally sensitive fluorophore and its use in detecting single-nucleotide polymorphisms.like most enzymes, dna polymerases undergo a large conformational change on the binding of a correct nucleotide. to determine how the conformational change contributes to substrate specificity, we labeled the t7 dna polymerase with a conformationally sensitive fluorophore at a position that provides a signal coincident with structural changes following nucleotide binding and distinguishes correct base pairs from incorrect ones by the sign of the fluorescence change. here we describe methods to d ...200818834851
differential accumulation of retroelements and diversification of nb-lrr disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean.the genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. the impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. we sequenced an approximately 1 million-bp region in soybean (glycine max) centered on the rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. these two regions were also compared with homologous regions in several related ...200818842825
unnatural substrate repertoire of a, b, and x family dna polymerases.as part of an effort to develop unnatural base pairs that are stable and replicable in dna, we examined the ability of five different polymerases to replicate dna containing four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs. the unnatural pairs were developed based on intensive studies using the klenow fragment of dna polymerase i from e. coli (kf) and found to be recognized to varying degrees. the five additional polymerases characterized here include fam ...200818847263
mechanism of muts searching for dna mismatches and signaling repair.dna mismatch repair is initiated by the recognition of mismatches by muts proteins. the mechanism by which muts searches for and recognizes mismatches and subsequently signals repair remains poorly understood. we used single-molecule analyses of atomic force microscopy images of muts-dna complexes, coupled with biochemical assays, to determine the distributions of conformational states, the dna binding affinities, and the atpase activities of wild type and two mutants of muts, with alanine subst ...200818854319
the diagnosis of genital herpes - beyond culture: an evidence-based guide for the utilization of polymerase chain reaction and herpes simplex virus type-specific serology.accurate identification of persons with genital herpes is necessary for optimal patient management and prevention of transmission. because of inherent inaccuracies, clinical diagnosis of genital herpes should be confirmed by laboratory testing for the causative agents herpes simplex virus type 1 (hsv-1) and hsv type 2 (hsv-2). further identification of the hsv type is valuable for counselling on the natural history of infection and risk of transmission. laboratory methods include antigen detecti ...200718923735
replisome dynamics and use of dna trombone loops to bypass replication blocks.replisomes are dynamic multiprotein machines capable of simultaneously replicating both strands of the dna duplex. this review focuses on the structure and function of the e. coli replisome, many features of which generalize to other bacteria and eukaryotic cells. for example, the bacterial replisome utilizes clamps and clamp loaders to coordinate the actions required of the trombone model of lagging strand synthesis made famous by bruce alberts. all cells contain clamps and clamp loaders and th ...200818931783
ssb as an organizer/mobilizer of genome maintenance complexes.when duplex dna is altered in almost any way (replicated, recombined, or repaired), single strands of dna are usually intermediates, and single-stranded dna binding (ssb) proteins are present. these proteins have often been described as inert, protective dna coatings. continuing research is demonstrating a far more complex role of ssb that includes the organization and/or mobilization of all aspects of dna metabolism. escherichia coli ssb is now known to interact with at least 14 other proteins ...200818937104
a full-length group 1 bacterial sigma factor adopts a compact structure incompatible with dna binding.the sigma factors are the key regulators of bacterial transcription initiation. through direct read-out of promoter dna sequence, they recruit the core rna polymerase to sites of initiation, thereby dictating the rna polymerase promoter-specificity. the group 1 sigma factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are autoregulated by an n-terminal sequence known as sigma1.1. we report the solution structure of thermoto ...200818940669
in vitro approaches to analysis of transcription termination.transcription termination is an important event in the transcription cycle that has been exploited in a variety of genetic regulatory mechanisms. analysis of transcription termination is greatly facilitated by in vitro approaches. we describe a basic protocol for analysis of transcription termination in vitro, and include descriptions of parameters that can be modified for specific types of experimental questions.200818948199
in vitro approaches to analysis of transcription termination.transcription termination is an important event in the transcription cycle that has been exploited in a variety of genetic regulatory mechanisms. analysis of transcription termination is greatly facilitated by in vitro approaches. we describe a basic protocol for analysis of transcription termination in vitro, and include descriptions of parameters that can be modified for specific types of experimental questions.200818948199
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