Prokaryotes

Mol Genet Metab, 2004 Sep-Oct, 83(1-2), 28 - 37
Propionic acidemia: mutation update and functional and structural effects of the variant alleles; Desviat LR et al.; Mutations in the PCCA or PCCB genes, encoding both subunits of propionyl-CoA carboxylase, result in propionic acidemia, a life-threatening inborn error of metabolism with autosomal recessive inheritance . To date, 41 mutations in the PCCA gene and 54 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements, and a variety of small insertions and deletions and splicing defects . A greater heterogeneity is observed in the PCCA gene, specially in Caucasians, with no prevalent mutations, while in the Japanese population three mutations account for more than half of the alleles studied . For the PCCB gene a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations . These two populations show a different mutational spectrum, only sharing some involving CpG dinucleotides probably as recurrent mutational events . Functional characterization of the mutant missense alleles has been accomplished using different prokaryotic and eukaryotic systems, and the structural consequences have been analyzed in the available crystal models . For the PCCA gene, the main molecular effect of the expressed mutations is related to protein instability, except two mutations in the active site predictably affecting ATP binding . In the PCCB gene the majority of the analyzed mutations are predicted to alter the active site conformation resulting in diminished activity . A few carboxy-terminal PCCB mutations affect the interaction between subunits and the assembly with PCCA to form a functional PCC oligomer . The amount of normal transcripts resulting from some PCCA and PCCB splicing mutations has also been analyzed . Overall, the data generated from the expression analysis reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.

Parasitol Today, 1993 Apr, 9(4), 122 - 6
ATP versus pyrophosphate: glycolysis revisited in parasitic protists; Mertens E; It is generally assumed that glycolysis is remarkably similar among all organisms, prokaryotic and eukaryotic . This view has been extended to protists . However, there is growing evidence that significant deviations from conventional glycolysis occur in protists . Very different species, some parasitic, rely on peculiar enzymes that use pyrophosphate as substrate . In this review, Emmanuel Mertens describes such unusual pyrophosphate metabolism in parasitic protists.

Parasitol Today, 1993 Oct, 9(10), 388 - 92
Genome analysis of Theileria parva; Morzaria SP et al.; Recent technological developments in the field of genome analyses have advanced our knowledge of the structures of prokaryotic and eukoryotic genomes . Examples of these range from small bacterial genomes, such as Escherichia coli, to the more complex genomes of Caenorhabditis elegans, Drosophila, humans and mouse . Here, Subhash Morzona and John Young review developments in mapping the genome of on economically important protozoan parasite o f cattle, Theileria parva . This map provides a framework for more detailed analysis of the genome structure o f this organism . The methodologies developed in constructing the map also have application to the mapping of other protozoan genomes.

Parasitol Today, 1993 Oct, 9(10), 381 - 4
The Trypanosoma cruzi ribosomal P protein family: Classification and antigenicity; Levin MJ et al.; The multi-copy ribosomal P proteins have been identified on the ribosomes of prokaryotic and eukaryotic cells, and their antigenicity is an important feature of human Trypanosoma cruzi infection . In this review, Mariano Levin, Martin Vazquez, Dan Kaplan and Alejandro Schijman give a rational basis for the classification of these proteins, and discuss their inter-relationship.

Parasitol Today, 1988 Dec, 4(12), 357 - 60
Polyamine biosynthesis and inhibition in Trichomonas vaginalis; Yarlett N; Polyomines - particularly putrescine, spermidine and spermine - are ubiquitous components of eukaryote and most prokaryote cells, and are essential for optimal cell proliferation . But since routes of polyamine synthesis may differ, for example between parasites and their hosts, selective inhibition of polyamine metabolism offers an attractive target for chemotherapy - as already shown with the success of difluoromethylomithine (DFMO) as an inhibitor of polyamine synthesis in African trypanosomes . Parasitology Today has featured a series of articles reviewing research on polyamine metabolism of various parasites (eg . vol . 3, pp 190-192, pp 312-315; vol . 4, pp 18-20) and here, Nigel Yorlett discusses these metabolic aspects of Trichomonas vaginalis (Fig . 1)-a common parasite of the urogenital tract.

Parasitol Today, 1988 Jan, 4(1), 18 - 20
Polyamine metabolism of filaria and allied parasites; Walter RD; Putrescine and the polyamines spermidine and spermine occur both in prokaroytes and in eukaryotes where they seem intimately involved in regulatory processes of cellular growth and differentiation . They seem to play an important role related to the biosynthesis of nucleic acids and proteins, although at the molecular level their precise function remains unclear . In general, prokaryotes utilize putrescine and spermidine while eukaryotes tend to have higher concentrations of spermidine and spermine compared to putrescine(1-3.) Differences in polyamine metabolism between parasites and their hosts suggest several potential targets for chemotherapeutic attack As Rolf Walter discusses here, such approaches have already been exploited for African trypanosomes and also offer some leads for the chemotherapy of helminth infections.

Acta Virol, 2004, 48(2), 97 - 107
Expression of herpes simplex virus 1 glycoprotein D in prokaryotic and eukaryotic cells; Mosko T et al.; Recombinant plasmids encoding either the full-length glycoprotein D (FLgD) or truncated gDs were constructed . The recombinant plasmids were expressed in Escherichia coli and BHK-21 cells . The strongest expression was obtained with the recombinant plasmid encoding a truncated gD which corresponded to the gD ectodomain . The cells transformed with this plasmid showed good exponential growth ensuring satisfactory yields of the expressed polypeptide in the form of the fusion protein . The fusion protein was biotinylated and efficiently purified . The shortest truncated gD, which contained the main continuous antigenic locus VII binding neutralization antibody and additional continuous antibody binding epitopes, still reacted with specific antibody as proven by immunoblot analysis . In addition, a shuttle vector for expression of FLgD in mammalian cells was constructed . This vector-transfected BHK-21 cells expressed gD for 40 days during 9 consecutive passages . The expression of gD began on day 2 and culminated at day 9 post transfection (p.t.).

J Mol Evol, 2004 Jun, 58(6), 712 - 21
Presequence acquisition during secondary endocytobiosis and the possible role of introns; Kilian O et al.; Targeting of nucleus-encoded proteins into chloroplasts is mediated by N-terminal presequences . During evolution of plastids from formerly free-living cyanobacteria by endocytobiosis, genes for most plastid proteins have been transferred from the plastid genome to the nucleus and subsequently had to be equipped with such plastid targeting sequences . So far it is unclear how the gene domains coding for presequences and the respective mature proteins may have been assembled . While land plant plastids are supposed to originate from a primary endocytobiosis event (a prokaryotic cyanobacterium was taken up by a eukaryotic cell), organisms with secondary plastids like diatoms experienced a second endocytobiosis step involving a eukaryotic alga taken up by a eukaryotic host cell . In this group of algae, apparently most genes encoding chloroplast proteins have been transferred a second time (from the nucleus of the endosymbiont to the nucleus of the secondary host) and thus must have been equipped with additional targeting signals . We have analyzed cDNAs and the respective genomic DNA fragments of seven plastid preproteins from the diatom Phaeodactylum tricornutum . In all of these genes we found single spliceosomal introns, generally located within the region coding for the N-terminal plastid targeting sequences or shortly downstream of it . The positions of the introns can be related to the putative phylogenetic histories of the respective genes, indicating that the bipartite targeting sequences in these secondary algae might have evolved by recombination events via introns.

J Mol Evol, 2004 Jun, 58(6), 692 - 700
Cytosine methylation is not the major factor inducing CpG dinucleotide deficiency in bacterial genomes; Wang Y et al.; CpG dinucleotide deficiency has been found in viruses, mitochondria, prokaryotes, and eukaryotes . The consensual explanation is that it is due to deamination of methylated cytosines, as established for vertebrate and plants . However, we still do not know whether C5 cytosine methylation is also the major cause of CpG deficiency in bacteria . By combining annotation and experimental data identifying the presence of C5 cytosine methyltransferases with analysis of CpG relative abundance in 67 bacterial species, we found that CpG relative abundance in most bacterial genomes that have cytosine C5 methyltransferases tends to be in the normal range (observed/expected values between 0.82 and 1.21) . In contrast, many bacterial species likely to be lacking C5 cytosine methylation showed CpG deficiency . Furthermore, when comparing genomes with one another, TpG and CpA relative abundances were found to be independent from CpG relative abundance . This contrasted with intragenome analyses, where C3pG1 relative abundance (the subscripts refer to position of a nucleotide in a codon) was found to be generally positively correlated with T3pG1 relative abundances when plotted against GC content in protein coding sequences (CDSs) . This suggests the existence of alternative mechanisms contributing to CpG deficiency in bacteria.

Nat Rev Mol Cell Biol, 2004 Oct, 5(10), 781 - 91
Pathways of chaperone-mediated protein folding in the cytosol; Young JC et al.; Cells are faced with the task of folding thousands of different polypeptides into a wide range of conformations . For many proteins, the folding process requires the action of molecular chaperones . In the cytosol of prokaryotic and eukaryotic cells, molecular chaperones of different structural classes form a network of pathways that can handle substrate polypeptides from the point of initial synthesis on ribosomes to the final stages of folding.

J Microbiol, 2004 Sep, 42(3), 200 - 4
A proteomic approach to study msDNA function in Escherichia coli; Jeong MA et al.; Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase . It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation . In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis . Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS . Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources . One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation . The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive .

Nucleic Acids Res . 2004 Sep 29;32(17):e133.
Osprey: a comprehensive tool employing novel methods for the design of oligonucleotides for DNA sequencing and microarrays; Gordon PM et al.; We have developed a software package called Osprey for the calculation of optimal oligonucleotides for DNA sequencing and the creation of microarrays based on either PCR-products or directly spotted oligomers . It incorporates a novel use of position-specific scoring matrices, for the sensitive and specific identification of secondary binding sites anywhere in the target sequence . Using accelerated hardware is faster and more efficient than the traditional pairwise alignments used in most oligo-design software . Osprey consists of a module for target site selection based on user input, novel utilities for dealing with problematic sequences such as repeats, and a common code base for the identification of optimal oligonucleotides from the target list . Overall, these improvements provide a program that, without major increases in run time, reflects current DNA thermodynamics models, improves specificity and reduces the user's data preprocessing and parameterization requirements . Using a TimeLogic hardware accelerator, we report up to 50-fold reduction in search time versus a linear search strategy . Target sites may be derived from computer analysis of DNA sequence assemblies in the case of sequencing efforts, or genome or EST analysis in the case of microarray development in both prokaryotes and eukaryotes.

Biochem J, 2005 Feb 1, 385(Pt 3), 703 - 13
The endo-beta-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours; Jam M et al.; Two beta-agarase genes, agaA and agaB, were functionally cloned from the marine bacterium Zobellia galactanivorans . The agaA and agaB genes encode proteins of 539 and 353 amino acids respectively, with theoretical masses of 60 and 40 kDa . These two beta-agarases feature homologous catalytic domains belonging to family GH-16 . However, AgaA displays a modular architecture, consisting of the catalytic domain (AgaAc) and two C-terminal domains of unknown function which are processed during secretion of the enzyme . In contrast, AgaB is composed of the catalytic module and a signal peptide similar to the N-terminal signature of prokaryotic lipoproteins, suggesting that this protein is anchored in the cytoplasmic membrane . Gel filtration and electrospray MS experiments demonstrate that AgaB is a dimer in solution, while AgaAc is a monomeric protein . AgaAc and AgaB were overexpressed in Escherichia coli and purified to homogeneity . Both enzymes cleave the beta-(1-->4) linkages of agarose in a random manner and with retention of the anomeric configuration . Although they behave similarly towards liquid agarose, AgaAc is more efficient than AgaB in the degradation of agarose gels . Given these organizational and catalytic differences, we propose that, reminiscent of the agarolytic system of Pseudoalteromonas atlantica, AgaA is specialized in the initial attack on solid-phase agarose, while AgaB is involved with the degradation of agarose fragments.

Anal Chem, 2004 Oct 1, 76(19), 5930 - 6
Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutations in DNA; Palecek E et al.; MutS protein plays an important role in the DNA repair system in prokaryotic and eukaryotic cells; it recognizes unpaired and mispaired bases in duplex DNA and can be used for detection of point mutations in vitro . We have shown that small amounts of this protein can be detected electrochemically at mercury and carbon electrodes without any labeling . Using constant current stripping analysis (CPSA) and mercury electrodes, tens of attomoles of this protein can be detected . The sensitivity of the determination at carbon electrodes is by more than 3 orders of magnitude lower . Using biotinylated DNA duplexes attached to magnetic beads, single-base mismatches and insertion/deletions were recognized by MutS . Picogram amounts of this protein were detected by CPSA after MutS releasing from the beads.

J Med Chem, 2004 Oct 7, 47(21), 5149 - 58
Inhibition of isoprene biosynthesis pathway enzymes by phosphonates, bisphosphonates, and diphosphates; Cheng F et al.; We have investigated the docking of a variety of inhibitors and substrates to the isoprene biosynthesis pathway enzymes farnesyl diphosphate synthase (FPPS), isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IPPI) and deoxyxylulose-5-phosphate reductoisomerase (DXR) using the Lamarckian genetic alogorithm program, AutoDock . The docked ligand structures are predicted with a approximately 0.8 A rms deviation from the structures determined crystallographically . The errors found are a function of the number of atoms in the ligand (R = 0.91, p < 0.0001) and, to a lesser extent, on the resolution of the crystallographic structure (R = 0.70, p < 0.008) . The structures of three isoprenoid diphosphates docked to the FPPS enzyme reveal strong electrostatic interactions with Mg(2+), lysine and arginine active site residues . Similar results are obtained with the docking of four IPPI inhibitors to the IPPI enzyme . The DXR substrate, deoxyxylulose-5-phosphate, is found to dock to Mn(2+)-NADPH-DXR in an almost identical manner as does the inhibitor fosimdomycin to Mn(2+)-DXR (ligand heavy atom rms deviation = 0.90 A) and is poised to interact with NADPH . Bisphosphonate inhibitors are found to bind to the allylic binding sites in both eukaryotic and prokaryotic FPPSs, in good accord with recent crystallographic results (a 0.4 A rms deviation from the X-ray structure with the E . coli enzyme) . Overall, these results show for the first time that the geometries of a broad variety of phosphorus-containing inhibitors and substrates of isoprene biosynthesis pathway enzymes can be well predicted by using computational methods, which can be expected to facilitate the design of novel inhibitors of these enzymes.

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 117 - 23
Abundance of 4Fe-4S motifs in the genomes of methanogens and other prokaryotes; Major TA et al.; The abundance of 4Fe-4S motifs of the form CX2CX2CX3C was analyzed in the open reading frames (ORFs) of 120 prokaryotic genomes . The abundance of ORFs containing the CX2CX2CX3C motif or isORFs correlated (r=0.82) with methanogenesis (p=0.0001), archaea (p=0.0173), anaerobiosis (p<0.0001) and genome size (p<0.0001) . Optimal growth temperature (hyperthermophily) did not correlate with the number of isORFs (p=0.6283) . Large numbers of CX2CX2CX3C motifs may be associated with unique physiologies: methanogenic archaea contained the greatest number of CX2CX2CX3C motifs found among the prokaryotic groups; however, only about 15% of the motifs were in genes directly involved in methanogenesis . Large numbers of CX2CX2CX3C motifs may also be associated with generalists such as Desulfitobacterium hafniense, which is an anaerobic bacterium containing multiple reductases.

Dev Comp Immunol, 2005, 29(2), 143 - 52
Cloning and characterization of chicken stromal cell derived factor-1; Read LR et al.; Stromal cell derived factor-1, SDF-1, belongs to the CXC family of chemokines and has been identified in mammals, amphibians, and fish . This chemokine has a diverse array of functions in organogenesis, hematopoeisis, B cell development and recruitment of immune system cells . Here, we report the cloning of the chicken SDF-1 ortholog and examine its temporal and spatial expression . The chicken SDF-1 cDNA contained an open reading frame encoding a predicted protein of 89 amino acids, which shared 40-75% identity to SDF-1 protein in other species . Protein folding simulation predicted a tertiary structure very similar to that obtained for human SDF-1 . Recombinant chicken SDF-1 was produced using a prokaryotic expression system and the recombinant protein was shown to be biologically active in a calcium flux assay . The SDF-1 gene was found to be expressed ubiquitously and constitutively in adult tissues and was present as early as the primitive streak stage of chicken embryos.

Trends Biochem Sci, 2004 Oct, 29(10), 535 - 41
A common folding mechanism in the cytochrome c family; Travaglini-Allocatelli C et al.; Of the globular proteins, cytochrome c (cyt c) has been used extensively as a model system for folding studies . Here we analyse the folding pathway of different cyt c proteins from prokaryotes and eukaryotes, and attempt to single out general correlations between structural determinants and folding mechanisms . Recent studies provide evidence that the folding pathway of several cyt c proteins involves the formation of a partially structured intermediate . Using state-of-the-art kinetic analysis on published data, we show that such a folding intermediate is an obligatory on-pathway species that might represent either a defined local minimum in the reaction coordinate or an unstable high-energy state . Available data also indicate that some essential structural features of the folding intermediate and transition states are highly conserved across this protein family . Thus, cyt c proteins share a consensus folding mechanism in spite of large differences in physico-chemical properties and thermodynamic stability . This novel outlook on the folding of cyt c can shed light on much published data and might offer a general scheme by which to plan new experiments.

Biol Chem, 2004 Aug, 385(8), 749 - 54
Distinctive functional features in prokaryotic and eukaryotic Cu,Zn superoxide dismutases; Gabbianelli R et al.; Bacterial and eukaryotic Cu,Zn superoxide dismutases show remarkable differences in the active site region and in their quaternary structure organization . We report here a functional comparison between four Cu,Zn superoxide dismutases from Gram-negative bacteria and the eukaryotic bovine enzyme . Our data indicate that bacterial dimeric variants are characterized by catalytic rates higher than that of the bovine enzyme, probably due to the solvent accessibility of their active site . Prokaryotic Cu,Zn superoxide dismutases also show higher resistance to hydrogen peroxide inactivation and lower HCO3- -dependent peroxidative activity . Moreover, unlike the eukaryotic enzyme, all bacterial variants are susceptible to inactivation by chelating agents and show variable sensitivity to proteolytic attack, with the E . coli monomeric enzyme showing higher rates of inactivation by EDTA and proteinase K . We suggest that differences between individual bacterial variants could be due to the influence of modifications at the dimer interface on the enzyme conformational flexibility.

Mol Vis, 2004 Sep 08, 10, 655 - 62
The IXI/V motif in the C-terminal extension of alpha-crystallins: alternative interactions and oligomeric assemblies; Pasta SY et al.; PURPOSE: Alpha-crystallin, a hetero-oligomer of alphaA- and alphaB-crystallin, is involved in maintaining eye lens transparency, primarily by its structural packing and chaperone activity . alphaA- and alphaB-crystallin share significant sequence homology, which is almost exclusively restricted to the central, conserved "alphaA-crystallin domain" . The flanking N-terminal domain and C-terminal extension are highly variable both in sequence and length . Mutations and age-related post-translational modifications of these proteins are associated with congenital and age-onset cataracts . Interestingly, most mutations or truncations in the C-terminal extensions of the alpha-crystallins and other alpha-sHsps like Hsp27 lead to pathology . It is therefore important to understand the structure/function relationship of this region . Sequence alignment of the C-terminal extensions of alphaA- and alphaB-crystallin with other homologues shows a conserved IXI/V motif . The purpose of this study was to investigate the role of this conserved motif, IPV in alphaA-crystallin and IPI in alphaB-crystallin (corresponding to residues 159-161 in both crystallins), in the structure and chaperone activity . METHODS: The isoleucine/valine residues in the IPV motif of alphaA-crystallin and the IPI motif of alphaB-crystallin were mutated to glycine and studied the secondary and tertiary structure of the mutant proteins using circular dichroism and fluorescence spectroscopy, and the quaternary structure using glycerol density gradient centrifugation and dynamic light scattering . Chaperone activity was studied at 37 degrees C and 25 degrees C using DTT induced aggregation of insulin as a model system . We have performed fluorescence resonance energy transfer (FRET) experiments to investigate the interactions of this motif in homo- and hetero-oligomers . Since alphaB-crystallin is devoid of Cys residues, we have introduced a Cys residue flanking the IPI motif (T162CalphaB-crystallin) to facilitate fluorescence labeling studies . RESULTS: Unlike in other homologues from plants or prokaryotes, mutation of the isoleucine/valine residues in alpha-crystallins does not result in oligomer dissociation or loss of chaperone activity . On the contrary, the mutant proteins retain their capacity to oligomerize and show enhanced chaperone activity at 37 degrees C . The mutants also exhibit significantly higher chaperone-like activity at 25 degrees C . FRET experiments show that the region spanning the IPI/V motif comes in proximity either to the beta-strands of the "alpha-crystallin" domain or the corresponding IPI/V region of another subunit . CONCLUSIONS: Our mutational studies show that the IPI/V motif has a propensity to participate in inter-subunit interactions, either with regions in the alpha-crystallin domain or with the corresponding IPI/V region on another monomer . These interactions are important in the structure and function of alpha-crystallins . This motif also appears to be important in the temperature dependent chaperone-like activity of the alpha-crystallins . The propensity of the IPI/V motif to form multiple inter-subunit interactions may contribute to the diversity in structure and function seen in the alpha-crystallin/sHsp family.

Nucleic Acids Res, 2004 Sep 24, 32(17), 5036 - 44 Print 2004.
Solving the riddle of codon usage preferences: a test for translational selection; dos Reis M et al.; Translational selection is responsible for the unequal usage of synonymous codons in protein coding genes in a wide variety of organisms . It is one of the most subtle and pervasive forces of molecular evolution, yet, establishing the underlying causes for its idiosyncratic behaviour across living kingdoms has proven elusive to researchers over the past 20 years . In this study, a statistical model for measuring translational selection in any given genome is developed, and the test is applied to 126 fully sequenced genomes, ranging from archaea to eukaryotes . It is shown that tRNA gene redundancy and genome size are interacting forces that ultimately determine the action of translational selection, and that an optimal genome size exists for which this kind of selection is maximal . Accordingly, genome size also presents upper and lower boundaries beyond which selection on codon usage is not possible . We propose a model where the coevolution of genome size and tRNA genes explains the observed patterns in translational selection in all living organisms . This model finally unifies our understanding of codon usage across prokaryotes and eukaryotes . Helicobacter pylori, Saccharomyces cerevisiae and Homo sapiens are codon usage paradigms that can be better understood under the proposed model.

J Biol Chem, 2004 Nov 5, 279(45), 47076 - 80 Epub 2004 Sep 23.
Functional characterization of a prokaryotic Kir channel; Enkvetchakul D et al.; The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells . A related gene family (KirBac) has recently been identified in prokaryotes . While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products . Here we present functional characterization of KirBac1.1 reconstituted in liposomes . Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+ -selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions . In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa approximately 6) . This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1895 - 902
A proposal for further integration of the cyanobacteria under the Bacteriological Code; Oren A; This taxonomic note reviews the present status of the nomenclature of the cyanobacteria under the Bacteriological Code . No more than 13 names of cyanobacterial species have been proposed so far in the International Journal of Systematic and Evolutionary Microbiology (IJSEM)/International Journal of Systematic Bacteriology (IJSB), and of these only five are validly published . The cyanobacteria (Cyanophyta, blue-green algae) are also named under the Botanical Code, and the dual nomenclature system causes considerable confusion . This note calls for a more intense involvement of the International Committee on Systematics of Prokaryotes (ICSP), its Judicial Commission and its Subcommittee on the Taxonomy of Photosynthetic Prokaryotes in the nomenclature of the cyanobacteria under the Bacteriological Code . The establishment of minimal standards for the description of new species and genera should be encouraged in a way that will be acceptable to the botanical authorities as well . This should be followed by the publication of an 'Approved List of Names of Cyanobacteria' in IJSEM . The ultimate goal is to achieve a consensus nomenclature that is acceptable both to bacteriologists and to botanists, anticipating the future implementation of a universal 'Biocode' that would regulate the nomenclature of all organisms living on Earth.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1427 - 8
Notification that new names and new combinations have appeared in volume 54, part 3, of the IJSEM; Cyanobacterial genes transmitted to the nucleus before divergence of red algae in the Chromista; Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan . nozaki@biol.s.u-tokyo.ac.jp

The plastids of red algae, green plants, and glaucophytes may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis . In contrast, the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events involving a phototrophic eukaryote and a eukaryotic host cell . Although phylogenetic analyses of multiple plastid genes from a wide range of eukaryotic lineages have been carried out, the phylogenetic positions of the secondary plastids of the Chromista (Heterokontophyta, Haptophyta and Cryptophyta) are ambiguous in a range of different analyses . This ambiguity may be the result of unusual substitutions or bias in the plastid genes established by the secondary endosymbiosis . In this study, we carried out phylogenetic analyses of five nuclear genes of cyanobacterial origin (6-phosphogluconate dehydrogenase {gnd}, oxygen-evolving-enhancer {psbO}, phosphoglycerate kinase {pgk}, delta-aminolevulinic acid dehydratase {aladh}, and ATP synthase gamma {atpC} genes), using the genome sequence data from the primitive red alga Cyanidioschyzon merolae 10D . The sequence data robustly resolved the origin of the cyanobacterial genes in the nuclei of the Chromista (Heterokontophyta and Haptophyta) and Dinophyta, before the divergence of the extant red algae (including Porphyra {Rhodophyceae} and Cyanidioschyzon {Cyadidiophyceae}) . Although it is likely that gnd genes in the Chromista were transmitted from the cyanobacterium-like ancestor of plastids in the primary endosymbiosis, other genes might have been transferred from nuclei of a red algal ancestor in the secondary endosymbiosis . Therefore, the results indicate that the Chromista might have originated from the ancient secondary endosymbiosis before the divergence of extant red algae.

J Mol Evol, 2004 Jul, 59(1), 59 - 71
Comparative analysis of the ribosomal components of the hydrogenosome-containing protist, Trichomonas vaginalis; Arisue N et al.; The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes . In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized . The sedimentation coefficient of the T . vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli . Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80 . This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E . coli (approximately 55) . N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features . Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T . vaginalis sequences are positioned within a eukaryotic clade . Comparison of deduced secondary structure models of the small and large subunit rRNAs of T . vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T . vaginalis, while variable regions are shortened or lost . These lines of evidence demonstrate that the T . vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.

J Mol Evol, 2004 Jul, 59(1), 51 - 8
New aspects on lanosterol 14alpha-demethylase and cytochrome P450 evolution: lanosterol/cycloartenol diversification and lateral transfer; Rezen T et al.; Sterol 14alpha-demethylase (CYP51) is a member of the cytochrome P450 superfamily, widely found in animals, fungi, and plants but present in few prokaryotic groups . CYP51 is currently believed to be the ancestral cytochrome P450 that has been transferred from prokaryotes to eukaryotic kingdoms . We propose an alternate view of CYP51 evolution that has an impact on understanding the evolution of the entire CYP superfamily . Two hundred forty-nine bacterial and four archaeal CYP sequences have been aligned and a bacterial CYP tree designed, showing a separation of two branches . Prokaryotic CYP51s cluster to the minor branch, together with other eukaryote-like CYPs . Mycobacterial and methylococcal CYP51s cluster together (100% bootstrap probability), while Streptomyces CYP51 remains on a distant branch . A CYP51 phylogenetic tree has been constructed from 44 sequences resulting in a ((plant, bacteria),(animal, fungi)) topology (100% bootstrap probability) . This is in accordance with the lanosterol/cycloartenol diversification of sterol biosynthesis . The lanosterol branch (nonphotosynthetic lineage) follows the previously proposed topology of animal and fungal orthologues (100% bootstrap probability), while plant and D . discoideum CYP51s belong to the cycloartenol branch (photosynthetic lineage), all in accordance with biochemical data . Bacterial CYP51s cluster within the cycloartenol branch (69% bootstrap probability), which is indicative of a lateral gene transfer of a plant CYP51 to the methylococcal/mycobacterial progenitor, suggesting further that bacterial CYP51s are not the oldest CYP genes . Lateral gene transfer is likely far more important than hitherto thought in the development of the diversified CYP superfamily . Consequently, bacterial CYPs may represent a mixture of genes with prokaryotic and eukaryotic origin.

IEEE Trans Nanobioscience, 2003 Mar, 2(1), 29 - 34
Parallel pattern identification in biological sequences on clusters; Huang CH et al.; Tandem repeats are ubiquitous sequence features in both prokaryotic and eukaryotic genomes . They are known to cause several inherited neurological diseases in humans . Identifying these patterns is a highly computation-intensive process . Previous parallel implementations use straightforward domain decomposition based on existing sequential algorithms and rely on parallel machines with low-latency interconnection network and fast hardware support for processor synchronization . Our research exploits the superior cost effectiveness and flexibility achieved through low-cost clusters to speed up biological computations by designing communication-efficient parallel algorithms for pattern identification . This paper presents a low communication-overhead parallel algorithm for pattern identification in biological sequences . Given a biological sequence of length n and a pattern of length m, we conclude an algorithm with five computation/communication phases, each requiring O(n) computation time and only O(p) message units . The low communication overhead of the algorithm is essential in achieving reasonable speedups on clusters, where the inter-processor communication latency is usually higher.

Physiology (Bethesda), 2004 Oct, 19, 293 - 9
A two-holed story: structural secrets about ClC proteins become unraveled?
Babini E, Pusch M.
ClC Cl(-) channels are found in almost all organisms, ranging from bacteria to mammals, in which nine Cl(-) channels belonging to the ClC family have been identified . The biophysical properties and physiological functions of ClC Cl(-) channels have been extensively reviewed . In this short review, we will focus on recent results obtained on the X-ray structure and functional properties of the prokaryotic ClC-ec1 protein and some results obtained on the role of the cytoplasmic COOH terminus of mammalian ClCs.

J Mol Biol, 2004 Oct 8, 343(1), 249 - 65
Inventory and comparative analysis of rice and Arabidopsis ATP-binding cassette (ABC) systems; Garcia O et al.; ATP-binding cassette (ABC) proteins constitute a large superfamily found in all kingdoms of living organisms . The recent completion of two draft sequences of the rice (Oryza sativa) genome allowed us to analyze and classify its ABC proteins and to compare to those in Arabidopsis thaliana . We identified a similar number of ABC proteins in rice and Arabidopsis (121 versus 120), despite the rice genome being more than three times the size of Arabidopsis . Both Arabidopsis and rice have representative members in all seven major subfamilies of ABC ATPases (A to G) commonly found in eukaryotes . This comparative analysis allowed the detection of 29 potential orthologous sequences in Arabidopsis and rice . However, plant share with prokaryotes a specific set of ABC systems that is not detected in animals . These ABC systems might be inherited from the cyanobacterial ancestor of chloroplasts . The present work provides the first complete inventory of rice ABC proteins and an updated inventory of those proteins in Arabidopsis.

J Mol Biol, 2004 Oct 8, 343(1), 1 - 28
STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer; Leipe DD et al.; Using sequence profile analysis and sequence-based structure predictions, we define a previously unrecognized, widespread class of P-loop NTPases . The signal transduction ATPases with numerous domains (STAND) class includes the AP-ATPases (animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators) and NACHT NTPases (e.g . NAIP, TLP1, Het-E-1) that have been studied extensively in the context of apoptosis, pathogen response in animals and plants, and transcriptional regulation in bacteria . We show that, in addition to these well-characterized protein families, the STAND class includes several other groups of (predicted) NTPase domains from diverse signaling and transcription regulatory proteins from bacteria and eukaryotes, and three Archaea-specific families . We identified the STAND domain in several biologically well-characterized proteins that have not been suspected to have NTPase activity, including soluble adenylyl cyclases, nephrocystin 3 (implicated in polycystic kidney disease), and Rolling pebble (a regulator of muscle development); these findings are expected to facilitate elucidation of the functions of these proteins . The STAND class belongs to the additional strand, catalytic E division of P-loop NTPases together with the AAA+ ATPases, RecA/helicase-related ATPases, ABC-ATPases, and VirD4/PilT-like ATPases . The STAND proteins are distinguished from other P-loop NTPases by the presence of unique sequence motifs associated with the N-terminal helix and the core strand-4, as well as a C-terminal helical bundle that is fused to the NTPase domain . This helical module contains a signature GxP motif in the loop between the two distal helices . With the exception of the archaeal families, almost all STAND NTPases are multidomain proteins containing three or more domains . In addition to the NTPase domain, these proteins typically contain DNA-binding or protein-binding domains, superstructure-forming repeats, such as WD40 and TPR, and enzymatic domains involved in signal transduction, including adenylate cyclases and kinases . By analogy to the AAA+ ATPases, it can be predicted that STAND NTPases use the C-terminal helical bundle as a "lever" to transmit the conformational changes brought about by NTP hydrolysis to effector domains . STAND NTPases represent a novel paradigm in signal transduction, whereby adaptor, regulatory switch, scaffolding, and, in some cases, signal-generating moieties are combined into a single polypeptide . The STAND class consists of 14 distinct families, and the evolutionary history of most of these families is riddled with dramatic instances of lineage-specific expansion and apparent horizontal gene transfer . The STAND NTPases are most abundant in developmentally and organizationally complex prokaryotes and eukaryotes . Transfer of genes for STAND NTPases from bacteria to eukaryotes on several occasions might have played a significant role in the evolution of eukaryotic signaling systems.

Int J Biochem Cell Biol, 2005 Jan, 37(1), 54 - 68
TB tools to tell the tale-molecular genetic methods for mycobacterial research; Machowski EE et al.; In spite of the availability of drugs and a vaccine, tuberculosis--one of man's medical nemeses--remains a formidable public health problem, particularly in the developing world . The persistent nature of the tubercle bacillus, with one third of the world's population is estimated to be infected, combined with the emergence of multi drug-resistant strains and the exquisite susceptibility of HIV-positive individuals, has underscored the urgent need for in-depth study of the biology of Mycobacterium tuberculosis address the resurgence of TB . In aiming to understand the mechanisms by which mycobacteria react to their immediate environments, molecular genetic tools have been developed from naturally occurring genetic elements . These include protein expressing genes, and episomal and integrating elements, which have been derived mainly from prokaryotic but also from eukaryotic organisms . Molecular genetic tools that had been established as routine procedures in other prokaryotic genera were thus mimicked . Knowledge of the underlying mechanisms greatly expedited the harnessing of these elements for mycobacteriological research and has brought us to a point where these molecular genetic tools are now employed routinely in laboratories worldwide.

FEMS Yeast Res, 2004 Oct, 5(1), 11 - 8
Therapeutic potential of yeast killer toxin-like antibodies and mimotopes; Magliani W et al.; This review focuses on the potential of yeast killer toxin (KT)-like antibodies (KTAbs), that mimic a wide-spectrum KT through interaction with specific cell wall receptors (KTR) and their molecular derivatives (killer mimotopes), as putative new tools for transdisease anti-infective therapy . KTAbs are produced during the course of experimental and natural infections caused by KTR-bearing micro-organisms . They have been produced by idiotypic vaccination with a KT-neutralizing mAb, also in their monoclonal and recombinant formats . KTAbs and KTAbs-derived mimotopes may exert a strong therapeutic activity against mucosal and systemic infections caused by eukaryotic and prokaryotic pathogenic agents, thus representing new potential wide-spectrum antibiotics.

Curr Biol, 2004 Sep 21, 14(18), R768 - 70
How do prokaryotic cells cycle?
Margolin W, Bernander R.
This issue of Current Biology features five reviews covering various key aspects of the eukaryotic cell cycle . The topics include initiation of chromosome replication, assembly of the mitotic spindle, cytokinesis, the regulation of cell-cycle progression, and cell-cycle modeling, focusing mainly on budding yeast, fission yeast and animal cell model systems . The reviews underscore common themes as well as key differences in the way these processes are carried out and regulated among the different model organisms . Consequently, an important question is how cell-cycle mechanisms and controls have evolved, particularly in the broader perspective of the three domains of life.

Mini Rev Med Chem, 2004 Sep, 4(7), 721 - 39
The relationship between inhibitors of eukaryotic and prokaryotic serine proteases; Konaklieva MI et al.; The ability to inhibit serine proteases is a major focus in the pharmaceutical industry . Serine proteases of medical importance range in phylogenetic diversity from the metallo-proteases, which play a role in pulmonary hypertension, and destruction of the lung parenchyma in emphysema, to those proteases (beta-lactamases), which play a role in the resistance of bacteria to beta-lactam antibiotics . In both the mammalian and microbial systems, the development of serine protease inhibitors has been a focal strategy spurring investigations in the area of serine protease dependent prodrugs that incorporate a bactericidal moiety as well as other classes of metalloprotease inhibitors.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2004 Aug, 26(4), 368 - 71
{Clone and expression of isocitrate lyase gene in Mycobacterium tuberculosis H37Rv}; Li DW et al.; OBJECTIVE: To construct recombinant plasmid with isocitrate lyase (ICL) gene of Mycobacterium tuberculosis H37Rv for stable and high level expression of ICL in prokaryotic expression system . METHODS: The recombinant plasmid with ICL gene (pET30 (a)-Rv0467) was constructed by polymerase chain reaction and cloning . The fusion protein was expressed in E . coli host strain BL21 (DE3) . Activity of the fusion protein was studied after it was purified with metal chelating chromatography . RESULTS: We constructed the plasmid which could highly express Mycobacterium tuberculosis H37Rv ICL . The plasmid was highly expressed in E . coli BL21 (DE3), in which the fusion protein accounted for 30% of total protein content . After having been purified by metal chelating chromatography, the purity of the soluble fusion protein was 90% . The fusion protein had activity of ICL . CONCLUSION: Using the prokaryotic expression system, the ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed, which build the basis for screening new anti-tuberculosis drugs with ICL as the target point.

Nat Rev Microbiol, 2004 Oct, 2(10), 802 - 8
Chlamydia and apoptosis: life and death decisions of an intracellular pathogen; Byrne GI et al.; The chlamydiae are important obligate intracellular prokaryotic pathogens that, each year, are responsible for millions of human infections involving the eye, genital tract, respiratory tract, vasculature and joints . The chlamydiae grow in cytoplasmic vesicles in susceptible host cells, which include the mucosal epithelium, vascular endothelium, smooth muscle cells, circulating monocytes and recruited or tissue-specific macrophages . One important pathogenic strategy that chlamydiae have evolved to promote their survival is the modulation of programmed cell death pathways in infected host cells . The chlamydiae can elicit the induction of host cell death, or apoptosis, under some circumstances and actively inhibit apoptosis under others . This subtle pathogenic mechanism highlights the manner in which these highly successful pathogens take control of infected cells to promote their own survival - even under the most adverse circumstances.

Bioinformatics . 2004 Sep 16; {Epub ahead of print}
Improving genome annotations using phylogenetic profile anomaly detection; Mikkelsen TS et al.; MOTIVATION: A promising strategy for refining genome annotations is to detect features that conflict with known functional or evolutionary relationships between groups of genes . Previous work in this area has been focused on investigating the absence of "housekeeping" genes or components of well-studied pathways . We have sought to develop a method for improving new annotations that can automatically synthesize and use the information available in a database of other annotated genomes . RESULTS: We show that a probabilistic model of phylogenetic profiles, trained from a database of curated genome annotations, can be used to reliably detect errors in new annotations . We use our method to identify 22 genes that were missed in previously published annotations of prokaryotic genomes . AVAILABILITY: The method was evaluated using MATLAB and open source software referenced in this work . Scripts and datasets are available from the authors upon request.

Arch Gerontol Geriatr, 1990 Mar-Apr, 10(2), 207 - 16
'Oxygen toxicity' induced by isoproterenol oxidation on living cells . An in vitro prokaryotic model; Giunta S et al.; Oxygen radical damage is a relevant problem in gerontological research . It has been implicated both in the aging process itself and in aging-related pathologies . Oxygen radicals from catecholamines seem to play an important role in central nervous system and cardiovascular system disorders during aging . Prokaryotic experimental systems have been shown to provide a simple and short term in vitro model for 'oxygen toxicity' from catecholamine oxidation which might be useful also in age-related research . In this paper we show that the synthetic sympathomimetic catecholamine, isoproterenol, oxidizes when added to bacteriological media and that this oxidation process causes bacterial growth inhibition . Both isoproterenol oxidation and the growth-inhibitory activity can be prevented by the addition of antioxidants . The addition of exogenous catalase (CAT), while unable to prevent isoproterenol oxidation, totally suppressed the bacterial growth inhibition; the addition of exogenous superoxide dismutase partially antagonized isoproterenol oxidation and suppressed bacterial growth inhibition although less efficiently than CAT . The model described suggests that besides 'oxygen toxicity' by endogenous natural catecholamines, iatrogenic tissue injury caused by the oxidation intermediates from this class of pharmacological compounds must also be considered.

J Biol Chem, 2004 Nov 12, 279(46), 47906 - 11 Epub 2004 Sep 14.
Lipopolysaccharide-free heat shock protein 60 activates T cells; Osterloh A et al.; A possible function of eukaryotic heat shock protein 60 (Hsp60) as endogenous danger signal has been controversially discussed in the past . Hsp60 was shown to induce the secretion of proinflammatory cytokines in professional antigen-presenting cells and to enhance the activation of T cells in primary stimulation . However, in vitro activation of macrophages by Hsp60 was attributed to contaminating endotoxin in the recombinant Hsp60 protein preparations . Here, we employ low endotoxin recombinant human Hsp60 and murine Hsp60 expressed by eukaryotic cell lines to dissect the Hsp60 protein-mediated effects from biologic effects that are mediated by prokaryotic contaminants in the Hsp60 protein preparation . The induction of tumor necrosis factor-alpha secretion in mouse macrophages is lost after endotoxin removal and is not mediated by Hsp60 expressed in eukaryotic systems . In contrast, the Hsp60-mediated enhancement of antigen-specific T cell activation does not correlate with endotoxin contamination . Moreover, Hsp60 that is expressed on the surface of different eukaryotic cell lines increases the activation of T cells in primary stimulation . Taken together, we provide evidence that endogenous Hsp60, which is thought to be released from dying infected cells in vivo, has a biological function that is not due to contaminating pathogen-associated molecules.

J Biol Chem, 2004 Nov 26, 279(48), 50437 - 45 Epub 2004 Nov 26.
Phage phi29 DNA replication organizer membrane protein p16.7 contains a coiled coil and a dimeric, homeodomain-related, functional domain; Munoz-Espin D et al.; The Bacillus subtilis phage varphi29-encoded membrane protein p16.7 is one of the few proteins known to be involved in prokaryotic membrane-associated DNA replication . Protein p16.7 contains an N-terminal transmembrane domain responsible for membrane localization . A soluble variant lacking the N-terminal membrane anchor, p16.7A, forms dimers in solution, binds to DNA, and has affinity for the varphi29 terminal protein . Here we show that the soluble N-terminal half of p16.7A can form a dimeric coiled coil . However, a second domain, located in the C-terminal half of the protein, has been characterized as being the main domain responsible for p16.7 dimerization . This 70-residue C-terminal domain, named p16.7C, also constitutes the functional part of the protein as it binds to DNA and terminal protein . Sequence alignments, secondary structure predictions, and spectroscopic analyses suggest that p16.7C is evolutionarily related to DNA binding homeodomains, present in many eukaryotic transcriptional regulator proteins . Based on the results, a structural model of p16.7 is presented.

Genes Dev, 2004 Sep 15, 18(18), 2183 - 94
The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease; Giaccia AJ et al.; The ability to sense and respond to changes in oxygen is essential for the survival of prokaryotic and eukaryotic organisms . Oxygen-sensing mechanisms have been developed to maintain cell and tissue homeostasis, as well as to adapt to the chronic low-oxygen conditions found in diseases such as cancer . This report on the first Keystone Meeting on the Biology of Hypoxia will summarize our current understanding of key genes and pathways involved in oxygen sensing that are required for normal development and that are dysregulated in disease states . It will also comment on future directions for this exciting field.

In Vivo, 2004 Jul-Aug, 18(4), 505 - 7
Effect of a trifluoromethyl ketone on the motility of proton pump-deleted mutant of Escherichia coli strain and its wild-type; Molnar A et al.; We have recently found that 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone {TF18} exhibited the most potent antibacterial activity among 30 trifluoromethyl ketones against various prokaryotes, such as Escherichia coli (E . coli) . In the present study, the inhibition of E . coli motility by TF18 was investigated . TF18 showed the lowest minimum inhibitory concentration (MIC) and highest inhibitory effect on the motility of E . coli strains . The wild-type E . coli was more sensitive to inhibition of motility than its proton pump-deleted mutant strain at subinhibitory concentrations . These data suggest that one of the targets of the antibacterial effect of the trifluoromethyl ketone is the proton pump system.

Plant Cell, 2004 Oct, 16(10), 2665 - 82 Epub 2004 Sep 14.
DNA gyrase is involved in chloroplast nucleoid partitioning; Cho HS et al.; DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes . Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana . NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar . NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles . Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles . Furthermore, 4',6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma . Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant . Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Sep, 20(5), 578 - 81
{Expression of GST-PRL-2 fusion protein in prokaryotic cells and preparation of Hen egg yolk immunoglobulin (IgY) against PRL-2}; Guo AL et al.; AIM: To express phosphatase 2 of regenerating liver (PRL-2) fusion protein in E.coli and to prepare specific hen egg yolk immunoglobulin(IgY) against PRL-2 . METHODS: A full-length human PRL-2 gene coding sequence was cloned into expression vectors pGEX-4T-2 and pET21a, then transformed into E.coli . Fusion protein GST-PRL-2 was expressed in E.coli via IPTG induction . The expressed proteins were purified from lysates through Glutathione Sepharose 4B and the Ni-NTA agarose columns, respectively . Egg-laying hens were immunized using the purified GST-PRL-2 with Freund's complete or incomplete adjuvant . The specificity of the resulting antibody was identified by Western blot . RESULTS: A high level of expression of target protein was detected by Western blot after IPTG induction and purified protein was obtained through Glutathione Sepharose 4B and the Ni-NTA affinity chromatography agarose columns, respectively . Western blot analysis showed that the anti-PRL-2 polyclonal antibody can recognize 6xHis-PRL-2 fusion protein . CONCLUSION: The hen egg yolk immunoglobulin(IgY) against PRL-2 expressed in E.coli has good specificity which provides an useful reagent for the detection of PRL-2.

Int Rev Cytol, 2004, 238, 59 - 118
Organelle nuclei in higher plants: structure, composition, function, and evolution; Sakai A et al.; Plant cells have two distinct types of energy-converting organelles: plastids and mitochondria . These organelles have their own DNAs and are regarded as descendants of endosymbiotic prokaryotes . The organelle DNAs associate with various proteins to form compact DNA-protein complexes, which are referred to as organelle nuclei or nucleoids . Various functions of organelle genomes, such as DNA replication and transcription, are performed within these compact structures . Fluorescence microscopy using the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole has played a pivotal role in establishing the concept of "organelle nuclei." This fluorochrome has also facilitated the isolation of morphologically intact organelle nuclei, which is indispensable for understanding their structure and composition . Moreover, development of an in vitro transcription?DNA synthesis system using isolated organelle nuclei has provided us with a means of measuring and analyzing the function of organelle nuclei . In addition to these morphological and biochemical approaches, genomics has also had a great impact on our ability to investigate the components of organelle nuclei . These analyses have revealed that organelle nuclei are not a vestige of the bacterial counterpart, but rather are a complex system established through extensive interaction between organelle and cell nuclear genomes during evolution . Extensive diversion or exchange during evolution is predicted to have occurred for several important structural proteins, such as major DNA-compacting proteins, and functional proteins, such as RNA and DNA polymerases, resulting in complex mechanisms to control the function of organelle genomes . Thus, organelle nuclei represent the most dynamic front of interaction between the three genomes (cell nuclear, plastid, and mitochondrial) constituting eukaryotic plant cells.

Gene, 2004 Sep 15, 339, 149 - 60
The origin and evolution of eucaryal HIS7 genes: from metabolon to bifunctional proteins?
Brilli M, Fani R.
The fifth step of histidine biosynthesis is catalysed by an imidazole glycerol-phosphate (IGP) synthase . In Archaea and Bacteria, the active form of IGP synthase is a stable 1:1 dimeric complex constituted by a glutamine amidotransferase (GAT) and a cyclase, the products of hisH and hisF . In Eucarya, the two activities are associated with a single bifunctional polypeptide encoded by HIS7 . In this work, we report a comparative analysis of the amino acid sequence of all the available HisH, HisF and HIS7 proteins, which allowed depicting a likely evolutionary pathway leading to the present-day bifunctional HIS7 genes . According to the model that we propose, the bifunctional HIS7 gene is the outcome of a gene fusion event between two independent ancestral cistrons encoding an amidotransferase and a cyclase, respectively . The phylogenetic distribution of the eucaryal HIS7 genes and the analysis of all the available prokaryotic counterparts (hisH and hisF) revealed the absence of such fusions in prokaryotes, suggesting that the fusion event very likely occurred in an early stage of eucaryal evolution and was fixed in the nucleated cells . The biological significance of this gene fusion is also discussed.

Trends Parasitol, 2004 Oct, 20(10), 462 - 8
Genomic filtering: an approach to discovering novel antiparasitics; McCarter JP; Genomic filtering is a rapid approach to identifying and prioritizing molecular targets for drug discovery . For infectious disease applications, comparative genomics filters allow the selection of pathogen-specific gene products, whereas functional genomics filters, such as RNA interference (RNAi), allow the selection of gene products essential for pathogen survival . The approach is especially applicable to antiparasitic drug discovery where the phylogenetic distance between parasite and host make the likelihood of drug cross-toxicity due to conservation of molecular targets greater than for more distantly related pathogens such as prokaryotes . This article discusses some of the inherent challenges of applying genomics to the early steps of drug discovery and describes one successful comparative and functional genomics filtering strategy that has been implemented to prioritize molecular targets and identify chemical leads for nematode control.

PLoS Biol . 2004 Sep;2(9):E277 . Epub 2004 Sep 07.
A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D-carboxypeptidase activity; Chang CI et al.; The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes . We have determined the crystal structure of PGRP-SA to 2.2-A resolution and analyzed its peptidoglycan (PG) recognition and signaling activities . We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs . Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation . Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity . As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.

J Biol Chem, 2004 Nov 26, 279(48), 49656 - 63 Epub 2004 Nov 26.
Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons; Tang HL et al.; Although initiation of translation at non-AUG codons occurs occasionally in prokaryotes and higher eukaryotes, it has not been reported in yeast until very recently . Evidence presented here shows that redundant ACG codons are recognized as alternative translation start sites for ALA1, the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase . ALA1 is shown to be a bifunctional gene that provides both cytoplasmic and mitochondrial activities . Unlike most bifunctional genes that contain alternative in-frame AUG initiators, there is only one AUG codon, designated AUG1, close to the 5'-end of the ALA1 open reading frame . Transcriptional mapping identified three overlapping transcripts, with 5'-ends at positions 54, 105, and 117 nucleotides upstream of AUG1, respectively . Site-specific mutagenesis demonstrated that the cytoplasmic and mitochondrial functions of ALA1 are provided by two protein isoforms with distinct amino termini; that is, a short cytoplasmic form initiated at AUG1 and a longer mitochondrial isoform initiated at two upstream in-frame ACG codons, i.e . ACG(-25) and ACG(-24) . These two ACG codons function redundantly in initiation of translation . Either codon can function in the absence of the other . The short transcript appears to serve as the template for the cytoplasmic form, whereas the longer transcripts are likely to code for both isoforms via alternative initiation . Because yeast ribosomes in general cannot efficiently recognize a non-AUG initiator, this unique feature of redundancy of non-AUG initiators in a single mRNA may in itself represent a novel paradigm for translation initiation from poor initiators.

FEBS Lett, 2004 Sep 10, 574(1-3), 101 - 5
A cyanobacterial gene encoding an ortholog of Pirin is induced under stress conditions; Hihara Y et al.; Pirin is a recently identified protein in eukaryotes as a transcription cofactor or as an apoptosis-related protein . Although Pirin is highly conserved from bacteria to human, there have been no reports on prokaryotic Pirin orthologs . We show here that pirA (sll1773) encoding an ortholog of Pirin together with an adjacent gene, pirB (ssl3389), was upregulated under high salinity and some other stress conditions in a cyanobacterium Synechocystis sp . PCC 6803 . Induction of the pirAB genes was not related to cell death and disruption of pirA did not affect the gene expression profile . Expression of the pirAB genes was negatively regulated by a LysR family transcriptional regulator encoded by pirR (slr1871) located immediately upstream of pirAB in the divergent direction . DNA microarray analysis indicated that PirR repressed expression of closely located ORFs, slr1870 and mutS (sll1772), in addition to pirAB and pirR itself.

Trends Plant Sci, 2004 Aug, 9(8), 371 - 7
The ALDH gene superfamily of Arabidopsis; Kirch HH et al.; Aldehyde dehydrogenases (ALDHs) represent a protein superfamily of NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes . The Arabidopsis genome contains 14 unique ALDH sequences encoding members of nine ALDH families, including eight known families and one novel family (ALDH22) that is currently known only in plants . Here, we identify members of the ALDH gene superfamily in Arabidopsis; provide a revised, unified nomenclature for these ALDH genes; analyze the molecular relationship among Arabidopsis ALDH genes and compare them to ALDH genes from other species, including prokaryotes and mammals; and describe the role of ALDHs in cytoplasmic male sterility, plant defense and abiotic stress tolerance.

BMC Bioinformatics . 2004 Sep 09;5(1):127.
Functionally specified protein signatures distinctive for each of the different blue copper proteins; Giri AV et al.; BACKGROUND: Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint . The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences . Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes . The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests . Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families . This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins . The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands {each from histidine} and one sulphur containing thiolate ligand {from cysteine}, with strong interactions between the copper center and these ligands . RESULTS: Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership . The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes . ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides . The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom . It was highly specific for each kind of blue copper protein and the false picks were minimized . The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section . CONCLUSIONS: These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences.

Plant Cell Physiol, 2004 Aug, 45(8), 960 - 7
ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase; Shimada H et al.; The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division . To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division . Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes . The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif . Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division . Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.

Mol Biol Evol, 2005 Jan, 22(1), 21 - 28 Epub 2004 Sep 8.
Unique Origin and Lateral Transfer of Prokaryotic Chlorophyll-b and Chlorophyll-d Light-Harvesting Systems; Chen M et al.; pcb genes, encoding proteins binding light-harvesting chlorophylls, were cloned and sequenced from the Chl d-containing cyanobacterium, Acaryochloris marina, and the Chl b-containing cyanobacterium, Prochloron didemni . Both organisms contained two tandem pcb genes . Peptide fingerprinting confirmed the expression of one of the A . marina pcb genes . Phylogenetic tree reconstruction using distance-matrix and maximum-likelihood methods indicated a single origin of the pcb gene family, whether occurring in Chl b-containing or Chl d-containing organisms . This may indicate widespread lateral transfer of the Pcb protein-based light-harvesting system.

Annu Rev Cell Dev Biol . 2004 Jul 2; {Epub ahead of print}
Cellular Length Control Systems; Marshall WF; The problem of organelle size control can be addressed most simply by considering cellular structures that are linear, so that their size can be defined by a single parameter: length . We compare existing studies on several linear biological structures including prokaryotic flagella and flagellar hooks, eukaryotic flagella, sarcomere thin filaments, and microvilli . In some cases, existing evidence strongly supports the idea that length control involves a molecular ruler, in which the size of the overall structure is compared with the size of an individual molecule . In other cases, length control is likely to involve a steady-state balance of assembly and disassembly, in which one or the other rate is inherently length dependent . The lessons learned from size control in linear structures should be applicable to organelles with more complex three-dimensional structures . Expected online publication date for the Annual Review of Cell and Developmental Biology Volume 20 is October 6, 2004 . Please see for revised estimates.

Annu Rev Cell Dev Biol . 2004 Jun 11; {Epub ahead of print}
New Developments in Mitochondrial Assembly; Koehler CM; The mitochondrion has developed an elaborate translocation system for the import of nuclear-coded proteins and the export of proteins coded on the mitochondrial genome . Precursor proteins contain targeting and sorting information to reach the mitochondrion, while the translocons recognize the information and direct the precursor to the correct compartment . The outer membrane contains the TOM (translocase of the outer membrane) complex for translocation and the SAM (sorting and assembly machinery) complex for assembly of outer membrane proteins with complex topologies . At the inner membrane, the TIM23 (translocase of the inner membrane) mediates the import of mitochondrial proteins with a typical N-terminal targeting sequence, and the TIM22 complex mediates the import of polytopic inner membrane proteins . Based on its prokaryotic origin, the inner membrane also contains several components that mediate the export and assembly of proteins from within the matrix . Together the translocation and assembly complexes coordinate assembly of the mitochondrion . Expected online publication date for the Annual Review of Cell and Developmental Biology Volume 20 is October 6, 2004 . Please see for revised estimates.

Annu Rev Genet . 2004 Aug 13; {Epub ahead of print}
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 . Expected online publication date for the Annual Review of Genetics Volume 38 is November 10, 2004 . Please see for revised estimates.

Annu Rev Microbiol . 2004 Mar 26; {Epub ahead of print}
Selection for Gene Clustering by Tandem Duplication; Reams AB et al.; In prokaryotic genomes, related genes are frequently clustered in operons and higher-order arrangements that reflect functional context . Organization emerges despite rearrangements that constantly shuffle gene and operon order . Evidence is presented that the tandem duplication of related genes acts as a driving evolutionary force in the origin and maintenance of clusters . Gene amplification can be viewed as a dynamic and reversible regulatory mechanism that facilitates adaptation to variable environments . Clustered genes confer selective benefits via their ability to be coamplified . During evolution, rearrangements that bring together related genes can be selected if they increase the fitness of the organism in which they reside . Similarly, the benefits of gene amplification can prevent the dispersal of existing clusters . Examples of frequent and spontaneous amplification of large genomic fragments are provided . The possibility is raised that tandem gene duplication works in concert with horizontal gene transfer as interrelated evolutionary forces for gene clustering . Expected online publication date for the Annual Review of Microbiology Volume 58 is September 8, 2004 . Please see for revised estimates.

Nucleic Acids Res, 2004 Sep 07, 32(16), 4725 - 31 Print 2004.
Gene co-regulation is highly conserved in the evolution of eukaryotes and prokaryotes; Snel B et al.; Differences between species have been suggested to largely reside in the network of connections among the genes . Nevertheless, the rate at which these connections evolve has not been properly quantified . Here, we measure the extent to which co-regulation between pairs of genes is conserved over large phylogenetic distances; between two eukaryotes Caenorhabditis elegans and Saccharomyces cerevisiae, and between two prokaryotes Escherichia coli and Bacillus subtilis . We first construct a reliable set of co-regulated genes by combining various functional genomics data from yeast, and subsequently determine conservation of co-regulation in worm from the distribution of co-expression values . For B.subtilis and E.coli, we use known operons and regulons . We find that between 76 and 80% of the co-regulatory connections are conserved between orthologous pairs of genes, which is very high compared with previous estimates and expectations regarding network evolution . We show that in the case of gene duplication after speciation, one of the two inparalogous genes tends to retain its original co-regulatory relationship, while the other loses this link and is presumably free for differentiation or sub-functionalization . The high level of co-regulation conservation implies that reliably predicted functional relationships from functional genomics data in one species can be transferred with high accuracy to another species when that species also harbours the associated genes.

Front Biosci, 2004 Sep 01, 9, 3262 - 7
A report on single exon genes (SEG) in eukaryotes; Sakharkar MK et al.; Single exon genes (SEG) are archetypical of prokaryotes . Hence, their presence in intron-rich, multi-cellular eukaryotic genomes is perplexing . Consequently, a study on SEG origin and evolution is important . Towards this goal, we took the first initiative of identifying and counting SEG in nine completely sequenced eukaryotic organisms--four of which are unicellular (E . cuniculi, S . cerevisiae, S . pombe, P . falciparum) and five of which are multi-cellular (C . elegans, A . thaliana, D . melanogaster, M . musculus, H . sapiens) . This exercise enabled us to compare their proportion in unicellular and multi-cellular genomes . The comparison suggests that the SEG fraction decreases with gene count (r = -0.80) and increases with gene density (r = 0.88) in these genomes . We also examined the distribution patterns of their protein lengths in different genomes.

Front Biosci, 2004 Sep 01, 9, 2964 - 71
Can ends justify the means? Digging deep for human fusion genes of prokaryotic origin; Yiting Y et al.; Gene fusion has been described as an important evolutionary phenomenon . This report focuses on identifying, analyzing, and tabulating human fusion proteins of prokaryotic origin . These fusion proteins are found to mimic operons, simulate protein-protein interfaces in prokaryotes, exhibiting multiple functions and alternative splicing in humans . The accredited biological functions for each of these proteins is made available as a database at http://sege.ntu.edu.sg/wester/fusion/

J Eukaryot Microbiol, 2004 Jul-Aug, 51(4), 394 - 401
The microtubule analog protein, FtsZ, in the endosymbiont of trypanosomatid protozoa; Motta MC et al.; Blastocrithidia culicis and Crithidia deanei are trypanosomatids that harbor an endosymbiotic bacterium in their cytoplasm . In prokaryotes, numerous proteins are essential for cell division, such as FtsZ, which is encoded by filament-forming temperature-sensitive (fts) genes . FtsZ is the prokaryotic homolog of eukaryotic tubulin and is present in bacteria and archaea, and has also been identified in mitochondria and chloroplasts . FtsZ plays a key role in the initiation of cytokinesis . It self-assembles into the Z ring, which establishes the division plane during septation . In this study, immunoblotting analysis using a FtsZ polyclonal antibody, revealed a 40-kDa band characteristic of FtsZ in endosymbiont fractions and in whole trypanosomatid homogenates, but not in whole cell extracts of aposymbiotic strains . Confocal microscopy and ultrastructural analysis revealed a specific and dispersed labeling over the endosymbiont . Bars and ring-like structures, which are suggestive of the presence of Z-rings, were never observed, even during the division of the symbiont . This peculiar distribution of FtsZ may represent an arrangement of cytoskeleton protein intermediate between prokaryotic and eukaryotic cells . The endosymbiont ftsz gene was completely sequenced after amplification of DNA from symbiont-bearing trypanosomatids or from pure endosymbiont fractions, using PCR and specific primers . The sequences obtained from the endosymbionts from C . deanei and B . culicis were very similar, and were most closely related to bacteria from the genus Pseudomonas.

Planta . 2004 Sep 4; {Epub ahead of print}
Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs; Romano A et al.; Medium-chain-length poly-3-( R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters . The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes {B . Rehm et al . (1998) J . Biol Chem 273:24044-24051} as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase . In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato ( Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment . Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column . The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines . We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.

Curr Opin Pharmacol, 2004 Oct, 4(5), 479 - 86
Structural understanding of efflux-mediated drug resistance: potential routes to efflux inhibition; McKeegan KS et al.; The active efflux of cytotoxic drugs mediated by multidrug transporters is the basis of multidrug resistance in prokaryotic and eukaryotic cells . Individual multidrug transporters can be extremely versatile, often exhibiting a staggering range of substrate specificity that can negate the effects of clinically relevant therapies . The effective treatment of bacterial, fungal and protozoan infections, along with certain cancer treatments, has been compromised by the presence of multidrug transporters . Traditionally, advances in the understanding of multidrug transporters have been made through biochemical analyses; more recently, however, fundamental advances have been made with the elucidation of several three dimensional structures of representative multidrug pumps . Biochemical and structural analysis of multidrug pumps could lead to the development of novel 'anti-efflux' therapies.

J Virol Methods, 2004 Oct, 121(1), 73 - 8
Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections; Rosati S et al.; Among animal lentiviruses, Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV) and Small ruminant lentiviruses (SRLV) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia . The detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life . Capsid antigen (CA) is the major viral core protein and specific antibodies against this antigen are usually first recognised in infected sheep, goat and horse, remaining detectable for long period . Transmembrane (TM) domain of envelope glycoprotein contains a well conserved motif known to form an immunodominant epitope in several lentiviruses . In this study a simple strategy was developed to express the entire CA and the TM epitope in a single fusion protein from equine, feline and small ruminant lentiviruses in prokaryotic system and evaluated the diagnostic utility of a purified preparation in an indirect ELISA for each of the three infections . Results demonstrate that, for FIV and SRLV infections, the combination of CA and TM fractions increases the sensitivity of diagnostic tests based only on CA . The corresponding CA/TM antigen from EIAV showed excellent agreement with Coggins test.

Biochemistry, 2004 Sep 14, 43(36), 11576 - 88
Photochromic biliproteins from the cyanobacterium Anabaena sp . PCC 7120: lyase activities, chromophore exchange, and photochromism in phytochrome AphA; Zhao KH et al.; Photochromic biliproteins can be switched by light between two states, initiated by Z/E photoisomerization of the linear tetrapyrrole chromophore . The cyanobacterium Anabaena sp . PCC 7120 contains three genes coding for such biliproteins, two coding for phytochromes (aphA/B) and one for the alpha subunit of phycoerythrocyanin (pecA) . (a) aphA was overexpressed in Escherichia coli with N-terminal His and S tags, and the protein was reconstituted by an optimized protocol with phycocyanobilin (PCB), to yield the photochromic chromoprotein, PCB-AphA, carrying the PCB chromophore . (b) AphA chromophorylation is autocatalytic such as in other phytochromes . (c) AphA chromophorylation is also possible by chromophore transfer from the PCB-carrying biliprotein, phycocyanin (CPC) . The autocatalytic transfer is very slow, and it is enhanced more than 100-fold by catalysis of PCB:CpcA lyase and alpha-CPC as donor . (d) Through deletion mutations of aphA, a short sequence IQPHGV {amino acids (aa) 26-31} was found essential for the lyase activity of AphA, indicating an interaction of the N terminus with the chromophore-binding domain around cysteine 259 . (e) A motif of at least 23 aa, starting with this sequence and located approximately 250 aa N terminal of the chromophore-binding cysteine, is proposed to relate to the lyase function in plant and most prokaryotic phytochromes . (f) Long-range interactions in AphA are further supported by blue-shifted absorptions (<or=12 nm) of both the Pr and Pfr forms of truncated chromoproteins.

Plant Physiol, 2004 Sep, 136(1), 2532 - 47 Epub 2004 Sep 03.
Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K+ homeostasis in pollen development; Sze H et al.; A combined bioinformatic and experimental approach is being used to uncover the functions of a novel family of cation/H(+) exchanger (CHX) genes in plants using Arabidopsis as a model . The predicted protein (85-95 kD) of 28 AtCHX genes after revision consists of an amino-terminal domain with 10 to 12 transmembrane spans (approximately 440 residues) and a hydrophilic domain of approximately 360 residues at the carboxyl end, which is proposed to have regulatory roles . The hydrophobic, but not the hydrophilic, domain of plant CHX is remarkably similar to monovalent cation/proton antiporter-2 (CPA2) proteins, especially yeast (Saccharomyces cerevisiae) KHA1 and Synechocystis NhaS4 . Reports of characterized fungal and prokaryotic CPA2 indicate that they have various transport modes, including K(+)/H(+) (KHA1), Na(+)/H(+)-K(+) (GerN) antiport, and ligand-gated ion channel (KefC) . The expression pattern of AtCHX genes was determined by reverse transcription PCR, promoter-driven beta-glucuronidase expression in transgenic plants, and Affymetrix ATH1 genome arrays . Results show that 18 genes are specifically or preferentially expressed in the male gametophyte, and six genes are highly expressed in sporophytic tissues . Microarray data revealed that several AtCHX genes were developmentally regulated during microgametogenesis . An exciting idea is that CHX proteins allow osmotic adjustment and K(+) homeostasis as mature pollen desiccates and then rehydrates at germination . The multiplicity of CHX-like genes is conserved in higher plants but is not found in animals . Only 17 genes, OsCHX01 to OsCHX17, were identified in rice (Oryza sativa) subsp . japonica, suggesting diversification of CHX in Arabidopsis . These results reveal a novel CHX gene family in flowering plants with potential functions in pollen development, germination, and tube growth.

Bioinformatics . 2004 Sep 3; {Epub ahead of print}
Genome properties: a system for the investigation of prokaryotic genetic content for microbiology, genome annotation and comparative genomics; Haft DH et al.; MOTIVATION: The presence or absence of metabolic pathways, and structures provides context that makes protein annotation far more reliable . Compiling such information across microbial genomes improves the functional classification of proteins and provides a valuable resource for comparative genomics . RESULTS: We have created Genome Properties to present key aspects of prokaryotic biology using standardized computational methods and controlled vocabularies . Properties reflect gene content, phenotype, phylogeny, and computational analyses . The results of searches using Hidden Markov Models (HMMs) allow many properties to be deduced automatically, especially for families of proteins (equivalogs) conserved in function since their last common ancestor . Additional properties are derived from curation, published reports, and other forms of evidence . Genome Properties has been applied to 156 complete prokaryotic genomes, and is easily mined to find differences between species, correlations between metabolic features and families of uncharacterized proteins, or relationships among properties . AVAILABILITY: Genome Properties can be found at http://www.tigr.org/Genome_Properties.

Acta Biochim Biophys Sin (Shanghai), 2004 Sep, 36(9), 589 - 96
Recombinant bivalent vaccine against foot-and-mouth disease virus serotype O/A infection in guinea pig; Yi JZ et al.; In this study, two DNA fragments encoding amino acid (141-160)-(21-140)-(141-160) of the VP1 of FMDV (foot-and-mouth disease virus) serotype O and (138-160)-(21-40)-(138-160) of the serotype A FMDV were chemically synthesized . These two tandem-repeat fragments were ligated and transfected into prokaryotic expression vector pTrcHis A to construct pTH-O-A . The other vector called pTH-O-scIgG-A was constructed similarly only that the two tandem-repeat DNA fragments were linked by the bovine-IgG heavy chain coding sequence . Guinea pigs immunized with the two bivalent vaccines pTH-O-A and pTH-O-scIgG-A showed both specific antibody activity and T cell proliferation responses . FMDV challenge tests showed that 85% and 70% of guinea pigs vaccinated twice with 200 mg of the fusion protein of pTH-O-A were protected from FMDV serotype O and serotype A infection respectively . 70% and 57% of the guinea pigs immunized with the fusion protein of pTH-O-scIgG-A were protected from FMDV serotype O and serotype A infection respectively.

Biophys J, 2004 Sep, 87(3), 1686 - 96
Exterior site occupancy infers chloride-induced proton gating in a prokaryotic homolog of the ClC chloride channel; Bostick DL et al.; The ClC family of anion channels mediates the efficient, selective permeation of Cl(-) across the biological membranes of living cells under the driving force of an electrochemical gradient . In some eukaryotes, these channels are known to exhibit a unique gating mechanism, which appears to be triggered by the permeant Cl(-) anion . We infer details of this gating mechanism by studying the free energetics of Cl(-) occupancy in the pore of a prokaryotic ClC homolog . These free energetics were gleaned from 30 ns of molecular dynamics simulation on an approximately 133,000-atom system consisting of a hydrated membrane embedded StClC transporter . The binding sites for Cl(-) in the transporter were determined for the cases where the putative gating residue, Glu(148), was protonated and unprotonated . When the glutamate gate is protonated, Cl(-) favorably occupies an exterior site, S(ext), to form a queue of anions in the pore . However, when the glutamate gate is unprotonated, Cl(-) cannot occupy this site nor, consequently, pass through the pore . An additional, previously undetected, site was found in the pore near the outer membrane that exists regardless of the protonation state of Glu(148) . Although this suggests that, for the prokaryotic homolog, protonation of Glu(148) may be the first step in transporting Cl(-) at the expense of H(+) transport in the opposite direction, an evolutionary argument might suggest that Cl(-) opens the ClC gate in eukaryotic channels by inducing the conserved glutamate's protonation . During an additional 20 ns free dynamics simulation, the newly discovered outermost site, S(out), and the innermost site, S(int), were seen to allow spontaneous exchange of Cl(-) ions with the bulk electrolyte while under depolarization conditions.

Antioxid Redox Signal, 2004 Oct, 6(5), 825 - 34
Bacterial heme oxygenases; Frankenberg-Dinkel N; The importance of heme oxygenases in heme catabolism, iron utilization, and cellular signaling has been recognized for many years and is a well studied process in eukaryotes . Through the accessibility of an increasing number of bacterial genomes, it has become evident that heme oxygenases are also widespread in prokaryotes . In these organisms, the heme oxygenase reaction serves a similar function as in eukaryotes . A major role of bacterial heme oxygenases has been attributed to acquisition of iron in prokaryotic pathogens, but other functions, such as involvement in phytobilin biosynthesis, have been described . This review summarizes the current state of the art on bacterial heme oxygenase research . The various biological roles of this enzyme in prokaryotes and their biochemical properties will be discussed.

Genome Biol . 2004;5(9):R64 . Epub 2004 Aug 26.
Comprehensive analysis of pseudogenes in prokaryotes: widespread gene decay and failure of putative horizontally transferred genes; Liu Y et al.; BACKGROUND: Pseudogenes often manifest themselves as disabled copies of known genes . In prokaryotes, it was generally believed (with a few well-known exceptions) that they were rare . RESULTS: We have carried out a comprehensive analysis of the occurrence of pseudogenes in a diverse selection of 64 prokaryote genomes . Overall, we find a total of around 7,000 candidate pseudogenes . Moreover, in all the genomes surveyed, pseudogenes occur in at least 1 to 5% of all gene-like sequences, with some genomes having considerably higher occurrence . Although many large populations of pseudogenes arise from large, diverse protein families (for example, the ABC transporters), notable numbers of pseudogenes are associated with specific families that do not occur that widely . These include the cytochrome P450 and PPE families (PF00067 and PF00823) and others that have a direct role in DNA transposition . CONCLUSIONS: We find suggestive evidence that a large fraction of prokaryote pseudogenes arose from failed horizontal transfer events . In particular, we find that pseudogenes are more than twice as likely as genes to have anomalous codon usage associated with horizontal transfer . Moreover, we found a significant difference in the number of horizontally transferred pseudogenes in pathogenic and non-pathogenic strains of Escherichia coli.

Environ Microbiol, 2004 Oct, 6(10), 1042 - 8
Intracellular manganese granules formed by a subsurface bacterium; Glasauer S et al.; The demonstrated ability of prokaryotes to form internal metal oxide particles during active metabolism has been restricted to Fe . Mineral-bound Mn(IV) is a known electron acceptor during dissimilatory metal reduction by Shewanella putrefaciens, yet no internal deposits of Mn have been reported to form during anaerobic respiration . We observed distinct nanometre-sized Mn-rich granules in the cytoplasm when either birnessite or pyrolusite (beta-MnO(2)) served as the electron acceptor during growth . During rapid Mn reduction, additional precipitates of Mn were also observed in the periplasm together with the cytoplasmic granules . The bacteria did not accumulate detectable Mn in the outer membrane during formation of the internal precipitates . This is the first report of an intracellular Mn solid produced by bacteria and coupled anaerobically to DR.

Shi Yan Sheng Wu Xue Bao, 2002 Jun, 35(2), 77 - 81
{Expression and characterization of carboxylesterase A2 in E . coli}; Huang J et al.; The most commonly observed change that has been linked to resistance development is the increase in activity of carboxylesterases . The putative mechanism involves an overproduction of this enzyme for the sequestration and the hydroxylation of various organophosphate and carbamate insecticides . Carboxylesterases A2 cDNA was amplified from Culex quinquefasciatus by RT-PCR and sequenced consequently . Target gene was inserted into pET-28a to create prokaryotic expression plasmid pET-EstA2 . When pET-EstA2 was transformed into E . coli BL21, the recombinant was induced by IPTG . A pure recombinant protein was obtained by affinity purification . Compared with carboxylesterase A2 purified from Culex quinquefasciatus, carboxylesterase A2, purified from the product of the transgenic of E . coli, has the same Km, but the Vm was higher than that of it, which shows that carboxylesterase A2, purified from the product of E . coli by affinity, is purer than that from Culex quinquefasciatus . The study on the expression and characterization of carboxylesterase A2 in E . coli is more useful for its future application.

J Bacteriol, 2004 Sep, 186(18), 6325 - 6
Recombinant cyclophilins lack nuclease activity; Manteca A et al.; Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis . Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.

Genetics, 2004 Aug, 167(4), 1585 - 95
The high-mobility group A-type protein CarD of the bacterium Myxococcus xanthus as a transcription factor for several distinct vegetative genes; Galbis-Martinez M et al.; CarD is the only reported prokaryotic protein showing structural and functional features typical of eukaryotic high-mobility group A transcription factors . In prokaryotes, proteins similar to CarD appear to be confined primarily to myxobacteria . In Myxococcus xanthus, CarD has been previously shown to act as a positive element in two different regulatory networks: one for light-induced synthesis of carotenoids and the other for starvation-induced fruiting body formation . We have now tested the effect of a loss-of-function mutation in the carD gene (carD1) on the expression of a random collection of lacZ-tagged genes, which are normally expressed in the dark during vegetative growth in rich medium . Our results indicate that CarD plays a significant role in the transcriptional regulation of various indicated genes . The carD1 mutation downregulates some genes and upregulates others . Also reported here is the isolation of several mutations that suppress the strong effect of carD1 on the expression of a particular vegetative gene . One of them (sud-2) also suppresses the effect of carD1 on other vegetative genes and on fruiting-body formation . Thus, CarD and the sud-2 gene product appear to participate in a single mechanism, which underlies various apparently diverse regulatory phenomena ascribed to CarD .

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2004 Jun, 18(2), 122 - 6
{Construction of full-length complementary DNA of hepatitis C virus genome from an HCV infected patient}; Mao HX et al.; BACKGROUND: To construct the full-length complementary DNA of HCV genome from an HCV infected patient . METHODS: Four HCV gene fragments (1.6, 3.5, 2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment, which was further cloned into a cassette vector with fixed 5-prime and 3-prime termini of HCV to make the full-length cDNA . The cDNA heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions . The prokaryotic expressed Core, NS3 protease, NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis . And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system . RESULTS: The cDNA covered the near full-length of HCV genome from the patient's serum . The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA . The prokaryotic expressed viral proteins could be identified by patient serum . In the NS3/4A-SEAP system, NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media . CONCLUSION: These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone.

Photochem Photobiol, 2004 Jul-Aug, 80, 31 - 5
Expressed sequence tag analysis of the dinoflagellate Lingulodinium polyedrum during dark phase; Tanikawa N et al.; To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light-dark cycle . A total of 4324 5'-end sequence tags were isolated . The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database . Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae . We also isolated three bioluminescence-related (luciferase and two luciferin-binding proteins {LBP}) and 37 photosynthesis-related genes . Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene . These cDNA clones and EST sequence data should provide a powerful resource for future genome-wide functional analyses for uncharacterized genes.

J Gen Physiol, 2004 Sep, 124(3), 203 - 10
A cyclic nucleotide modulated prokaryotic K+ channel; Nimigean CM et al.; A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K(+) channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus . The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified . Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer . As followed by a radioactive (86)Rb(+) flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K(+) channel . Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations . Since this bacterial homologue does not have the "C-linker" sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.

Trends Biochem Sci, 2004 Sep, 29(9), 469 - 77
Ancestral lipid biosynthesis and early membrane evolution; Pereto J et al.; Archaea possess unique membrane phospholipids that generally comprise isoprenoid ethers built on sn-glycerol-1-phosphate (G1P) . By contrast, bacterial and eukaryal membrane phospholipids are fatty acid esters linked to sn-glycerol-3-phosphate (G3P) . The two key dehydrogenase enzymes that produce G1P and G3P, G1PDH and G3PDH, respectively, are not homologous . Various models propose that these enzymes originated during the speciation of the two prokaryotic domains, and the nature (and even the very existence) of lipid membranes in the last universal common ancestor (cenancestor) is subject to debate . G1PDH and G3PDH belong to two separate superfamilies that are universally distributed, suggesting that members of both superfamilies existed in the cenancestor . Furthermore, archaea possess homologues to known bacterial genes involved in fatty acid metabolism and synthesize fatty acid phospholipids . The cenancestor seems likely to have been endowed with membrane lipids whose synthesis was enzymatic but probably non-stereospecific.

Curr Opin Plant Biol, 2004 Oct, 7(5), 499 - 505
Plant response regulators implicated in signal transduction and circadian rhythm; Mizuno T; The so-called 'response regulators' were originally discovered as common components of the widespread histidine (His)-->aspartate (Asp) phosphorelay signal transduction system in prokaryotes . Through the course of evolution, higher plants have also come to employ such prokaryotic response regulators (RRs) for their own signal transduction, such as the elicitation of plant hormone (e.g . cytokinin) responses . Furthermore, plants have evolved their own atypical variants of response regulators, pseudo response regulators (PRRs), which are used to modulate sophisticated biological processes, including circadian rhythms and other light-signal responses . Recent studies using the model plant Arabidopsis thaliana have begun to shed light on the interesting functions of these plant response regulators.

Expert Opin Biol Ther, 2004 Sep, 4(9), 1533 - 40
CpG DNA: immunomodulation and remodelling of the asthmatic airway; Jain VV et al.; Asthma is a disorder of increasing severity and prevalence . Present understanding of the pathogenesis of asthma emphasises its inflammatory nature . Unbridled inflammation appears to induce irreversible changes, collectively known as airway remodelling . CpG oligonucleotides are a class of compounds that have been developed from studies of prokaryotic DNA . Bacterial DNA, for example, contains sequence motifs based on the dinucleotides cytosine-guanine (CpG); these motifs are suppressed in mammalian DNA and induce (as part of the innate immune system) inflammation, characterised by the induction of T helper type 1 and regulatory responses . The pattern of inflammation promoted by CpG DNA tends to suppress the cytokine and cellular responses characteristic of asthma and atopic disorders . This has led to the investigation and development of CpG DNA as a novel therapeutic approach for the treatment and prevention of these disorders . In addition to suppressing acute and persistent airway inflammation, CpG DNA also reduces the development of subepithelial collagen deposition, goblet cell hyperplasia/metaplasia, and other aspects of airway remodelling . In this paper, the effects of CpG DNA on asthma inflammation and remodelling are reviewed.

Plant Physiol, 2004 Sep, 136(1), 2587 - 608 Epub 2004 Aug 27.
AraPerox . A database of putative Arabidopsis proteins from plant peroxisomes; Reumann S et al.; To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S {2004} Plant Physiol 135: 783-800) . About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively . To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags . The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in beta-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid alpha-oxidation, as well as NADP-dependent dehydrogenases and reductases . In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases . Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid . Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases . The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.

BMC Mol Biol . 2004 Aug 26;5(1):14.
Sequence periodicity of Escherichia coli is concentrated in intergenic regions; Hosid S et al.; BACKGROUND: Sequence periodicity with a period close to the DNA helical repeat is a very basic genomic property . This genomic feature was demonstrated for many prokaryotic genomes . The Escherichia coli sequences display the period close to 11 base pairs . RESULTS: Here we demonstrate that practically only ApA/TpT dinucleotides contribute to overall dinucleotide periodicity in Escherichia coli . The noncoding sequences reveal this periodicity much more prominently compared to protein-coding sequences . The sequence periodicity of ApC/GpT, ApT and GpC dinucleotides along the Escherichia coli K-12 is found to be located as well mainly within the intergenic regions . CONCLUSIONS: The observed concentration of the dinucleotide sequence periodicity in the intergenic regions of E . coli suggests that the periodicity is a typical property of prokaryotic intergenic regions . We suppose that this preferential distribution of dinucleotide periodicity serves many biological functions; first of all, the regulation of transcription.

Proc Natl Acad Sci U S A, 2004 Sep 7, 101(36), 13186 - 91 Epub 2004 Aug 25.
A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major; Vickers TJ et al.; Glyoxalase I forms part of the glyoxalase pathway that detoxifies reactive aldehydes such as methylglyoxal, using the spontaneously formed glutathione hemithioacetal as substrate . All known eukaryotic enzymes contain zinc as their metal cofactor, whereas the Escherichia coli glyoxalase I contains nickel . Database mining and sequence analysis identified putative glyoxalase I genes in the eukaryotic human parasites Leishmania major, Leishmania infantum, and Trypanosoma cruzi, with highest similarity to the cyanobacterial enzymes . Characterization of recombinant L . major glyoxalase I showed it to be unique among the eukaryotic enzymes in sharing the dependence of the E . coli enzyme on nickel . The parasite enzyme showed little activity with glutathione hemithioacetal substrates but was 200-fold more active with hemithioacetals formed from the unique trypanosomatid thiol trypanothione . L . major glyoxalase I also was insensitive to glutathione derivatives that are potent inhibitors of all other characterized glyoxalase I enzymes . This substrate specificity is distinct from that of the human enzyme and is reflected in the modification in the L . major sequence of a region of the human protein that interacts with the glycyl-carboxyl moiety of glutathione, a group that is conjugated to spermidine in trypanothione . This trypanothione-dependent glyoxalase I is therefore an attractive focus for additional biochemical and genetic investigation as a possible target for rational drug design.

Proc Natl Acad Sci U S A, 2004 Aug 31, 101(35), 12783 - 8 Epub 2004 Aug 24.
Multiple pathways process stalled replication forks; Michel B et al.; Impairment of replication fork progression is a serious threat to living organisms and a potential source of genome instability . Studies in prokaryotes have provided evidence that inactivated replication forks can restart by the reassembly of the replication machinery . Several strategies for the processing of inactivated replication forks before replisome reassembly have been described . Most of these require the action of recombination proteins, with different proteins being implicated, depending on the cause of fork arrest . The action of recombination proteins at blocked forks is not necessarily accompanied by a strand-exchange reaction and may prevent rather than repair fork breakage . These various restart pathways may reflect different structures at stalled forks . We review here the different strategies of fork processing elicited by different kinds of replication impairments in prokaryotes and the variety of roles played by recombination proteins in these processes .

J Biol Chem, 2004 Nov 19, 279(47), 48821 - 9 Epub 2004 Aug 23.
FtsZ fiber bundling is triggered by a conformational change in bound GTP; Marrington R et al.; Polymer formation by the essential FtsZ protein plays a crucial role in the cytokinesis of most prokaryotes . Lateral associations between these FtsZ polymers to form bundles or sheets are widely predicted to be extremely important for FtsZ function in vivo . We have carried out a study in vitro of FtsZ polymer formation and bundling using linear dichroism (LD) to assess structural properties of the polymers . We demonstrate proof-of-principle experiments to show that LD can be used as a technique to follow FtsZ polymerization, and we present the LD spectra of FtsZ polymers . Our subsequent examination of FtsZ polymer bundling induced by calcium reveals a substantial increase in the LD signal indicative of increased polymer length and rigidity . We also detect a specific conformational change in the guanine moiety associated with bundling, whereas the conformation and configuration of the FtsZ monomers within the polymer remain largely unchanged . We demonstrate that other divalent cations can induce this conformational change in FtsZ-bound GTP coincident with polymer bundling . Therefore, we present "flipping" of the guanine moiety in FtsZ-bound GTP as a mechanism that explains the link between reduced GTPase activity, increased polymer stability, and polymer bundling.

FEBS Lett, 2004 Aug 27, 573(1-3), 73 - 7
Correlations between genomic GC levels and optimal growth temperatures in prokaryotes; Musto H et al.; In prokaryotes, GC levels range from 25% to 75%, and Topt from approximately 0 degrees C to >100 degrees C . When all species are considered together, no correlation is found between the two variables . Correlations are found, however, when Families of prokaryotes are analysed . Indeed, when Families comprising at least 10 species were studied (a set of 20 Families), positive correlations are found for 15 of them . Furthermore, a comparative analysis by independent contrasts made within the Families in order to control for phylogenetic non-independence showed qualitatively equivalent results . We conclude that Topt is one of the factors that influences genomic GC in prokaryotes.

J Mol Biol, 2004 Sep 10, 342(2), 383 - 401
Acetylation of the chemotaxis response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli; Barak R et al.; Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure . Here, we overproduced Acs from E.coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes . Such characterization is essential for revealing the function of CheY acetylation in chemotaxis . The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation . The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient approximately 3) . By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation . Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electro-spray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with non-acetylated CheY being the most abundant species . By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites . Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues . Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region.

Arch Biochem Biophys, 2004 Oct 1, 430(1), 16 - 21
Oxidative remodeling of plastid carotenoids; Camara B et al.; Carotenoids are isoprenoid pigmented compounds that are present in representatives from practically all eukaryotic and prokaryotic taxa . In plants, carotenoids are synthesized and normally sequestered in plastids as lipophilic C40 constituents . However, they are also subjected to oxidative remodeling initiated by specific carotenoid cleavage dioxygenases . Primary products resulting from these reactions undergo modifications involving oxido-reduction, dehydratation rearrangement, and glycosylation . This review focuses on only a few of these derivatives for which the enzymes and genes involved have been characterized . The compartmentation of this metabolism and its significance have also been considered.

Anal Biochem, 2004 Sep 15, 332(2), 330 - 6
Thermally denaturing high-performance liquid chromatography analysis of primase activity; Koepsell S et al.; Prokaryotic primase, a DNA-dependent RNA polymerase, is a target of interest for the development of novel antibiotics . A new assay was developed to evaluate the inhibition of primase activity while avoiding the limitations of existing assays that require the incorporation of radiolabeled nucleotides into the growing primer followed by electrophoretic separation and autoradiography or scintillation counting . These existing technologies are either time consuming or unable to give detailed information on the kinetics, size, and nature of the primers synthesized . To address these issues in a nonradioactive manner, a thermally denaturing high-performance liquid chromatography (HPLC) assay was developed that was able to (1) measure the two modes of primase activity (de novo and overlong primer synthesis), (2) quantitate de novo primer synthesis kinetics yielding a rate constant of 0.00251 s(-1), and (3) determine that dNTPs inhibited primase activity with an IC50 of 9.5 microM . In addition, the differential elution properties of short DNA and RNA oligonucleotides on an alkylated nonporous polystyrene-divinylbenzene copolymer microsphere bead column were determined . The thermally denaturing HPLC assay provides rapid quantitative analysis of primase function and qualitative analysis of activity with regard to the nature of the primers synthesized.

Chem Biol, 2004 Aug, 11(8), 1157 - 63
Engineering a ligand-dependent RNA transcriptional activator; Buskirk AR et al.; RNA has recently been shown to play diverse roles in gene regulation, including the small molecule-dependent inhibition of translation in prokaryotes . To create an artificial genetic switch that acts at the level of transcription, we fused a small molecule binding aptamer to a previously evolved RNA that activates transcription when localized to a promoter . We designed a conformational shift in which a helical element required for transcriptional activation was stabilized upon ligand binding . Selection and screening in S . cerevisiae optimized the linker region, generating an RNA that is 10-fold more active in the presence of tetramethylrosamine (TMR) . TMR increases the activity of this evolved RNA in a graded, dose-dependent manner . Our results exemplify a strategy for controlling the activity of laboratory-evolved RNAs in living cells.

Phys Rev E Stat Nonlin Soft Matter Phys . 2004 Jul;70(1 Pt 1):011909 . Epub 2004 Jul 15.
Dynamics of gene regulatory networks with cell division cycle; Chen L et al.; This paper focuses on modeling and analyzing the nonlinear dynamics of gene regulatory networks with the consideration of a cell division cycle with duplication process of DNA, in particular for switches and oscillators of synthetic networks . We derive two models that may correspond to the eukaryotic and prokaryotic cells, respectively . A biologically plausible three-gene model ( lac, tetR, and cI ) and a repressilator as switch and oscillator examples are used to illustrate our theoretical results . We show that the cell cycle may play a significant role in gene regulation due to the nonlinear dynamics of a gene regulatory network although gene expressions are usually tightly controlled by transcriptional factors.

Ann Univ Mariae Curie Sklodowska {Med}, 2003, 58(2), 390 - 3
Copper and metallothioneins in cancer cells; Florianczyk B; Metallothioneins (Mts) are intracellular proteins whose biological function is zinc and copper regulation as well as detoxification of toxic metals . Another function of these proteins is sweeping away free radicals . MT synthesis induction is stimulated by such factors as metallic ions, free radicals, cytokines, lymphokines and stress . An increased intracellular metallothionein expression was found in many human and animal neoplasms . Copper functions as cofactor in various redox enzymes . At the same time, copper is very toxic to both eukaryotic and prokaryotic cells . Copper ions can bind to proteins and nucleic acids and cause the oxidation of lipids and proteins . The formation of deleterious free radicals is also enhanced by copper ions . For cell viability, regulation of intracellular copper activity is thus crucially important and mechanisms must exist for the homeostasis of copper . An elevated copper level is noted in many types of neoplastic tissue.

Biosci Biotechnol Biochem, 2004 Aug, 68(8), 1758 - 67
A Genome-wide view of the Escherichia coli BasS-BasR two-component system implicated in iron-responses; Hagiwara D et al.; Histidine-to-aspartate (His-->Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes . Determination of the entire genomic sequence of Escherichia coli revealed that this gram-negative bacterium has 29 His-kinases and 32 response regulators . Of the 29 His-kinases, 23 have already been experimentally characterized at least to some extent in terms of their physiological functions . No physiological stimulus has yet been identified for each of the remaining 6 His-kinases (BasS, CreC, RstB, YfhK, YehU, and YpdA) . Here we characterized the BasS-BasR two-component system with reference to its physiological function, taking genetic approaches together with genome-wide transcriptome profiling . First we showed that the hypothetical yfbE operon that appears to be implicated in the modification of lipopolysaccharides is regulated at the level of transcription in response to external iron, and then we showed that the BasS-BasR system is essential for this iron-dependent induction of yfbE . Another PhoQ-PhoP two-component system was also implicated in the full induction of yfbE in response to iron, but it was not essential . To gain more insight into the BasS-BasR system, we conducted genome-wide transcriptome analysis by microarray, finding that many of the uncovered putative iron-induced and BasS-BasR-dependent genes are somehow associated with acidic and/or anaerobic growth conditions . In this respect, it was found that mutant cells defective in the BasS-BasR system were sensitive to mild-acid growth conditions in the presence of a relatively high concentration of iron . These results are discussed with regard to a comprehensive picture of the His-->Asp phosphorelay signaling network in E . coli.

Protein Sci, 2004 Sep, 13(9), 2547 - 52
Substrate-induced asymmetry and channel closure revealed by the apoenzyme structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase; Morris VK et al.; Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in prokaryotic coenzyme A (CoA) biosynthesis, directing the transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) to yield dephospho-CoA (dPCoA) . The crystal structures of Escherichia coli PPAT bound to its substrates, product, and inhibitor revealed an allosteric hexameric enzyme with half-of-sites reactivity, and established an in-line displacement catalytic mechanism . To provide insight into the mechanism of ligand binding we solved the apoenzyme (Apo) crystal structure of PPAT from Mycobacterium tuberculosis . In its Apo form, PPAT is a symmetric hexamer with an open solvent channel . However, ligand binding provokes asymmetry and alters the structure of the solvent channel, so that ligand binding becomes restricted to one trimer.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Aug, 24(8), 913 - 6
{Cloning, expression and identification of human survivin in E . coli}; Bie J et al.; OBJECTIVE: To efficiently express and identify recombinant human survivin in E . coli . METHODS: Survivin cDNA was amplified by reverse transcriptional (RT)-PCR and cloned into the prokaryotic expression vector pBV220, followed by expression of the recombinant plasmid in E.coli strain BL21 (Gold) . To obtain survivin protein, DEAE-Sepharose Fast-Flow ion exchange chromatography and Sephacryl S-200 gel filtration were performed . Western blot analysis was used for detecting the expressed product . RESULTS: Survivin protein was expressed in E.coli in the form of inclusion body at the expression level over 30% of the total cell protein . After ion exchange chromatography and gel filtration, the recombinant protein reached a purity over 95% and exhibited specific reaction with mouse anti-human antibody . CONCLUSION: Survivin protein with high purity can be obtained by the method described above to facilitate further study of the anti-apoptosis function of survivin.

J Bacteriol, 2004 Sep, 186(17), 5950 - 5
Analysis of chimeric chemoreceptors in Bacillus subtilis reveals a role for CheD in the function of the McpC HAMP domain; Kristich CJ et al.; Motile prokaryotes use a sensory circuit for control of the motility apparatus in which ligand-responsive chemoreceptors regulate phosphoryl flux through a modified two-component signal transduction system . The chemoreceptors exhibit a modular architecture, comprising an N-terminal sensory module, a C-terminal output module, and a HAMP domain that connects the N- and C-terminal modules and transmits sensory information between them via an unknown mechanism . The sensory circuits mediated by two chemoreceptors of Bacillus subtilis have been studied in detail . McpB is known to regulate chemotaxis towards the attractant asparagine in a CheD-independent manner, whereas McpC requires CheD to regulate chemotaxis towards the attractant proline . Although CheD is a phylogenetically widespread chemotaxis protein, there exists only a limited understanding of its function . We have constructed chimeras between McpB and McpC to probe the role of CheD in facilitating sensory transduction by McpC . We found that McpC can be converted to a CheD-independent receptor by the replacement of one-half of its HAMP domain with the corresponding sequence from McpB, suggesting that McpC HAMP domain function is complex and may require intermolecular interactions with the CheD protein . When considered in combination with the previous observation that CheD catalyzes covalent modification of the C-terminal modules of B . subtilis receptors, these results suggest that CheD may interact with chemoreceptors at multiple, functionally distinct sites.

J Bacteriol, 2004 Sep, 186(17), 5790 - 8
Mycoplasma hyopneumoniae p65 surface lipoprotein is a lipolytic enzyme with a preference for shorter-chain fatty acids; Schmidt JA et al.; Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine . p65 is an immunodominant surface lipoprotein of M . hyopneumoniae that is specifically recognized during disease . Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes . To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons . After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein . The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP) . Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester . Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39 degrees C . Calcium ions did not increase the activity of recombinant GST-p65 . Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M . hyopneumoniae in vitro . Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC . The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases . This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.

J Bacteriol, 2004 Sep, 186(17), 5614 - 20
A regulatory trade-off as a source of strain variation in the species Escherichia coli; King T et al.; There are few existing indications that strain variation in prokaryotic gene regulation is common or has evolutionary advantage . In this study, we report on isolates of Escherichia coli with distinct ratios of sigma factors (RpoD, sigmaD, or sigma70 and RpoS or sigmaS) that affect transcription initiated by RNA polymerase . Both laboratory E . coli K-12 lineages and nondomesticated isolates exhibit strain-specific endogenous levels of RpoS protein . We demonstrate that variation in genome usage underpins intraspecific variability in transcription patterns, resistance to external stresses, and the choice of beneficial mutations under nutrient limitation . Most unexpectedly, RpoS also controlled strain variation with respect to the metabolic capability of bacteria with more than a dozen carbon sources . Strains with higher sigmaS levels were more resistant to external stress but metabolized fewer substrates and poorly competed for low concentrations of nutrients . On the other hand, strains with lower sigmaS levels had broader nutritional capabilities and better competitive ability with low nutrient concentrations but low resistance to external stress . In other words, RpoS influenced both r and K strategist functions of bacteria simultaneously . The evolutionary principle driving strain variation is proposed to be a conceptually novel trade-off that we term SPANC (for "self-preservation and nutritional competence") . The availability of multiple SPANC settings potentially broadens the niche occupied by a species consisting of individuals with narrow specialization and reveals an evolutionary advantage offered by polymorphic regulation . Regulatory diversity is likely to be a significant contributor to complexity in a bacterial world in which multiple sigma factors are a universal feature.

J Med Chem, 2004 Aug 26, 47(18), 4439 - 52
Structural basis for the binding of didemnins to human elongation factor eEF1A and rationale for the potent antitumor activity of these marine natural products; Marco E et al.; Didemnins and tamandarins are closely related marine natural products with potent inhibitory effects on protein synthesis and cell viability . On the basis of available biochemical and structural evidence and results from molecular dynamics simulations, a model is proposed that accounts for the strong and selective binding of these compounds to human elongation factor eEF1A in the presence of GTP . We suggest that the p-methoxyphenyl ring of these cyclic depsipeptides is inserted into the same pocket in eEF1A that normally lodges either the 3' terminal adenine of aminoacylated tRNA, as inferred from two prokaryotic EF-Tu.GTP.tRNA complexes, or the aromatic side chain of Phe/Tyr-163 from the nucleotide exchange factor eEF1Balpha, as observed in several X-ray crystal structures of a yeast eEF1A:eEF1Balpha complex . This pocket, which has a strong hydrophobic character, is formed by two protruding loops on the surface of eEF1A domain 2 . Further stabilization of the bound depsipeptide is brought about by additional crucial interactions involving eEF1A domain 1 in such a way that the molecule fits snugly at the interface between these two domains . In the GDP-bound form of eEF1A, this binding site exists only as two separate halves, which accounts for the much greater affinity of didemnins for the GTP-bound form of this elongation factor . This binding mode is entirely different from those seen in the complexes of the homologous prokaryotic EF-Tu with kirromycin-type antibiotics or the cyclic thiazolyl peptide antibiotic GE2270A . Interestingly, the set of interactions used by didemnins to bind to eEF1A is also distinct from that used by eEF1Balpha or eEF1Bbeta, thus establishing a competition for binding to a common site that goes beyond simple molecular mimicry . The model presented here is consistent with both available biochemical evidence and known structure-activity relationships for these two classes of natural compounds and synthetic analogues and provides fertile ground for future research.

Cell Mol Life Sci, 2004 Aug, 61(16), 2020 - 30
Acetyl-coenzyme A synthetase (AMP forming); Starai VJ et al.; Acetyl-coenzyme A synthetase (AMP forming; Acs) is an enzyme whose activity is central to the metabolism of prokaryotic and eukaryotic cells . The physiological role of this enzyme is to activate acetate to acetyl-coenzyme A (Ac-CoA) . The importance of Acs has been recognized for decades, since it provides the cell the two-carbon metabolite used in many anabolic and energy generation processes . In the last decade researchers have learned how carefully the cell monitors the synthesis and activity of this enzyme . In eukaryotes and prokaryotes, complex regulatory systems control acs gene expression as a function carbon flux, with a second layer of regulation exerted posttranslationally by the NAD+/sirtuin-dependent protein acetylation/deacetylation system . Recent structural work provides snapshots of the dramatic conformational changes Acs undergoes during catalysis . Future work on the regulation of acs gene expression will expand our understanding of metabolic integration, while structure/function studies will reveal more details of the function of this splendid molecular machine.

Proc R Soc Lond B Biol Sci, 2004 Sep 7, 271(1550), 1783 - 9
Widespread vertical transmission and associated host sex-ratio distortion within the eukaryotic phylum Microspora; Terry RS et al.; Vertical transmission (VT) and associated manipulation of host reproduction are widely reported among prokaryotic endosymbionts . Here, we present evidence for widespread use of VT and associated sex-ratio distortion in a eukaryotic phylum . The Microspora are an unusual and diverse group of eukaryotic parasites that infect all animal phyla . Following our initial description of a microsporidian that feminizes its crustacean host, we survey the diversity and distribution of VT within the Microspora . We find that vertically transmitted microsporidia are ubiquitous in the amphipod hosts sampled and that they are also diverse, with 11 species of microsporidia detected within 16 host species . We found that infections were more common in females than males, suggesting that host sex-ratio distortion occurs in five out of eight parasite species tested . Phylogenetic reconstruction demonstrates that VT occurs in all major lineages of the phylum Microspora and that sex-ratio distorters are found on multiple branches of the phylogenetic tree . We propose that VT is either an ancestral trait or evolves with peculiar frequency in this phylum . If the association observed here between VT and host sex-ratio distortion holds true across other host taxa, these eukaryotic parasites may join the bacterial endosymbionts in their importance as sex-ratio distorters.

Mikrobiologiia, 2004 May-Jun, 73(3), 398 - 405
{Obtaining of intrapopulational dissociants of some bacilli and the use of DIR-PCR for their identification}; Tsygankova SV et al.; The paper is the first to suggest methods for rapid obtaining and genotypic identification of phenotypic (colonial-morphological) dissociants of bacterial cultures . For revelation of the potential dissociation ability and obtaining of dissociants, the use of bacterial cyst-like refractile cells (CRC) is recommended . These cells are characterized by enhanced variability; upon their first passage, an abrupt increase in the dissociation index is observed as a result of the emergence of cells that form morphologically different types of colonies . The approaches elaborated were tested with Bacillus cereus, B . subtilis, and B . licheniformis, for which colonial-morphological dissociants of various types were obtained after the first passage of CRC (both of those formed in the developmental cycle of bacteria and of those arising as a result of artificial increase of the concentration of anabiosis autoinducers in the cultivation medium) . The genomic distinctions between dissociants of B . cereus and B . subtilis were estimated using polymerase chain reaction with a primer system designed based on the analysis of nucleotide sequences of complete prokaryotic genomes available in the GenBank database (DIR-PCR) . The application of the suggested method allowed distinctions between the genomes of dissociants of Bacillus cereus and B . subtilis to be revealed, which is in agreement with the hypothesis that suggests reversible intragenomic rearrangements to be the basis of bacterial dissociation into subpopulations.

J Huazhong Univ Sci Technolog Med Sci, 2004, 24(2), 107 - 11, 123
Construction, expression and identification of a recombinant BCG vaccine encoding human mycobacterium tuberculosis heat shock protein 65; Dai W et al.; Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection . The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M . tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR) . These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M . tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65 . Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained . The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating . The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble . Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M . tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.

Ann Univ Mariae Curie Sklodowska {Med}, 2003, 58(1), 85 - 8
Copper in the organism--transport and storage in the cells; Florianczyk B; Copper functions as cofactor in various redox enzymes . At the same time, copper is very toxic to both eukaryotic and prokaryotic cells . Copper ions can bind to proteins and nucleic acids and cause the oxidation of lipids and proteins . The formation of deleterious free radicals is also enhanced by copper ions . For cell viability, regulation of intracellular copper activity is thus crucially important and mechanisms must exist for the homeostasis of copper.

Zhonghua Yi Xue Za Zhi, 2004 Jun 17, 84(12), 1024 - 8
{Construction and expression of the fusion protein DT389-hIL-13 and its cytotoxicity to glioma cell lines}; Tuo HZ et al.; OBJECTIVE: To construct a fusion protein toxin DT389-hIL-13 which comprises the N-terminal 389 amino acids of diphtheria toxin (DT389) and human interlukin 13 (hIL-13), and to explore its cytotoxicity on U251 glioma cells . METHODS: The cDNA of hIL-13 gene was amplified by PCR and linked with the 3'-terminus of the gene encoding the N-terminal 389 amino acids, which correspond to the enzyme domain and transmembrane domain of diphtheria toxin . The tandem constructed gene was then inserted into an E . coli expression vector pET30a . The resulted expression vector was transformed into E . coli BL21 and induced by IPTG . The expressed protein was analyzed by SDS-PAGE and Western blot analysis . U(251) glioma cells were cultured DT389-hIl-13 was added into the culture . The cytotoxicity was determined using colorimetric MTS proliferation assay . RESULTS: The expression plasmid pET30a/DT389-hIL13 was constructed with correct sequence . The recombinant protein was successfully expressed in E . coli in manner of inclusion body and with a relative molecular weight of about 55 000, which reacted well with both anti-diphtheria toxin and anti-hIL-13 polyclonal antibody in Western blot assay . The purified recombinant chimeric toxin was found to effectively inhibit the prolifieration of glioblastoma multiforme cells bearing high affinity hIL-13 receptors, and resulted in dose-dependent relationship with 50% inhibition concentration (IC(50)) of 5 x 10(-)11mol/L . CONCLUSION: Prokaryotic expression system can be recruited to produce recombinant chimeric toxin DT389-hIL-13 . The results may lay a foundation for preparing specific the agent targets for tumors overexpressing IL-13 receptor.

Biochemistry, 2004 Aug 24, 43(33), 10701 - 9
Short variable sequence acquired in evolution enables selective inhibition of various inward-rectifier K+ channels; Ramu Y et al.; Tertiapin (TPN), a small protein toxin originally isolated from honey bee venom, inhibits only certain eukaryotic inward-rectifier K(+) (Kir) channels with high affinity . We found that a short ( approximately 10 residues) sequence in Kir channels, located in the N-terminal part of the linker between the two transmembrane segments, is essential for high-affinity inhibition by TPN and that variability in the region underlies the great variation of TPN affinities among eukaryotic Kir channels . This short variable region is however not present in a bacterial Kir channel (KirBac1.1) or in many other types of prokaryotic and eukaryotic K(+) channels . Thus, the acquisition in evolution of the variable region in eukaryotic Kir channels has created the opportunity to selectively target the numerous types of Kir channel that play important physiological roles . We also show that TPN sensitivity can be readily conferred onto some Kir channels that currently have no known inhibitors by replacing their variable region with that from a TPN-sensitive channel . In heterologous expression systems, such acquired toxin sensitivity will allow currents carried by mutant channels to be readily isolated from interfering background currents . Finally we show that, in the heteromeric GIRK1/4 channels, the GIRK4 and not GIRK1 subunit confers the high affinity for TPN.

Science, 2004 Aug 13, 305(5686), 997 - 1000
Discovery of symbiotic nitrogen-fixing cyanobacteria in corals; Lesser MP et al.; Colonies of the Caribbean coral Montastraea cavernosa exhibit a solar-stimulated orange-red fluorescence that is spectrally similar to a variety of fluorescent proteins expressed by corals . The source of this fluorescence is phycoerythrin in unicellular, nonheterocystis, symbiotic cyanobacteria within the host cells of the coral . The cyanobacteria coexist with the symbiotic dinoflagellates (zooxanthellae) of the coral and express the nitrogen-fixing enzyme nitrogenase . The presence of this prokaryotic symbiont in a nitrogen-limited zooxanthellate coral suggests that nitrogen fixation may be an important source of this limiting element for the symbiotic association.

World J Gastroenterol, 2004 Sep 15, 10(18), 2675 - 9
Construction of prokaryotic expression system of ltB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity; Yan J et al.; AIM: To construct ltB-ureB fusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein . METHODS: The ureB gene from a clinical Helicobacter pylori (H pylori) strain Y06 and the ltB gene from Escherichia coli (E . coli) strain 44851 were linked into ltB-ureB fusion gene by PCR . The fusion gene sequence was analyzed after T-A cloning . A prokaryotic recombinant expression vector pET32a inserted with ltB-ureB fusion gene (pET32a-ltB-ureB) was constructed . Expression of the recombinant LTB-UreB protein (rLTB-UreB) in E . coli BL21DE3 induced by isopropylthio-beta-D-galactoside (IPTG) at different concentrations was detected by SDS-PAGE . Western blot assays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of H pylori and a self-prepared rabbit anti-rUreB serum, respectively, and determine the antigenicity of the recombinant protein on inducing specific antibody in rabbits . GM1-ELISA was used to demonstrate the adjuvanticity of rLTB-UreB . Immunoreaction of rLTB-UreB to the UreB antibody positive sera from 125 gastric patients was determined by using ELISA . RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ltB-ureB fusion gene were 100% . IPTG with different dosages of 0.1-1.0 mmol/L could efficiently induce pET32a-ltB-ureB-E.coli BL21DE3 to express the rLTB-UreB . The output of the target recombinant protein expressed by pET32a-ureB-E.coli BL21DE3 was approximately 35% of the total bacterial proteins . rLTB-UreB mainly presented in the form of inclusion body . Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori and anti-rUreB serum as well as induce rabbit to produce specific antibody . The strong ability of rLTB-UreB binding bovine GM1 indicated the existence of adjuvanticity of the recombinant protein . All the UreB antibody positive sera from the patients (125/125) were positive for rLTB-UreB . CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established . The expressed rLTB-UreB showed qualified immunogenicity, antigenicity and adjuvanticity . All the results mentioned above laid a firm foundation for further development of H pylori genetically engineered vaccine.

J Virol, 2004 Sep, 78(17), 9270 - 6
Glycan-controlled epitopes of prion protein include a major determinant of susceptibility to sheep scrapie; Moudjou M et al.; A key feature of prion encephalopathies is the accumulation of a misfolded form of the host glycoprotein PrP . Cell-free and cell culture studies have shown that the efficiency of conversion of PrP into the disease-associated form is influenced by its amino acid sequence and also by its carbohydrate moiety . Here, we characterize four novel glycoform-dependent monoclonal antibodies raised against prokaryotic recombinant sheep PrP . We demonstrate that these antibodies discriminate the PrP monoglycosylated species, since two of them recognize molecules that have the first Asn glycosylation site occupied (mono1) while the other two recognize molecules glycosylated at the second site (mono2) . Remarkably, the recognition of PrP by the anti-mono2 antibodies was strongly influenced by the amino acid present at position 171, i.e., either Gln or Arg . This polymorphism is known to be the main determinant of susceptibility and resistance to scrapie in sheep . Altogether, our findings lead us to propose that each glycan chain controls the accessibility of PrP determinants located close upstream from their attachment site . The monoglycoform-assigned and the allotype-restricted antibodies described here, the first to date, should provide further opportunities to investigate the involvement of each glycan chain in PrP conversion in relation to prion strain diversity and the basis of the resistance conferred by the Arg-171 amino acid.

J Virol, 2004 Sep, 78(17), 9016 - 29
Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model; Cheung AK; Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems . In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes . These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication . Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model . The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.

J Biol Chem, 2004 Oct 22, 279(43), 45013 - 9 Epub 2004 Aug 11.
Conformational transitions induced by the binding of MgATP to the vitamin B12 ATP-binding cassette (ABC) transporter BtuCD; Oloo EO et al.; ATP-binding cassette transporters use the free energy of ATP hydrolysis to transport structurally diverse molecules across prokaryotic and eukaryotic membranes . Computer simulation studies of the "real-time" dynamics of the ATP binding process in BtuCD, the vitamin B12 importer from Escherichia coli, demonstrate that the docking of ATP to the catalytic pockets progressively draws the two cytoplasmic nucleotide-binding cassettes toward each other . Movement of the cassettes into closer opposition in turn induces conformational rearrangement of alpha-helices in the transmembrane domain . The shape of the translocation pathway consequently changes in a manner that could aid the vectorial movement of vitamin B12 . These results suggest that ATP binding may indeed represent the power stroke in the catalytic mechanism . Moreover, occlusion of ATP at one catalytic site is mechanically coupled to opening of the nucleotide-binding pocket at the second site . We propose that this asymmetry in nucleotide binding behavior at the two catalytic pockets may form the structural basis by which the transporter is able to alternate ATP hydrolysis from one site to the other.

BMC Bioinformatics . 2004 Aug 12;5(1):109.
The Hotdog fold: wrapping up a superfamily of thioesterases and dehydratases; Dillon SC et al.; BACKGROUND: The Hotdog fold was initially identified in the structure of Escherichia coli FabA and subsequently in 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas sp . strain CBS . Since that time structural determinations have shown a number of other apparently unrelated proteins also share the Hotdog fold . RESULTS: Using sequence analysis we unify a large superfamily of HotDog domains . Membership includes numerous prokaryotic, archaeal and eukaryotic proteins involved in several related, but distinct, catalytic activities, from metabolic roles such as thioester hydrolysis in fatty acid metabolism, to degradation of phenylacetic acid and the environmental pollutant 4-chlorobenzoate . The superfamily also includes FapR, a non-catalytic bacterial homologue that is involved in transcriptional regulation of fatty acid biosynthesis.We have defined 17 subfamilies, with some characterisation . Operon analysis has revealed numerous HotDog domain-containing proteins to be fusion proteins, where two genes, once separate but adjacent open-reading frames, have been fused into one open-reading frame to give a protein with two functional domains . Finally we have generated a Hidden Markov Model library from our analysis, which can be used as a tool for predicting the occurrence of HotDog domains in any protein sequence . CONCLUSIONS: The HotDog domain is both an ancient and ubiquitous motif, with members found in the three branches of life.

Philos Trans R Soc Lond B Biol Sci, 2004 Aug 29, 359(1448), 1249 - 66; discussion 1266-7
Life at low water activity; Grant WD; Two major types of environment provide habitats for the most xerophilic organisms known: foods preserved by some form of dehydration or enhanced sugar levels, and hypersaline sites where water availability is limited by a high concentration of salts (usually NaCl) . These environments are essentially microbial habitats, with high-sugar foods being dominated by xerophilic (sometimes called osmophilic) filamentous fungi and yeasts, some of which are capable of growth at a water activity (a(w)) of 0.61, the lowest a(w) value for growth recorded to date . By contrast, high-salt environments are almost exclusively populated by prokaryotes, notably the haloarchaea, capable of growing in saturated NaCl (a(w) 0.75) . Different strategies are employed for combating the osmotic stress imposed by high levels of solutes in the environment . Eukaryotes and most prokaryotes synthesize or accumulate organic so-called 'compatible solutes' (osmolytes) that have counterbalancing osmotic potential . A restricted range of bacteria and the haloarchaea counterbalance osmotic stress imposed by NaCl by accumulating equivalent amounts of KCl . Haloarchaea become entrapped and survive for long periods inside halite (NaCl) crystals . They are also found in ancient subterranean halite (NaCl) deposits, leading to speculation about survival over geological time periods.

Environ Microbiol, 2004 Sep, 6(9), 879 - 86
Quantifying the accessibility of the metagenome by random expression cloning techniques; Gabor EM et al.; The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host . The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism . In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion . Genes of the last category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs . To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analysed for the presence of expression signals functional in E . coli hosts, using bioinformatics tools . Our study reveals significant differences in the predicted expression modes between distinct taxonomic groups of organisms and suggests that about 40% of the enzymatic activities may be readily recovered by random cloning in E . coli.

Nature, 1979 Jul 5, 280(5717), 17 - 9
A unifying model for the G1 period in prokaryotes and eukaryotes; Cooper S; A model to explain the cell division cycle in both prokaryotes and eukaryotes is presented . No specific 'G1 functions' take place during the G1 period, which is merely part of a larger period for the preparation of DNA synthesis which began at the previous initiation of DNA synthesis . A G1 period exists merely because the doubling time of the cells is greater than the sum of the S and G2 periods.

J Invest Dermatol, 2004 Sep, 123(3), 589 - 91
Congenital erythropoietic porphyria: report of a novel mutation with absence of clinical manifestations in a homozygous mutant sibling; Ged C et al.; In a Palestinian family, four siblings were shown to express typical and severe congenital erythropoietic porphyria (CEP) . A new mutation of the uroporphyrinogen III synthase (UROS) gene was evidenced by systematic sequencing of the UROS gene: the substitution of serine by proline at the amino acid residue 47 (S47P) was present at the homozygous state in the four patients . The mother was heterozygous, the father was not examined . Surprisingly, in one unaffected sister, UROS activity was markedly deficient and UROS gene analysis showed a homozygous mutant profile . The deleterious role of the mutant S47P protein on UROS activity was demonstrated by prokaryotic expression . This observation is the first report of a healthy status associated with homozygosity for a mutation of UROS gene in a severely affected family . We then draw hypotheses to explain the protective phenotype in the homozygous healthy subject.

J Biol Chem, 2004 Oct 22, 279(43), 44384 - 93 Epub 2004 Aug 09.
The iron superoxide dismutase from the filamentous cyanobacterium Nostoc PCC 7120 . Localization, overexpression, and biochemical characterization; Regelsberger G et al.; The nitrogen-fixing filamentous cyanobacterium Nostoc PCC 7120 (formerly named Anabaena PCC 7120) possesses two genes for superoxide dismutase, a unique membrane-associated manganese superoxide dismutase (MnSOD) and a soluble iron superoxide dismutase (FeSOD) . A phylogenetic analysis of FeSODs shows that cyanobacterial enzymes form a well separated cluster with filamentous species found in one subcluster and unicellular species in the other . Activity staining, inhibition patterns, and immunogold labeling show that FeSOD is localized in the cytosol of vegetative cells and heterocysts (nitrogenase containing specialized cells formed during nitrogen-limiting conditions) . The recombinant Nostoc FeSOD is a homodimeric, acidic enzyme exhibiting the characteristic iron peak at 350 nm in its ferric state, an almost 100% occupancy of iron per subunit, a specific activity using the ferricytochrome assay of (2040 +/- 90) units mg(-1) at pH 7.8, and a dissociation constant Kd of the azide-FeSOD complex of 2.1 mM . Using stopped flow spectroscopy it was shown that the decay of superoxide in the presence of various FeSOD concentrations is first-order in enzyme concentration allowing the calculation of the catalytic rate constants, which increase with decreasing pH: 5.3 x 10(9) M(-1) s(-1) (pH 7) to 4.8 x 10(6) M(-1) s(-1) (pH 10) . FeSOD and MnSOD complement each other to keep the superoxide level low in Nostoc PCC 7120, which is discussed with respect to the fact that Nostoc PCC 7120 exhibits oxygenic photosynthesis and oxygen-dependent respiration within a single prokaryotic cell and also has the ability to form differentiated cells under nitrogen-limiting conditions.

World J Gastroenterol, 2004 Sep 1, 10(17), 2560 - 2
Cloning and expression and immunogenicity of Helicobacter pylori BabA2 gene; Bai Y et al.; AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori (H pylori) and to study the immunogenicity of BabA . METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain . Furthermore, BabA immunogenicity was studied by animal test . RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank . The BabA recombinant protein accounted for 34.8% of the total bacterial protein . The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA.CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of H pylori infection .

Acta Crystallogr D Biol Crystallogr, 1997 Sep, 53(Pt 5), 513 - 23
Preliminary analysis of amphibian red cell m ferritin in a novel tetragonal unit cell; Ha Y; The ferritins are a multigene family of proteins that concentrate and store iron in all prokaryotic and eukaryotic cells . 24 monomeric subunits which fold as four-helix bundles assemble to form a protein shell with 432 cubic symmetry and an external diameter of approximately 130 A . The iron is stored inside the protein shell as a mineralized core approximately 80 A in diameter . Recombinant amphibian red cell M ferritin crystallizes in approximately 2 M (NH(4))(2)SO(4) at pH 4.6 in a space group that has not been reported previously . Electron microscopy, precession photography, Patterson and Fourier maps of the native protein and a UO(2)(2+) derivative, and simulations were used to determine that the unit-cell dimensions are a = b = 169.6, c = 481.2 A, alpha = beta = gamma = 90 degrees and the space group is P4(1)2(1)2 or P4(3)2(1)2 . A preliminary model of the structure was obtained by molecular replacement, with amphibian red cell L ferritin as the model . In contrast to previously determined ferritin crystal structures which have intermolecular contacts at the twofold and threefold molecular axes, M ferritin crystals have a novel intermolecular interaction mediated by interdigitation of the DE loops of two molecules at the fourfold molecular axes.

Biorheology, 2004, 41(3-4), 309 - 13
Hydrostatic pressure-induced changes in cellular protein synthesis; Lammi MJ et al.; Hydrostatic pressure is a well-known effector of cellular protein synthesis . High continuous hydrostatic pressure inhibits protein synthesis in general . It has been known for a long time that 30S ribosomal subunit is associated with the effects of pressure on protein synthesis in prokaryotes, however, the mechanisms of action are still not completely understood . Our new data suggest that synthesis of eukaryotic elongation factor-2 (eEF-2) is decreased under 30 MPa continuous hydrostatic pressure . Thus, eEF-2 may have a role in the synthesis of pressure-regulated proteins in eukaryotic cells . The presence of pressure-sensitive proteins indicate that hydrostatic pressure can induce very specific responses in stressed cells . Accumulation of heat shock protein 70 and 90 beta occurs under high pressure, independent of the general inhibition of protein synthesis, although this response appears clearly weaker than during heat stress.

Plant Physiol, 2004 Aug, 135(4), 2046 - 54 Epub 2004 Aug 06.
AtPng1p . The first plant transglutaminase; Della Mea M et al.; Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants . A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase . AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain . AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions . The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies . Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction . The purified protein links polyamines-spermine (Spm) > spermidine (Spd) > putrescine (Put)-and biotin-cadaverine to dimethylcasein in a calcium-dependent manner . Analyses of the gamma-glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of gamma-glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase . The Ca(2+)- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol . To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.

J Bioinform Comput Biol, 2004 Jun, 2(2), 353 - 73
Multivariate entropy distance method for prokaryotic gene identification; Ouyang Z et al.; A new simple method is found for efficient and accurate identification of coding sequences in prokaryotic genome . The method employs a Shannon description of artificial language for DNA sequences . It consists in translating a DNA sequence into a pseudo-amino acid sequence with 20 fundamental words according to the universal genetic code . With an entropy-density profile (EDP), the method maps a sequence of finite length to a vector and then analyzes its position in the 20-dimensional phase space depending on its nature . It is found that the ratio of the relative distance to an averaged coding and non-coding EDP over a small number (up to one) of open reading frames (ORFs) can serve as a good coding potential . An iterative algorithm is designed for finding a set of "root" sequences using this coding potential . A multivariate entropy distance (MED) algorithm is then proposed for the identification of prokaryotic genes; it has a feature to combine the use of a coding potential and an EDP-based sequence similarity analysis . The current version of MED is unsupervised, parameter-free and simple to implement . It is demonstrated to be able to detect 95-99% genes with 10-30% of additional genes when tested against the RefSeq database of NCBI and to detect 97.5-99.8% of confirmed genes with known functions . It is also shown to be able to find a set of (functionally known) genes that are missed by other well-known gene finding algorithms . All measurements show that the MED algorithm reaches a similar performance level as the algorithms like GeneMark and Glimmer for prokaryotic gene prediction.

Mol Biol Evol, 2004 Nov, 21(11), 2149 - 60 Epub 2004 Aug 05.
The evolutionary repertoires of the eukaryotic-type ABC transporters in terms of the phylogeny of ATP-binding domains in eukaryotes and prokaryotes; Igarashi Y et al.; ABC (ATP-binding cassette) transporters play an important role in the communication of various substrates across cell membranes . They are ubiquitous in prokaryotes and eukaryotes, and eukaryotic types (EK-types) are distinguished from prokaryotic types (PK-types) in terms of their genes and domain organizations . The EK-types and PK-types mainly consist of exporters and importers, respectively . Prokaryotes have both the EK-types and the PK-types . The EK-types in prokaryotes are usually called "bacterial multidrug ABC transporters," but they are not well characterized in comparison with the multidrug ABC transporters in eukaryotes . Thus, an exhaustive search of the EK-types among diverse organisms and detailed sequence classification and analysis would elucidate the evolutionary history of EK-types . It would also help shed some light on the fundamental repertoires of the wide variety of substrates through which multidrug ABC transporters in eukaryotes communicate . In this work, we have identified the EK-type ABC transporters in 126 prokaryotes using the profiles of the ATP-binding domain (NBD) of the EK-type ABC transporters from 12 eukaryotes . As a result, 11 clusters were identified from 1,046 EK-types ABC transporters . In particular, two large novel clusters emerged, corresponding to the bacterial multidrug ABC transporters related to the ABCB and ABCC families in eukaryotes, respectively . In the genomic context, most of these genes are located alone or adjacent to genes from the same clusters . Additionally, to detect functional divergences in the NBDs, the Kullback-Leibler divergence was measured among these bacterial multidrug transporters . As a result, several putative functional regions were identified, some corresponding to the predicted secondary structures . We also analyzed a phylogeny of the EK-type ABC transporters in both prokaryotes and eukaryotes, which revealed that the EK-type ABC transporters in prokaryotes have certain repertoires corresponding to the conventional ABC protein groups in eukaryotes . On the basis of these findings, we propose an updated evolutionary hypothesis in which the EK-type ABC transporters in both eukaryotes and prokaryotes consisted of several kinds of ABC transporters in putative ancestor cells before the divergence of eukaryotic and prokaryotic cells.

Curr Opin Biotechnol, 2004 Aug, 15(4), 364 - 73
Prokaryotic expression of antibodies and affibodies; Fernandez LA; Recent advances have been made in the development of systems for the display and expression of recombinant antibodies and affibodies in filamentous phages, Escherichia coli and other prokaryotic cells . Emphasis has been placed on improving phage and phagemid vectors, alternative systems for expression in different cellular compartments (e.g . the outer membrane, periplasm, cytoplasm and extracellular secretion) and novel multimerization systems for generating bivalent or multivalent binding molecules.

Structure (Camb), 2004 Aug, 12(8), 1383 - 94
Structure of Escherichia coli AMP nucleosidase reveals similarity to nucleoside phosphorylases; Zhang Y et al.; AMP nucleosidase (AMN) catalyzes the hydrolysis of AMP to form adenine and ribose 5-phosphate . The enzyme is found only in prokaryotes, where it plays a role in purine nucleoside salvage and intracellular AMP level regulation . Enzyme activity is stimulated by ATP and suppressed by phosphate . The structure of unliganded AMN was determined at 2.7 A resolution, and structures of the complexes with either formycin 5'-monophosphate or inorganic phosphate were determined at 2.6 A and 3.0 A resolution, respectively . AMN is a biological homohexamer, and each monomer is composed of two domains: a catalytic domain and a putative regulatory domain . The overall topology of the catalytic domain and some features of the substrate binding site resemble those of the nucleoside phosphorylases, demonstrating that AMN is a new member of the family . The structure of the regulatory domain consists of a long helix and a four-stranded sheet and has a novel topology.

Acta Biochim Biophys Sin (Shanghai), 2004 Aug, 36(8), 571 - 6
An effective method for raising antisera against beta-defensins: double-copy protein expression of mBin1b in E . coli; Xiao LQ et al.; Bin1b is a rat epididymis specific beta-defensin which may have fertility related functions in addition to its antimicrobial activity . beta-defensins are cysteine-rich cationic antimicrobial peptides that have their important implications in innate and adaptive immunity . Though considerable numbers of new beta-defensins have been discovered, few corresponding antibodies have been reported . The small peptide with special structure and antimicrobial nature of beta-defensins make them very difficult to express in prokaryotic system . Here we adopted a double-copy protein expression scheme based on which not only the mBin1b protein was successfully expressed but also the immunity of the antigen was enhanced . The validity of the antisera was verified by using Western blotting and immunohistochemical analyses . It will be a useful tool for deeply investigating the roles of Bin1b and also provide a simple but effective method in raising antisera against other members of the beta-defensin gene family.

J Biol Chem, 2004 Oct 8, 279(41), 42750 - 7 Epub 2004 Aug 03.
Mass spectrometry of ribosomes from Saccharomyces cerevisiae: implications for assembly of the stalk complex; Hanson CL et al.; The acidic ribosomal P proteins form a distinct protuberance on the 60 S subunit of eukaryotic ribosomes . In yeast this structure is composed of two heterodimers (P1alpha-P2beta and P1beta-P2alpha) attached to the ribosome via P0 . Although for prokaryotic ribosomes the isolation of a pentameric stalk complex comprising the analogous proteins is well established, its observation has not been reported for eukaryotic ribosomes . We used mass spectrometry to examine the composition of the stalk proteins on ribosomes from Saccharomyces cerevisiae . The resulting mass spectra reveal a noncovalent complex of mass 77,291 +/- 7 Da assigned to the pentameric stalk . Tandem mass spectrometry confirms this assignment and is consistent with the location of the P2 proteins on the periphery of the stalk complex, shielding the P1 proteins, which in turn interact with P0 . No other oligomers are observed, confirming the specificity of the pentameric complex . At lower m/z values the spectra are dominated by individual proteins, largely from the stalk complex, giving rise to many overlapping peaks . To define the composition of the stalk proteins in detail we compared spectra of ribosomes from strains in which genes encoding either or both of the interacting stalk proteins P1alpha or P2beta are deleted . This enables us to define novel post-translational modifications at very low levels, including a population of P2alpha molecules with both phosphorylation and trimethylation . The deletion mutants also reveal interactions within the heterodimers, specifically that the absence of P1alpha or P2beta destabilizes binding of the partner protein on the ribosome . This implies that assembly of the stalk complex is not governed solely by interactions with P0 but is a cooperative process involving binding to partner proteins for additional stability on the ribosome.

Asia Pac J Clin Nutr . 2004;13(Suppl):S14.
The regulatory architecture of the human genome; Mattick JS; The draft human genome sequence has provided the first detailed view of the landscape of human genetic programming, with the emphasis to date being on identifying protein-coding genes and determining their biochemical and biological function . However, complex dynamical objects cannot be described just in terms of their components, but must rather be addressed in terms of their integrated function, which includes both the assembly and control of the system . It is these (largely hidden) ontological, physiological and metabolic networks that ultimately determine the emergent effects of variation in endogenous genetic programming and its intersection with environmental variables, including nutrition . Such considerations have motivated the first tentative steps to describe complex cellular and organismal phenomena, including metabolic networks and protein interaction networks, in terms of "scale-free" networks, a concept derived from the connection characteristics of modern electronic networks . However, analysis of integrated systems suggests that regulatory networks which control function are in fact "accelerating" networks, i.e., that regulation must scale non-linearly (usually quadratically) with function . This has been confirmed by analysis of regulatory genes in prokaryotes, which scale quadratically with genome size, the observed upper limit of which (about 12Mb) correlates with the extrapolated point at which new regulators are predicted to exceed new functional genes, suggesting that protein-based regulatory systems have reached their limit in these organisms . The current orthodoxy holds that genes are generally synonymous with proteins, and therefore that proteins not only fulfil the structural and functional roles within cells, but are also the main agents by which cellular dynamics are controlled, in conjunction with cis-regulatory elements and environmental signals . This is true in prokaryotes, whose genomes are very largely comprised of contiguous protein coding sequences . It is assumed that this is also true in multicellular organisms, despite the fact the proportion of protein-coding sequences declines as a function of complexity and is only a small minority of the genomic programming of complex organisms like mammals . This assumption has led to several logical extensions and subsidiary assumptions, in particular that the increased complexity of eukaryotes is explained by the combinatorics of regulatory factors intersecting with more complex promoters, with the corollary that the majority of non-protein-coding sequences in eukaryotic genomes are either cis-regulatory elements or evolutionary debris (i.e . junk) . This may not be correct . Around 98% of the transcriptional output of the human genome is non-protein-coding RNA (derived from introns of protein-coding genes and from non-protein-coding genes, of which increasing numbers are being discovered), and at least half of the human genome is transcribed . Therefore either the human genome is replete with useless transcription, or these RNAs are fulfilling some unexpected function . In addition it is becoming evident that a significant proportion of the noncoding regions of the human genome is under evolutionary constraint, some of it much more highly conserved than proteins . Such observations and the increasing number of complex genetic phenomena being shown to be directed by regulatory RNAs, suggests that the complex organisms may have evolved a more advanced genetic operating system, which occupies the majority of our genome sequence, and in which ncRNA signals constitute a highly parallel network of digital, feed-forward regulatory signals that control differentiation and development . Variation in this regulatory architecture may be equally if not more important than variation in the (protein) components in determining the differences between individuals and species, including quantitative trait variation, sensitivity to environmental parameters and susceptibility to disease.

Protein Expr Purif, 2004 Sep, 37(1), 47 - 52
Prokaryotic expression, polyclonal antibody preparation, and sub-cellular localization analysis of Na+, K+-ATPase beta2 subunit; Chen TF et al.; Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule . To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl portion segment of NKA1b2 . The coding region for amino acids 190-290 at the carboxyl portion of NKA1b2 (NKA1b2-CP) was sub-cloned into the vector pGEX-4T-2 and introduced into the Escherichia coli BL21(DE3) cell for efficient soluble expression . The amino acid sequence of expressed protein was determined using mass spectrometry following Mascot analysis . After purification, GST-NKA-beta2-CP was used to immunize the adult rabbits following standard protocols . The produced antiserum could detect the NKA1b2 protein expressed not only in the prokaryotic cells (E . coli) but also in the eukaryotic cells (COS7) transfected with NKA1b2 expression vector (pEGFP-NKA1b2) . Furthermore, the antiserum was used for determining the localization of NKA1b2 in primary culture of neonatal rat neurons using immunohistochemical technique . Results demonstrated that NKA1b2 was localized both in the cytoplasm and cellular membrane . The preparation of anti-NKA-beta2-CP polyclonal antibody will facilitate further functional study on NKA1b2.

Protein Expr Purif, 2004 Sep, 37(1), 1 - 7
Engineering, cloning, and expression of genes encoding the multimeric luteinizing-hormone-releasing hormone linked to T cell determinants in Escherichia coli; Gupta JC et al.; Two synthetic genes were designed and engineered to encode for multimeric luteinizing-hormone-releasing hormone (LHRH) peptides linked to T cell determinants . These genes were cloned into the prokaryotic expression vectors under control of strong inducible promoters, to overexpress the multimeric LHRH peptides as recombinant proteins . Multimeric LHRH-T cell peptides were expressed as insoluble inclusion bodies in Escherichia coli cultures . Cell extracts containing the recombinant proteins showed immunoreactivity on Western blots with monoclonal antibody recognizing the native hormonal peptide . These gene constructs have potential applications in therapy of sex-steroid-hormone-dependent cancers.

Angew Chem Int Ed Engl, 2004 Jul 5, 43(27), 3526 - 48
Sulfotransferases: structure, mechanism, biological activity, inhibition, and synthetic utility; Chapman E et al.; The sulfonation (also known as sulfurylation) of biomolecules has long been known to take place in a variety of organisms, from prokaryotes to multicellular species, and new biological functions continue to be uncovered in connection with this important transformation . Early studies of sulfotransferases (STs), the enzymes that catalyze sulfonation, focused primarily on the cytosolic STs, which are involved in detoxification, hormone regulation, and drug metabolism . Although known to exist, the membrane-associated STs were not studied as extensively until more recently . Involved in the sulfonation of complex carbohydrates and proteins, they have emerged as central players in a number of molecular-recognition events and biochemical signaling pathways . STs have also been implicated in many pathophysiological processes . As a result, much interest in the complex roles of STs and in their targeting for therapeutic intervention has been generated . Progress in the elucidation of the structures and mechanisms of sulfotransferases, as well as their biological activity, inhibition, and synthetic utility, are discussed in this Review.

Proc Natl Acad Sci U S A, 2004 Aug 10, 101(32), 11605 - 10 Epub 2004 Aug 03.
Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions; Amit R et al.; Holliday junctions form during DNA repair and homologous recombination processes . These processes entail branch migration, whereby the length of two arms of a cruciform increases at the expense of the two others . Branch migration is carried out in prokaryotic cells by the RuvAB motor complex . We study RuvAB-catalyzed branch migration by following the motion of a small paramagnetic bead tethered to a surface by two opposing arms of a single cruciform . The bead, pulled under the action of magnetic tweezers, exerts tension on the cruciform, which in turn transmits the force to a single RuvAB complex bound at the crossover point . This setup provides a unique means of measuring several kinetic parameters of interest such as the translocation rate, the processivity, and the force on the substrate against which the RuvAB complex cannot effect translocation . RuvAB-catalyzed branch migration proceeds with a small, discrete number of rates, supporting the view that the monomers comprising the RuvB hexameric rings are not functionally homogeneous and that dimers or trimers constitute the active subunits . The most frequently encountered rate, 98 +/- 3 bp/sec, is approximately five times faster than previously estimated . The apparent processivity of branch migration between pauses of inactivity is approximately 7,000 bp . Branch migration persists against opposing forces up to 23 pN.

J Biol Chem, 2004 Sep 24, 279(39), 40677 - 82 Epub 2004 Jul 29.
The mechanism of potent GTP cyclohydrolase I inhibition by 2,4-diamino-6-hydroxypyrimidine: requirement of the GTP cyclohydrolase I feedback regulatory protein; Kolinsky MA et al.; Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in a biological system . Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor . Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH . Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP) . Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4 . Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system . We further demonstrate that L-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP . These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of L-Phe .

Eur J Biochem, 2004 Aug, 271(16), 3449 - 56
Functional characterization of the evolutionarily divergent fern plastocyanin; Navarro JA et al.; Plastocyanin (Pc) is a soluble copper protein that transfers electrons from cytochrome b(6)f to photosystem I (PSI), two protein complexes that are localized in the thylakoid membranes in chloroplasts . The surface electrostatic potential distribution of Pc plays a key role in complex formation with the membrane-bound partners . It is practically identical for Pcs from plants and green algae, but is quite different for Pc from ferns . Here we report on a laser flash kinetic analysis of PSI reduction by Pc from various eukaryotic and prokaryotic organisms . The reaction of fern Pc with fern PSI fits a two-step kinetic model, consisting of complex formation and electron transfer, whereas other plant systems exhibit a mechanism that requires an additional intracomplex rearrangement step . The fern Pc interacts inefficiently with spinach PSI, showing no detectable complex formation . This can be explained by assuming that the unusual surface charge distribution of fern Pc impairs the interaction . Fern PSI behaves in a similar way as spinach PSI in reaction with other Pcs . The reactivity of fern Pc towards several soluble c-type cytochromes, including cytochrome f, has been analysed by flavin-photosensitized laser flash photolysis, demonstrating that the specific surface motifs for the interaction with cytochrome f are conserved in fern Pc.

Eur J Biochem, 2004 Aug, 271(16), 3297 - 309
Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases; Zamocky M; Molecular phylogeny among catalase-peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed . Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected . Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity . The N-terminal and C-terminal domains of catalase-peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history . The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase-peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily . From the results, it is obvious that the duplication event in the gene for catalase-peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed . Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution.

J Bioinform Comput Biol, 2003 Oct, 1(3), 475 - 93
A comprehensive whole genome bacterial phylogeny using correlated peptide motifs defined in a high dimensional vector space; Stuart GW et al.; As whole genome sequences continue to expand in number and complexity, effective methods for comparing and categorizing both genes and species represented within extremely large datasets are required . Methods introduced to date have generally utilized incomplete and likely insufficient subsets of the available data . We have developed an accurate and efficient method for producing robust gene and species phylogenies using very large whole genome protein datasets . This method relies on multidimensional protein vector definitions supplied by the singular value decomposition (SVD) of a large sparse data matrix in which each protein is uniquely represented as a vector of overlapping tetrapeptide frequencies . Quantitative pairwise estimates of species similarity were obtained by summing the protein vectors to form species vectors, then determining the cosines of the angles between species vectors . Evolutionary trees produced using this method confirmed many accepted prokaryotic relationships . However, several unconventional relationships were also noted . In addition, we demonstrate that many of the SVD-derived right basis vectors represent particular conserved protein families, while many of the corresponding left basis vectors describe conserved motifs within these families as sets of correlated peptides (copeps) . This analysis represents the most detailed simultaneous comparison of prokaryotic genes and species available to date.

Proc Natl Acad Sci U S A, 2004 Aug 10, 101(32), 11646 - 51 Epub 2004 Aug 02.
In different organisms, the mode of interaction between two signaling proteins is not necessarily conserved; Park SY et al.; Although interfaces mediating protein-protein interactions are thought to be under strong evolutionary constraints, binding of the chemotaxis histidine kinase CheA to its phosphorylation target CheY suggests otherwise . The structure of Thermotoga maritima CheA domain P2 in complex with CheY reveals a different association than that observed for the same Escherichia coli proteins . Similar regions of CheY bind CheA P2 in the two systems, but the CheA P2 domains differ by an approximately 90 degrees rotation . CheA binds CheY with identical affinity in T . maritima and E . coli at the vastly different temperatures where the respective organisms live . Distinct sets of P2 residues mediate CheY binding in the two complexes; conservation patterns of these residues in CheA and compensations in CheY delineate two families of prokaryotic chemotaxis systems . A protein complex that has the same components and general function in different organisms, but an altered structure, indicates unanticipated complexity in the evolution of protein-protein interactions and cautions against extrapolating structural data from homologs.

Proc Natl Acad Sci U S A, 2004 Aug 10, 101(32), 11821 - 6 Epub 2004 Aug 02.
Targeting cell division: small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality; Margalit DN et al.; FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells . Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis . We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ . Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association . These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively . We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents . Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target.

Nucleic Acids Res, 2004 Aug 02, 32(13), 4081 - 9 Print 2004.
Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo; Martin F et al.; Aminoacyl-tRNA synthetases (aaRSs) are enzymes that are highly specific for their tRNA substrates . Here, we describe the expansion of a class IIb aaRS-tRNA specificity by a genetic selection that involves the use of a modified tRNA displaying an amber anticodon and the argE(amber) and lacZ(amber) reporters . The study was performed on Escherichia coli aspartyl-tRNA synthetase (AspRS) and amber tRNA(Asp) . Nine AspRS mutants able to charge the amber tRNA(Asp) and to suppress the reporter genes were selected from a randomly mutated library . All the mutants exhibited a new amber tRNA(Asp) specificity in addition to the initial native tRNA(Asp) . Six mutations were found in the anticodon-binding site located in the N-terminal OB-fold . The strongest suppressor was a mutation of residue Glu-93 that contacts specifically the anticodon nucleotide 34 in the crystal structure . The other mutations in the OB-fold were found at close distance from the anticodon in the so-called loop L45 and strand S1 . They concern residues that do not contact tRNA(Asp) in the native complex . In addition, this study shows that suppressors can carry mutations located far from the anticodon-binding site . One such mutation was found in the synthetase hinge-module where it increases the tRNA(Asp)-charging rate, and two other mutations were found in the prokaryotic-specific insertion domain and the catalytic core . These mutants seem to act by indirect effects on the tRNA acceptor stem binding and on the conformation of the active site of the enzyme . Altogether, these data suggest the existence of various ways for modifying the mechanism of tRNA discrimination.

Genome Res, 2004 Aug, 14(8), 1575 - 84
Stress-induced DNA duplex destabilization (SIDD) in the E . coli genome: SIDD sites are closely associated with promoters; Wang H et al.; We present the first analysis of stress-induced DNA duplex destabilization (SIDD) in a complete chromosome, the Escherichia coli K12 genome . We used a newly developed method to calculate the locations and extents of stress-induced destabilization to single-base resolution at superhelix density sigma = -0.06 . We find that SIDD sites in this genome show a statistically highly significant tendency to avoid coding regions . And among intergenic regions, those that either contain documented promoters or occur between divergently transcribing coding regions, and hence may be inferred to contain promoters, are associated with strong SIDD sites in a statistically highly significant manner . Intergenic regions located between convergently transcribing genes, which are inferred not to contain promoters, are not significantly enriched for destabilized sites . Statistical analysis shows that a strongly destabilized intergenic region has an 80% chance of containing a promoter, whereas an intergenic region that does not contain a strong SIDD site has only a 24% chance . We describe how these observations may illuminate specific mechanisms of regulation, and assist in the computational identification of promoter locations in prokaryotes .

Biotechnol Bioeng, 2004 Aug 20, 87(4), 478 - 84
BioLogic gates enable logical transcription control in mammalian cells; Kramer BP et al.; The architecture of gene regulatory networks is reminiscent of electronic circuits . Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks . Gene circuit engineers have pioneered the construction of artificial gene regulatory networks with the intention to pave the way for the construction of therapeutic gene circuits for next-generation gene therapy approaches . However, due to the lack of a critical amount of eukaryotic cell-compatible gene regulation systems, the field has so far been limited to prokaryotes . Recent development of several mammalian cell-compatible expression control systems laid the foundations for the assembly of transcription control modules that can respond to several inputs . Herein, three approaches to evoke combinatorial transcription control have been followed: (i) construction of artificial promoters with up to three operator sites for regulatory proteins, and (ii) parallel and (iii) serial linking of two gene regulation systems . We have combined tetracycline-, streptogramin-, macrolide-, and butyrolactone transcription control systems to engineer BioLogic gates of the NOT IF-, AND-, NOT IF IF-, NAND-, OR-, NOR-, and INVERTER-type in mammalian cells, which are able to respond to up to three different small molecule inputs . BioLogic gates enable logical transcriptional control in mammalian cells and, in combination with modern transduction technologies, could serve as versatile tools for regulated gene expression and as building blocks for complex artificial gene regulatory networks for applications in gene therapy, tissue engineering, and biotechnology.

Methods Mol Med, 2004, 102, 155 - 72
Evaluation of autoimmunity to transaldolase in multiple sclerosis; Niland B et al.; Transaldolase is a target of autoimmunity mediated by T cells and antibody (Ab) in patients with multiple sclerosis . Functional T-cell assays, T- and B-cell epitope mapping, and detection of transaldolase-specific antibodies in patients with multiple sclerosis are described . Recombinant transaldolase was produced in a prokaryotic expression vector for use in Western blot analysis of sera of these patients . Overlapping transaldolase peptides 15 amino acids (aa) long were synthesized onto cellulose membranes to map immunodominant B-cell epitopes . Amino acid sequence homologies between viral peptides and immunodominant B-cell epitopes of transaldolase were identified using a computer-based algorithm . Direct assessment of molecular mimicry between transaldolase B-cell epitopes and related viral peptides is also shown . T-cell epitopes are mapped in a T-cell proliferation assay using multiple sclerosis patient and control donor cells . Autoantigen-specific T cells are identified by MHC-peptide tetramer staining using flow cytometry analysis.

Biochem J, 2004 Nov 15, 384(Pt 1), 179 - 90
Guanidinium chloride denaturation of the dimeric Bacillus licheniformis BlaI repressor highlights an independent domain unfolding pathway; Vreuls C et al.; The Bacillus licheniformis 749/I BlaI repressor is a prokaryotic regulator that, in the absence of a beta-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase . The BlaI repressor is composed of two structural domains . The 82-residue NTD (N-terminal domain) is a DNA-binding domain, and the CTD (C-terminal domain) containing the next 46 residues is a dimerization domain . Recent studies have shown the existence of the monomeric, dimeric and tetrameric forms of BlaI in solution . In the present study, we analyse the equilibrium unfolding of BlaI in the presence of GdmCl (guanidinium chloride) using different techniques: intrinsic and ANS (8-anilinonaphthalene-l-sulphonic acid) fluorescence, far- and near-UV CD spectroscopy, cross-linking, analytical ultracentrifugation, size exclusion chromatography and NMR spectroscopy . In addition, the intact NTD and CTD were purified after proteolysis of BlaI by papain, and their unfolding by GdmCl was also studied . GdmCl-induced equilibrium unfolding was shown to be fully reversible for BlaI and for the two isolated fragments . The results demonstrate that the NTD and CTD of BlaI fold/unfold independently in a four-step process, with no significant co-operative interactions between them . During the first step, the unfolding of the BlaI CTD occurs, followed in the second step by the formation of an 'ANS-bound' intermediate state . Cross-linking and analytical ultracentrifugation experiments suggest that the dissociation of the dimer into two partially unfolded monomers takes place in the third step . Finally, the unfolding of the BlaI NTD occurs at a GdmCl concentration of approx . 4 M . In summary, it is shown that the BlaI CTD is structured, more flexible and less stable than the NTD upon GdmCl denaturation . These results contribute to the characterization of the BlaI dimerization domain (i.e . CTD) involved in the induction process.

Sheng Li Ke Xue Jin Zhan, 2004 Apr, 35(2), 119 - 24
{Recent advances of protein phosphorylation in proteome}; Yang C et al.; With the advent of post-genomic era, it is an important task to study the expression, modification and interaction of the total proteins in cells, tissue and organs for proteomics in biological organism . The protein phosphorylation is one of the most popular mechanisms in signal transduction and enzyme modulation . It is estimated that about 2% of the total genes encoded 500 kinases and 100 phosphatases in human genome . As the key point of expression modulation in prokaryotic and eukaryotic cells, the protein phosphorylation and dephosphorylation may help to reveal the status of the life system at the molecular level . The phosphorylation in proteome remains challenging for functional genomics . In the present review, some new advances such as identification and characterization of protein phosphorylation in proteome are summarized.

J Biol Chem, 2004 Oct 1, 279(40), 41664 - 9 Epub 2004 Jul 28.
A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) mediates ATPase activity; Kidd JF et al.; The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ABC superfamily of transporter proteins . Recently, crystal structures of intact, prokaryotic members of this family have been described . These structures suggested that ATP binding and hydrolysis occurs at two sites formed at the interface between their nucleotide binding domains (NBDs) . In contrast to the prokaryotic family members, the NBDs of CFTR are asymmetric (both structurally and functionally), and previous to the present studies, it was not clear whether both NBDs are required for ATP hydrolysis . In order to assess the relative roles of the two NBDs of human CFTR, we purified and reconstituted NBD1 and NBD2, separately and together . We found that NBD1 and NBD2 by themselves exhibited relatively low ATPase activity . Co-assembly of NBD1 and NBD2 exhibited a 2-3-fold enhancement in catalytic activity relative to the isolated domains and this increase reflected enhanced ATP turnover (V(max)) . These data provide the first direct evidence that heterodimerization of the NBD1 and NBD2 domains of CFTR is required to generate optimal catalytic activity.

Biochem J, 2004 Nov 1, 383(Pt . 3), 551 - 9
Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay; Gul S et al.; DNA ligases are key enzymes involved in the repair and replication of DNA . Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP . This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention . We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases . The assay has been used to determine a number of kinetic constants for S . aureus DNA ligase catalysis . These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM) . A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)) . In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1 . Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate . The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Annu Rev Phytopathol, 2004, 42, 83 - 105
Evolution of plant parasitism among nematodes; Baldwin JG et al.; Despite extraordinary diversity of free-living species, a comparatively small fraction of nematodes are parasites of plants . These parasites represent at least three disparate clades in the nematode tree of life, as inferred from rRNA sequences . Plant parasites share functional similarities regarding feeding, but many similarities in feeding structures result from convergent evolution and have fundamentally different developmental origins . Although Tylenchida rRNA phylogenies are not fully resolved, they strongly support convergent evolution of sedentary endoparasitism and plant nurse cells in cyst and root-knot nematodes . This result has critical implications for using model systems and genomics to identify and characterize parasitism genes for representatives of this clade . Phylogenetic studies reveal that plant parasites have rich and complex evolutionary histories that involve multiple transitions to plant parasitism and the possible use of genes obtained by horizontal transfer from prokaryotes . Developing a fuller understanding of plant parasitism will require integrating more comprehensive and resolved phylogenies with appropriate choices of model organisms and comparative evolutionary methods.

Biochim Biophys Acta, 2004 Jul 23, 1658(1-2), 37 - 43
The protein import and assembly machinery of the mitochondrial outer membrane; Taylor RD et al.; The process of mitochondrial protein import has been studied for many years . Despite this attention, many processes associated with mitochondrial biogenesis are poorly understood . Insight into one of these processes, assembly of beta-barrel proteins into the mitochondrial outer membrane, will be discussed . This review focuses on recent data that suggest that assembly of beta-barrel proteins into the outer mitochondrial membrane is dependent on a newly identified protein complex termed the sorting and assembly machinery (SAM complex) . Members of the SAM complex have been identified in both eukaryotic and prokaryotic organisms, suggesting that the process of beta-barrel assembly into membranes has been conserved through evolution.

Biochim Biophys Acta, 2004 Jul 23, 1658(1-2), 2 - 13
NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter; Padan E et al.; Na(+)/H(+) antiporters are ubiquitous membrane proteins that are involved in homeostasis of H(+) and Na(+) throughout the biological kingdom . Corroborating their role in pH homeostasis, many of the Na(+)/H(+) antiporter proteins are regulated directly by pH . The pH regulation of NhaA, the Escherichia coli Na(+)/H(+) antiporter (EcNhaA), as of other, both eukaryotic and prokaryotic Na(+)/H(+) antiporters, involves a pH sensor and conformational changes in different parts of the protein that transduce the pH signal into a change in activity . Thus, residues that affect the pH response, the translocation or both activities cluster in separate domains along the antiporter molecules . Importantly, in the NhaA family, these domains are conserved . Helix-packing model of EcNhaA based on cross-linking data suggests, that in the three dimensional structure of NhaA, residues that affect the pH response may be in close proximity, forming a single pH sensitive domain . Therefore, it is suggested that, despite considerable differences in the primary structure of the antiporters from the bacterial NhaA to the mammalian NHEs, their three-dimensional architectures are conserved . Test of this possibility awaits the atomic resolution of the 3D structure of the antiporters.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Apr 30, 22(2), 94 - 7
{Soluble expression of Plasmodium falciparum glutamate dehydrogenase in Escherichia coli, and its purification and identification}; Li Y et al.; OBJECTIVE: To make soluble expression of Plasmodium falciparum (FCC1/HN) glutamate dehydrogenase (GDH) in Escherichia coli, purification and immunocompetence identification of the recombinant non-fusion GDH . METHODS: The GDH gene was cloned into prokaryotic expression vector pET23 (a) to form recombinant expression vector pET23 (a)/GDH . pET23(a)/GDH was transformed into E . coli BL21 (DE3) . Induced by IPTG (isopropyl-beta D-thiogalactoside), GDH was highly expressed in the supernatant after sonication . The soluble recombinant GDH was purified by Source-Q and Source-S chromatography . Enzyme-linked immunosorbent assay and Western blotting were carried out to identify the immunocompetence of the purified product . RESULTS: SDS-PAGE analysis showed that the soluble GDH protein accounted for approximately 15% of the total bacterial protein . By two-step ion-exchange chromatography, the purity of GDH reached more than 90% and the GDH possessed high antigenicity . CONCLUSION: The soluble expression of GDH results in an integral three-dimensional structure epitope with high biological activity.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Apr 30, 22(2), 76 - 9
{Expression of proteinase cathepsin L1 gene of Schistosoma japonicum in Escherichia coli}; Yi B et al.; OBJECTIVE: To express the proteinase cathepsin L1 gene of Schistosoma japonicum (SjCL1) in Escherichia coli JM109 cells . METHODS: The SjCL1 gene was amplified from the recombinant plasmid pcDNA3-SjCL1 by PCR . The gene was cloned into a prokaryotic expression vector pGEX4T-1 to construct a recombinant plasmid pGEX-SjCL1 . The E . coli JM109 cells were transformed with the recombinant plasmid pGEX-SjCL1 and the transformants were induced by IPTG to express the recombinant protein, the target protein was then identified by SDS-PAGE and Western blotting . RESULTS: A 1 kb length PCR product was obtained and a recombinant plasmid pGEX-SjCL1 was constructed . The expression product was detected by SDS-PAGE and Western blotting and an expression band about 62000 was found . CONCLUSION: The SjCL1 gene is effectively expressed in the E . coli JM109 cells.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1415 - 21
Recommended standards for the description of new species of anoxygenic phototrophic bacteria; Imhoff JF et al.; Recommended standards for the description of new species of the anoxygenic phototrophic bacteria are proposed in accordance with Recommendation 30b of the International Code of Nomenclature of Bacteria . These standards include information on the natural habitat, ecology and phenotypic properties including morphology, physiology and pigments and on genetic information and nucleic acid data . The recommended standards were supported by the Subcommittee on the taxonomy of phototrophic bacteria of the International Committee on Systematics of Prokaryotes . They are considered as guidelines for authors to prepare descriptions of new species.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1243 - 55
'Candidatus Phytoplasma', a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects; IRPCM Phytoplasma/Spiroplasma Working Team--Phytoplasma Taxonomy Group; The trivial name 'phytoplasma' has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms . Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes . It is proposed here to accommodate phytoplasmas within the novel genus 'Candidatus (Ca.) Phytoplasma' . Given the diversity within 'Ca . Phytoplasma', several subtaxa are needed to accommodate organisms that share <97.5% similarity among their 16S rRNA gene sequences . This report describes the properties of 'Ca . Phytoplasma', a taxon that includes the species 'Ca . Phytoplasma aurantifolia' (the prokaryote associated with witches'-broom disease of small-fruited acid lime), 'Ca . Phytoplasma australiense' (associated with Australian grapevine yellows), 'Ca . Phytoplasma fraxini' (associated with ash yellows), 'Ca . Phytoplasma japonicum' (associated with Japanese hydrangea phyllody), 'Ca . Phytoplasma brasiliense' (associated with hibiscus witches'-broom in Brazil), 'Ca . Phytoplasma castaneae' (associated with chestnut witches'-broom in Korea), 'Ca . Phytoplasma asteris' (associated with aster yellows), 'Ca . Phytoplasma mali' (associated with apple proliferation), 'Ca . Phytoplasma phoenicium' (associated with almond lethal disease), 'Ca . Phytoplasma trifolii' (associated with clover proliferation), 'Ca . Phytoplasma cynodontis' (associated with Bermuda grass white leaf), 'Ca . Phytoplasma ziziphi' (associated with jujube witches'-broom), 'Ca . Phytoplasma oryzae' (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence doree, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively) . Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97.5% similar to those of other 'Ca . Phytoplasma' species, are characterized by distinctive biological, phytopathological and genetic properties . These include 'Ca . Phytoplasma pyri' (associated with pear decline), 'Ca . Phytoplasma prunorum' (associated with European stone fruit yellows), 'Ca . Phytoplasma spartii' (associated with spartium witches'-broom), 'Ca . Phytoplasma rhamni' (associated with buckthorn witches'-broom), 'Ca . Phytoplasma allocasuarinae' (associated with allocasuarina yellows), 'Ca . Phytoplasma ulmi' (associated with elm yellows) and an additional taxon for the stolbur phytoplasma . Conversely, some organisms, despite their 16S rRNA gene sequence being <97.5% similar to that of any other 'Ca . Phytoplasma' species, are not presently described as Candidatus species, due to their poor overall characterization.

FEBS Lett, 2004 Jul 30, 571(1-3), 161 - 5
Human peroxiredoxin 5 is a peroxynitrite reductase; Dubuisson M et al.; Peroxiredoxins are an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes . Peroxiredoxin 5, which is the last discovered mammalian member, was previously shown to reduce peroxides with the use of reducing equivalents derived from thioredoxin . We report here that human peroxiredoxin 5 is also a peroxynitrite reductase . Analysis of peroxiredoxin 5 mutants, in which each of the cysteine residues was mutated, suggests that the nucleophilic attack on the O-O bond of peroxynitrite is performed by the N-terminal peroxidatic Cys(47) . Moreover, with the use of pulse radiolysis, we show that human peroxiredoxin 5 reduces peroxynitrite with an unequalled high rate constant of (7+/-3)x10(7) M(-1)s(-1).

Biochem J, 2004 Nov 1, 383(Pt . 3), 419 - 28
Crystal structures of the antitermination factor NusB from Thermotoga maritima and implications for RNA binding; Bonin I et al.; NusB is a prokaryotic transcription factor involved in antitermination processes, during which it interacts with the boxA portion of the mRNA nut site . Previous studies have shown that NusB exhibits an all-helical fold, and that the protein from Escherichia coli forms monomers, while Mycobacterium tuberculosis NusB is a dimer . The functional significance of NusB dimerization is unknown . We have determined five crystal structures of NusB from Thermotoga maritima . In three crystal forms the protein appeared monomeric, whereas the two other crystal forms contained assemblies, which resembled the M . tuberculosis dimers . In solution, T . maritima NusB could be cross-linked as dimers, but it migrated as a monomer in gel-filtration analyses, suggesting a monomer/dimer equilibrium with a preference for the monomer . Binding to boxA-like RNA sequences could be detected by gel-shift analyses and UV-induced cross-linking . An N-terminal arginine-rich sequence is a probable RNA binding site of the protein, exhibiting aromatic residues as potential stacking partners for the RNA bases . Anions located in various structures support the assignment of this RNA binding site . The proposed RNA binding region is hidden in the subunit interface of dimeric NusB proteins, such as NusB from M . tuberculosis, suggesting that such dimers have to undergo a considerable conformational change or dissociate for engagement with RNA . Therefore, in certain organisms, dimerization may be employed to package NusB in an inactive form until recruitment into antitermination complexes.

Appl Microbiol Biotechnol . 2004 Jul 24; {Epub ahead of print}
Recombinant expression systems in the pharmaceutical industry; Schmidt FR; In terms of downstream processing efficiency, secretory expression systems offer potential advantages for the production of recombinant proteins, compared with inclusion body forming cytosolic systems . However, for high-volume therapeutics like insulin, the product yields of the majority of the potentially available secretory systems is not yet fully competitive . Current strategies to improve productivity and secretion efficiency comprise: (1) enhancement of gene expression rates, (2) optimization of secretion signal sequences, (3) coexpression of chaperones and foldases, (4) creation of protease deficient mutants to avoid premature product degradation and (5) subsequent breeding and mutagenesis . For the production of non-glycosylated proteins and proteins, which are natively glycosylated but are also pharmacologically active without glycosylation, prokaryotes, which usually lack metabolic pathways for glycosylation, are theoretically the most suitable organisms and offer two alternatives: either Escherichia coli strains are conditioned to be efficient secreters or efficient native secreters like Bacillus species are accordingly developed . To fully exploit the secretory capacity of fungal species, a deeper understanding of their posttranslational modification physiology will be necessary to steer the degree and pattern of glycosylation, which influences both folding and secretion efficiency . Insect and mammalian cells display posttranslational modification patterns very similar or identical to humans, but in view of the entailed expenditures, their employment can only be justified if their modification machinery is required to ensure a desired pharmacological activity.

Naturwissenschaften, 2004 Aug, 91(8), 371 - 3 Epub 2004 Jul 24.
The bacterium Xenorhabdus nematophila inhibits phospholipases A2 from insect, prokaryote, and vertebrate sources; Park Y et al.; The bacterium, Xenorhabdus nematophila, is a virulent insect pathogen . Part of its pathogenicity is due to impairing cellular immunity by blocking biosynthesis of eicosanoids, the major recognized signal transduction system in insect cellular immunity . X . nematophila inhibits the first step in eicosanoid biosynthesis, phospholipase A(2) (PLA(2)) . Here we report that the bacterium inhibits PLA(2) from two insect immune tissues, hemocytes and fat body, as well as PLA(2)s selected to represent a wide range of organisms, including prokaryotes, insects, reptiles, and mammals . Our finding on a bacterial inhibitor of PLA(2) activity contributes new insight into the chemical ecology of microbe-host interactions, which usually involve actions rather than inhibitors of PLA(2)s.

Med Sci Monit, 2004 Aug, 10(8), BR294 - 9 Epub 2004 Jul 23.
Cytotoxic and genotoxic effects of ss-(triphenylpho-s-phonio)ethyl carboxylate and of N,N'-bis(dihexylphos-phinoylmethyl)-1,4-diaminocyclohexane; Ilinskaya O et al.; BACKGROUND: Several organophosphorous compounds (OPs) are now being tested therapeutically . Cholinesterase inhibition, which in large doses makes these agents effective pesticides, may also be useful in other doses for treating dementia . Metrifonate, for example, has been used to treat schistosomiasis and is undergoing trials for the treatment of primary degenerative dementia . MATERIAL/METHODS: Here we report the characterization of newly synthesized OPs from the group of phosphobetaines {beta-(triphenylphosphonio)ethyl carboxylate, PB} and of alpha-aminophosphoryl compounds {N,N'-bis(dihexylphosphinoylmethyl)-1,4-diaminocyclohexane, AP} according to their toxic and genotoxic properties determined in prokaryotic and eukaryotic test systems . RESULTS: The absence of toxicity towards Gram-negative bacteria and of genotoxicity in Ames mutagenicity assay and in SOS-chromotest did not exclude the cytotoxic effect of PB towards NIH3T3 mouse fibroblasts, which supports the notion of an extremely diverse interspecies response to OPs . In contrast, AP demonstrated toxic properties detected by antibacterial effect as well as by the inhibition of the proliferation and respiration of fibroblasts . The enzymatic transformation of the compound is necessary to reveal the genotoxic properties of AP . The role of mammalian microsomal enzymes and of bacterial C-P lyase in the formation of AP genotoxic metabolites is under discussion . CONCLUSIONS: Neither toxicity nor genotoxicity of PB was found in bacterial tests . Cytotoxic and mutagenic effects of AP were detected . The data contribute to the investigation of the biological activity of novel organophosphates which could be useful for the future development of OP-based therapeutics.

Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11310 - 5 Epub 2004 Jul 26.
RNA dynamics in live Escherichia coli cells; Golding I et al.; We describe a method for tracking RNA molecules in Escherichia coli that is sensitive to single copies of mRNA, and, using the method, we find that individual molecules can be followed for many hours in living cells . We observe distinct characteristic dynamics of RNA molecules, all consistent with the known life history of RNA in prokaryotes: localized motion consistent with the Brownian motion of an RNA polymer tethered to its template DNA, free diffusion, and a few examples of polymer chain dynamics that appear to be a combination of chain fluctuation and chain elongation attributable to RNA transcription . We also quantify some of the dynamics, such as width of the displacement distribution, diffusion coefficient, chain elongation rate, and distribution of molecule numbers, and compare them with known biophysical parameters of the E . coli system.

J Gen Physiol, 2004 Aug, 124(2), 151 - 61
Gating transitions in bacterial ion channels measured at 3 micros resolution; Shapovalov G et al.; Ion channels of high conductance (>200 pS) are widespread among prokaryotes and eukaryotes . Two examples, the Escherichia coli mechanosensitive ion channels Ec-MscS and Ec-MscL, pass currents of 125-300 pA . To resolve temporal details of conductance transitions, a patch-clamp setup was optimized for low-noise recordings at a time resolution of 3 micros (10-20 times faster than usual) . Analyses of the high-resolution recordings confirm that Ec-MscL visits many subconductance states and show that most of the intersubstate transitions occur more slowly than the effective resolution of 3 micros . There is a clear trend toward longer transition times for the larger transitions . In Ec-MscS recordings, the majority of the observed full conductance transitions are also composite . We detected a short-lived (approximately 20 micros) Ec-MscS substate at 2/3 of full conductance; transitions between 2/3 and full conductance did not show fine structure and had a time course limited by the achieved resolution . Opening and closing transitions in MscS are symmetrical and are not preceded or followed by smaller, rapid currents ("anticipations" or "regrets") . Compared with other, lower-conductance channels, these measurements may detect unusually early states in the transitions from fully closed to fully open . Increased temporal resolution at the single-molecule level reveals that some elementary steps of structural transitions are composite and follow several alternative pathways, while others still escape resolution . High-bandwidth, low-noise single-channel measurements may provide details about state transitions in other high-conductance channels; and similar procedures may also be applied to channel- and nanopore-based single-molecule DNA measurements.

Parasitol Today, 1996, 12(1), 7 - 14
Progress towards an amebiasis vaccine; Stanley SL Jr; Amebiasis (infection by Entamoeba histolytica) remains a major health problem in much of the developing world . Morbidity and mortality from amebic dysentery and amebic liver abscess have persisted despite the availability of effective anti-amebic therapy, suggesting a need for alternative measures of disease control . Through the application of recombinant DNA technology, several E . histolytica antigens have now been expressed in prokaryotic systems and tested in animal models as vaccines to prevent invasive amebiasis . In this review, Sam Stanley Jr discusses why a vaccine for amebiasis may be feasible, and describes the recent development of several promising recombinant E . histolytica antigen-based parenteral and oral vaccine candidates.

Indian J Exp Biol, 2004 Jan, 42(1), 9 - 25
Genetics of metal resistance in acidophilic prokaryotes of acidic mine environments; Banerjee PC; Acidophilic bacteria inhabiting acidic mine regions cause natural leaching of sulphidic ores . They are now exploited in industrial operations for leaching of metals and beneficiation of low-grade and recalcitrant ores . Recent trends emphasize application of thermoacidophiles and genetic engineering of ore-leaching bacteria for greater success in this area . This requires an in-depth understanding on the molecular genetics of these bacteria and construction of cloning vectors for them . Metal resistance is considered as the most suitable phenotypic trait for cloning vectors of bio-mining chemolithoautotrophic (viz . Acidithiobacillus ferrooxidans) and heterotrophic (Acidiphilium and Acidocella species) bacteria of mine environments . These bacteria take part in ore-leaching either directly or indirectly, exhibit low to high level of resistance/tolerance to various metals under different conditions . Majority of these bacteria contain one or more plasmids--the genetic elements that usually carry metal resistant genes . But none of the At . ferrooxidans plasmids has been definitely proved to harbour metal-resistant genes which have mostly been found in the chromosome of this bacterium . Plasmids of acidophilic heterotrophs of the genera Acidiphilium and Acidocella, on the other hand, carry metal resistant genes . While genes bestowing arsenic resistance in Acidiphilium multivorum are similar to those analyzed from other sources, the metal (Cd and Zn)-resistance conferring cloned plasmid DNA fragments from Acidiphilium symbioticum KM2 and Acidocella GS19h strains were found to have no sequence similarity with the reported Cd- and Zn-resistant genes . Such observations indicate some novel aspects of metal resistance in acidophilic bacteria.

Protein Sci, 2004 Aug, 13(8), 2170 - 83
Proteome-wide functional classification and identification of prokaryotic transmembrane proteins by transmembrane topology similarity comparison; Arai M et al.; We propose a new method for classifying and identifying transmembrane (TM) protein functions in proteome-scale by applying a single-linkage clustering method based on TM topology similarity, which is calculated simply from comparing the lengths of loop regions . In this study, we focused on 87 prokaryotic TM proteomes consisting of 31 proteobacteria, 22 gram-positive bacteria, 19 other bacteria, and 15 archaea . Prior to performing the clustering, we first categorized individual TM protein sequences as "known," "putative" (similar to "known" sequences), or "unknown" by using the homology search and the sequence similarity comparison against SWISS-PROT to assess the current status of the functional annotation of the TM proteomes based on sequence similarity only . More than three-quarters, that is, 75.7% of the TM protein sequences are functionally "unknown," with only 3.8% and 20.5% of them being classified as "known" and "putative," respectively . Using our clustering approach based on TM topology similarity, we succeeded in increasing the rate of TM protein sequences functionally classified and identified from 24.3% to 60.9% . Obtained clusters correspond well to functional superfamilies or families, and the functional classification and identification are successfully achieved by this approach . For example, in an obtained cluster of TM proteins with six TM segments, 109 sequences out of 119 sequences annotated as "ATP-binding cassette transporter" are properly included and 122 "unknown" sequences are also contained.

J Bioinform Comput Biol, 2004 Mar, 2(1), 1 - 19
Prokaryote phylogeny without sequence alignment: from avoidance signature to composition distance; Hao B et al.; This is a review of a new and essentially simple method of inferring phylogenetic relationships from complete genome data without using sequence alignment . The method is based on counting the appearance frequency of oligopeptides of a fixed length (up to K = 6) in the collection of protein sequences of a species . It is a method without fine adjustment and choice of genes . Applied to prokaryotic genomes it has led to results comparable with the bacteriologists' systematics as reflected in the latest 2002 outline of the Bergey's Manual of Systematic Bacteriology . The method has also been used to compare chloroplast genomes and to the phylogeny of Coronaviruses including human SARS-CoV . A key point in our approach is subtraction of a random background from the original counts by using a Markov model of order K-2 in order to highlight the shaping role of natural selection . The implications of the subtraction procedure is specially analyzed and further development of the new approach is indicated . Copyright Imperial College Press

Bioinformatics, 2004 Dec 12, 20(18), 3462 - 5 Epub 2004 Jul 22.
ORFcurator: molecular curation of genes and gene clusters in prokaryotic organisms; Rosenfeld JA et al.; SUMMARY: The ability to detect clusters of functionally related genes in multiple microbial genomes has enormous potential for enhancing studies on gene function and microbial evolution . The staggering amount of new genome sequence data presents a largely untapped resource for gene cluster discovery . To date, gene cluster analysis has not been fully automated, and one must rely on manual, tedious and time-consuming manipulation of sequences . To facilitate accurate and rapid identification of conserved gene clusters, we developed a database-driven web application, called ORFcurator . We used ORFcurator to find clusters containing any genes similar to those of the 14-gene Widespread Colonization Island of Actinobacillus actinomycetemcomitans . From 126 genomes, ORFcurator identified all 73 clusters previously determined by manual searching . AVAILABILITY: ORFcurator and all associated scripts are freely available as supplementary information . SUPPLEMENTARY INFORMATION: http://www.genomecurator.org/ORFcurator/

Expert Rev Vaccines, 2004 Aug, 3(4), 463 - 76
Bacterial viruses as human vaccines?
Clark JR, March JB.
Bacteriophages (or phages) are viruses of bacteria, consisting of nucleic acid packaged within a protein coat . In eukaryotic hosts, phages are unable to replicate and in the absence of a suitable prokaryotic host, behave as inert particulate antigens . In recent years, work has shown that whole phage particles can be used to deliver vaccines in the form of immunogenic peptides attached to modified phage coat proteins or as delivery vehicles for DNA vaccines, by incorporating a eukaryotic promoter-driven vaccine gene within their genome . While both approaches are promising by themselves, in future there is also the exciting possibility of creating a hybrid phage combining both components to create phage that are cheap, easy and rapid to produce and that deliver both protein and DNA vaccines via the oral route in the same construct.

Methods Mol Biol, 2004, 267, 169 - 82
Cell-free protein synthesis with prokaryotic combined transcription-translation; Swartz JR et al.; Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells . One model system is prokaryotic cell-free protein synthesis . This technology offers an attractive and convenient approach to produce properly folded recombinant DNA (rDNA) proteins on a laboratory scale, screen PCR fragment libraries in a high-throughput format, express pharmaceutical proteins, incorporate labeled or unnatural amino acids into proteins, and activate microbial physiology to allow for investigation of biological systems . We describe the preparation of materials necessary for the expression, quantification, and purification of rDNA proteins from active Escherichia coli extracts.

Methods Mol Biol, 2004, 267, 3 - 14
Host cell compatibility in protein expression; Greene JJ; The expression of cloned genes in prokaryotic or eukaryotic host cells provides the means not only for the study of gene function but also for the production of substantial amounts of protein and nonprotein molecules for commercial and investigational use . In the case of proteins, strategies for determining the most appropriate vector-host combination for the expression of an exogenous gene depend on a diverse range of factors that relate ultimately to the properties of the gene and its product . The approach used in the downstream purification of the product is another factor that impinges on this selection . However, among the most important considerations in the choice of vector and host in ensuring the maximal amount of expression is the compatibility of the host cells to translate the RNA transcript, to ensure the proper folding of the product, and to sustain the protein in the intact and functional state.

Q Rev Biophys, 2003 Nov, 36(4), 429 - 53
Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination; Prevost C et al.; Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence . This process is important for both DNA repair and DNA segregation . In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA . These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments . DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA . In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination . In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove . The possible role and location of two flexible loops of RecA are discussed.

Am Nat, 2004 Jul, 164(1), 1 - 12 Epub 2004 May 03.
Mobile gene cassettes: a fundamental resource for bacterial evolution; Michael CA; Horizontal gene transfer increases genetic diversity in prokaryotes to a degree not allowed by the limitations of reproduction by binary fission . The integron/gene cassette system is one of the most recently characterized examples of a system that facilitates horizontal gene transfer . This system, discovered in the context of multidrug resistance, is recognized in a clinical context for its role in allowing pathogens to adapt to the widespread use of antibiotics . Recent studies suggest that gene cassettes are common and encode functions relevant to many adaptive traits . To estimate the diversity of mobile cassettes in a natural environment, a molecular technique was developed to provide representative distributions of cassette populations at points within a sampling area . Subsequently, statistical methods analogous to those used for calculating species diversity were employed to assess the diversity of gene cassettes within the sample area in addition to gaining an estimate of cassette pool size . Results indicated that the number of cassettes within a 5x10-m sample area was large and that the overall mobile cassette metagenome was likely to be orders of magnitude larger again . Accordingly, gene cassettes appear to be capable of mobilizing a significant genetic resource and consequently have a substantial impact on bacterial adaptability.

Plant Physiol, 2004 Jul, 135(3), 1269 - 79
Metabolic responses to the reduction in palmitate caused by disruption of the FATB gene in Arabidopsis; Bonaventure G et al.; Disruption of the FATB gene in Arabidopsis results in a two-thirds reduction in saturated fatty acids, largely palmitate, in the leaf extra-plastidic phospholipids and a reduction in the growth rate of the mutant compared to wild type (Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB {2003} Plant Cell 15: 1020-1033) . In this study, we report that although fatb-ko seedlings grow more slowly than wild type, the rate of fatty acid synthesis in leaves of the mutant increases by 40% . This results in approximately the same amount of palmitate exported from the plastid as in wild type but an increase in oleate export of about 55% . To maintain constant amounts of fatty acids in leaves, thereby counterbalancing their higher rate of production, the mutant also increases its rate of fatty acid degradation . Although fatb-ko leaves have higher rates of fatty acid synthesis and turnover, the relative proportions of membrane lipids are similar to wild type . Thus, homeostatic mechanisms to preserve membrane compositions compensate for substantial changes in rates of fatty acid and glycerolipid metabolism in the mutant . Pulse-chase labeling studies show that in fatb-ko leaves there is a net increase in the synthesis of both prokaryotic and eukaryotic lipids and consequently of their turnover . The net loss of palmitate from phosphatidylcholine plus phosphatidylethanolamine is similar for wild type and mutant, suggesting that mechanisms are not present that can preferentially preserve the saturated fatty acids . In summary, the leaf cell responds to the loss of saturated fatty acid production in the fatb-ko mutant by increasing both fatty acid synthesis and degradation, but in doing so the mechanisms for increased fatty acid turnover contribute to the lowering of the percentage of saturated fatty acids found in eukaryotic lipids.

J Biol Chem, 2004 Sep 17, 279(38), 39505 - 12 Epub 2004 Jul 20.
A trimeric quaternary structure is conserved in bacterial and human glutamate transporters; Gendreau S et al.; Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system . They are known to be multimers; however, the number of subunits forming a functional transporter is controversial . We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP . Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits . In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters . Overexpression and purification of ecgltP in E . coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles . Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.

J Invertebr Pathol, 2004 Jul, 86(3), 77 - 86
Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea ariakensis Gould; Sun J et al.; Moribund specimens of the oyster, Crassostrea ariakensis Gould, aged 2-3 years were collected from Hailing Bay in Yangxi County of Guangdong Province from February to May and November to December in the years 2001, 2002, and 2003 . A massive infection by an obligate intracellular prokaryote, specifically a rickettsia-like organism (RLO), was found . Here we report investigations of this RLO in the tissues of the oyster C . ariakensis Gould and describe the histology, ultrastructure, and morphogenesis of this pathogen in C . ariakensis Gould . Light microscopic observations of stained tissues revealed cytoplasmic inclusion bodies typical of prokaryote infection in about 87% (26/30) of the oysters . Most inclusions were observed in epithelial cells and connective tissues of the gill, mantle, and digestive gland of most of the infected oysters . The shape, size, and color of inclusions from different tissues were polymorphic . Electron microscopic examination of digestive gland, gill, and mantle tissues showed that the RLOs were intracytoplasmic . RLOs were often round, dumb-bell-shaped (undergoing binary fission), or occasionally rod-shaped and ranged from approximately 0.58 to 1.20microm in size . The organisms exhibited an ultrastructure characteristic of prokaryotic bacteria-like cells, including a trilaminar cell wall, electron-dense periplasmic ribosome zone, and a DNA nucleoid . Reproductive stages, including transverse binary fission, were observed by TEM . These stages were frequently observed within membrane-bound cytoplasmic vacuoles . Hexagonal phage-like particles in the cytoplasm of RLOs were also observed.

J Immunol Methods, 2004 Jul, 290(1-2), 3 - 28
Binding proteins from alternative scaffolds; Nygren PA et al.; The use of so-called protein scaffolds for the generation of novel binding proteins via combinatorial engineering has recently emerged as a powerful alternative to natural or recombinant antibodies . This concept requires an extraordinary stable protein architecture tolerating multiple substitutions or insertions at the primary structural level . With respect to broader applicability it should involve a type of polypeptide fold which is observed in differing natural contexts and with distinct biochemical functions, so that it is likely to be adaptable to novel molecular recognition purposes . The quickly growing number of approaches can be classified into three groups: carrier proteins for the display of single variegated loops, scaffolds providing rigid elements of secondary structure, and protein frameworks supporting a group of conformationally variable loops in a fixed spatial arrangement . Generally, such artificial receptor proteins should be based on monomeric and small polypeptides that are robust, easily engineered, and efficiently produced in inexpensive prokaryotic expression systems . Today, progress in protein library technology allows for the parallel development of immunoglobulin (Ig) as well as scaffold-based affinity reagents . Both biomolecular tools have the potential to complement each other, thus expanding the possibility to find an affinity reagent suitable for a given application . The repertoire of protein scaffolds hitherto recruited for combinatorial protein engineering purposes will probably be further expanded in the future, including both additional natural proteins and de novo designed proteins, contributing to the collection of libraries available at present . In this review both the structural features and the practical use of scaffold proteins will be discussed and exemplified.

Prog Biophys Mol Biol, 2004 Sep, 86(1), 5 - 43
Quantitative analysis of signaling networks; Sauro HM et al.; The response of biological cells to environmental change is coordinated by protein-based signaling networks . These networks are to be found in both prokaryotes and eukaryotes . In eukaryotes, the signaling networks can be highly complex, some networks comprising of 60 or more proteins . The fundamental motif that has been found in all signaling networks is the protein phosphorylation/dephosphorylation cycle--the cascade cycle . At this time, the computational function of many of the signaling networks is poorly understood . However, it is clear that it is possible to construct a huge variety of control and computational circuits, both analog and digital from combinations of the cascade cycle . In this review, we will summarize the great versatility of the simple cascade cycle as a computational unit and towards the end give two examples, one prokaryotic chemotaxis circuit and the other, the eukaryotic MAPK cascade.

Brief Bioinform, 2004 Jun, 5(2), 131 - 49
Computational approaches for the analysis of gene neighbourhoods in prokaryotic genomes; Rogozin IB et al.; Gene order in prokaryotes is conserved to a much lesser extent than protein sequences . Only some operons, primarily those that encode physically interacting proteins, are conserved in all or most of the bacterial and archaeal genomes . Nevertheless, even the limited conservation of operon organisation that is observed provides valuable evolutionary and functional clues through multiple genome comparisons . With the rapid growth in the number and diversity of sequenced prokaryotic genomes, functional inferences for uncharacterized genes located in the same conserved gene neighborhood with well-studied genes are becoming increasingly important . In this review, we discuss various computational approaches for identification of conserved gene strings and construction of local alignments of gene orders in prokaryotic genomes.

Brief Bioinform, 2004 Jun, 5(2), 118 - 30
Probabilistic methods of identifying genes in prokaryotic genomes: connections to the HMM theory; Azad RK et al.; In this paper, we review developments in probabilistic methods of gene recognition in prokaryotic genomes with the emphasis on connections to the general theory of hidden Markov models (HMM) . We show that the Bayesian method implemented in GeneMark, a frequently used gene-finding tool, can be augmented and reintroduced as a rigorous forward-backward (FB) algorithm for local posterior decoding described in the HMM theory . Another earlier developed method, prokaryotic GeneMark.hmm, uses a modification of the Viterbi algorithm for HMM with duration to identify the most likely global path through hidden functional states given the DNA sequence . GeneMark and GeneMark.hmm programs are worth using in concert for analysing prokaryotic DNA sequences that arguably do not follow any exact mathematical model . The new extension of GeneMark using the FB algorithm was implemented in the software program GeneMark.fba . Given the DNA sequence, this program determines an a posteriori probability for each nucleotide to belong to coding or non-coding region . Also, for any open reading frame (ORF), it assigns a score defined as a probabilistic measure of all paths through hidden states that traverse the ORF as a coding region . The prediction accuracy of GeneMark.fba determined in our tests was compared favourably to the accuracy of the initial (standard) GeneMark program . Comparison to the prokaryotic GeneMark.hmm has also demonstrated a certain, yet species-specific, degree of improvement in raw gene detection, ie detection of correct reading frame (and stop codon) . The accuracy of exact gene prediction, which is concerned about precise prediction of gene start (which in a prokaryotic genome unambiguously defines the reading frame and stop codon, thus, the whole protein product), still remains more accurate in GeneMarkS, which uses more elaborate HMM to specifically address this task.

Folia Microbiol (Praha), 2004, 49(3), 247 - 58
Evolutionary relatedness between glycolytic enzymes most frequently occurring in genomes; Oslancova A et al.; More than 100 sequenced genomes were searched for genes coding for the enzymes involved in glycolysis in an effort to find the most frequently occurring ones . Triosephosphate isomerase (TIM), glyceraldehyde-3-phosphate dehydrogenase (GAPD), phosphoglycerate kinase (PGK) and enolase (ENOL) were found to be present in 90 investigated genomes all together . The final set consisted of 80 prokaryotic and 10 eukaryotic genomes . Of the 80 prokaryotic genomes, 73 were from Bacteria, 7 from Archaea . Two microbial genomes were also from Eucarya (yeasts) . Eight genomes of nonmicrobial origin were included for comparison . The amino acid sequences of TIMs, GAPDs, PGKs and ENOLs were collected and aligned, and their individual as well as concatenated evolutionary trees were constructed and discussed . The trees clearly demonstrate a closer relatedness between Eucarya and Archaea (especially the concatenated tree) but they do not support the hypothesis that eukaryotic glycolytic enzymes should be closely related to their alpha-proteobacterial counterparts . Phylogenetic analyses further reveal that although the taxonomic groups (e.g., alpha-proteobacteria, gamma-proteobacteria, firmicutes, actinobacteria, etc.) form their more or less compact clusters in the trees, the inter-clade relationships between the trees are not conserved at all . On the other hand, several examples of conservative relatedness separating some clades of the same taxonomic groups were observed, e.g., Buchnera along with Wigglesworthia and the rest of gamma-proteobacteria, or mycoplasmas and the rest of firmicutes . The results support the view that these glycolytic enzymes may have their own evolutionary history.

Apoptosis, 2004 May, 9(3), 299 - 307
BAX Inhibitor-1, an ancient cell death suppressor in animals and plants with prokaryotic relatives; Huckelhoven R; BAX Inhibitor-1 (BI-1) was originally described as testis enhanced gene transcript in mammals . Functional screening in yeast for human proteins that can inhibit the cell death provoking function of BAX, a proapoptotic Bcl-2 family member, led to functional characterisation and renaming of BI-1 . The identification of functional homologues of BI-1 in plants and yeast widened the understanding of BI-1 function as an ancient suppressor of programmed cell death . BI-1 is one of the few cell death suppressors conserved in animals and plants . Computer predictions and experimental data together suggest that BI-1 is a membrane spanning protein with 6 to 7 transmembrane domains and a cytoplasmic C-terminus sticking in the endoplasmatic reticulum and nuclear envelope . Proteins similar to BI-1 are present in other eukaryotes, bacteria, and even viruses encode BI-1 like proteins . BI-1 is involved in development, response to biotic and abiotic stress and probably represents an indispensable cell protectant . BI-1 appears to suppress cell death induced by mitochondrial dysfunction, reactive oxygen species or elevated cytosolic Ca(2+) levels . This review focuses on the present understanding about BI-1 and suggests potential directions for further analyses of this increasingly noticed protein.

Mol Pharmacol, 2004 Oct, 66(4), 807 - 16 Epub 2004 Jul 16.
Mutation in nucleotide-binding domains of sulfonylurea receptor 2 evokes Na-ATP-dependent activation of ATP-sensitive K+ channels: implication for dimerization of nucleotide-binding domains to induce channel opening; Yamada M et al.; The ATP-sensitive K+ (KATP) channel is composed of a sulfonylurea receptor (SUR) and a pore-forming subunit, Kir6.2 . SUR is an ATP-binding cassette (ABC) protein with two nucleotide-binding domains (NBD1 and NBD2) . Intracellular ATP inhibits KATP channels through Kir6.2 and activates them through NBDs . However, it is still unknown how ATP-bound NBDs activate KATP channels . A prokaryotic ABC protein, MJ0796, which is entirely NBD, forms a dimer in the presence of Na-ATP when its glutamate at position 171 is substituted with glutamine . Mg2+ or K+ destabilizes the dimer . We made the corresponding mutation in the NBD1 (D834N) and/or NBD2 (E1471Q) of SUR2A and SUR2B . As measured in the inside-out configuration of the patch-clamp method, SUR2x(D834N, E1471)/Kir6.2 channels mediated significantly larger currents in the presence of internal 1 mM Na-ATP than K-ATP alone or Mg-ATP . The response to Na-ATP resulted from an increase in the open probability but not single-channel amplitude of the channels and was abolished by glibenclamide (10(-5) M) . In the presence of 1 mM Mg2+ -free ATP, Na+ increased the activity of the channels in a concentration-dependent manner . The Na-ATP-dependent activation was never observed with KATP channels including either the wild-type SUR2x, SUR2x(D834N), or SUR2x(E1471) . Nicorandil activated SUR2x(D834N, E1471Q)/Kir6.2 channels more strongly in the presence of Na-ATP than K-ATP alone, whereas the reverse was true for wild-type SUR2x/Kir6.2 channels . Therefore, it is likely that NBDs of SUR2x dimerize in response to ATP and nicorandil . The dimerization induces the opening of the KATP channel, probably by causing a conformational change of SUR2x.

J Biol Chem, 2004 Sep 17, 279(38), 39999 - 40006 Epub 2004 Jul 16.
Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana; Sun Y et al.; Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance . Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development . To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined . p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization . When produced in either prokaryotic or eukaryotic cells the p26 alpha-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers . The amino terminus was, therefore, required for efficient dimer formation . Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning . Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei . A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress . However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.

Microbiology, 2004 Jul, 150(Pt 7), 2247 - 56
Comparative analysis of eukaryotic-type protein phosphatases in two streptomycete genomes; Shi L et al.; Inspection of the genomes of Streptomyces coelicolor A3(2) and Streptomyces avermitilis reveals that each contains 55 putative eukaryotic-type protein phosphatases (PPs), the largest number ever identified from any single prokaryotic organism . Unlike most other prokaryotic genomes that have only one or two superfamilies of eukaryotic-type PPs, the streptomycete genomes possess the eukaryotic-type PPs that belong to four superfamilies: 2 phosphoprotein phosphatases and 2 low-molecular-mass protein tyrosine phosphatases in each species, 49 Mg(2+)- or Mn(2+)-dependent protein phosphatases (PPMs) and 2 conventional protein tyrosine phosphatases (CPTPs) in S . coelicolor A3(2), and 48 PPMs and 3 CPTPs in S . avermitilis . Sixty-four percent of the PPs found in S . coelicolor A3(2) have orthologues in S . avermitilis, indicating that they originated from a common ancestor and might be involved in the regulation of more conserved metabolic activities . The genes of eukaryotic-type PP unique to each surveyed streptomycete genome are mainly located in two arms of the linear chromosomes and their evolution might be involved in gene acquisition or duplication to adapt to the extremely variable soil environments where these organisms live . In addition, 56 % of the PPs from S . coelicolor A3(2) and 65 % of the PPs from S . avermitilis possess at least one additional domain having a putative biological function . These include the domains involved in the detection of redox potential, the binding of cyclic nucleotides, mRNA, DNA and ATP, and the catalysis of phosphorylation reactions . Because they contained multiple functional domains, most of them were assigned functions other than PPs in previous annotations . Although few studies have been conducted on the physiological functions of the PPs in streptomycetes, the existence of large numbers of putative PPs in these two streptomycete genomes strongly suggests that eukaryotic-type PPs play important regulatory roles in primary or secondary metabolic pathways . The identification and analysis of such a large number of putative eukaryotic-type PPs from S . coelicolor A3(2) and S . avermitilis constitute a basis for further exploration of the signal transduction pathways mediated by these phosphatases in industrially important strains of streptomycetes.

Metab Eng, 2004 Jul, 6(3), 239 - 44
Alternative pathways of galactose assimilation: could inverse metabolic engineering provide an alternative to galactosemic patients?
Lai K, Klapa MI.
The galactose assimilation pathway has been extensively studied as an example of a genetic regulatory switch . Besides the importance of this pathway as a tool in basic biological research, unraveling its structure and regulation is also of major medical importance . Impairment of galactose assimilation is the cause of the genetic metabolic disease known as "galactosemia", while the in vivo activity of the pathway affects the production of glycans . The latter have been connected to tumor metastasis, anti-cancer drug resistance and various cardiovascular diseases . Despite the vast amount of studies, however, galactose assimilation and its interaction with other parts of the metabolic network have not been fully elucidated yet . In yeast and higher eukaryotes, it is still being studied as comprising only the linear Leloir pathway . Recent observations, however, indicate that alternative pathways of galactose assimilation identified in prokaryotes and fungi might also be present in yeast . Such a result is valuable per se, because it could lead to the discovery of these pathways in humans . Even more importantly, these pathways provide alternative phenotypes with known genetic fingerprints that can be used in the context of classical and inverse metabolic engineering to examine and treat the mechanisms of defects of galactose assimilation.

Philos Trans R Soc Lond B Biol Sci, 2004 Apr 29, 359(1444), 623 - 38
Prokaryote diversity and taxonomy: current status and future challenges; Oren A; The prokaryotes are by far the most abundant organisms inhabiting planet Earth . They are also by far the most diverse, both metabolically and phylogenetically; they encompass the Bacteria and the Archaea, two out of the three major divisions of living organisms . The current prokaryote species classification is based on a combination of genomic and phenotypic properties . The recommended cut-off value of 70% DNA-DNA similarity to delineate species signifies an extremely broad species definition for the prokaryotes compared with the higher eukaryotes . The number of validly named species of prokaryotes is currently slightly more than 6200 . However, on the basis of small-subunit rDNA characterization of whole communities and other approaches, the more exact number of species present can be inferred to be at least two orders of magnitude larger . Classic culturing methods based on colony formation on agar are generally unsatisfactory for the recovery of bacteria from the environment . Many of the most abundant prokaryotes in nature have not yet been brought into culture . Some of these may thrive by means of as yet unknown modes of energy generation . Several novel methods have recently enabled the isolation of some interesting organisms of environmental significance . A better coverage of the prokaryote diversity on Earth depends on such innovative approaches, combined with appropriate funding.

Acta Biochim Biophys Sin (Shanghai), 2004 Apr, 36(4), 309 - 13
Expression of a modified Cry1Ie gene in E . coli and in transgenic tobacco confers resistance to corn borer; Liu YJ et al.; The wild-type Cry1Ie gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants . Modified Cry1Ie gene (designated as Cry1Iem) was cloned into prokaryotic expression vector pET28b and its expression in E . coli was confirmed by SDS-PAGE analysis . Bioassays using crude expression products in E . coli revealed that Cry1Iem protein had a similar toxicity to corn borer as wild-type Cry1Ie . Cry1Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301 . Transgenic tobacco plants carrying Cry1Iem showed insecticidal activity against corn borer.

J Biol Chem, 2004 Sep 10, 279(37), 38220 - 7 Epub 2004 Jul 13.
Mutational analysis and characterization of nocardicin C-9' epimerase; Kelly WL et al.; The biosynthetic gene cluster for the nocardicin A producer Nocardia uniformis subsp . tsuyamanensis ATCC 21806 was recently identified . Nocardicin A is the most potent of a series of monocyclic beta-lactam antibiotics produced by this organism . Its activity has been attributed to a syn-configured oxime moiety and a d-homoseryl side chain attached through an unusual ether linkage to the core nocardicin framework . Notably present in the nocardicin biosynthetic gene cluster is nocJ, encoding a protein with sequence similarity to the pyridoxal 5'-phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid deaminases . Insertional mutagenesis of nocJ abolished nocardicin A production, while the l-homoseryl isomer, isonocardicin A, was still observed . Expression of the disrupted nocJ gene in trans was sufficient to restore production of nocardicin A in the disruption mutant . Heterologous expression, purification, and in vitro characterization of NocJ by UV spectroscopy, cofactor reduction, chiral HPLC analysis of the products and their exchange behavior in deuterium oxide led to confirmation of its role as the PLP-dependent nocardicin C-9' epimerase responsible for interconversion of the nocardicin homoseryl side chain in both nocardicin A with isonocardicin A, and nocardicin C with isonocardicin C . NocJ is the first member of a new class of beta-lactam aminoacyl side chain epimerases, the first two classes being the evolutionarily distinct prokaryotic PLP-dependent isopenicillin N epimerase and the fungal isopenicillin N epimerase two protein system.

Biochemistry, 2004 Jul 20, 43(28), 9243 - 55
Isolation and characterization of the human tRNA-(N1G37) methyltransferase (TRM5) and comparison to the Escherichia coli TrmD protein; Brule H et al.; A human TRM5 cDNA has been cloned and recombinant tRNA-N(1)G37 methyltransferase was produced . The recombinant enzyme methylates the N1 position of guanosine 37 (G37) in selected tRNA transcripts utilizing S-adenosyl methionine . The effects of RNA sequence and structure on the methylation reaction in comparison between the Escherichia coli TrmD and human TRM5 recombinant enzymes are presented . G37-methylation by TRM5 occurs regardless of the nature of the nucleotide at position 36 . TRM5 also methylates inosine at position 37 unlike TrmD, which recognizes the G36pG37 motif preferentially and does not methylate inosine . New evidence is presented concerning TrmD showing that with some tRNA species, A at position 36 is also recognized . The TRM5 enzyme is sensitive to subtle changes in the tRNA-protein tertiary interaction leading to loss of activity . The TrmD enzyme is more tolerant of alterations in tRNA-protein tertiary interactions as long as the core tRNA structure and the G36pG37 are present . The TRM5 enzyme does not have an absolute requirement for magnesium ions, whereas TrmD requires magnesium to express activity . TRM5 demonstrates much higher affinity for substrates with K(m) values for tRNA that are nanomolar . TrmD has K(m) values for tRNA in the micromolar range . Recombinant TRM5 appears to function as a 60 772 Da monomer, while recombinant TrmD functions as a homodimer of 30 586 Da subunits . Bioinformatic analysis of the human TRM5 genomic locus (KIAA1393) have identified TRM5 homologues in eukaryotes and archaea; however, no significantly homologous regions were identified in any prokaryotes including the TrmD gene.

Anim Biotechnol, 2004 May, 15(1), 77 - 88
The interferon-alpha genes from three chicken lines and its effects on H9N2 influenza viruses; Xia C et al.; The interferon-alpha genes from three chicken lines were cloned by a direct PCR technique, and the effects of recombinant protein expressed in a prokaryotic system on highly pathogenic H9N2 influenza viruses were investigated . The cloned ChIFN-alpha gene encoded a protein of 193 amino acids with a signal sequence of 31 amino acids and mature peptides of 162 amino acids . Comparison of ChIFN-alpha sequences, detected six amino acids substitutions at positions 50, 58, 65, 81, 181, and 183 . Homology analysis indicated that ChIFN-alpha genes could be subdivided into two lineages, SH-ChIFN-alpha and WJ-ChIFN-alpha . In addition, both SH-ChIFN-alpha and WJ-ChIFN-alpha were expressed with the N-terminal 6 consecutive histidine residues in a high-level prokaryotic expression system . Recombinant chicken interferon-alpha (rChIFN-alpha) protein has anti-VSV activity of more than 1 x 10(8) U/mg . Moreover, High concentration (10,000U) of rSH-ChIFN-alpha resulted in over 40% inhibition of the H9N2 virus infection in chicken embryos (Ovo), and 100% inhibition from one day-old to five day-old chickens (Vivo) . The results suggested that rChIFN-alpha is a potential agent against many Chicken viral strains.

Indian J Exp Biol, 2003 Aug, 41(8), 797 - 804
Circadian clock genes in Drosophila: recent developments; Subramanian P et al.; Circadian rhythms provide a temporal framework to living organisms and are established in a majority of eukaryotes and in a few prokaryotes . The molecular mechanisms of circadian clock is constantly being investigated in Drosophila melanogaster . The core of the clock mechanism was described by a transcription-translation feedback loop model involving period (per), timeless (tim), dclock and cycle genes . However, recent research has identified multiple feedback loops controlling rhythm generation and expression . Novel mutations of timeless throw more light on the functions of per and tim products . Analysis of pdf neuropeptide gene (expressed in circadian pacemaker cells in Drosophila), indicate that PDF acts as the principal circadian transmitter and is involved in output pathways . The product of cryptochrome is known to function as a circadian photoreceptor as well as component of the circadian clock . This review focuses on the recent progress in the field of molecular rhythm research in the fruit fly . The gene(s) and the gene product(s) that are involved in the transmission of environmental information to the clock, as well as the timing signals from the clock outward to cellular functions are remain to be determined.

Plant Physiol, 2004 Jul, 135(3), 1243 - 55 Epub 2004 Jul 09.
Cloning and characterization of two NAD kinases from Arabidopsis . identification of a calmodulin binding isoform; Turner WL et al.; NAD kinase (NADK; ATP:NAD 2'-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryotes and eukaryotes, generates the important pyridine nucleotide NADP from substrates ATP and NAD . The role of NADKs in plants is poorly understood, and cDNAs encoding plant NADKs have not previously been described to our knowledge . We have cloned two cDNAs from Arabidopsis predicted to encode NADK isoforms, designated NADK1 and NADK2, respectively . Expressed as recombinant proteins in bacteria, both NADK1 and NADK2 were catalytically active, thereby confirming their identity as NADKs . Transcripts for both isoforms were detected in all tissues examined and throughout development . Although the predicted catalytic regions for NADK1 and NADK2 show sequence similarity to NADKs from other organisms, NADK2 possesses a large N-terminal extension that appears to be unique to plants . Using recombinant glutathione-S-transferase fusion proteins and calmodulin (CaM)-affinity chromatography, we delineated a Ca2+-dependent CaM-binding domain to a 45-residue region within the N-terminal extension of NADK2 . Although recombinant NADK2 was not responsive to CaM in vitro, immunoblot analysis suggests that native NADK2 is a CaM-binding protein . In Arabidopsis crude extracts, CaM-dependent NADK activity was much greater than CaM-independent activity throughout development, particularly in young seedlings . A native CaM-dependent NADK was partially purified from Arabidopsis seedlings (Km NAD=0.20 mM, Km Mg2+ -ATP=0.17 mM) . The enzyme was fully activated by conserved CaM (S0.5 = 2.2 nm) in the presence of calcium but displayed differential responsiveness to eight CaM-like Arabidopsis proteins . Possible roles for NADKs in plants are discussed in light of our observations.

Bioinformatics, 2004 Dec 12, 20(18), 3308 - 17 Epub 2004 Jul 09.
Accuracy improvement for identifying translation initiation sites in microbial genomes; Zhu HQ et al.; MOTIVATION: At present the computational gene identification methods in microbial genomes have a high prediction accuracy of verified translation termination site (3' end), but a much lower accuracy of the translation initiation site (TIS, 5' end) . The latter is important to the analysis and the understanding of the putative protein of a gene and the regulatory machinery of the translation . Improving the accuracy of prediction of TIS is one of the remaining open problems . RESULTS: In this paper, we develop a four-component statistical model to describe the TIS of prokaryotic genes . The model incorporates several features with biological meanings, including the correlation between translation termination site and TIS of genes, the sequence content around the start codon; the sequence content of the consensus signal related to ribosomal binding sites (RBSs), and the correlation between TIS and the upstream consensus signal . An entirely non-supervised training system is constructed, which takes as input a set of annotated coding open reading frames (ORFs) by any gene finder, and gives as output a set of organism-specific parameters (without any prior knowledge or empirical constants and formulas) . The novel algorithm is tested on a set of reliable datasets of genes from Escherichia coli and Bacillus subtillis . MED-Start may correctly predict 95.4% of the start sites of 195 experimentally confirmed E.coli genes, 96.6% of 58 reliable B.subtillis genes . Moreover, the test results indicate that the algorithm gives higher accuracy for more reliable datasets, and is robust to the variation of gene length . MED-Start may be used as a postprocessor for a gene finder . After processing by our program, the improvement of gene start prediction of gene finder system is remarkable, e.g . the accuracy of TIS predicted by MED 1.0 increases from 61.7 to 91.5% for 854 E.coli verified genes, while that by GLIMMER 2.02 increases from 63.2 to 92.0% for the same dataset . These results show that our algorithm is one of the most accurate methods to identify TIS of prokaryotic genomes . AVAILABILITY: The program MED-Start can be accessed through the website of CTB at Peking University: http://ctb.pku.edu.cn/main/SheGroup/MED_Start.htm.

Proc Natl Acad Sci U S A, 2004 Jul 20, 101(29), 10566 - 71 Epub 2004 Jul 08.
The voltage-gated Na+ channel NaVBP has a role in motility, chemotaxis, and pH homeostasis of an alkaliphilic Bacillus; Ito M et al.; The prokaryotic voltage-gated Na(+) channel, NaChBac, is one of a growing channel superfamily of unknown function . Here we show that Na(V)BP, the NaChBac homologue encoded by ncbA in alkaliphilic Bacillus pseudofirmus OF4, is a voltage-gated Na(+) channel potentiated by alkaline pH . Na(V)BP has roles in motility, chemotaxis, and pH homeostasis at high pH . Reduced motility of bacteria lacking functional Na(V)BP was reversed by restoration of the native channel but not by a mutant Na(V)BP engineered to be Ca(2+)-selective . Motile ncbA mutant cells and wild-type cells treated with a channel inhibitor exhibited behavior opposite to the wild type in response to chemoeffectors . Mutants lacking functional Na(V)BP were also defective in pH homeostasis in response to a sudden alkaline shift in external pH under conditions in which cytoplasmic {Na(+)} is limiting for this crucial process . The defect was exacerbated by mutation of motPS, the motility channel genes . We hypothesize that activation of Na(V)BP at high pH supports diverse physiological processes by a combination of direct and indirect effects on the Na(+) cycle and the chemotaxis system.

Curr Biol, 2004 Jul 13, 14(13), R505 - 7
Prokaryotic development: a new player on the cell cycle circuit; Stephens C; A genetic regulatory circuit recently described in the bacterium Caulobacter crescentus generates reciprocal oscillations in the abundance of two key transcription factors to control landmark events in the cell cycle.

Curr Biol, 2004 Jul 13, 14(13), 1167 - 73
FtsZ exhibits rapid movement and oscillation waves in helix-like patterns in Escherichia coli; Thanedar S et al.; Prokaryotes contain cytoskeletal proteins such as the tubulin-like FtsZ, which forms the Z ring at the cell center for cytokinesis, and the actin-like MreB, which forms a helix along the long axis of the cell and is required for shape maintenance . Using time-lapse analysis of Escherichia coli cells expressing FtsZ-GFP, we found that FtsZ outside of the Z ring also localized in a helix-like pattern and moved very rapidly within this pattern . The movement occurred independently of the presence of Z rings and was most easily detectable in cells lacking Z rings . Moreover, we observed oscillation waves of FtsZ-GFP in the helix-like pattern, particularly in elongated cells, and the period of this oscillation was similar to that of the Min proteins . The MreB helix was not required for the rapid movement of FtsZ or the oscillation of MinD . The results suggest that FtsZ not only forms the Z ring but also is part of a highly dynamic, potentially helical cytoskeleton in bacterial cells.

BMC Microbiol . 2004 Jul 08;4(1):26.
Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR; Zhong S et al.; BACKGROUND: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements . Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient . Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E . coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced . RESULTS: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units . Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655 . Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome . Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement . Two other E . coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR . They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively . CONCLUSION: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes . Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step . Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes . The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks.

Mol Biol Evol, 2004 Oct, 21(10), 1884 - 94 Epub 2004 Jul 07.
Successful lateral transfer requires codon usage compatibility between foreign genes and recipient genomes; Medrano-Soto A et al.; We present evidence supporting the notion that codon usage (CU) compatibility between foreign genes and recipient genomes is an important prerequisite to assess the selective advantage of imported functions, and therefore to increase the fixation probability of horizontal gene transfer (HGT) events . This contrasts with the current tendency in research to predict recent HGTs in prokaryotes by assuming that acquired genes generally display poor CU . By looking at the CU level (poor, typical, or rich) exhibited by putative xenologs still resembling their original CU, we found that most alien genes predominantly present typical CU immediately upon introgression, thereby suggesting that the role of CU amelioration in HGT has been overemphasized . In our strategy, we first scanned a representative set of 103 complete prokaryotic genomes for all pairs of candidate xenologs (exported/imported genes) displaying similar CU . We applied additional filtering criteria, including phylogenetic validations, to enhance the reliability of our predictions . Our approach makes no assumptions about the CU of foreign genes being typical or atypical within the recipient genome, thus providing a novel unbiased framework to study the evolutionary dynamics of HGT.

Appl Environ Microbiol, 2004 Jul, 70(7), 4129 - 35
Contribution of SAR11 bacteria to dissolved dimethylsulfoniopropionate and amino acid uptake in the North Atlantic ocean; Malmstrom RR et al.; SAR11 bacteria are abundant in marine environments, often accounting for 35% of total prokaryotes in the surface ocean, but little is known about their involvement in marine biogeochemical cycles . Previous studies reported that SAR11 bacteria are very small and potentially have few ribosomes, indicating that SAR11 bacteria could have low metabolic activities and could play a smaller role in the flux of dissolved organic matter than suggested by their abundance . To determine the ecological activity of SAR11 bacteria, we used a combination of microautoradiography and fluorescence in situ hybridization (Micro-FISH) to measure assimilation of (3)H-amino acids and {(35)S}dimethylsulfoniopropionate (DMSP) by SAR11 bacteria in the coastal North Atlantic Ocean and the Sargasso Sea . We found that SAR11 bacteria were often abundant in surface waters, accounting for 25% of all prokaryotes on average . SAR11 bacteria were typically as large as, if not larger than, other prokaryotes . Additionally, more than half of SAR11 bacteria assimilated dissolved amino acids and DMSP, whereas about 40% of other prokaryotes assimilated these compounds . Due to their high abundance and activity, SAR11 bacteria were responsible for about 50% of amino acid assimilation and 30% of DMSP assimilation in surface waters . The contribution of SAR11 bacteria to amino acid assimilation was greater than would be expected based on their overall abundance, implying that SAR11 bacteria outcompete other prokaryotes for these labile compounds . These data suggest that SAR11 bacteria are highly active and play a significant role in C, N, and S cycling in the ocean.

Brief Funct Genomic Proteomic, 2002 Jul, 1(2), 204 - 12
Ribosome display: cell-free protein display technology; He M et al.; Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries . It uses the principle of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes), through the formation of stable protein-ribosome-mRNA (PRM) complexes . This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA, which is then converted and amplified as DNA for further manipulation, including repeated cycles or protein expression . Ribosome display has a number of advantages over cell-based systems such as phage display; in particular, it can display very large libraries without the restriction of bacterial transformation . It is also suitable for generating toxic, proteolytically sensitive and unstable proteins, and allows the incorporation of modified amino acids at defined positions . In combination with polymerase chain reaction (PCR)-based methods, mutations can be introduced efficiently into the selected DNA pool in subsequent cycles, leading to continuous DNA diversification and protein selection (in vitro protein evolution) . Both prokaryotic and eukaryotic ribosome display systems have been developed and each has its own distinctive features . In this paper, ribosome display systems and their application in selection and evolution of proteins are reviewed.

Biochim Biophys Acta, 2004 Jul 6, 1673(1-2), 29 - 44
Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways; West CM et al.; The Skp1 protein, best known as a subunit of E3(SCF)-ubiquitin ligases, is subject to complex glycosylation in the cytoplasm of the cellular slime mold Dictyostelium . Pro143 of this protein is sequentially modified by a prolyl hydroxylase and five soluble glycosyltransferases (GT), to yield the structure Galalpha1,Galalpha1,3Fucalpha1,2Galbeta1,3GlcNAcalpha1-HyPro143 . These enzymes are unusual in that they are expressed in the cytoplasmic compartment of the cell, rather than the secretory pathway where complex glycosylation of proteins usually occurs . The first enzyme in the pathway appears to be related to the soluble animal prolyl 4-hydroxylases (P4H), which modify the transcriptional factor subunit HIF-1alpha in the cytoplasm, and more distantly to the P4Hs that modify collagen and other proteins in the rER, based on biochemical and informatics analyses . The soluble alphaGlcNAc-transferase acting on Skp1 has been cloned and is distantly related to the mucin-type polypeptide N-acetyl-alpha-galactosaminyltransferase in the Golgi of animals . Its characterization has led to the discovery of a family of related polypeptide N-acetyl-alpha-glucosaminyltransferases in the Golgi of selected lower eukaryotes . The Skp1 GlcNAc is extended by a bifunctional diglycosyltransferase that sequentially and apparently processively adds beta1,3Gal and alpha1,2Fuc . Though this structure is also formed in the animal secretory pathway, the GTs involved are dissimilar . Conceptual translation of available genomes suggests the existence of this kind of complex cytoplasmic glycosylation in other eukaryotic microorganisms, including diatoms, oomycetes, and possibly Chlamydomonas and Toxoplasma, and an evolutionary precursor of this pathway may also occur in prokaryotes.

World J Gastroenterol, 2004 Jul 15, 10(14), 2029 - 33
Prokaryotic expression and renaturation of engineering chimeric Fab antibody against human hepatoma; Xing JL et al.; AIM: To express chimeric Fd (cFd) and chimeric light chain (cL) in E.coli respectively and refold them into chimeric Fab (cFab) antibody . METHODS: cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL . Then, the competent E.coli cells were transformed by the recombinant vectors and induced by IPTG . Moreover, a large quantity of cFd and cL expression products were prepared and mixed with equal molar to refold into cFab by gradient dialysis . The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting, ELISA and HPLC . RESULTS: High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of total bacteria proteins, respectively . Their relative molecular masses were all 24 ku or so, and both of them mainly existed in the form of inclusion bodies . In addition, cFd and cL were successfully refolded into cFab by gradient dialysis, with about 59.45% of recovery when the starting total protein concentration was 100 microg/mL . The renatured cFab could specifically bind to related antigen with high affinity . CONCLUSION: The cFab antibody against human hepatoma was highly and efficiently expressed and refolded, which laid a solid foundation for studying its application in the treatment of hepatoma.

Cytogenet Genome Res, 2004, 105(2-4), 435 - 41
Sequence-specific modification of mouse genomic DNA mediated by gene targeting techniques; Sangiuolo F et al.; The major impact of the human genome sequence is the understanding of disease etiology with deduced therapy . The completion of this project has shifted the interest from the sequencing and identification of genes to the exploration of gene function, signalling the beginning of the post-genomic era . Contrasting with the spectacular progress in the identification of many morbid genes, today therapeutic progress is still lagging behind . The goal of all gene therapy protocols is to repair the precise genetic defect without additional modification of the genome . The main strategy has traditionally been focused on the introduction of an expression system designed to express a specific protein, defective in the transfected cell . But the numerous deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders . Among these one is represented by gene repair based on homologous recombination (HR) . Simply stated, the process involves targeting the mutation in situ for gene correction and for restoration of a normal gene function . Homologous recombination is an efficient means for genomic manipulation of prokaryotes, yeast and some lower eukaryotes . By contrast, in higher eukaryotes it is less efficient than in the prokaryotic system, with non-homologous recombination being 10-50 fold higher . However, recent advances in gene targeting and novel strategies have led to the suggestion that gene correction based on HR might be used as clinical therapy for genetic disease . This site-specific gene repair approach could represent an alternative gene therapy strategy in respect to those involving the use of retroviral or lentiviral vectors to introduce therapeutic genes and linked regulatory sequences into random sites within the target cell genome . In fact, gene therapy approaches involving addition of a gene by viral or nonviral vectors often give a short duration of gene expression and are difficult to target to specific populations of cells . The purpose of this paper is to review oligonucleotide-based gene targeting technologies and their applications on modifying the mouse genome .

J Mol Biol, 2004 Jul 23, 340(5), 957 - 64
Periodic transcriptional organization of the E.coli genome; Kepes F; The organization of transcription within the prokaryotic nucleoid may be expected to both depend on and determine the structure of the chromosome . Indeed, immunofluorescence localization of transcriptional regulators has revealed foci in actively transcribing Escherichia coli cells . Furthermore, structural and biochemical approaches suggest that there are approximately 50 independent loop domains per genome . Here I show that in four E.coli strains, genes that are controlled by a sequence-specific transcriptional regulator tend to locate next to the gene encoding this regulator, or at regular distances that are multiples of 1/50th of the chromosome length . This periodicity is consistent with a solenoidal epi-organization of the chromosome, which would gather into foci the interacting partners; the regulator molecules and their DNA binding sites . Binding at genuine regulatory sites on DNA would thus be optimized by co-transcriptionally translating regulators in their vicinity.

Biochemistry, 2004 Jul 13, 43(27), 8616 - 24
Heme A synthase does not incorporate molecular oxygen into the formyl group of heme A; Brown KR et al.; Heme A is an obligatory cofactor in all eukaryotic and many prokaryotic cytochrome c oxidases . The final step in heme A biosynthesis requires the oxidation of the C8 methyl substituent on pyrrole ring D to an aldehyde, a reaction catalyzed by heme A synthase . To effect this transformation, heme A synthase is proposed to utilize a heme B cofactor, oxidizing the substrate via successive monooxygenase reactions . Consistent with this hypothesis, the activity of heme A synthase is found to be strictly dependent on molecular oxygen . Surprisingly, when cells expressing heme A synthase were incubated with (18)O(2), no significant incorporation of label was observed in heme A, the C8 alcohol intermediate, or the C8 overoxidized byproduct . Conversely, when the cells were grown in H(2)(18)O, partial labeling was observed at every heme oxygen position . These results suggest that the oxygen on the heme A aldehyde is derived from water . Although our data do not allow us to exclude the possibility of exchange with water inside of the cell, the results seem to question a mechanism utilizing successive monooxygenase reactions and support instead a mechanism of heme O oxidation via electron transfer.

Plant Cell Rep, 2004 Oct, 23(4), 224 - 30 Epub 2004 Jul 02.
Transgene expression in strawberries driven by a heterologous phloem-specific promoter; Zhao Y et al.; Strawberry is susceptible to diseases caused by phytoplasmas, mycoplasma-like prokaryotes restricted to sieve elements in the phloem tissue of infected plants . One strategy to improve strawberry resistance to phytoplasmas involves transgenic expression of anti-microbial peptide genes in phloem . For targeted phloem-specific expression, we constructed a binary vector with an expression cassette bearing the beta-glucuronidase (GUS) reporter gene (uidA) under control of the Arabidopsis sucrose-H+ symporter gene (AtSUC2) promoter . Transgenic strawberry lines were generated with high efficiencies by a modified transformation protocol, which combines the adoption of a 3-day pre-selection period following transformation, and the addition of 10-microM thidiazuron to the regeneration medium . Histological GUS activity indicated that the reporter gene was expressed specifically in phloem of leaves, petioles, and roots of transgenic plants . The results suggest that the transformation protocol and the AtSUC2 promoter may be useful for engineering phytoplasma-resistant transgenic strawberries.

Nat Struct Mol Biol, 2004 Aug, 11(8), 791 - 6 Epub 2004 Jul 04.
Crystal structure of a Rad51 filament; Conway AB et al.; Rad51, the major eukaryotic homologous recombinase, is important for the repair of DNA damage and the maintenance of genomic diversity and stability . The active form of this DNA-dependent ATPase is a helical filament within which the search for homology and strand exchange occurs . Here we present the crystal structure of a Saccharomyces cerevisiae Rad51 filament formed by a gain-of-function mutant . This filament has a longer pitch than that seen in crystals of Rad51's prokaryotic homolog RecA, and places the ATPase site directly at a new interface between protomers . Although the filament exhibits approximate six-fold symmetry, alternate protein-protein interfaces are slightly different, implying that the functional unit of Rad51 within the filament may be a dimer . Additionally, we show that mutation of His352, which lies at this new interface, markedly disrupts DNA binding.

Eur J Biochem, 2004 Jul, 271(14), 2855 - 62
How mammalian transcriptional repressors work; Thiel G et al.; Research on the regulation of transcription in mammals initially focused on the mechanism of transcriptional activation and 'positive control' of gene regulation . In contrast, transcriptional repression and 'negative control' of gene transcription was viewed rather as part of the 'prokaryotic book of biology' . However, results obtained in recent years have shown convincingly that transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and may play a key role in many biological processes . In particular, the fact that human diseases, such as Rett and ICF syndromes as well as some human forms of cancer, are connected with the activities of human repressor proteins indicates that transcriptional repression and gene silencing is essential for maintenance of the cellular integrity of a multicellular organism . The wide range of diseases caused by aberration in transcriptional repression sheds light on the importance of understanding how mammalian transcriptional repressor proteins work.

Indian J Exp Biol, 2004 Mar, 42(3), 235 - 43
Transglutaminases, thioredoxins and protein disulphide isomerase: diverse enzymes with a common goal of cross-linking proteins in lower organisms; Rao RU et al.; Prokaryotes and various eukaryotes have remarkable ability to survive under adverse physiologic conditions and protect themselves from environmental stresses . An important mechanism by which they accomplish this is by synthesizing rigid and biochemically inert structures around them . In general, these structures are highly stable and resistant to mechanical and chemical insults . Biochemically, they are composed of complex carbohydrates, such as chitin and heavily crosslinked scaffold of proteins to form complex structures, such as sheath, cuticle, and epicuticle . Transglutaminases (TGases) are a family of enzymes that share catalytic function with thioredoxin and protein disulphide isomerases (PDI) and catalyze protein crosslink reaction by establishing epsilon-(gamma-glutamyl)lysine isopeptide bonds . The isopeptide bonds thus formed are of great physiologic significance because once formed, they cannot be hydorlysed by any known enzymes of the eukaryote system and exhibit high resistance to reducing agents, detergents, and chaotropic agents . Therefore, it is likely that protective structures viz., sheath, cuticle, epicuticle, and viral core proteins synthesized by microorganisms involve active participation of TGases . In this review, we briefly describe the current knowledge of non-mammalian TGases and their possible role in growth, development, and survival of small organisms . Special reference is made to filarial nematode and bacterial TGases since they are the most well-characterized and studied enzymes among non-mammalian TGases.

Mol Cells, 2004 Jun 30, 17(3), 422 - 9
Characterization of the plastid-encoded carboxyltransferase subunit (accD) gene of potato; Lee SS et al.; The plastid accD gene encoding the carboxyltransferase b subunit of acetyl-coenzyme A carboxylase (ACCase) was cloned from potato . Potato accD (saccD) is 2487 bp in length with a 614 bp 5 cent upstream promoter region and an ORF of 1524 bp, corresponding to a polypeptide of 507 amino acids . The N-terminal region lacks recognizable motifs, while the C-terminal regions contains five motifs . Among these is motif II, PLIIVCASGGARMQE, the sole motif present in all available accD sequences of plants and animals, and of E . coli, suggesting that this motif may correspond to the catalytic site . saccD has the typical prokaryotic promoter signatures, TTGACA and TATCAA, which are -35 and -10-like sequences for plastid-encoded RNA polymerase (PEP), at positions -184 and -160, respectively . However, it seems to be transcribed by the nucleus-encoded RNA polymerase because it is expressed in tuber and root, and in the dark (under crippled PEP conditions) and its transcription initiation sites do not correspond to those of PEP . saccD is expressed in all potato tissues, i.e., leaf, stem, root, and tuber, and its transcript is produced at a similar rate in the light and dark, at different developmental stages, and during growth in the presence of different sugars and carbon sources . Taken together, our results suggest that potato accD is a housekeeping gene constitutively expressed in both chloroplast and amyloplast.

Proc Natl Acad Sci U S A, 2004 Jul 13, 101(28), 10284 - 9 Epub 2004 Jul 01.
The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity; Doublie S et al.; In prokaryotes, two DNA glycosylases recognize and excise oxidized pyrimidines: endonuclease III (Nth) and endonuclease VIII (Nei) . The oxidized purine 8-oxoguanine, on the other hand, is recognized by Fpg (also known as MutM), a glycosylase that belongs to the same family as Nei . The recent availability of the human genome sequence allowed the identification of three human homologs of Escherichia coli Nei . We report here the crystal structure of a human Nei-like (NEIL) enzyme, NEIL1 . The structure of NEIL1 exhibits the same overall fold as E . coli Nei, albeit with an unexpected twist . Sequence alignments had predicted that NEIL1 would lack a zinc finger, and it was therefore expected to use a different DNA-binding motif instead . Our structure revealed that, to the contrary, NEIL1 contains a structural motif composed of two antiparallel beta-strands that mimics the antiparallel beta-hairpin zinc finger found in other Fpg/Nei family members but lacks the loops that harbor the zinc-binding residues and, therefore, does not coordinate zinc . This "zincless finger" appears to be required for NEIL1 activity, because mutating a very highly conserved arginine within this motif greatly reduces the glycosylase activity of the enzyme.

Nat Biotechnol, 2004 Jul, 22(7), 911 - 7
Analysis of genomic context: prediction of functional associations from conserved bidirectionally transcribed gene pairs; Korbel JO et al.; Several widely used methods for predicting functional associations between proteins are based on the systematic analysis of genomic context . Efforts are ongoing to improve these methods and to search for novel aspects in genomes that could be exploited for function prediction . Here, we use gene expression data to demonstrate two functional implications of genome organization: first, chromosomal proximity indicates gene coregulation in prokaryotes independent of relative gene orientation; and second, adjacent bidirectionally transcribed genes (that is,'divergently' organized coding regions) with conserved gene orientation are strongly coregulated . We further demonstrate that such bidirectionally transcribed gene pairs are functionally associated and derive from this a novel genomic context method that reliably predicts links between >2,500 pairs of genes in approximately 100 species . Around 650 of these functional associations are supported by other genomic context methods . In most instances, one gene encodes a transcriptional regulator, and the other a nonregulatory protein . In-depth analysis in Escherichia coli shows that the vast majority of these regulators both control transcription of the divergently transcribed target gene/operon and auto-regulate their own biosynthesis . The method thus enables the prediction of target processes and regulatory features for several hundred transcriptional regulators.

Nucleic Acids Res, 2004 Jun 30, 32(11), 3503 - 10 Print 2004.
Multiple elements required for translation of plastid atpB mRNA lacking the Shine-Dalgarno sequence; Hirose T et al.; The mechanism of translational initiation differs between prokaryotes and eukaryotes . Prokaryotic mRNAs generally contain within their 5'-untranslated region (5'-UTR) a Shine-Dalgarno (SD) sequence that serves as a ribosome-binding site . Chloroplasts possess prokaryotic-like translation machinery, and many chloroplast mRNAs have an SD-like sequence, but its position is variable . Tobacco chloroplast atpB mRNAs contain no SD-like sequence and are U-rich in the 5'-UTR (-20 to -1 with respect to the start codon) . In vitro translation assays with mutated mRNAs revealed that an unstructured sequence encompassing the start codon, the AUG codon and its context are required for translation . UV crosslinking experiments showed that a 50 kDa protein (p50) binds to the 5'-UTR . Insertion of an additional initiation region (SD-sequence and AUG) in the 5'-UTR, but not downstream, arrested translation from the authentic site; however, no inhibition was observed by inserting only an AUG triplet . We hypothesize for translational initiation of the atpB mRNA that the ribosome enters an upstream region, slides to the start codon and forms an initiation complex with p50 and other components.

FEBS Lett, 2004 Jul 2, 569(1-3), 43 - 8
Polymerization of nucleotide-free, GDP- and GTP-bound cell division protein FtsZ: GDP makes the difference; Huecas S et al.; Stable, more than 98% nucleotide-free apo-FtsZ was prepared from purified Methanococcus jannaschhi FtsZ . This facilitates the study of the functional mechanisms of this FtsZ, an assembling GTPase, which shares a common fold with eukaryotic tubulin . Apo-FtsZ underwent cooperative magnesium-induced polymerization with a similar critical concentration and morphology related to that of reconstituted GTP-bound FtsZ, suggesting that the binding of GTP contributes insignificantly to the stability of the FtsZ polymers . On the other hand, reconstituted GDP-FtsZ polymerized with a larger critical concentration than GTP-FtsZ, indicating that GDP binding destabilizes FtsZ polymers . Upon GTP hydrolysis by FtsZ polymers, in the absence of a continued GTP supply and under macromolecular crowding conditions enhancing FtsZ polymerization, the straight GTP polymers disappeared and were replaced by characteristic helically curved GDP-bound polymers . These results suggest that the roles of GTP binding and hydrolysis by this archaeal FtsZ are simply to facilitate disassembly . In a physiological situation in GTP excess, GDP-bound FtsZ subunits could again bind GTP, or trigger disassembly, or be recognized by FtsZ filament depolymerizing proteins, allowing the Z-ring dynamics during prokaryotic cell division.

Plant J, 2004 Jul, 39(2), 170 - 81
Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen, resulting in male sterility in Arabidopsis thaliana; Ariizumi T et al.; A novel male-sterile mutant of Arabidopsis thaliana was isolated by means of T-DNA tagging . Pollen abortion of the mutant was evident after microspore release, and pollen grains were completely absent at anthesis . Transmission electron microscope analysis revealed that primexine was coarsely developed, and that although sporopollenin was produced, it was not deposited onto the microspore plasma membrane . The sporopollenin that failed to be deposited aggregated and accumulated within the locule and on the locule wall . Finally, as no exine formation was observed, the mutant was named nef1 . The plastoglobuli within the plastids of the tapetum were reduced, and lipid accumulation was considerably decreased . The mutant had a significantly altered leaf chloroplast ultrastructure and showed various growth defects . Lipid analysis revealed that the total lipid content in nef1 was lower than that in the wild type, which indicated that Nef1 was involved in lipid metabolism . Cloning of the full-length Nef1 indicated that the gene encodes a novel plant protein of 1123 amino acids with limited sequence similarities to membrane proteins or transporter-like proteins, and the NEF1 is predicted to be a plastid integral membrane protein . Motif analysis revealed that NEF1 contains prokaryotic membrane lipoprotein lipid attachment sites that are involved in maintaining cell envelope integrity . It is predicted that the Nef1 encodes a membrane protein that maintains the envelope integrity in the plastids.

Crit Rev Biotechnol, 2003, 23(4), 233 - 66
Polysaccharide lyases: recent developments as biotechnological tools; Michaud P et al.; Polysaccharide lyases, which are polysaccharide cleavage enzymes, act mainly on anionic polysaccharides . Produced by prokaryote and eukaryote organisms, these enzymes degrade (1,4) glycosidic bond by a beta elimination mechanism and have unsaturated oligosaccharides as major products . New polysaccharides are cleaved only by their specific polysaccharide lyases . From anionic polysaccharides controlled degradations, various biotechnological applications were investigated . This review catalogues the degradation of bacterial, plant and animal polysaccharides (neutral and anionic) by this family of carbohydrate acting enzymes.

Cell Mol Life Sci, 2004 Jul, 61(13), 1562 - 78
The ultimate nanoscale mincer: assembly, structure and active sites of the 20S proteasome core; Heinemeyer W et al.; 20S proteasomes constitute the proteolytic core of large protease complexes found in all branches of life . Among these, the eukaryotic 26S proteasome ubiquitously poses as a vital final entity in regulated degradation of intracellular proteins . The composition of 20S core particles has been disclosed in detail, facilitated by groundbreaking studies on ancestral prokaryotic 20S proteasomes of low complexity and culminated in the crystal structure determination of the much more complex eukaryotic particles . This article first summarizes insights into the structural organization of the 20S core followed by characterization of its proteolytic activities, which are confined to the central cavity of the particle . In eukaryotes they reside in three different subunit types differing in their preference for cleavage sites in substrates as well as in their importance for the proteasome's cellular function . The second part reviews current knowledge on the biogenesis pathways of 20S core particles, which have to ensure not only the fixed subunit arrangement but also activation of proteolytic subunits in a late assembly state.

Acta Biochim Pol, 2004, 51(2), 281 - 98
Signalling: basics and evolution; Williams RJ; Signalling concerns the transfer of information from one body, a source, to another, a receiver in order to stimulate activity . The problem arises with the word information . It is defined as what is transferred in a sequence of things, say between people, e.g . words or signs . The idea of signalling between people is then obvious but it is not clear in cell biology . Information transfer, signalling, is required for the organisation of all cellular activity but we must ask what is transferred and how is it transmitted and received? Sometimes it is assumed that all information, i.e . organisation in a cell, is represented in the DNA sequence . This is incorrect . We shall show that the environment is a second source of information concerning material and energy . The receiving party from both DNA and the environment is general metabolism . The metabolism then signals back and sends information to both DNA and uptake from the environment . Even then energy is needed with machinery to send out all signals . This paper examines the way signalling evolved from prokaryotes through to man . In this process the environmental information received increased to the extent that finally the brain is a phenotypic as much as a genotypic organ within a whole organism . By phenotypic we mean it is organised by and interactive with information from the environment.

Biotechnol Lett, 2004 Jul, 26(13), 1051 - 5
Cloning, sequence and expression of the lactate dehydrogenase gene from the human malaria parasite, Plasmodium vivax; Turgut-Balik D et al.; Increased drug resistance to anti-malarials highlights the need for the development of new therapeutics for the treatment of malaria . To this end, the lactate dehydrogenase (LDH) gene was cloned and sequenced from genomic DNA of Plasmodium vivax ( PvLDH) Belem strain . The 316 amino acid protein-coding region of the PvLDH gene was inserted into the prokaryotic expression vector pKK223-3 and a 34 kDa protein with LDH activity was expressed in E . coli . Structural differences between human LDHs and PfLDH make the latter an attractive target for inhibitors leading to novel anti-malarial drugs . The sequence similarity between PvLDH and PfLDH (90% residue identity and no insertions or deletions) indicate that the same approach could be applied to Plasmodium vivax, the most common human malaria parasite in the world.

Cytogenet Genome Res, 2004, 105(1), 54 - 6
The evolutionary conservation of the human chitotriosidase gene in rodents and primates; Gianfrancesco F et al.; Chitinases have been identified in a variety of organisms ranging from prokaryotes to eukaryotes, known to specifically degrade chitin, an abundant polymer of N-acetylglucosamine . Recently a human chitinolytic enzyme called CHIT1 was discovered . CHIT1 is expressed by activated macrophages and hydrolyzes artificial chitotrioside substrates, but its specific function in humans is unknown, since it is generally believed that man completely lacks endogenous chitin and endogenous substrates for chitinases . An intriguing question is whether the chitotriosidase activity is just an evolutionary remnant or it has a physiological function in man . To test these hypotheses we utilized a "phylogenomic" approach performing accurate sequence analyses of this gene, coding for CHIT1, in rodents and primates . Inspecting the sequences available in public databases, we determined that this gene is conserved in rodents (mouse and rat) and primates (chimpanzee, gorilla, orangutan, gibbon, baboon, a common marmoset and black macaque) . Moreover we found that a 24-base pair duplication that determines an enzymatically inactive human protein is not present in primates, suggesting that this polymorphism was created during human evolution . These results indicate that chitotriosidase is conserved across the evolutionary scale . Such conservation of the CHIT1 gene argues in favour of an important biological role .

J Gen Virol, 2004 Jul, 85(Pt 7), 2035 - 44
Identification of the thymidylate synthase within the genome of white spot syndrome virus; Li Q et al.; Thymidylate synthase (TS) (EC 2.1.1.45) is essential for the de novo synthesis of dTMP in prokaryotic and eukaryotic organisms . Within the white spot syndrome virus (WSSV) genome, an open reading frame (WSV067) that encodes a 289 amino acid polypeptide showed significant homology to all known TSs from species including mammals, plants, fungi, protozoa, bacteria and DNA viruses . In this study, WSV067 was expressed in Escherichia coli, and the purified recombinant protein showed TS activity in dUMP-folate-binding assays using ultraviolet difference spectroscopy . RT-PCR and Western blot analyses showed that WSV067 was a genuine and early gene . Phylogenetic analysis revealed that WSSV-TS was more closely related to the TSs of eukaryotes than to those from prokaryotes.

J Biol Chem, 2004 Sep 3, 279(36), 37779 - 88 Epub 2004 Jun 24.
Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter; Roosbeek S et al.; In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters . These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2 . To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2 . We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry . We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells . The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues . This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells . The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1 . Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.

Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1175 - 84
Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding; Sasaki Y et al.; Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA . Two physically distinct types of enzymes are found in nature . Heteromeric ACCase composed of four subunits is usually found in prokaryotes, and homomeric ACCase composed of a single large polypeptide is found in eukaryotes . Most plants have both forms, the heteromeric form in plastids, in which de novo fatty acids are synthesized, and the homomeric form in cytosol . This review focuses on the structure and regulation of plant heteromeric ACCase and its manipulation for plant breeding.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W517 - 21
LOCnet and LOCtarget: sub-cellular localization for structural genomics targets; Nair R et al.; LOCtarget is a web server and database that predicts and annotates sub-cellular localization for structural genomics targets; LOCnet is one of the methods used in LOCtarget that can predict sub-cellular localization for all eukaryotic and prokaryotic proteins . Targets are taken from the central registration database for structural genomics, namely, TargetDB . LOCtarget predicts localization through a combination of four different methods: known nuclear localization signals (PredictNLS), homology-based transfer of experimental annotations (LOChom), inference through automatic text analysis of SWISS-PROT keywords (LOCkey) and de novo prediction through a system of neural networks (LOCnet) . Additionally, we report predictions from SignalP . The final prediction is based on the method with the highest confidence . The web server can be used to predict sub-cellular localization of proteins from their amino acid sequence . The LOCtarget database currently contains localization predictions for all eukaryotic proteins from TargetDB and is updated every week . The server is available at http://www.rostlab.org/services/LOCtarget/.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W318 - 20
PredictRegulon: a web server for the prediction of the regulatory protein binding sites and operons in prokaryote genomes; Yellaboina S et al.; An interactive web server is developed for predicting the potential binding sites and its target operons for a given regulatory protein in prokaryotic genomes . The program allows users to submit known or experimentally determined binding sites of a regulatory protein as ungapped multiple sequence alignments . It analyses the upstream regions of all genes in a user-selected prokaryote genome and returns the potential binding sites along with the downstream co-regulated genes (operons) . The known binding sites of a regulatory protein can also be used to identify its orthologue binding sites in phylogeneticaly related genomes where the trans-acting regulator protein and cognate cis-acting DNA sequences could be conserved . PredictRegulon can be freely accessed from a link on our world wide web server: http://www.cdfd.org.in/predictregulon/.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W204 - 7
A suite of web-based programs to search for transcriptional regulatory motifs; Liu Y et al.; The identification of regulatory motifs is important for the study of gene expression . Here we present a suite of programs that we have developed to search for regulatory sequence motifs: (i) BioProspector, a Gibbs-sampling-based program for predicting regulatory motifs from co-regulated genes in prokaryotes or lower eukaryotes; (ii) CompareProspector, an extension to BioProspector which incorporates comparative genomics features to be used for higher eukaryotes; (iii) MDscan, a program for finding protein-DNA interaction sites from ChIP-on-chip targets . All three programs examine a group of sequences that may share common regulatory motifs and output a list of putative motifs as position-specific probability matrices, the individual sites used to construct the motifs and the location of each site on the input sequences . The web servers and executables can be accessed at http://seqmotifs.stanford.edu.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W45 - 7
CVTree: a phylogenetic tree reconstruction tool based on whole genomes; Qi J et al.; Composition Vector Tree (CVTree) implements a systematic method of inferring evolutionary relatedness of microbial organisms from the oligopeptide content of their complete proteomes . Since the first bacterial genomes were sequenced in 1995 there have been several attempts to infer prokaryote phylogeny from complete genomes . Most of them depend on sequence alignment directly or indirectly and, in some cases, need fine-tuning and adjustment . The composition vector method circumvents the ambiguity of choosing the genes for phylogenetic reconstruction and avoids the necessity of aligning sequences of essentially different length and gene content . This new method does not contain 'free' parameter and 'fine-tuning' . A bootstrap test for a phylogenetic tree of 139 organisms has shown the stability of the branchings, which support the small subunit ribosomal RNA (SSU rRNA) tree of life in its overall structure and in many details . It may provide a quick reference in prokaryote phylogenetics whenever the proteome of an organism is available, a situation that will become commonplace in the near future.

BMC Biol . 2004 Jun 23;2(1):15.
Inter-genomic displacement via lateral gene transfer of bacterial trp operons in an overall context of vertical genealogy; Xie G et al.; BACKGROUND: The growing conviction that lateral gene transfer plays a significant role in prokaryote genealogy opens up a need for comprehensive evaluations of gene-enzyme systems on a case-by-case basis . Genes of tryptophan biosynthesis are frequently organized as whole-pathway operons, an attribute that is expected to facilitate multi-gene transfer in a single step . We have asked whether events of lateral gene transfer are sufficient to have obscured our ability to track the vertical genealogy that underpins tryptophan biosynthesis . RESULTS: In 47 complete-genome Bacteria, the genes encoding the seven catalytic domains that participate in primary tryptophan biosynthesis were distinguished from any paralogs or xenologs engaged in other specialized functions . A reliable list of orthologs with carefully ascertained functional roles has thus been assembled and should be valuable as an annotation resource . The protein domains associated with primary tryptophan biosynthesis were then concatenated, yielding single amino-acid sequence strings that represent the entire tryptophan pathway . Lateral gene transfer of several whole-pathway trp operons was demonstrated by use of phylogenetic analysis . Lateral gene transfer of partial-pathway trp operons was also shown, with newly recruited genes functioning either in primary biosynthesis (rarely) or specialized metabolism (more frequently) . CONCLUSIONS: (i) Concatenated tryptophan protein trees are congruent with 16S rRNA subtrees provided that the genomes represented are of sufficiently close phylogenetic spacing . There are currently seven tryptophan congruency groups in the Bacteria . Recognition of a succession of others can be expected in the near future, but ultimately these should coalesce to a single grouping that parallels the 16S rRNA tree (except for cases of lateral gene transfer) . (ii) The vertical trace of evolution for tryptophan biosynthesis can be deduced . The daunting complexities engendered by paralogy, xenology, and idiosyncrasies of nomenclature at this point in time have necessitated an expert-assisted manual effort to achieve a correct analysis . Once recognized and sorted out, paralogy and xenology can be viewed as features that enrich evolutionary histories.

Can J Microbiol, 2004 Apr, 50(4), 225 - 38
Sequence analysis of bacterial redox enzyme maturation proteins (REMPs); Turner RJ et al.; The twin-arginine protein transport (Tat) system is a remarkable molecular machine dedicated to the translocation of fully folded proteins across energy-transducing membranes . Complex cofactor-containing Tat substrates acquire their cofactors prior to export, and substrate proteins actually require to be folded before transport can proceed . Thus, it is very likely that mechanisms exist to prevent wasteful export of immature Tat substrates or to curb competition between immature and mature substrates for the transporter . Here we assess the primary sequence relationships between the accessory proteins implicated in this process during assembly of key respiratory enzymes in the model prokaryote Escherichia coli . For each respiratory enzyme studied, a redox enzyme maturation protein (REMP) was assigned . The main finding from this review was the hitherto unexpected link between the Tat-linked REMP DmsD and the nitrate reductase biosynthetic protein NarJ . The evolutionary link between Tat transport and cofactor insertion processes is discussed.

Evolution Int J Org Evolution, 2004 May, 58(5), 946 - 55
The rate and pattern of cladogenesis in microbes; Martin AP et al.; Theories of macroevolution rarely have been extended to include microbes; however, because microbes represent the most ancient and diverse assemblage of organismal diversity, such oversight limits our understanding of evolutionary history . Our analysis of phylogenetic trees for microbes suggests that macroevolution may differ between prokaryotes and both micro- and macroeukaryotes (mainly plants and animals) . Phylogenetic trees inferred for prokaryotes and some microbial eukaryotes conformed to expectations assuming a constant rate of cladogenesis over time and among lineages: nevertheless, microbial eukaryote trees exhibited more variation in rates of cladogenesis than prokaryote trees . We hypothesize that the contrast of macroevolutionary dynamics between prokaryotes and many eukaryotes is due, at least in part, to differences in the prevalence of lateral gene transfer (LGT) between the two groups . Inheritance is predominantly, if not wholly, vertical within eukaryotes, a feature that allows for the emergence and maintenance of heritable variation among lineages . By contrast, frequent LGT in prokaryotes may ameliorate heritable variation in rate of cladogenesis resulting from the emergence of key innovations; thus, the inferred difference in macroevolution might reflect exclusivity of key innovations in eukaryotes and their promiscuous nature in prokaryotes.

J Biol Chem, 2004 Aug 27, 279(35), 36951 - 61 Epub 2004 Jun 21.
The spacious active site of a Y-family DNA polymerase facilitates promiscuous nucleotide incorporation opposite a bulky carcinogen-DNA adduct: elucidating the structure-function relationship through experimental and computational approaches; Perlow-Poehnelt RA et al.; Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo{a}pyrene (BP) . A tumorigenic metabolite of BP, (+)-anti-benzo-{a}pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-{BP}-N(2)-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems . The 10S (+)-trans-anti-{BP}-N(2)-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions . Dpo4 belongs to the DinB branch of the Y-family, which also includes E . coli pol IV and eukaryotic pol kappa . We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-{BP}-N(2)-dG adduct by Dpo4 . Dpo4 is able to bypass the 10S (+)-trans-anti-{BP}-N(2)-dG adduct, albeit to a lesser extent than unmodified guanine, and the V(max) values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar . Computational studies suggest that 10S (+)-trans-anti-{BP}-N(2)-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion.

Prog Nucleic Acid Res Mol Biol, 2004, 78, 305 - 37
Cooperation of endo- and exoribonucleases in chloroplast mRNA turnover; Bollenbach TJ et al.; Chloroplasts were acquired by eukaryotic cells through endosymbiosis and have retained their own gene expression machinery . One hallmark of chloroplast gene regulation is the predominance of posttranscriptional control, which is exerted both at the gene-specific and global levels . This review focuses on how chloroplast mRNA stability is regulated, through an examination of poly(A)-dependent and independent pathways . The poly(A)-dependent pathway is catalyzed by polynucleotide phosphorylase (PNPase), which both adds and degrades destabilizing poly(A) tails, whereas RNase II and PNPase may both participate in the poly(A)-independent pathway . Each system is initiated through endonucleolytic cleavages that remove 3' stem-loop structures, which are catalyzed by the related proteins CSP41a and CSP41b and possibly an RNase E-like enzyme . Overall, chloroplasts have retained the prokaryotic endonuclease-exonuclease RNA degradation system despite evolution in the number and character of the enzymes involved . This reflects the presence of the chloroplast within a eukaryotic host and the complex responses that occur to environmental and developmental cues.

Biochemistry, 2004 Jun 29, 43(25), 8234 - 46
Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies; Wang H et al.; Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites . The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket . This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122) . Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively . MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM . Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA) . Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step . Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively . Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0 . Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 {k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03}, which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0 . Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity . From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)) . However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonation of the thiolate-imidazolium ion . In addition, substitutions of the triad aspartate are not tolerated . The enzyme appears, therefore, to be engineered to rapidly form a stable acetylated species poised to react with an arylamine substrate.

Nat Genet, 2004 Jul, 36(7), 760 - 6 Epub 2004 Jun 20.
Biased biological functions of horizontally transferred genes in prokaryotic genomes; Nakamura Y et al.; Horizontal gene transfer is one of the main mechanisms contributing to microbial genome diversification . To clarify the overall picture of interspecific gene flow among prokaryotes, we developed a new method for detecting horizontally transferred genes and their possible donors by Bayesian inference with training models for nucleotide composition . Our method gives the average posterior probability (horizontal transfer index) for each gene sequence, with a low horizontal transfer index indicating recent horizontal transfer . We found that 14% of open reading frames in 116 prokaryotic complete genomes were subjected to recent horizontal transfer . Based on this data set, we quantitatively determined that the biological functions of horizontally transferred genes, except mobile element genes, are biased to three categories: cell surface, DNA binding and pathogenicity-related functions . Thus, the transferability of genes seems to depend heavily on their functions.

Nucleic Acids Res, 2004 Jun 18, 32(11), 3282 - 93 Print 2004.
Variable and conserved elements of human ribosomes surrounding the mRNA at the decoding and upstream sites; Graifer D et al.; This study is centred upon an important biological problem concerning the structural organization of mammalian ribosomes that cannot be studied by X-ray analysis because 80S ribosome crystals are still unavailable . Here, positioning of the mRNA on 80S ribosomes was studied using mRNA analogues containing the perfluorophenylazide cross-linker on either the guanosine or an uridine residue . The modified nucleotides were directed to positions from -9 to +6 with respect to the first nucleotide of the P site bound codon by a tRNA cognate to the triplet targeted to the P site . Upon mild UV-irradiation, the modified nucleotides at positions +4 to +6 cross-linked to protein S15 and 18S rRNA nucleotides A1823-A1825 . In addition, modified guanosines in positions +5 and +6 also cross-linked to G626, and in position +1 to G1702 . Cross-linking from the upstream positions was mainly to protein S26 that has no prokaryotic homologues . These findings indicate that the tail of mammalian S15 comes closer to the decoding site than that of its prokaryotic homologue S19, and that the environments of the upstream part of mRNA on 80S and 70S ribosomes differ . On the other hand, the results confirm the widely accepted idea regarding the conserved nature of the decoding site of the small subunit rRNA.

Toxicon, 2004 Jun 15, 43(8), 951 - 60
Characterization of the outer pore region of the apamin-sensitive Ca2+-activated K+ channel rSK2; Jager H et al.; We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K+ channels . Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2 . In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K+ channel SK1 and SK3 like . Directed by the results of the toxin receptor on the ShakerK+ channel, we changed single amino acids of the SK2 K+ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3 . Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK+ channel to improve Shaker K+ channel CTX sensitivity . Interestingly SK2 V342G became CTX sensitive with a Kd of 19 nM and was also KTX sensitive Kd=97 nM . SK2 S344E (KdCTX = 105 nM,KdKTX = 144 nM) and G348D (KdCTX = 31 nM,Kd KTX = 89 nM) became also CTX and KTX sensitive with a lower affinity . The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (Kd = 6 nM,Kd = 48 nM, and Kd = 12 nM) . Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K+ channel pore is very similar to those of voltage-gated K+ channels such as the Shaker K+ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel . From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore.We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K+ channels . Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2 . In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K+ channel SK1 and SK3 like . Directed by the results of the toxin receptor on the ShakerK+ channel, we changed single amino acids of the SK2 K+ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3 . Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK+ channel to improve Shaker K+ channel CTX sensitivity . Interestingly SK2 V342G became CTX sensitive with a Kd of 19 nM and was also KTX sensitive Kd = 97 nM . SK2 S344E (KdCTX = 105 nM,KdKTX = 144 nM) and G348D (KdCTX = 31 nM,Kd KTX = 89 nM) became also CTX and KTX sensitive with a lower affinity . The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (Kd = 6 nM,Kd = 48 nM, and Kd = 12 nM) . Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K+ channel pore is very similar to those of voltage-gated K+ channels such as the Shaker K+ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel . From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore .

Res Microbiol, 2004 Jun, 155(5), 376 - 86
Shaping bacterial genomes with integrative and conjugative elements; Burrus V et al.; Integrative and conjugative elements (ICEs) are self-transmissible mobile genetic elements that are increasingly recognized to contribute to lateral gene flow in prokaryotes . ICEs, like most temperate bacteriophages integrate into the genome and like conjugative plasmids disseminate by conjugative transfer to new hosts . Thought of schematically, the structure of ICEs is similar to that of other types of the mobile elements; ICEs have a backbone composed of three modules ensuring maintenance, dissemination and regulation . This backbone can acquire additional functions probably through the action of insertion sequences, transposons and specific recombinases . Previously, ICEs were thought of as only vectors for transfer of antibiotic resistance genes, but it is now evident that ICEs can mediate the transfer of a very diverse set of functions . ICEs allow bacteria to rapidly adapt to new environmental conditions and to colonize new niches . Like phages and conjugative plasmids they also likely mediate the transfer of virulence determinants . ICEs shape the bacterial genome, promoting variability between strains of the same species and distributing genes between unrelated bacterial genera . Finally, we propose that by utilizing conserved integration sites, ICEs may promote the mobilization of genomic islands.

Virology, 2004 Jul 1, 324(2), 412 - 8
Functional analysis of virion host shutoff protein of pseudorabies virus; Lin HW et al.; During lytic infection, the virion host shutoff (vhs) protein of alphaherpesviruses causes the degradation of mRNAs nonspecifically . In this work, we cloned the vhs gene (UL41 open reading frame) of pseudorabies virus (PRV; TNL strain) by PCR, and its nucleotide sequences were determined . The PCR product of vhs gene was subcloned into the prokaryotic pET32b expression vector, and production of the recombinant vhs protein was examined by SDS-PAGE . Result of Western blotting demonstrated that our recombinant vhs protein reacted with antiserum against a synthetic peptide of 17 amino acids of the vhs protein . After purification with nickel-chelate affinity chromatography, the purified recombinant vhs protein exhibited in vitro ribonuclease activity as expected . We further cloned the vhs gene into eukaryotic expression vectors and investigated the intracellular function of vhs protein by DNA transfection . By transient transfection and CAT assay, we found the CAT activity was reduced in the presence of vhs, indicating that degradation of mRNA of the CAT gene was caused by the vhs . Furthermore, our results showed that the plaque formation of pseudorabies virus was blocked by exogenous vhs . Taken together, we have cloned the vhs gene of pseudorabies virus (TNL strain) and conducted functional analysis of the recombinant vhs protein in vitro as well as in vivo.

Beijing Da Xue Xue Bao, 2004 Jun 18, 36(3), 268 - 71
{Prokaryotic expression, purification and preparation of polyclonal antibody and immunohistochemistry analysis of RS21-C6 molecule}; Li Y et al.; OBJECTIVE: To express a thymocyte development related molecule RS21-C6 protein prepare its polyclonal antibodies and study its distribution in thymus by immunohistochemistry analysis . METHODS: RS21-C6 recombinant protein was expressed in E.coli system and then purified by Ni-NTA chromatographic method . Polyclonal antibodies were prepared by immunized rabbits with the purified recombinant protein . Immunohistochemistry analysis was performed to show the distribution of RS21-C6 in mouse thymus tissue . RESULTS: RS21-C6 recombinant protein was successfully expressed and purified . Then the high titer polyclonal antibody was prepared and its specificity was confirmed . The immunohistochemistry analysis demonstrated that RS21-C6 had an intense expression in the medullary region of thymus while scattered in the cortical region and the cortical medullary junction region of thymus . It also showed that RS21-C6 was co-expressed in thymocyte and thymic stromal cells . CONCLUSION: RS21-C6 molecule is broadly and highly expressed in thymus which suggests that this molecule may perform various functions in thymocytes at different developmental stages.

Curr Opin Microbiol, 2004 Jun, 7(3), 221 - 6
Prokaryotic diversity and its limits: microbial community structure in nature and implications for microbial ecology; Curtis TP et al.; Recent advances in the estimation of prokaryotic diversity have brought us insight into two questions: what is the extent of prokaryotic diversity, and perhaps more importantly, why bother finding out . In this review, we highlight the insights about the extent of diversity that may be gained by considering patterns that occur, or are likely to occur, in the relative abundance of prokaryotic taxa . We posit that global reservoirs of diversity are an important driving force behind patterns in localised diversity seen in leaves, intestines and wastewater treatment reactors . Thus, where the reservoir community is very large and relatively even, chance alone will prevent physically identical communities from having the same, or sometimes even stable, communities . By contrast, communities that tend to be similar (even when not physically identical) and stable are observed where the source diversity is low . Thus the relationship between structure and function in a community can only be understood, predicted and engineered through an understanding of the source of diversity from which the community is drawn.

Curr Opin Genet Dev, 2004 Apr, 14(2), 107 - 12
Links between DNA replication and recombination in prokaryotes; McGlynn P; Recombination plays a crucial role in underpinning genome duplication, ensuring that replication blocks are removed or bypassed, and that the replication machinery is subsequently reloaded back onto the DNA . Recent studies have identified a surprising variety of ways in which damaged replication forks are repaired and have shown that the mechanism used depends on the nature of the original blocking lesion . Indeed, an emerging theme is that a single recombination enzyme or complex can perform highly varied tasks, depending on the context of the recombination reaction.

Nat Biotechnol, 2004 Jul, 22(7), 877 - 82 Epub 2004 Jun 13.
Cold-shock induced high-yield protein production in Escherichia coli; Qing G et al.; Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability . Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock . Several proteins were produced with very high yields, including E . coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM) . The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy . We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref . 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

J Biol Chem, 2004 Sep 10, 279(37), 38960 - 8 Epub 2004 Jun 12.
Crystal structure of the oxygen-dependant coproporphyrinogen oxidase (Hem13p) of Saccharomyces cerevisiae; Phillips JD et al.; Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyzes the sixth step of the heme biosynthetic pathway . Unusually for heme biosynthetic enzymes, CPO exists in two evolutionarily and mechanistically distinct families, with eukaryotes and some prokaryotes employing members of the highly conserved oxygen-dependent CPO family . Here, we report the crystal structure of the oxygen-dependent CPO from Saccharomyces cerevisiae (Hem13p), which was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 A . The protein adopts a novel structure that is quite different from predicted models and features a central flat seven-stranded anti-parallel sheet that is flanked by helices . The dimeric assembly, which is seen in different crystal forms, is formed by packing of helices and a short isolated strand that forms a beta-ladder with its counterpart in the partner subunit . The deep active-site cleft is lined by conserved residues and has been captured in open and closed conformations in two different crystal forms . A substratesized cavity is completely buried in the closed conformation by the approximately 8-A movement of a helix that forms a lid over the active site . The structure therefore suggests residues that likely play critical roles in catalysis and explains the deleterious effect of many of the mutations associated with the disease hereditary coproporphyria.

Gene, 2004 Jun 23, 335, 25 - 35
Two novel asparaginyl endopeptidase-like cysteine proteinases from the protist Trichomonas vaginalis: their evolutionary relationship within the clan CD cysteine proteinases; Leon-Felix J et al.; Cysteine proteinases (CPs) are important virulence factors of the protozoan parasite Trichomonas vaginalis . A total of six genes coding for cathepsin L-like CPs belonging to clan CA have been identified in T . vaginalis . At least 23 distinct spots with proteolytic activity have been detected by two-dimensional (2-D) substrate gel electrophoresis from in vitro grown parasites; however, only few of them have been characterized . In this work, we detected six spots with proteolytic activity and molecular weights between 25 and 35 kDa . The six proteinases correspond to two distinct CP families: the papain-like family, represented by four spots with pIs between 4.5 and 5.5; and the legumain-like family represented by two spots with pI 6.3 and 6.5 . Next, we obtained two cDNAs encoding for legumain-like CPs from T . vaginalis, which were named Tvlegu-1 and Tvlegu-2 . The size of these cDNA clones were 1225 and 1364 bp, which encoded for 388 and 415 amino acids, respectively . Their putative translation products have molecular masses of 42.8 and 47.2 kDa, corresponding to inactive legumain-like CP precursors . The two sequences share approximately 40% identity at the amino acid level . These protein products can be classified within a branch of the legumain-like family in clan CD cysteine proteinases due to their sensitivity to specific proteinases inhibitors, their DNA sequences, and phylogenetic reconstruction . However, they do not correspond either to the typical asparaginyl endopeptidase or the glycosylphosphatidylinositol (GPI): protein transamidase subfamilies . These results suggest that the TVLEGU-1 and TVLEGU-2 peptidases are likely to be part of a new subfamily within the legumain-like family of clan CD cysteine proteinases . Furthermore, they could be one of the missing links between prokaryotic and eukaryotic CPs in clan CD enzymes.

Biochemistry (Mosc), 2004 May, 69(5), 471 - 84
Photosynthetic units of phototrophic organisms; Boichenko VA; Photoautotrophic organisms play a key role in the biosphere of the Earth, converting solar energy of the 350-1000 nm range into biochemically available form . In contemporary aquatic and terrestrial ecosystems, the dominating groups are the oxygen evolving cyanobacteria, algae, and higher plants . Anoxygenic phototrophic microorganisms occupy mainly ecological niches with extreme environmental conditions . Despite diverse evolution of all these taxonomic groups, their photosynthetic apparatus has a similar molecular design and identical principles of operation . This review covers recent data about features of the structural and functional organization of pigment-protein complexes of the basic types of photosynthetic units in prokaryotes and eukaryotes . A correspondence between the optical properties of various photosynthetic units and the natural light conditions is discussed.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 206 - 9
{Prokaryotic expression of human calcyclin-binding protein and preparation of mouse polyclonal antibody against hCacyBP}; Cheng Y et al.; AIM: To express hCacyBP in E.coli and prepare mouse polyclonal antibody against hCacyBP so as to study tissue distribution of hCacyBP and evaluate its role during formation of multidrug resistance to gastric cancer . METHODS: hCacyBP gene was subcloned into an expression vector pET28a, and then the recombinant vector was transformed into E . coli BL21 . The recombinant protein was expressed in BL21 under IPTG induction . Using purified hCacyBP as immunogen, mouse polyclonal antibody against hCacyBP was prepared . hCacyBP was detected by Western blot in 10 kinds of rabbit tissues . Expression and distribution of hCacyBP in SGC7901/VCR and SGC7901 cells were detected by Western blot and immunohistochemical staining . RESULTS: hCacyBP was successfully expressed in E . coli . The Western blot analysis showed that hCacyBP was expressed in all 10 kinds of rabbit tissues, but expression of brain and liver tissues were higher as compared with other tissues . Expression and distribution of hCacyBP in both SGC7901/VCR and SGC7901 cells had no significant difference . CONCLUSION: CacyBP expressed widespreadly in varied tissues . Polyclonal antibody against hCacyBP that we prepared has high specificity, which provides a powerful tool for studying the function of hCacyBP.

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