FEMS Microbiol Lett, 2005 Jan 1, 242(1), 117 - 26 Organisation of the S10, spc and alpha ribosomal protein gene clusters in prokaryotic genomes; Coenye T et al.; Although it is well known that there is no long range colinearity in gene order in bacterial genomes, it is thought that there are several regions that are under strong structural constraints during evolution, in which gene order is extremely conserved . One such region is the str locus, containing the S10-spc-alpha operons . These operons contain genes coding for ribosomal proteins and for a number of housekeeping genes . We compared the organisation of these gene clusters in 111 sequenced prokaryotic genomes (99 bacterial and 12 archaeal genomes) . We also compared the organisation to the phylogeny based on 16S ribosomal RNA gene sequences and the sequences of the ribosomal proteins L22, L16 and S14 . Our data indicate that there is much variation in gene order and content in these gene clusters, both in bacterial as well as in archaeal genomes . Our data indicate that differential gene loss has occurred on multiple occasions during evolution . We also noted several discrepancies between phylogenetic trees based on 16S rRNA gene sequences and sequences of ribosomal proteins L16, L22 and S14, suggesting that horizontal gene transfer did play a significant role in the evolution of the S10-spc-alpha gene clusters. FEMS Microbiol Lett, 2005 Jan 1, 242(1), 73 - 9 Fluorescent detection of beta-lactamase activity in living Escherichia coli cells via esterase supplementation; Nord O et al.; The TEM-1 beta-lactamase protein fragment complementation assay was investigated for its applicability in affinity protein-based interaction studies in Escherichia coli, using an affibody-based model system . Results from co-transformation experiments showed that an ampicillin resistant phenotype was specifically associated with cognate affibody-target pairings . Attempts to monitor beta-lactamase complementation in vitro with the fluorescent beta-lactamase substrates CCF2/AM and CCF2 showed that E . coli lacks an esterase activity necessary for activation of the esterified and membrane-permeable CCF2/AM form of the substrate . Interestingly, supplementation of the assay reaction with a purified fungal lipase (cutinase) resulted in efficient activation of CCF2/AM in vitro . Further, periplasmic expression of cutinase allowed for fluorescent discrimination between beta-lactamase positive and negative living E . coli cells using the CCF2/AM substrate, which should open the way for novel applications for this prokaryotic host in protein interaction studies. DNA Seq, 2004 Aug, 15(4), 314 - 8 Cloning, characterization and prokaryotic expression of cytosolic malate dehydrogenase from Oryza sativa; Lin CF et al.; cDNA sequences of malate dehydrogenase (MDH) were cloned from various species, and MDH was identified to play an important role in cell energy metabolism . Here, we present the isolation and characterization of its homologue (OscMDH) in Oryza sativa . Comparison of the results to the genome details indicated that OscMDH consisted of seven exons . Sequence alignment showed that the deduced amino acid sequence of OscMDH shared a significant similarity with cMDH protein in Zea mays, as well as with other cMDH gene products . The different expression patterns of OscMDH in different tissues revealed that OscMDH mRNA was highly transcribed in either young panicle or immature seed, while its abundance was much low in green leaf and root . A nearly 56-kDa fusion protein generated by adding a Trx-His-tag at the N-terminal of OscMDH was induced by IPTG in Escherichia coli BL21 and an obvious MDH activity was detected in the protein by native PAGE analysis . All these results suggest that OscMDH encodes a cytosolic MDH in rice. DNA Seq, 2004 Aug, 15(4), 299 - 302 Isolation, tissue distribution and prokaryotic expression of a novel human X-linked gene LHFPL1; Huang C et al.; We report here the cloning and characterization of a novel human lipoma HMGIC fusion partner-like 1 (LHFPL1) gene, isolated from human brain cDNA library, and mapped to Xq23 by browsing the UCSC genomic database . LHFPL1 contains an ORF with length of 660bp, encoding a protein with a signal peptide sequence and three transmembrane regions, and its predicted molecular weight is 23.7kDa which coincides with the result of prokaryotic expression . LHFPL1 protein is postulated to be localized at endoplasmic reticulum . RT-PCR amplification in seventen human tissues revealed that LHFPL1 is expressed widely in all tissues, especially highly in lung, thymus, skeleton muscle, colon and ovary . In addition, it was demonstrated that LHFPL1 is also transcribed in six liver tumor cell lines. Cell Mol Life Sci, 2005 Jan, 62(1), 4 - 9 Is chlamydial heat shock protein 60 a risk factor for oncogenesis? Di Felice V, David S, Cappello F, Farina F, Zummo G. Heat shock protein 60 (HSP60) plays an important role in the protein folding of prokaryotic and eukaryotic cells . Most of the papers published on chlamydial HSP60 concern its role in immune response during infection . In the last decade, exposure to Chlamydia trachomatis has been consistently associated with the development of cervical and ovarian cancer . Moreover, it has been suggested that chlamydial HSP60 may have an anti-apoptotic effect during persistent infection . We hypothesize that the accumulation of exogenous chlamydial HSP60 in the cytoplasm of actively replicating eukaryotic cells may interfere with the regulation of the apoptotic pathway . The concomitant expression of viral oncoproteins and/or the presence of mutations may lead to the ability to survive apoptotic stimuli, loss of replicative senescence, uncontrolled proliferation and, finally neoplastic transformation. Med Hypotheses, 2005, 64(3), 505 - 11 The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor - an alternative gene therapy; Celec P et al.; BACKGROUND:: Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer) . Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis . In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease . However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation . AIM:: To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis . METHODS AND RESULTS:: Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF(121), VEGF(164), VEGF(189)) . Selection of recombinants was achieved by cultivating E . coli cells on ampicillin-added medium . The expression of target genes in the T7 expression system was induced by isopropyl-beta-d-thiogalactoside (IPTG) . Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms . Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy . CONCLUSION AND HYPOTHESIS:: In summary, E . coli strain expressing VEGF was prepared and its biological effect confirmed . Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use . The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine . The main advantage of the described approach lies in the enhanced regulation control - bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics) . The hypothesis of alternative gene therapy should be proved in further studies. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1997 Dec, 11(4), 319 - 21 {cDNA cloning, sequencing and expression of recombinant human prorelaxin}; Ding C et al.; In order to accelerate the development of biological recombinant human prorelaxin in our country, direct cloning of human prorelaxin gene from freshly prepared corpus luteum of Chinese woman by RT-PCR method was conducted . Isolated prorelaxin gene contains B, A chains and connective peptide . The amplified products were cloned and confirmed by DNA sequencing using Sanger Dideoxy method . It was subcloned into LKB2, a prokaryotic expression vector offering promoter LacI control, and highly expressed in E . coli . The results suggest that the PCR amplified DNA fragment shared identical sequence with known prorelaxin gene reported abroad . The SDS-PAGE analysis revealed that the expressed protein accounted for about 30% of the total cell protein, its MW was approximately 20 kD and conformed soluble protein. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1997 Dec, 11(4), 311 - 4 {Research of a screening strategy for high prokaryotic expression clone}; Li F et al.; The low expression in E . coli can be often explained by the beginning sequence that causes low translational initiation . A simple selection procedure is then used to enrich those sequences from the bank that lead to high levels of translation . The selection procedure is based on the use of a translationally coupled tetracycline resistance gene . The basic steps are as follows: (1) Construction of selection vector pBV223; (2) Screening high expression clones through different tetracycline concentrations . Several clones have been selected in 60 microg/ml concentration of tetracycline . So this system will provide an important way in preparation of some cytokines which have a low expression level in E . coli but have significant economic value. J Synchrotron Radiat, 2005 Jan 1, 12(Pt 1), 8 - 12 Epub 2004 Dec 23. High-throughput production of Pyrococcus furiosus proteins: considerations for metalloproteins; Jenney FE Jr; Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life . As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol . Its 1.9 million basepair genome encodes approximately 2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank . The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds . The immediate objective of this work is to obtain recombinant forms of all P . furiosus proteins in their functional states for structural determination . Proteins successfully produced by overexpression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors . Analyses of the P . furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category . The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble . This work describes development of the production pipeline with attention to prediction and incorporation of cofactors. Antimicrob Agents Chemother, 2005 Jan, 49(1), 249 - 55 Antisense phosphorodiamidate morpholino oligomer length and target position effects on gene-specific inhibition in Escherichia coli; Deere J et al.; Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner . PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli . Shorter PMOs generally inhibited luciferase greater than longer PMOs . Conversely, in bacterial cell-free protein synthesis reactions, longer PMOs inhibited equally or more than shorter PMOs . Overlapping, isometric (10-base) PMOs complementary to the region around the start codon of luciferase inhibited to different extents in bacterial cell-free protein expression reactions . Including the anti-start codon in PMOs was not required for maximal inhibition . PMOs targeted to 5' nontranslated or 3' coding regions within luciferase mRNA did not inhibit, except for one PMO targeted to the ribosome-binding site . Inhibition of luciferase expression correlated negatively with the predicted secondary structure of mRNA regions targeted by PMO but did not correlate with C+G content of targeted regions . The effects of PMO length and position were corroborated by using PMOs (6 to 20 bases) targeted to acpP, a gene required for viability . Because inhibition by PMOs of approximately 11 bases was unexpected based on previous results in eukaryotes, we tested an 11-base PMO in HeLa cells and reticulocyte cell-free protein synthesis reactions . The 11-base PMO significantly inhibited luciferase expression in HeLa cells, although less than did a 20-base PMO . In reticulocyte cell-free reactions, there was a trend toward more inhibition with longer PMOs . These studies indicate that strategies for designing PMOs are substantially different for prokaryotic than eukaryotic targets. Proc R Soc Lond B Biol Sci, 2004 Dec 22, 271(1557), 2551 - 8 Does a tree-like phylogeny only exist at the tips in the prokaryotes? Creevey CJ, Fitzpatrick DA, Philip GK, Kinsella RJ, O'connell MJ, Pentony MM, Travers SA, Wilkinson M, McInerney JO. The extent to which prokaryotic evolution has been influenced by horizontal gene transfer (HGT) and therefore might be more of a network than a tree is unclear . Here we use supertree methods to ask whether a definitive prokaryotic phylogenetic tree exists and whether it can be confidently inferred using orthologous genes . We analysed an 11-taxon dataset spanning the deepest divisions of prokaryotic relationships, a 10-taxon dataset spanning the relatively recent gamma-proteobacteria and a 61-taxon dataset spanning both, using species for which complete genomes are available . Congruence among gene trees spanning deep relationships is not better than random . By contrast, a strong, almost perfect phylogenetic signal exists in gamma-proteobacterial genes . Deep-level prokaryotic relationships are difficult to infer because of signal erosion, systematic bias, hidden paralogy and/or HGT . Our results do not preclude levels of HGT that would be inconsistent with the notion of a prokaryotic phylogeny . This approach will help decide the extent to which we can say that there is a prokaryotic phylogeny and where in the phylogeny a cohesive genomic signal exists. J Mol Histol, 2004 Aug, 35(6), 545 - 53 RNA interference: The molecular immune system; Bagasra O et al.; Introduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS) . The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer . siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo- and endo-nucleases as well as other proteins . Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner . RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals . In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression . In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21- to 23-nt-long siRNAs . However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi . siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells . Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements . Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described . Recent data in C . elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed . However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined . Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells . RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins . RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla . We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements . The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed. Mol Microbiol, 2005 Jan, 55(1), 5 - 15 Exploring prokaryotic diversity: there are other molecular worlds; Fernandez LA; Summary Prokaryotes are the major source of biological diversity on earth . This is not simply because of the large number of species present, or because of their diverse growth conditions and environmental niches populated by them, but because of the wealth of genes, metabolic pathways and molecular processes that are only found in prokaryotic cells . Therefore, Bacteria and Archaea (and their phages) cannot be considered any longer as miniaturized models of Eukaryotes, but as a genuine source of unique biological processes that are mediated by unique sets of genes and molecular devices . A true understanding of complex biological phenomena will require a deeper knowledge of this vast prokaryotic world . The second European Molecular Biology Organization (EMBO) conference on Molecular Microbiology entitled 'Exploring Prokaryotic Diversity' explored many aspects of this newly emerging interest in the prokaryotic world. Wei Sheng Yan Jiu, 2004 Sep, 33(5), 596 - 9 {High-effective expression and construction of a prokaryote expression vector pET30a-glutathione-S-transferase A1 gene from human liver cDNA library and sequence determination}; Chen P et al.; OBJECTIVE: To express highly effective on IPTG-induced and construct an prokaryote expression vector carrying human glutathione-S- transferase (GST) A1 gene and to determine partial sequence analysis . METHODS: The GSTA1 cDNA was amplified and extracted from human liver total RNAs by RT-PCR approach and recombined with prokaryote expression vector pET30a . The recombined vector pET30a-GSTA1 was verified using PCR, restriction analysis and sequencing determination and induced with IPTG in 35 degrees C . RESULTS: Human GSTA1 gene was recombined corrected with pET30a, compared with Genbank , in code 512 T-->C, amino acid Met-->Thr; alignment core is 99% . The expressed fusion protein,with molecular weight of about 34.4 KDa, were about 41.8% (1h), 68% (2h), 68.3% (3h), 83% (4h) of the total cell protein by SDS-PAGE . CONCLUSION: The protein expression of GSTA1 was demonstrated by Western blotting, it is correct. Cell Cycle . 2004 Dec 20;3(12) {Epub ahead of print} Comparative Genomics, Evolution and Origins of the Nuclear Envelope and Nuclear Pore Complex; Mans BJ et al.; The presence of a distinct nucleus, the compartment for confining the genome, transcription and RNA maturation, is a central (and eponymous) feature that distinguishes eukaryotes from prokaryotes . Structural integrity of the nucleus is maintained by the nuclear envelope (NE) . A crucial element of this structure is the nuclear pore complex (NPC), a macromolecular machine with over 90 protein components, which mediates nucleo-cytoplasmic communication . We investigated the provenance of the conserved domains found in these perinuclear proteins and reconstructed a parsimonious scenario for NE and NPC evolution by means of comparative-genomic analysis of their components from the available sequences of 28 sequenced eukaryotic genomes . We show that the NE and NPC proteins were tinkered together from diverse domains, which evolved from prokaryotic precursors at different points in eukaryotic evolution, divergence from pre-existing eukaryotic paralogs performing other functions, and de novo . It is shown that several central components of the NPC, in particular, the RanGDP import factor NTF2, the HEH domain of Src1p-Man1, and, probably, also the key domains of karyopherins and nucleoporins, the HEAT/ARM and WD40 repeats, have a bacterial, most likely, endosymbiotic origin . The specialized immunoglobulin (Ig) domain in the globular tail of the animal lamins, and the Ig domains in the nuclear membrane protein GP210 are shown to be related to distinct prokaryotic families of Ig domains . This suggests that independent, late horizontal gene transfer events from bacterial sources might have contributed to the evolution of perinuclear proteins in some of the major eukaryotic lineages . Snurportin 1, one of the highly conserved karyopherins, contains a cap-binding domain which is shown to be an inactive paralog of the guanylyl transferase domain of the mRNA-capping enzyme, exemplifying recruitment of paralogs of pre-exsiting proteins for perinuclear functions . It is shown that several NPC proteins containing super-structure- forming alpha-helical and beta-propeller modules are most closely related to corresponding proteins in the cytoplasmic vesicle biogenesis and coating complexes . From these observations, we infer an autogenous scenario of nuclear evolution in which the nucleus emerged in the primitive eukaryotic ancestor (the "prekaryote") as part of cell compartmentalization triggered by archaeo-bacterial symbiosis . A pivotal event in this process was the radiation of Ras-superfamily GTPases yielding Ran, the key regulator of nuclear transport . A primitive NPC with approximately 20 proteins and a Src1p-Man1-like membrane protein with a DNA-tethering HEH domain are inferred to have been integral perinuclear components in the las common ancestor of modern eukaryotes. Genetics, 2004 Dec, 168(4), 2245 - 60 Intragenic spatial patterns of codon usage bias in prokaryotic and eukaryotic genomes; Qin H et al.; To study the roles of translational accuracy, translational efficiency, and the Hill-Robertson effect in codon usage bias, we studied the intragenic spatial distribution of synonymous codon usage bias in four prokaryotic (Escherichia coli, Bacillus subtilis, Sulfolobus tokodaii, and Thermotoga maritima) and two eukaryotic (Saccharomyces cerevisiae and Drosophila melanogaster) genomes . We generated supersequences at each codon position across genes in a genome and computed the overall bias at each codon position . By quantitatively evaluating the trend of spatial patterns using isotonic regression, we show that in yeast and prokaryotic genomes, codon usage bias increases along translational direction, which is consistent with purifying selection against nonsense errors . Fruit fly genes show a nearly symmetric M-shaped spatial pattern of codon usage bias, with less bias in the middle and both ends . The low codon usage bias in the middle region is best explained by interference (the Hill-Robertson effect) between selections at different codon positions . In both yeast and fruit fly, spatial patterns of codon usage bias are characteristically different from patterns of GC-content variations . Effect of expression level on the strength of codon usage bias is more conspicuous than its effect on the shape of the spatial distribution. J Biol Chem . 2004 Dec 20; {Epub ahead of print} AtNAP1 represents an atypical SufB protein in arabidopsis plastids; Xu XM et al.; The assembly of iron-sulfur (Fe-S) clusters involves several pathways and in prokaryotes the mobilization of sulfur (SUF) system is paramount for Fe-S biogenesis and repair during oxidative stress . The prokaryotic SUF system consist of six proteins: SufC is an ABC/ATPase which forms a complex with SufB and SufD, SufA acts as a scaffold protein and SufE and SufS are involved in sulfur mobilization from cysteine . Despite the importance of Fe-S proteins in higher plant plastids, little is known regarding plastidic Fe-S cluster assembly . We have recently shown that Arabidopsis harbors an evolutionary conserved plastidic SufC protein (AtNAP7) capable of hydrolyzing ATP and interacting with the SufD homolog AtNAP6 . Based on this and the prokaryotic SUF system we speculated that a SufB-like protein may exist in plastids . Here we demonstrate that the Arabidopsis plastid-localized SufB homolog AtNAP1 can complement SufB deficiency in E . coli during oxidative stress . Furthermore, we demonstrate that AtNAP1 can interact with AtNAP7 inside living chloroplasts suggesting the presence of a plastidic AtNAP1/AtNAP6/AtNAP7 complex and remarkable evolutionary conservation of the SUF system . However, in contrast to prokaryotic SufB proteins with no associated ATPase activity we show that AtNAP1 is a Fe-stimulated ATPase and that AtNAP1 is capable of forming homodimers . Our results suggest that AtNAP1 represents an atypical plastidic SufB-like protein important for Fe-S cluster assembly and for regulating Fe homeostasis in Arabidopsis. Mol Cell, 2004 Dec 22, 16(6), 881 - 91 A mechanism for the potent inhibition of eukaryotic acetyl-coenzyme a carboxylase by soraphen a, a macrocyclic polyketide natural product; Shen Y et al.; Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism . Soraphen A, a macrocyclic polyketide natural product, is a nanomolar inhibitor against the biotin carboxylase (BC) domain of human, yeast, and other eukaryotic ACCs . Here we report the crystal structures of the yeast BC domain, alone and in complex with soraphen A . Soraphen has extensive interactions with an allosteric site, about 25 A from the active site . The specificity of soraphen is explained by large structural differences between the eukaryotic and prokaryotic BC in its binding site, confirmed by our studies on the effects of single-site mutations in this binding site . Unexpectedly, our structures suggest that soraphen may bind in the BC dimer interface and inhibit the BC activity by disrupting the oligomerization of this domain . Observations from native gel electrophoresis confirm this structural insight . The structural information provides a foundation for structure-based design of new inhibitors against these enzymes. Biochemistry, 2004 Dec 28, 43(51), 16106 - 18 Evaluation of the DNA binding tendencies of the transition state regulator AbrB; Bobay BG et al.; Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors . One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity . Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes . MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets . MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner . UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes . General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed . It is likely that these parameters contribute to the differential binding proclivities of AbrB . In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D98 - 102 TRACTOR_DB: a database of regulatory networks in gamma-proteobacterial genomes; Gonzalez AD et al.; Experimental data on the Escherichia coli transcriptional regulatory system has been used in the past years to predict new regulatory elements (promoters, transcription factors (TFs), TFs' binding sites and operons) within its genome . As more genomes of gamma-proteobacteria are being sequenced, the prediction of these elements in a growing number of organisms has become more feasible, as a step towards the study of how different bacteria respond to environmental changes at the level of transcriptional regulation . In this work, we present TRACTOR_DB (TRAnscription FaCTORs' predicted binding sites in prokaryotic genomes), a relational database that contains computational predictions of new members of 74 regulons in 17 gamma-proteobacterial genomes . For these predictions we used a comparative genomics approach regarding which several proof-of-principle articles for large regulons have been published . TRACTOR_DB may be currently accessed at or at Contact Email id is tractor@cifn.unam.mx. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D501 - 4 NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins; Pruitt KD et al.; The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database provides a non-redundant collection of sequences representing genomic data, transcripts and proteins . Although the goal is to provide a comprehensive dataset representing the complete sequence information for any given species, the database pragmatically includes sequence data that are currently publicly available in the archival databases . The database incorporates data from over 2400 organisms and includes over one million proteins representing significant taxonomic diversity spanning prokaryotes, eukaryotes and viruses . Nucleotide and protein sequences are explicitly linked, and the sequences are linked to other resources including the NCBI Map Viewer and Gene . Sequences are annotated to include coding regions, conserved domains, variation, references, names, database cross-references, and other features using a combined approach of collaboration and other input from the scientific community, automated annotation, propagation from GenBank and curation by NCBI staff. Mol Immunol, 2005 Mar, 42(5), 589 - 98 Cloning, in vitro expression and bioactivity of duck interleukin-2; Zhou JY et al.; In this report, the cDNA sequences of Shaoxing (SX) and Muscovy (MV) duck IL-2 were cloned, then recombinant duck IL-2 (rduIL-2) was produced in prokaryotic expression system . In vitro bioactivity of rduIL-2 was determined by lymphocyte proliferation assay and in vivo bioactivity of rduIL-2 was assessed by vaccine immunization . Monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) specific for rduIL-2 were generated and characterized by ELISA, Western blot and neutralizing assays . The cDNA contains an open reading frame (ORF) of 420-base pairs encoding a protein of 140 amino acids (aa) with a putative signal peptide of 21aa . The His-duIL-2 fusion protein was recognized in Western blot by mAb against chicken IL-2 (chIL-2), but not by mAbs against human IL-2 and mouse IL-2 . Recombinant duIL-2 induces in vitro proliferation of Con A-stimulated duck splenocytes in MTT assay and strengthens duck immune responses induced by vaccinating the inactivated oil emulsion vaccine against avian influenza virus . Polyclonal antibodies and mAb 2B3 against rduIL-2 were shown to have effective neutralizing ability by inhibiting the biological activities of both recombinant duIL-2 and endogenous duIL-2 . Despite the fact that duck and chicken IL-2s only share identity of 55.0-56.7% in amino acid sequence, duck and chicken IL-2 molecules displayed similar cross-priming activity in in vitro lymphocyte proliferation assays . The results, at the first time, indicated that rduIL-2 has the potential to be used as an immunoadjuvant for enhancing vaccine efficacy and an immunotherapeutic, and the mAbs against rduIL-2 further facilitate basic immunobiological studies of the role of IL-2 in avian immune system. Expert Rev Vaccines, 2004 Dec, 3(6), 673 - 9 New recombinant vaccines based on the use of prokaryotic antigen-display systems; De Berardinis P et al.; A major challenge in vaccine design has been to identify antigen presentation systems that elicit strong T- and B-cell responses . In the authors' laboratory, two new delivery vehicles derived from nonpathogenic prokaryotic organisms were recently designed and investigated . Conserved antigenic determinants were inserted into the N-terminal region of the major pVIII coat protein of bacteriophage fd virions or on the surface of an icosahedral scaffold formed by the acyltransferase component (E2 protein) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus . The data indicate that the antigenic determinant displayed by either fd virions or on the surface of the E2 lattice are accessible to the immune system, and are able to trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo . These systems offer the potential for safe and inexpensive vaccines to elicit full-spectrum immune responses. Biotechnol Lett, 2004 Nov, 26(21), 1629 - 34 Expression of an antibody-targeted plasminogen activator in Escherichia coli using chemically induced stress responses; Yu M et al.; A recombinant gene coding for an antibody-targeted urokinase-type plasminogen activator was constructed for the purpose of enhancing the thrombolytic specificity of urokinase . The recombinant gene was cloned into prokaryotic expression vector pTrcHisA, and transformed into Escherichia coli strain Rosetta (DE3) . Less than 4mg of the desired protein/l could be obtained in the form of inclusion bodies . Of various inducers and enhancers of stress responses, the heat-shock enhances, streptomycin, the osmotic stress inducers, -arabinose and sucrose, and the cold-shock enhancer, tetracycline, simulated the expression of the antibody-targeted plasminogen activator by 2- 5-fold. Plant Mol Biol, 2004 Sep, 56(2), 225 - 39 Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces; Fujimoto S et al.; We identified a novel nucleoplasm localized protein in Arabidopsis called AT-hook motif nuclear localized protein 1 (AHL1), which was isolated by visual screening of transformants using random GFP::cDNA fusions . AHL1 contains an AT-hook motif and unknown conserved PPC (plants and prokaryotes conserved) domain that includes a hydrophobic region . Approximately 30 paralogues were identified in the Arabidopsis genome . Proteins with PPC-like domains are found in Bacteria, Archaea and the plant kingdom, but in Bacteria and Archaea the PPC containing proteins of do not have an AT-hook motif . Thus, the PPC domain is evolutionary conserved and has a new function such as AT-rich DNA binding . AHL1 was mainly localized in the nucleoplasm, but little in the nucleolus and heterochromatic region, and was concentrated in the boundary region between euchromatin and heterochromatin . Biochemically, AHL1 was also found in the nuclear matrix fraction . In the M phase, AHL1 was localized on the chromosomal surface . The AT-hook motif was essential for matrix attachment region (MAR) binding, and the hydrophobic region of the PPC was indispensable for nuclear localization . Our results suggest that AHL1 is a novel plant MAR binding protein, which is related to the positioning of chromatin fibers in the nucleus by the presence of an AT-hook motif and PPC domain . In addition, AHL1 is located on the surface of chromosomes during mitosis. Plant Mol Biol, 2004 May, 55(1), 17 - 32 EST-analysis of the thermo-acidophilic red microalga Galdieria sulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts; Weber AP et al.; When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first . However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0-4 and temperatures of up to 56 degrees C . This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols . This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology . The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G . sulphuraria a valuable model for physical studies on the photosynthetic apparatus . In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes . Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation . To begin to explore the unique biology of G . sulphuraria , 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated . Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism . For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases . Sequence data and additional information are available at http://genomics.msu.edu/galdieria. Biomed Environ Sci, 2004 Sep, 17(3), 350 - 8 Identification of a new peptide deformylase gene from enterococcus faecium and establishment of a new screening model targeted on PDF for novel antibiotics; Tang XB et al.; OBJECTIVE: To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF . METHODS: A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E . coli B121(DE3) . Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics . RESULT: A new PDF gene of Enterococcus faecium was identified successfully . Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples . CONCLUSION: A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy . Positive samples screened may be antibacterial agents of broad spectrum. Nature, 2004 Dec 16, 432(7019), 910 - 3 Genome sequence of Silicibacter pomeroyi reveals adaptations to the marine environment; Moran MA et al.; Since the recognition of prokaryotes as essential components of the oceanic food web, bacterioplankton have been acknowledged as catalysts of most major biogeochemical processes in the sea . Studying heterotrophic bacterioplankton has been challenging, however, as most major clades have never been cultured or have only been grown to low densities in sea water . Here we describe the genome sequence of Silicibacter pomeroyi, a member of the marine Roseobacter clade (Fig . 1), the relatives of which comprise approximately 10-20% of coastal and oceanic mixed-layer bacterioplankton . This first genome sequence from any major heterotrophic clade consists of a chromosome (4,109,442 base pairs) and megaplasmid (491,611 base pairs) . Genome analysis indicates that this organism relies upon a lithoheterotrophic strategy that uses inorganic compounds (carbon monoxide and sulphide) to supplement heterotrophy . Silicibacter pomeroyi also has genes advantageous for associations with plankton and suspended particles, including genes for uptake of algal-derived compounds, use of metabolites from reducing microzones, rapid growth and cell-density-dependent regulation . This bacterium has a physiology distinct from that of marine oligotrophs, adding a new strategy to the recognized repertoire for coping with a nutrient-poor ocean. J Bacteriol, 2005 Jan, 187(1), 336 - 48 Cold shock of a hyperthermophilic archaeon: Pyrococcus furiosus exhibits multiple responses to a suboptimal growth temperature with a key role for membrane-bound glycoproteins; Weinberg MV et al.; The hyperthermophilic archaeon, Pyrococcus furiosus, was grown on maltose near its optimal growth temperature, 95 degrees C, and at the lower end of the temperature range for significant growth, 72 degrees C . In addition, cultures were shocked by rapidly dropping the temperature from 95 to 72 degrees C . This resulted in a 5-h lag phase, during which time little growth occurred . Transcriptional analyses using whole-genome DNA microarrays representing 2,065 open reading frames (ORFs) in the P . furiosus genome showed that cells undergo three very different responses at 72 degrees C: an early shock (1 to 2 h), a late shock (5 h), and an adapted response (occurring after many generations at 72 degrees C) . Each response involved the up-regulation in the expression of more than 30 ORFs unique to that response . These included proteins involved in translation, solute transport, amino acid biosynthesis, and tungsten and intermediary carbon metabolism, as well as numerous conserved-hypothetical and/or membrane-associated proteins . Two major membrane proteins were evident after one-dimensional sodium dodecyl sulfate-gel analysis of cold-adapted cells, and staining revealed them to be glycoproteins . Their cold-induced expression evident from the DNA microarray analysis was confirmed by quantitative PCR . Termed CipA (PF0190) and CipB (PF1408), both appear to be solute-binding proteins . While the archaea do not contain members of the bacterial cold shock protein (Csp) family, they all contain homologs of CipA and CipB . These proteins are also related phylogenetically to some cold-responsive genes recently identified in certain bacteria . The Cip proteins may represent a general prokaryotic-type cold response mechanism that is present even in hyperthermophilic archaea. J Bacteriol, 2005 Jan, 187(1), 143 - 54 Impact of mutS inactivation on foreign DNA acquisition by natural transformation in Pseudomonas stutzeri; Meier P et al.; In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches . The mutS gene of naturally transformable Pseudomonas stutzeri ATCC 17587 (genomovar 2) was identified and characterized . The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatch-binding motif similar to those in MutS of Escherichia coli . A mutS::aac mutant showed 20- to 163-fold-greater spontaneous mutability . Transformation experiments with DNA fragments of rpoB containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in mutS+ . The mutS+ gene of strain ATCC 17587 did not complement an E . coli mutant but partially complemented a P . stutzeri JM300 mutant (genomovar 4) . The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by mutS::aac, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation . Expression of mutS+ from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection . Remarkably, mutS::aac altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA . We concluded that in P . stutzeri upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR. Angew Chem Int Ed Engl, 2004 Dec 17, 44(1), 34 - 66 Expanding the genetic code; Wang L et al.; Although chemists can synthesize virtually any small organic molecule, our ability to rationally manipulate the structures of proteins is quite limited, despite their involvement in virtually every life process . For most proteins, modifications are largely restricted to substitutions among the common 20 amino acids . Herein we describe recent advances that make it possible to add new building blocks to the genetic codes of both prokaryotic and eukaryotic organisms . Over 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive, and redox-active amino acids, glycosylated amino acids, and amino acids with keto, azido, acetylenic, and heavy-atom-containing side chains . By removing the limitations imposed by the existing 20 amino acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties. J Mol Evol, 2004 Dec, 59(6), 849 - 58 Mutation exposed: a neutral explanation for extreme base composition of an endosymbiont genome; Wernegreen JJ et al.; The influence of neutral mutation pressure versus selection on base composition evolution is a subject of considerable controversy . Yet the present study represents the first explicit population genetic analysis of this issue in prokaryotes, the group in which base composition variation is most dramatic . Here, we explore the impact of mutation and selection on the dynamics of synonymous changes in Buchnera aphidicola, the AT-rich bacterial endosymbiont of aphids . Specifically, we evaluated three forms of evidence . (i) We compared the frequencies of directional base changes (AT-->GC vs . GC-->AT) at synonymous sites within and between Buchnera species, to test for selective preference versus effective neutrality of these mutational categories . Reconstructed mutational changes across a robust intraspecific phylogeny showed a nearly 1:1 AT-->GC:GC-->AT ratio . Likewise, stationarity of base composition among Buchnera species indicated equal rates of AT-->GC and GC-->AT substitutions . The similarity of these patterns within and between species supported the neutral model . (ii) We observed an equivalence of relative per-site AT mutation rate and current AT content at synonymous sites, indicating that base composition is at mutational equilibrium . (iii) We demonstrated statistically greater equality in the frequency of mutational categories in Buchnera than in parallel mammalian studies that documented selection on synonymous sites . Our results indicate that effectively neutral mutational pressure, rather than selection, represents the major force driving base composition evolution in Buchnera . Thus they further corroborate recent evidence for the critical role of reduced N(e) in the molecular evolution of bacterial endosymbionts. Proc Natl Acad Sci U S A, 2004 Dec 21, 101(51), 17747 - 52 Epub 2004 Dec 14. Massive horizontal transfer of mitochondrial genes from diverse land plant donors to the basal angiosperm Amborella; Bergthorsson U et al.; Several plants are known to have acquired a single mitochondrial gene by horizontal gene transfer (HGT), but whether these or any other plants have acquired many foreign genes is entirely unclear . To address this question, we focused on Amborella trichopoda, because it was already known to possess one horizontally acquired gene and because it was found in preliminary analyses to contain several more . We comprehensively sequenced the mitochondrial protein gene set of Amborella, sequenced a variable number of mitochondrial genes from 28 other diverse land plants, and conducted phylogenetic analyses of these sequences plus those already available, including the five sequenced mitochondrial genomes of angiosperms . Results indicate that Amborella has acquired one or more copies of 20 of its 31 known mitochondrial protein genes from other land plants, for a total of 26 foreign genes, whereas no evidence for HGT was found in the five sequenced genomes . Most of the Amborella transfers are from other angiosperms (especially eudicots), whereas others are from nonangiosperms, including six striking cases of transfer from (at least three different) moss donors . Most of the transferred genes are intact, consistent with functionality and/or recency of transfer . Amborella mtDNA has sustained proportionately more HGT than any other eukaryotic, or perhaps even prokaryotic, genome yet examined. Zhonghua Wai Ke Za Zhi, 2004 Oct 7, 42(19), 1170 - 3 {Molecular cloning and expression of human PDGF-B chain mature peptide gene.}; Chen JT et al.; OBJECTIVE: To acquire sufficient PDGF-BB protein and provide the basis for the further studies of its role on the fracture healing and trauma reconstruction and its clinical applications . METHODS: Constructed the prokaryotic expression vector pQE-PDGF-B with the gene rearrangement technique, and the monomeric form of recombinant PDGF-B expressed in E . coli M15 . RESULTS: PDGF-B mature peptide gene was inserted into prokaryotic expression vector pQE30, which was confirmed by PCR, enzyme digestion and sequencing identification; the expressed products of pQE-PDGF-B in E . coli showed a single protein on SDS-PAGE, and their expression level was about 15% of the total bacterial protein . The molecular weight of the purified PDGF-B protein was about 15 KDs on SDS-PAGE . CONCLUSIONS: The construction of recombinant plasmid and preparation of the monomeric protein of PDGF-B provides a solid foundation for further studying the function of PDGF-BB and producing biologically PDGF-BB protein. Proc Natl Acad Sci U S A, 2004 Dec 28, 101(52), 18228 - 33 Epub 2004 Dec 13. Identification of a SulP-type bicarbonate transporter in marine cyanobacteria; Price GD et al.; Cyanobacteria possess a highly effective CO(2)-concentrating mechanism that elevates CO(2) concentrations around the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) . This CO(2)-concentrating mechanism incorporates light-dependent, active uptake systems for CO(2) and HCO(-)(3) . Through mutant studies in a coastal marine cyanobacterium, Synechococcus sp . strain PCC7002, we identified bicA as a gene that encodes a class of HCO(-)(3) transporter with relatively low transport affinity, but high flux rate . BicA is widely represented in genomes of oceanic cyanobacteria and belongs to a large family of eukaryotic and prokaryotic transporters presently annotated as sulfate transporters or permeases in many bacteria (SulP family) . Further gain-of-function experiments in the freshwater cyanobacterium Synechococcus PCC7942 revealed that bicA expression alone is sufficient to confer a Na(+)-dependent, HCO(3)(-) uptake activity . We identified and characterized three cyanobacterial BicA transporters in this manner, including one from the ecologically important oceanic strain, Synechococcus WH8102 . This study presents functional data concerning prokaryotic members of the SulP transporter family and represents a previously uncharacterized transport function for the family . The discovery of BicA has significant implications for understanding the important contribution of oceanic strains of cyanobacteria to global CO(2) sequestration processes. Biochem Biophys Res Commun, 2005 Jan 21, 326(3), 687 - 94 A novel rhythm of microcystin biosynthesis is described in the cyanobacterium Microcystis panniformis Komárek et al; Bittencourt-Oliveira Mdo C et al.; The presence of microcystins (MCY) in the cyanobacteria Microcystis panniformis Komarek et al . is reported for the first time . This strain of cyanobacterium has been isolated from Barra Bonita, an eutrophicated water reservoir in Sao Paulo state, Brazil . The identification of M . panniformis was confirmed by both traditional morphological analysis and the phycocyanin intergenic spacer sequences . MCY-LR and {Asp(3)}-MCY-LR were identified in this strain after HPLC purification and extensive ESI-MS/MS analysis . Their levels in this strain were determined by HPLC and ranged from 0.25 to 2.75 and 0.08 to 0.75 fmol/cell, respectively . Analyzing the levels of MCY-LR and {Asp(3)}-MCY-LR in different times during the light:dark (L:D) cycle, it was found that levels of MCYs per cell were at least threefold as high during the day-phase than during the night-phase . This may be associated to the biological clock since prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a timing mechanism that is independent of the cell division cycle . Our findings also showed the same pattern under light:light (L:L) cycle. Biochem Biophys Res Commun, 2005 Jan 21, 326(3), 618 - 23 Overexpression of a eukaryotic glutathione reductase gene from Brassica campestris improved resistance to oxidative stress in Escherichia coli; Yoon HS et al.; Glutathione reductase (GR) plays an essential role in a cell's defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant glutathione . We constructed a recombinant plasmid based on the expression vector pET-18a that overexpresses a eukaryotic GR from Brassica campestris (BcGR) in Escherichia coli . For comparative analyses, E . coli GR (EcGR) was also subcloned in the same manner . The transformed E . coli with the recombinant constructs accumulated a high level of GR transcripts upon IPTG induction . Also, Western blot analysis showed overproduction of the BcGR protein in a soluble fraction of the transformed E . coli extract . When treated with oxidative stress generating reagents such as paraquat, salicylic acid, and cadmium, the BcGR overproducing E . coli exhibited a higher level of growth and survival rate than the control E . coli strain, but it was not as high as the E . coli strain transformed with the inducible EcGR . The translated amino acid sequences of BcGR and EcGR share 37.3% identity but all the functionally known important residues are conserved . It appears that eukaryotic BcGR functions in a prokaryotic system by providing protection against oxidative damages in E . coli. BMC Plant Biol . 2004 Dec 13;4(1):20 {Epub ahead of print} Molecular characterization and functional expression of flavonol 6-hydroxylase; Anzellotti D et al.; BACKGROUND: Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants . Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations . The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase . ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis . RESULTS: A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein . This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity . The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4-trimethylquercetin as the preferred substrate . The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C . americanum genome . CONCLUSIONS: Recombinant F6H has been functionally expressed and characterized at the molecular level . The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme . The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction. Acta Biochim Biophys Sin (Shanghai), 2004 Dec, 36(12), 845 - 50 Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell proliferation; Hou WH et al.; Human canstatin, a 24 kD fragment of the alpha2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth . To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan . The recombinant mouse canstatin efficiently expressed in E . coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody . The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins . The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93% . The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed . In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells . Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. Microbiol Mol Biol Rev, 2004 Dec, 68(4), 603 - 16 New insights into type II NAD(P)H:quinone oxidoreductases; Melo AM et al.; Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the two-electron transfer from NAD(P)H to quinones, without any energy-transducing site . NDH-2 accomplish the turnover of NAD(P)H, regenerating the NAD(P)(+) pool, and may contribute to the generation of a membrane potential through complexes III and IV . These enzymes are usually constituted by a nontransmembrane polypeptide chain of approximately 50 kDa, containing a flavin moiety . There are a few compounds that can prevent their activity, but so far no general specific inhibitor has been assigned to these enzymes . However, they have the common feature of being resistant to the complex I classical inhibitors rotenone, capsaicin, and piericidin A . NDH-2 have particular relevance in yeasts like Saccharomyces cerevisiae and in several prokaryotes, whose respiratory chains are devoid of complex I, in which NDH-2 keep the balance and are the main entry point of electrons into the respiratory chains . Our knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms . The latter showed that most organisms contain genes that potentially encode NDH-2 . An overview of this development is presented, with special emphasis on microbial enzymes and on the identification of three subfamilies of NDH-2. J Biol Chem . 2004 Dec 20; {Epub ahead of print} The high resolution crystal structure of a native thermostable serpin reveals the complex mechanism underpinning the stressed to relaxed transition; Fulton KF et al.; Serpins (serine protease inhibitors) fold into a native metastable state and utilize a complex conformational change to inhibit target proteases . An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperatures . However, the prokaryote serpin, thermopin, from Thermobifida fusca is able to function in a heated environment . We have determined the 1.8 A X-ray crystal structure of thermopin in the native, inhibitory conformation . A structural comparison with the previously determined 1.5 A structure of cleaved thermopin provides detailed insight into the complex mechanism of conformational change in serpins . Flexibility in the shutter region and electrostatic interactions at the top of the Abeta sheet (the breach) involving the C-terminal tail, a unique structural feature of thermopin, are postulated to be important for controlling inhibitory activity and triggering conformational change, respectively, in the native state . We discuss the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures. J Biol Chem . 2004 Dec 7; {Epub ahead of print} Molecular structure of human galactokinase: Implications for type II galactosemia; Thoden JB et al.; Galactokinase functions in the Leloir pathway for galactose metabolism by catalyzing the MgATP dependent phosphorylation of the C-1 hydroxyl group of a-D-galactose . The enzyme is known to belong to the GHMP superfamily of small molecule kinases and has attracted significant research attention for well over forty years . Approximately 20 mutations have now been identified in human galactokinase, which result in the diseased state referred to as Type II galactosemia . Here we report the three-dimensional architecture of human galactokinase with bound a-D-galactose and MgAMPPNP . The overall fold of the molecule can be described in terms of two domains with the active site wedged between them . The N-terminal domain is dominated by a six-stranded mixed b-sheet while the C-terminal motif contains six a-helices and two layers of anti-parallel b-sheet . Those residues specifically involved in sugar binding include Arg 37, Glu 43, His 44, Asp 46, Gly 183, Asp 186, and Tyr 236 . The C-1 hydroxyl group of a-D-galactose sits within 3.3 A of the g-phosphorus of the nucleotide and 3.4 A of the guanidinium group of Arg 37 . The carboxylate side chain of Asp 186 lies within ~3.2 A of the C-2 hydroxyl group of a-D-galactose and the guanidinium group of Arg 37 . Both Arg 37 and Asp 186 are strictly conserved among both prokaryotic and eukaryotic galactokinases . In addition to providing molecular insight into the active site geometry of the enzyme, the model also provides a structural framework upon which to more fully understand the consequences of the those mutations known to give rise to Type II galactosemia. IUBMB Life, 2004 Sep, 56(9), 529 - 34 Bicarbonate stimulated adenylyl cyclases; Cann M; Bicarbonate ion is fundamental to the biology of all living organisms . HCO(3)(-) is vital to such diverse physiological processes as carbon fixation, cellular homeostasis, sperm maturation, and nucleotide synthesis . A defined subset of adenylyl cyclases identified in eukaryotes and prokaryotes are directly activated by HCO(3)(-) . As such, cAMP represents the first identified biological effector for fluctuations in intracellular inorganic carbon levels . The identification of a signal transduction pathway activated by HCO(3)(-) has far reaching implications for understanding how the cell responds to fluctuations in this essential anion. IUBMB Life, 2004 Sep, 56(9), 527 - 8 Non-metazoan class III nucleotidyl cyclases: novel forms and functions; Schaap P; Nucleotidyl cyclases synthesize cAMP or cGMP in response to various forms of sensory input . Their regulation and function in mammalian hormone action and neurotransmission has been well characterized over the past 40 years . More recently a much broader repertoire of adenylyl- and guanylyl cyclase forms is emerging from studies of more basal eukaryotes and prokaryotes . These cyclases play crucial roles in the development, physiology and pathogenicity of these organisms . This series of reviews highlights both unique and highly conserved aspects of the activation mechanisms of these enzymes, as compared to those of their mammalian descendants. Eur J Histochem, 2004 Jul-Sep, 48(3), 261 - 5 HSP10 selective preference for myeloid and megakaryocytic precursors in normal human bone marrow; Cappello F et al.; Heat shock proteins (HSPs) constitute a heterogeneous family of proteins involved in cell homeostasis . During cell life they are involved in harmful insults, as well as in immune and inflammatory reactions . It is known that they regulate gene expression, and cell proliferation, differentiation and death . HSP60 is a mitochondrial chaperonin, highly preserved during evolution, responsible of protein folding . Its function is strictly dependent on HSP10 in both prokaryotic and eukaryotic elements . We investigated the presence and the expression of HSP60 and HSP10 in a series of 20 normal human bone marrow specimens (NHBM) by the means of immunohistochemistry . NHBM showed no expression of HSP60, probably due to its being below the detectable threshold, as already demonstrated in other normal human tissues . By contrast, HSP10 showed a selective positivity for myeloid and megakaryocytic lineages . The positivity was restricted to precursor cells, while mature elements were constantly negative . We postulate that HSP10 plays a role in bone marrow cell differentiation other than being a mitochondrial co-chaperonin . The present data emphasize the role of HSP10 during cellular homeostasis and encourage further investigations in this field. DNA Repair (Amst), 2005 Feb 3, 4(2), 279 - 83 How are specialized (low-fidelity) eukaryotic polymerases selected and switched with high-fidelity polymerases during translesion DNA synthesis? Fischhaber PL, Friedberg EC. Specialized DNA polymerases are required in both prokaryotic and eukaryotic cells for bypassing sites of template DNA damage that arrest high-fidelity DNA replication . Recent studies in the literature provide hints of the complexity of DNA switching between polymerases for translesion DNA synthesis (TLS) and those for normal DNA replication. J Theor Biol, 2005 Feb 21, 232(4), 559 - 67 A fractal method to distinguish coding and non-coding sequences in a complete genome based on a number sequence representation; Zhou LQ et al.; A fractal method to distinguish coding and non-coding sequences in a complete genome is proposed, based on different statistical behaviors between these two kinds of sequences . We first propose a number sequence representation of DNA sequences . Multifractal analysis is then performed on the measure representation of the obtained number sequence . The three exponents C(-1), C(1) and C(2) are selected from the result of multifractal analysis . Each DNA may be represented by a point in the three-dimensional space generated by these three-component vectors . It is shown that points corresponding to coding and non-coding sequences in the complete genome of many prokaryotes are roughly distributed in different regions . Fisher's discriminant algorithm can be used to separate these two regions in the spanned space . If the point (C(-1),C(1),C(2)) for a DNA sequence is situated in the region corresponding to coding sequences, the sequence is discriminated as a coding sequence; otherwise, the sequence is classified as a non-coding one . For all 51 prokaryotes we considered , the average discriminant accuracies p(c),p(nc),q(c) and q(nc) reach 72.28%, 84.65%, 72.53% and 84.18%, respectively. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Aug, 22(4), 204 - 8 {Transient expression of Echinococcus granulosus Eg95 DNA vaccine and induction of immune response in mice}; Lin RY et al.; OBJECTIVE: To detect the in vitro expression of pcDNA3-Eg95 and to observe the immunological effect of the Eg95 DNA vaccine in mice . METHODS: The eukaryotic recombinant plasmid pcDNA3-Eg95 was transfected into HeLa cells with liposome-mediated method . RT-PCR, ELISA and Western blotting were used to analyze the expression of Eg95 mRNA and Eg95 protein, respectively . The BALB/c mice were immunized by pcDNA3-Eg95 . Anti-Eg95 IgG and IgG2a in murine serum were determined by ELISA . The proliferation activity of spleen T lymphocytes was tested using MTT assay . RESULTS: Using RT-PCR method, the expression of Eg95 mRNA was confirmed in vitro . The results of ELISA and Western blotting showed that there was a specific Eg95 protein, which can be specifically recognized by anti-sera of Eg95-prokaryotic-expressing protein in pcDNA3-Eg95 transfected HeLa cell lysis . The specific IgG was induced during the 3rd week and continued to increase until week 10 . IgG2a was detected after 2 weeks and maintained a higher level till week 10 . There was a significant difference of IgG2a level between pcDNA3-Eg95 immunized group and pcDNA3 control (P<0.01) . In spleen T cell proliferation response, the stimulation index (SI) in pcDNA3-Eg95 group was higher than that of vector control (P<0.01) . CONCLUSION: Eg95 DNA vaccine can induce significant cellular and humoral immune response in mice. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Feb, 30(1), 105 - 14 {The structural, transcriptional and homology analysis of two frr genes in rice.}; Hu X et al.; Two rice (Oryza sativa subsp . japonica cv . Nipponbare) ribosome recycling factor genes--OsfrrA and OsfrrB had been identified and characterized in this study . The gene OsfrrA is located on chromosome 4 while OsfrrB on chromosome 7 . No other homologue is found in rice organelle genomes . Both genes are unique in rice genome and constitutively expressed . The N-terminal character of their encoded protein products suggests that the proteins are transferred to mitochondrion and chloroplast respectively and carry out their functions . The sequence conservation and the constitutive expression profile of the two genes strongly imply their indispensable role in plant growth . In addition, these sequences share phylogenetic homology to some extent with other prokaryotic and eukaryotic RRFs, providing further evidence for the endosymbiotic theory, and implying the potential value of RRFs in molecular evolution research. J Clin Microbiol, 2004 Dec, 42(12), 5439 - 43 Genetic bases of the rifampin resistance phenotype in Brucella spp; Marianelli C et al.; Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens . Its bactericidal activity is due to its ability to bind to the beta subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene . Mutations of the rpoB gene have been characterized in rifampin-resistant (Rif(r)) strains of Escherichia coli and Mycobacterium tuberculosis . The genetic bases of Rif(r) in Brucella spp . are still unknown . In the present study, the nucleotide sequences of the rpoB gene of the Rif(r) vaccine strain Brucella abortus RB51 and of 20 Rif(r) clones derived in our laboratory from two Brucella melitensis isolates were determined . These sequences were then compared to those of the respective rifampin-susceptible (Rif(s)) parental strains and to the published B . melitensis strain 16M . All Rif(r) strains carried one or more missense mutations mapping in two regions of the rpoB gene . These two "hot" regions were investigated in eight additional Rif(r) Brucella laboratory mutants and in 20 reference Rif(s) Brucella strains . rpoB mutations were found in all Rif(r) mutants . In contrast, no missense mutations were found in any analyzed Rif(s) strains . Our results represent the first from a study of the molecular characterization of rpoB mutations in resistant Brucella strains and provide an additional proof of the association of specific rpoB mutations with the development of the Rif(r) phenotype in prokaryotes . In addition, because of the relationship between Rif(r) and the attenuation of virulence in Brucella spp., studies of virulence in these mutants may provide useful information about the genetic basis of pathogenesis in Brucella. Microbiology, 2004 Dec, 150(Pt 12), 4189 - 97 Evolution of the PPM-family protein phosphatases in Streptomyces: duplication of catalytic domain and lateral recruitment of additional sensory domains; Zhang W et al.; Originally identified from eukaryotes, the Mg2+- or Mn2+-dependent protein phosphatases (PPMs) are a diverse group of enzymes whose members include eukaryotic PP2C and some prokaryotic serine/threonine phosphatases . In a previous study, unexpectedly large numbers of PPMs were identified in two Streptomyces genomes . In this work, a phylogenetic analysis was performed with all the PPMs available from a wide variety of microbial sources to determine the evolutionary origin of the Streptomyces PPM proteins . Consistent with earlier hypotheses, the results suggested that the microbial PPMs were relatively recent additions from eukaryotic sources . Results also indicated that the Streptomyces PPMs were divided into two major subfamilies at an early stage of their emergence in Streptomyces genomes . The first subfamily, which contains only six Streptomyces PPMs, possesses a catalytic domain whose sequence and architecture are similar to that of eukaryotic PPMs; the second subfamily contains 89 Streptomyces PPMs that lack the 5a and 5b catalytic domain motifs, similar to the PPMs SpoIIE and RsbU of Bacillus subtilis . Significant gene duplication was observed for the PPMs in the second subfamily . In addition, more than half (54 %) of the Streptomyces PPMs from the second subfamily were found to have at least one additional sensory domain, most commonly the PAS or the GAF domain . Phylogenetic analysis showed that these domains tended to be clustered according to the putative physiological functions rather than taxonomic relationship, implying that they might have arisen as a result of domain recruitment in a late evolutionary stage . This study provides an insight into how Streptomyces spp . may have expanded their PPM-based signal transduction networks to enable them to respond to a greater range of environmental changes. Virology, 2005 Jan 5, 331(1), 47 - 62 A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity; Lee C et al.; Reverse genetic systems were developed for a highly virulent 'atypical' porcine reproductive and respiratory syndrome virus (PRRSV) . The full-length genome of 15395 nucleotides was assembled as a single cDNA clone and placed under either the prokaryotic T7 or eukaryotic CMV promoter . Transfection of cells with the RNA transcripts or the DNA clone induced cytopathic effects and produced infectious progeny . The reconstituted virus was stable and grew to the titer of the parental virus in cells . Upon infection, pigs produced clinical signs and lung pathology typical for PRRSV and induced viremia and specific antibodies . Previously, we showed that the PRRSV nucleocapsid (N) protein forms homodimers via both noncovalent and covalent interactions and that cysteine at position 23 is responsible for the covalent interaction . The functional significance of cysteines of N for PRRSV infectivity was assessed using the infectious cDNA clone . Each cysteine of N at positions 23, 75, and 90 was replaced with serine and the individual mutation was incorporated into the cDNA clone such that three independent cysteine mutants were constructed . When transfected, the wild type and C75S clones induced cytopathic effects and produced infectious virus with indistinguishable plaque morphology . In contrast, the C23S mutation completely abolished infectivity of the clone, indicating that C23-mediated N protein homodimerization plays a critical role in PRRSV infectivity . Unexpectedly, the C90S mutation also appeared to be lethal for virus infectivity . Genome replication and mRNA transcription were both positive for the replication-defective C23S and C90S mutants . The data suggest that, in addition to homodimerization, the PRRSV N protein may also undergo heterodimerization with another structural protein using cysteine 90 and that the N protein heterodimerization is essential for PRRSV infectivity. Curr Opin Struct Biol, 2004 Dec, 14(6), 741 - 7 Three's company: component structures bring a closer view of tripartite drug efflux pumps; Eswaran J et al.; Bacterial multidrug resistance is a serious clinical problem and is commonly conferred by tripartite efflux 'pumps' in the prokaryotic cell envelope . Crystal structures of the three components of a drug efflux pump have now been solved: the outer membrane TolC exit duct in the year 2000, the inner membrane AcrB antiporter in 2002 and the periplasmic adaptor MexA in 2004 . These structures have enhanced our understanding of the principles underlying pump assembly and operation, and present pumps as new drug targets. J Mol Biol, 2005 Jan 21, 345(3), 433 - 9 Supramolecular assemblies of human frataxin are formed via subunit-subunit interactions mediated by a non-conserved amino-terminal region; O'Neill HA et al.; The mitochondrial protein frataxin is emerging as a novel mechanism to promote iron metabolism while also providing anti-oxidant protection . Recombinant frataxin proteins from different species are able to form large molecular assemblies that store Fe(III) as a stable mineral in vitro . Furthermore, monomeric and assembled forms of frataxin donate Fe(II) to the Fe-S cluster scaffold protein IscU, {3Fe-4S}1+ aconitase, and ferrochelatase in vitro . However, little is known about the speciation of frataxin in vivo, and the physiologically relevant form(s) of the protein remains undefined . Here, we report that human heart mitochondria contain frataxin species of increasing negative surface charge and molecular mass, ranging from monomer to polymers of >1 MDa . Moreover, we show that the main partner protein of frataxin, IscU, binds in a stable manner to frataxin oligomers . These results suggest that assembly is a physiologic property of frataxin . Biochemical analyses further reveal that, unlike the prokaryotic and yeast frataxin homologues, which require iron-protein interactions for assembly, human frataxin uses stable subunit-subunit interactions involving a non-conserved amino-terminal region . We propose that human frataxin is a modular protein that depends on self-assembly to accomplish its diverse functions. Biotechnol Bioeng, 2005 Jan 20, 89(2), 195 - 205 Engineering HlyA hypersecretion in Escherichia coli based on proteomic and microarray analyses; Lee PS et al.; Escherichia coli is a common host for recombinant protein production for biotechnology applications . Secretion to the extracellular media has the potential to reduce protein aggregation and to simplify downstream purification . However, the complexity of the mechanisms of protein secretion has confounded prior attempts to engineer enhanced secretion phenotypes . Here, mutagenesis was used to perturb E . coli W3110 cells secreting HlyA via a Type I pathway . An activity assay identified a mutant secreting fourfold more active alpha-hemolysin than the parent strain . The mutant was characterized using both high-density microarrays for mRNA profiling and a proteomics strategy for protein expression . The relative mRNA and protein expression levels of tRNA-synthetases were decreased in the mutant compared to the parent . A mathematical model of prokaryotic translation was used to design a variant of the hlyA gene that encodes the same amino acid sequence but uses rare codons to slow the rate of translation by altering five bases . Analysis of the parent strain transformed with a plasmid containing this variant gene resulted in the recovery of, and further improvement upon, the selected hypersecretion phenotype . These results present one of the first successful metabolic engineering attempts based on molecular information provided by mRNA and protein expression profiling approaches and resulting in a phenotype useful to the biotechnology community . (c) 2004 Wiley Periodicals, Inc. Biotechnol Bioeng, 2005 Jan 5, 89(1), 9 - 17 Broad-spectrum protein biosensors for class-specific detection of antibiotics; Weber CC et al.; The dramatically increasing prevalence of multi-drug-resistant human pathogenic bacteria and related mortality requires two key actions: (i) decisive initiatives for the detection of novel antibiotics and (ii) a global ban for use of antibiotics as growth promotants in stock farming . Both key actions entail technology for precise, high-sensitive detection of antibiotic substances either to detect and validate novel anti-infective structures or to enforce the non-use of clinically relevant antibiotics . We have engineered prokaryotic antibiotic response regulators into a molecular biosensor configuration able to detect tetracycline, streptogramin, and macrolide antibiotics in spiked liquids including milk and serum at ng/mL concentrations and up to 2 orders of magnitude below current Swiss and EC threshold values . This broad-spectrum, class-specific, biosensor-based assay has been optimized for use in a storable ready-to-use and high-throughput-compatible ELISA-type format . At the center of the assay is an antibiotic sensor protein whose interaction with specific DNA fragments is responsive to a particular class of antibiotics . Binding of biosensor protein to the cognate DNA chemically linked to a solid surface is converted into an immuno-based colorimetric readout correlating with specific antibiotics concentrations . (c) 2004 Wiley Periodicals, Inc. J Am Soc Nephrol, 2004 Dec, 15(12), 2972 - 80 Calcineurin Abeta is central to the expression of the renal type II Na/Pi co-transporter gene and to the regulation of renal phosphate transport; Moz Y et al.; The sensing and response to extracellular phosphate (Pi) concentration is preserved from prokaryotes to mammals and ensures an adequate supply of Pi in the face of large differences in its availability . In mammals, the kidneys are central to Pi homeostasis . Renal Pi reabsorption is mediated by a Na/Pi co-transporter that is regulated by a renal Pi sensing system and humoral factors . The signal transduction by which Pi regulates type II Na/Pi activity is largely unknown . It is shown that calcineurin inhibitors specifically and dramatically decrease type II Na/Pi gene expression in a proximal tubule cell line and in vivo . Mice with genetic deletion of the calcineurin Abeta gene had a marked decrease in type II Na/Pi mRNA levels and remarkably did not show the expected increase in type II Na/Pi mRNA levels after the challenge of a low-Pi diet . In contrast, the regulation of renal 25(OH)-vitamin D 1alpha-hydroxylase gene expression by Pi was intact . This is the first demonstration that calcineurin has a crucial role in the signal transduction pathway regulating renal Pi homeostasis both in vitro and in vivo . These results suggest that the use of calcineurin inhibitors contributes to the renal Pi wasting seen in renal transplant patients. J Comput Biol, 2004, 11(4), 519 - 43 The analysis of stress-induced duplex destabilization in long genomic DNA sequences; Benham CJ et al.; We present a method for calculating predicted locations and extents of stress-induced DNA duplex destabilization (SIDD) as functions of base sequence and stress level in long DNA molecules . The base pair denaturation energies are assigned individually, so the influences of near neighbors, methylated bases, adducts, or lesions can be included . Sample calculations indicate that copolymeric energetics give results that are close to those derived when full near-neighbor energetics are used; small but potentially informative differences occur only in the calculated SIDD properties of moderately destabilized regions . The method presented here for analyzing long sequences calculates the destabilization properties within windows of fixed length N, with successive windows displaced by an offset distance d(o) . The final values of the relevant destabilization parameters for each base pair are calculated as weighted averages of the values computed for each window in which that base pair appears . This approach implicitly assumes that the strength of the direct coupling between remote base pairs that is induced by the imposed stress attenuates with their separation distance . This strategy enables calculations of the destabilization properties of DNA sequences of any length, up to and including complete chromosomes . We illustrate its utility by calculating the destabilization properties of the entire E . coli genomic DNA sequence . A preliminary analysis of the results shows that promoters are associated with SIDD regions in a highly statistically significant manner, suggesting that SIDD attributes may prove useful in the computational prediction of promoter locations in prokaryotes. Anal Bioanal Chem . 2004 Dec 2; {Epub ahead of print} Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE-FPLC with ICP-(ORS)MS detection; Rodriguez-Cea A et al.; Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes . The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC) . Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step . The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution . Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450 . Trouts were treated intraperitoneally with beta-naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4+/-0.1 nmol mL(-1) P-450 enzymes was obtained by ultracentrifugation . Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery . The solubilized cytochromes P-450 were further separated by AE-FPLC, with UV detection, or coupled to ICP-MS with an octapole reaction system, ICP-(ORS)MS (monitoring Fe signals at masses 54, 56, and 57) . A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes . ICP-(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins. Nucleic Acids Res, 2004, 32(21), 6321 - 6 Print 2004. Functional clues for hypothetical proteins based on genomic context analysis in prokaryotes; Doerks T et al.; Three integrated genomic context methods were used to annotate uncharacterized proteins in 102 bacterial genomes . Of 7853 orthologous groups with unknown function containing 45,110 proteins, 1738 groups could be linked to functionally associated partners . In many cases, those partners are uncharacterized themselves (hinting at newly identified modules) or have been described in general terms only . However, we were able to assign pathways, cellular processes or physical complexes for 273 groups (encompassing 3624 previously functionally uncharacterized proteins). Genome Biol . 2004;5(12):R97 . Epub 2004. Hidden localization motifs: naturally occurring peroxisomal targeting signals in non-peroxisomal proteins; Neuberger G et al.; BACKGROUND: Can sequence segments coding for subcellular targeting or for posttranslational modifications occur in proteins that are not substrates in either of these processes? Although considerable effort has been invested in achieving low false-positive prediction rates, even accurate sequence-analysis tools for the recognition of these motifs generate a small but noticeable number of protein hits that lack the appropriate biological context but cannot be rationalized as false positives . RESULTS: We show that the carboxyl termini of a set of definitely non-peroxisomal proteins with predicted peroxisomal targeting signals interact with the peroxisomal matrix protein receptor peroxin 5 (PEX5) in a yeast two-hybrid test . Moreover, we show that examples of these proteins - chicken lysozyme, human tyrosinase and the yeast mitochondrial ribosomal protein L2 (encoded by MRP7) - are imported into peroxisomes in vivo if their original sorting signals are disguised . We also show that even prokaryotic proteins can contain peroxisomal targeting sequences . CONCLUSIONS: Thus, functional localization signals can evolve in unrelated protein sequences as a result of neutral mutations, and subcellular targeting is hierarchically organized, with signal accessibility playing a decisive role . The occurrence of silent functional motifs in unrelated proteins is important for the development of sequence-based function prediction tools and the interpretation of their results . Silent functional signals have the potential to acquire importance in future evolutionary scenarios and in pathological conditions. Appl Environ Microbiol, 2004 Dec, 70(12), 7445 - 55 Characterization of C1-metabolizing prokaryotic communities in methane seep habitats at the Kuroshima Knoll, southern Ryukyu Arc, by analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA genes; Inagaki F et al.; Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment . 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (approximately 34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site . In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA) . In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g {wet weight}), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g {wet weight}) was most abundant at the surface of the MC core . These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity . The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g {wet weight}) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g {wet weight}) . The mmoX gene was never detected . In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C1 metabolism . Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur. Appl Environ Microbiol, 2004 Dec, 70(12), 7161 - 72 Development of a universal microarray based on the ligation detection reaction and 16S rrna gene polymorphism to target diversity of cyanobacteria; Castiglioni B et al.; The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds . In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health . Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important . Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments . Our approach is based on the use of a ligation detection reaction coupled to a universal array . Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia . These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences . For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested . The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene . Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample . Our universal array method shows great potential for rapid and reliable identification of cyanobacteria . It can be easily adapted to future development and could thus be applied both in research and environmental monitoring. Appl Environ Microbiol, 2004 Dec, 70(12), 6998 - 7009 Microarray and functional gene analyses of sulfate-reducing prokaryotes in low-sulfate, acidic fens reveal cooccurrence of recognized genera and novel lineages; Loy A et al.; Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems . Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 microM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schloppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities . 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens . Members of the family "Syntrophobacteraceae" were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schloppnerbrunnen I . These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens . Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives . In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens . These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats. Genome Res, 2004 Dec, 14(12), 2469 - 77 Computing prokaryotic gene ubiquity: rescuing the core from extinction; Charlebois RL et al.; The genomic core concept has found several uses in comparative and evolutionary genomics . Defined as the set of all genes common to (ubiquitous among) all genomes in a phylogenetically coherent group, core size decreases as the number and phylogenetic diversity of the relevant group increases . Here, we focus on methods for defining the size and composition of the core of all genes shared by sequenced genomes of prokaryotes (Bacteria and Archaea) . There are few (almost certainly less than 50) genes shared by all of the 147 genomes compared, surely insufficient to conduct all essential functions . Sequencing and annotation errors are responsible for the apparent absence of some genes, while very limited but genuine disappearances (from just one or a few genomes) can account for several others . Core size will continue to decrease as more genome sequences appear, unless the requirement for ubiquity is relaxed . Such relaxation seems consistent with any reasonable biological purpose for seeking a core, but it renders the problem of definition more problematic . We propose an alternative approach (the phylogenetically balanced core), which preserves some of the biological utility of the core concept . Cores, however delimited, preferentially contain informational rather than operational genes; we present a new hypothesis for why this might be so. Mol Biol Evol . 2004 Dec 1; {Epub ahead of print} Clusters of Co-Expressed Genes in Mammalian Genomes are Conserved by Natural Selection; Singer GA et al.; Genes that belong to the same functional pathways are often packaged into operons in prokaryotes . However, aside from examples in nematode genomes, this form of transcriptional regulation appears to be absent in eukaryotes . Nevertheless, a number of recent studies have shown that gene order in eukaryotic genomes is not completely random, and that genes with similar expression patterns tend to be clustered together . What remains unclear is whether coexpressed genes have been gathered together by natural selection to facilitate their regulation, or if the genes are co-expressed simply by virtue of their being close together in the genome . Here, we show that gene expression clusters tend to contain fewer chromosomal breakpoints between human and mouse than expected by chance, which indicates that they are being held together by natural selection . This conclusion applies to clusters defined on the basis of broad (housekeeping) expression, or on the basis of correlated transcription profiles across tissues . Contrary to a previous reports, we find that genes with high expression are not clustered to a greater extent than expected by chance and are not conserved during evolution. Front Biosci, 2005 Jan 1, 10, 462 - 77 Print 2005 Jan 1. Cytoplasmic membrane iron permease systems in the bacterial cell envelope; Koster W; Iron is a vital nutrient for the vast majority of organisms . Gram-positive bacteria, Gram-negative bacteria and archaea established different strategies to utilize iron from various sources . This article concentrates on the cytoplasmic membrane-associated uptake systems involved in the acquisition of iron . Genetic, biochemical and structural data as well as computational analyses were taken into consideration . Besides divalent metal transporters of the Nramp type and ferrous iron transport proteins of the Feo type, four distinct families of ABC transporters related to iron uptake are known . Their components mediate the transfer of iron, siderophores, heme and vitamin B12 into the cytosol of prokaryotes. Sichuan Da Xue Xue Bao Yi Xue Ban, 2004 Nov, 35(6), 753 - 6 {Construction and prokaryotic expression of a recombinant immunotoxin fused with mouse interleukin 18 and truncated Pseudomonas exotoxin}; Li H et al.; OBJECTIVE: To construct a new recombinant immunotoxin expression vector by fusion of mouse interleukin 18 (mIL-18) gene and a truncated form of A (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli . METHODS: mIL-18 cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR) . The mIL-18 cDNA was subcloned into a PE38 gene inserted fusion protein expression plasmid . The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing . After transformed into E . coli BL21 and induced by IPTG, the expressed product was obtained and detected the molecular weight and specificity by SDS-PAGE and Western-blotting . RESULTS: The new recombinant immunotoxin expression vector was constructed successfully . The IL-18-PE38 fusion protein was expressed in E . coli BL21, and the molecular weight of the expression product was identical to the expected value . In addition, the protein expressed could react with the specific antibody against mIL-18 . CONCLUSION: IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cell and may have some potential value in the treatment of autoimmune disease and T cell leukemias. Biophys Chem, 2004 Dec 20, 112(2-3), 233 - 44 The phylogeny of persistence in DNA; Poland D; We continue our study, Poland {Biophysical Chemistry 110 (2004) 59-2}, of the distribution of C or G (C-G for short) in the DNA of select organisms, in particular, the tendency for C-G to cluster on all scales with respect to the number of bases considered . We previously found that if we counted the number of C-G bases in consecutive, nonoverlapping boxes containing a total of m bases, then the width of the distribution function describing how many C-G bases are in a box increases with respect to m dramatically relative to the width expected for a random distribution . The relative width of the C-G composition distribution function was found to vary accurately as a power law with respect to m, the size of the box, over a very wide range of m values . We express the power law in terms of a characteristic exponent gamma, that is, the relative widths of the distributions vary as m(gamma) . The enhanced relative width of the distribution functions is a direct consequence of the tendency for boxes of similar composition to follow one another . This tendency represents persistence in composition from box to box and hence we refer to gamma as the persistence exponent . The occurrence of a power law means that the tendency for C-G to cluster is present on all scales of sequence length (box size) up to the total length of the chromosome which for bacteria is the entire genome . The persistence exponent gamma that characterizes the power law is thus an important parameter describing the distribution of C-G on all scales from individual base pairs up to the total length of the DNA sample considered . In the present paper, we determine the characteristic exponent gamma and the associated fractal dimension of DNA samples for a selection of species representing all of the major types of organism, that is, we explore the phylogeny of the exponent gamma . Here we treat six prokaryotes and six eukaryotes which, together with the species we have previously treated, brings the total number of species we have examined to 15 . We find the power law form for the C-G distribution for all of the species treated and hence this behavior seems to be ubiquitous . The values of the characteristic exponent gamma that we find tend to cluster around the value gamma=0.20 with no obvious pattern with respect to phylogeny . The extreme values that we obtain are gamma=0.057 (yeast) and gamma=0.386 (human) . We conclude by showing that the persistence of C-G clustering on the scale of the length of a chromosome is dramatically illustrated by interpreting the C-G distribution as a random walk. Nucleosides Nucleotides Nucleic Acids, 2004 Oct, 23(8-9), 1475 - 9 Mechanistic studies of dUTPases; Kovari J et al.; The essential enzyme dUTPase is responsible for preventive DNA repair via exclusion of uracil . Lack or inhibition of the enzyme induces thymine-less cell death in cells performing active DNA synthesis, serving therefore as an important chemotherapeutic target . In the present work, employing differential circular dichroism spectroscopy, we show that D . mel . dUTPase, a recently described eukaryotic model, has a similar affinity of binding towards alpha,beta-imino-dUTP as compared to the prokaryotic E . coli enzyme . However, in contrast to the prokaryotic dUTPase, the nucleotide exerts significant protection against tryptic digestion at a specific tryptic site 20 A far from the active site in the fly enzyme . This result indicates that binding of the nucleotide in the active site induces an allosteric conformational change within the central threefold channel of the homotrimer exclusively in the eukaryotic enzyme . Nucleotide binding induced allosterism in the D . mel . dUTPase, but not in the E . coli enzyme, might be associated with the altered hydropathy of subunit interfaces in these two proteins. Annu Rev Genet, 2004, 38, 771 - 92 Comparative genomic structure of prokaryotes; Bentley SD et al.; Recent advances in DNA-sequencing technologies have made available an enormous resource of data for the study of bacterial genomes . The broad sample of complete genomes currently available allows us to look at variation in the gross features and characteristics of genomes while the detail of the sequences reveal some of the mechanisms by which these genomes evolve . This review aims to describe bacterial genome structures according to current knowledge and proposed hypotheses . We also describe examples where mechanisms of genome evolution have acted in the adaptation of bacterial species to particular niches. Biochem Biophys Res Commun, 2005 Jan 7, 326(1), 87 - 92 An enzymatic cycling method for the determination of serum total bile acids with recombinant 3alpha-hydroxysteroid dehydrogenase; Zhang GH et al.; A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay . We constructed a prokaryotic expression system to prepare the recombinant 3alpha-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay . The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm . The enzymatic cycling method could detect 0.22 micromol/L total bile acids in serum . Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively . The calibration curve for total bile acids in serum was linear between 0.5 and 180 micromol/L . This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase . In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3alpha-hydroxysteroid dehydrogenase as the tool enzyme. Bioinformatics . 2004 Nov 25; {Epub ahead of print} A fuzzy guided genetic algorithm for operon prediction; Jacob E et al.; MOTIVATION: The operon structure of the prokaryotic genome is a critical input for the reconstruction of regulatory networks at the whole genome level . As experimental methods for detection of operons are difficult and time consuming, efforts are being put into developing computational methods that can use available biological information to predict operons . Method: A genetic algorithm is developed to evolve a starting population of putative operon maps of the genome into progressively better predictions . Fuzzy scoring functions based on multiple criteria are used for assessing the "fitness" of the newly evolved operon maps and guiding their evolution . RESULTS: The algorithm organizes the whole genome into operons . The fuzzy guided genetic algorithm-based approach makes it possible to use diverse biological information like genome sequence data, functional annotations and conservation across multiple genomes, to guide the organization process . This approach does not require any prior training with experimental operons . The predictions from this algorithm for E . coli K12 and B . subtilis are evaluated against experimentally discovered operons for these organisms . The accuracy of the method is evaluated using an ROC analysis . The area under the ROC curve is around 0.9, which indicates excellent accuracy . SUPPLEMENTARY INFORMATION: List of predicted operons for E . coli K12 and B . subtilis . The fuzzy rule base for generating fitness scores for putative operons. Nucleic Acids Res . 2004 Nov;32(20):e165. In situ imaging and isolation of proteins using dsDNA oligonucleotides; Dellaire G et al.; As proteomics initiatives mature, the need will arise for the multiple visualization of proteins and supramolecular complexes within their true context, in situ . Single-stranded DNA and RNA aptamers can be used for low resolution imaging of cellular receptors and cytoplasmic proteins by light microscopy (LM) . These techniques, however, cannot be applied to the imaging of nuclear antigens as these single-stranded aptamers bind endogenous RNA and DNA with high affinity . To overcome this problem, we have developed a novel method for the in situ detection of proteins using double-stranded DNA oligonucleotides . To demonstrate this system we have utilized the prokaryotic DNA-binding proteins LacI and TetR as peptide tags to image fusion proteins in situ using dsDNA oligonucleotides encoding either the Lac or Tet operator . Using fluorescent and fluorogold dsDNA oligonucleotides, we localized within the nucleus a TetR-PML fusion protein within promyelocytic leukaemia protein (PML) bodies by LM and a LacI-SC35 fusion protein within nuclear speckles by correlative light and electron microscopy (LM/EM) . Isolation of LacI-SC35 was also accomplished by using biotinylated dsDNA and streptavidin sepharose . The use of dsDNA oligonucleotides should complement existing aptamer in situ detection techniques by allowing the multiple detection and localization of nuclear proteins in situ and at high resolution. Antimicrob Agents Chemother, 2004 Dec, 48(12), 4495 - 504 A mutation in Escherichia coli DNA gyrase conferring quinolone resistance results in sensitivity to drugs targeting eukaryotic topoisomerase II; Gruger T et al.; Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases . Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV . In Escherichia coli, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase . One common site for resistance-conferring mutations alters Ser83, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones . In the present study we demonstrate that the mutation of Ser83 to Trp in DNA gyrase (Gyr(S83W)) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes . Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of Gyr(S83W), and the enzyme caused elevated levels of DNA cleavage in the presence of these agents . The DNA sequence preference for Gyr(S83W)-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases . Introduction of the Gyr(S83W) mutation in E . coli strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin . Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes. Environ Microbiol, 2004 Dec, 6(12), 1228 - 43 Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes; Eiler A et al.; The diversity of freshwater bacterioplankton communities has not been extensively studied despite their key role in foodwebs and the cycling of carbon and associated major elements . In order to explore and characterize the composition of bacterioplankton associated with cyanobacterial blooms, large 16S rRNA clone libraries from four lakes experiencing such blooms were analysed . The four libraries contained 1461 clones, of which 559 were prokaryotic sequences of non-cyanobacterial origin . These clones were classified into 158 operational taxonomic units affiliated mainly with bacterial divisions commonly found in freshwater systems, e.g . Proteobacteria, Bacteriodetes, Actinobacteria, Verrucomicrobia and Planctomycetes . Richness and evenness of non-cyanobacterial clones were similar to other clone libraries obtained for freshwater bacterioplankton, suggesting that bacterial communities accompanying cyanobacterial blooms are as diverse as non-bloom communities . Many of the identified operational taxonomic units grouped with known freshwater clusters but the libraries also contained novel clusters of bacterial sequences that may be characteristic for cyanobacterial blooms . About 25% of the operational taxonomic units were detected in more than one lake . Even so, 16S rRNA heterogeneity analysis demonstrated large differences in community composition between lakes regardless of their similar characteristics and close proximity . Hence even the similar environmental conditions created by different cyanobacterial blooms may foster very dissimilar bacterial communities, which could indicate that the genetic diversity in lake bacteria have been underestimated in the past. Cell Mol Biol (Noisy-le-grand), 2004 Jul, 50(5), 517 - 24 Culture-independent analysis of bacterial species from an anaerobic mat from Lake Fryxell, Antarctica: prokaryotic diversity revisited; Stackebrandt E et al.; Using a lake sediment mat sample from Lake Fryxell, Antarctica, different DNA extraction and purification methods were compared by denaturing gradient gel electrophoresis (DGGE) . Based on the analyses of cloned 16S rRNA gene sequences a high degree of as yet