J Bacteriol, 1999 Sep, 181(18), 5871 - 5 A single-ring mitochondrial chaperonin (Hsp60-Hsp10) can substitute for GroEL-GroES in vivo; Nielsen KL et al.; Chaperonins participate in the facilitated folding of a variety of proteins in vivo . To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an Escherichia coli strain engineered so that the groE operon is under strict regulatory control . We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES. FEBS Lett, 1999 Sep 17, 458(2), 117 - 23 HtpG is essential for the thermal stress management in cyanobacteria; Tanaka N et al.; The heat shock protein (Hsp) HtpG is a member of the Hsp90 protein family . We cloned a single-copy gene encoding a homologue of HtpG from the unicellular cyanobacterium Synechococcus sp . PCC 7942 . Sequence alignment with HtpGs from other prokaryotes revealed unique features in the cyanobacterial HtpG primary sequence . A monocistronic mRNA of the htpG gene increased transiently in response to heat shock . In order to elucidate the role of HtpG in vivo, we inactivated the htpG gene by targeted mutagenesis . Although the mutation did not affect the photoautotrophic growth at 30 and 42 degrees C, the mutant cells were unable to grow at 45 degrees C . They lost both basal and acquired thermotolerances . These results indicate that HtpG plays an essential role for the thermal stress management in cyanobacteria, the first such an example for either a photosynthetic or a prokaryotic organism. Nucleic Acids Res, 1999 Oct 1, 27(19), 3911 - 20 Heuristic approach to deriving models for gene finding; Besemer J et al.; Computer methods of accurate gene finding in DNA sequences require models of protein coding and non-coding regions derived either from experimentally validated training sets or from large amounts of anonymous DNA sequence . Here we propose a new, heuristic method producing fairly accurate inhomogeneous Markov models of protein coding regions . The new method needs such a small amount of DNA sequence data that the model can be built 'on the fly' by a web server for any DNA sequence >400 nt . Tests on 10 complete bacterial genomes performed with the GeneMark.hmm program demonstrated the ability of the new models to detect 93.1% of annotated genes on average, while models built by traditional training predict an average of 93.9% of genes . Models built by the heuristic approach could be used to find genes in small fragments of anonymous prokaryotic genomes and in genomes of organelles, viruses, phages and plasmids, as well as in highly inhomogeneous genomes where adjustment of models to local DNA composition is needed . The heuristic method also gives an insight into the mechanism of codon usage pattern evolution. Clin Immunol, 1999 Sep, 92(3), 256 - 64 Molecular definition and characterization of recombinant bovine CB8 and CB10: immunogenicity and arthritogenicity; Tang B et al.; Theoretically, the ability to produce recombinant type II collagen (CII) peptide fragments in a prokaryotic expression system would be extremely useful for preparing adequate amounts of CII peptides suitable for therapeutic uses . Bacteria do not contain the enzymes involved in the extensive posttranslational modifications that occur during the biosynthesis of CII, such as the hydroxylation of prolyl and lysyl residues and glycosylation of hydroxylysyl residues . As these posttranslational modifications may play a role in the immune and arthritogenic response to CII, it was unclear whether collagen expressed in Escherichia coli would be immunologically comparable to tissue-derived CII . Therefore, we prepared recombinant proteins for CB8 and CB10 by cloning CB8 (CII 403-551) and CB10 (CII 552-897) genes from bovine chondrocytes by RT-PCR technique and expressing them in an E . coli expression system . Characterization of these recombinant proteins revealed that both rCB8 and rCB10 stimulated T cell proliferation in a T cell determinant-specific manner . The T cells from mice immunized with rCB8 respond specifically to a synthetic peptide, CII 445-453, the CB8 T cell determinant . Conversely, rCB10-primed T cells respond strongly to CII 610-618, the CB10 T cell determinant . Recombinant CB8-induced autoantibodies that bound to mouse CB8 as effectively and in the same topographic distribution as tissue-derived CB8 . Finally, when rCB8 and rCB10 proteins were used to immunize B10.RIII (H-2(r)) mice, rCB8 induced arthritis in 33% of the mice, very similar to the incidence induced by tissue-derived CB8 peptide . As was found to be the case with tissue-derived CB10, rCB10 was completely ineffective in inducing arthritis . Pathological changes of arthritic joints in the mice immunized with rCB8 were similar to those observed in mice immunized with tissue-derived CB8 . Thus, these recombinant CII peptides expressed in E . coli can induce an effective immunologic response and suggest that functionally useful CII peptides can be generated by the prokaryotic expression system . Biosci Biotechnol Biochem, 1999 Jul, 63(7), 1171 - 80 Kinetic studies on the function of all the conserved tryptophans involved inside and outside the QW motifs of squalene-hopene cyclase: stabilizing effect of the protein structure against thermal denaturation; Sato T et al.; Site-directed mutagenesis experiments were carried out to identify the responsibility of the eight QW motifs for the reaction catalyzed by squalene-hopene cyclase (SHC) . Alterations of the conserved tryptophans, which are responsible for the stacking structure with glutamine, into aliphatic amino acids gave a significantly lower temperature for the catalytic optimum as for the mutageneses of QW motifs 4, 5a and 5b, which are specifically present in SHCs . However, there was no change in the optimal temperatures of the mutated SHCs targeted at the other five motifs 1, 2, 3, 5c and 6 . Thus, reinforcement against heat denaturation can be proposed as a function of the three QW motifs 4, 5a and 5b, but no function could be identified for the QW motifs 1, 2, 3, 5c and 6, although they are commonly found in all the families of prokaryotic SHCs and eukaryotic oxidosqualene cyclases . On the other hand, the three conserved tryptophans of W169, W312 and W489, which are located inside the putative central cavity and outside the QW motifs, were identified as components of the active sites, but also had a function against thermal denaturation . The other two tryptophan residues of W142 and W558, which are located outside the QW motifs, were found not to be active sites, but also had a role for stabilizing the protein structure . It is noteworthy that the mutants replaced by phenylalanine had higher temperatures for the catalytic optimum than those replaced by aliphatic amino acids . The catalytic optimal pH values for all the mutants remained unchanged with an identical value of 6.0. Blood, 1999 Sep 15, 94(6), 1855 - 63 Promoter elements of vav drive transgene expression in vivo throughout the hematopoietic compartment; Ogilvy S et al.; To develop a method for targeting expression of genes to the full hematopoietic system, we have used transgenic mice to explore the transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it . Previously, we showed that a cluster of elements surrounding its promoter could drive hematopoietic-specific expression of a bacterial lacZ reporter gene, but the expression was confined to lymphocytes and was sporadically silenced . Those limitations are ascribed here to the prokaryotic reporter gene . With a human CD4 (hCD4) cell surface reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types . In multiple lines, hCD4 appeared on most, if not all, B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid cells . Moreover, high levels appeared on both lineage-committed progenitors and the more primitive preprogenitors . In the fetus, expression was evident in erythroid cells of the definitive but not the primitive type . These results indicate that a prokaryotic sequence can inactivate a transcription unit and that the vav promoter region constitutes a potent transgenic vector for the entire definitive hematopoietic compartment. J Exp Med, 1999 Sep 6, 190(5), 717 - 24 Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family; Agranoff D et al.; Mammalian natural resistance-associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria . Eukaryotic Nramp homologues transport divalent cations such as Fe(2+), Mn(2+), Zn(2+), and Cu(2+) . Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guerin {BCG}) also encode an Nramp homologue (Mramp) . RNA encoding Mramp induces approximately 20-fold increases in (65)Zn(2+) and (55)Fe(2+) uptake when injected into Xenopus laevis oocytes . Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5 . Mramp-mediated (65)Zn(2+) and (55)Fe(2+) transport is abolished by an excess of Mn(2+) and Cu(2+), confirming that Mramp interacts with a broad range of divalent transition metal cations . Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M . tuberculosis are upregulated in response to increases in ambient Fe(2+) and Cu(2+) between <1 and 5 microM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase . Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5-70 microM Cu(2+) . M . bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp . Mramp exemplifies a novel prokaryotic class of metal ion transporter . Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria. Biochem J, 1999 Sep 15, 342 Pt 3, 721 - 8 Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases; Arimitsu E et al.; Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity . Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues . The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species . Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues . The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively . The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes . In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families . The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved . Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins. Mol Microbiol, 1999 Sep, 33(5), 895 - 903 The plasmid prophage N15: a linear DNA with covalently closed ends; Rybchin VN et al.; Coliphage N15 is a temperate bacteriophage whose prophage is a linear plasmid molecule with covalently closed ends (telomeres) . The N15 prophage provided the first example of such DNA in prokaryotes and, up to now, it is the only known example of a linear plasmid in Escherichia coli . The linear N15 mature phage DNA has single-stranded cohesive ends . The phage and plasmid prophage DNAs are circularly permuted . The nucleotide structure of the telomere-forming site tel RL in phage DNA corresponds to the structures of the terminal hairpin loops . It suggests a unique mechanism for conversion of the circular phage DNA to the linear plasmid form, which is performed by the prokaryotic telomerase (protelomerase) . The results of a comparison of the protelomerase with integrases lead us to suggest that these proteins may have evolved from a common ancestor . The mechanism of plasmid N15 replication is unknown . We propose that the protelomerase participates in linear plasmid replication, acting as a resolvase of replicative intermediates that are tail-to-tail linear dimers . The sequence analysis of the N15 DNA showed that it represents an evolutionary 'link' between plasmids F, P1, P4 and lambdoid bacteriophages. Hybridoma, 1999 Jun, 18(3), 243 - 9 Expression and characterization of anti-NCA-95 scFv (CEA 79 scFv) in a prokaryotic expression vector modified to contain a Sfi I and Not I site; Yi K et al.; The CEA 79 antibody has been used in bone marrow scintigraphy for the differential diagnosis of skeletal tumors and the evaluation of the bone marrow status of patients with various hematological disorders . The specific localization of radio-labeled CEA 79 antibody in bone marrow depends on its reactivity with NCA-95 (nonspecific cross-reacting antigen-95) present on the surface and in the cytosol of human granulocytes and myelopoietic cells . To make a CEA 79 scFv molecule that would be less immunogenic and more penetrating than the intact mouse immunoglobulin, we constructed a pRSET Sfi I/Not I expression vector . The scFv gene was then excised from a pCANTAB 5 E phage display vector by digestion with Sfi I and Not I and inserted into the pRSET Sfi I/Not I expression vector . Upon transformation of a BL21(DE3)pLysS strain of E . coli, CEA 79 scFv became expressed in inclusion bodies requiring a renaturation process for solubilization . The final yield of CEA 79 scFv was 5 mg per a liter of culture . The refolded CEA 79 scFv exhibited an affinity (Kd = 2.1 x 10(-9) M) equivalent to that of the original CEA 79 antibody (K(d) = 3.3 x 10(-9) M) and the same immunoreactivity to CEA and NCA-95 in Western blots and in immunohistochemical staining experiments. Nat Biotechnol, 1999 Sep, 17(9), 906 - 9 A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes; Knoblauch M et al.; A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes . The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 microm . The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection . We demonstrate injection of Lucifer Yellow and Lucifer Yellow-dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms . Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures . Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA containing its gene into individual chloroplasts. Nat Biotechnol, 1999 Sep, 17(9), 884 - 8 Rapid identification of DNA-binding proteins by mass spectrometry; Nordhoff E et al.; We report a protocol for the rapid identification of DNA-binding proteins . Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract . Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . The determined molecular masses are often sufficient for identification . If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches . Apart from protein identification, the protocol also yields information on posttranslational modifications . The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA. FEBS Lett, 1999 Sep 3, 457(3), 527 - 33 H(+)-PPases: a tightly membrane-bound family Baltscheffsky M, Schultz A, Baltscheffsky H. The earliest known H(+)-PPase (proton-pumping inorganic pyrophosphatase), the integrally membrane-bound H(+)-PPi synthase (proton-pumping inorganic pyrophosphate synthase) from Rhodospirillum rubrum, is still the only alternative to H(+)-ATP synthase in biological electron transport phosphorylation . Cloning of several higher plant vacuolar H(+)-PPase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton-pumping enzymes . The bacterial H(+)-PPi synthase and two algal vacuolar H(+)-PPases are homologous with this family, as deduced from their cloned genes . The prokaryotic and algal homologues differ more than the H(+)-PPases from higher plants, facilitating recognition of functionally significant entities . Primary structures of H(+)-PPases are reviewed and compared with H(+)-ATPases and soluble PPases. FEBS Lett, 1999 Sep 3, 457(3), 527 - 33 H+ -PPases: a tightly membrane-bound family; Baltscheffsky M et al.; The earliest known H+-PPase (proton-pumping inorganic pyrophosphatase), the integrally membrane-bound H+-PPi synthase (proton-pumping inorganic pyrophosphate synthase) from Rhodospirillum rubrum, is still the only alternative to H+-ATP synthase in biological electron transport phosphorylation . Cloning of several higher plant vacuolar H+-PPase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton-pumping enzymes . The bacterial H+-PPi synthase and two algal vacuolar H+-PPases are homologous with this family, as deduced from their cloned genes . The prokaryotic and algal homologues differ more than the H+-PPases from higher plants, facilitating recognition of functionally significant entities . Primary structures of H+-PPases are reviewed and compared with H+-ATPases and soluble PPases. Orig Life Evol Biosph, 1999 Aug, 29(4), 425 - 35 Directed molecular evolution; Harris LF et al.; We propose the existence of a relationship of stereochemical complementarity between gene sequences that code for interacting components: nucleic acid-nucleic acid, protein-protein and protein-nucleic acid . Such a relationship would impose evolutionary constraints on the DNA sequences themselves, thus retaining these sequences and governing the direction of the evolutionary process . Therefore, we propose that prebiotic, template-directed autocatalytic synthesis of mutally cognate peptides and polynucleotides resulted in their amplification and evolutionary conservation in contemporary prokaryotic and eukaryotic organisms as a genetic regulatory apparatus . If this proposal is correct, then the relationships between the sequences in DNA coding for these interactions constitute a life code of which the genetic code is only one aspect of the many related interactions encoded in DNA. Orig Life Evol Biosph, 1999 Aug, 29(4), 391 - 404 The antibiotic viomycin as a model peptide for the origin of the co-evolution of RNA and proteins; Wank H et al.; Viomycin is an RNA-binding peptide antibiotic which inhibits prokaryotic protein synthesis and group I intron self-splicing . This antibiotic enhances the activity of the ribozyme derived from the Neurospora crassa VS RNA, and at sub-inhibitory concentrations it induces the formation of group I intron oligomers . Here, we address the question whether viomycin exerts specificity in the promotion of RNA-RNA interactions . In an in vitro selection experiment we tested the ability of viomycin to specifically select molecules out of an RNA pool . Group I intron RNA was incubated with a pool of random sequence RNA, or with a pool of RNA molecules which had previously been enriched for viomycin-binding RNAs . Viomycin was added in order to select viomycin-binding RNAs and to guide their interaction with the intron RNA resulting in recombinant molecules . Viomycin was indeed capable of specifically selecting RNA molecules which contain viomycin-binding sites promoting recombination . These results suggest that small peptides are able to play the role of selector molecules in a putative 'RNA World' launching the co-evolution of RNA and proteins into an 'RNA-protein World'. EMBO J, 1999 Sep 1, 18(17), 4597 - 607 Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6 A resolution; Komori H et al.; The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer . A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor . We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F . The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains . Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities . The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization . The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication. Trends Microbiol, 1999 Sep, 7(9), 377 - 81 The influence of dsRNA viruses on the biology of plant pathogenic fungi; McCabe PM et al.; Double-stranded RNA viruses are ubiquitous in fungi . They are non-infective and, like most prokaryotic plasmids, are only transmitted to compatible strains via cell fusion . Most are cryptic, but some with an established phenotype, such as the hypoviruses of the chestnut-blight fungus, have been studied for their potential as biological control agents of fungi. EMBO J, 1999 Sep 1, 18(17), 4875 - 81 Natural synthesis of a DNA-binding protein from the C-terminal domain of DNA gyrase A in Borrelia burgdorferi; Knight SW et al.; We have identified a 34 kDa DNA-binding protein with an HU-like activity in the Lyme disease spirochete Borrelia burgdorferi . The 34 kDa protein is translated from an abundant transcript initiated within the gene encoding the A subunit of DNA gyrase . Translation of the 34 kDa protein starts at residue 499 of GyrA and proceeds in the same reading frame as full-length GyrA, resulting in an N-terminal-truncated protein . The 34 kDa GyrA C-terminal domain, although not homologous, substitutes for HU in the formation of the Type 1 complex in Mu transposition, and complements an HU-deficient strain of Escherichia coli . This is the first example of constitutive expression of two gene products in the same open reading frame from a single gene in a prokaryotic cellular system. Eur J Biochem, 1999 Aug, 263(3), 726 - 35 Overexpression, purification and biochemical characterization of the wound-induced leucine aminopeptidase of tomato; Gu YQ et al.; Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A) . While the expression of LapA genes are well characterized, the specificity of the LAP-A enzyme has not been studied . The LAP-A preprotein and mature polypeptide were overexpressed in Escherichia coli . PreLAP-A was not processed and was inactive accumulating in inclusion bodies . In contrast, 55-kDa mature LAP-A subunits assembled into an active, 357-kDa enzyme in E . coli . LAP-A from E . coli cultures was purified to apparent homogeneity and characterized relative to its animal (porcine LAP) and prokaryotic (E . coli PepA) homologues . Similar to the porcine and E . coli enzymes, the tomato LAP-A had high temperature and pH optima . Mn2+ was a strong activator for all three enzymes, while chelators, zinc ion, and the slow-binding aminopeptidase inhibitors (amastatin and bestatin) strongly inhibited activities of all three LAPs . The substrate specificities of porcine, E . coli and tomato LAPs were determined using amino-acid-p-nitroanilide and -beta-naphthylamide substrates . The tomato LAP-A preferentially hydrolyzed substrates with N-terminal Leu, Met and Arg residues . LAP-A had substantially lower levels of activity on other chromogenic substrates . Several differences in substrate specificities for the animal, plant and prokaryotic enzymes were noted. Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10129 - 33 Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry; Griffey RH et al.; We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains . High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously . Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum . In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex . Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain . We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS-MS protection assay . We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity . The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant . An A1409-G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin . These observations demonstrate the power of MS-based methods to provide molecular insights into small molecule/RNA interactions useful in the design of selective new antimicrobial drugs. Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10104 - 8 Heightened sensitivity of a lattice of membrane receptors; Duke TA et al.; Receptor proteins in both eukaryotic and prokaryotic cells have been found to form two-dimensional clusters in the plasma membrane . In this study, we examine the proposition that such clusters might show coordinated responses because of the spread of conformational states from one receptor to its neighbors . A Monte Carlo simulation was developed in which receptors flipped in probabilistic fashion between an active and an inactive state . Conformational energies depended on (i) ligand binding, (ii) a chemical modification of the receptor conferring adaptation, and (iii) the activity of neighboring receptors . Rate constants were based on data from known biological receptors, especially the bacterial Tar receptor, and on theoretical constraints derived from an analogous Ising model . The simulated system showed a greatly enhanced sensitivity to external signals compared with a corresponding set of uncoupled receptors and was operational over a much wider range of ambient concentrations . These and other properties should make a lattice of conformationally coupled receptors ideally suited to act as a "nose" by which a cell can detect and respond to extracellular stimuli. Proc R Soc Lond B Biol Sci, 1999 Aug 7, 266(1428), 1571 - 7 The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells; Vellai T et al.; Eukaryotes have long been thought to have arisen by evolving a nucleus, endomembrane, and cytoskeleton . In contrast, it was recently proposed that the first complex cells, which were actually proto-eukaryotes, arose simultaneously with the acquisition of mitochondria . This so-called symbiotic association hypothesis states that eukaryotes emerged when some ancient anaerobic archaebacteria (hosts) engulfed respiring alpha-proteobacteria (symbionts), which evolved into the first energy-producing organelles . Therefore, the intracellular compartmentalization of the energy-converting metabolism that was bound originally to the plasma membrane appears to be the key innovation towards eukaryotic genome and cellular organization . The novel energy metabolism made it possible for the nucleotide synthetic apparatus of cells to be no longer limited by subsaturation with substrates and catalytic components . As a consequence, a considerable increase has occurred in the size and complexity of eukaryotic genomes, providing the genetic basis for most of the further evolutionary changes in cellular complexity . On the other hand, the active uptake of exogenous DNA, which is general in bacteria, was no longer essential in the genome organization of eukaryotes . The mitochondrion-driven scenario for the first eukaryotes explains the chimera-like composition of eukaryotic genomes as well as the metabolic and cellular organization of eukaryotes. Cell Death Differ, 1999 Aug, 6(8), 805 - 12 Bacterial death induced by expression of the intracellular portion of human Fas; Yang Y et al.; In attempting to produce the intracellular portion of human Fas (IC175 - 319) as a GST-fusion protein we found that expression of GST-IC175 - 319, but not GST alone or GST-IC231 - 298 (containing the Fas death domain), rapidly caused the death of host E . coli cells . Expression of GST-IC175 - 319 with a single amino acid substitution (V238N) corresponding to the mouse lprcg mutation, or E245A, which abolishes the ability of Fas to self-associate, did not kill bacteria . Deletional analysis identified a 20-amino acids region (Asp210 - Lys230) as essential for the killing activity, and introduction of a single amino acid substitution (T225P) in this 20 amino acid region markedly decreased the ability of Fas- IC175 - 319 to cause bacterial death . These data indicate that Fas can deliver a death signal in prokaryotic organisms by a means that shares some features with eukaryotic cells, and raise the possibility that certain mechanisms leading to programmed cell death may be conserved from bacteria to mammalian cells. Res Microbiol, 1999 Jul-Aug, 150(6), 375 - 84 Horizontal gene transfers in the environment: natural transformation as a putative process for gene transfers between transgenic plants and microorganisms; Bertolla F et al.; Horizontal gene transfers among bacteria, such as natural transformation or conjugation, may have played an important role in bacterial evolution . They are thought to have been involved in promoting genome plasticity which permitted bacteria to adapt very efficiently to any change in their environment and to colonize a wide range of ecosystems . Evidence that some genes were transferred from eukaryotes, and in particular, from plants to bacteria, was obtained from nucleotide and protein sequence analyses . However, numerous factors, including some which are endogenous to the bacterial cells, tend to limit the extent of transfer, particularly among phylogenetically distant organisms . The goal of this paper is to give an overview of the potentials and limits of natural interkingdom gene transfers, with particular focus on prokaryote-originating sequences which fit the nuclear genome of transgenic plants. Anal Chem, 1999 Aug 15, 71(16), 3436 - 40 Multiplexed screening of neutral mass-tagged RNA targets against ligand libraries with electrospray ionization FTICR MS: a paradigm for high-throughput affinity screening; Hofstadler SA et al.; We demonstrate that binding of mixtures of aminoglycosides can be measured simultaneously against multiple RNA targets of identical length and similar (or identical) molecular weight . Addition of a neutral mass tag to one of the RNA targets shifts the detected peaks to a higher mass/charge ratio, where complexes with small molecules can be identified unambiguously . An appropriately placed neutral mass tag does not alter RNA--ligand binding . The utility of this strategy is demonstrated with model RNAs corresponding to the decoding region of the prokaryotic and eukaryotic rRNAs and a mixture of five aminoglycosides . Complexes are observed between the aminoglycoside library and the prokaryotic rRNA model, while no aminoglycoside was observed to bind to the mass-tagged eukaryotic rRNA model . The differential binding data is consistent with the eukaryotic A-site rRNA having a different conformation compared with the prokaryotic A-site that prevents entry and binding of neomycin-class aminoglycosides . Mass spectrometric analysis of neutral mass-tagged macromolecular targets represents a new high-throughput screening paradigm in which the interaction of multiple targets against a collection of small molecules can be evaluated in parallel. Braz J Med Biol Res, 1999 Sep, 32(9), 1063 - 71 Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae; Padula M et al.; In the present study, we analyzed DNA damage induced by phycocyanin (PHY) in the presence of visible light (VL) using a set of repair endonucleases purified from Escherichia coli . We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species . Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes . High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at detectable levels . DNA repair after PHY photosensitization was also investigated . Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants . The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type . Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant . The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells . Moreover, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae. Biochemistry, 1999 Aug 24, 38(34), 10949 - 57 A molecular envelope of the ligand-binding domain of a glutamate receptor in the presence and absence of agonist; Abele R et al.; Solution scattering studies were performed on a ligand-binding domain (S1S2) of a glutamate receptor ion channel (GluR) in order to study GluR-binding and signal-transduction mechanisms . The core of the ligand-binding domain is homologous to prokaryotic periplasmic binding proteins (PBP), whose binding mechanism involves a dramatic cleft closure: the "Venus flytrap" . Several models of GluR function have proposed that a similar cleft closure is induced by agonist binding . We have directly tested this putative functional homology by measuring the radius of gyration of S1S2 in the presence and absence of saturating concentrations of agonists . In contrast to the PBP, S1S2 shows no reduction in radius of gyration upon agonist binding, excluding a comparably large conformational change . Furthermore, we determined an ab initio molecular envelope for our S1S2 construct, which also contains the peptides that connect the PBP homology core to the three transmembrane domains and to an N-terminal domain . By fitting an atomic model of the ligand-binding domain core to the envelope of our extended construct, we were able to establish the likely position of these connecting peptides . Their positions relative to one another and to the expected sites of an agonist-induced conformational change suggest that ion channel gating and desensitization may involve more subtle and complex mechanisms than have been assumed based on the structural homology to the PBP. Hum Genet, 1999 Jun, 104(6), 454 - 9 NME6: a new member of the nm23/nucleoside diphosphate kinase gene family located on human chromosome 3p21.3; Mehus JG et al.; The NME (nm23/nucleoside diphosphate kinase) gene family in human is involved in the phosphorylation of nucleoside diphosphates and a variety of regulatory phenomena associated with development, oncogenic transformation, and metastasis . Here we report the cDNA sequence for a sixth member of this family, NME6 . The cDNA sequence predicts a 186-residue protein that includes the characteristic active site motif of a nucleoside diphosphate (NDP) kinase, as well as the other residues previously identified as crucial for nucleotide binding and catalysis . The NME6 protein sequence is only 34-41% identical to the five previously reported human NME proteins, and is similarly related to prokaryotic and primitive eukaryotic NDP kinases . Compared to typical proteins of this family such as NME1 and NME2, NME6 has three additional residues located in the Kpn loop, and a 22-residue extension at the COOH-terminal . Using radiation hybrid mapping, the NME6 gene was localized to chromosome 3p21.3 . The 1.3-kb transcript of NME6 is expressed at a moderately low level in many human tissues, and is most abundant in kidney, prostate, ovary, intestine, and spleen . Homologous cDNAs were also cloned and sequenced for rat and mouse . The sequence of the first 171 residues of the mouse homologue (Nm23-M6) is 94% identical to the deduced human NME6 protein. FEBS Lett, 1999 Jul 30, 456(1), 49 - 53 2.0 A X-ray structure of the ternary complex of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue; Stammers DK et al.; The X-ray crystal structure of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 A resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change . The first 100 residues of the 158 residue peptide contain a betaalpha betabeta alphabeta motif present in several other proteins including nucleoside diphosphate kinase . Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad-spectrum anti-infective agents. Plant Cell, 1999 Aug, 11(8), 1565 - 78 Polyadenylation occurs at multiple sites in maize mitochondrial cox2 mRNA and is independent of editing status; Lupold DS et al.; Polyadenylation of nucleus-encoded transcripts has a well-defined role in gene expression . The extent and function of polyadenylation in organelles and prokaryotic systems, however, are less well documented . Recent reports of polyadenylation-mediated RNA destabilization in Escherichia coli and in vascular plant chloroplasts prompted us to look for polyadenylation in plant mitochondria . Here, we report the use of reverse transcription-polymerase chain reaction to map multiple polyadenylate addition sites in maize mitochondrial cox2 transcripts . The lack of sequence conservation surrounding these sites suggests that polyadenylation may occur at many 3' termini created by endoribonucleolytic and/or exoribonucleolytic activities, including those activities involved in 3' end maturation . Endogenous transcripts could be efficiently polyadenylated in vitro by using maize mitochondrial lysates with an activity that added AMP more efficiently than GMP . Polyadenylated substrates were tested for stability in maize mitochondrial S100 extracts, and we found that, compared with nonpolyadenylated RNAs, the polyadenylated substrates were less stable . Taken together with the low abundance of polyadenylated RNAs in maize mitochondria, our results are consistent with a degradation-related process . The fact that polyadenylation does not dramatically destabilize plant mitochondrial transcripts, at least in vitro, is in agreement with results obtained for animal mitochondria but differs from those obtained for chloroplasts and E . coli . Because fully edited, partially edited, and unedited transcripts were found among the cloned polyadenylated cox2 cDNAs, we conclude that RNA editing and polyadenylation are independent processes in maize mitochondria. Biochem Biophys Res Commun, 1999 Aug 19, 262(1), 7 - 13 Quantification of the human granulocytic ehrlichiosis agent based on analysis of rRNA isolated from control and infected HL-60 cells; Wu JM et al.; Human granulocytic ehrlichiosis (HGE) is an emerging vector-borne disease caused by an Ehrlichia species similar or identical to E . equi and E . phagocytophila . Previous studies have shown that the pathogen can be cultivated in vitro in permissive cells such as human promyelocytic HL-60 leukemia cells . The mechanism(s) of its infection and propagation in target cells, however, is not well understood, due in part to lack of a method capable of quantitatively determining the amount of the infectious agent . Although several assays currently exist for the HGE agent, they are mostly qualitative and have a number of limitations . In this report, size differences between prokaryotic and eukaryotic rRNAs are utilized to quantitatively assay the HGE agent in HL-60 cells . By comparing the integrated intensity of agarose gel resolved HGE-specific rRNA in host cells, with identically prepared and analyzed rRNA isolated from known quantities of E . coli (JM 109), it is possible to calculate the E . coli-equivalence of the HGE agent present in HL-60 cells according to the equation: Y (E . coli, in viable cells x 10(8)) = -2.573 + 0.11X (% infection by the HGE agent in HL-60 cells) . The method described is reproducible, sensitive, and is not limited by availability of antisera . Furthermore, since the assay has no designer primer and repeated amplification requirements, it can be easily disseminated to and standardized in other laboratories . Eur J Biochem, 1999 Aug, 264(1), 74 - 84 Mitochondrial thioredoxin reductase in bovine adrenal cortex its purification, properties, nucleotide/amino acid sequences, and identification of selenocysteine; Watabe S et al.; Mitochondrial thioredoxin reductase was purified from bovine adrenal cortex . The enzyme is a first protein component in the mitochondrial thioredoxin-dependent peroxide reductase system . The purified reductase exhibited an apparent molecular mass of 56 kDa on SDS/PAGE, whereas the native protein was about 100 kDa, suggesting a homodimeric structure . It catalysed NADPH-dependent reduction of 5, 5'dithiobis(2-nitrobenzoic acid) and thioredoxins from various origins but not glutathione, oxidized dithiothreitol, DL-alpha-lipoic acid, or insulin . Amino acid and nucleotide sequence analyses revealed that it had a presequence composed of 21 amino acids which had features characteristic of a mitochondrial targeting signal . The amino acid sequence of the mature protein was similar to that of bovine cytosolic thioredoxin reductase (57%) and of human glutathione reductase (34%) and less similar to that of Escherichia coli (19%) or yeast (17%) enzymes . Human and bovine cytosolic thioredoxin reductase were recently identified to contain selenocysteine (Sec) as one of their amino acid constituents . We also identified Sec in the C-terminal region of mitochondrial (mt)-thioredoxin reductase by means of MS and amino acid sequence analyses of the C-terminal fragment . The four-amino acid motif, Gly-Cys-Sec-Gly, which is conserved among all Sec-containing thioredoxin reductases, probably functions as the third redox centre of the enzyme, as the mitochondrial reductase was inhibited by 1-chloro-2,4-dinitrobenzene, which was reported to modify Sec and Cys covalently . It is known that mammalian thioredoxin reductase is different from bacterial or yeast enzyme in, for example, their subunit molecular masses and domain structures . These two different types of enzymes with similar activity are suggested to have evolved convergently . Our data clearly show that mitochondria, which might have originated from symbiotic prokaryotes, contain thioredoxin reductase similar to the cytosolic enzyme and different from the bacterial one. RNA, 1999 Aug, 5(8), 1014 - 20 Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis; Frolova LY et al.; Although the primary structures of class 1 polypeptide release factors (RF1 and RF2 in prokaryotes, eRF1 in eukaryotes) are known, the molecular basis by which they function in translational termination remains obscure . Because all class 1 RFs promote a stop-codon-dependent and ribosome-dependent hydrolysis of peptidyl-tRNAs, one may anticipate that this common function relies on a common structural motif(s) . We have compared amino acid sequences of the available class 1 RFs and found a novel, common, unique, and strictly conserved GGQ motif that should be in a loop (coil) conformation as deduced by programs predicting protein secondary structure . Site-directed mutagenesis of the human eRF1 as a representative of class 1 RFs shows that substitution of both glycyl residues in this motif, G183 and G184, causes complete inactivation of the protein as a release factor toward all three stop codons, whereas two adjacent amino acid residues, G181 and R182, are functionally nonessential . Inactive human eRF1 mutants compete in release assays with wild-type eRF1 and strongly inhibit their release activity . Mutations of the glycyl residues in this motif do not affect another function, the ability of eRF1 together with the ribosome to induce GTPase activity of human eRF3, a class 2 RF . We assume that the novel highly conserved GGQ motif is implicated directly or indirectly in the activity of class 1 RFs in translation termination. J Microbiol Methods, 1999 Aug, 37(2), 187 - 92 Magnetic capture-hybridization method for purification and probing of mRNA for neutral protease of Bacillus cereus; Bach HJ et al.; A magnetic capture-hybridization method was assessed for the isolation of prokaryotic mRNA for the neutral protease of B . cereus from liquid culture . A biotin-labeled specific probe was hybridized to the mRNA transcripts and subsequently captured by streptavidin-coated paramagnetic beads . mRNA was detected by dot-blot hybridization with a ds DIG-labeled DNA-probe . The magnetic capture hybridization is a rapid and simple method and has a promising potential for gene expression studies in complex samples. Microb Ecol, 1999 Aug, 38(2), 93 - 113 Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population; Fogel GB et al.; > Abstract Determination of the relative abundance of a specific prokaryote in an environmental sample is of major interest in applied and environmental microbiology . Relative abundance can be calculated using knowledge of SSU rDNA copy number, amount of SSU rDNA in the sample, and a weighted average estimate of the genome sizes for organisms in the original sample . By surveying the literature, we provide estimates of genome size and SSU rDNA copy number for 303 and 101 prokaryotes, respectively . This compilation can be used to make reasonable estimates for a wide range of organisms in the calculation of relative abundance . A statistical analysis suggests that no correlation exists between genome size and SSU rDNA copy number . A phylogenetic analysis is used to offer insights into the evolution of both genome size and SSU rDNA copy number.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p93.html J Mol Evol, 1999 Aug, 49(2), 250 - 9 Origin and evolution of paralogous rRNA gene clusters within the flatworm family Dugesiidae (Platyhelminthes, Tricladida); Carranza S et al.; Analysis of the 18S rDNA sequences of five species of the family Dugesiidae (phylum Platyhelminthes, suborder Tricladida, infraorder Paludicola) and eight species belonging to families Dendrocoelidae and Planaridae and to the infraorder Maricola showed that members of the family Dugesiidae have two types of 18S rDNA genes, while the rest of the species have only one . The duplication event also affected the ITS-1, 5.8S, ITS-2 region and probably the 28S gene . The mean divergence value between the type I and the type II sequences is 9% and type II 18S rDNA genes are evolving 2.3 times more rapidly than type I . The evolutionary rates of type I and type II genes were calibrated from biogeographical data, and an approximate date for the duplication event of 80-120 million years ago was calculated . The type II gene was shown, by RT-PCR, to be transcribed in adult individuals of Schmidtea polychroa, though at very low levels . This result, together with the fact that most of the functionally important positions for small-subunit rRNA in prokaryotes have been conserved, indicates that the type II gene is probably functional. Biochemistry, 1999 Aug 10, 38(32), 10324 - 35 Structure of the KcsA potassium channel from Streptomyces lividans: a site-directed spin labeling study of the second transmembrane segment; Gross A et al.; KcsA is a prokaryotic potassium channel . The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers . Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry . To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure . Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure {Doyle, D., et al . (1998) Science 280, 69-77} . The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale . Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines . At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low . This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements . However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature . These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer. Dis Aquat Organ, 1999 Jun 23, 37(1), 61 - 72 Epitheliocystis agents in sea bream Sparus aurata: morphological evidence for two distinct chlamydia-like developmental cycles; Crespo S et al.; The morphology of membrane-bound intracellular inclusions, or 'cysts', of epitheliocystis from sea bream Sparus aurata is described . Inclusions under the light microscope appear either granular or amorphous . Granular inclusions do not elicit a proliferative host reaction and contain the 3 distinctive developmental stages of chlamydial organisms: the highly pleomorphic reproductive form or reticulate body, the condensing form or intermediate body and the infective non-dividing rather uniform elementary body . Amorphous inclusions may elicit a proliferative host reaction and contain prokaryotic organisms which differ morphologically from those reported within granular cysts . More or less elongated electron-lucent organisms divide by fission to give rise to electron-dense non-dividing small cells with a dense nucleoid . Vacuolated and non-vacuolated small cells are reported . The morphology and developmental cycle of sea bream epitheliocystis agents would support their chlamydial nature; however, the immunohistochemical study conducted on gill samples which carried both inclusions failed to demonstrate the expression of lipopolysaccharide (LPS) chlamydial antigen . The different stages of the 2 distinct developmental cycles described in the present study are compared with electron microscope observations of epitheliocystis organisms reported from different host species . The hypothesis that epitheliocystis infection in the sea bream might be caused by a unique highly pleomorphic chlamydia-like agent, the life history of which includes 2 entirely different developmental cycles, is discussed. J Bacteriol, 1999 Aug, 181(16), 4873 - 8 The adherence-associated lipoprotein P100, encoded by an opp operon structure, functions as the oligopeptide-binding domain OppA of a putative oligopeptide transport system in Mycoplasma hominis; Henrich B et al.; Mycoplasma hominis, a cell-wall-less prokaryote, was shown to be cytoadherent by the participation of a 100-kDa membrane protein (P100) . To identify the gene encoding P100, peptides of P100 were partially sequenced to enable the synthesis of P100-specific oligonucleotides suitable as probes for the detection of the P100 gene . With this strategy, we identified a genomic region of about 10 . 4 kb in M . hominis FBG carrying the P100 gene . Analysis of the complete deduced protein sequence suggests that P100 is expressed as a pre-lipoprotein with a structure in the N-terminal region common to peptide-binding proteins and an ATP- or GTP-binding P-loop structure in the C-terminal region . Downstream of the P100 gene, an additional four open reading frames putatively encoding the four core domains of an active transport system, OppBCDF, were localized . The organization of the P100 gene and oppBCDF in a transcriptionally active operon structure was demonstrated in Northern blot and reverse transcription-PCR analyses, as all gene-specific probes detected a common RNA of 9.5 kb . Primer extension analysis revealed that the transcriptional initiation site was localized 323 nucleotides upstream of the methionine-encoding ATG of the P100 gene . The peptide-binding character of the P100 protein was confirmed by fluorescence spectroscopy and strongly suggests that the cytoadherence-mediating lipoprotein P100 represents OppA, the substrate-binding domain of a peptide transport system in M . hominis. FEMS Microbiol Lett, 1999 Aug 1, 177(1), 75 - 82 A plasmid vector for isolation of strong promoters in Escherichia coli; Carbonelli DL et al.; In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed . It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria . Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin . The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs . Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters . According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications. Planta, 1999 Aug 12, 209(2), 202 - 206 Priority of light/dark entrainment over temperature in setting the circadian rhythms of the prokaryote Synechococcus RF-1; Lin RF et al.; Light/dark (L/D) and temperature are two major factors in the entrainment of circadian rhythms . The input pathways of these two environmental factors for the entrainment of circadian rhythms in Synechococcus RF-1 are different since the overt rhythms in mutant CR-1, one of the circadian-rhythm mutants of Synechococcus RF-1, could be established by temperature cycles but not by L/D . Therefore, it was of interest to investigate the phases of Synechococcus RF-1 cells entrained simultaneously by L/D and temperature . The circadian rhythms of nitrogenase activity and protein synthesis in RF-1 cells entrained by L/D, and by lowered or raised temperatures differed in their peaks of activity . Comparison of the phases of RF-1 cells entrained by L/D and temperature independently, and by L/D and temperature simultaneously indicated that L/D entrainment has priority over the temperature effect. Gene Ther, 1999 Mar, 6(3), 309 - 13 Control of parvovirus DNA replication by a tetracycline-regulated repressor; Maxwell IH et al.; Autonomous parvoviruses are small, single strand DNA viruses which preferentially replicate in transformed and tumor cells, causing cell death by expression of the cytotoxic nonstructural protein, NS1 . Several parvoviruses of the rodent group, including LuIII, efficiently infect human transformed cell lines . The potential for systemic use of these viruses in targeting metastases might be enhanced if NS1 expression and viral replication could be controlled by an innocuous drug such as tetracycline . We therefore substituted prokaryotic tetracycline operator sequences for part of P4 of LuIII, the promoter responsible for transcription of the mRNAs for nonstructural proteins . The resulting construct unexpectedly showed constitutive expression in transiently transfected cells, as indicated by efficient excision and amplification of viral replicative form (RF) DNA . This was apparently due to self-stimulatory transcriptional transactivation by NS1 . This problem was overcome by cotransfection with a plasmid expressing a chimera of the repressor of the tetracycline operon with a KRAB transrepression domain . These conditions allowed efficient control of transcription and RF amplification by the tetracycline derivative, doxycycline . These observations form a basis for developing a therapeutic agent based on a drug-controlled parvovirus. Biochim Biophys Acta, 1999 Aug 5, 1428(2-3), 305 - 13 Determination of different amino sugar 2'-epimerase activities by coupling to N-acetylneuraminate synthesis; Rodriguez-Aparicio LB et al.; A new procedure for quantitating the amount of N-acetyl-D-mannosamine (ManNAc) or ManNAc-6-phosphate produced by 2'-epimerase activities involved in sialic acid metabolism has been developed . The ManNAc generated by the action of N-acetyl-D-glucosamine (GlcNAc) and UDP-GlcNAc 2'-epimerases is condensed with pyruvate through the action of N-acetylneuraminate lyase and the sialic acid released is measured by the thiobarbituric acid assay . For the analysis of prokaryotic GlcNAc-6-phosphate 2'-epimerase, ManNAc-6-phosphate can also be evaluated by this coupled assay after dephosphorylation of the sugar phosphate . This system provides a sensitive, rapid, reproducible, specific and simple procedure (feasible with commercial reagents) for measuring amino sugar 2'-epimerases from eukaryotic and prokaryotic sources . The technique reported here permitted us to detect UDP-GlcNAc 2'-epimerase and GlcNAc 2'-epimerase in mammalian cell extracts and GlcNAc-6-phosphate 2'-epimerase in bacterial extracts. Biochimie, 1999 Jun, 81(6), 631 - 43 Mutants of Chlamydomonas: tools to study thylakoid membrane structure, function and biogenesis; de Vitry C et al.; The unicellular green alga Chlamydomonas reinhardtii is a model system for the study of photosynthesis and chloroplast biogenesis . C . reinhardtii has a photosynthesis apparatus similar to that of higher plants and it grows at rapid rate (generation time about 8 h) . It is a facultative phototroph, which allows the isolation of mutants unable to perform photosynthesis and its sexual cycle allows a variety of genetic studies . Transformation of the nucleus and chloroplast genomes is easily performed . Gene transformation occurs mainly by homologous recombination in the chloroplast and heterologous recombination in the nucleus . Mutants are precious tools for studies of thylakoid membrane structure, photosynthetic function and assembly . Photosynthesis mutants affected in the biogenesis of a subunit of a protein complex usually lack the entire complex; this pleiotropic effect has been used in the identification of the other subunits, in the attribution of spectroscopic signals and also as a 'genetic cleaning' process which facilitates both protein complex purification, absorption spectroscopy studies or freeze-fracture analysis . The cytochrome b6f complex is not required for the growth of C . reinhardtii, unlike the case of photosynthetic prokaryotes in which the cytochrome complex is also part of the respiratory chain, and can be uniquely studied in Chlamydomonas by genetic approaches . We describe in greater detail the use of Chlamydomonas mutants in the study of this complex. FEBS Lett, 1999 Jul 9, 454(3), 283 - 7 Biochemical characterization of recombinant polypeptides corresponding to the predicted betaalphaalpha fold in Aux/IAA proteins; Morgan KE et al.; The plant hormone indoleacetic acid (IAA or auxin) transcriptionally activates a select set of early genes . The Aux/IAA class of early auxin-responsive genes encodes a large family of short-lived, nuclear proteins . Aux/IAA polypeptides homo- and heterodimerize, and interact with auxin-response transcription factors (ARFs) via C-terminal regions conserved in both protein families . This shared region contains a predicted betaalphaalpha motif similar to the prokaryotic beta-ribbon DNA binding domain, which mediates both protein dimerization and DNA recognition . Here, we show by circular dichroism spectroscopy and by chemical cross-linking experiments that recombinant peptides corresponding to the predicted betaalphaalpha region of three Aux/IAA proteins from Arabidopsis thaliana contain substantial alpha-helical secondary structure and undergo homo- and heterotypic interactions in vitro . Our results indicate a similar biochemical function of the plant betaalphaalpha domain and suggest that the betaalphaalpha fold plays an important role in mediating combinatorial interactions of Aux/IAA and ARF proteins to specifically regulate secondary gene expression in response to auxin. Parasitol Res, 1999 Aug, 85(8-9), 779 - 82 Chloramphenicol-sensitive mitochondrial translation in Trypanosoma brucei; Nabholz CE et al.; We developed an in organello system to label newly synthesized mitochondrially encoded proteins of Trypanosoma brucei . Highly purified mitochondria, prepared under isotonic conditions, were incubated with radioactive methionine and cysteine in a suitable translation buffer . Analysis of mitochondrial extracts on TRIS-Tricine gels revealed a subset of labeled, NP-40-insoluble proteins . The labeling of these proteins was resistant to the cytosol-specific translation inhibitor cycloheximide . The proteins, however, were not labeled in the presence of chloramphenicol or erythromycin, inhibitors of prokaryotic type translation, or puromycin, a general translation inhibitor . These results indicate that isotonically isolated mitochondria of T . brucei are capable of protein synthesis. Trends Plant Sci, 1999 Aug, 4(8), 302 - 307 The endosymbiotic origin of the protein import machinery of chloroplastic envelope membranes; Reumann S et al.; Chloroplasts have evolved a complex proteinaceous machinery to import nuclear-encoded proteins . The origin of this machinery, following the endosymbiotic events leading to chloroplasts, is an intriguing, unresolved problem . Given that cyanobacteria are the probable ancestors of chloroplasts, the genome sequence of Synechocystis sp . PCC 6803 offers a valuable resource to identify putative homologs for components of this protein import machinery and to gain insights into its possibly endosymbiotic origin . Detailed computational sequence analysis reveals that Synechocystis sp . PCC 6803 has homologs of three of the known membrane proteins of the chloroplastic translocon, namely Toc75, Tic20 and Tic22, as well as a homolog of the putative component Tic55 . Thus, the chloroplastic protein import machinery is mainly derived from the endosymbiotic cyanobacterium, but, interestingly, not from any of the four main protein secretion systems of prokaryotes . The relatively high sequence variability between chloroplastic and Synechocystis proteins suggests that the ancestral proteins had different physiological roles and were modified significantly to fulfill the new demand of importing proteins into the evolving chloroplast . The fact that some chloroplastic protein import components are not related to any Synechocystis proteins (Toc159, Tic110 and Toc34) indicates that the chloroplastic protein import apparatus is of a dual evolutionary origin. Trends Microbiol, 1999 Aug, 7(8), 328 - 33 Apicomplexan plastids as drug targets; McFadden GI et al.; Prokaryotic metabolic pathways in the relict plastid of apicomplexan parasites make this organelle a promising target for drug development . The parasiticidal activity of several herbicides and antibacterial antibiotics is suspected to be a result of their ability to inhibit key plastid activities. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9218 - 23 A model for a umuDC-dependent prokaryotic DNA damage checkpoint; Opperman T et al.; The products of the Escherichia coli umuDC operon are required for translesion synthesis, the mechanistic basis of most mutagenesis caused by UV radiation and many chemicals . The UmuD protein shares homology with LexA, the repressor of SOS-regulated loci, and similarly undergoes a facilitated autodigestion on interaction with the RecA/single-stranded DNA nucleoprotein filaments formed after a cell experiences DNA damage . This cleavage, in which Ser-60 of UmuD acts as the nucleophile, produces UmuD', the form active in translesion synthesis . Expression of the noncleavable UmuD(S60A) protein and UmuC was found to increase survival after UV irradiation, despite the inability of the UmuD(S60A) protein to participate in translesion synthesis; this survival increase is uvr(+) dependent . Additional observations that expression of the UmuD(S60A) protein and UmuC delayed the resumption of DNA replication and cell growth after UV irradiation lead us to propose that the uncleaved UmuD protein and UmuC delay the resumption of DNA replication, thereby allowing nucleotide excision repair additional time to repair the damage accurately before replication is attempted . After a UV dose of 20 J/m(2), uncleaved UmuD is the predominant form for approximately 20 min, after which UmuD' becomes the predominant form, suggesting that the umuDC gene products play two distinct and temporally separated roles in DNA damage tolerance, the first in cell-cycle control and the second in translesion synthesis over unrepaired or irreparable lesions . The relationship of these observations to the eukaryotic DNA damage checkpoint is discussed. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8937 - 42 Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis; Bellamine A et al.; Sterol 14alpha-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms . Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14alpha-demethylases might be present in this bacterium . Using genomic DNA from mycobacterial strain H(37)Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14alpha-demethylase . Expression of the M . tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca . 50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14alpha-demethylases . It catalyzes 14alpha-demethylation of lanosterol, 24, 25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and (1)H NMR analysis . Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity . Structural requirements of a 14alpha-methyl group and Delta(8(9))-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol . These substrate requirements are similar to those established for plant and animal P450 14alpha-demethylases . From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes. Eur J Biochem, 1999 Jul, 263(1), 6 - 13 Structural maintenance of chromosomes (SMC) proteins: conserved molecular properties for multiple biological functions; Strunnikov AV et al.; The evolutionarily-conserved eukaryotic SMC (structural maintenance of chromosomes) proteins are ubiquitous chromosomal components in prokaryotes and eukaryotes . The eukaryotic SMC proteins form two kind of heterodimers: the SMC1/SMC3 and the SMC2/SMC4 types . These heterodimers constitute an essential part of higher order complexes, which are involved in chromatin and DNA dynamics . The two most prominent and best-characterized complexes are cohesin and condensin, necessary for sister chromatid cohesion and chromosome condensation . Here we discuss these functions together with additional roles in gene dosage compensation and DNA recombination. EMBO J, 1999 Aug 2, 18(15), 4108 - 17 A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli; Templin MF et al.; The first gene of a family of prokaryotic proteases with a specificity for L,D-configured peptide bonds has been identified in Escherichia coli . The gene named ldcA encodes a cytoplasmic L, D-carboxypeptidase, which releases the terminal D-alanine from L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine containing turnover products of the cell wall polymer murein . This reaction turned out to be essential for survival, since disruption of the gene results in bacteriolysis during the stationary growth phase . Owing to a defect in muropeptide recycling the unusual murein precursor uridine 5'-pyrophosphoryl N-acetylmuramyl-tetrapeptide accumulates in the mutant . The dramatic decrease observed in overall cross-linkage of the murein is explained by the increased incorporation of tetrapeptide precursors . They can only function as acceptors and not as donors in the crucial cross-linking reaction . It is concluded that murein recycling is a promising target for novel antibacterial agents. J Neurochem, 1999 Aug, 73(2), 700 - 11 Biochemical characterization of a lysosomal protease deficient in classical late infantile neuronal ceroid lipofuscinosis (LINCL) and development of an enzyme-based assay for diagnosis and exclusion of LINCL in human specimens and animal models; Sohar I et al.; Classical late-infantile neuronal ceroid lipofuscinosis (LINCL), a progressive and fatal neurodegenerative disease of childhood, results from mutations in a gene (CLN2) that encodes a protein with significant sequence similarity to prokaryotic pepstatin-insensitive acid proteases . We have developed a sensitive protease activity assay that allows biochemical characterization of the CLN2 gene product in various human biological samples, including solid tissues (brain and chorionic villi), blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells), and cultured cells (lymphoblasts, fibroblasts, and amniocytes) . The enzyme has a pH optimum of 3.5 and is rapidly inactivated at neutral pH . A survey of fibroblasts and lymphoblasts demonstrated that lack of activity was associated with LINCL arising from mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs' disease) . A study conducted using blood samples collected from classical LINCL families whose affliction was confirmed by genetic analysis indicates that the assay can distinguish homozygotes, heterozygotes, and normal controls and thus is useful for diagnosis and carrier testing . Analysis of archival specimens indicates that several specimens previously classified as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2 . Conversely, a specimen previously classified as juvenile NCL lacks pepinase activity and is associated with mutations in CLN2 . In addition, several animals with NCL-like neurodegenerative symptoms {mutant strains of mice (nclf and mnd), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle} were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL . Subcellular fractionation experiments indicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate . Taken together, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity. Appl Environ Microbiol, 1999 Aug, 65(8), 3627 - 32 Seasonal variations in microbial populations and environmental conditions in an extreme acid mine drainage environment; Edwards KJ et al.; Microbial populations, their distributions, and their aquatic environments were studied over a year (1997) at an acid mine drainage (AMD) site at Iron Mountain, Calif . Populations were quantified by fluorescence in situ hybridizations with group-specific probes . Probes were used for the domains Eucarya, Bacteria, and Archaea and the two species most widely studied and implicated for their role in AMD production, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans . Results show that microbial populations, in relative proportions and absolute numbers, vary spatially and seasonally and correlate with geochemical and physical conditions (pH, temperature, conductivity, and rainfall) . Bacterial populations were in the highest proportion (>95%) in January . Conversely, archaeal populations were in the highest proportion in July and September ( approximately 50%) and were virtually absent in the winter . Bacterial and archaeal populations correlated with conductivity and rainfall . High concentrations of dissolved solids, as reflected by high conductivity values (up to 125 mS/cm), occurred in the summer and correlated with high archaeal populations and proportionally lower bacterial populations . Eukaryotes were not detected in January, when total microbial cell numbers were lowest (<10(5) cells/ml), but eukaryotes increased at low-pH sites ( approximately 0.5) during the remainder of the year . This correlated with decreasing water temperatures (50 to 30 degrees C; January to November) and increasing numbers of prokaryotes (10(8) to 10(9) cells/ml) . T . ferrooxidans was in highest abundance (>30%) at moderate pHs and temperatures ( approximately 2.5 and 20 degrees C) in sites that were peripheral to primary acid-generating sites and lowest (0 to 5%) at low-pH sites (pH approximately 0.5) that were in contact with the ore body . L . ferrooxidans was more widely distributed with respect to geochemical conditions (pH = 0 to 3; 20 to 50 degrees C) but was more abundant at higher temperatures and lower pHs ( approximately 40 degrees C; pH approximately 0.5) than T . ferrooxidans. Dev Comp Immunol, 1999 Jun-Jul, 23(4-5), 375 - 90 Alpha2-macroglobulin: an evolutionarily conserved arm of the innate immune system; Armstrong PB et al.; All animals and plants have immune systems that protect them from the diversity of pathogens that would otherwise threaten their survival . The different components of the immune system may inactivate the pathogens themselves or promote the inactivation and clearance of toxic products produced by the pathogens . An important category of virulence factors of bacterial and prokaryotic pathogens are the proteases, which act to facilitate the invasion of the pathogens and to promote their destructive growth in the host organism . The present review concentrates on the comparative biology of an evolutionarily conserved arm of the immune system, the protein, alpha2-macroglobulin . alpha2-Macroglobulin is an abundant protein of the plasma of vertebrates and members of several invertebrate phyla and functions as a broad-spectrum protease-binding protein . Protease-conjugated alpha2-macroglobulin is selectively bound by cells contacting the body fluids and alpha2-macroglobulin and its protease cargo are then internalized and degraded in secondary lysosomes of those cells . In addition to this function as an agent for protease clearance, alpha2-macroglobulin binds a variety of other ligands, including several peptide growth factors and modulates the activity of a lectin-dependent cytolytic pathway in arthropods. Dev Comp Immunol, 1999 Jun-Jul, 23(4-5), 329 - 44 Antimicrobial peptides in insects; structure and function; Bulet P et al.; Antimicrobial peptides appear to be ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms . During the past 15 years a multitude of these peptides have been isolated largely from insects . In spite of great differences in size, amino acid composition and structure, most of the antimicrobial peptides from insects can be grouped into one of three categories . The largest category in number contains peptides with intramolecular disulfide bonds forming hairpin-like beta-sheets or alpha-helical-beta-sheet mixed structures . The second most important group is composed of peptides forming amphipathic alpha-helices . The third group comprises peptides with an overrepresentation in proline and/or glycine residues . In general, the insect antimicrobial peptides have a broad range of activity and are not cytotoxic . Despite a wealth of information on structural requirements for their antimicrobial activity, the mode of action of these peptides is not yet fully understood . However, some data suggest the existence of two types of mode of action: 1 . through peptide-lipid interaction or 2 . through receptor-mediated recognition processes . This review presents the main results obtained during the last four years in the field of antimicrobial peptides from insects with a special focus on the proline-rich and cysteine-rich peptides. Tidsskr Nor Laegeforen, 1999 Jun 30, 119(17), 2528 - 30 {Chlorination of drinking water--possible cancer risk from a by-product}; Holme JA et al.; In a recent study, 3-chloro-4-(dichloromethyl)-5-hydroxy-2{5H}-furanone (MX; CAS no 77439-76-0) which is formed during chlorination of water containing organic substances, was found to be carcinogenic in the rat, at multiple sites in both sexes . MX is known as a potent bacterial mutagen . Epidemiological studies have suggested an association between chlorinated water consumption and a moderate increase in the risk of cancer . Although MX is a strong mutagen in prokaryotes, its genotoxic effects in mammalian cells are not so large, and more variable results are obtained . Very low concentrations of MX are found in drinking water (ng/L), whereas the genotoxic and carcinogenic effects in experimental animals of this compound are detectable at relatively high doses (mg/kg body weight) . Relative to the risk for infectious diseases from the consumption of contaminated drinking water, the possible cancer risk associated with MX exposure appears to be low . Even so, efforts should be made to reduce disinfection byproduct formation by removing organic matter before chlorination. Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1313 - 6 Misunderstanding the Bacteriological Code; Tindall BJ; The Bacteriological Code contains Principles and Rules governing the naming of prokaryotic taxa . However, interpretation of the Code is not always easy, nor is the dynamic link between the names of taxa and a particular taxonomic opinion always fully appreciated. Structure Fold Des, 1999 Jul 15, 7(7), 829 - 40 The aspartate receptor cytoplasmic domain: in situ chemical analysis of structure, mechanism and dynamics; Bass RB et al.; BACKGROUND: Site-directed sulfhydryl chemistry and spectroscopy can be used to probe protein structure, mechanism and dynamics in situ . The aspartate receptor of bacterial chemotaxis is representative of a large family of prokaryotic and eukaryotic receptors that regulate histidine kinases in two-component signaling pathways, and has become one of the best characterized transmembrane receptors . We report here the use of cysteine and disulfide scanning to probe the helix-packing architecture of the cytoplasmic domain of the aspartate receptor . RESULTS: A series of designed cysteine pairs have been used to detect proximities between cytoplasmic helices in the full-length, membrane-bound receptor by measurement of disulfide-bond formation rates . Upon mild oxidation, 25 disulfide bonds from rapidly between three specific pairs of helices, whereas other helix pairs yield no detectable disulfide-bond formation . Further constraints on helix packing are provided by 14 disulfide bonds that retain receptor function in an in vitro kinase regulation assay . Of these functional disulfides, seven lock the receptor in the conformation that constitutively stimulates kinase activity ('lock-on'), whereas the remaining seven retain normal kinase regulation . Finally, disulfide-trapping experiments in the absence of bound kinase reveal large-amplitude relative motions of adjacent helices, including helix translations and rotations of up to 19 A and 180 degrees, respectively . CONCLUSIONS: The 25 rapidly formed and 14 functional disulfide bonds identify helix-helix contacts and their register in the full-length, membrane-bound receptor-kinase complex . The results reveal an extended, rather than compact, domain architecture in which the observed helix-helix interactions are best described by a four-helix bundle arrangement . A cluster of six lock-on disulfide bonds pinpoints a region of the four-helix bundle critical for kinase activation, whereas the signal-retaining disulfides indicate that signal-induced rearrangements of this region are small enough to be accommodated by disulfide-bond flexibility (< or = 1.2 A) . In the absence of bound kinase, helix packing within the cytoplasmic domain is highly dynamic. J Pept Res, 1999 May, 53(5), 578 - 89 NMR structural characterization of cecropin A(1-8) - magainin 2(1-12) and cecropin A (1-8) - melittin (1-12) hybrid peptides; Oh D et al.; In order to elucidate the structure-antibiotic activity relationships of the peptides, the three-dimensional structures of two hybrid peptides, CA(1-8) - MA(1-12) and CA(1-8) - ME(1-12) in trifluoroethanol-containing aqueous solution were investigated by NMR spectroscopy . Both CA(1-8) - MA(1-12) and CA(1-8) - ME(1-12) have strong antibacterial activity but only CA(1-8) - ME(1-12) has hemolytic activity against human erythrocytes . CA(1-8) - MA(1-12) has a hydrophobic 310-helix of only two turns combined with one short helix in the N-terminus with a flexible hinge section in between . CA(1-8) - MA(1-12) has a severely bent structure in the middle of the peptide . These structural features as well as the low hydrophobicity of CA(1-8) - MA(1-12) seem to be crucial for the selective lysis against the membrane of prokaryotic cells . CA(1-8) - ME(1-12) has an alpha-helical structure of about three turns in the melittin domain and a flexible structure with one turn in the cecropin domain connected with a flexible hinge section in between, and these might be the structural features required for membrane disruption against prokaryotic and eukaryotic cells . The central hinge region (Gly9-Ile10-Gly11) in an amphipathic antibacterial peptide is considered to play an important role in providing the conformational flexibility required for ion channel formation of the C-terminal hydrophobic alpha-helix on cell membrane. Anal Chem, 1999 Jul 15, 71(14), 2732 - 8 Microorganism identification by mass spectrometry and protein database searches; Demirev PA et al.; A method for rapid identification of microorganisms is presented, which exploits the wealth of information contained in prokaryotic genome and protein sequence databases . The method is based on determining the masses of a set of ions by MALDI TOF mass spectrometry of intact or treated cells . Subsequent correlation of each ion in the set to a protein, along with the organismic source of the protein, is performed by searching an Internet-accessible protein database . Convoluting the lists for all ions and ranking the organisms corresponding to matched ions results in the identification of the microorganism . The method has been successfully demonstrated on B . subtilis and E . coli, two organisms with completely sequenced genomes . The method has been also tested for identification from mass spectra of mixtures of microorganisms, from spectra of an organism at different growth stages, and from spectra originating at other laboratories . Experimental factors such as MALDI matrix preparation, spectral reproducibility, contaminants, mass range, and measurement accuracy on the database search procedure are addressed too . The proposed method has several advantages over other MS methods for microorganism identification. J Biochem (Tokyo), 1999 Aug, 126(2), 430 - 6 The cyanogenic glucoside, prunasin (D-mandelonitrile-beta-D-glucoside), is a novel inhibitor of DNA polymerase beta; Mizushina Y et al.; A DNA polymerase beta (pol . beta) inhibitor has been isolated independently from two organisms; a red perilla, Perilla frutescens, and a mugwort, Artemisia vulgaris . These molecules were determined by spectroscopic analyses to be the cyanogenic glucoside, D-mandelonitrile-beta-D-glucoside, prunasin . The compound inhibited the activity of rat pol . beta at 150 microM, but did not influence the activities of calf DNA polymerase alpha and plant DNA polymerases, human immunodefficiency virus type 1 reverse transcriptase, calf terminal deoxynucleotidyl transferase, or any prokaryotic DNA polymerases, or DNA and RNA metabolic enzymes examined . The compound dose-dependently inhibited pol . beta activity, the IC(50) value being 98 microM with poly dA/oligo dT(12-18) and dTTP as the DNA template and substrate, respectively . Inhibition of pol . beta by the compound was competitive with the substrate, dTTP . The inhibition was enhanced in the presence of fatty acid, and the IC(50) value decreased to approximately 40 microM . In the presence of C(10)-decanoic acid, the K(i) value for substrate dTTP decreased by 28-fold, suggesting that the fatty acid allowed easier access of the compound to the substrate-binding site. Nippon Ishinkin Gakkai Zasshi, 1999, 40(3), 119 - 23 {Mechanism of action of anti-fungal drugs}; Nakashima S; Clinical application of recently developed anti-fungal drugs including fluconazole and itraconazole has provided great advantages in the treatment of deep mycoses . However, pathogenic fungi belong to eukaryotes including humans and are phylogenetically apart from prokaryotes, i.e . bacteria . In other words, the components and metabolic pathways of fungi and mammals are very similar . This sometimes makes it difficult to treat severe mycoses with the anti-fungal drugs available at present . Therefore, new drugs which are more selective for fungal components, such as new azoles, are desired by clinicians and some of them are now under clinical trial . Fungal factors involved in dimorphic change including transcription factors and members of MAP kinase cascades as well as virulence factors including proteases, phospholipases and catalase have recently been identified . These factors and enzymes responsible for cell wall construction could be selective targets to develop new anti-fungal drugs. Biophys J, 1999 Aug, 77(2), 775 - 88 Evolutionary relationship between K(+) channels and symporters; Durell SR et al.; The hypothesis is presented that at least four families of putative K(+) symporter proteins, Trk and KtrAB from prokaryotes, Trk1,2 from fungi, and HKT1 from wheat, evolved from bacterial K(+) channel proteins . Details of this hypothesis are organized around the recently determined crystal structure of a bacterial K(+) channel: i . e., KcsA from Streptomyces lividans . Each of the four identical subunits of this channel has two fully transmembrane helices (designated M1 and M2), plus an intervening hairpin segment that determines the ion selectivity (designated P) . The symporter sequences appear to contain four sequential M1-P-M2 motifs (MPM), which are likely to have arisen from gene duplication and fusion of the single MPM motif of a bacterial K(+) channel subunit . The homology of MPM motifs is supported by a statistical comparison of the numerical profiles derived from multiple sequence alignments formed for each protein family . Furthermore, these quantitative results indicate that the KtrAB family of symporters has remained closest to the single-MPM ancestor protein . Strong sequence evidence is also found for homology between the cytoplasmic C-terminus of numerous bacterial K(+) channels and the cytoplasm-resident TrkA and KtrA subunits of the Trk and KtrAB symporters, which in turn are homologous to known dinucleotide-binding domains of other proteins . The case for homology between bacterial K(+) channels and the four families of K(+) symporters is further supported by the accompanying manuscript, in which the patterns of residue conservation are demonstrated to be similar to each other and consistent with the known 3D structure of the KcsA K(+) channel. Protein Expr Purif, 1999 Jul, 16(2), 359 - 68 High-level expression and purification of biologically active recombinant pokeweed antiviral protein; Rajamohan F et al.; Pokeweed antiviral protein (PAP) from the leaves of the pokeweed plant, Phytolacca americana, is a naturally occurring single-chain ribosome-inactivating protein, which catalytically inactivates both prokaryotic and eukaryotic ribosomes . The therapeutic potential of PAP has gained considerable interest in recent years due to the clinical use of native PAP as the active moiety of immunoconjugates against cancer and AIDS . The clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of a homogenously pure and active PAP preparation with minimal batch to batch variability from its natural source . Previous methods for expression of recombinant PAP in yeast, transgenic plants and Escherichia coli have resulted in either unacceptably low yields or were too toxic to the host system . Here, we report a successful strategy which allows high level expression of PAP as inclusion bodies in E . coli . Purification of refolded recombinant protein from solubilized inclusion bodies by size-exclusion chromatography yielded biologically active recombinant PAP (final yield: 10 to 12 mg/L) . The ribosome depurinating in vitro N-glycosidase activity and cellular anti-HIV activity of recombinant PAP were comparable to those of the native PAP . This expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PAP sufficient to carry out advanced clinical trials . To our knowledge, this is the first large-scale expression and purification of biologically active recombinant PAP from E . coli . J Biol Chem, 1999 Jul 30, 274(31), 21665 - 72 Yeast and rat Coq3 and Escherichia coli UbiG polypeptides catalyze both O-methyltransferase steps in coenzyme Q biosynthesis; Poon WW et al.; Ubiquinone (coenzyme Q or Q) is a lipid that functions in the electron transport chain in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes . Q-deficient mutants of Saccharomyces cerevisiae harbor defects in one of eight COQ genes (coq1-coq8) and are unable to grow on nonfermentable carbon sources . The biosynthesis of Q involves two separate O-methylation steps . In yeast, the first O-methylation utilizes 3, 4-dihydroxy-5-hexaprenylbenzoic acid as a substrate and is thought to be catalyzed by Coq3p, a 32.7-kDa protein that is 40% identical to the Escherichia coli O-methyltransferase, UbiG . In this study, farnesylated analogs corresponding to the second O-methylation step, demethyl-Q(3) and Q(3), have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro . Both yeast and rat Coq3p recognize the demethyl-Q(3) precursor as a substrate . In addition, E . coli UbiGp was purified and found to catalyze both O-methylation steps . Futhermore, antibodies to yeast Coq3p were used to determine that the Coq3 polypeptide is peripherally associated with the matrix-side of the inner membrane of yeast mitochondria . The results indicate that one O-methyltransferase catalyzes both steps in Q biosynthesis in eukaryotes and prokaryotes and that Q biosynthesis is carried out within the matrix compartment of yeast mitochondria. Cell Res, 1999 Jun, 9(2), 135 - 44 Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells; Xu CS et al.; The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 degrees C for 30 min, recovery for 12 h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography . Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels . It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells . Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found . 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked . A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed. FEMS Microbiol Lett, 1999 Jul 1, 176(1), 169 - 76 Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in Synechocystis sp . PCC 6803; Lee SW et al.; The biosynthetic pathway for the pteridine moiety of cyanopterine, as well as tetrahydrobiopterine, has been investigated in Synechocystis sp . PCC 6803 . Open reading frames slr0426, slr1626, slr0078 and sll0330 of the organism putatively encoding GTP cyclohydrolase I, dihydroneopterine aldolase, 6-pyruvoyltetrahydropterine synthase and sepiapterine reductase, respectively, have been cloned into T7-based vectors for expression in Escherichia coli . The recombinant proteins have been purified to homogeneity and demonstrated to possess expected genuine activities except that of sll0330 . Our result is the first direct evidence for the functional assignment of the open reading frames in Synechocystis sp . PCC 6803 . Furthermore, the 6-pyruvoyltetrahydropterine synthase gene is demonstrated for the first time in prokaryotes . Based on the result, biosynthesis of cyanopterine is discussed. FEMS Microbiol Lett, 1999 Jul 1, 176(1), 111 - 6 The cytoplasmic helical linker domain of receptor histidine kinase and methyl-accepting proteins is common to many prokaryotic signalling proteins; Aravind L et al.; Mutations in the cytoplasmic linker regions of receptor histidine kinase and chemoreceptor proteins have been shown previously to significantly impair receptor functions . Here we demonstrate significant sequence similarities between these regions in numerous histidine kinases, methyl-accepting proteins, adenylyl cyclases and other prokaryotic signalling proteins . It is suggested that these 'HAMP domains' possess roles of regulating the phosphorylation or methylation of homodimeric receptors by transmitting the conformational changes in periplasmic ligand-binding domains to cytoplasmic signalling kinase and methyl-acceptor domains. Mol Microbiol, 1999 Aug, 33(3), 599 - 611 Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases; Aleksenko A et al.; The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms . However, in animals and fungi, unlike prokaryotes, at least the first two activities are grouped on a multifunctional enzyme . Here, we report cloning, mapping and transcriptional characterization of some pyrimidine biosynthesis genes in the filamentous fungus Aspergillus nidulans . The first two steps of the pathway are performed by a multifunctional enzyme comprising the activities of carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) . This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN, which has a high degree of nucleotide identity with the Ura2 gene in Saccharomyces cerevisiae . The enzyme of the third step, dihydroorotase (DHOase), is encoded by a separate locus, pyrD . However, the pyrABCN gene apparently contains an evolutionary remnant of a DHOase-encoding sequence, similarly to the Ura2 gene of Saccharomyces cerevisiae . The pyrABCN gene is transcribed as a single 7 kb mRNA species . The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF genes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation . Derepression of pyrABCN and pyrD under pyrimidine starvation is noticeably enhanced in pyrE mutants that accumulate dihydroorotic acid . The pyrABCN gene maps to the distal portion of the right arm of the chromosome VIII, whereas the pyrD gene, in contrast to early genetic data, is closely linked to the brlA gene and located to the right of it . Our data on mitotic recombination should help to verify the genetic map of the chromosome VIII . Comparison of amino acid sequences of active dihydroorotases with related enzymes and with their non-functional homologues in yeast and Aspergillus indicates that the active dihydroorotases from fungi are more similar to ureases and enzymes of the pyrimidine degradation pathway . The 'silent' dihydroorotase domains of the multifunctional enzymes from fungi and active DHOase domains of the multifunctional enzymes in higher eukaryotes are more closely related to bacterial dehydroorotases. Ann N Y Acad Sci, 1999 May 18, 870, 330 - 8 Elucidating sequence codes: three codes for evolution; Trifonov EN; The sequences are related to evolution in several ways . First, they carry traces of a distant past . Two sequence features point to the earliest sequence organization . The universal hidden GCU-periodical pattern in mRNA suggests the earliest codons: GCU and its nine-point-change derivatives . They code for seven amino acids that by several criteria are also the oldest . Together it makes the earliest form of the triplet code, still recognizable in the extant sequences . Another feature present in the sequences, apparently, since separation of prokaryotes and eukaryotes, is hidden genome segmentation . Both protein-coding and noncoding sequences appear to have been formed by fusion of standard size units, about 360 bp (120 aa) in eukaryotes and 450 bp (150 aa) in prokaryotes . Presumably, the units have been functioning at some stage of evolution as autonomous single-gene size elements . There are sequence designs that promote evolution . One such design suitable for fast adaptation is the tandem repetition of identical sequences, so that their copy numbers in the repeat arrays would modulate (tune) the expression of nearby genes . The tandem repeat expansion diseases illustrate this mechanism in a dramatic way: overtuning of the respective gene expression leads to the disease. Ann N Y Acad Sci, 1999 May 18, 870, 100 - 7 The distribution of rates of spontaneous mutation over viruses, prokaryotes, and eukaryotes; Drake JW; Although mutation has chaotic aspects, spontaneous mutation rates assume certain characteristic values when expressed per genome per genome duplication . The rate among lytic RNA viruses is roughly 1, while the rate among retroelements is roughly 0.2 . The rate among viral and cellular microbes with DNA chromosomes is close to 0.0034 . Mutation rates among higher eukaryotes, estimated from specific-locus studies, vary greatly . Most of this variation can be suppressed if the rates are expressed per cell division instead of per sexual generation, and if the genome size is taken to be only a little larger than the sum of the protein-encoding sequences; then, the mutation rate is roughly 0.01 . The reasons for different characteristic mutation rates among different organism groups remain mysterious and pose a substantial challenge to students of evolution. FEBS Lett, 1999 Jul 2, 454(1-2), 75 - 80 Evolutionary aspects of inorganic pyrophosphatase; Sivula T et al.; Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other . Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases . Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases . Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site . Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases. Plant Mol Biol, 1999 May, 40(2), 297 - 305 Cloning and expression of a prokaryotic sucrose-phosphate synthase gene from the cyanobacterium Synechocystis sp . PCC 6803; Lunn JE et al.; Sucrose is one of several low-molecular-weight compounds that cyanobacteria accumulate in response to osmotic stress and which are believed to act as osmoprotectants . The genome of the cyanobacterium Synechocystis sp . PCC 6803 contains a 2163 bp open reading frame (ORF) that shows similarity to genes from higher plants encoding sucrose-phosphate synthase (SPS), the enzyme responsible for sucrose synthesis . The deduced amino acid sequence shows 35-39% identity with known higher-plant SPS sequences . The putative Synechocystis sps gene was cloned from genomic DNA by PCR amplification and expressed as a His6-tagged amino-terminal fusion protein in Escherichia coli . The expressed protein was purified and shown to be a functional SPS enzyme, confirming the identity of the ORF, which is the first sps gene to be cloned from a prokaryotic organism . The Synechocystis SPS has a molecular mass of 81.5 kDa, which is smaller than the typical higher-plant SPS subunit (117-119 kDa), and lacks the phosphorylation site motifs associated with light- and osmotic stress-induced regulation of SPS in higher plants . The enzyme has Km values for UDPG1c and Fru6P of 2.9 mM and 0.22 mM, respectively, with a Vmax of 17 micromol per minute per mg protein and a pH optimum of 8.5 . Unlike the higher-plant enzyme, ADPG1c, CDPG1c and GDPG1c can substitute for UDPG1c as the glucosyl donor with Km values of 2.5, 7.2 and 1.8 mM, respectively . The enzyme is activated by Mg2+ but not by G1c6P, and is only weakly inhibited by inorganic phosphate . The purified protein was used to raise a high-titre antiserum, which recognises a low-abundance 81 kDa protein in Synechocystis sp . PCC 6803 extracts . There was no apparent increase in expression of the 81 kDa protein when the cells were exposed to moderate salt stress, and SPS activity was very low in extracts from both unstressed and salt-stressed cells . These results and the lack of evidence for sucrose accumulation in Synechocystis sp . PCC6803 lead to the conclusion that expression of the sps gene plays no obvious role in adaptation to osmotic stress in this species. Annu Rev Biophys Biomol Struct, 1999, 28, 295 - 317 The proteasome; Bochtler M et al.; Proteasomes are large multisubunit proteases that are found in the cytosol, both free and attached to the endoplasmic reticulum, and in the nucleus of eukaryotic cells . Their ubiquitous presence and high abundance in these compartments reflects their central role in cellular protein turnover . Proteasomes recognize, unfold, and digest protein substrates that have been marked for degradation by the attachment of a ubiquitin moiety . Individual subcomplexes of the complete 26S proteasome are involved in these different tasks: The ATP-dependent 19S caps are believed to unfold substrates and feed them to the actual protease, the 20S proteasome . This core particle appears to be more ancient than the ubiquitin system . Both prokaryotic and archaebacterial ancestors have been identified . Crystal structures are now available for the E . coli proteasome homologue and the T . acidophilum and S . cerevisiae 20S proteasomes . All three enzymes are cylindrical particles that have their active sites on the inner walls of a large central cavity . They share the fold and a novel catalytic mechanism with an N-terminal nucleophilic threonine, which places them in the family of Ntn (N terminal nucleophile) hydrolases . Evolution has added complexity to the comparatively simple prokaryotic prototype . This minimal proteasome is a homododecamer made from two hexameric rings stacked head to head . Its heptameric version is the catalytic core of archaebacterial proteasomes, where it is sandwiched between two inactive antichambers that are made up from a different subunit . In eukaryotes, both subunits have diverged into seven different subunits each, which are present in the particle in unique locations such that a complex dimer is formed that has six active sites with three major specificities that can be attributed to individual subunits . Genetic, biochemical, and high-resolution electron microscopy data, but no crystal structures, are available for the 19S caps . A first step toward a mechanistic understanding of proteasome activation and regulation has been made with the elucidation of the X-ray structure of the alternative, mammalian proteasome activator PA28. Nature, 1999 Jul 8, 400(6740), 159 - 62 Chlorophyll b and phycobilins in the common ancestor of cyanobacteria and chloroplasts; Tomitani A et al.; Photosynthetic organisms have a variety of accessory pigments, on which their classification has been based . Despite this variation, it is generally accepted that all chloroplasts are derived from a single cyanobacterial ancestor . How the pigment diversity has arisen is the key to revealing their evolutionary history . Prochlorophytes are prokaryotes which perform oxygenic photosynthesis using chlorophyll b, like land plants and green algae (Chlorophyta), and were proposed to be the ancestors of chlorophyte chloroplasts . However, three known prochlorophytes (Prochloron didemni, Prochlorothrix hollandica and Prochlorococcus marinus) have been shown to be not the specific ancestors of chloroplasts, but only diverged members of the cyanobacteria, which contain phycobilins but lack chlorophyll b . Consequently it has been proposed that the ability to synthesize chlorophyll b developed independently several times in prochlorophytes and in the ancestor of chlorophytes . Here we have isolated the chlorophyll b synthesis genes (chlorophyll a oxygenase) from two prochlorophytes and from major groups of chlorophytes . Phylogenetic analyses show that these genes share a common evolutionary origin . This indicates that the progenitors of oxygenic photosynthetic bacteria, including the ancestor of chloroplasts, had both chlorophyll b and phycobilins. Am J Physiol, 1999 Jul, 277(1 Pt 1), G201 - 8 Modulation of host cell membrane fluidity: a novel mechanism for preventing bacterial adhesion; Ismaili A et al.; Adhesion of bacterial enteropathogens to host mucosal surfaces is a critical primary step in the pathogenesis of diarrheal disease . We investigated the effects of altering the physical properties of eukaryotic cells on bacterial adhesion with the use of a series of three structurally dissimilar membrane fluidizers and several Escherichia coli as test strains . Lipid fluidity of the cell plasma membrane was measured by steady-state fluorescence anisotropy employing the probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene . There was a dose-dependent and reversible inhibition of bacterial adhesion with increasing membrane fluidity . Time course experiments indicated that increasing membrane fluidity during the early stages of bacterial adhesion was essential for inhibition of attachment . None of the fluidizers affected the viability of either eukaryotic or prokaryotic cells . These findings demonstrate, for the first time, that changes in plasma membrane physical properties of epithelial cells can prevent microbial adhesion . This also suggests that altering the membrane properties of host cells could form a basis for novel strategies to prevent bacterial adhesion during infection in vivo. Biochim Biophys Acta, 1999 Jul 15, 1419(2), 299 - 306 Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP/ADP translocase; Alexeyev MF et al.; The Rickettsia prowazekii ATP/ADP translocase (Tlc) is the first member of a new family of ATP/ADP exchangers that includes both prokaryotic and eukaryotic proteins . We optimized the codon usage for expression of tlc in Escherichia coli by means of gene synthesis, expressed the synthetic gene in E . coli, and purified a modified Tlc that contained a C-terminal tag of 10 consecutive histidine residues by immobilized metal affinity chromatography . Although codon usage in R . prowazekii is very different from E . coli, the optimization of the codon usage by itself was insufficient to improve expression . However, the change of the cloning vector from pET11a to pT7-5 led to a 3-10-fold increase in the specific ATP transport rate by cells expressing the synthetic construct . The authenticity of the purified protein was confirmed by N-terminal amino acid sequencing and a matrix assisted laser desorption/ionization mass spectrometry. EMBO J, 1999 Jul 15, 18(14), 4076 - 84 Mechanism of DNA segregation in prokaryotes: ParM partitioning protein of plasmid R1 co-localizes with its replicon during the cell cycle; Jensen RB et al.; The parA locus of plasmid R1 encodes a prokaryotic centromere-like system that mediates genetic stabilization of plasmids by an unknown mechanism . The locus codes for two proteins, ParM and ParR, and a centromere-like DNA region (parC) to which the ParR protein binds . We showed recently that ParR mediates specific pairing of parC-containing DNA molecules in vitro . To obtain further insight into the mechanism of plasmid stabilization, we examined the intracellular localization of the components of the parA system . We found that ParM forms discrete foci that localize to specific cellular regions in a simple, yet dynamic pattern . In newborn cells, ParM foci were present close to both cell poles . Concomitant with cell growth, new foci formed at mid-cell . A point mutation that abolished the ATPase activity of ParM simultaneously prevented cellular localization and plasmid partitioning . A parA-containing plasmid localized to similar sites, i.e . close to the poles and at mid-cell, thus indicating that the plasmid co-localizes with ParM . Double labelling of single cells showed that plasmid DNA and ParM indeed co-localize . Thus, our data indicate that parA is a true partitioning system that mediates pairing of plasmids at mid-cell and subsequently moves them to the cell poles before cell division. Nat Struct Biol, 1999 Jul, 6(7), 634 - 8 Structure of the most conserved internal loop in SRP RNA; Schmitz U et al.; The signal recognition particle (SRP) directs translating ribosomes to the protein translocation apparatus of endoplasmic reticulum (ER) membrane or the bacterial plasma membrane . The SRP is universally conserved, and in prokaryotes consists of two essential subunits, SRP RNA and SRP54, the latter of which binds to signal sequences on the nascent protein chains . Here we describe the solution NMR structure of a 28-mer RNA composing the most conserved part of SRP RNA to which SRP54 binds . Central to this function is a six-nucleotide internal loop that assumes a novel Mg2+-dependent structure with unusual cross-strand interactions; besides a cross-strand A/A stack, two guanines form hydrogen bonds with opposite-strand phosphates . The structure completely explains the phylogenetic conservation of the loop bases, underlining its importance for SRP54 binding and SRP function. Structure Fold Des, 1999 Jun 15, 7(6), 619 - 27 Crystal structure of human bleomycin hydrolase, a self-compartmentalizing cystei