Publications
| Title | Abstract | Year(sorted ascending) Filter | PMID Filter |
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| protective effect of avian myelomonocytic growth factor in infection with marek's disease virus. | marek's disease virus (mdv) is a herpesvirus that induces t lymphomas in chickens. the aim of this study was to assess the role of the macrophage activator chicken myelomonocytic growth factor (cmgf) in controlling mdv infection. b13/b13 chickens, which are highly susceptible to md, were either treated with cmgf delivered via a live fowlpox virus (fp/cmgf) or treated with the parent vector (fp/m3) or were left as untreated controls. seven days later, when challenged with the very virulent rb-1b ... | 2002 | 11773382 |
| demystified... molecular pathology in oncology. | in the past 10 years, molecular biology has found major applications in pathology, particularly in oncology. this has been a field of enormous expansion, where pure science has found a place in clinical practice and is now of everyday use in any academic unit. this demystified review will discuss the techniques used in molecular pathology and then provide examples of how these can be used in oncology. | 2002 | 12456768 |
| tumor necrosis factor gene polymorphisms, leukocyte function, and sepsis susceptibility in blunt trauma patients. | the tumor necrosis factor alpha (tnf-alpha) -308 g/a and tnf-beta nco1 polymorphisms have been described to be associated with an increased risk for sepsis in critically ill patients. functional consequences associated with these polymorphisms remain unclear. we compared the genotype distribution of these tnf polymorphisms with susceptibility to severe sepsis and leukocyte function in blunt trauma patients (n = 70; mean injury severity score, 24 points [range, 4 to 57). severe sepsis was defined ... | 2002 | 12414751 |
| estimating the number of integrations in transformed plants by quantitative real-time pcr. | when generating transformed plants, a first step in their characterization is to obtain, for each new line, an estimate of how many copies of the transgene have been integrated in the plant genome because this can deeply influence the level of transgene expression and the ease of stabilizing expression in following generations. this task is normally achieved by southern analysis, a procedure that requires relatively large amounts of plant material and is both costly and labour-intensive. moreove ... | 2002 | 12398792 |
| helix-hairpin-helix motifs confer salt resistance and processivity on chimeric dna polymerases. | helix-hairpin-helix (hhh) is a widespread motif involved in sequence-nonspecific dna binding. the majority of hhh motifs function as dna-binding modules with typical occurrence of one hhh motif or one or two (hhh)(2) domains in proteins. we recently identified 24 hhh motifs in dna topoisomerase v (topo v). although these motifs are dispensable for the topoisomerase activity of topo v, their removal narrows the salt concentration range for topoisomerase activity tenfold. here, we demonstrate the ... | 2002 | 12368475 |
| ifret: an improved fluorescence system for dna-melting analysis. | fluorescence resonance energy transfer (fret) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of dna hybridization status. the process involves measuring changes in fluorescence as fret donor and acceptor moieties are brought closer together or moved farther apart as a result of dna hybridization/denaturation. in the present study, we introduce a new version of fret, which we term induced fret (ifret), that is ideally suited for mel ... | 2002 | 12213777 |
| microbial communities from methane hydrate-bearing deep marine sediments in a forearc basin. | microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the nankai trough near japan were characterized with cultivation-dependent and -independent techniques. acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial ... | 2002 | 12147470 |
| interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of dna binding. | previous structural studies on native t5 5' nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of dna binding was proposed in which single-stranded dna threads through the archway. we investigated the kinetic and substrate-binding characteristics of wild-type and ... | 2002 | 12084915 |
| p53 mutation in breast cancer. correlation with cell kinetics and cell of origin. | several studies have investigated the expression of the cytokeratins (cks), vimentin, the epithelial growth factor receptor (egfr), the oestrogen receptor (er), and the progesterone receptor (pgr), in breast cancer, but no study has directly compared p53 mutations with these phenotypic and differentiation markers in the same case. the present study was designed to provide some of this information. | 2002 | 12037031 |
| 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 |
| phi29 dna polymerase residues tyr59, his61 and phe69 of the highly conserved exoii motif are essential for interaction with the terminal protein. | phage phi29 encodes a dna-dependent dna polymerase belonging to the eukaryotic-type (family b) subgroup of dna polymerases that use a protein as the primer for initiation of dna synthesis. in one of the most important motifs present in the 3'-->5' exonucleolytic domain of proofreading dna polymerases, the exoii motif, phi29 dna polymerase contains three amino acid residues, y59, h61 and f69, which are highly conserved among most proofreading dna polymerases. these residues have recently been sho ... | 2002 | 11884636 |
| real-time pcr in virology. | the use of the polymerase chain reaction (pcr) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. real-time pcr has engendered wider acceptance of the pcr due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. there are currently five main chemistries used for the detection of pcr ... | 2002 | 11884626 |
| auto-protective redox buffering systems in stimulated macrophages. | macrophages, upon encounter with micro-organisms or stimulated by cytokines, produce various effector molecules aimed at destroying the foreign agents and protecting the organism. reactive oxygen species (ros) and reactive nitrogen species (rns) are front line molecules exerting strong cytotoxic activities against micro-organisms and many cells, including macrophages themselves. using cells of the murine macrophage cell line (raw 264.7) stimulated in vitro with lipopolysaccharide (lps) and/or in ... | 2002 | 11914132 |
| marking the start site of rna polymerase iii transcription: the role of constraint, compaction and continuity of the transcribed dna strand. | the effects of breaks in the individual strands of an rna polymerase iii promoter on initiation of transcription have been examined. single breaks have been introduced at 2 bp intervals in a 24 bp segment that spans the transcriptional start site of the u6 snrna gene promoter. their effects on transcription are asymmetrically distributed: transcribed (template) strand breaks downstream of bp-14 (relative to the normal start as +1) systematically shift the start site, evidently by disrupting the ... | 2002 | 11847118 |
| thermal acclimation and stress in the american lobster, homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitin. | using homologous molecular probes, we examined the influence of equivalent temperature shifts on the in vivo expression of genes coding for a constitutive heat shock protein (hsc70), heat shock proteins (hsps) (hsp70 and hsp90), and polyubiquitin, after acclimation in the american lobster, homarus americanus. we acclimated sibling, intermolt, juvenile male lobsters to thermal regimes experienced during overwintering conditions (0.4 +/- 0.3 degrees c), and to ambient pacific ocean temperatures (1 ... | 2002 | 11892992 |
| the phage n4 virion rna polymerase catalytic domain is related to single-subunit rna polymerases. | in vitro, bacteriophage n4 virion rna polymerase (vrnap) recognizes in vivo sites of transcription initiation on single-stranded templates. n4 vrnap promoters are comprised of a hairpin structure and conserved sequences. here, we show that vrnap consists of a single 3500 amino acid polypeptide, and we define and characterize a transcriptionally active 1106 amino acid domain (mini-vrnap). biochemical and genetic characterization of this domain indicates that, despite its peculiar promoter specifi ... | 2002 | 12411499 |
| a phylogenomic approach to bacterial phylogeny: evidence of a core of genes sharing a common history. | it has been claimed that complete genome sequences would clarify phylogenetic relationships between organisms, but up to now, no satisfying approach has been proposed to use efficiently these data. for instance, if the coding of presence or absence of genes in complete genomes gives interesting results, it does not take into account the phylogenetic information contained in sequences and ignores hidden paralogies by using a blast reciprocal best hit definition of orthology. in addition, concaten ... | 2002 | 12097345 |
| extremophiles 2002. | 2003 | 12813059 | |
| evolutionary connection between the catalytic subunits of dna-dependent rna polymerases and eukaryotic rna-dependent rna polymerases and the origin of rna polymerases. | the eukaryotic rna-dependent rna polymerase (rdrp) is involved in the amplification of regulatory micrornas during post-transcriptional gene silencing. this enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. no evolutionary relationship between rdrp and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery. | 2003 | 12553882 |
| cleavage of dna without loss of genetic information by incorporation of a disaccharide nucleoside. | a ribose residue inserted between the 3'-oh of one nucleotide and the 5'-phosphate group of the next nucleotide, functions as a site-specific cleavage site within dna. this extra ribose does not interrupt helix formation and it protects duplex dna against cleavage by restriction enzymes. cleavage can be obtained with periodate and all ribose fragments can be removed with sodium hydroxide. as a result of this, an intact natural oligodeoxynucleotide is obtained after ligation reaction, which means ... | 2003 | 14627809 |
| dissimilar mispair-recognition spectra of arabidopsis dna-mismatch-repair proteins msh2*msh6 (mutsalpha) and msh2*msh7 (mutsgamma). | besides orthologs of other eukaryotic mismatch-repair (mmr) proteins, plants encode msh7, a paralog of msh6. the arabidopsis thaliana recognition heterodimers atmsh2*msh6 (atmutsalpha) and atmsh2*msh3 (atmutsbeta) were previously found to bind the same subsets of mismatches as their counterparts in other eukaryotes--respectively, base-base mismatches and single extra nucleotides, loopouts of extra nucleotides (one or more) only--but atmsh2*msh7 (atmutsgamma) bound well only to a g/t mismatch. to ... | 2003 | 14530450 |
| methods for the in vitro determination of an individual disposition towards th1- or th2-reactivity by the application of appropriate stimulatory antigens. | in this study we performed several methods for the determination of cytokines (rt-pcr for the demonstration of cytokine mrna and flow cytometry for the analysis of intracellular cytokines) and compared them with a recently established test system stimulating peripheral blood mononuclear cells (pbmc) with th1- and th2-relevant recall antigens and analysing type 1 and type 2 cytokines by elisa. aim of the study was therefore to evaluate the reliability of th1/th2 cytokine profiles in two individua ... | 2003 | 12974758 |
| expression of cyclins a, e and topoisomerase ii alpha correlates with centrosome amplification and genomic instability and influences the reliability of cytometric s-phase determination. | the progression of normal cells through the cell cycle is meticulously regulated by checkpoints guaranteeing the exact replication of the genome during s-phase and its equal division at mitosis. a prerequisite for this achievement is synchronized dna-replication and centrosome duplication. in this context the expression of cyclins a and e has been shown to play a principal role. | 2003 | 12875657 |
| structure, function and evolution of the signal recognition particle. | the signal recognition particle (srp) is a ribonucleoprotein particle essential for the targeting of signal peptide-bearing proteins to the prokaryotic plasma membrane or the eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. srp binds to the signal peptide emerging from the exit site of the ribosome and forms a ribosome nascent chain (rnc)-srp complex. the rnc-srp complex then docks in a gtp-dependent manner with a membrane-anchored srp receptor and the protein is tr ... | 2003 | 12853463 |
| many paths to methyltransfer: a chronicle of convergence. | s-adenosyl-l-methionine (adomet) dependent methyltransferases (mtases) are involved in biosynthesis, signal transduction, protein repair, chromatin regulation and gene silencing. five different structural folds (i-v) have been described that bind adomet and catalyze methyltransfer to diverse substrates, although the great majority of known mtases have the class i fold. even within a particular mtase class the amino-acid sequence similarity can be as low as 10%. thus, the structural and catalytic ... | 2003 | 12826405 |
| nucleic acid recognition by ob-fold proteins. | the ob-fold domain is a compact structural motif frequently used for nucleic acid recognition. structural comparison of all ob-fold/nucleic acid complexes solved to date confirms the low degree of sequence similarity among members of this family while highlighting several structural sequence determinants common to most of these ob-folds. loops connecting the secondary structural elements in the ob-fold core are extremely variable in length and in functional detail. however, certain features of l ... | 2003 | 12598368 |
| organ-specific expression of brassinosteroid-biosynthetic genes and distribution of endogenous brassinosteroids in arabidopsis. | brassinosteroids (brs) are steroidal plant hormones that are essential for growth and development. there is only limited information on where brs are synthesized and used. we studied the organ specificity of br biosynthesis in arabidopsis, using two different approaches: we analyzed the expression of br-related genes using real-time quantitative reverse transcriptase-polymerase chain reaction, and analyzed endogenous brs using gas chromatography-mass spectrometry. before starting this study, we ... | 2003 | 12529536 |
| identification of lpea, a psaa-like membrane protein that promotes cell entry by listeria monocytogenes. | the intracellular life of listeria monocytogenes starts by a complex process of entry involving several bacterial ligands and eukaryotic receptors. in this work, we identified in silico from the sequence of the genome of l. monocytogenes a previously unknown gene designated lpea (for lipoprotein promoting entry) encoding a 35-kda protein homologous to psaa, a lipoprotein belonging to the lrai family and implicated in the cell adherence of streptococcus pneumoniae and related species. by construc ... | 2003 | 12496198 |
| mutational analysis of the chlamydia trachomatis dnak promoter defines the optimal -35 promoter element. | a long-standing question in the biology of the intracellular bacterium, chlamydia, has been the structure of the promoter recognized by its rna polymerase. the 'rna polymerase sigma subunit paradox' refers to the difficulty reconciling the conservation between the rna polymerases of chlamydia and escherichia coli, especially at the level of the promoter-recognition sigma subunit, with the general lack of homology between chlamydial promoters and the e.coli sigma(70) consensus promoter. while the ... | 2003 | 12527761 |
| the sigma70 family of sigma factors. | members of the sigma70 family of sigma factors are components of the rna polymerase holoenzyme that direct bacterial or plastid core rna polymerase to specific promoter elements that are situated 10 and 35 base-pairs upstream of transcription-initiation points. members of the sigma70 family also function as contact points for some activator proteins, such as phob and lambda(cl), and play a role in the initiation process itself. the primary sigma factor, which is essential for general transcripti ... | 2003 | 12540296 |
| expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis. | oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. in addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. here, we studied both lipoxygenase pathways in human atherosclerosis. the 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients ... | 2003 | 12552108 |
| site-specific protein modification to identify the mutl interface of muth. | we have mapped the region for the protein interaction site of the escherichia coli mismatch repair protein muth for its activator protein mutl by a site-specific protein modification approach. for this purpose we generated a cysteine-free variant of muth and 12 variants thereof, each containing a single cysteine residue at surface positions selected on the basis of available structural and sequence information for muth. all muth variants displayed wild type activity both in vivo and in vitro. th ... | 2003 | 12560476 |
| whole genome analysis of genetic alterations in small dna samples using hyperbranched strand displacement amplification and array-cgh. | structural genetic alterations in cancer often involve gene loss or gene amplification. with the advent of microarray approaches for the analysis of the genome, as exemplified by array-cgh (comparative genomic hybridization), scanning for gene-dosage alterations is limited only by issues of dna microarray density. however, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient dna for array-cgh analysis. we sought to deve ... | 2003 | 12566408 |
| the feci extracytoplasmic-function sigma factor of escherichia coli interacts with the beta' subunit of rna polymerase. | transcription of the ferric citrate transport system of escherichia coli k-12 is mediated by the extracytoplasmic-function (ecf) sigma factor feci, which is activated by ferric citrate in the growth medium. by using a bacterial two-hybrid system, it was shown in vivo that feci binds to the beta' subunit of rna polymerase. the inactive mutant protein feci(k155e) displayed reduced binding to beta', and small deletions along the entire feci protein led to total impairment of beta' binding. in vitro ... | 2003 | 12618442 |
| luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial rna polymerase. | the binding of sigma factors to core rna polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic rna polymerases, sigma factor binding is a promising target for drug discovery. a homogeneous assay for sigma binding to rna polymerase (escherichia coli) based on luminescence resonance energy transfer (lret) ... | 2003 | 12620834 |
| leucine biosynthesis in fungi: entering metabolism through the back door. | after exploring evolutionary aspects of branched-chain amino acid biosynthesis, the review focuses on the extended leucine biosynthetic pathway as it operates in saccharomyces cerevisiae. first, the genes and enzymes specific for the leucine pathway are considered: leu4 and leu9 (encoding the alpha-isopropylmalate synthase isoenzymes), leu1 (isopropylmalate isomerase), and leu2 (beta-isopropylmalate dehydrogenase). emphasis is given to the unusual distribution of the branched-chain amino acid pa ... | 2003 | 12626680 |
| increased rectal mucosal expression of interleukin 1beta in recently acquired post-infectious irritable bowel syndrome. | background and aims: chronic bowel disturbances resembling irritable bowel syndrome (ibs) develop in approximately 25% of patients after an episode of infectious diarrhoea. although we have previously shown that psychosocial factors operating at the time of, or prior to, the acute illness appear to predict the development of post-infectious ibs (pi-ibs), our finding of an increased inflammatory cell number in the rectum persisting for at least three months after the acute infection suggested tha ... | 2003 | 12631663 |
| processive dna synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations. | dna polymerases replicate dna by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations. here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable bacillus dna polymerase i. the 6-bp extension involves a 20-a translocation of the dna duplex, representing the largest molecular movement observed in a protein crystal. in addition, we obtained the structure of ... | 2003 | 12649320 |
| high-efficiency generation of antibiotic-resistant strains of streptococcus pneumoniae by pcr and transformation. | we designed a method by which to generate antibiotic-resistant strains of streptococcus pneumoniae at frequencies 4 orders of magnitude greater than the spontaneous mutation rate. the method is based on the natural ability of this organism to be genetically transformed with pcr products carrying sequences homologous to its chromosome. the genes encoding the targets of ciprofloxacin (parc, encoding the parc subunit of dna topoisomerase iv), rifampin (rpob, encoding the beta subunit of rna polymer ... | 2003 | 12654655 |
| induced nucleotide specificity in a gtpase. | in signal-recognition particle (srp)-dependent protein targeting to the bacterial plasma membrane, two gtpases, ffh (a subunit of the bacterial srp) and ftsy (the bacterial srp receptor), act as gtpase activating proteins for one another. the molecular mechanism of this reciprocal gtpase activation is poorly understood. in this work, we show that, unlike other gtpases, free ftsy exhibits only low preference for gtp over other nucleotides. on formation of the srp.ftsy complex, however, the nucleo ... | 2003 | 12663860 |
| methionine regeneration and aminotransferases in bacillus subtilis, bacillus cereus, and bacillus anthracis. | the conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the ia subfamily (l. c. berger, j. wilson, p. wood, and b. j. berger, j. bacteriol. 183:4421-4434, 2001). the genome of bacillus subtilis has been found to contain no subfamily ia aminotransferase sequences. instead, the analogous enzymes in b. subtilis were found to be members of the if subfamily. th ... | 2003 | 12670965 |
| structural basis of replication origin recognition by the dnaa protein. | escherichia coli dnaa binds to 9 bp sequences (dnaa boxes) in the replication origin, oric, to form a complex initiating chromosomal dna replication. in the present study, we determined the crystal structure of its dna-binding domain (domain iv) complexed with a dnaa box at 2.1 a resolution. dnaa domain iv contains a helix-turn-helix motif for dna binding. one helix and a loop of the helix- turn-helix motif are inserted into the major groove and 5 bp (3' two-thirds of the dnaa box sequence) are ... | 2003 | 12682358 |
| yeast dna polymerase eta makes functional contacts with the dna minor groove only at the incoming nucleoside triphosphate. | dna polymerase eta (pol eta) functions in the proficient bypass of a variety of dna lesions. relative to the replicative polymerases, pol eta has a greater tolerance for distorted dna geometries and possesses a low fidelity. x-ray crystal structures and studies with nucleotide analogs have implicated interactions with the dna minor groove as being crucial for the high fidelity of replicative dna polymerases. to determine whether pol eta also makes such functionally important contacts with the dn ... | 2003 | 12692307 |
| structure-function studies of escherichia coli rpoh (sigma32) by in vitro linker insertion mutagenesis. | the sigma factor rpoh (sigma(32)) is the key regulator of the heat shock response in escherichia coli. many structural and functional properties of the sigma factor are poorly understood. to gain further insight into rpoh regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique. thirty-one distinct insertions were obtained, and their sigma fact ... | 2003 | 12700252 |
| the signal recognition particle binds to protein l23 at the peptide exit of the escherichia coli ribosome. | the signal recognition particle (srp) from escherichia coli, composed of ffh protein and 4.5s rna, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. srp binds at the peptide exit of the large ribosomal subunit. structural details of the interaction are not known. here, the position of ffh or srp on the ribosome was probed by using site-specific uv-induced crosslinking by p-azidophenacyl bromide (azp) attached to a number of cysteine residues engi ... | 2003 | 12702815 |
| an unusual mechanism of bacterial gene expression revealed for the rnase p protein of thermus strains. | the rnase p protein gene (rnpa) completely overlaps the rpmh gene (encoding ribosomal protein l34) out of frame in the thermophilic bacterium thermus thermophilus. this results in the synthesis of an extended rnase p protein (c5) of 163 aa and, by inference, of 240 aa in the related strain thermus filiformis. start codons of rnpa and rpmh, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between l34 and c5 translation and, acc ... | 2003 | 12719542 |
| rna-structural mimicry in escherichia coli ribosomal protein l4-dependent regulation of the s10 operon. | ribosomal protein l4 regulates the 11-gene s10 operon in escherichia coli by acting, in concert with transcription factor nusa, to cause premature transcription termination at a rho-independent termination site in the leader sequence. this process presumably involves l4 interaction with the leader mrna. here, we report direct, specific, and independent binding of ribosomal protein l4 to the s10 mrna leader in vitro. most of the binding energy is contributed by a small hairpin structure within th ... | 2003 | 12738792 |
| rna polymerase mutations that impair conversion to a termination-resistant complex by q antiterminator proteins. | bacteriophage lambda q-protein stably binds and modifies rna polymerase (rnap) to a termination-resistant form. we describe amino acid substitutions in rnap that disrupt q-mediated antitermination in vivo and in vitro. the positions of these substitutions in the modeled rnap/dna/rna ternary elongation complex, and their biochemical properties, suggest that they do not define a binding site for q in rnap, but instead act by impairing interactions among core rnap subunits and nucleic acids that ar ... | 2003 | 12756229 |
| an intersubunit contact stimulating transcription initiation by e coli rna polymerase: interaction of the alpha c-terminal domain and sigma region 4. | the c-terminal domain of the escherichia coli rna polymerase (rnap) alpha subunit (alphactd) stimulates transcription initiation by interacting with upstream (up) element dna and a variety of transcription activators. here we identify specific substitutions in region 4.2 of sigma 70 (sigma(70)) and in alphactd that decrease transcription initiation from promoters containing some, but not all, up elements. this decrease in transcription derives from a decrease in the initial equilibrium constant ... | 2003 | 12756230 |
| comparative thermal denaturation of thermus aquaticus and escherichia coli type 1 dna polymerases. | thermal denaturations of the type 1 dna polymerases from thermus aquaticus (taq polymerase) and escherichia coli (pol 1) have been examined using differential scanning calorimetry and cd spectroscopy. the full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in taq) and a 5' nuclease domain. removal of the 5' nuclease domains produces the 'large fragment' domains of pol 1 and taq, termed klenow and klentaq respectively. although the hi ... | 2003 | 12786603 |
| limitations of taqman pcr for detecting divergent viral pathogens illustrated by hepatitis a, b, c, and e viruses and human immunodeficiency virus. | recent events illustrate the imperative to rapidly and accurately detect and identify pathogens during disease outbreaks, whether they are natural or engineered. particularly for our primary goal of detecting bioterrorist releases, detection techniques must be both species-wide (capable of detecting all known strains of a given species) and species specific. due to classification restrictions on the publication of data for species that may pose a bioterror threat, we illustrate the challenges of ... | 2003 | 12791858 |
| prevalence of human t-lymphotropic virus type 1 among blood donors from mashhad, iran. | the city of mashhad is the capital of khorasan, the northeastern province of iran, which has been recognized as an area where human t-lymphotropic virus type 1 (htlv-1) infection is endemic. all serum samples from blood donors are routinely screened for htlv-1 by using enzyme-linked immunosorbent assay (elisa). in the present study, 28,926 donors (81.86% male and 18.14% female) with a mean age of 32 years (range, 18 to 65 years) were screened in a 6 months period (july to december 1999). of thes ... | 2003 | 12791885 |
| identification and characterization of transposable elements of paracoccus pantotrophus. | we studied diversity and distribution of transposable elements residing in different strains (dsm 11072, dsm 11073, dsm 65, and lmd 82.5) of a soil bacterium paracoccus pantotrophus (alpha-proteobacteria). with application of a shuttle entrapment vector pmec1, several novel insertion sequences (iss) and transposons (tns) have been identified. they were sequenced and subjected to detailed comparative analysis, which allowed their characterization (i.e., identification of transposase genes, termin ... | 2003 | 12813068 |
| ralstonia eutropha h16 encodes two and possibly three intracellular poly[d-(-)-3-hydroxybutyrate] depolymerase genes. | intracellular poly[d-(-)-3-hydroxybutyrate] (phb) depolymerases degrade phb granules to oligomers and monomers of 3-hydroxybutyric acid. recently an intracellular phb depolymerase gene (phaz1) from ralstonia eutropha was identified. we now report identification of candidate phb depolymerase genes from r. eutropha, namely, phaz2 and phaz3, and their characterization in vivo. phaz1 was used to identify two candidate depolymerase genes in the genome of ralstonia metallidurans. phaz1 and these genes ... | 2003 | 12813072 |
| mismatch recognition-coupled stabilization of msh2-msh6 in an atp-bound state at the initiation of dna repair. | mismatch repair proteins correct errors in dna via an atp-driven process. in eukaryotes, the msh2-msh6 complex recognizes base pair mismatches and small insertion/deletions in dna and initiates repair. both msh2 and msh6 proteins contain walker atp-binding motifs that are necessary for repair activity. to understand how these proteins couple atp binding and hydrolysis to dna binding/mismatch recognition, the atpase activity of saccharomyces cerevisiae msh2-msh6 was examined under pre-steady-stat ... | 2003 | 12820877 |
| pilin-like proteins in the extremely thermophilic bacterium thermus thermophilus hb27: implication in competence for natural transformation and links to type iv pilus biogenesis. | the extreme thermophile thermus thermophilus hb27 exhibits high frequencies of natural transformation. although we recently reported identification of the first competence genes in thermus, the molecular basis of dna uptake is unknown. a pilus-like structure is assumed to be involved. twelve genes encoding prepilin-like proteins were identified in three loci in the genome of t. thermophilus. mutational analyses, described in this paper, revealed that one locus, which contains four genes that enc ... | 2003 | 12839734 |
| processing of dna lesions by archaeal dna polymerases from sulfolobus solfataricus. | spontaneous damage to dna as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80 degrees c are endowed with powerful dna repair mechanisms to maintain genome stability. of particular interest is the processing of dna lesions during replication, which can result in fixed mutations. the hyperthermophilic crenarchaeon sulfolobus solfataricus has two functional dna polymerase ... | 2003 | 12853619 |
| assessing functional divergence in ef-1alpha and its paralogs in eukaryotes and archaebacteria. | a number of methods have recently been published that use phylogenetic information extracted from large multiple sequence alignments to detect sites that have changed properties in related protein families. in this study we use such methods to assess functional divergence between eukaryotic ef-1alpha (eef-1alpha), archaebacterial ef-1alpha (aef-1alpha) and two eukaryote-specific ef-1alpha paralogs-eukaryotic release factor 3 (erf3) and hsp70 subfamily b suppressor 1 (hbs1). overall, the evolutio ... | 2003 | 12853641 |
| a new thermus sp. class-iis enzyme sub-family: isolation of a 'twin' endonuclease tspdti with a novel specificity 5'-atgaa(n(11/9))-3', related to tspgwi, taqii and tth111ii. | the tspdti restriction endonuclease, which shows a novel recognition specificity 5'-atgaa(n(11/9))-3', was isolated from thermus sp. dt. tspdti appears to be a 'twin' of restriction endonuclease tspgwi from thermus sp. gw, as we have previously reported. tspgwi was isolated from the same location as tspdti, it recognizes a related sequence 5'-acgga(n(11/9))-3' and has conserved cleavage positions. both enzymes resemble two other class-iis endonucleases from thermus sp.: taqii and tth111ii. n-ter ... | 2003 | 12853651 |
| identification of the agr locus of listeria monocytogenes: role in bacterial virulence. | listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. we have recently adapted signature-tagged transposon mutagenesis (stm) to identify genes involved in the virulence of l. monocytogenes. a new round of stm allowed us to identify a new locus encoding a protein homologous to agra, the well-studied response regulator of staphylococcus aureus and part of a two-component system involved in bacterial virulence ... | 2003 | 12874326 |
| lack of sugar discrimination by human pol mu requires a single glycine residue. | dna polymerase mu (pol mu) is a novel family x dna polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. we show here that human pol mu is not able to discriminate against the 2'-oh group of the sugar moiety. it inserts rntps with an efficiency that is <10-fold lower than that of dntps, in sharp contrast with the >1000-fold discrimination characteristic of most dna-dependent dna polymerases. the lack of sugar discrimination by po ... | 2003 | 12888504 |
| structures of escherichia coli dna mismatch repair enzyme muts in complex with different mismatches: a common recognition mode for diverse substrates. | we have refined a series of isomorphous crystal structures of the escherichia coli dna mismatch repair enzyme muts in complex with g:t, a:a, c:a and g:g mismatches and also with a single unpaired thymidine. in all these structures, the dna is kinked by approximately 60 degrees upon protein binding. two residues widely conserved in the muts family are involved in mismatch recognition. the phenylalanine, phe 36, is seen stacking on one of the mismatched bases. the same base is also seen forming a ... | 2003 | 12907723 |
| targeted gene evolution in escherichia coli using a highly error-prone dna polymerase i. | we present a system for random mutagenesis in escherichia coli for the evolution of targeted genes. to increase error rates of dna polymerase i (pol i) replication, we introduced point mutations in three structural domains that govern pol i fidelity. expression of error-prone pol i in vivo results in strong mutagenesis of a target sequence encoded in a pol i-dependent plasmid (8.1 x 10-4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence. mu ... | 2003 | 12909725 |
| enzymatic repair of an expanded genetic information system. | the excision repair machinery of a thermophilic bacterium has been shown to recognize and repair an expanded genetic base pair. native thermus aquaticus dna polymerase will remove a mispaired natural base and replace it with a non-natural base to form an expanded base pair. in addition, dna ligase will recognize a nick formed by polymerase between two non-natural base pairs and covalently attach the two strands, thus demonstrating complete repair of a bifurcated base-paired model duplex. these r ... | 2003 | 12930955 |
| a novel sensor of nadh/nad+ redox poise in streptomyces coelicolor a3(2). | we describe the identification of rex, a novel redox-sensing repressor that appears to be widespread among gram-positive bacteria. in streptomyces coelicolor rex binds to operator (rop) sites located upstream of several respiratory genes, including the cydabcd and rex-hemacd operons. the dna-binding activity of rex appears to be controlled by the redox poise of the nadh/nad+ pool. using electromobility shift and surface plasmon resonance assays we show that nadh, but not nad+, inhibits the dna-b ... | 2003 | 12970197 |
| cdna cloning, expression and characterization of an allergenic l3 ribosomal protein of aspergillus fumigatus. | aspergillus fumigatus (afu) is an important fungal pathogen causing allergic and invasive respiratory disorders. a plethora of multi-functional allergens/antigens secreted by afu have been implicated in pathogenesis. the present study was undertaken to identify and characterize novel afu allergen/antigen by cdna library approach. cdna library of afu was immunoscreened with pooled sera of allergic bronchopulmonary aspergillosis (abpa) patients. the cdna clone, ts1, reacting significantly with spe ... | 2003 | 12974759 |
| thermodynamics of the binding of thermus aquaticus dna polymerase to primed-template dna. | dna binding of the type 1 dna polymerase from thermus aquaticus (taq polymerase) and its klentaq large fragment domain have been studied as a function of temperature. equilibrium binding assays were performed from 5 to 70 degrees c using a fluorescence anisotropy assay and from 10 to 60 degrees c using isothermal titration calorimetry. in contrast to the usual behavior of thermophilic proteins at low temperatures, taq and klentaq bind dna with high affinity at temperatures down to 5 degrees c. t ... | 2003 | 14500822 |
| crystallization of the gmppcp complex of the ng domains of thermus aquaticus ffh and ftsy. | the gtpases ffh and ftsy are components of the prokaryotic signal recognition particle protein-targeting pathway. the two proteins interact in a gtp-dependent manner, forming a complex that can be stabilized by use of the non-hydrolyzable gtp analog gmppcp. crystals of the complex of the ng gtpase domains of the two proteins have been obtained from ammonium sulfate solutions. crystals grow with several different morphologies, predominately as poorly diffracting plates and needle clusters, but oc ... | 2003 | 14501130 |
| mutations in the dna mismatch repair proteins muts and mutl of oxazolidinone-resistant or -susceptible enterococcus faecium. | mutations in muts and mutl, which encode dna mismatch repair (mmr) proteins, can confer hypermutator phenotypes and may facilitate the emergence of mutational antibiotic resistance in bacteria. linezolid-resistant enterococci (lre) rarely emerge during therapy and contain mutations in 23s rrna genes. as enterococci with defective mmr could be prone to the development of oxazolidinone resistance mutations, we investigated 13 clinical isolates of enterococcus faecium, including 2 lre, for mutation ... | 2003 | 14506009 |
| molecular characterization of brucella abortus chromosome ii recombination. | large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. the recently completed brucella melitensis 16m and brucella suis 1330 genomes have facilitated the investigation of such events in the brucella spp. suppressive subtractive hybridization (ssh) was employed in identifying genomic differences between b. melitensis 16m and brucella abortus 2308. analysis of 45 ssh clones revealed several deletions on chromosomes of b. abortus ... | 2003 | 14526025 |
| phylogenetic diversity, abundance, and axial distribution of bacteria in the intestinal tract of two soil-feeding termites (cubitermes spp.). | the hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal ph and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. in this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microb ... | 2003 | 14532056 |
| real-time pcr based on sybr-green i fluorescence: an alternative to the taqman assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. | real-time pcr is increasingly being adopted for rna quantification and genetic analysis. at present the most popular real-time pcr assay is based on the hybridisation of a dual-labelled probe to the pcr product, and the development of a signal by loss of fluorescence quenching as pcr degrades the probe. though this so-called 'taqman' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. | 2003 | 14552656 |
| cold-sensitive mutants of taq dna polymerase provide a hot start for pcr. | although the thermophilic bacterium thermus aquaticus grows optimally at 70 degrees c and cannot grow at moderate temperatures, its dna polymerase i has significant activity at 20-37 degrees c. this activity is a bane to some pcrs, since it catalyzes non-specific priming. we report mutations of klentaq (an n-terminal deletion variant) dna polymerase that have markedly reduced activity at 37 degrees c yet retain apparently normal activity at 68 degrees c and resistance at 95 degrees c. the first ... | 2003 | 14576300 |
| enhancement of dna, cdna synthesis and fidelity at high temperatures by a dimeric single-stranded dna-binding protein. | bacterial single-stranded dna-binding proteins (ssbs) are required for dna replication and repair. we have over-expressed and purified the native form and two his-tagged fusions of the ssb from thermus thermophilus (tthssb). the three proteins were found as dimers in solution. they bound in vitro to single-stranded dna specifically over a temperature range of 4-80 degrees c, and the wild-type protein could withstand incubation at 94 degrees c for 2 min. addition of tthssb to pcr halved the elong ... | 2003 | 14602905 |
| crystal structure of bacillus subtilis alpha-amylase in complex with acarbose. | the crystal structure of bacillus subtilis alpha-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation. after comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process. | 2003 | 14617662 |
| functional characterization of helicobacter pylori dnab helicase. | helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. not much is known regarding dna replication in h.pylori that is important for cell survival. here we report the cloning, expression and characterization of h.pylori dnab (hpdnab) helicase both in vitro and in vivo. among the dnab homologs, only escherichia coli dnab has been studied extensively. hpdnab showed strong 5' to 3' helicase and atpase activity. interestingly, h.pylori does not have an obvious dnac h ... | 2003 | 14627816 |
| dna bending and unbending by muts govern mismatch recognition and specificity. | dna mismatch repair is central to the maintenance of genomic stability. it is initiated by the recognition of base-base mismatches and insertion/deletion loops by the family of muts proteins. subsequently, atp induces a unique conformational change in the muts-mismatch complex but not in the muts-homoduplex complex that sets off the cascade of events that leads to repair. to gain insight into the mechanism by which muts discriminates between mismatch and homoduplex dna, we have examined the conf ... | 2003 | 14634210 |
| functional analysis of nsp3 phosphoprotein mutants of sindbis virus. | alphavirus nsp3 phosphoprotein is essential for virus replication and functions initially within polyprotein p123 or p23 components of the short-lived minus-strand replicase, and upon polyprotein cleavage, mature nsp3 likely functions also in plus-strand synthesis. we report the identification of a second nsp3 mutant from among the a complementation group of sindbis virus (sin) heat-resistant strain, ts rna-negative mutants. the ts138 mutant possessed a change of g4303 to c, predicting an ala68- ... | 2003 | 14645567 |
| crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication. | targeting of secretory and membrane proteins by the signal recognition particle (srp) is evolutionarily conserved, and the multidomain protein srp54 acts as the key player in srp-mediated protein transport. binding of a signal peptide to srp54 at the ribosome is coordinated with gtp binding and subsequent complex formation with the srp receptor. because these functions are localized to distinct domains of srp54, communication between them is essential. we report the crystal structures of srp54 f ... | 2003 | 14657338 |
| donation of catalytic residues to rna polymerase active center by transcription factor gre. | during transcription elongation, rna polymerase (rnap) occasionally loses its grip on the growing rna end and backtracks on the dna template. prokaryotic gre factors rescue the backtracked ternary elongating complex through stimulation of an intrinsic endonuclease activity, which removes the disengaged 3' rna segment. by using rna-protein crosslinking in defined ternary elongating complexes, site-directed mutagenesis, discriminative biochemical assays, and docking of the two protein structures, ... | 2003 | 14668436 |
| structure of mth11/mth rpp29, an essential protein subunit of archaeal and eukaryotic rnase p. | we have determined the solution structure of mth11 (mth rpp29), an essential subunit of the rnase p enzyme from the archaebacterium methanothermobacter thermoautotrophicus (mth). rnase p is a ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5' leader sequence during maturation of trnas in all three domains of life. in eubacteria, this enzyme is made up of two subunits: a large rna ( approximately 120 kda) responsible for mediating catalysis, and a small protein cofactor ... | 2003 | 14673079 |
| neighbouring bases in template influence base-pairing of isoguanine. | assuming that the efficiency of the incorporation of 5-methyl-2'-deoxyisocytosine-5' triphosphate (dmictp) and dttp opposite isoguanine (ig) is a measure of the proportion of the keto and enol tautomers of ig in the thermus aquaticus dna polymerase active centre, we studied the influence of temperature and ig-neighbouring bases in the template on base-pairing of ig. on the basis of experiments with four sequences (3'-txt-5', 3'-gxg-5', 3'-cxc-5', 3'-cxt-5', where x=ig) at 37, 50, 65 and 80 degre ... | 2003 | 12387728 |
| 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 |
| surfaces of spo0a and rna polymerase sigma factor a that interact at the spoiig promoter in bacillus subtilis. | in bacillus subtilis, the dna binding protein spo0a activates transcription from two classes of promoters, those used by rna polymerase containing the primary sigma factor, sigma(a) (e.g., spoiig), and those used by rna polymerase containing the secondary sigma factor, sigma(h) (e.g., spoiia). several single amino acid substitutions in region 4 of sigma(a) define positions in sigma(a) that are specifically required for spo0a-dependent promoter activation. similarly, several single amino acid sub ... | 2004 | 14679239 |
| thermostability of multidomain proteins: elongation factors ef-tu from escherichia coli and bacillus stearothermophilus and their chimeric forms. | recombinant mesophilic escherichia coli (ec) and thermophilic bacillus stearothermophilus (bst) elongation factors ef-tus, their isolated g-domains, and six chimeric ef-tus composed of domains of either ef-tu were prepared, and their gdp/gtp binding activities and thermostability were characterized. bstef-tu and bstg-domain bound gdp and gtp with affinities in nanomolar and submicromolar ranges, respectively, fully comparable with those of ecef-tu. in contrast, the ecg-domain bound the nucleotid ... | 2004 | 14691225 |
| interactions between the 2.4 and 4.2 regions of sigmas, the stress-specific sigma factor of escherichia coli, and the -10 and -35 promoter elements. | the sigmas subunit of escherichia coli rna polymerase holoenzyme (esigmas) is a key factor of gene expression upon entry into stationary phase and in stressful conditions. the selectivity of promoter recognition by esigmas and the housekeeping esigma70 is as yet not clearly understood. we used a genetic approach to investigate the interaction of sigmas with its target promoters. starting with down-promoter variants of a sigmas promoter target, osmep, altered in the -10 or -35 elements, we isolat ... | 2004 | 14704342 |
| the single-stranded dna-binding protein of deinococcus radiodurans. | deinococcus radiodurans r1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of dna damage without induced mutation. single-stranded dna-binding (ssb) protein is an essential protein in all organisms and is involved in dna replication, recombination and repair. the published genomic sequence from deinococcus radiodurans includes a putative single-stranded dna-binding protein gene (ssb; dr0100) requiring a translational frameshift for synthesis ... | 2004 | 14718065 |
| heterodimeric gtpase core of the srp targeting complex. | two structurally homologous guanosine triphosphatase (gtpase) domains interact directly during signal recognition particle (srp)-mediated cotranslational targeting of proteins to the membrane. the 2.05 angstrom structure of a complex of the ng gtpase domains of ffh and ftsy reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. the structure explains the coordinate activation of the two gtpases. conformational changes coupled to forma ... | 2004 | 14726591 |
| photo-cross-linking of a purified preinitiation complex reveals central roles for the rna polymerase ii mobile clamp and tfiie in initiation mechanisms. | the topological organization of a tata binding protein-tfiib-tfiif-rna polymerase ii (rnap ii)-tfiie-promoter complex was analyzed using site-specific protein-dna photo-cross-linking of gel-purified complexes. the cross-linking results for the subunits of rnap ii were used to determine the path of promoter dna against the structure of the enzyme. the results indicate that promoter dna wraps around the mobile clamp of rnap ii. cross-linking of tfiif and tfiie both upstream of the tata element and ... | 2004 | 14729958 |
| identification and mapping of self-assembling protein domains encoded by the escherichia coli k-12 genome by use of lambda repressor fusions. | self-assembling proteins and protein fragments encoded by the escherichia coli genome were identified from e. coli k-12 strain mg1655. libraries of random dna fragments cloned into a series of lambda repressor fusion vectors were subjected to selection for immunity to infection by phage lambda. survivors were identified by sequencing the ends of the inserts, and the fused protein sequence was inferred from the known genomic sequence. four hundred sixty-three nonredundant open reading frame-encod ... | 2004 | 14973045 |
| a novel strategy to engineer dna polymerases for enhanced processivity and improved performance in vitro. | mechanisms that allow replicative dna polymerases to attain high processivity are often specific to a given polymerase and cannot be generalized to others. here we report a protein engineering-based approach to significantly improve the processivity of dna polymerases by covalently linking the polymerase domain to a sequence non-specific dsdna binding protein. using sso7d from sulfolobus solfataricus as the dna binding protein, we demonstrate that the processivity of both family a and family b p ... | 2004 | 14973201 |
| efficiency and pattern of uv pulse laser-induced rna-rna cross-linking in the ribosome. | escherichia coli ribosomes were irradiated with a krf excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10-40 mj for a 1 cm2 area, corresponding to fluences of 4.5 to 18 x 10(9) w m(-2), to determine strand breakage yields and the frequency and pattern of rna-rna cross- linking in the 16s rrna. samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample. the yield of strand breaks per photon was intensity dep ... | 2004 | 14999094 |
| proteomic analysis of thioredoxin-targeted proteins in escherichia coli. | thioredoxin, a ubiquitous and evolutionarily conserved protein, modulates the structure and activity of proteins involved in a spectrum of processes, such as gene expression, apoptosis, and the oxidative stress response. here, we present a comprehensive analysis of the thioredoxin-linked escherichia coli proteome by using tandem affinity purification and nanospray microcapillary tandem mass spectrometry. we have identified a total of 80 proteins associated with thioredoxin, implicating the invol ... | 2004 | 15004283 |
| palm mutants in dna polymerases alpha and eta alter dna replication fidelity and translesion activity. | we isolated active mutants in saccharomyces cerevisiae dna polymerase alpha that were associated with a defect in error discrimination. among them, l868f dna polymerase alpha has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (wt) polymerase alpha. in vivo, mutant dna polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that dna polymerase alpha-dependent replication errors are recognized and repair ... | 2004 | 15024063 |