Mol Biol Evol, 2004 Aug, 21(8), 1477 - 81 Epub 2004 Feb 12. Intracellular spheroid bodies of Rhopalodia gibba have nitrogen-fixing apparatus of cyanobacterial origin; Prechtl J et al.; Nitrogen fixation is not regarded as a eukaryotic invention . The process has only been reported as being carried out by bacteria . These prokaryotes typically interact with their eukaryotic hosts as extracellular and temporary nonobligate nitrogen-fixing symbionts . However, intracellular permanent "spheroid bodies" have been reported within the fresh-water diatom Rhopalodia gibba, and these, too, have been speculated as being able to provide nitrogen to their host diatom . These spheroid bodies have gram-negative characteristics with thylakoids . We demonstrate that they fix nitrogen under light conditions . We also show that phylogenetic analyses of their 16rRNA and nif D genes predict that their genome is closely related to that of Cyanothece sp . ATCC 51.142, a free-living diazotrophic cyanobacterium . We suggest that the intracellular spheroid bodies of Rhopalodia gibba may represent a vertically transmitted, permanent endosymbiotic stage in the transition from a free-living diazotrophic cyanobacterium to a nitrogen-fixing eukaryotic organelle. Bioinformatics, 2004 Jun 12, 20(9), 1373 - 80 Epub 2004 Feb 12. A hint to search for metalloproteins in gene banks; Andreini C et al.; MOTIVATION: With the advent of genome sequencing, a huge database of protein primary sequences has been accumulating . In parallel, a number of tools to investigate and expand upon this information, e.g . reconstructing and building relationships between protein families and superfamilies, have been developed . Metalloproteins are proteins capable of binding one or more metal ions, which are required for their biological function or for regulation of their activities or for structural purposes . Sometimes, metal binding can be observed in vitro but not be physiologically relevant . At present, there is a lack of specific tools to address the matter of the identification of metalloproteins in databases of gene sequences . RESULTS: In the present work, an approach exploiting metal-binding patterns (MBPs) of metalloproteins present in the Protein Data Bank to search gene banks for new metalloproteins is presented and applied to copper proteins . Nearly 100 different MBPs have been identified and then used for subsequent applications . The ensemble of sequences of the whole PDB is used to assess the potentiality and limits of the method and to identify levels of confidence for the predictions output by the search . It appears that copper-binding capabilities are identified with a confidence >90% when the percentage of identical amino acids aligned around the MBP by PHI-BLAST is at least 20% with respect to the entire protein domain length . If this percentage is between 10% and 20%, the level of confidence is approximately 50% . Application of the methodology to the entire genome sequences of Pyrococcus furiosus, Escherichia coli, Drosophila melanogaster and Homo sapiens suggests some differentiation between prokaryotes and eukaryotes . SUPPLEMENTARY INFORMATION: A table reporting statistics on the MBP identified; a list of all hits retrieved for the four organisms considered; a figure showing the number of hits for the four organisms as a function of I(d)(Global). Bioinformatics, 2004 Jul 10, 20(10), 1527 - 34 Epub 2004 Feb 12. Gap statistics for whole genome shotgun DNA sequencing projects; Wendl MC et al.; MOTIVATION: Investigators utilize gap estimates for DNA sequencing projects . Standard theories assume sequences are independently and identically distributed, leading to appreciable under-prediction of gaps . RESULTS: Using a statistical scaling factor and data from 20 representative whole genome shotgun projects, we construct regression equations that relate coverage to a normalized gap measure . Prokaryotic genomes do not correlate to sequence coverage, while eukaryotes show strong correlation if the chaff is ignored . Gaps decrease at an exponential rate of only about one-third of that predicted via theory alone . Case studies suggest that departure from theory can largely be attributed to assembly difficulties for repeat-rich genomes, but bias and coverage anomalies are also important when repeats are sparse . Such factors cannot be readily characterized a priori, suggesting upper limits on the accuracy of gap prediction . We also find that diminishing coverage probability discussed in other studies is a theoretical artifact that does not arise for the typical project. Methods, 2004 Mar, 32(3), 219 - 26 Lab scale and medium scale production of recombinant allergens in Escherichia coli; Wallner M et al.; Recombinant products have become invaluable tools for diagnostic as well as therapeutic purposes in modern medicine . Especially in cases where raw naturally derived products are difficult to standardize, well-defined recombinant single components represent the matter of choice . In the recent past, much effort has been undertaken to define individual proteins derived from various sources like pollen, spores of moulds, pet dander, and food causing Type 1 allergic reactions in humans . Therefore, methods for cloning, sequencing, and expressing cDNAs coding for allergens in Escherichia coli became of great interest to allergologists . For the recombinant production of allergens, suitable expression systems, growing conditions, and purification steps have to be established for each individual product . Finally, the purified recombinant allergen has to be carefully investigated for the biochemical, biophysical, and immunological properties . In the following paper, several prokaryotic expression systems, purification strategies, and analytical methods will be presented and pitfalls discussed. Structure (Camb), 2004 Feb, 12(2), 205 - 14 DNA binding and degradation by the HNH protein ColE7; Hsia KC et al.; The bacterial toxin ColE7 bears an HNH motif which has been identified in hundreds of prokaryotic and eukaryotic endonucleases, involved in DNA homing, restriction, repair, or chromosome degradation . The crystal structure of the nuclease domain of ColE7 in complex with a duplex DNA has been determined at 2.5 A resolution . The HNH motif is bound at the minor groove primarily to DNA phosphate groups at and beyond the 3' side of the scissile phosphate, with little interaction with ribose groups and bases . This result provides a structural basis for sugar- and sequence-independent DNA recognition and the inhibition mechanism by inhibitor Im7, which blocks the substrate binding site but not the active site . Structural comparison shows that two families of endonucleases bind and bend DNA in a similar way to that of the HNH ColE7, indicating that endonucleases containing a "betabetaalpha-metal" fold of active site possess a universal mode for protein-DNA interactions. Mar Biotechnol (NY), 2001 May, 3(3), 241 - 5 Effects of insert size on transposition efficiency of the sleeping beauty transposon in mouse cells; Karsi A et al.; Transposon vectors are widely used in prokaryotic and lower eukaryotic systems . However, they were not available for use in vertebrate animals until the recent reconstitution of a synthetic fish transposon, Sleeping Beauty ( SB) . The reacquisition of transposability of the SB transposase fostered great enthusiasm for using transposon vectors as tools in vertebrate animals, particularly for gene transfer to facilitate accelerated integration of transgenes into chromosomes . Here, we report the effects of insert sizes on transposition efficiency of SB . A significant effect of insert size on efficiency of transposition by SB was found . The SB transposase enhanced the integration efficiency effectively for SB transposon up to approximately 5.6 kb, but lost its ability to enhance the integration efficiency when the transposon size was increased to 9.1 kb . This result indicates that the SB transposon system is highly applicable for transferring small genes, but may not be applicable for transferring very large genes. Mar Biotechnol (NY), 2002 Dec, 4(6), 546 - 58 Synthesis of the Neurotoxin Quinolinic Acid in Apoptotic Tissue from Suberites domuncula: Cell Biological, Molecular Biological, and Chemical Analyses; Schroder HC et al.; Sessile marine animals, such as sponges, are prone to infection by prokaryotic as well as by eukaryotic attacking organisms . Using the sponge Suberites domuncula we document for the first time that in its apoptotic tissue a toxic compound is produced that very likely controls the elimination of the dying tissue . Apoptosis was induced by exposing the sponges to 2,2?-dipyridyl or by maintaining them under nonaeration conditions . After that treatment at least one eukaryotic epibiont (Bittium sp.) could be found grazing on apoptotic tissue . Cell proliferation assays demonstrated that aqueous extracts from unaffected sponge tissue displayed no cytotoxicity . However, addition of an extract from apoptotic tissue to neuronal cells from rat brain exerted strong toxicity . The underlying compound was identified as quinolinic acid; quantitative determination showed that quinolinic acid is present only in apoptotic tissue (4.8 mg/g dry wet weight) . The complementary DNA encoding the key enzyme of the quinolinic acid pathway, 3-hydroxyanthranilate 3,4-dioxygenase, was cloned and characterized . The expression of this gene is up-regulated in apoptotic tissue . These data suggest that a complex molecular network controls apoptotic elimination of sponge tissue, which results in the synthesis of the bioactive compound quinolinic acid that controls the elimination of the tissue, perhaps via differential effects on grazing epibionts. J Struct Biol, 2004 Mar, 145(3), 254 - 62 Cell organization and ultrastructure of a magnetotactic multicellular organism; Keim CN et al.; Magnetotactic multicellular aggregates and many-celled magnetotactic prokaryotes have been described as spherical organisms composed of several Gram-negative bacteria capable to align themselves along magnetic fields and swim as a unit . Here we describe a similar organism collected in a large hypersaline lagoon in Brazil . Ultrathin sections and freeze fracture replicas showed that the cells are arranged side by side and face both the external environment and an internal acellular compartment in the center of the organism . This compartment contains a belt of filaments linking the cells, and numerous membrane vesicles . The shape of the cells approaches a pyramid, with the apex pointing to the internal compartment, and the basis facing the external environment . The contact region of two cells is flat and represents the pyramid faces, while the contacts of three or more cells contain cell projections and represent the edges . Freeze-fracture replicas showed a high concentration of intramembrane particles on the edges and also in the region of the outer membrane that faces the external environment . Dark field optical microscopy showed that the whole organism performs a coordinated movement with either straight or helicoidal trajectories . We conclude that the organisms described in this work are, in fact, highly organized prokaryotic multicellular organisms. Gene, 2004 Feb 18, 327(1), 61 - 73 Homogeneity and long-term stability of tetracycline-regulated gene expression with low basal activity by using the rtTA2S-M2 transactivator and insulator-flanked reporter vectors; Qu Z et al.; Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture . Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA . These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability . Moreover, they also require high concentrations of Doxycycline (Dox) . The 5 amino acid substitutions in the rtTA variant rtTA2S-M2 confer exquisite sensitivity to Dox . Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells . However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined . We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture . Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE-green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture . Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated . The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness . However, inclusion of flanking double copies of a 'minimal core element' of the chicken beta-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression . Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter . Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox. J Biomol NMR, 2004 Apr, 28(4), 357 - 67 Using codon optimization, chaperone co-expression, and rational mutagenesis for production and NMR assignments of human eIF2 alpha; Ito T et al.; Producing a well behaved sample at high concentration is one of the main hurdles when starting a new project on an interesting protein . Especially when one attempts to overexpress a eukaryotic protein in bacteria, some difficulties are encountered, such as low expression level, low solubility, or even lack of folded structure . Overexpression in prokaryotic systems is highly desirable for cost-effective production of different isotope-labeled samples needed for NMR studies . Here we describe generally applicable methods for obtaining highly concentrated protein samples efficiently . This approach was developed as we tried to produce a NMR-suitable sample of the 35 kDa human translation initiation factor eIF2 alpha, a protein that expresses poorly in E . coli and has very low solubility . First, an E . coli codon-optimized gene was synthesized on a thermal cycler, which increased the expression level by a factor of two . Second, we used co-expression of bacterial chaperone proteins, which largely increased the fraction of correctly folded protein found in the soluble phase . Third, we used rational mutagenesis guided by both the sequence alignment among homologues and the homology of one domain to a known fold for improving solubility and stability of the target protein by tenfold . Combining all these methods made it possible to produce from a one-liter preparation a 0.5 mM sample of human eIF2 alpha that showed well-resolved NMR spectra and enabled nearly complete assignment of the protein . These methods may be generally useful for studies of other eukaryotic proteins that are otherwise difficult to express and exhibit poor solubility. Bull Math Biol, 2004 Mar, 66(2), 261 - 99 Qualitative simulation of the initiation of sporulation in Bacillus subtilis; De Jong H et al.; Under conditions of nutrient deprivation, the Gram positive soil bacterium Bacillus subtilis can abandon vegetative growth and form a dormant, environmentally-resistant spore instead . The decision to either divide or sporulate is controlled by a large and complex genetic regulatory network integrating various environmental, cell-cycle, and metabolic signals . Although sporulation in B . subtilis is one of the best-understood model systems for prokaryotic development, very little quantitative data on kinetic parameters and molecular concentrations are available . A qualitative simulation method is used to model the sporulation network and simulate the response of the cell to nutrient deprivation . Using this method, we have been able to reproduce essential features of the choice between vegetative growth and sporulation, in particular the role played by competing positive and negative feedback loops. Biochim Biophys Acta, 2004 Feb 15, 1608(2-3), 75 - 96 The low molecular mass subunits of the photosynthetic supracomplex, photosystem II; Shi LX et al.; The photosystem II (PSII) complex is located in the thylakoid membrane of higher plants, algae and cyanobacteria and drives the water oxidation process of photosynthesis, which splits water into reducing equivalents and molecular oxygen by solar energy . Electron and X-ray crystallography analyses have revealed that the PSII core complex contains between 34 and 36 transmembrane alpha-helices, depending on the organism . Of these helices at least 12-14 are attributed to low molecular mass proteins . However, to date, at least 18 low molecular mass (<10 kDa) subunits are putatively associated with the PSII complex . Most of them contain a single transmembrane span and their protein sequences are conserved among photosynthetic organisms . In addition, these proteins do not have any similarity to any known functional proteins in any type of organism, and only two of them bind a cofactor . These findings raise intriguing questions about why there are so many small protein subunits with single-transmembrane spans in the PSII complex, and their possible functions . This article reviews our current knowledge of this group of proteins . Deletion mutations of the low molecular mass subunits from both prokaryotic and eukaryotic model systems are compared in an attempt to understand the function of these proteins . From these comparisons it seems that the majority of them are involved in stabilization, assembly or dimerization of the PSII complex . The small proteins may facilitate fast dynamic conformational changes that the PSII complex needs to perform an optimal photosynthetic activity. Environ Microbiol, 2004 Mar, 6(3), 274 - 87 Diversity of prokaryotes and methanogenesis in deep subsurface sediments from the Nankai Trough, Ocean Drilling Program Leg 190; Newberry CJ et al.; Diversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co-enzyme M reductase (mcrA) genes . Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan . DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea . Sequences of Bacteria were dominated by an uncultured and deeply branching 'deep sediment group' (53% of sequences) . Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota . There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis . These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria . The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales . The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis . These substrates also gave the highest rates in 14C-radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments . Thus, this research demonstrates the importance of the 'deep sediment group' of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub-seafloor sediments. J Exp Biol, 2004 Feb, 207(Pt 6), 963 - 71 Desiccation and rehydration elicit distinct heat shock protein transcript responses in flesh fly pupae; Hayward SA et al.; Heat shock proteins (Hsps) are a ubiquitous component of the cellular response to stress in both prokaryotic and eukaryotic organisms, but their role and function during desiccation stress in terrestrial arthropods has received limited attention . Molecular responses to rehydration are arguably as important as those to desiccation in maintaining cellular integrity and enzyme activity, but the role of Hsps during stress recovery is poorly understood and has never been addressed with respect to rehydration in insects . This study identifies distinct differences in the Hsp response to desiccation and rehydration in the flesh fly Sarcophaga crassipalpis, as well as differences in the desiccation responses of diapausing and nondiapausing pupae . In nondiapausing pupae, the expression of two inducible Hsps (Hsp23 and Hsp70) is upregulated by desiccation, but the water loss threshold for Hsp expression changes at different rates of dehydration . Continued desiccation results in the prolonged expression of both Hsp23 and Hsp70, which may contribute to the delayed adult eclosion noted in samples desiccated for more than 3 days at <5% relative humidity/25 degrees C . In diapausing pupae, hsp23 and hsp70 transcripts are already highly expressed and are not further upregulated by desiccation stress . Both of the constitutive Hsps investigated, Hsp90 and Hsc70, were unresponsive to desiccation in both nondiapausing and diapausing pupae . However, both Hsp90 and Hsc70 were upregulated upon rehydration in nondiapausing and diapausing pupae . These results indicate distinct roles for the different Hsps during desiccation stress and rehydration/stress recovery . The response to desiccation recovery (rehydration) is similar to the Hsp response to cold recovery identified in S . crassipalpis: Hsp90 and Hsc70 are upregulated in both cases. Appl Environ Microbiol, 2004 Feb, 70(2), 804 - 13 Impact of virioplankton on archaeal and bacterial community richness as assessed in seawater batch cultures; Winter C et al.; During cruises in the tropical Atlantic Ocean (January to February 2000) and the southern North Sea (December 2000), experiments were conducted to monitor the impact of virioplankton on archaeal and bacterial community richness . Prokaryotic cells equivalent to 10 to 100% of the in situ abundance were inoculated into virus-free seawater, and viruses equivalent to 35 to 360% of the in situ abundance were added . Batch cultures with microwave-inactivated viruses and without viruses served as controls . The apparent richness of archaeal and bacterial communities was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA gene fragments . Although the estimated richness of the prokaryotic communities generally was greatly reduced within the first 24 h of incubation due to confinement, the effects of virus amendment were detected at the level of individual operational taxonomic units (OTUs) in the T-RFLP patterns of both groups, Archaea and BACTERIA: One group of OTUs was detected in the control samples but was absent from the virus-treated samples . This negative response of OTUs to virus amendment probably was caused by viral lysis . Additionally, we found OTUs not responding to the amendments, and several OTUs exhibited variable responses to the addition of inactive or active viruses . Therefore, we conclude that individual members of pelagic archaeal and bacterial communities can be differently affected by the presence of virioplankton. Protein Expr Purif, 2004 Mar, 34(1), 158 - 65 First structural investigation of the restriction ribonuclease RegB: NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra; Saida F et al.; The bacteriophage T4 genome-encoded ribonuclease RegB is the unique well-defined restriction endoribonuclease . This protein cleaves with an almost absolute specificity its RNA substrate in the middle of the GGAG tetranucleotide mainly found in the Shine-Dalgarno sequence (required for the prokaryotic initiation of the translation) . This protein has no significant homology to any known ribonuclease and its structure has never been investigated . The extreme toxicity of this ribonuclease prevents the expression of large quantities for structural studies . Here, we show that the toxicity of RegB can be bypassed by using the RegB H48A point mutant and explain why resolving the structure of this mutant is relevant . For nuclear magnetic resonance (NMR) purposes, we report the preparation of highly pure (13)C/(15)N double-labelled 1.2mM samples of RegB H48A using a high yield expression procedure in minimal medium (30 mg/L) . We also present a set of solution conditions that maintain the concentrated samples of this protein stable for long periods at the NMR-required temperature . Finally, we present the first (1)H/(15)N and (1)H/(13)C two-dimensional NMR spectra of RegB H48A . These spectra show that the protein is folded and that the full structural analysis of RegB by NMR is feasible. Protein Expr Purif, 2004 Mar, 34(1), 68 - 74 Non-fusion expression in Escherichia coli, purification, and characterization of a novel Ca2+- and phospholipid-binding protein annexin B1; Zhang Y et al.; Annexin B1 is a novel member of the annexin family of Ca2+- and phospholipid-binding proteins from Cysticercus cellulosae . To obtain high quality annexin B1 for biochemical and biophysical analyses, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters P(R) and P(L) . After induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form . The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps . The final yield was about 25 mg/L bacterial culture . Western blot analysis showed that recombinant annexin B1 was specifically recognized by serum of pigs infected with cysticercosis . Secondary structure predictions from circular dichroism spectroscopy indicated that alpha-helix is the main secondary structure of the protein . In anticoagulant assays, the recombinant non-fusion protein exhibited dose-dependent effects in modified kaolin partial thromboplastin time (KPTT) prolongation and doubled the clotting time of control human plasma at 60 microg/ml . The expression, purification, and initial characterization of annexin B1 set an important stage for further characterization of the protein. Bioinformatics, 2004 May 1, 20(7), 1074 - 80 Epub 2004 Feb 05. Recognition and analysis of protein-coding genes in severe acute respiratory syndrome associated coronavirus; Sharma R et al.; MOTIVATION: The recent outbreak of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) has necessitated an in-depth molecular understanding of the virus to identify new drug targets . The availability of complete genome sequence of several strains of SARS virus provides the possibility of identification of protein-coding genes and defining their functions . Computational approach to identify protein-coding genes and their putative functions will help in designing experimental protocols . RESULTS: In this paper, a novel analysis of SARS genome using gene prediction method GeneDecipher developed in our laboratory has been presented . Each of the 18 newly sequenced SARS-CoV genomes has been analyzed using GeneDecipher . In addition to polyprotein 1ab(1), polyprotein 1a and the four genes coding for major structural proteins spike (S), small envelope (E), membrane (M) and nucleocapsid (N), six to eight additional proteins have been predicted depending upon the strain analyzed . Their lengths range between 61 and 274 amino acids . Our method also suggests that polyprotein 1ab, polyprotein 1a, S, M and N are proteins of viral origin and others are of prokaryotic . Putative functions of all predicted protein-coding genes have been suggested using conserved peptides present in their open reading frames . AVAILABILITY: Detailed results of GeneDecipher analysis of all the 18 strains of SARS-CoV genomes are available at http://www.igib.res.in/sarsanalysis.html Bioinformatics, 2004 Mar 22, 20(5), 673 - 81 Epub 2004 Feb 05. Comparison of various algorithms for recognizing short coding sequences of human genes; Gao F et al.; MOTIVATION: Since the early 1980s of the twentieth century, there has been great progress in the development of computational gene-finding algorithms . Some problems, however, have not yet been solved currently . Recognizing short genes in prokaryotes and short exons in eukaryotes is one of such problems . The paper is devoted to assessing various algorithms, including those currently available and the new ones proposed here, in order to find the best algorithm to solve the issue . RESULTS: The databases consisting of phase-specific coding and non-coding sequences of human genes with length of 192, 162, 129, 108, 87, 63 and 42 bp, respectively, have been established . Based on the databases and a standard benchmark, 19 algorithms were evaluated, which include the methods of Markov models with orders of 1 through 5, codon usage, hexamer usage, codon preference, amino acid usage, codon prototype, Fourier transform and 8 Z curve methods with various numbers of parameters . Consequently, the Z curve methods with 69 and 189 parameters are the best ones among them, based on the databases constructed here . In addition to the highest recognition accuracy confirmed by 10-fold cross-validation tests, the Z curve methods are much simpler computationally than the second best one, the fifth-order Markov chain model, in which 12 288 parameters are used . We hope that the Z curve methods presented in this paper would be beneficial to the further development of gene-finding algorithms . AVAILABILITY: The programs of various Z curve methods are available on request. Bioinformatics, 2004 Apr 12, 20(6), 984 - 5 Epub 2004 Feb 05. Exploring genome architecture through GOV: a WWW-based gene order visualizer; Sakharkar KR et al.; MOTIVATION: The past decade has seen extension in the methods of sequence analysis from single gene based to analyzing multiple genes and proteins simultaneously . Consequently, there is a need for software tools that will allow mining of these enormous datasets at genome level effectively . A key challenge is to make them user-friendly, available to a larger community and integrate with public domain software without much hassle . RESULTS: A web-based interactive computational tool is described for visualization and comparison of gene order from prokaryotic and selected viral genome data . Many intriguing similarities and differences in gene order of multiple genomes can be compared and revealed . The interface facilitates easy extraction of the nucleotide sequence of the gene of interest and BLAST analysis against GenBank at NCBI to provide insights into gene functions and orthologs of the gene in other species. Mol Microbiol, 2004 Feb, 51(4), 1143 - 54 Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro; Merickel SK et al.; In vitro studies have demonstrated that Hin-catalysed site-specific DNA inversion occurs within a tripartite invertasome complex assembled at a branch on a supercoiled DNA molecule . Multiple DNA exchanges within a recombination complex (processive recombination) have been found to occur with particular substrates or reaction conditions . To investigate the mechanistic properties of the Hin recombination reaction in vivo, we have analysed the topology of recombination products generated by Hin catalysis in growing cells . Recombination between wild-type recombination sites in vivo is primarily limited to one exchange . However, processive recombination leading to knotted DNA products is efficient on substrates containing recombination sites with non-identical core nucleotides . Multiple exchanges are limited by a short DNA segment between the Fis-bound enhancer and closest recombination site and by the strength of Fis-Hin interactions, implying that the enhancer normally remains associated with the recombining complex throughout a single exchange reaction, but that release of the enhancer leads to multiple exchanges . This work confirms salient mechanistic aspects of the reaction in vivo and provides strong evidence for the propensity of plectonemically branched DNA in prokaryotic cells . We also demonstrated that a single DNA exchange resulting in inversion in vitro is accompanied by a loss of four negative supercoils. Genome Res, 2004 Feb, 14(2), 201 - 8 A motif co-occurrence approach for genome-wide prediction of transcription-factor-binding sites in Escherichia coli; Bulyk ML et al.; Various computational approaches have been developed for predicting cis-regulatory DNA elements in prokaryotic genomes . We describe a novel method for predicting transcription-factor-binding sites in Escherichia coli . Our method takes advantage of the principle that transcription factors frequently coregulate gene expression, but without requiring prior knowledge of which groups of genes are coregulated . Using position weight matrices for 49 known transcription factors, we examined spacings between pairs of matrix hits . These pairs were assigned probabilities according to the overrepresentation of their separation distance . The functions of many open reading frames (ORFs) downstream from predicted binding sites are unknown, and may correspond to novel regulon members . For five predictions, knockouts with mutated replacements of the predicted binding sites were created in E . coli MG1655 . Quantitative real-time PCR (RT-PCR) indicates that for each of the knockouts, at least one gene immediately downstream exhibits a statistically significant change in mRNA expression . This approach may be useful in analyzing binding sites in a variety of organisms. J Bacteriol, 2004 Feb, 186(4), 1165 - 74 clpB, a novel member of the Listeria monocytogenes CtsR regulon, is involved in virulence but not in general stress tolerance; Chastanet A et al.; Clp-HSP100 ATPases are a widespread family of ubiquitous proteins that occur in both prokaryotes and eukaryotes and play important roles in the folding of newly synthesized proteins and refolding of aggregated proteins . They have also been shown to participate in the virulence of several pathogens, including Listeria monocytogenes . Here, we describe a member of the Clp-HSP100 family of L . monocytogenes that harbors all the characteristics of the ClpB subclass, which is absent in the closely related gram-positive model organism, Bacillus subtilis . Transcriptional analysis of clpB revealed a heat shock-inducible sigma(A)-type promoter . Potential binding sites for the CtsR regulator of stress response were identified in the promoter region . In vivo and in vitro approaches were used to show that expression of clpB is repressed by CtsR, a finding indicating that clpB is a novel member of the L . monocytogenes CtsR regulon . We showed that ClpB is involved in the pathogenicity of L . monocytogenes since the DeltaclpB mutant is significantly affected by virulence in a murine model of infection; we also demonstrate that this effect is apparently not due to a defect in general stress resistance . Indeed, ClpB is not involved in tolerance to heat, salt, detergent, puromycin, or cold stress, even though its synthesis is inducible by heat shock . However, ClpB was shown to play a role in induced thermotolerance, allowing increased resistance of L . monocytogenes to lethal temperatures . This work gives the first example of a clpB gene directly controlled by CtsR and describes the first role for a ClpB protein in induced thermotolerance and virulence in a gram-positive organism. J Mol Biol, 2004 Feb 13, 336(2), 539 - 49 Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli; Zmijewski MA et al.; Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes . Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception . Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues . The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function . The M . mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2 . Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M . mazei DnaK . The M . mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison . However, in vivo analyses indicate that there are also significant differences . The M . mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M . mazei dnaK gene . Thus, while the data from in vitro tests demonstrate functional similarities between the M . mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ. Environ Microbiol, 2004 Feb, 6(2), 121 - 30 Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity; Taylor MW et al.; Biodiversity is fundamental to both eukaryote and prokaryote ecology, yet investigations of diversity often differ markedly between the two disciplines . Host specificity - the association of organisms with only a few (specialism) or many (generalism) host species - is recognized within eukaryote ecology as a key determinant of diversity . In contrast, its implications for microbial diversity have received relatively little attention . Here we explore the relationship between microbial diversity and host specificity using marine sponge-bacteria associations . We used a replicated, hierarchical sampling design and both 16S rDNA- and rpoB-based denaturing gradient gel electrophoresis (DGGE) to examine whether three co-occurring sponges from temperate Australia -Cymbastela concentrica, Callyspongia sp . and Stylinos sp . - contained unique, specialized communities of microbes . Microbial communities varied little within each species of sponge, but variability among species was substantial . Over five seasons, the microbial community in C . concentrica differed significantly from other sponges, which were more similar to seawater . Overall, three types of sponge-associated bacteria were identified via 16S rDNA sequencing of excised DGGE bands: 'specialists'- found on only one host species, 'sponge associates'- found on multiple hosts but not in seawater, and 'generalists' from multiple hosts and seawater . Analogous to other high diversity systems, the degree of specificity of prokaryotes to host eukaryotes could have a potentially significant effect on estimates of marine microbial diversity. Mol Microbiol, 2004 Jan, 51(2), 567 - 77 RcaE is a complementary chromatic adaptation photoreceptor required for green and red light responsiveness; Terauchi K et al.; The recent discovery of large numbers of phytochrome photoreceptor genes in both photosynthetic and non-photosynthetic prokaryotes has led to efforts to understand their physiological roles in environmental acclimation . One receptor in this class, RcaE, is involved in controlling complementary chromatic adaptation, a process that regulates the transcription of operons encoding light-harvesting proteins in cyanobacteria . Although all previously identified phytochrome responses are maximally sensitive to red and far red light, complementary chromatic adaptation is unique in that it is responsive to green and red light . Here, we present biochemical and genetic evidence demonstrating that RcaE is a photoreceptor and that it requires the cysteine at position 198 to ligate an open chain tetrapyrrole covalently in a manner analogous to chromophore attachment in plant phytochromes . Furthermore, although the wild-type rcaE gene can rescue red and green light photoresponses of an rcaE null mutant, a gene in which the codon for cysteine 198 is converted to an alanine codon rescues the red light but not the green light response . Thus, RcaE is a photoreceptor that is required for both green and red light responsiveness during complementary chromatic adaptation and is the first identified phytochrome class sensor that is involved in sensing and responding to green and red light rather than red and far red light. Bioorg Med Chem, 2004 Feb 15, 12(4), 683 - 92 Analogues of thiolactomycin as potential anti-malarial and anti-trypanosomal agents; Jones SM et al.; A series of analogues of the naturally occurring antibiotic thiolactomycin (TLM) have been synthesised and evaluated for their ability to inhibit the growth of the malaria parasite, Plasmodium falciparum . Thiolactomycin is an inhibitor of Type II fatty acid synthase which is found in plants and most prokaryotes, but not an inhibitor of Type I fatty acid synthase in mammals . A number of the analogues showed inhibition equal to or greater than TLM . The introduction of hydrophobic alkyl groups at the C3 and C5 positions of the thiolactone ring lead to increased inhibition, the best showing a fourteenfold increase in activity over TLM . In addition, some of the analogues showed activity when assayed against the parasitic protozoa, Trypanosoma cruzi and Trypanosoma brucei. J Am Chem Soc, 2004 Feb 11, 126(5), 1363 - 8 Mapping enzyme active sites in complex proteomes; Adam GC et al.; Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited . To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity . Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform . We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes . For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1) . These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes . More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites. J Exp Bot, 2004 Mar, 55(397), 547 - 56 Epub 2004 Jan 30. Differential expression of three genes encoding an ethylene receptor in rice during development, and in response to indole-3-acetic acid and silver ions; Yau CP et al.; Five ethylene receptor genes, OS-ERS1, OS-ERS2, OS-ETR2, OS-ETR3, and OS-ETR4 were isolated and characterized from rice . The genomic structure of OS-ERS1 and OS-ERS2 revealed that the introns within the coding sequences occurred in conserved positions to those of At-ETR1 and At-ERS1, whereas each of the OS-ETR2, OS-ETR3, and OS-ETR4 genes contained 1 intron within its coding region located at a position equivalent to those of At-ERS2, At-ETR2, and At-EIN4 . Deduced amino acid sequences of OS-ERS1, OS-ERS2, OS-ETR2, OS-ETR3, and OS-ETR4 showed that they exhibited significant homology to the prokaryotic two-component signal transducer and a wide range of ethylene receptors in a variety of plant species . Northern analysis revealed that the level of OS-ETR2 mRNA was markedly elevated either by the exogenous application of IAA or by ethylene treatment in young etiolated rice seedlings, whereas the OS-ERS1 transcript level was only slightly induced under the same experimental conditions . Pretreatment with silver prevented IAA-induced and ethylene-induced accumulation of both mRNAs (OS-ERS1 and OS-ETR2) . However, the abundance of OS-ERS2 mRNA was shown to be down-regulated by both IAA and ethylene treatments, indicating that it was not positively regulated by ethylene . Analysis of the expression of the three ethylene receptor genes in different tissues of rice has unravelled their corresponding tissue-specificity in which OS-ERS1 was constitutively expressed in considerable amounts in all tissues studied, while OS-ERS2 and OS-ETR2 exhibited differential expression patterns in different tissues of rice . Moreover, higher levels of these three mRNAs were commonly observed in anthers when compared with their corresponding levels in other tissues, suggesting the important role played by ethylene involved in the regulation of pollen development in rice . Among the five ethylene receptor genes, the expression levels of both OS-ETR3 and OS-ETR4 were too low to be detected by the northern blot analysis . Results from RT-PCR illustrated that both mRNAs were present in young green rice seedlings and anthers. Curr Med Chem, 2004 Jan, 11(2), 199 - 219 The vesosome-- a multicompartment drug delivery vehicle; Kisak ET et al.; Assembling structures to divide space controllably and spontaneously into subunits at the nanometer scale is a significant challenge, although one that biology has solved in two distinct ways: prokaryotes and eukaryotes . Prokaryotes have a single compartment delimited by one or more lipid-protein membranes . Eukaryotes have nested-membrane structures that provide internal compartments--such as the cell nucleus and cell organelles in which specialized functions are carried out . We have developed a simple method of creating nested bilayer compartments in vitro via the "interdigitated" bilayer phase formed by adding ethanol to a variety of saturated phospholipids . At temperatures below the gel-liquid crystalline transition, T(m), the interdigitated lipid-ethanol sheets are rigid and flat; when the temperature is raised above T(m), the sheets become flexible and close on themselves and the surrounding solution to form closed compartments . During this closure, the sheets can entrap other vesicles, biological macromolecules, or colloidal particles . The result is efficient and spontaneous encapsulation without disruption of even fragile materials to form biomimetic nano-environments for possible use in drug delivery, colloidal stabilization, or as microreactors . The vesosome structure can take full advantage of the 40 years of progress in liposome development including steric stabilization, pH loading of drugs, and intrinsic biocompatibility . However, the multiple compartments of the vesosome give better protection to the interior contents in serum, leading to extended release of model compounds in comparison to unilamellar liposomes. Bioinformatics, 2004 May 1, 20(7), 993 - 1005 Epub 2004 Jan 29. Effects of choice of DNA sequence model structure on gene identification accuracy; Azad RK et al.; MOTIVATION: Markov chain models of DNA sequences have frequently been used in gene finding algorithms . Performance of the algorithm critically depends on the model structure and parameters . Still, the issue of choosing the model structure has not been studied with sufficient attention . RESULTS: We have assessed performance of several types of Markov chain models, both fixed order (FO) models and models with interpolation, within the framework of the GeneMark algorithm . The performance was measured in two ways: (i) the accuracy of detection of protein-coding potential in artificial DNA sequences and (ii) the accuracy of identifying genes in real prokaryotic genomes . We observed that the models built by deleted interpolation (DI) slightly outperformed other models in detecting protein-coding potential in artificial DNA sequences with GC content in the medium range and also in detecting genes in real genomes with medium GC content . For artificial and real genomic DNA with high or low GC content, we observed that the models built by DI were in some cases slightly outperformed by FO models. Curr Oncol Rep, 2004 Mar, 6(2), 88 - 95 Antitumor applications of stimulating toll-like receptor 9 with CpG oligodeoxynucleotides; Krieg AM; Tumor immunotherapy has evolved from the use of crude bacterial extracts to chemically synthesized ligands for specific immune receptors, such as Toll-like receptors (TLRs) . One of the most promising targets for therapeutic immune activation is TLR9, which detects unmethylated CpG dinucleotides present in viral and prokaryotic genomes, which are generally methylated in host DNA . This review describes the immune effects of synthetic CpG oligonucleotides as TLR9 ligands and their applications in cancer immunotherapy. J Biol Chem, 2004 Apr 9, 279(15), 14570 - 8 Epub 2004 Jan 28. Post-translational formylglycine modification of bacterial sulfatases by the radical S-adenosylmethionine protein AtsB; Fang Q et al.; C(alpha)-Formylglycine (FGly) is the catalytic residue of sulfatases . FGly is generated by post-translational modification of a cysteine (prokaryotes and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif . AtsB of Klebsiella pneumoniae is directly involved in FGly generation from serine . AtsB is predicted to belong to the newly discovered radical S-adenosylmethionine (SAM) superfamily . By in vivo and in vitro studies we show that SAM is the critical co-factor for formation of a functional AtsB.SAM.sulfatase complex and for FGly formation by AtsB . The SAM-binding site of AtsB involves (83)GGE(85) and possibly also a juxtaposed FeS center coordinated by Cys(39) and Cys(42), as indicated by alanine scanning mutagenesis . Mutation of these and other conserved cysteines as well as treatment with metal chelators fully impaired FGly formation, indicating that all three predicted FeS centers are crucial for AtsB function . It is concluded that AtsB oxidizes serine to FGly by a radical mechanism that is initiated through reductive cleavage of SAM, thereby generating the highly oxidizing deoxyadenosyl radical, which abstracts a hydrogen from the serine-C(beta)H(2)-OH side chain. Biophys J, 2004 Feb, 86(2), 846 - 60 Conduction mechanisms of chloride ions in ClC-type channels; Corry B et al.; The conduction properties of ClC-0 and ClC-1 chloride channels are examined using electrostatic calculations and three-dimensional Brownian dynamics simulations . We create an open-state configuration of the prokaryotic ClC Cl(-) channel using its known crystallographic structure as a basis . Two residues that are occluding the channel are slowly pushed outward with molecular dynamics to create a continuous ion-conducting path with the minimum radius of 2.5 A . Then, retaining the same pore shape, the prokaryotic ClC channel is converted to either ClC-0 or ClC-1 by replacing all the nonconserved dipole-containing and charged amino acid residues . Employing open-state ClC-0 and ClC-1 channel models, current-voltage curves consistent with experimental measurements are obtained . We find that conduction in these pores involves three ions . We locate the binding sites, as well as pinpointing the rate-limiting steps in conduction, and make testable predictions about how the single channel current across ClC-0 and ClC-1 will vary as the ionic concentrations are increased . Finally, we demonstrate that a ClC-0 homology model created from an alternative sequence alignment fails to replicate any of the experimental observations. Trends Genet, 2004 Feb, 20(2), 95 - 102 Driving change: the evolution of alternative genetic codes; Santos MA et al.; Pioneering studies in the 1960s that elucidated the genetic code suggested that all extant forms of life use the same genetic code . This early presumption has subsequently been challenged by the discovery of deviations of the universal genetic code in prokaryotes, eukaryotic nuclear genomes and mitochondrial genomes . These studies have revealed that the genetic code is still evolving despite strong negative forces working against the fixation of mutations that result in codon reassignment . Recent data from in vitro, in vivo and in silico comparative genomics studies are revealing significant, previously overlooked links between modified nucleosides in tRNAs, genetic code ambiguity, genome base composition, codon usage and codon reassignment. Vet Res, 2003 Nov-Dec, 34(6), 761 - 75 Antigenic and genetic characterisation of lipoprotein lppC from Mycoplasma mycoides subsp . mycoides SC; Pilo P et al.; Lipoprotein lppC, an immunodominant antigen, and its corresponding gene lppC were characterised in Mycoplasma mycoides subspecies mycoides small colony (SC) type, the etiological agent of contagious bovine pleuropneumonia (CBPP) . The lppC gene was found in the type strain of M . mycoides subsp . mycoides SC and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains . Southern blot analysis indicated the presence of at least four copies of lppC in the genome of M . mycoides subsp . mycoides SC, of which only one seems to be functional . Genes homologous to lppC have also been detected in closely related mycoplasmas such as M . mycoides subsp . mycoides large colony (LC) type and in M . sp . bovine group 7 . lppC is encoded as a precursor with a consensus sequence for a prokaryotic signal peptidase II . The amino acid sequence of lppC and its precursor showed similarity to both LppB (at the N-terminal domain) and LppQ (at the C-terminal domain), two lipoproteins described previously in M . mycoides subsp . mycoides SC . The N-terminal domain of the mature lppC seems to be surface exposed . The C-terminal domain presented an integral membrane structure made up of five repeated units, rich in hydrophobic and aromatic amino acids, which may have pore forming potential in the mycoplasmal membrane . A recombinant peptide representing the N-terminal half of lppC was obtained following cloning in vector pETHIS-1 and expression in Escherichia coli hosts . The recombinant protein was used on immunoblots for serological analysis of sera from cattle that were naturally or experimentally infected with M . mycoides subsp . mycoides SC. Arch Microbiol, 2004 Mar, 181(3), 171 - 81 Epub 2004 Jan 24. Protein phosphorylation on tyrosine in bacteria; Cozzone AJ et al.; Protein phosphorylation on tyrosine has been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes . This covalent modification is catalyzed by autophosphorylating ATP-dependent protein-tyrosine kinases that exhibit structural and functional features similar, but not identical, to those of their eukaryotic counterparts . The reversibility of the reaction is effected by two main classes of protein-tyrosine phosphatases: one includes conventional eukaryotic-like phosphatases and dual-specific phosphatases, and the other comprises acidic phosphatases of low molecular weight . Less frequently, a third class concerns enzymes of the polymerase-histidinol phosphatase type . In terms of genomic organization, the genes encoding a protein-tyrosine phosphatase and a protein-tyrosine kinase in a bacterial species are most often located next to each other on the chromosome . In addition, these genes are generally part of large operons that direct the coordinate synthesis of proteins involved in the production or regulation of exopolysaccharides and capsular polysaccharides . Recent data provide evidence that there exists a direct relationship between the reversible phosphorylation of proteins on tyrosine and the production of these polysaccharidic polymers, which are also known to be important virulence factors . Therefore, a new concept has emerged suggesting the existence of a biological link between protein-tyrosine phosphorylation and bacterial pathogenicity. Nat Med, 2004 Feb, 10(2), 187 - 92 Epub 2004 Jan 25. Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration; Heikenwalder M et al.; DNA containing unmethylated cytidyl guanosyl (CpG) sequences, which are underrepresented in mammalian genomes but prevalent in prokaryotes, is endocytosed by cells of the innate immune system, including macrophages, monocytes and dendritic cells, and activates a pathway involving Toll-like receptor-9 (TLR9) . CpG-containing oligodeoxynucleotides (CpG-ODN) are potent stimulators of innate immunity, and are currently being tested as adjuvants of antimicrobial, antiallergic, anticancer and antiprion immunotherapy . Little is known, however, about the consequences of repeated CpG-ODN administration, which is advocated for some of these applications . Here we report that daily injection of 60 microg CpG-ODN dramatically alters the morphology and functionality of mouse lymphoid organs . By day 7, lymphoid follicles were poorly defined; follicular dendritic cells (FDC) and germinal center B lymphocytes were suppressed . Accordingly, CpG-ODN treatment for > or =7 d strongly reduced primary humoral immune responses and immunoglobulin class switching . By day 20, mice developed multifocal liver necrosis and hemorrhagic ascites . All untoward effects were strictly dependent on CpG and TLR9, as neither the CpG-ODN treatment of Tlr9(-/-) mice nor the repetitive challenge of wild-type mice with nonstimulatory ODN (AT-ODN) or with the TLR3 agonist polyinosinic:cytidylic acid (polyI:C) were immunotoxic or hepatotoxic. J Mol Evol, 2004 Jan, 58(1), 1 - 11 Whole proteome prokaryote phylogeny without sequence alignment: a K-string composition approach; Qi J et al.; A systematic way of inferring evolutionary relatedness of microbial organisms from the oligopeptide content, i.e., frequency of amino acid K-strings in their complete proteomes, is proposed . The new method circumvents the ambiguity of choosing the genes for phylogenetic reconstruction and avoids the necessity of aligning sequences of essentially different length and gene content . The only "parameter" in the method is the length K of the oligopeptides, which serves to tune the "resolution power" of the method . The topology of the trees converges with K increasing . Applied to a total of 109 organisms, including 16 Archaea, 87 Bacteria, and 6 Eukarya, it yields an unrooted tree that agrees with the biologists' "tree of life" based on SSU rRNA comparison in a majority of basic branchings, and especially, in all lower taxa. J Leukoc Biol, 2004 Jun, 75(6), 939 - 50 Epub 2004 Jan 23. Diversity in the Sir2 family of protein deacetylases; Buck SW et al.; The silent information regulator (Sir2) family of protein deacetylases (Sirtuins) are nicotinamide adenine dinucleotide (NAD)(+)-dependent enzymes that hydrolyze one molecule of NAD(+) for every lysine residue that is deacetylated . The Sirtuins are phylogenetically conserved in eukaryotes, prokaryotes, and Archeal species . Prokaryotic and Archeal species usually have one or two Sirtuin homologs, whereas eukaryotes typically have multiple versions . The founding member of this protein family is the Sir2 histone deacetylase of Saccharomyces cerevisiae, which is absolutely required for transcriptional silencing in this organism . Sirtuins in other organisms often have nonhistone substrates and in eukaryotes, are not always localized in the nucleus . The diversity of substrates is reflected in the various biological activities that Sirtuins function, including development, metabolism, apoptosis, and heterochromatin formation . This review emphasizes the great diversity in Sirtuin function and highlights its unusual catalytic properties. Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 7 - 13 Exploring prokaryotic taxonomy; Lilburn TG et al.; Techniques drawn from exploratory data analysis, using tools found in the S-Plus statistical software package, have been used to inspect and maintain the Bergey's Taxonomic Outline and to move towards an automated and community-based means of working on the outline . These techniques can be used to classify sequences from unnamed and uncultured organisms, to visualize errors in the taxonomy or in the curation of the sequences, to suggest emendations to the taxonomy or to the classification of extant species and to complement the visualization of phylogenies based on treeing methods . A dataset of more than 9200 aligned small-subunit rRNA sequences was analysed in the context of the current taxonomic outline . The use of the algorithm in exploring and modifying the taxonomy is illustrated with an example drawn from the family Comamonadaceae. FEBS Lett, 2004 Jan 16, 557(1-3), 45 - 8 Bacillus subtilis YxaG is a novel Fe-containing quercetin 2,3-dioxygenase; Bowater L et al.; The Bacillus subtilis genome contains genes for three hypothetical proteins belonging to the bicupin family, two of which we have previously shown to be Mn(II)-dependent oxalate decarboxylases . We have now shown that the third, YxaG, exhibits quercetin 2,3-dioxygenase activity and that it contains Fe ions . This contrasts with the eukaryotic enzyme which contains a Cu ion . YxaG is the first prokaryotic carbon monoxide-forming enzyme that utilises a flavonol to be characterised and is only the second example of a prokaryotic dioxygenolytic carbon monoxide-forming enzyme known to contain a cofactor . It is proposed to rename the B . subtilis gene qdoI. Mol Biol Evol, 2004 Apr, 21(4), 681 - 90 Epub 2004 Jan 22. Deriving the genomic tree of life in the presence of horizontal gene transfer: conditioned reconstruction; Lake JA et al.; The horizontal gene transfer (HGT) being inferred within prokaryotic genomes appears to be sufficiently massive that many scientists think it may have effectively obscured much of the history of life recorded in DNA . Here, we demonstrate that the tree of life can be reconstructed even in the presence of extensive HGT, provided the processes of genome evolution are properly modeled . We show that the dynamic deletions and insertions of genes that occur during genome evolution, including those introduced by HGT, may be modeled using techniques similar to those used to model nucleotide substitutions that occur during sequence evolution . In particular, we show that appropriately designed general Markov models are reasonable tools for reconstructing genome evolution . These studies indicate that, provided genomes contain sufficiently many genes and that the Markov assumptions are met, it is possible to reconstruct the tree of life . We also consider the fusion of genomes, a process not encountered in gene sequence evolution, and derive a method for the identification and reconstruction of genome fusion events . Genomic reconstructions of a well-defined classical four-genome problem, the root of the multicellular animals, show that the method, when used in conjunction with paralinear/logdet distances, performs remarkably well and is relatively unaffected by the recently discovered big genome artifact. J Virol Methods, 2004 Mar 15, 116(2), 161 - 7 Expression and purification of turkey coronavirus nucleocapsid protein in Escherichia coli; Loa CC et al.; Purification of turkey coronavirus (TCoV) nucleocapsid (N) protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study . Turkey coronavirus was partially purified from infected intestine of turkey embryo by sucrose gradient ultracentrifugation and RNA was extracted . The N protein gene was amplified from the extracted RNA by reverse transcription-polymerase chain reaction and cloned . The recombinant expression construct (pTri-N) was identified by polymerase chain reaction and sequencing analysis . Expression of histidine-tagged fusion N protein with a molecular mass of 57 kd was determined by Western blotting analysis . By chromatography on nickel-agarose column, the expressed N protein was purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis . The protein recovery could be 2.5 mg from 100 ml of bacterial culture . The purified N protein was recognized by antibody to TCoV in Western blotting assay . The capability of the recombinant N protein to differentiate positive serum of turkey infected with TCoV from normal turkey serum was evident in enzyme-linked immunosorbent assays (ELISA) . These results indicated that the expressed N protein is a superior source of TCoV antigen for development of antibody-capture ELISA for detection of antibodies to TCoV. Bioinformatics, 2004 Jan 22, 20(2), 276 - 8 PGTdb: a database providing growth temperatures of prokaryotes; Huang SL et al.; Included in Prokaryotic Growth Temperature database (PGTdb) are a total of 1334 temperature data from 1072 prokaryotic organisms, Bacteria and Archaea: PGTdb integrates microbial growth temperature data from literature survey with their nucleotide/protein sequence and protein structure data from related databases . A direct correlation is observed between the average growth temperature of an organism and the melting temperature of proteins from the organism . Therefore, this database is useful not only for microbiologists to obtain cultivation condition, but also for biochemists and structure biologists to study the correlation between protein sequences/structures and their thermostability . In addition, the taxonomy and ribosomal RNA sequence(s) of an organism are linked through NCBI Taxonomy and the Ribosomal RNA Operon Copy Number Database umdb, respectively . PGTdb is the only integrated database on the Internet to provide the growth temperature data of the prokaryotes and the combined information of their nucleotide/protein sequences, protein structures, taxonomy and phylogeny . AVAILABILITY: http://pgtdb.csie.ncu.edu.tw FEMS Immunol Med Microbiol, 2004 Jan 15, 40(1), 11 - 9 Simple sequence repeats (microsatellites): mutational mechanisms and contributions to bacterial pathogenesis . A meeting review; Bayliss CD et al.; This review summarises the presentations and discussions that took place during a European Science Foundation-funded workshop whose purpose was to gain current perspectives on the mutational mechanisms of simple sequence repeats and the contribution of localised hypermutation in such repeats to bacterial pathogenesis . In vitro biophysical and biochemical assays of mutational mechanisms were covered as well as genetic studies in various eukaryotic and prokaryotic organisms . Presentations on bacterial pathogenesis elaborated investigations of the use of repeats for typing of strains, epidemiological investigations of mutation rates and functions of loci whose expression is controlled by simple sequence repeats . This review tabulates current perspectives on the cis- and trans-acting factors for mutation of simple sequence repeats and the orientations of mononucleotide repeats in some bacterial species that utilise repeats for adaptation. FEMS Microbiol Lett, 2004 Jan 15, 230(1), 137 - 42 Cloning and characterization of preferentially expressed genes in an aluminum-tolerant mutant derived from Penicillium chrysogenum IFO4626; Sugimoto M et al.; cDNAs expressed preferentially in an Al-tolerant microorganism were isolated by subtraction hybridization with cDNAs of Al-sensitive Penicillium chrysogenum IFO4626 as driver cDNA and cDNAs of the Al-tolerant mutant derived from the wild cells by UV irradiation as tester cDNA . Northern blot analysis revealed that mRNA levels of six genes were increased significantly in the Al-tolerant mutant after exposure to Al stress when compared with the wild cells . Two genes accumulated in both the presence and absence of Al stress and four genes were induced by Al stress in the Al-tolerant mutant . cDNA fragments were amplified by rapid amplification of cDNA ends and sequenced to obtain full-length cDNAs of the six genes . Two genes were novel or predicted ones and the others showed significant homology to known genes, ADP/ATP translocase, enolase, cysteine synthase, and glucoamylase, which are induced by environmental stresses in prokaryotic and eukaryotic cells . These enzyme activities increased in the Al-tolerant mutant when compared to those in the wild cells, showing that not only the levels of gene expression but also the levels of enzyme activities increased in the Al-tolerant mutant. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 489 - 94 Cloning of a gene encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase from Bordetella sp . 10d; Murakami S et al.; Bordetella sp . 10d produces a novel dioxygenase catalyzing the meta-cleavage of 4-amino-3-hydroxybenzoic acid, 4-amino-3-hydroxybenzoate 2,3-dioxygenase (4A3HBA23D) . A gene encoding 4A3HBA23D was cloned and named ahdA . The deduced amino acid sequence of ahdA showed 29.2-24.2% identities to those of prokaryotic and eukaryotic 3-hydoxybenzoate 3,4-dioxygenases in reported meta-cleavage dioxygenases . However, no identities were observed in the amino-terminal sequences of the first 29 amino acid residues . An ORF was found downstream of ahdA . The deduced amino acid sequence of the ORF showed identities to those of LysR family regulators involved in protocatechuate metabolism and contained motifs conserved in the regulators . On the basis of these results, the ORF was named ahdR encoding a putative LysR family regulator . The transcription start point of ahdA was localized 414-bp upstream of the start codon of ahdA . Two DNA-binding motifs of LysR family regulators were found upstream of the transcription start point . These observations suggest that a LysR family regulator encoded by ahdR regulates the expression of ahdA. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 476 - 82 N(6)-Methyldeoxyadenosine, a nucleoside commonly found in prokaryotes, induces C2C12 myogenic differentiation; Charles MP et al.; N(6)-methyl-2(')-deoxyadenosine (MedAdo) is a nucleoside naturally found in prokaryotic DNA . Interestingly, the N(6)-methylation of adenine in DNA seems to have been counter-selected during the course of evolution since MedAdo has not been detected in mammalian DNA until now . We show here that treatment with MedAdo induces myogenesis in C2C12 myoblasts . The presence of MedAdo in C2C12 DNA was investigated using a method based on HPLC coupled to electrospray ionization tandem mass spectrometry which is several thousand fold more sensitive than assays used previously . By this procedure, MedAdo is detected in the DNA from MedAdo-treated cells but remains undetectable in the DNA from control cells . Furthermore, MedAdo regulates the expression of p21, myogenin, mTOR, and MHC . Interestingly, in the pluripotent C2C12 cell line, MedAdo drives the differentiation towards myogenesis only . Thus, the biological effect of MedAdo is suppressed in the presence of BMP-2 which transdifferentiates C2C12 from myogenic into osteogenic lineage cells . Taken together these results point to MedAdo as a novel inducer of myogenesis and further extends the differentiation potentialities of this methylated nucleoside . Furthermore, these data raise the intriguing possibility that the biological effects of MedAdo on cell differentiation may have led to its counter-selection in eukaryotes. BioDrugs, 2004, 18(1), 51 - 62 Expression systems for the production of recombinant pharmaceuticals; Sodoyer R; The new generation of biological products are largely the result of genetic engineering . The qualitative and quantitative demand for recombinant proteins is steadily increasing . Molecular biologists are constantly challenged by the need to improve and optimise the existing expression systems, and also develop novel approaches to face the demands of producing the complex proteins of tomorrow . This continuous evolution is paralleled by growing concerns about the safety of these novel pharmaceuticals, with health authorities setting high standards for certification . One of the strategies used by researchers in this field involves sourcing new genetic elements for incorporation into expression systems by systematically analysing the rich natural diversity of microorganisms and plant-based expression systems . There are, in addition, numerous tools for modifying microorganisms and for re-engineering existing biological pathways or processes to meet the needs of the pharmaceutical industry . The aim of this review is to present the conventional and alternative expression systems, focusing on prokaryotic expression systems and briefly exploring other complementary recombinant protein production systems and their unique features. BioDrugs, 2004, 18(1), 37 - 50 A multi-model approach to nucleic acid-based drug development; Gautherot I et al.; With the advent of functional genomics and the shift of interest towards sequence-based therapeutics, the past decades have witnessed intense research efforts on nucleic acid-mediated gene regulation technologies . Today, RNA interference is emerging as a groundbreaking discovery, holding promise for development of genetic modulators of unprecedented potency . Twenty-five years after the discovery of antisense RNA and ribozymes, gene control therapeutics are still facing developmental difficulties, with only one US FDA-approved antisense drug currently available in the clinic . Limited predictability of target site selection models is recognized as one major stumbling block that is shared by all of the so-called complementary technologies, slowing the progress towards a commercial product . Currently employed in vitro systems for target site selection include RNAse H-based mapping, antisense oligonucleotide microarrays, and functional screening approaches using libraries of catalysts with randomized target-binding arms to identify optimal ribozyme/DNAzyme cleavage sites . Individually, each strategy has its drawbacks from a drug development perspective . Utilization of message-modulating sequences as therapeutic agents requires that their action on a given target transcript meets criteria of potency and selectivity in the natural physiological environment . In addition to sequence-dependent characteristics, other factors will influence annealing reactions and duplex stability, as well as nucleic acid-mediated catalysis . Parallel consideration of physiological selection systems thus appears essential for screening for nucleic acid compounds proposed for therapeutic applications . Cellular message-targeting studies face issues relating to efficient nucleic acid delivery and appropriate analysis of response . For reliability and simplicity, prokaryotic systems can provide a rapid and cost-effective means of studying message targeting under pseudo-cellular conditions, but such approaches also have limitations . To streamline nucleic acid drug discovery, we propose a multi-model strategy integrating high-throughput-adapted bacterial screening, followed by reporter-based and/or natural cellular models and potentially also in vitro assays for characterization of the most promising candidate sequences, before final in vivo testing. Trends Cell Biol, 1995 Dec, 5(12), 453 - 7 Two-component signal-transduction systems in budding yeast MAP a different pathway? Morgan BA, Bouquin N, Johnston LH. Until recently, two-component signal-transduction pathways were thought to be exclusively found in bacteria . Some eukaryotic examples have now been characterized but, at least in the budding yeast Saccharomyces cerevisiae, it appears that this type of signal-transduction pathway is not utilized as extensively as in bacteria . Further, the few eukaryotic examples described suggest that two-component signal-transduction pathways might not be freestanding, as in prokaryotes, but might effect gene expression by regulating eukaryotic mitogen-activated protein (MAP) kinase pathways. Trends Cell Biol, 1993 Oct, 3(10), 335 - 9 Protein translocation across membranes: common themes in divergent organisms; High S et al.; Specific signal sequences are required for the translocation of proteins into and across both the endoplasmic reticulum of eukaryotes and the plasma membrane of prokaryotes . The similar properties of these signals, together with their ability to function when transferred between systems, suggested that the mechanisms of translocation in the two cases may be fundamentally similar . Indeed, recent findings have revealed striking similarities between essential components of the prokaryotic and eukaryotic translocation systems, suggesting that both are derived from a common ancestor. Trends Cell Biol, 1993 Dec, 3(12), 421 - 6 Unusual routes of protein secretion: the easy way out; Kuchler K; Increasing numbers of polypeptides are being discovered that lack a cleavable hydrophobic signal sequence and are released from cells without passing through the classical secretory pathway . This article reviews the current knowledge of these alternative secretion pathways in prokaryotes and eukaryotes and discusses whether the mechanisms described in bacteria and yeast can be used as paradigms to explain unusual secretory phenomena in animal cells. In Silico Biol . 2003 Sep 16;3(4):0037 {Epub ahead of print} YACOP: Enhanced gene prediction obtained by a combination of existing methods; Tech M et al.; The performance of gene-predicting tools varies considerably if evaluated with respect to the parameters sensitivity and specificity or their capability to identify the correct start codon . We were interested to validate tools for gene prediction and to implement a metatool named YACOP, which combines existing tools and has a higher performance . YACOP parses and combines the output of the three gene-predicting systems Criticia, Glimmer and ZCURVE . It outperforms each of the programs tested with its high sensitivity and specificity values combined with a larger number of correctly predicted gene starts . Performance of YACOP and the gene-finding programs was tested by comparing their output with a carefully selected set of annotated genomes . We found that the problem of identifying genes in prokaryotic genomes by means of computational analysis was solved satisfactorily . In contrast, the correct localization of the start codon still appeared to be a problem, as in all cases under test at least 7.8% and up to 32.3% of the positions given in the annotations differed from the locus predicted by any of the programs tested . YACOP can be downloaded from http://www.g2l.bio.uni-goettingen.de. Nat Struct Mol Biol, 2004 Feb, 11(2), 149 - 56 Epub 2004 Jan 11. A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase; Kise Y et al.; Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cells . The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine . In contrast, the closely related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS) . Here, we determined the crystal structure of human mini TrpRS at a resolution of 2.3 A and compared the structure with those of prokaryotic TrpRS and human mini TyrRS . Deletion of the tRNA anticodon-binding (TAB) domain insertion, consisting of eight residues in the human TrpRS, abolished the enzyme's apoptotic activity for endothelial cells, whereas its translational catalysis and cell-binding activities remained unchanged . Thus, we have identified the inserted peptide motif that activates the angiostatic signaling. J Bacteriol, 2004 Feb, 186(3), 683 - 91 ATP/ADP translocases: a common feature of obligate intracellular amoebal symbionts related to Chlamydiae and Rickettsiae; Schmitz-Esser S et al.; ATP/ADP translocases catalyze the highly specific transport of ATP across a membrane in an exchange mode with ADP . Such unique transport proteins are employed by plant plastids and have among the prokaryotes so far only been identified in few obligate intracellular bacteria belonging to the Chlamydiales and the Rickettsiales . In this study, 12 phylogenetically diverse bacterial endosymbionts of free-living amoebae and paramecia were screened for the presence of genes encoding ATP/ADP transport proteins . The occurrence of ATP/ADP translocase genes was found to be restricted to endosymbionts related to rickettsiae and chlamydiae . We showed that the ATP/ADP transport protein of the Parachlamydia-related endosymbiont of Acanthamoeba sp . strain UWE25, a recently identified relative of the important human pathogens Chlamydia trachomatis and Chlamydophila pneumoniae, is functional when expressed in the heterologous host Escherichia coli and demonstrated the presence of transcripts during the chlamydial developmental cycle . These findings indicate that the interaction between Parachlamydia-related endosymbionts and their amoeba hosts concerns energy parasitism similar to the interaction between pathogenic chlamydiae and their human host cells . Phylogenetic analysis of all known ATP/ADP translocases indicated that the genes encoding ATP/ADP translocases originated from a chlamydial ancestor and were, after an ancient gene duplication, transferred horizontally to rickettsiae and plants. J Bacteriol, 2004 Feb, 186(3), 623 - 30 A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell; So AK et al.; A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes . In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes . These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth . Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes . Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp . and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity . The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle. J Biol Chem, 2004 Apr 2, 279(14), 13601 - 6 Epub 2004 Jan 16. The Bacillus subtilis counterpart of the mammalian 3-methyladenine DNA glycosylase has hypoxanthine and 1,N6-ethenoadenine as preferred substrates; Aamodt RM et al.; The AAG family of 3-methyladenine DNA glycosylases was initially thought to be limited to mammalian cells, but genome sequencing efforts have revealed the presence of homologous proteins in certain prokaryotic species as well . Here, we report the first molecular characterization of a functional prokaryotic AAG homologue, i.e . YxlJ, termed bAag, from Bacillus subtilis . The B . subtilis aag gene was expressed in Escherichia coli, and the protein was purified to homogeneity . As expected, B . subtilis Aag was found to be a DNA glycosylase, which releases 3-alkylated purines and hypoxanthine, as well as the cyclic etheno adduct 1,N(6)-ethenoadenine from DNA . However, kinetic analysis showed that bAag removed hypoxanthine much faster than human AAG with a 10-fold higher value for k(cat), whereas the rate of excision of 1, N(6)-ethenoadenine was found to be similar . In contrast, it was found that bAag removes 3-methyladenine and 3-methylguanine approximately 10-20 times more slowly than human AAG, and there was hardly any detectable excision of 7-methylguanine . It thus appears that bAag has a minor role in the repair of DNA alkylation damage and an important role in preventing the mutagenic effects of deaminated purines and cyclic etheno adducts in Bacillus subtilis. Cancer Res, 2004 Jan 1, 64(1), 1 - 6 In vitro characterization of enzymatic properties and inhibition of the p53R2 subunit of human ribonucleotide reductase; Shao J et al.; p53R2 is a newly identified subunit of ribonucleotide reductase (RR) and plays a crucial role in supplying precursors for DNA repair in a p53-dependent manner . In our current work, all three human RR subunit proteins (p53R2, hRRM2, and hRRM1) were prokaryotically expressed and highly purified . Using an in vitro {(3)H}CDP reduction assay, the activity of RR reconstituted with either p53R2 or hRRM2 was found to be time, concentration, and hRRM1 dependent . The kinetic activity of p53R2-containing RR was about 20-50% lower than that of hRRM2-containing RR . Using a synthetic heptapeptide to inhibit RR activity, it was shown that p53R2 bound to hRRM1 through the same COOH-terminal heptapeptide as hRRM2 . However, hRRM2 had a 4.76-fold higher binding affinity for hRRM1 than p53R2, which may explain the reduced RR activity of p53R2 relative to hRRM2 . Of interest, p53R2 was 158-fold more susceptible to the iron chelator deferoxamine mesylate than hRRM2, although the iron content of the two proteins determined by atomic absorption spectrometer was almost the same . To the contrary, p53R2 was 2.50-fold less sensitive than hRRM2 to the radical scavenger hydroxyurea, whereas EPR showed similar spectra of the tyrosyl radical in two proteins . Triapine, a new RR inhibitor, was equally potent for p53R2 and hRRM2 . These inhibition studies showed that the iron center and tyrosyl radical are involved in RR activity for both p53R2 and hRRM2 . The susceptibility differences to RR inhibitors between p53R2 and hRRM2 may lead to a new direction in drug design for human cancer treatment. Biochim Biophys Acta, 2003 Dec 30, 1635(2-3), 83 - 92 Structural analysis of sphingophospholipids derived from Sphingobacterium spiritivorum, the type species of genus Sphingobacterium; Naka T et al.; The unique feature of the genus Sphingobacterium is the presence of sphingophospholipids and ceramides, besides diacylglycerophospholipids . As major cellular lipid components, five kinds of sphingophospholipids were purified from Sphingobacterium spiritivorum ATCC 33861(T), the type species of genus Sphingobacterium . They were identified as ceramide phosphorylethanolamines (CerPE-1 and CerPE-2), ceramide phosphoryl-myo-inositols (CerPI-1 and CerPI-2), and ceramide phosphorylmannose (CerPM-1) . The ceramide of CerPE-1, CerPI-1, and CerPM-1 was composed of 15-methylhexadecasphinganine (isoheptadeca sphinganine, iso-C17:0) and 13-methyltetradecanoic acid (isopentadecanoic acid, iso-C15:0), whereas that of CerPE-2 and CerPI-2 was composed of isoheptadeca sphinganine and 2-hydroxy-13-methyltetradecanoic acid (2-hydroxy isopentadecanoic acid, 2-OH iso-C15:0) . These sphingophospholipids were also found in cellular lipids of Sphingobacterium multivorum ATCC 33613(T), Sphingobacterium mizutaii ATCC 33299(T), Sphingobacterium faecium IFO 15299(T), Sphingobacterium thalpophilum ATCC 43320(T), and Sphingobacterium antarcticum ATCC 51969(T) . To our knowledge, the existence of CerPM-1 is a novel sphingophospholipid through eukaryotic and prokaryotic cells. Eur J Biochem, 2004 Feb, 271(3), 534 - 44 Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli; Croitoru V et al.; Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes . No clear function has been assigned to the smallest of these three factors, IF1 . Therefore, to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA . Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal infA+ gene, followed by deletion of this infA+ gene . Using this approach, the six arginine residues of IF1 were altered to leucine or aspartate . Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis . All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration . Differences in growth phenotypes of the mutants were observed in both minimal and rich media . Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA+ allele . Several of these recombinants showed reduced growth rate and a partial cold-sensitive phenotype . This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1. J Biol Chem, 2004 Apr 23, 279(17), 17932 - 44 Epub 2004 Jan 14. Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme; Kovari J et al.; dUTPase is responsible for preventive DNA repair via exclusion of uracil . Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis . Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment . Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro . The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 degrees C, 23 degrees C lower than Escherichia coli dUTPase . In contrast to the bacterial enzyme, Mg(2+) binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature . A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis . In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer . Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase . The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces. J Biol Chem, 2004 Mar 26, 279(13), 12363 - 8 Epub 2004 Jan 13. Yeast Nfs1p is involved in thio-modification of both mitochondrial and cytoplasmic tRNAs; Nakai Y et al.; The IscS protein is a pyridoxal phosphate-containing cysteine desulfurase involved in iron-sulfur cluster biogenesis . In prokaryotes, IscS is also involved in various metabolic functions, including thio-modification of tRNA . By contrast, the eukaryotic ortholog of IscS (Nfs1) has thus far been shown to be functional only in mitochondrial iron-sulfur cluster biogenesis . We demonstrate here that yeast Nfs1p is also required for the post-transcriptional thio-modification of both mitochondrial (mt) and cytoplasmic (cy) tRNAs in vivo . Depletion of Nfs1p resulted in an immediate impairment of the 2-thio-modification of 5-carboxymethylaminomethyl-2-thiouridine at the wobble positions of mt-tRNA(UUU)(Lys) and mt-tRNA(UUG)(Gln) . In addition, we observed a severe reduction in the 2-thio-modification of 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) of cy-tRNA(UUU)(Lys2) and cy-tRNA(UUC)(Glu3), although the effect was somewhat delayed compared with that seen in mt-tRNAs . Mass spectrometry analysis revealed an increase in 5-methoxycarbonylmethyluridine concomitant with a decrease in mcm(5)s(2)U in cy-tRNAs that were prepared from Nfs1p-depleted cells . These results suggest that Nfs1p is involved in the 2-thio-modification of both 5-carboxymethylaminomethyl-2-thiouridine in mt-tRNAs and mcm(5)s(2)U in cy-tRNAs. Biotechnol Lett, 2003 Dec, 25(23), 1973 - 5 Expression and characterization of recombinant NH2-terminal cell binding fragment of vitronectin in E . coli; Jang JH et al.; A recombinant peptide fragment of vitronectin (rVN143), that includes the Arg-Gly-Asp (RGD) cell recognition site, was expressed in Escherichia coli using a prokaryotic expression system . The addition of recombinant rVN143 peptide enhances cell adhesion and proliferation similar (approximately 70%) to those of native VN. Nat Struct Mol Biol, 2004 Jan, 11(1), 29 - 35 Epub 2003 Dec 29. Adenine riboswitches and gene activation by disruption of a transcription terminator; Mandal M et al.; A class of riboswitches that recognizes guanine and discriminates against other purine analogs was recently identified . RNAs that carry the consensus sequence and structural features of guanine riboswitches are located in the 5' untranslated region (UTR) of numerous prokaryotic genes, where they control the expression of proteins involved in purine salvage and biosynthesis . We report that three representatives of this riboswitch class bind adenine with values for apparent dissociation constant (apparent K(d)) that are several orders of magnitude lower than those for binding guanine . Because preference for adenine is attributable to a single nucleotide substitution, the RNA most likely recognizes its ligand by forming a Watson-Crick base pair . In addition, the adenine riboswitch associated with the ydhL gene of Bacillus subtilis functions as a genetic 'on' switch, wherein adenine binding causes a structural rearrangement that precludes formation of an intrinsic transcription terminator stem. Protein Sci, 2004 Feb, 13(2), 545 - 8 Epub 2004 Jan 10. Solution structure of a BolA-like protein from Mus musculus; Kasai T et al.; The BolA-like proteins are widely conserved from prokaryotes to eukaryotes . The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown . Here we determined the structure of a mouse BolA-like protein . The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2) . This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold . The conserved residues in the BolA-like proteins are assembled on the one side of the protein. J Gen Physiol, 2004 Feb, 123(2), 109 - 19 Epub 2004 Jan 12. Ionic currents mediated by a prokaryotic homologue of CLC Cl- channels; Accardi A et al.; CLC-ec1 is an E . coli homologue of the CLC family of Cl- channels, which are widespread throughout eukaryotic organisms . The structure of this membrane protein is known, and its physiological role has been described, but our knowledge of its functional characteristics is severely limited by the absence of electrophysiological recordings . High-density reconstitution and incorporation of crystallization-quality CLC-ec1 in planar lipid bilayers failed to yield measurable CLC-ec1 currents due to porin contamination . A procedure developed to prepare the protein at a very high level of purity allowed us to measure macroscopic CLC-ec1 currents in lipid bilayers . The current is Cl- selective, and its pH dependence mimics that observed with a 36Cl- flux assay in reconstituted liposomes . The unitary conductance is estimated to be <0.2 pS . Surprisingly, the currents have a subnernstian reversal potential in a KCl gradient, indicating imperfect selectivity for anions over cations . Mutation of a conserved glutamate residue found in the selectivity filter eliminates the pH-dependence of both currents and 36Cl- flux and appears to trap CLC-ec1 in a constitutively active state . These effects correlate well with known characteristics of eukaryotic CLC channels . The E148A mutant displays nearly ideal Cl- selectivity. Cell, 2004 Jan 9, 116(1), 25 - 38 Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus; Robinson NP et al.; Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin . The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes . However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus . Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6 . We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs . These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity. Biochemistry, 2004 Jan 20, 43(2), 289 - 99 A minimal kinetic model for a viral DNA packaging machine; Yang Q et al.; Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses . These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism . Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA . Allosteric interactions between the multiple catalytic sites have been reported . This study probes these catalytic interactions using enzyme kinetic, photoaffinity labeling, and vanadate inhibition studies . The ensemble of data forms the basis for a minimal kinetic model for lambda terminase . The model incorporates an ADP-driven conformational reorganization of the terminase subunits assembled on viral DNA, which is central to the activation of a catalytically competent packaging machine . The proposed model provides a unifying mechanism for allosteric interaction between the multiple catalytic sites of the holoenzyme and explains much of the kinetic data in the literature . Given that similar packaging mechanisms have been proposed for viruses as dissimilar as lambda and the herpes viruses, the model may find general utility in our global understanding of the enzymology of virus assembly. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2003 Dec, 20(4), 634 - 7 {E . coli-based production of recombinant FALL-39}; Feng Y et al.; This study was aimed at constructing a prokaryotic expression system to resolve the difficulties in acquiring antibacterial peptide and to meet the needs of research and drug development . Total RNA was extracted from human pulmonary gland epithelial cell line SPC-A-1, and a cDNA encoding mature FALL-39 peptide was amplified by RT-PCR . The recombinant prokaryotic expression vector pGEX-1 lambda T-FALL-39 was constructed . Using affinity chromatography, thrombin cleaving and AU-PAGE elution, we obtained the purified FALL-39 . MIC, MEC, MBC analyses demonstrated that the FALL-39 had strong antibacterial activity. J Biol Chem, 2004 Mar 26, 279(13), 13119 - 28 Epub 2004 Jan 08. Crystal structure of Thermotoga maritima alpha-L-fucosidase . Insights into the catalytic mechanism and the molecular basis for fucosidosis; Sulzenbacher G et al.; Fucosylated glycoconjugates are involved in numerous biological events, and alpha-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance . Deficiency in alpha-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration . To gain insight into alpha-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima alpha-l-fucosidase . This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (beta/alpha)(8)-like domain and a C-terminal beta-sandwich domain . The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp(244), and the Bronsted acid/base, Glu(266) . Because T . maritima alpha-l-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis. Comput Methods Programs Biomed, 2004 Jan, 73(1), 55 - 60 A graphical tool for parasite genome annotation; Nilsson D et al.; A graphical tool to facilitate rapid primary annotation of genomic sequence has been developed . Within a single interface the user can import sequences or database entries, run feature prediction programs and similarity searches, filter results, add additional manually found features and notes, and finally export annotations for database submission . Integrated rule-based feature corroboration and a novel decision support heuristic using ORF orientation, length and base-composition further enhances the efficiency of the annotation process without compromising flexibility . The program has been explicitly tailored to use in protozoan parasite genome projects, but can constitute a useful tool for prokaryote annotation as well . It is successfully being used by our lab in the Trypanosoma cruzi genome project, and can be obtained from the authors upon request. Mol Biol (Mosk), 2003 Nov-Dec, 37(6), 931 - 43 {First class transcription termination factors--functional analogs of aminoacyl tRNA}; Kiselev LL; Reviewed and discussed are the recent data demonstrating profound functional similarity between class-1 translation termination factors (RF1 and RF2 in prokaryotes, aRF1 and eRF1 in Archaea and eukaryotes, respectively) and aminoacyl-tRNA as regards their roles in the course of translation on the ribosome . Functional analogy of these two components of the cell protein-synthesizing machinery was suggested long ago; however, numerous experimental proofs have been obtained only recently . This similarity implies that decoding of the genetic information by the ribosomal machine is performed similarly at all stages of translation, though tRNA plays the main role at initiation and elongation, while the protein is most important for termination . Earlier it was found that nucleic acids (ribozymes) can operate like the protein enzymes, and now we have got evidence for the reverse: a protein (translation termination factor) can act like a nucleic acid (tRNA) . Thus one can speak of "exchange" of molecular functions between proteins and nucleic acids . Therefore, the profound chemical difference between proteins and nucleic acids is not an insuperable barrier to their mutual functional replacement in certain situations. Appl Environ Microbiol, 2004 Jan, 70(1), 240 - 7 New molecular screening tools for analysis of free-living diazotrophs in soil; Burgmann H et al.; Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse . Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment . However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes . We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification . These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting . The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils . These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set . Furthermore, they were better suited to distinguish between diazotroph populations in the different soils . Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities. Protein Expr Purif, 2004 Feb, 33(2), 238 - 45 A novel multi-affinity tag system to produce high levels of soluble and biotinylated proteins in Escherichia coli; Ashraf SS et al.; We describe here a novel multi-affinity tag vector that can be used to produce high levels of soluble, in vivo biotinylated proteins in Escherichia coli . This system combines the solubility-enhancing ability of maltose-binding protein (MBP), the versatility of the hexahistidine tag (His(6)), and the site-specific in vivo biotinylation of a 15-amino acid tag (AviTag) . We used this multi-tag system in an attempt to improve expression levels of two prokaryotic proteins-elongation factor Tu (TufB) and DNA gyrase subunit A (GyrA)-as well as two eukaryotic nuclear receptors-glucocorticoid receptor (GR) and small heterodimer partner (SHP) . The multi-tag system not only vastly improved the expression of the two prokaryotic proteins tested, but also yielded complete, site-specific, in vivo biotinylation of these proteins . The results obtained from the TufB expression and purification are presented and discussed in detail . The nuclear receptors, though soluble as fusion partners, failed to remain soluble once the MBP tag was cleaved . Despite this limitation of the system, the multi-affinity tag approach is a useful system that can improve expression of some otherwise insoluble or poorly expressing proteins, to obtain homogeneous, purified, fully biotinylated protein for downstream applications. Int Rev Cytol, 2003, 232, 217 - 62 Organization, developmental dynamics, and evolution of plastid nucleoids; Sato N et al.; The plastid is a semiautonomous organelle essential in photosynthesis and other metabolic activities of plants and algae . Plastid DNA is organized into the nucleoid with various proteins and RNA, and the nucleoid is subject to dynamic changes during the development of plant cells . Characterization of the major DNA-binding proteins of nucleoids revealed essential differences in the two lineages of photosynthetic eukaryotes, namely nucleoids of green plants contain sulfite reductase as a major DNA-binding protein that represses the genomic activity, whereas the prokaryotic DNA-binding protein HU is abundant in plastid nucleoids of the rhodophyte lineage . In addition, current knowledge on DNA-binding proteins, as well as the replication and transcription systems of plastids, is reviewed from comparative and evolutionary points of view . A revised hypothesis on the discontinuous evolution of plastid genomic machinery is presented: despite the cyanobacterial origin of plastids, the genomic machinery of the plastid genome is fundamentally different from its counterpart in cyanobacteria. Proc Natl Acad Sci U S A, 2004 Jan 20, 101(3), 881 - 5 Epub 2004 Jan 06. Global gene repression by KaiC as a master process of prokaryotic circadian system; Nakahira Y et al.; A kaiABC clock gene cluster was previously identified from cyanobacterium Synechococcus elongatus PCC 7942, and the feedback regulation of kai genes was proposed as the core mechanism generating circadian oscillation . In this study, we confirmed that the Kai-based oscillator is the dominant circadian oscillator functioning in cyanobacteria . We probed the nature of this regulation and found that excess KaiC represses not only kaiBC but also the rhythmic components of all genes in the genome . This result strongly suggests that the KaiC protein primarily coordinates genomewide gene expression, including its own expression . We also found that a promoter derived from E . coli is feedback controlled by KaiC and restores the complete circadian rhythm in kaiBC-inactivated arrhythmic mutants, provided it can express kaiB and kaiC genes at an appropriate level . Unlike eukaryotic models, specific regulation of the kaiBC promoter is not essential for cyanobacterial circadian oscillations. Biochem J, 2004 May 1, 379(Pt 3), 541 - 52 Modelling and bioinformatics studies of the human Kappa-class glutathione transferase predict a novel third glutathione transferase family with similarity to prokaryotic 2-hydroxychromene-2-carboxylate isomerases; Robinson A et al.; The Kappa class of GSTs (glutathione transferases) comprises soluble enzymes originally isolated from the mitochondrial matrix of rats . We have characterized a Kappa class cDNA from human breast . The cDNA is derived from a single gene comprising eight exons and seven introns located on chromosome 7q34-35 . Recombinant hGSTK1-1 was expressed in Escherichia coli as a homodimer (subunit molecular mass approximately 25.5 kDa) . Significant glutathione-conjugating activity was found only with the model substrate CDNB (1-chloro-2,4-ditnitrobenzene) . Hyperbolic kinetics were obtained for GSH (parameters: K(m)app, 3.3+/-0.95 mM; V(max)app, 21.4+/-1.8 micromol/min per mg of enzyme), while sigmoidal kinetics were obtained for CDNB (parameters: S0.5app, 1.5+/-1.0 mM; V(max)app, 40.3+/-0.3 micromol/min per mg of enzyme; Hill coefficient, 1.3), reflecting low affinities for both substrates . Sequence analyses, homology modelling and secondary structure predictions show that hGSTK1 has (a) most similarity to bacterial HCCA (2-hydroxychromene-2-carboxylate) isomerases and (b) a predicted C-terminal domain structure that is almost identical to that of bacterial disulphide-bond-forming DsbA oxidoreductase (root mean square deviation 0.5-0.6 A) . The structures of hGSTK1 and HCCA isomerase are predicted to possess a thioredoxin fold with a polyhelical domain (alpha(x)) embedded between the beta-strands (betaalphabetaalpha(x)betabetaalpha, where the underlined elements represent the N and C motifs of the thioredoxin fold), as occurs in the bacterial disulphide-bond-forming oxidoreductases . This is in contrast with the cytosolic GSTs, where the helical domain occurs exclusively at the C-terminus (betaalphabetaalphabetabetaalphaalpha(x)) . Although hGSTK1-1 catalyses some typical GST reactions, we propose that it is structurally distinct from other classes of cytosolic GSTs . The present study suggests that the Kappa class may have arisen in prokaryotes well before the divergence of the cytosolic GSTs. Microb Ecol, 2003 Aug, 46(2), 200 - 15 Structure and seasonal dynamics of hyporheic zone microbial communities in free-stone rivers of the western United States; Feris KP et al.; The hyporheic zone of a river is characterized by being nonphotic, exhibiting chemical/redox gradients, and having a heterotrophic food web based on the consumption of organic carbon entrained from surface waters . Hyporheic microbial communities constitute the base of food webs in these environments and are important for maintaining a functioning lotic ecosystem . While microbial communities of rivers dominated by fine-grained sediments are relatively well studied, little is known about the structure and seasonal dynamics of microbial communities inhabiting the predominantly gravel and cobble hyporheic zones of rivers of the western United States . Here, we present the first molecular analysis of hyporheic microbial communities of three different stream types (based on mean base discharge, substratum type, and drainage area), in Montana . Utilizing 16S rDNA phylogeny, DGGE pattern analysis, and qPCR, we have analyzed the prokaryotic communities living on the 1.7 to 2.36 mm grain-size fraction of hyporheic sediments from three separate riffles in each stream . DGGE analysis showed clear seasonal community patterns, indicated similar community composition between different riffles within a stream (95.6-96.6% similarity), and allowed differentiation between communities in different streams . Each river supported a unique complement of species; however, several phylogenetic groups were conserved between all three streams including Pseudomonads and members of the genera Aquabacterium, Rhodoferax, Hyphomicrobium, and Pirellula . Each group showed pronounced seasonal trends in abundance, with peaks during the Fall . The Hyphomicrobium group was numerically dominant throughout the year in all three streams . This work provides a framework for investigating the effects of various environmental factors and anthropogenic effects on microbial communities inhabiting the hyporheic zone. J Mol Evol, 2003 Oct, 57(4), 453 - 66 A survey of codon and amino acid frequency bias in microbial genomes focusing on translational efficiency; Merkl R; Unequal use of syn