| a dual-specificity aminoacyl-trna synthetase in the deep-rooted eukaryote giardia lamblia. | cysteinyl-trna (cys-trna) is essential for protein synthesis. in most organisms the enzyme responsible for the formation of cys-trna is cysteinyl-trna synthetase (cysrs). the only known exceptions are the euryarchaea methanococcus jannaschii and methanobacterium thermoautotrophicum, which do not encode a cysrs. deviating from the accepted concept of one aminoacyl-trna synthetase per amino acid, these organisms employ prolyl-trna synthetase as the enzyme that carries out cys-trna formation. to da ... | 2000 | 11078517 |
| conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: implications for c type cytochrome biogenesis and folding. | cytochrome c(552) from hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of escherichia coli. the b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 m lower than for the wild-type protein. the reduction potential is 75 mv lower than that of the recombinant wild-type protein. the heme can be re ... | 2000 | 10792037 |
| evidence for horizontal gene transfer in evolution of elongation factor tu in enterococci. | the elongation factor tu, encoded by tuf genes, is a gtp binding protein that plays a central role in protein synthesis. one to three tuf genes per genome are present, depending on the bacterial species. most low-g+c-content gram-positive bacteria carry only one tuf gene. we have designed degenerate pcr primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. the amplified dna fragments were ... | 2000 | 11092850 |
| the 2 a crystal structure of leucyl-trna synthetase and its complex with a leucyl-adenylate analogue. | leucyl-, isoleucyl- and valyl-trna synthetases are closely related large monomeric class i synthetases. each contains a homologous insertion domain of approximately 200 residues, which is thought to permit them to hydrolyse ('edit') cognate trna that has been mischarged with a chemically similar but non-cognate amino acid. we describe the first crystal structure of a leucyl-trna synthetase, from the hyperthermophile thermus thermophilus, at 2.0 a resolution. the overall architecture is similar t ... | 2000 | 10811626 |
| rubrerythrin and rubredoxin oxidoreductase in desulfovibrio vulgaris: a novel oxidative stress protection system. | evidence is presented for an alternative to the superoxide dismutase (sod)-catalase oxidative stress defense system in desulfovibrio vulgaris (strain hildenborough). this alternative system consists of the nonheme iron proteins, rubrerythrin (rbr) and rubredoxin oxidoreductase (rbo), the product of the rbo gene (also called desulfoferrodoxin). a deltarbo strain of d. vulgaris was found to be more sensitive to internal superoxide exposure than was the wild type. unlike rbo, expression of plasmid- ... | 2001 | 11114906 |
| characterization and evolution of anthranilate 1,2-dioxygenase from acinetobacter sp. strain adp1. | the two-component anthranilate 1,2-dioxygenase of the bacterium acinetobacter sp. strain adp1 was expressed in escherichia coli and purified to homogeneity. this enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of o(2) and consumption of one nadh. the terminal oxygenase component formed an alpha(3)beta(3) hexamer of 54- and 19-kda subunits. biochemical analyses demonstrated one rieske-type [2fe-2s] center and one mononuclear nonheme iron center in each larg ... | 2001 | 11114907 |
| initiation factor 2 of myxococcus xanthus, a large version of prokaryotic translation initiation factor 2. | we have isolated the structural gene for translation initiation factor if2 (infb) from the myxobacterium myxococcus xanthus. the gene (3.22 kb) encodes a 1,070-residue protein showing extensive homology within its g domain and c terminus to the equivalent regions of if2 from escherichia coli. the protein cross-reacts with antibodies raised against e. coli if2 and was able to complement an e. coli infb mutant. the m. xanthus protein is the largest if2 known to date. this is essentially due to a l ... | 2001 | 11114918 |
| vima gene downstream of reca is involved in virulence modulation in porphyromonas gingivalis w83. | a 0.9-kb open reading frame encoding a unique 32-kda protein was identified downstream of the reca gene of porphyromonas gingivalis. reverse transcription-pcr and northern blot analysis showed that both the reca gene and this open reading frame are part of the same transcriptional unit. this cloned fragment was insertionally inactivated using the ermf-ermam antibiotic resistance cassette to create a defective mutant by allelic exchange. when plated on brucella blood agar, the mutant strain, desi ... | 2001 | 11119521 |
| molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. | phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (rpps) revealed the monophyletic origin of these genes. the most deeply branching class, exemplified by tet and otra, consisted of genes from the antibiotic-producing organisms streptomyces rimosus and streptomyces lividans. with a high degree of confidence, the corresponding genes of the other seven classes (tet m, tet s, tet o, tet w, tet q, tet t, and tetb p) formed phylogenetically distinct sepa ... | 2001 | 11133424 |
| ihfa gene of the bacterium myxococcus xanthus and its role in activation of carotenoid genes by blue light. | myxococcus xanthus responds to blue light by producing carotenoids. several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes. we had already reported the isolation of a carotenoid-less, tn5-induced strain (mr508), whose mutant site was unlinked to the indicated regulatory genes. here, we show that omegamr508::tn5 affects all known light-inducible promoters in different ways. it blocks the activation o ... | 2001 | 11133949 |
| performance of the cobas amplicor hcv monitor test, version 2.0, an automated reverse transcription-pcr quantitative system for hepatitis c virus load determination. | a clinical evaluation of an automated quantitative pcr assay, the cobas amplicor hcv monitor test, version 2.0 (v2.0), was carried out to assess the performance of this test in comparison with that of the previous, manual version, the amplicor hcv monitor test, and with that of nested pcr. serial dilutions of serum samples infected with genotype 1b, 2a, or 3, as well as synthetic rna transcripts and serum samples derived from 87 patients with chronic hepatitis c and infected with genotype 1a, 1b ... | 2000 | 10834978 |
| three-dimensional cryo-electron microscopy localization of ef2 in the saccharomyces cerevisiae 80s ribosome at 17.5 a resolution. | using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome small middle dotef2 complex at 17.5 a resolution by three-dimensional (3d) cryo-electron microscopy. ef2 is directly visible in the 3d map and the overall arrangement of the complex from saccharomyces cerevisiae corresponds to that previously seen in escherichia coli. however, pronounced differences were found in two prominent regions. first, in the yeast system the interaction be ... | 2000 | 10835368 |
| the location of protein s8 and surrounding elements of 16s rrna in the 70s ribosome from combined use of directed hydroxyl radical probing and x-ray crystallography. | ribosomal protein s8, which is essential for the assembly of the central domain of 16s rrna, is one of the most thoroughly studied rna-binding proteins. to map its surrounding rna in the ribosome, we carried out directed hydroxyl radical probing of 16s rrna using fe(ii) tethered to nine different positions on the surface of protein s8 in 70s ribosomes. hydroxyl radical-induced cleavage was observed near the classical s8-binding site in the 620 stem, and flanking the other s8-footprinted regions ... | 2000 | 10836793 |
| characterization of an atp-dependent dna ligase from the thermophilic archaeon methanobacterium thermoautotrophicum. | we report the production, purification and characterization of a dna ligase encoded by the thermophilic archaeon methanobacterium thermoautotrophicum. the 561 amino acid mth: ligase catalyzed strand-joining on a singly nicked dna in the presence of a divalent cation (magnesium, manganese or cobalt) and atp (k(m) 1.1 microm). datp can substitute for atp, but ctp, gtp, utp and nad(+) cannot. mth: ligase activity is thermophilic in vitro, with optimal nick-joining at 60 degrees c. mutational analys ... | 2000 | 10871342 |
| importance of discriminator base stacking interactions: molecular dynamics analysis of a73 microhelix(ala) variants. | transfer of alanine from escherichia coli alanyl-trna synthetase (alars) to rna minihelices that mimic the amino acid acceptor stem of trna(ala) has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the 'discriminator' base at position 73. solution nmr has indicated that this single-stranded nucleotide is predominantly stacked onto g1 of the first base pair of the alanine acceptor stem helix. we report the activity of a ... | 2000 | 10871402 |
| arginines 29 and 59 of elongation factor g are important for gtp hydrolysis or translocation on the ribosome. | gtp hydrolysis by elongation factor g (ef-g) is essential for the translocation step in protein elongation. the low intrinsic gtpase activity of ef-g is strongly stimulated by the ribosome. here we show that a conserved arginine, r29, of escherichia coli ef-g is crucial for gtp hydrolysis on the ribosome, but not for gtp binding or ribosome interaction, suggesting that it may be directly involved in catalysis. another conserved arginine, r59, which is homologous to the catalytic arginine of g(al ... | 2000 | 10880458 |
| common fold in helix-hairpin-helix proteins. | helix-hairpin-helix (hhh) is a widespread motif involved in non-sequence-specific dna binding. the majority of hhh motifs function as dna-binding modules, however, some of them are used to mediate protein-protein interactions or have acquired enzymatic activity by incorporating catalytic residues (dna glycosylases). from sequence and structural analysis of hhh-containing proteins we conclude that most hhh motifs are integrated as a part of a five-helical domain, termed (hhh)(2) domain here. it t ... | 2000 | 10908318 |
| crystal structure of a repair enzyme of oxidatively damaged dna, mutm (fpg), from an extreme thermophile, thermus thermophilus hb8. | the mutm [formamidopyrimidine dna glycosylase (fpg)] protein is a trifunctional dna base excision repair enzyme that removes a wide range of oxidatively damaged bases (n-glycosylase activity) and cleaves both the 3'- and 5'-phosphodiester bonds of the resulting apurinic/apyrimidinic site (ap lyase activity). the crystal structure of mutm from an extreme thermophile, thermus thermophilus hb8, was determined at 1.9 a resolution with multiwavelength anomalous diffraction phasing using the intrinsic ... | 2000 | 10921868 |
| visualization of trna movements on the escherichia coli 70s ribosome during the elongation cycle. | three-dimensional cryomaps have been reconstructed for trna-ribosome complexes in pre- and posttranslocational states at 17-a resolution. the positions of trnas in the a and p sites in the pretranslocational complexes and in the p and e sites in the posttranslocational complexes have been determined. of these, the p-site trna position is the same as seen earlier in the initiation-like fmet-trna(f)(met)-ribosome complex, where it was visualized with high accuracy. now, the positions of the a- and ... | 2000 | 10931859 |
| cloning of the soda gene from corynebacterium melassecola and role of superoxide dismutase in cellular viability. | the soda gene encoding the corynebacterium melassecola manganese-cofactored superoxide dismutase (sod) has been cloned in escherichia coli and sequenced. the gene is transcribed monocistronically; the predicted polypeptide is 200 amino acids long and associates in a homotetrameric, manganese-dependent form, able to complement an sod-deficient e. coli mutant. a second open reading frame, coding for a putative 217-amino-acid protein with high homology to peptide methionine sulfoxide reductases fro ... | 2001 | 11157941 |
| tarp: a nuclear protein expressed in prostate and breast cancer cells derived from an alternate reading frame of the t cell receptor gamma chain locus. | previously, we identified the expression of a prostate-specific form of t cell receptor gamma chain (tcrgamma) mrna in the human prostate and demonstrated that it originates from epithelial cells and not from infiltrating t lymphocytes. here, we show that this prostate-specific transcript is also expressed in three breast cancer cell lines and breast cancer tissues. analysis of the cdna sequence predicts that this transcript can encode two protein products of 7 and 13 kda, and in vitro translati ... | 2000 | 10931945 |
| role of the tat ransport system in nitrous oxide reductase translocation and cytochrome cd1 biosynthesis in pseudomonas stutzeri. | by transforming n2o to n2, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. the signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the tat pathway. we have identified tat genes of pseudomonas stutzeri and functionally analyzed the unlinked tatc and tate loci. a tatc mutant retained n2o reductase in the cytoplasm in the unprocessed form and lacking the meta ... | 2001 | 11160097 |
| new host-vector system for thermus spp. based on the malate dehydrogenase gene. | a thermus thermophilus hb27 strain was constructed in which the malate dehydrogenase (mdh) gene was deleted. the deltamdh colonies are recognized by a small-colony phenotype. wild-type phenotype is restored by transformation with thermus plasmids or integration vector containing an intact mdh gene. the wild-type phenotype provides a positive selection tool for the introduction of plasmid dna into thermus spp., and because mdh levels can be readily quantified, this host-vector system is a conveni ... | 2001 | 11160114 |
| identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme b and leucine biosynthesis in methanoarchaea. | two putative methanococcus jannaschii isocitrate dehydrogenase genes, mj1596 and mj0720, were cloned and overexpressed in escherichia coli, and their gene products were tested for the ability to catalyze the nad- and nadp-dependent oxidative decarboxylation of dl-threo-3-isopropylmalic acid, threo-isocitrate, erythro-isocitrate, and homologs of threo-isocitrate. neither enzyme was found to use any of the isomers of isocitrate as a substrate. the protein product of the mj1596 gene, designated aks ... | 2000 | 10940051 |
| characterization of rnase p from thermotoga maritima. | the protein subunit of rnase p from a thermophilic bacterium, thermotoga maritima, was overexpressed in and purified from escherichia coli. the cloned protein was reconstituted with the rna subunit transcribed in vitro. the temperature optimum of the holoenzyme is near 50 degrees c, with no enzymatic activity at 65 degrees c or above. this finding is in sharp contrast to the optimal growth temperature of t.maritima, which is near 80 degrees c. however, in heterologous reconstitution experiments ... | 2001 | 11160919 |
| a hydrogen peroxide-forming nadh oxidase that functions as an alkyl hydroperoxide reductase in amphibacillus xylanus. | the amphibacillus xylanus nadh oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-nadh, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (fad) or the small disulfide-containing salmonella enterica ahpc protein. the enzyme has two disulfide bonds, cys128-cys131 and cys337-cys340, which can act as redox centers in addition to the enzyme-bound fad (k. ohnishi, y. niimura, m. hidaka, h. masaki, h. suzuki, t. uozumi, and t. ni ... | 2000 | 10960086 |
| connection between poly-beta-hydroxybutyrate biosynthesis and growth on c(1) and c(2) compounds in the methylotroph methylobacterium extorquens am1. | several dna regions containing genes involved in poly-beta-hydroxybutyrate (phb) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph methylobacterium extorquens am1. genes involved in phb biosynthesis include those encoding beta-ketothiolase (phaa), nadph-linked acetoacetyl coenzyme a (acetyl-coa) reductase (phab), and phb synthase (phac). phaa and phab are closely link ... | 2001 | 11208803 |
| experimental evolution of enzyme temperature activity profile: selection in vivo and characterization of low-temperature-adapted mutants of pyrococcus furiosus ornithine carbamoyltransferase. | we have obtained mutants of pyrococcus furiosus ornithine carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. the mutants were double ones, still complementing at 15 degrees c, a temperature already in the psychrophilic range. their kinetic analysis is reported. | 2001 | 11208811 |
| thermostable chitosanase from bacillus sp. strain ck4: cloning and expression of the gene and characterization of the enzyme. | a thermostable chitosanase gene from the environmental isolate bacillus sp. strain ck4, which was identified on the basis of phylogenetic analysis of the 16s rrna gene sequence and phenotypic analysis, was cloned, and its complete dna sequence was determined. the thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kda enzyme. the deduced amino acid sequence of the chitosanase from bacillu ... | 2000 | 10966383 |
| structure and activity of clpb from escherichia coli. role of the amino-and -carboxyl-terminal domains. | clpb is a member of a protein-disaggregating multi-chaperone system in escherichia coli. the mechanism of protein-folding reactions mediated by clpb is currently unknown, and the functional role of different sequence regions in clpb is under discussion. we have expressed and purified the full-length clpb and three truncated variants with the n-terminal, c-terminal, and a double n- and c-terminal deletion. we studied the protein concentration-dependent and atp-induced oligomerization of clpb, cas ... | 2000 | 10982797 |
| genome of the extremely radiation-resistant bacterium deinococcus radiodurans viewed from the perspective of comparative genomics. | the bacterium deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, uv radiation, oxidizing agents, and electrophilic mutagens. d. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. these characte ... | 2001 | 11238985 |
| physical and functional interaction between the eukaryotic orthologs of prokaryotic translation initiation factors if1 and if2. | to initiate protein synthesis, a ribosome with bound initiator methionyl-trna must be assembled at the start codon of an mrna. this process requires the coordinated activities of three translation initiation factors (if) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eif). the factors eif1a and eif5b from eukaryotes show extensive amino acid sequence similarity to the factors if1 and if2 from prokaryotes. by a combination of two-hybrid, coimmunoprecipitation, and in ... | 2000 | 10982835 |
| uv-induced crosslinks in the 16s rrnas of escherichia coli, bacillus subtilis and thermus aquaticus and their implications for ribosome structure and photochemistry. | sixteen long-range crosslinks are induced in escherichia coli 16s rrna by far-uv irradiation. crosslinking patterns in two other organisms, bacillus subtilis and thermus aquaticus, were investigated to determine if the number and location of crosslinks in e.coli occur because of unusually photoreactive nucleotides at particular locations in the rrna sequence. thirteen long-range crosslinks in b.subtilis and 15 long-range crosslinks in t.aquaticus were detected by gel electrophoresis and 10 cross ... | 2000 | 11000271 |
| peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in escherichia coli cell homogenates and intact cells. | an assay was developed to determine the activity of peptide deformylase (pdf) inhibitors under conditions as close as possible to the physiological situation. the assay principle is the detection of n-terminal [35s]methionine labeling of a protein that contains no internal methionine. if pdf is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the n-terminal methionine label. in the presence of a pdf inhibitor, ... | 2001 | 11257015 |
| peptide deformylase as an antibacterial drug target: target validation and resistance development. | new inhibitors of peptide deformylase (pdf) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) the inhibition of escherichia coli growth could be counteracted by overexpression of pdf from different organisms, including e. coli, streptococcus pneumoniae, and haemophi ... | 2001 | 11257016 |
| hydrogen peroxide-forming nadh oxidase belonging to the peroxiredoxin oxidoreductase family: existence and physiological role in bacteria. | amphibacillus xylanus and sporolactobacillus inulinus nadh oxidases belonging to the peroxiredoxin oxidoreductase family show extremely high peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of the small disulfide redox protein, ahpc (peroxiredoxin). in order to investigate the distribution of this enzyme system in bacteria, 15 bacterial strains were selected from typical aerobic, facultatively anaerobic, and anaerobic bacteria. ahpc-linked alkyl hydroper ... | 2001 | 11274101 |
| elongation of repetitive dna by dna polymerase from a hyperthermophilic bacterium thermus thermophilus. | short repetitive dna sequences are believed to be one of the primordial genetic elements that served as a source of complex large dna found in the genome of modern organisms. however, the mechanism of its expansion (increase in repeat number) during the course of evolution is unclear. we demonstrate that the dna polymerase of the hyperthermophilic bacterium thermus thermophilus can elongate oligodna with several tandem repeats to very long dna in vitro. for instance, 48mer repetitive oligodna (t ... | 2000 | 11024180 |
| two-dimensional gel electrophoresis analyses of ph-dependent protein expression in facultatively alkaliphilic bacillus pseudofirmus of4 lead to characterization of an s-layer protein with a role in alkaliphily. | the large majority of proteins of alkaliphilic bacillus pseudofirmus of4 grown at ph 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant ph-dependent variation. a new surface layer protein (slpa) was identified in these studies. although the prominence of some apparent breakdown products of slpa in gels from ph 10.5-grown cells led to discovery of the alkaliphile s-layer, the largest and major slpa forms were present in large amounts in gels from ... | 2000 | 11029415 |
| analysis of the levels of conservation of the j domain among the various types of dnaj-like proteins. | dnaj-like proteins are defined by the presence of an approximately 73 amino acid region termed the j domain. this region bears similarity to the initial 73 amino acids of the escherichia coli protein dnaj. although the structures of the j domains of e coli dnaj and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the j domains of the various dnaj-like proteins has yet been attempted. a multiple alignment of 2 ... | 2000 | 11048657 |
| a dna ligase from a hyperthermophilic archaeon with unique cofactor specificity. | a gene encoding dna ligase (lig(tk)) from a hyperthermophilic archaeon, thermococcus kodakaraensis kod1, has been cloned and sequenced, and its protein product has been characterized. lig(tk) consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 da. sequence comparison with previously reported dna ligases and the presence of conserved motifs suggested that lig(tk) was an atp-dependent dna ligase. phylogenetic analysis indicated that lig ... | 2000 | 11053387 |
| trna aminoacylation by arginyl-trna synthetase: induced conformations during substrates binding. | the 2.2 a crystal structure of a ternary complex formed by yeast arginyl-trna synthetase and its cognate trna(arg) in the presence of the l-arginine substrate highlights new atomic features used for specific substrate recognition. this first example of an active complex formed by a class ia aminoacyl-trna synthetase and its natural cognate trna illustrates additional strategies used for specific trna selection. the enzyme specifically recognizes the d-loop and the anticodon of the trna, and the ... | 2000 | 11060012 |
| conservation of the binding site for the arginine repressor in all bacterial lineages. | the arginine repressor argr/ahrc is a transcription factor universally conserved in bacterial genomes. its recognition signal (the arg box), a weak palindrome, is also conserved between genomes, despite a very low degree of similarity between individual sites within a genome. thus, the arginine repressor is different from two other universal transcription factors - hrca, whose recognition signal is very strongly conserved both within and between genomes, and lexa/dinr, whose signal is strongly c ... | 2001 | 11305941 |
| crystal structure combined with genetic analysis of the thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch. | ribosome recycling factor (rrf), in concert with elongation factor ef-g, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. the crystal structure of thermus thermophilus rrf was determined at 2.6 a resolution. it is a trna-like l-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2). although the individual domain structures are similar to those of thermotoga mar ... | 2000 | 11073219 |
| cloning and functional characterization of an nad(+)-dependent dna ligase from staphylococcus aureus. | a staphylococcus aureus mutant conditionally defective in dna ligase was identified by isolation of complementing plasmid clones that encode the s. aureus liga gene. orthologues of the putative s. aureus nad(+)-dependent dna ligase could be identified in the genomes of bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif i, kxdg with lysine 112, which is believed to be the proposed site of adenylation. dna se ... | 2001 | 11325928 |
| evidence against an interaction between the mrna downstream box and 16s rrna in translation initiation. | based on the complementarity of the initial coding region (downstream box [db]) of several bacterial and phage mrnas to bases 1469 to 1483 in helix 44 of 16s rrna (anti-downstream box [adb]), it has been proposed that db-adb base pairing enhances translation in a way that is similar to that of the shine-dalgarno (sd)/anti-shine-dalgarno (asd) interaction. computer modeling of helix 44 on the 30s subunit shows that the topography of the 30s ribosome does not allow a simultaneous db-adb interactio ... | 2001 | 11344158 |
| crystal structure of yeast initiation factor 4a, a dead-box rna helicase. | the eukaryotic translation initiation factor 4a (eif4a) is a member of the dea(d/h)-box rna helicase family, a diverse group of proteins that couples an atpase activity to rna binding and unwinding. previous work has provided the structure of the amino-terminal, atp-binding domain of eif4a. extending those results, we have solved the structure of the carboxyl-terminal domain of eif4a with data to 1.75 a resolution; it has a parallel alpha-beta topology that superimposes, with minor variations, o ... | 2000 | 11087862 |
| specific interaction between the ribosome recycling factor and the elongation factor g from mycobacterium tuberculosis mediates peptidyl-trna release and ribosome recycling in escherichia coli. | once the translating ribosomes reach a termination codon, the nascent polypeptide chain is released in a factor-dependent manner. however, the p-site-bound deacylated trna and the ribosomes themselves remain bound to the mrna (post-termination complex). the ribosome recycling factor (rrf) plays a vital role in dissociating this complex. here we show that the mycobacterium tuberculosis rrf (mturrf) fails to rescue escherichia coli lj14, a strain temperature-sensitive for rrf (frr(ts)). more inter ... | 2001 | 11387230 |
| recombinant thermus aquaticus rna polymerase, a new tool for structure-based analysis of transcription. | the three-dimensional structure of dna-dependent rna polymerase (rnap) from thermophilic thermus aquaticus has recently been determined at 3.3 a resolution. currently, very little is known about t. aquaticus transcription and no genetic system to study t. aquaticus rnap genes is available. to overcome these limitations, we cloned and overexpressed t. aquaticus rnap genes in escherichia coli. overproduced t. aquaticus rnap subunits assembled into functional rnap in vitro and in vivo when coexpres ... | 2001 | 11114902 |
| gene cluster of rhodothermus marinus high-potential iron-sulfur protein: oxygen oxidoreductase, a caa(3)-type oxidase belonging to the superfamily of heme-copper oxidases. | the respiratory chain of the thermohalophilic bacterium rhodothermus marinus contains an oxygen reductase, which uses hipip (high potential iron-sulfur protein) as an electron donor. the structural genes encoding the four subunits of this hipip:oxygen oxidoreductase were cloned and sequenced. the genes for subunits ii, i, iii, and iv (named rcoxa to rcoxd) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstre ... | 2001 | 11133964 |
| characterization of a heme-dependent catalase from methanobrevibacter arboriphilus. | recently it was reported that methanogens of the genus methanobrevibacter exhibit catalase activity. this was surprising, since methanobrevibacter species belong to the order methanobacteriales, which are known not to contain cytochromes and to lack the ability to synthesize heme. we report here that methanobrevibacter arboriphilus strains az and dh1 contained catalase activity only when the growth medium was supplemented with hemin. the heme catalase was purified and characterized, and the enco ... | 2001 | 11425719 |
| natural transformation in mesophilic and thermophilic bacteria: identification and characterization of novel, closely related competence genes in acinetobacter sp. strain bd413 and thermus thermophilus hb27. | the mesophile acinetobacter sp. strain bd413 and the extreme thermophile thermus thermophilus hb27 display high frequencies of natural transformation. in this study we identified and characterized a novel competence gene in acinetobacter sp. strain bd413, coma, whose product displays significant similarities to the competence proteins coma and comec in neisseria and bacillus species. transcription of coma correlated with growth phase-dependent transcriptional regulation of the recently identifie ... | 2001 | 11425734 |
| molecular characterization of desulfovibrio gigas neelaredoxin, a protein involved in oxygen detoxification in anaerobes. | desulfovibrio gigas neelaredoxin is an iron-containing protein of 15 kda, having a single iron site with a his(4)cys coordination. neelaredoxins and homologous proteins are widespread in anaerobic prokaryotes and have superoxide-scavenging activity. to further understand its role in anaerobes, its genomic organization and expression in d. gigas were studied and its ability to complement escherichia coli superoxide dismutase deletion mutant was assessed. in d. gigas, neelaredoxin is transcribed a ... | 2001 | 11443075 |
| identification of the outer membrane porin of thermus thermophilus hb8: the channel-forming complex has an unusually high molecular mass and an extremely large single-channel conductance. | the outer membrane of the thermophilic bacterium thermus thermophilus was isolated using sucrose step gradient centrifugation. its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 ns in 1 m kcl. the channel protein was purified by preparative sodium dodecyl sulfate (sds)-polyacylamide gel electrophoresis. it has a high molecular mass of 185 kda, and its channel-forming ability resists boiling in sds for 10 min. | 2001 | 11133980 |
| a subunit of human nuclear rnase p has atpase activity. | human nuclear rnase p purified from hela cells has atpase activity. this activity is associated with one of the protein subunits of the enzyme, rpp20. thus, human nuclear rnase p, which contains several proteins and one essential rna, has at least one other enzymatic activity in addition to cleavage of phosphoester bonds in rna. the amino acid sequence of rpp20 has a signature motif found in an atpase-containing subunit of a family of protein complexes (abc transporters) that mediate a variety o ... | 2001 | 11149958 |
| assessment, by transcription-mediated amplification, of virologic response in patients with chronic hepatitis c virus treated with peginterferon alpha-2a. | transcription-mediated amplification (tma) is an isothermal, autocatalytic target amplification method which has the potential to detect less than 50 hepatitis c virus (hcv) rna copies/ml (10 iu/ml). the tma assay was used to assess the presence of residual hcv rna in plasma from patients treated with polyethylene glycol-modified interferon alpha-2a (peginterferon alpha-2a) who showed a virologic relapse after the end of therapy. stored end-of-treatment and end-of-follow-up plasma samples from 1 ... | 2001 | 11474002 |
| the kink-turn: a new rna secondary structure motif. | analysis of the haloarcula marismortui large ribosomal subunit has revealed a common rna structure that we call the kink-turn, or k-turn. the six k-turns in h.marismortui 23s rrna superimpose with an r.m.s.d. of 1.7 a. there are two k-turns in the structure of thermus thermophilus 16s rrna, and the structures of u4 snrna and l30e mrna fragments form k-turns. the structure has a kink in the phosphodiester backbone that causes a sharp turn in the rna helix. its asymmetric internal loop is flanked ... | 2001 | 11483524 |
| functional and evolutionary relationship between arginine biosynthesis and prokaryotic lysine biosynthesis through alpha-aminoadipate. | our previous studies revealed that lysine is synthesized through alpha-aminoadipate in an extremely thermophilic bacterium, thermus thermophilus hb27. sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from alpha-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. in the present study, we cloned an argd homolog of t. thermophilus hb27 which was not included in the previously cloned lysine bios ... | 2001 | 11489859 |
| peplomycin, a bleomycin derivative, induces myofibroblasts in pulmonary fibrosis. | to analyse the mechanism by which a bleomycin derivative, peplomycin (plm) induces pulmonary fibrosis, we investigated differentiation of rat pulmonary fibroblasts to myofibroblasts (mf). in intraperitoneally plm (5 mg/kg/day)-injected rats, the peripheries of lungs adjacent to the pleura revealed advanced fibrosis with a small number of alpha-smooth muscle actin (alpha-sma)-positive mf, which ultrastructurally possessed abundant microfilaments and cellular organelles. in the fibrotic tissue, th ... | 2001 | 11493347 |
| resistance of streptococcus pneumoniae to deformylase inhibitors is due to mutations in defb. | resistance to peptide deformylase inhibitors in escherichia coli or staphylococcus aureus is due to inactivation of transformylase activity. knockout experiments in streptococcus pneumoniae r6x indicate that the transformylase (fmt) and deformylase (defb) genes are essential and that a def paralog (defa) is not. actinonin-resistant mutants of s. pneumoniae atcc 49619 harbor mutations in defb but not in fmt. reintroduction of the mutated defb gene into wild-type s. pneumoniae r6x recreates the re ... | 2001 | 11502510 |
| primary structure of a novel subunit in ba3-cytochrome oxidase from thermus thermophilus. | the bax-type cytochrome c oxidase from thermus thermophilus is known as a two subunit enzyme. deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit iia" spanning the membrane. the hydrophobic n-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. its complete amino acid sequence was determined by a combination of automated edman degradation of both the deformylated and the cyanoge ... | 2000 | 11152118 |
| hybrid protein between ribosomal protein s16 and rimm of escherichia coli retains the ribosome maturation function of both proteins. | the rimm protein in escherichia coli is associated with free 30s ribosomal subunits but not with 70s ribosomes and is important for efficient maturation of the 30s subunits. a mutant lacking rimm shows a sevenfold-reduced growth rate and a reduced translational efficiency. here we show that a double alanine-for-tyrosine substitution in rimm prevents it from associating with the 30s subunits and reduces the growth rate of e. coli approximately threefold. several faster-growing derivatives of the ... | 2001 | 11514519 |
| recombinational transfer of 100-kilobase genomic dna to plasmid in bacillus subtilis 168. | transformation of bacillus subtilis by a plasmid requires a circular multimeric form. in contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. a recombinational transfer system was constructed with this intrinsic b. subtilis recombinational repair pathway. the vector, pgets103, a derivative of the theta-type replicating plasmid ptb19 of thermophilic bacillus, had the full length of escherichia coli plasmid pbr322. a multimeric form of pg ... | 2001 | 11514534 |
| integrity of thermus thermophilus cytochrome c552 synthesized by escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmabcdefgh: biochemical, spectral, and structural characterization of the recombinant protein. | we describe the design of escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the thermus thermophilus cytochrome c552 gene. key features are (1) construction of a plasmid-borne, chimeric cyca gene encoding an escherichia coli-compatible, n-terminal signal sequence (metlysileseriletyralathrleu alaalaleuserleualaleuproalaglyala) followed by the amino acid sequence of mature thermus cytochrome c552; and (2) coexpression of the chimeric cyca gene with plasmi ... | 2000 | 11152119 |
| production of recombinant alpha-galactosidases in thermus thermophilus. | a thermus thermophilus selector strain for production of thermostable and thermoactive alpha-galactosidase was constructed. for this purpose, the native alpha-galactosidase gene (agat) of t. thermophilus th125 was inactivated to prevent background activity. in our first attempt, insertional mutagenesis of agat by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (alpha-galactoside) and galactose as sole carbohydrate sources due to a polar effec ... | 2001 | 11526023 |
| hepatitis c virus 3'x region interacts with human ribosomal proteins. | to identify proteins that can bind the 3' untranslated region (utr) of hepatitis c virus (hcv) we screened human cdna libraries using the saccharomyces cerevisiae three-hybrid system. screening with an rna sequence derived from the 3'-terminal 98 nucleotides (3'x region) of an infectious clone of hcv (h77c) yielded clones of human ribosomal proteins l22, l3, s3, and ml3, a mitochondrial homologue of l3. we performed preliminary characterization of the binding between the 3'x region and these pro ... | 2001 | 11152508 |
| development of a reverse transcription-pcr-dna enzyme immunoassay for detection of "norwalk-like" viruses and hepatitis a virus in stool and shellfish. | outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. the analysis of environmental samples by newer diagnostic techniques such as reverse transcription-pcr (rt-pcr) amplification of nucleic acid has begun to identify human enteric viruses (predominantly "norwalk-like" viruses [nlvs]) as the cause of many of these outbreaks. to streamline nlv detection from environmental samples such as shellfish, we have developed an rt-pcr-oligoprobe amplificat ... | 2001 | 11157239 |
| the crystal structure of the ttcsaa protein: an export-related chaperone from thermus thermophilus. | the csaa protein was first characterized in bacillus subtilis as a molecular chaperone with export-related activities. here we report the 2.0 angstrom-resolution crystal structure of the thermus thermophilus csaa protein, designated ttcsaa. atomic structure and experiments in solution revealed a homodimer as the functional unit. the structure of the ttcsaa monomer is reminiscent of the well known oligonucleotide-binding fold, with the addition of extensions at the n- and c-termini that form an e ... | 2001 | 11157762 |
| structure of the emapii domain of human aminoacyl-trna synthetase complex reveals evolutionary dimer mimicry. | the emapii (endothelial monocyte-activating polypeptide ii) domain is a trna-binding domain associated with several aminoacyl-trna synthetases, which becomes an independent domain with inflammatory cytokine activity upon apoptotic cleavage from the p43 component of the multisynthetase complex. it comprises a domain that is highly homologous to bacterial trna-binding proteins (trbp), followed by an extra domain without homology to known proteins. trbps, which may represent ancient trna chaperones ... | 2001 | 11157763 |
| mutations in the listerial prob gene leading to proline overproduction: effects on salt tolerance and murine infection. | the observed sensitivity of listeria monocytogenes to the toxic proline analogue l-azetidine-2-carboxylic acid (az) suggested that proline synthesis in listeria may be regulated by feedback inhibition of gamma-glutamyl kinase (gk), the first enzyme of the proline biosynthesis pathway, encoded by the prob gene. taking advantage of the epicurian coli mutator strain xl1-red, we performed random mutagenesis of the recently described proba operon and generated three independent mutations in the liste ... | 2001 | 11571156 |
| multiple regulatory mechanisms act on the 5' untranslated region of the s-layer gene from thermus thermophilus hb8. | the role of the 5' untranslated region (5'utr) of the s-layer gene from thermus thermophilus was analyzed through the isolation of delta 5'utr mutants. in these mutants the half-life of spla mrna was strongly reduced and slpa transcription was no longer subjected to growth phase-dependent repression. overproduction and detachment of the external envelopes of the mutants were observed in stationary phase. | 2001 | 11157968 |
| crystal structure of the holliday junction migration motor protein ruvb from thermus thermophilus hb8. | we report here the crystal structure of the ruvb motor protein from thermus thermophilus hb8, which drives branch migration of the holliday junction during homologous recombination. ruvb has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in atp binding and hydrolysis. dna is likely to interact with a large basic cleft, which encompasses the atp-binding pocket and domain boundaries, whereas the junction-recognition protein ruva may bind a ... | 2001 | 11171970 |
| characterization of a brucella species 25-kilobase dna fragment deleted from brucella abortus reveals a large gene cluster related to the synthesis of a polysaccharide. | in the present study we completed the nucleotide sequence of a brucella melitensis 16m dna fragment deleted from b. abortus that accounts for 25,064 bp and show that the other brucella spp. contain the entire 25-kb dna fragment. two short direct repeats of four nucleotides, detected in the b. melitensis 16m dna flanking both sides of the fragment deleted from b. abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication. in addition to omp31, co ... | 2001 | 11598046 |
| occurrence of transsulfuration in synthesis of l-homocysteine in an extremely thermophilic bacterium, thermus thermophilus hb8. | a cell extract of an extremely thermophilic bacterium, thermus thermophilus hb8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with o-acetyl-l-homoserine and l-cysteine as substrates but not beta-synthesis with dl-homocysteine and l-serine (or o-acetyl-l-serine). the amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. the syntheses of cystathionine beta-lyase (ec 4.4.1.8) and ... | 2001 | 11222609 |
| crystal structure of the lrp-like transcriptional regulator from the archaeon pyrococcus furiosus. | the lrpa protein from the hyperthermophilic archaeon pyrococcus furiosus belongs to the lrp/asnc family of transcriptional regulatory proteins, of which the escherichia coli leucine-responsive regulatory protein is the archetype. its crystal structure has been determined at 2.9 a resolution and is the first for a member of the lrp/asnc family, as well as one of the first for a transcriptional regulator from a hyperthermophile. the structure consists of an n-terminal domain containing a helix-tur ... | 2001 | 11230123 |
| influence of a sulfhydryl cross-link across the allosteric-site interface of e. coli phosphofructokinase. | to assess the role of quaternary stability on the properties of escherichia coli phosphofructokinase (pfk), a disulfide bond has been introduced across the subunit interface containing the allosteric binding sites in e. coli phosphofructokinase by changing n288 to cysteine. n288 is located in close proximity to the equivalent residue on an adjacent subunit. although sds-page of oxidized n288c indicates monomeric protein, blocking the six native cysteine residues with n-ethyl maleimide (nem) reve ... | 2001 | 11604525 |
| differential effects of replacing escherichia coli ribosomal protein l27 with its homologue from aquifex aeolicus. | the rpma gene, which encodes 50s ribosomal subunit protein l27, was cloned from the extreme thermophile aquifex aeolicus, and the protein was overexpressed and purified. comparison of the a. aeolicus protein with its homologue from escherichia coli by circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas the e. coli protein is unstructured under the same conditions. a mutant of e. coli ... | 2001 | 11673426 |
| clue to damage recognition by uvrb: residues in the beta-hairpin structure prevent binding to non-damaged dna. | uvrb, the ultimate damage-recognizing component of bacterial nucleotide excision repair, contains a flexible beta-hairpin rich in hydrophobic residues. we describe the properties of uvrb mutants in which these residues have been mutated. the results show that y101 and f108 in the tip of the hairpin are important for the strand-separating activity of uvrb, supporting the model that the beta-hairpin inserts between the two dna strands during the search for dna damage. residues y95 and y96 at the b ... | 2001 | 11689453 |
| crystal structure of hiv-1 reverse transcriptase in complex with a polypurine tract rna:dna. | we have determined the 3.0 a resolution structure of wild-type hiv-1 reverse transcriptase in complex with an rna:dna oligonucleotide whose sequence includes a purine-rich segment from the hiv-1 genome called the polypurine tract (ppt). the ppt is resistant to ribonuclease h (rnase h) cleavage and is used as a primer for second dna strand synthesis. the 'rnase h primer grip', consisting of amino acids that interact with the dna primer strand, may contribute to rnase h catalysis and cleavage spec ... | 2001 | 11250910 |
| brucella abortus genes identified following constitutive growth and macrophage infection. | the chronicity of brucella abortus infection in humans and animals depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. although no human vaccine exists for brucella, vaccine development in other bacteria has been based on deletions of selective nutritional as well as regulatory systems. our goal is to develop a vaccine for brucella. to further this aim, we have used a green fluorescent protein (gfp) reporter system to identify constitut ... | 2001 | 11705955 |
| prediction of structural domains of tap reveals details of its interaction with p15 and nucleoporins. | vertebrate tap is a nuclear mrna export factor homologous to yeast mex67p. the middle domain of tap binds directly to p15, a protein related to the nuclear transport factor 2 (ntf2), whereas its c-terminal domain interacts with various nucleoporins, the components of the nuclear pore complex (npc). here, we report that the middle domain of tap is also similar to ntf2, as well as to regions in ras-gap sh3 domain binding protein (g3bp) and some plant protein kinases. based on the known three-dimen ... | 2000 | 11256625 |
| rac, a stable ribosome-associated complex in yeast formed by the dnak-dnaj homologs ssz1p and zuotin. | the yeast cytosol contains multiple homologs of the dnak and dnaj chaperone family. our current understanding of which homologs functionally interact is incomplete. zuotin is a dnaj homolog bound to the yeast ribosome. we have now identified the dnak homolog ssz1p/pdr13p as zuotin's partner chaperone. zuotin and ssz1p form a ribosome-associated complex (rac) that is bound to the ribosome via the zuotin subunit. rac is unique among the eukaryotic dnak-dnaj systems, as the 1:1 complex is stable, e ... | 2001 | 11274393 |
| the hunt for living gold. the search for organisms in extreme environments yields useful enzymes for industry. | | 2001 | 11713183 |
| bacterial diversity and community structure in an aerated lagoon revealed by ribosomal intergenic spacer analyses and 16s ribosomal dna sequencing. | we investigated the bacterial community structure in an aerated plug-flow lagoon treating pulp and paper mill effluent. for this investigation, we developed a composite method based on analyses of pcr amplicons containing the ribosomal intergenic spacer (ris) and its flanking partial 16s rrna gene. community percent similarity was determined on the basis of ris length polymorphism. a community succession was evident in the lagoon, indicated by a progressive community transition through seven sam ... | 2001 | 11282606 |
| purification and characterization of the recombinant thermus sp. strain t2 alpha-galactosidase expressed in escherichia coli. | the nucleotide sequence of the thermus sp. strain t2 dna coding for a thermostable alpha-galactosidase was determined. the deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (m(r), 53,514). the observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from thermus brockianus was over 70%. thermus sp. strain t2 alpha-galactosidase was expressed in its active form in escherichia coli and purified. native polyacrylamide gel ele ... | 2001 | 11282611 |
| crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and if3. | the small ribosomal subunit is responsible for the decoding of genetic information and plays a key role in the initiation of protein synthesis. we analyzed by x-ray crystallography the structures of three different complexes of the small ribosomal subunit of thermus thermophilus with the a-site inhibitor tetracycline, the universal initiation inhibitor edeine and the c-terminal domain of the translation initiation factor if3. the crystal structure analysis of the complex with tetracycline reveal ... | 2001 | 11296217 |
| rna tertiary interactions in the large ribosomal subunit: the a-minor motif. | analysis of the 2.4-a resolution crystal structure of the large ribosomal subunit from haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes rna tertiary and quaternary structures. this motif is termed the a-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at c-g base pairs, where they form hydrogen bonds with one or both of the 2' ohs of ... | 2001 | 11296253 |
| attached and unattached bacterial communities in a 120-meter corehole in an acidic, crystalline rock aquifer. | the bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. one hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the mineral park mine near kingman, ariz. every fourth 1.5-m core was acquired using mi ... | 2001 | 11319087 |
| oxaloacetate synthesis in the methanarchaeon methanosarcina barkeri: pyruvate carboxylase genes and a putative escherichia coli-type bifunctional biotin protein ligase gene (bpl/bira) exhibit a unique organization. | evidence is presented that, in methanosarcina barkeri oxaloacetate synthesis, an essential and major co(2) fixation reaction is catalyzed by an apparent alpha(4)beta(4)-type acetyl coenzyme a-independent pyruvate carboxylase (pyc), composed of 64.2-kda biotinylated and 52.9-kda atp-binding subunits. the purified enzyme was most active at 70 degrees c, insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and severely inhibited by atp, adp, and excess mg(2+). it showed ... | 2001 | 11371547 |
| novel posttranslational activation of the lys2-encoded alpha-aminoadipate reductase for biosynthesis of lysine and site-directed mutational analysis of conserved amino acid residues in the activation domain of candida albicans. | the alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi. the alpha-aminoadipate reductase (aar) of this pathway catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde by a complex mechanism involving two gene products, lys2p and lys5p. the lys2 and lys5 genes encode, respectively, a 155-kda inactive aar and a 30-kda phosphopantetheinyl transferase (pptase) which transfers a phosphopantetheinyl group from coenzyme a (coa) to lys2p for th ... | 2001 | 11717270 |
| spontaneous erythromycin resistance mutation in a 23s rrna gene, rrla, of the extreme thermophile thermus thermophilus ib-21. | spontaneous, erythromycin-resistant mutants of thermus thermophilus ib-21 were isolated and found to carry the mutation a2058g in one of two 23s rrna operons. the heterozygosity of these mutants indicates that a2058g confers a dominant or codominant phenotype in this organism. this mutation provides a valuable tool for the genetic manipulation of the 23s rrna genes of thermus. | 2001 | 11418580 |
| using surface-bound rubidium ions for protein phasing. | rubidium is a monovalent metal that can be used as a counterion in protein solutions. x-ray anomalous scattering from rubidium ions bound to the protein surface was used for phasing of the crystal structure of the hsp60 apical domain from thermus thermophilus. multiple-wavelength anomalous dispersion (mad) data were collected from a crystal obtained from a solution containing 0.2 m rubidium salt. one molecule of protein (147 amino acids) binds one well ordered and one poorly ordered rb atom. pha ... | 2001 | 11418770 |
| rad54 protein stimulates the postsynaptic phase of rad51 protein-mediated dna strand exchange. | rad54 and rad51 are important proteins for the repair of double-stranded dna breaks by homologous recombination in eukaryotes. as previously shown, rad51 protein forms nucleoprotein filaments on single-stranded dna, and rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded dna partner. here we demonstrate that saccharomyces cerevisiae rad54 protein has an additional role in the postsynaptic phase of dna strand exchange by stimulat ... | 2001 | 11459988 |
| high stability of a ferredoxin from the hyperthermophilic archaeon a. ambivalens: involvement of electrostatic interactions and cofactors. | the ferredoxin from the thermophilic archaeon acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (t(m)) of 122 degrees c (ph 7). to gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various phs. thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (guhcl) concentrations, yields a linear correlation between unfolding enthalpies (deltah[t(m)]) and t( ... | 2001 | 11468351 |
| high-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification. | single nucleotide polymorphisms (snps) are the foundation of powerful complex trait and pharmacogenomic analyses. the availability of large snp databases, however, has emphasized a need for inexpensive snp genotyping methods of commensurate simplicity, robustness, and scalability. we describe a solution-based, microtiter plate method for snp genotyping of human genomic dna. the method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification o ... | 2001 | 11511324 |
| a conformational change in the ribosomal peptidyl transferase center upon active/inactive transition. | the ribosome is a dynamic particle that undergoes many structural changes during translation. we show through chemical probing with dimethyl sulfate (dms) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in ph, temperature, and monovalent ion concentration, consistent with observations made by elson and coworkers over 30 years ago. moreover, we have found that the ph-dependent dms reactivity of a2451 in the center of the 23s rrna peptid ... | 2001 | 11517305 |
| deletion of the gre3 aldose reductase gene and its influence on xylose metabolism in recombinant strains of saccharomyces cerevisiae expressing the xyla and xks1 genes. | saccharomyces cerevisiae ferments hexoses efficiently but is unable to ferment xylose. when the bacterial enzyme xylose isomerase (xi) from thermus thermophilus was produced in s. cerevisiae, xylose utilization and ethanol formation were demonstrated. in addition, xylitol and acetate were formed. an unspecific aldose reductase (ar) capable of reducing xylose to xylitol has been identified in s. cerevisiae. the gre3 gene, encoding the ar enzyme, was deleted in s. cerevisiae cen.pk2-1c, yielding y ... | 2001 | 11722921 |
| xylulokinase overexpression in two strains of saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate. | fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes xyl1 (xylose reductase) and xyl2 (xylitol dehydrogenase). xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. the gene xks1 encodes the xylulose-phosphorylating enzyme xylulokinase. in this study, we determi ... | 2001 | 11526030 |
| diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in streptococcus pneumoniae. | mechanisms of resistance were studied in 22 macrolide-resistant mutants selected in vitro from 5 parental strains of macrolide-susceptible streptococcus pneumoniae by serial passage in various macrolides (t. a. davies, b. e. dewasse, m. r. jacobs, and p. c. appelbaum, antimicrob. agents chemother., 44:414-417, 2000). portions of genes encoding ribosomal proteins l22 and l4 and 23s rrna (domains ii and v) were amplified by pcr and analyzed by single-strand conformational polymorphism analysis to ... | 2002 | 11751122 |
| complete polar lipid composition of thermoplasma acidophilum ho-62 determined by high-performance liquid chromatography with evaporative light-scattering detection. | polar ether lipids of thermoplasma acidophilum ho-62 were purified by high-performance liquid chromatography with an evaporative light-scattering detector. structures of purified lipids were investigated by capillary gas chromatography, mass spectrometry, and nuclear magnetic resonance. three types of ether lipids were found: phospholipids, glycolipids, and phosphoglycolipids. the two phospholipids had glycerophosphate as the phosphoester moiety. the seven glycolipids had different combinations ... | 2002 | 11751835 |
| cloning, sequencing, and expression of the cold-inducible hutu gene from the antarctic psychrotrophic bacterium pseudomonas syringae. | a promoter-fusion study with a tn 5-based promoter probe vector had earlier found that the hutu gene which encodes the enzyme urocanase for the histidine utilization pathway is upregulated at a lower temperature (4 degrees c) in the antarctic psychrotrophic bacterium pseudomonas syringae. to examine the characteristics of the urocanase gene and its promoter elements from the psychrotroph, the complete hutu and its upstream region from p. syringae were cloned, sequenced, and analyzed in the prese ... | 2002 | 11772602 |