J Mol Biol, 2001 Mar 9, 306(5), 1037 - 58 Structural origins of aminoglycoside specificity for prokaryotic ribosomes; Lynch SR et al.; Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit . Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes . The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously . Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure . A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form . This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic . The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide . Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide . These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics. J Mol Biol, 2001 Mar 9, 306(5), 1023 - 35 Structure of a eukaryotic decoding region A-site RNA; Lynch SR et al.; The aminoglycoside antibiotics target a region of highly conserved nucleotides in the aminoacyl-tRNA site (A site) of 16 S RNA on the 30 S subunit . The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1A have been determined . Here, the structure of a eukaryotic decoding region A-site oligonucleotide has been determined using homonuclear and heteronuclear NMR spectroscopy, and compared to the unbound prokaryotic rRNA structure . The two structures are similar, with a U1406-U1495 base-pair, a C1407-G1494 Watson-Crick base-pair, and a G1408-A1493 base-pair instead of the A1408-A1493 base-pair of the prokaryotic structure . The two structures differ in the orientation of the 1408 position with respect to A1493; G1408 is rotated toward the major groove, which is the binding pocket for aminoglycosides . The structures also differ in the stacking geometry of G1494 on A1493, which could have slight long-range conformational effects. Anal Biochem, 2001 Mar, 290(2), 272 - 6 Expression of recombinant extracellular domain of the type II transforming growth factor-beta receptor: utilization in a modified enzyme-linked immunoabsorbent assay to screen TGF-beta agonists and antagonists; Fahey MS et al.; TGF-beta is a ubiquitous protein that exhibits a broad spectrum of biological activity . The prokaryotic expression and purification of the extracellular domain of the type II TGF-beta receptor (T beta R-II-ED), without the need for fusion protein cleavage and refolding, is described . The recombinant T beta R-II-ED fusion protein bound commercially available TGF-beta 1 and displayed an affinity of 11.1 nM . In a modified ELISA, receptor binding to TGF-beta1 was inhibited by TGF-beta 3 . The technique lends itself to high-throughput screening of combinatorial libraries for the identification of TGF-beta agonists and antagonists and this, in turn, may have important therapeutic implications . Anal Biochem, 2001 Mar, 290(2), 260 - 71 Selection-marker-free modification of the murine beta-casein gene using a lox2272 {correction of lox2722} site; Kolb AF; Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome . Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site . DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci . In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the beta-casein gene in murine embryonic stem cells . The incompatibility of the heterospecific lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy . The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach . A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification . This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene . RNA, 2001 Feb, 7(2), 194 - 206 Mechanism of ribosome recruitment by hepatitis C IRES RNA; Kieft JS et al.; Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5' untranslated region, called the internal ribosome entry site (IRES) . In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle . Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3) . We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex . Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date. Acta Microbiol Immunol Hung, 2001, 48(1), 115 - 27 The place of viruses in the "tree of life"; Sinkovics JG; Ribozymal entry into vesicle containing autocatalytically replicating oligopeptides engendered RNA proliferation and enzyme synthesis within units whose RNA genomes derived from ancestors of viroids . There is good reason to consider the coexistence of proto- or spheroplastic forms of ancient prokaryotes and archaeons . Predecessors of extant mycoplasmavirus L3 or archaeal fuselloviruses could induce cell fusions among these entities . The possibility that the first eukaryotic cells arose consequentially to virally mediated fusions of prokaryotic and archaeal proto- or spheroplasts is presented . Retrotransposons and endogenous retroviruses might have emerged in theropod dinosaurs when Aves evolved; and directed the development of syncytiotrophoblasts in the placentae of the first mammals . As viruses coevolved with their hosts descendants of ancient viruses diverged from one another . Certain phenotypical features could connect extant phages and eukaryotic viruses to common ancestors. Plant Cell Physiol, 2001 Feb, 42(2), 121 - 8 Quality control of photosystem II; Yamamoto Y; Photosystem II is particularly vulnerable to excess light . When illuminated with strong visible light, the reaction center D1 protein is damaged by reactive oxygen molecules or by endogenous cationic radicals generated by photochemical reactions, which is followed by proteolytic degradation of the damaged D1 protein . Homologs of prokaryotic proteases, such as ClpP, FtsH and DegP, have been identified in chloroplasts, and participation of the thylakoid-bound FtsH in the secondary degradation steps of the photodamaged D1 protein has been suggested . We found that cross-linking of the D1 protein with the D2 protein, the alpha-subunit of cytochrome b(559), and the antenna chlorophyll-binding protein CP43, occurs in parallel with the degradation of the D1 protein during the illumination of intact chloroplasts, thylakoids and photosystem II-enriched membranes . The cross-linked products are then digested by a stromal protease(s) . These results indicate that the degradation of the photodamaged D1 protein proceeds through membrane-bound proteases and stromal proteases . Moreover, a 33-kDa subunit of oxygen-evolving complex (OEC), bound to the lumen side of photosystem II, regulates the formation of the cross-linked products of the D1 protein in donor-side photoinhibition of photosystem II . Thus, various proteases and protein components in different compartments in chloroplasts are implicated in the efficient turnover of the D1 protein, thus contributing to the control of the quality of photosystem II under light stress conditions. Genome Res, 2001 Mar, 11(3), 356 - 72 Genome alignment, evolution of prokaryotic genome organization, and prediction of gene function using genomic context; Wolf YI et al.; Gene order in prokaryotes is conserved to a much lesser extent than protein sequences . Only several operons, primarily those that code for physically interacting proteins, are conserved in all or most of the bacterial and archaeal genomes . Nevertheless, even the limited conservation of operon organization that is observed can provide valuable evolutionary and functional clues through multiple genome comparisons . A program for constructing gapped local alignments of conserved gene strings in two genomes was developed . The statistical significance of the local alignments was assessed using Monte Carlo simulations . Sets of local alignments were generated for all pairs of completely sequenced bacterial and archaeal genomes, and for each genome a template-anchored multiple alignment was constructed . In most pairwise genome comparisons, <10% of the genes in each genome belonged to conserved gene strings . When closely related pairs of species (i.e., two mycoplasmas) are excluded, the total coverage of genomes by conserved gene strings ranged from <5% for the cyanobacterium Synechocystis sp to 24% for the minimal genome of Mycoplasma genitalium, and 23% in Thermotoga maritima . The coverage of the archaeal genomes was only slightly lower than that of bacterial genomes . The majority of the conserved gene strings are known operons, with the ribosomal superoperon being the top-scoring string in most genome comparisons . However, in some of the bacterial-archaeal pairs, the superoperon is rearranged to the extent that other operons, primarily those subject to horizontal transfer, show the greatest level of conservation, such as the archaeal-type H+-ATPase operon or ABC-type transport cassettes . The level of gene order conservation among prokaryotic genomes was compared to the cooccurrence of genomes in clusters of orthologous genes (COGs) and to the conservation of protein sequences themselves . Only limited correlation was observed between these evolutionary variables . Gene order conservation shows a much lower variance than the cooccurrence of genomes in COGs, which indicates that intragenome homogenization via recombination occurs in evolution much faster than intergenome homogenization via horizontal gene transfer and lineage-specific gene loss . The potential of using template-anchored multiple-genome alignments for predicting functions of uncharacterized genes was quantitatively assessed . Functions were predicted or significantly clarified for approximately 90 COGs (approximately 4% of the total of 2414 analyzed COGs) . The most significant predictions were obtained for the poorly characterized archaeal genomes; these include a previously uncharacterized restriction-modification system, a nuclease-helicase combination implicated in DNA repair, and the probable archaeal counterpart of the eukaryotic exosome . Multiple genome alignments are a resource for studies on operon rearrangement and disruption, which is central to our understanding of the evolution of prokaryotic genomes . Because of the rapid evolution of the gene order, the potential of genome alignment for prediction of gene functions is limited, but nevertheless, such predictions information significantly complements the results obtained through protein sequence and structure analysis. Curr Opin Plant Biol, 2001 Apr, 4(2), 157 - 61 Protein expression in plastids; Heifetz PB et al.; The genome of the plastid has generated much interest as a target for plant transformation . The characteristics of plastid transgenes both reflect the prokaryotic origin of plastid organelles and provide a unique set of features that are currently lacking in genes introduced into the plant nucleus . Recent progress has been made in understanding plastid expression of recombinant proteins. Science, 2001 Jan 19, 291(5503), 498 - 501 Crystal structure of an initiation factor bound to the 30S ribosomal subunit; Carter AP et al.; Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis . In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3 . Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit . Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1 . The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other . The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit. Trends Genet, 2001 Mar, 17(3), 113 - 20 How many genes in Arabidopsis come from cyanobacteria? An estimate from 386 protein phylogenies; Rujan T et al.; It is well known that chloroplasts and mitochondria donated many genes to nuclear chromosomes during evolution - but how many is "many"? A sample of 3961 Arabidopsis nuclear protein-coding genes was compared with the complete set of proteins from yeast and 17 reference prokaryotic genomes, including one cyanobacterium (the lineage from which plastids arose) . The analysis of 386 phylogenetic trees distilled from these data suggests that between approximately 400 (1.6%) and approximately 2200 (9.2%) of Arabidopsis nuclear genes stem from cyanobacteria . The degree of conservation preserved in protein sequences in addition to lateral gene transfer between free-living prokaryotes pose substantial challenges to genome phylogenetics. Environ Microbiol, 2000 Apr, 2(2), 227 - 37 Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica; Bowman JP et al.; 16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica . From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of >0.98) . A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G+C Gram-positive division . Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria (Desulfosarcina group, Syntrophus and Geobacterl Pelobacter/Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes) . Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material . Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments. Insect Biochem Mol Biol, 2001 Mar 15, 31(4-5), 491 - 6 Expression of host S-adenosylmethionine decarboxylase gene and polyamine composition in aphid bacteriocytes; Nakabachi A et al.; Differential cDNA display and quantitative RT-PCR revealed that mRNA of host S-adenosylmethionine decarboxylase (SAMDC) was abundant only in the aphid endosymbiotic system well organized in young hosts, suggesting that SAMDC plays some important roles in the system . SAMDC is a key enzyme to synthesize polyamines that are known to be involved in a large array of biological events including protein synthesis, DNA stabilization, DNA replication, and cell proliferation . As the first step to investigate roles of polyamines in the endosymbiotic system, polyamine composition in bacteriocytes was determined by high performance liquid chromatography . As a result, we found that bacteriocytes contained virtually an only single polyamine, spermidine . The spermidine content of bacteriocytes fluctuated with time in the course of development and aging of the host aphid . This is the first report of polyamine assessment in a prokaryote-eukaryote endocellular symbiotic system, which demonstrated a unique polyamine composition. J Virol, 2001 Mar, 75(6), 2921 - 8 Spontaneous activation of the lytic cycle in cells infected with a recombinant Kaposi's sarcoma-associated virus; Delecluse HJ et al.; The genetic analysis of human herpesvirus 8 (HHV8), also termed Kaposi's sarcoma-associated virus, has been hampered by severe difficulties in producing infectious viral particles and modifying the viral genome . In this article, we report the successful cloning of the HHV8 complete genome onto a prokaryotic F-plasmid replicon which allows the propagation of the recombinant viral DNA in Escherichia coli . The insertion of the F-plasmid into the HHV8 genome interrupts the ORF56 gene, whose expression product-by homology with the Epstein-Barr virus BSLF1 gene--is supposed to be necessary for lytic DNA replication . After introduction of the recombinant HHV8 DNA into 293 cells, early viral antigens are expressed, suggesting that spontaneous lytic replication is initiated . However, completion of the lytic program is prevented by the absence of the ORF56 protein, and a quasi-latent state is established . Upon reintroduction of the ORF56 viral gene, the block is overcome and infectious HHV8 virions are produced . As the recombinant HHV8 genome can be easily modified in E . coli, this experimental system opens the way to an extensive genetic analysis of other HHV8 functions. Bioinformatics, 2001 Jan, 17(1), 95 - 7 Transcription-associated protein families are primarily taxon-specific; Coulson RM et al.; The mechanisms controlling gene regulation appear to be fundamentally different in eukaryotes and prokaryotes (Struhl (1999) CELL, 98, 1-4) . To investigate this diversity further, we have analysed the distribution of all known transcription-associated proteins (TAPs), as reflected by sequence database annotations . Our results for the primary phylogenetic domains (Archaea, Bacteria and Eukaryota) show that TAP families are mostly taxon-specific and very few transcriptional regulators are common across these domains. Adv Biochem Eng Biotechnol, 2001, 71, 81 - 123 Biochemical and molecular basis of microbial synthesis of polyhydroxyalkanoates in microorganisms; Steinbuchel A et al.; Intensive research on the physiology, biochemistry, and molecular genetics of the metabolism of polyhydroxyalkanoates (PHA) during the last 15 years has revealed a dramatic increase of our knowledge on the biosynthesis of these polyesters in bacteria . This mainly very basic research has revealed several new, hitherto not described enzymes and pathways . In addition, many genes encoding the enzymes of these pathways and in particular the key enzyme of PHA biosynthesis, PHA synthase, were cloned and characterized at a molecular level . This knowledge was utilized to establish PHA biosynthesis in many prokaryotic and eukaryotic organisms, which were unable to synthesize PHAs, and to apply the methodology of metabolic engineering, thus opening new perspectives for the production of various PHAs by fermentation biotechnology or agriculture in economically feasible processes . This contribution summarizes the properties of PHA synthases and gives an overview on the genes for these enzymes and other enzymes of PHA biosynthesis that have been cloned and are available . It also summarizes our current knowledge on the regulation at the enzyme and gene level of PHA biosynthesis in bacteria. Adv Biochem Eng Biotechnol, 2001, 71, 125 - 57 Physiology, regulation, and limits of the synthesis of poly(3HB); Babel W et al.; The properties of poly(3-hydroxybutyrate) combined with the fact that it can be produced easily by numerous prokaryotes from renewable resources and even from potentially toxic waste products using well-known fermentation processes have generated keen interest in this biopolyester as a substitute for chemo-synthetic petroleum-derived polymers in many applications . However, the high price of poly(3HB) compared with the conventional synthetic materials currently in use has restricted its availability in a wide range of applications . If the economic viability of poly(3HB) production and its competitiveness are to be improved, more must be found out about the phenotypic optimization and the upper limits of bacterial systems as the factory of poly(3HB) . In this chapter, two aspects of poly(3HB) are reviewed--poly(3HB) formation as a physiological response to external limitations and overcoming internal bottlenecks, and poly(3HB) as a commercially attractive polyester . From a physiological viewpoint, the ability to synthesize and degrade poly(3HB) is considered an investment in the future and provides organisms with a selective advantage . Poly(3HB) is presented as a strategic survival polymer, and it is shown that growth-associated synthesis is not as rare as reported . The influence of the efficiency and velocity of cell multiplication and product formation, of poly(3HB) content and of productivity on the overall yield, and finally on the economics of the whole process are discussed and evaluated from the technological or consumer's point of view . The specific production rate and poly(3HB) content appear to be more important than the yield coefficients. Environ Microbiol, 2000 Dec, 2(6), 632 - 43 Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes; Ziemer CJ et al.; A model rumen system, dual-flow continuous culture fermenters, was evaluated by two comparative criteria in two experiments using ribosomal (r)RNA-targeted DNA probes to compare key microbial groups in samples . The initial experiment measured temporal changes in population structure during adaptation of ruminal microbial populations in fermenters over 240 h . The fermenter inoculum contained 34.9% Bacteria, 60.1% Eukarya and 6.8% Archaea measured as a fraction of total small subunit (SSU) rRNA quantified using a universal probe . The cellulolytic bacterial genus Fibrobacter comprised 9.5% of total SSU rRNA in the inoculum . After 240 h of fermenter operation, the average abundance was 80.9% Bacteria, 6.1% Eukarya, 5.1% Archaea and Fibrobacter genus accounted for 6.6% of the total SSU rRNA . Divergence between ruminal and fermenter population structure was evaluated in the second experiment and samples were classified as ruminal, inoculum or fermenter (96, 120, 144 and 168 h of fermenter operation) . Fermenter samples had higher relative abundances of Bacteria (84.5%) and Archaea (2.1%) and lower relative abundances of Eukarya (1.8%) than ruminal samples (average 48.0% Bacteria, 1.3% Archaea and 61.5% Eukarya) . The relative abundance of Fibrobacter was similar in all samples, averaging 2.5% . The ruminal and fermenter samples had similar proportions of F . succinogenes and F . succinogenes subgroup 3 (as a percentage of Fibrobacter SSU rRNA) . Fibrobacter succinogenes subgroup 1 and F . intestinalis proportions of Fibrobacter were lower in fermenter samples (8.2% and 0.7% respectively) than in ruminal samples (28.4% and 2.2% respectively) . Fermenters were able to maintain a core prokaryotic community structure similar to the native microbial community in the rumen . Although protozoa populations were lost, maintenance of Fibrobacter and archaeal populations indicated that the model system supported a functional community structure similar to the rumen . This model rumen system may serve as a suitable tool for studying aspects of ruminal microbial ecology and may resolve some of the relationships between microbial community structure and function by providing control of experimental conditions. Nature, 2001 Feb 1, 409(6820), 637 - 41 The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase; Gomis-Ruth FX et al.; The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown . In prokaryotes, trans-membrane passage by bacterial conjugation, is the main route for horizontal gene transfer . It is the means for rapid acquisition of new genetic information, including antibiotic resistance by pathogens . Trans-kingdom gene transfer from bacteria to plants or fungi and even bacterial sporulation are special cases of conjugation . An integral membrane DNA-binding protein, called TrwB in the Escherichia coli R388 conjugative system, is essential for the conjugation process . This large multimeric protein is responsible for recruiting the relaxosome DNA-protein complex, and participates in the transfer of a single DNA strand during cell mating . Here we report the three-dimensional structure of a soluble variant of TrwB . The molecule consists of two domains: a nucleotide-binding domain of alpha/beta topology, reminiscent of RecA and DNA ring helicases, and an all-alpha domain . Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase . A central channel, 20 A in width, traverses the hexamer. Nature, 2001 Feb 1, 409(6820), 607 - 10 Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity; Moon-van der Staay SY et al.; Picoplankton--cells with a diameter of less than 3 microm--are the dominant contributors to both primary production and biomass in open oceanic regions . However, compared with the prokaryotes, the eukaryotic component of picoplankton is still poorly known . Recent discoveries of new eukaryotic algal taxa based on picoplankton cultures suggest the existence of many undiscovered taxa . Conventional approaches based on phenotypic criteria have limitations in depicting picoplankton composition due to their tiny size and lack of distinctive taxonomic characters . Here we analyse, using an approach that has been very successful for prokaryotes but has so far seldom been applied to eukaryotes, 35 full sequences of the small-subunit (18S) ribosomal RNA gene derived from a picoplanktonic assemblage collected at a depth of 75 m in the equatorial Pacific Ocean, and show that there is a high diversity of picoeukaryotes . Most of the sequences were previously unknown but could still be assigned to important marine phyla including prasinophytes, haptophytes, dinoflagellates, stramenopiles, choanoflagellates and acantharians . We also found a novel lineage, closely related to dinoflagellates and not previously described. Nature, 2001 Feb 1, 409(6820), 603 - 7 Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton; Lopez-Garcia P et al.; Phylogenetic information from ribosomal RNA genes directly amplified from the environment changed our view of the biosphere, revealing an extraordinary diversity of previously undetected prokaryotic lineages . Using ribosomal RNA genes from marine picoplankton, several new groups of bacteria and archaea have been identified, some of which are abundant . Little is known, however, about the diversity of the smallest planktonic eukaryotes, and available information in general concerns the phytoplankton of the euphotic region . Here we recover eukaryotes in the size fraction 0.2-5 microm from the aphotic zone (250-3,000 m deep) in the Antarctic polar front . The most diverse and relatively abundant were two new groups of alveolate sequences, related to dinoflagellates that are found at all studied depths . These may be important components of the microbial community in the deep ocean . Their phylogenetic position suggests a radiation early in the evolution of alveolates. Ann R Coll Surg Engl, 2001 Jan, 83(1), 1 - 9 Hunterian Lecture . What can we learn about mechanisms of mutation from a study of craniosynostosis? Moloney D. Mutation may be defined simply as structural change affecting the genetic material . The generation of genetic variety by spontaneous mutational events has been the driving force behind evolution--without such mutation our complex human genome could not have evolved . However, as doctors, we more frequently encounter mutation in the context of human disease, whether in somatic cells as a cause of cancer, or in the germline as a cause of inheritable disease . In these contexts, the processes of mutagenesis are relevant to every field of medicine . Scientific study of mutational mechanisms has logically been founded in the relatively simple genetic systems of the prokaryotes and such lowly eukaryotes as the fruit-fly . The study of human clinical genetics approaches the problem from quite the opposite direction--from that of the most highly evolved genetic system . Whilst this approach may be dependent less on logical progression and more on phenomenology, it nevertheless provides a complementary avenue for the observation and study of mutational mechanisms . The genetic research described in this article is firmly rooted in such phenomenology, based as it is on rare craniosynostosis syndromes . Over the past decade, there has been a deluge of molecular discoveries in the field of craniosynostosis . This promises improvements in classification, prognostication, pre-natal diagnosis, and perhaps ultimately for potential avenues for cure . However, exciting as these clinical prospects are, the research presented here has a different focus: it investigates the mechanistic basis underlying the craniosynostosis mutations, in the hope that such study may lead to insights applicable generally to the field of mutagenesis. Cell Mol Life Sci, 1999 Nov 30, 56(9-10), 825 - 42 ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae; Van Dyck L et al.; Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria . Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle . Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins . Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae . Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity . ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes . Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence. Cell Mol Life Sci, 1999 Nov 30, 56(9-10), 719 - 28 Evolution of bacterial pathogenesis; Ziebuhr W et al.; The evolution of bacteria is associated with continuous generation of novel genetic variants . The major driving forces in this process are point mutations, genetic rearrangements, and horizontal gene transfer . A large number of human and animal bacterial pathogens have evolved the capacity to produce virulence factors that are directly involved in infection and disease . Additionally, many bacteria express resistance traits against antibiotics . Both virulence factors and resistance determinants are subject to intrastrain genetic and phenotypic variation . They are often encoded on unstable DNA regions . Thus, they can be readily transferred to bacteria of the same species or even to non-related prokaryotes . This review article focuses on the main mechanisms of bacterial microevolution responsible for the rapid emergence of variants with novel virulence and resistance properties . In addition, processes of macroevolution are described with special emphasis on gene transfer and fixation of adaptive mutations in the genome of pathogens. Proc R Soc Lond B Biol Sci, 2001 Jan 22, 268(1463), 113 - 9 Antiquity of the biological sulphur cycle: evidence from sulphur and carbon isotopes in 2700 million-year-old rocks of the Belingwe Belt, Zimbabwe; Grassineau NV et al.; Sulphur and carbon isotopic analyses on small samples of kerogens and sulphide minerals from biogenic and non-biogenic sediments of the 2.7 x 10(9) years(Ga)-old Belingwe Greenstone Belt (Zimbabwe) imply that a complex biological sulphur cycle was in operation . Sulphur isotopic compositions display a wider range of biological fractionation than hitherto reported from the Archaean . Carbon isotopic values in kerogen record fractionations characteristic of rubisco activity methanogenesis and methylotrophy and possibly anoxygenic photosynthesis . Carbon and sulphur isotopic fractionations have been interpreted in terms of metabolic processes in 2.7 Ga prokaryote mat communities, and indicate the operation of a diverse array of metabolic processes . The results are consistent with models of early molecular evolution derived from ribosomal RNA. Analyst, 2001 Jan, 126(1), 52 - 7 Searching the Porphyromonas gingivalis genome with peptide fragmentation mass spectra; Chen W et al.; An approach is described for genomic database searching based on experimentally observed proteolytic fragments, e.g., isolated from 1D or 2D gels or analyzed directly, that can be applied to unfinished prokaryotic genomic data in the absence of annotations or previously assigned open reading frames (ORFs) . This variation on the database search is in contrast to the more familiar use of peptide mass spectral fragmentation data to search fully annotated inferred protein databases, e.g., OWL or SWISS-PROT . We compared the SEQUEST search results from a six reading frame translation of the Porphyromonas gingivalis genome DNA sequence with those from computationally derived ORFs created using publicly available genomics software tools . The ORF approach eliminated many of the artifacts present in output from the six reading frame search . The method was applied to uninterpreted tandem mass spectrometric data derived from proteins secreted by the periodontal pathogen Porphyromonas gingivalis in response to the gingival epithelial cell environment, a model system for the study of host-pathogen interactions relevant to human periodontal disease. Philos Trans R Soc Lond B Biol Sci, 2001 Jan 29, 356(1405), 53 - 60 DNA polymerase iota and related rad30-like enzymes; McDonald JP et al.; Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood . This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases . Members of this so-called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea . Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA . All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA . Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes . Humans possess two Rad30 paralogs, Rad30A and Rad30B . The RAD30A gene encodes DNA polymerase eta and defects in the protein lead to the xeroderma pigmentosum variant (XP-V) phenotype in humans . Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol iota . Based upon in vitro studies, it appears that Pol iota has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error-prone DNA polymerase. Crit Rev Biochem Mol Biol, 2000, 35(6), 393 - 432 Nucleotide pyrophosphatases/phosphodiesterases on the move; Bollen M et al.; Nucleotide pyrophosphatases/phosphodiesterases (NPPs) release nucleoside 5'-monophosphates from nucleotides and their derivatives . They exist both as membrane proteins, with an extracellular active site, and as soluble proteins in body fluids . The only well-characterized NPPs are the mammalian ecto-enzymes NPP1 (PC-1), NPP2 (autotaxin) and NPP3 (B10; gp130(RB13-6)) . These are modular proteins consisting of a short N-terminal intracellular domain, a single transmembrane domain, two somatomedin-B-like domains, a catalytic domain, and a C-terminal nuclease-like domain . The catalytic domain of NPPs is conserved from prokaryotes to mammals and shows remarkable structural and catalytic similarities with the catalytic domain of other phospho-/sulfo-coordinating enzymes such as alkaline phosphatases . Hydrolysis of pyrophosphate/phosphodiester bonds by NPPs occurs via a nucleotidylated threonine . NPPs are also known to auto(de)phosphorylate this active-site threonine, a process accounted for by an intrinsic phosphatase activity, with the phosphorylated enzyme representing the catalytic intermediate of the phosphatase reaction . NPP1-3 have been implicated in various processes, including bone mineralization, signaling by insulin and by nucleotides, and the differentiation and motility of cells . While it has been established that most of these biological effects of NPPs require a functional catalytic site, their physiological substrates remain to be identified. Tissue Cell, 2000 Dec, 32(6), 494 - 500 Study of the microbial aggregation in Mycobacterium using image analysis and electron microscopy; Borrego S et al.; Cellular aggregation, which occurs in both prokaryotes and eukaryotes, is controlled by the hydrophobicity as well as the electrokinetic potential of the cell surface and substratum . It is known that the Mycobacterium genus form aggregates, but the influence of sugar on the cellular aggregation has not been reported for this genus . The mutant strain Mycobacterium sp . MB-3683 that transforms sterol to androstenedione (AD), a steroidal precursor used by the pharmaceutical industries, was employed in this study . This strain was cultivated in a synthetic medium on three sugars (glycerol, glucose and fructose) at different concentrations, and at 144 h microbial growth, cellular aggregation, hydrophobicity, lipid content, fatty acid composition, and width of cellular walls were measured . It was observed that at different sugar concentrations, similar growth and pH were obtained . However, in fructose, the aggregation level was significantly high, followed by glycerol and glucose (fructose < glycerol < glucose) . These results were confirmed using electron microscopy and the aggregate area quantified by image analysis . Hydrophobicity was the highest in fructose and the lowest in glucose . The total lipids, in contrast to cellular hydrophobicity, were higher in glucose than glycerol . Although, the hydrophilic-lipophilic balance (HLB) of principal fatty acids isolated was similar regardless of sugar used . In glycerol and fructose, the paraffins were observed, which are responsible for the high cellular hydrophobicity detected above . The width of cell wall of the organisms grown on glucose and fructose was similar, but in glycerol the walls were very thin . There is a correspondence between cell wall width and lipid content. Proc R Soc Lond B Biol Sci, 2000 Dec 22, 267(1461), 2517 - 21 Intraspecific phylogenetic congruence among multiple symbiont genomes; Funk DJ et al.; Eukaryotes often form intimate endosymbioses with prokaryotic organisms . Cases in which these symbionts are transmitted cytoplasmically to host progeny create the potential for co-speciation or congruent evolution among the distinct genomes of these partners . If symbionts do not move horizontally between different eukaryotic hosts, strict phylogenetic congruence of their genomes is predicted and should extend to relationships within a single host species . Conversely, even rare 'host shifts' among closely related lineages should yield conflicting tree topologies at the intraspecific level . Here, we investigate the historical associations among four symbiotic genomes residing within an aphid host: the mitochondrial DNA of Uroleucon ambrosiae aphids, the bacterial chromosome of their Buchnera bacterial endosymbionts, and two plasmids associated with Buchnera . DNA sequence polymorphisms provided a significant phylogenetic signal and no homoplasy for each data set, yielding completely and significantly congruent phylogenies for these four genomes and no evidence of horizontal transmission . This study thus provides the first evidence for strictly vertical transmission and 'co-speciation' of symbiotic organisms at the intraspecific level, and represents the lowest phylogenetic level at which such coevolution has been demonstrated . These results may reflect the obligate nature of this intimate mutualism and indicate opportunities for adaptive coevolution among linked symbiont genomes. Nature, 2001 Jan 11, 409(6817), 219 - 23 Projection structure of a ClC-type chloride channel at 6.5 A resolution; Mindell JA et al.; Virtually all cells in all eukaryotic organisms express ion channels of the ClC type, the only known molecular family of chloride-ion-selective channels . The diversity of ClC channels highlights the multitude and range of functions served by gated chloride-ion conduction in biological membranes, such as controlling electrical excitability in skeletal muscle, maintaining systemic blood pressure, acidifying endosomal compartments, and regulating electrical responses of GABA (gamma-aminobutyric acid)-containing interneurons in the central nervous system . Previously, we expressed and purified a prokaryotic ClC channel homologue . Here we report the formation of two-dimensional crystals of this ClC channel protein reconstituted into phospholipid bilayer membranes . Cryo-electron microscopic analysis of these crystals yields a projection structure at 6.5 A resolution, which shows off-axis water-filled pores within the dimeric channel complex. Nature, 2001 Jan 11, 409(6817), 211 - 5 The protein-protein interaction map of Helicobacter pylori; Rain JC et al.; With the availability of complete DNA sequences for many prokaryotic and eukaryotic genomes, and soon for the human genome itself, it is important to develop reliable proteome-wide approaches for a better understanding of protein function . As elementary constituents of cellular protein complexes and pathways, protein-protein interactions are key determinants of protein function . Here we have built a large-scale protein-protein interaction map of the human gastric pathogen Helicobacter pylori . We have used a high-throughput strategy of the yeast two-hybrid assay to screen 261 H . pylori proteins against a highly complex library of genome-encoded polypeptides . Over 1,200 interactions were identified between H . pylori proteins, connecting 46.6% of the proteome . The determination of a reliability score for every single protein-protein interaction and the identification of the actual interacting domains permitted the assignment of unannotated proteins to biological pathways. Scand J Plast Reconstr Surg Hand Surg, 2000 Dec, 34(4), 289 - 99 Bone-inductive efficacy of recombinant human bone morphogenetic protein-2 expressed in Escherichia coli: an experimental study in rat mandibular defects; Kimura M et al.; Because to our knowledge the efficacy of prokaryotically expressed recombinant human bone morphogenetic proteins (rhBMP) to promote orthotopic osteogenesis has not previously been investigated, our aim was to test the efficacy of rhBMP-2 produced in Escherichia coli to promote bone healing in a standardised experimental bone healing model in rat mandibles . Different doses of rhBMP-2 were delivered in an absorbable collagen sponge carrier, and microporous barrier membranes were placed over half the number of defects in each treatment group, thereby making intraosseous cells the only recruitment source for new osteogenic cells . Results were evaluated by computerised image analysis after 12 and 24 days . The relative efficacy of rhBMP-2 preparations of different purity was also compared . E coli-produced rhBMP-2 stimulated bone healing, but its efficacy was estimated to be about one order of magnitude less than that of rhBMP-2 expressed in eukaryotic cells . We conclude that bacterially expressed rhBMP-2 is osteogenic in vivo, although higher doses will be required than of rhBMP-2 expressed in mammalian cell lines. Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 582 - 6 {Construction of fusion recombinant plasmid coding for RGD peptide and urokinase B chain, its expression in Escherichia coli and preliminary characterization of its biological activity}; Zhu FX et al.; A synthetic RGD (Arg-Gly-Asp) peptide-coding sequence was fused with urokinase B-chain cDNA and then cloned into the prokaryotic expression vector pBV220 . The fused gene was expressed in E . coli DH5 alpha under the control of PRPL promoter by 42 degrees C induction . The expression level of the fusion protein was over 9.2% of the total bacterial proteins as a or of inactive inclusion body . The purified fusion protein was obtained with similar antigenicity as urokinase shown by Western blotting . Its in vitro fibrinolysis and anti-platelet aggregation activity was also evaluated by bioassay. Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 566 - 9 {Fusion and expression of the gene encoding human Mn-SOD to anti-CEA single-chain antibody in Escherichia coli}; He HJ et al.; The gene encoding human manganese-superoxide dismutase (Mn-SOD) was fused to anti-carcinoembryonic antigen single-chain antibody gene to construct the fusion gene, then was ligated into prokaryotic expression vector pET-22b(+), The fusion gene was expressed in E . coli at high level, accounting for 24% of the total bacteria soluble protein; and was characterized by SDS-PAGE and Western-blot analysis; the expression product had the CEA-binding ability in RIA, and also had the SOD activity by pyrogallol autoxidation assay . So, the Mn-SOD moiety retains substantial enzymatic activity, where the ScFv moiety can deliver the fusion protein to tumor, Mn-SOD is a potential tumor-suppressor gene, maybe the fusion protein can provide a new pathway to tumor therapy. Environ Microbiol, 1999 Dec, 1(6), 517 - 23 Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds; Anton J et al.; A fluorescence in situ hybridization (FISH) protocol suitable for the identification of prokaryotes inhabiting hypersaline environments was developed and applied to several crystallizer ponds with salinities above 36% from a multipond solar saltern in Alicante, Spain . Two morphotypes were abundant in these environments: rods and square or square-like prokaryotes that could be affiliated to Bacteria and Archaea, respectively, by FISH with domain-specific probes . FISH with a newly designed probe proved that the archaeal 16S rDNA sequence most frequently recovered from the crystallizers, SPhT, originated from the dominant square-like prokaryotes . These uncultured prokaryotes have the morphology of Walsby's square bacteria . Additionally, FISH with a probe targeted to the genus Haloarcula, members of which are frequently isolated from this environment, indicated that this genus accounts for less than 0.1% of the total prokaryotic community. Environ Microbiol, 1999 Feb, 1(1), 65 - 74 Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment; Sahm K et al.; In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell . Here, we report evidence that quantification of SRB rRNA by slot-blot hybridization is a valuable tool for a more realistic assessment of SRB abundance in the natural environment . The distribution of SRB was investigated in a coastal marine sediment by hybridization of membrane-immobilized rRNA with oligonucleotide probes . As represented by general probe-target groups, SRB rRNA contributed between 18% and 25% to the prokaryotic rRNA pool . The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulphobacter could not be detected . The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates (SRR) measured with 35SO4(2-) tracer in whole-core incubations . While SRB abundance was highest near the surface, peaking at around 1.5 cm, measured sulphate reduction rates were lowest in this region . A second peak of SRB rRNA was observed at the transition zone from oxidized to reduced sediment, directly above the sulphate reduction maximum . Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment . Cellular sulphate reduction rates calculated on the basis of these estimated cell numbers were between 0.01 and 0.09 fmol SO4(2-) cell(-1) day(-1), which is below the rates that have been determined for pure cultures (0.2-50 fmol SO4(2-) cell(-1) day(-1)) growing exponentially at nearoptimal temperature with a surplus of substrates. Cell, 2001 Jan 26, 104(2), 259 - 68 Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells; Constantinou A et al.; During homologous recombination, DNA strand exchange leads to Holliday junction formation . The movement, or branch migration, of this junction along DNA extends the length of the heteroduplex joint . In prokaryotes, branch migration and Holliday junction resolution are catalyzed by the RuvA and RuvB proteins, which form a complex with RuvC resolvase to form a "resolvasome" . Mammalian cell-free extracts have now been fractionated to reveal analogous activities . An ATP-dependent branch migration activity, which migrates junctions through >2700 bp, cofractionates with the Holliday junction resolvase during several chromatographic steps . Together, the two activities promote concerted branch migration/resolution reactions similar to those catalyzed by E . coli RuvABC, highlighting the preservation of this essential pathway in recombination and DNA repair from prokaryotes to mammals. Genome Biol . 2001;2(1):REVIEWS3001 . Epub 2001 Jan 09. The ring-type polymerase sliding clamp family; Bruck I et al.; Ring-type polymerases consist of a DNA polymerase, a ring-shaped sliding clamp protein and a clamp-loading complex . Sliding clamp proteins are found in all organisms and are called proliferating cell nuclear antigen (PCNA) in eukaryotes and the beta clamp in prokaryotes . Both PCNA and beta form a ring around DNA, which is made up of two subunits of three domains each in beta but three subunits of two domains each in PCNA . Despite this difference and a lack of detectable sequence homology, the structures of the two rings are very similar . The sliding clamp slides along DNA and tethers the polymerase to the DNA, enabling rapid and processive DNA replication. Genome Biol . 2000;1(6):REVIEWS3003 . Epub 2000 Dec 08. Cytochromes P450: a success story; Werck-Reichhart D et al.; SUMMARY: Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins . The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants . Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution . P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron . Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates . The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising . They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics . In plants, chemical defense seems to be a major reason for P450 diversification . In prokaryotes, P450s are soluble proteins . In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes . The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization . P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable. J Invest Dermatol, 2001 Feb, 116(2), 286 - 95 A novel gene expressed in human keratinocytes with long-term in vitro growth potential is required for cell growth; Aurelian L et al.; The herpes simplex virus large subunit of ribonucleotide reductase differs from its counterparts in eukaryotic and prokaryotic cells and in other viruses in that it contains a unique domain that codes for a distinct serine-threonine protein kinase that activates the Ras/MEK/MAPK mitogenic pathway and is required for virus growth . Previous studies suggested that ribonucleotide reductase protein kinase was co-opted from a cellular gene . Cellular genes similar to ribonucleotide reductase protein kinase were not cloned, however, and their function is unknown . Here we report that a novel gene (H11) that codes for a protein similar to herpes simplex virus 2 ribonucleotide reductase protein kinase, is expressed in skin tissues, cultured keratinocytes, and the keratinocyte cell line A431 . The protein is phosphorylated and it associates with the plasma membrane . H11 is expressed in keratinocytes with long-term in vitro growth potential and is coexpressed with high levels of adhesion molecules involved in signal transduction, such as beta1 integrin . Antisense oligonucleotides that inhibit H11 expression inhibit DNA synthesis and keratinocyte proliferation, suggesting that H11 expression is required for cell growth. Infect Immun, 2001 Mar, 69(3), 1679 - 86 Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray; Ang S et al.; To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used . Total RNAs of H . pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin . Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method . Gene expression of all ORFs was measured by densitometry . Among the 1,534 ORFs, 53 ORFs were highly expressed (> or = 30% of rRNA control in densitometry ratios) . There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation . A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition . The remaining 952 ORFs were not detectable under either pH condition . These data were highly reproducible and comparable to those obtained by the RNA slot blot method . Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale. EMBO J, 2001 Feb 15, 20(4), 819 - 27 An active role for a structured B-linker in effector control of the sigma54-dependent regulator DmpR; O'Neill E et al.; The activities of many prokaryotic sigma54-dependent transcriptional activators are controlled by the N-terminal A-domain of the protein, which is linked to the central transcriptional activation domain via a short B-linker . It used to be thought that these B-linkers simply serve as flexible tethers . Here we show that the B-linker of the aromatic-responsive regulator DmpR and many other regulators of the family contain signature heptad repeats with regularly spaced hydrophobic amino acids . Mutant analysis of this region of DmpR demonstrates that B-linker function is dependent on the heptad repeats and is critical for activation of the protein by aromatic effectors . The phenotypes of DmpR mutants refute the existing model that the level of ATPase activity directly controls the level of transcription it promotes . The mutant analysis also shows that the B-linker is involved in repression of ATPase activity and that allosteric changes upon effector binding are transduced to alleviate both B-linker repression of ATP hydrolysis and A-domain repression of transcriptional activation . The mechanistic implications of these findings for DmpR and other family members are discussed. EMBO J, 2001 Feb 15, 20(4), 713 - 22 A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II; Haussuhl K et al.; Although light is the ultimate substrate in photosynthesis, it can also be harmful and lead to oxidative damage of the photosynthetic apparatus . The main target for light stress is the central oxygen-evolving photosystem II (PSII) and its D1 reaction centre protein . Degradation of the damaged D1 protein and its rapid replacement by a de novo synthesized copy represent the important repair mechanism of PSII crucial for plant survival under light stress conditions . Here we report the isolation of a single-copy nuclear gene from Arabidopsis thaliana, encoding a protease that performs GTP-dependent primary cleavage of the photodamaged D1 protein and hence catalysing the key step in the repair cycle in plants . This protease, designated DegP2, is a homologue of the prokaryotic Deg/Htr family of serine endopeptidases and is associated with the stromal side of the non-appressed region of the thylakoid membranes . Increased expression of DegP2 under high salt, desiccation and light stress conditions was measured at the protein level. ALTEX, 1997, 14(3), 107 - 110 Generation of a Translational Extract from Eukaryotic Cells that have Grown as Monolayers; Favre D; The present article relates to the rapid and efficient generation of an in vitro translational extract that is obtained from the cytoplasmic fraction of eukaryotic cells that have grown as monolayers . The procedure is totally devoid of the use of animals or animal fluids . The cytoplasmic extract that is obtained allows efficient protein synthesis of exogenously added mRNAs . The latter mRNAs can be purified from prokaryotic or eukaryotic cells, or can be transcribed in vitro by employing any convenient RNA polymerase (for example, the bacteriophage SP6, T3 or T7 RNA polymerase) . The described cytoplasmic preparation appears to be applicable to a large number of different eukaryotic cell lines . The cytoplasmic extract can be prepared freshly on a daily basis, or can be frozen and subsequently thawed for further use, without loss of activity . The preparation of the translation extract does not require living animals. Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 301 - 3 Crystallization and preliminary X-ray diffraction analysis of a {2Fe-2S} ferredoxin (FdVI) from Rhodobacter capsulatus; Armengaud J et al.; A {2Fe-2S} ferredoxin found in the photosynthetic bacterium Rhodobacter capsulatus has been purified in recombinant form from Escherichia coli . This protein, called FdVI, resembles ferredoxins involved in iron-sulfur cluster biosynthesis in various prokaryotic and eukaryotic cells . Purified recombinant FdVI was recovered in high yields and appeared to be indistinguishable from the genuine R . capsulatus ferredoxin based on UV-visible absorption and EPR spectroscopy and mass spectrometry . FdVI has been crystallized in the oxidized state by a sitting-drop vapour-diffusion technique using sodium formate as precipitant . Seeding larger drops from a previous hanging-drop-grown small crystal resulted in the formation of long red-brown prismatic needles . Preliminary X-ray diffraction analysis indicated that FdVI crystals are orthorhombic and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 45.87, b = 49.83, c = 54.29 A. Trends Microbiol, 2001 Feb, 9(2), 71 - 9 The structure and function of drug pumps; Borges-Walmsley MI et al.; Resistance to drugs has emerged in biological systems as diverse as cancer cells undergoing chemotherapy and microbial pathogens undergoing treatment with antimicrobials . This medical problem is escalating and there is an urgent need for the development of new classes of drugs . In the case of pathogenic bacteria, we are rapidly approaching a scenario where there will be no effective antibiotics in the armoury of drugs available for treating the infectious diseases that these bacteria cause, returning us to the pre-antibiotic era when infectious diseases were rife because they were untreatable . One of the most frequently employed resistance strategies in both prokaryotes and eukaryotes is the transmembrane-protein-catalysed extrusion of drugs from the cell, with these proteins acting like bilge pumps, reducing the intracellular drug concentration to subtoxic levels . There is currently much scientific interest in understanding how these pumps operate, so that we might design transport inhibitors that would block them, allowing a renaissance for drugs that are no longer effective owing to their efflux. Biochem Soc Trans, 2000 Dec, 28(6), 675 - 7 Towards the cloning of GLY1; Miquel M; The Arabidopsis mutants designated gly1 exhibit a reduced carbon flux through the prokaryotic pathway that is compensated for by an increased carbon flux through the eukaryotic pathway . Biochemical approaches reveal that the gly1 phenotype cannot be explained by a deficiency in the enzymes of the prokaryotic pathway . The chemical complementation of the mutant phenotype by exogenous glycerol treatment of gly1 plants suggests a lesion affecting the glycerol 3-phosphate supply within the chloroplast . As an alternative to the biochemical study of the gly1 mutants we set out to map the GLY1 locus . The gly1 mutant being an EMS (ethyl methane sulphonate) mutant, we used a strategy based on the polymorphism existing between Arabidopsis ecotypes, here Columbia (gly1 background) and Landsberg erecta . We mapped gly1 on chromosome II . During the process of chromosome walking, the complete sequence of chromosome II was released, allowing us to make assumptions on candidate genes based on map location . We are currently sequencing the putative genes. Biochem Soc Trans, 2000 Dec, 28(6), 631 - 2 The molecular basis of the high linoleic acid content in Petunia seed oil: analysis of a seed-specific linoleic acid mutant; Verwoert I et al.; From a random transposon mutagenesis experiment, using Petunia line W138, a seed-specific linoleic acid mutant was isolated . The tagged gene was cloned and identified as a microsomal Delta(12) desaturase . Expression of the gene, however, was constitutive and not, as might have been expected, seed-specific . Moreover, self-fertilized homozygous mutants still contain 40% 18:2 in the seed lipid fraction . This suggests that at least two (seed-specific) Delta(12) desaturase genes are responsible for the high linoleic acid content in Petunia seed oil . Five members of the microsomal Delta(12) desaturase gene family have been identified and isolated . Data are presented on the molecular characterization and tissue-specific expression of these genes, which suggest that, in Petunia, the flux through the prokaryotic and eukaryotic pathways of lipid synthesis might be different from the situation found in Arabidopsis. Biopolymers, 2000, 55(4), 288 - 96 Peptide methionine sulfoxide reductase: biochemistry and physiological role; Brot N et al.; The oxidation of methionine to methionine sulfoxide both in vivo and in vitro can lead to the loss of biological activity in a variety of proteins . This loss of activity can be reversed by an enzyme called methionine sulfoxide reductase . The gene for this enzyme has been cloned and sequenced from a variety of prokaryotic and eukaryotic cells, and the deduced amino acid sequence is very highly conserved . The mechanism of action of the bovine enzyme has been shown to involve a critical cysteine residue located at position 72 of the protein . In addition to its role as a "repair" enzyme, other evidence suggests that the enzyme may be involved in bacterial adherence and regulation of protein activity. Bioessays, 2001 Feb, 23(2), 179 - 83 High local protein concentrations at promoters: strategies in prokaryotic and eukaryotic cells; Droge P et al.; The speed of chemical reactions is proportional to the concentration of molecules involved . Since proteins catalyze most of the essential reactions inside a living cell, their concentration should be as high as possible . An economical way to achieve this is through the establishment of small cell compartments . We propose that within these compartments, two types of local concentration effects are at work . (1) With local concentration type I reactions, multimeric proteins bound to a specific DNA sequence have an increased local concentration for a second DNA site sufficiently close-by, or for proteins bound to such a site . (2) For type II effects, DNA can be used as a scaffold to build unique nucleoprotein complexes that would otherwise not exist free in solution . These complexes are proficient in establishing longer-range interactions with similarly unique complexes located far away on the genome . We discuss the consequences of these local concentration effects in the light of the markedly different sizes of prokaryotic and eukaryotic cells and of their genomes. Plant J, 2001 Jan, 25(2), 127 - 35 Heterologous complementation of yeast reveals a new putative function for chloroplast m-type thioredoxin; Issakidis-Bourguet E et al.; In the chloroplast of higher plants, two types of thioredoxins (TRX), namely TRX m which shows high similarity to prokaryotic thioredoxins and TRX f which is more closely related to eukaryotic thioredoxins, have been found and biochemically characterized, but little is known about their physiological specificity with respect to their target(s) . Here, we tested, in vivo, the ability of organelle-specific TRX from Arabidopsis thaliana to compensate for TRX deficiency of a Saccharomyces cerevisiae mutant strain . Seven plant organellar TRX (four of the m type, two of the f type and a newly discovered TRX x of prokaryotic type) were expressed in yeast in a putative mature form . None of these heterologous TRX were able to restore growth on sulphate or methionine sulphoxide of the mutant cells . When we tested their ability to rescue the oxidant-hypersensitive phenotype of the TRX-deficient strain, we found that TRX m and TRX x, but not TRX f, affected the tolerance to oxidative stress induced by either hydrogen peroxide or an alkyl hydroperoxide . Athm1, Athm2, Athm4 and Athx induced hydrogen peroxide tolerance like the endogenous yeast thioredoxins . Unexpectedly, Athm3 had a hypersensitizing effect towards oxidative stress . The presence of functional heterologous TRX was checked in the recombinant clones tested, supporting distinct abilities for organelle-specific plant TRX to compensate for TRX deficiency in yeast . We propose a new function for the prokaryotic-type chloroplastic TRX as an anti-oxidant and provide in vivo evidence for different roles of chloroplastic TRX isoforms. Mol Biochem Parasitol, 2001 Jan 15, 112(1), 61 - 9 Tetracycline regulated gene expression in Leishmania donovani; Yan S et al.; The prokaryotic tetracycline-responsive repressor/operator system has proven to be useful for studying the function of essential genes and the expression of toxic gene products in a number of organisms, including Trypanosoma brucei . We report here the adaptation of this system for use in Leishmania . The inducible promoter construct contains a bleomycin resistance-luciferase fusion (BLE-LUC) gene driven by an rRNA promoter with two copies of the TetO sequence inserted two nucleotides upstream of the transcriptional start site . This construct showed regulation of BLE-LUC expression by two orders of magnitude when targeted into the rDNA locus in the reverse orientation relative to transcription of the rRNA genes in a Leishmania donovani cell line expressing TETR . The luciferase expression level in the absence of tetracycline was approximately 50-fold lower than that in the tubulin locus (where it is transcribed by pol II), while the expression level in the presence of tetracycline was approximately five-fold higher than that from the tubulin locus . There was no linear relationship between the level of TETR expression and the regulation, and changing of positions of operator did not increase regulation. Gene, 2000 Dec 30, 261(1), 93 - 105 A test of translational selection at 'silent' sites in the human genome: base composition comparisons in alternatively spliced genes; Iida K et al.; Natural selection appears to discriminate among synonymous codons to enhance translational efficiency in a wide range of prokaryotes and eukaryotes . Codon bias is strongly related to gene expression levels in these species . In addition, between-gene variation in silent DNA divergence is inversely correlated with codon bias . However, in mammals, between-gene comparisons are complicated by distinctive nucleotide-content bias (isochores) throughout the genome . In this study, we attempted to identify translational selection by analyzing the DNA sequences of alternatively spliced genes in humans and in Drosophila melanogaster . Among codons in an alternatively spliced gene, those in constitutively expressed exons are translated more often than those in alternatively spliced exons . Thus, translational selection should act more strongly to bias codon usage and reduce silent divergence in constitutive than in alternative exons . By controlling for regional forces affecting base-composition evolution, this within-gene comparison makes it possible to detect codon selection at synonymous sites in mammals . We found that GC-ending codons are more abundant in constitutive than alternatively spliced exons in both Drosophila and humans . Contrary to our expectation, however, silent DNA divergence between mammalian species is higher in constitutive than in alternative exons. Trends Genet, 2001 Jan, 17(1), 10 - 3 Evolution of prokaryotic gene order: genome rearrangements in closely related species; Suyama M et al.; Conservation of gene order in prokaryotes has become important in predicting protein function because, over the evolutionary timescale, genomes are shuffled so that local gene-order conservation reflects the functional constraints within the protein . Here, we compare closely related genomes to identify the rate with which gene order is disrupted and to infer the genes involved in the genome rearrangement. FEBS Lett, 2001 Jan 12, 488(1-2), 29 - 33 Novel type Arabidopsis thaliana H(+)-PPase is localized to the Golgi apparatus; Mitsuda N et al.; Vacuolar H(+)-PPase, a membrane bound proton-translocating pyrophosphatase found in various species including plants, some protozoan and prokaryotes, has been demonstrated to be localized to the vacuolar membrane in plants . Using a GUS reporter system and a green fluorescent protein (GFP) fusion protein, we investigated the tissue distribution and the subcellular localization, respectively, of a novel type H(+)-PPase encoded by AVP2/AVPL1 identified in the Arabidopsis thaliana genome . We showed that AVP2/AVPL1 is highly expressed at the trichome and the filament of stamen . Furthermore, the fluorescence of GFP-tagged AVP2/AVPL1 showed small dot-like structures that were observed throughout the cytoplasm of various Arabidopsis cells under a fluorescent microscope . The distribution of this dot-like fluorescent pattern was apparently affected by a treatment with brefeldin A . Moreover, we demonstrated that most dot-like fluorescent structures colocalized with a Golgi resident protein . These findings suggest that this novel type H(+)-PPase resides on the Golgi apparatus rather than the vacuolar membrane. Mol Cell, 2000 Dec, 6(6), 1275 - 85 Function of transcription cleavage factors GreA and GreB at a regulatory pause site; Marr MT et al.; Gre proteins of prokaryotes, and SII proteins of eukaryotes and archaea, are transcription elongation factors that promote an endogenous transcript cleavage activity of RNA polymerases; this process promotes elongation through obstructive regions of DNA, including transcription pauses that act as sites of genetic regulation . We show that a regulatory pause in the early part of the late gene operon of bacteriophage lambda is subject to such cleavage and resynthesis . In cells lacking the cleavage factors GreA and GreB, the pause is prolonged, and RNA polymerase occupies a variant position at the pause site . Furthermore, GreA and GreB are required to mediate efficient function of the lambda gene Q antiterminator at this site . Thus, cleavage factors are necessary for the natural progression of RNA polymerase in vivo. Biochem Biophys Res Commun, 2001 Jan 19, 280(2), 491 - 502 Analysis of the Desulfovibrio gigas transcriptional unit containing rubredoxin (rd) and rubredoxin-oxygen oxidoreductase (roo) genes and upstream ORFs; Silva G et al.; Rubredoxin-oxygen oxidoreductase, an 86-kDa homodimeric flavoprotein, is the final component of a soluble electron transfer chain that couples NADH oxidation with oxygen reduction to water from the sulfate-reducing bacterium Desulfovibrio gigas . A 4.2-kb fragment of D . gigas chromosomal DNA containing the roo gene and the rubredoxin gene was sequenced . Additional open reading frames designated as ORF-1, ORF-2, and ORF-3 were also identified in this DNA fragment . ORF-1 encodes a protein exhibiting homology to several proteins of the short-chain dehydrogenase/reductase family of enzymes . The N-terminal coenzyme-binding pattern and the active-site pattern characteristic of short chain dehydrogenase/reductase proteins are conserved in ORF-1 product . ORF-2 does not show any significant homology with any known protein, whereas ORF-3 encodes a protein having significant homologies with the branched-chain amino acid transporter AzlC protein family . Northern blot hybridization analysis with rd and roo-specific probes identified a common 1.5-kb transcript, indicating that these two genes are cotranscribed . The transcription start site was identified by primer extension analysis to be a guanidine 87 bp upstream the ATG start codon of rubredoxin . The transcript size indicates that the rd-roo mRNA terminates downstream the roo-coding unit . Putative -10 and -35 regulator regions of a varsigma(70)-type promoter, having similarity with E . coli varsigma(70) promoter elements, are found upstream the transcription start site . Rubredoxin-oxygen oxidoreductase and rubredoxin genes are shown to be constitutively and abundantly expressed . Using the data available from different prokaryotic genomes, the rubredoxin genomic organization and the first tentative to understand the phylogenetic relationships among the flavoprotein family are reported in this study . Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 99 - 103 Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11; Chen D et al.; Monoclonal antibody (MAb) 3H11 can bind specifically to different cancer cells from different tissues . MAb 3H11 labeled with radioactive isotopes has been used clinically to detect primary cancer and metastatic cancer . Molecular cloning of the antigen recognized by MAb 3H11 is important in studying tumor occurrence and in developing new biotherapy for cancer . Using MAb 3H11, we screened cDNA library made from the human gastric cancer cell line MGC 803, which reacts with MAb 3H11, and isolated one positive clone specifically recognized by the antibody . The insert cDNA fragment was 0.5 kb . After recombining with glutathione-S-transferase expression vector pGEX-4T, the cDNA fragment could be expressed into a fusion protein that specifically reacted with MAb 3H11 . Moreover, the fusion protein could competitively inhibit MAb 3H11 binding to MGC 803 cells . Based on the nucleotide sequence of the cDNA fragment, the full length of the cDNA (2156 bp) was obtained by Rapid-Amplification-cDNA-End (RACE) and nested PCR . Its reading frame was 1767 bp encoding a protein of 589 amino acids . Sequence analysis indicated that there is no highly homologous gene in the GenBank . Northern blot and RT-PCR showed that the mRNA of MAb 3H11 antigen was extensively distributed in embryonic tissue and in different cancerous tissues, but not in corresponding normal tissues . Moreover, in producing antibodies to the antigen expressed prokaryotically, we found that the immunogenicity of the antigen was low in mammalian . Thus we believe that this novel antigen acts as an expression regulator in embryo cells and regains expression in tumor cells . In addition, this antigen is characterized by low differentiation and high proliferation . Molecular function of the antigen needs to be investigated . Biochem Biophys Res Commun, 2000 Dec 29, 279(3), 803 - 7 Targeting of active human cytochrome P4501A1 (CYP1A1) to the periplasmic space of Escherichia coli; Kaderbhai MA et al.; Native human cytochrome P4501A1 (CYP1A1) was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase . The chimeric P450 construct was placed under the transcriptional control of the native phoA promoter in a prokaryotic expression vector . Induction of the hemoprotein by heterologous expression in E . coli following growth in a phosphate-limited medium resulted in abundant synthesis of recombinant CYP1A1 as detected by reduced CO-difference spectra . Furthermore, the signal-appended CYP1A1 was translocated across the bacterial inner membrane by the sec-dependent pathway and processed to yield authentic, heme-incorporated P450 within the periplasmic space . In vitro and whole-cell metabolic activity studies showed that the periplasmically-located CYP1A1 competently catalysed NADPH-dependent benzo{a}pyrene 3-hydroxylation and 7-ethoxyresorufin O-deethylation . The means to localise cytochromes P450 in the periplasm offers an ability to produce high levels of protein, attributable to the less hostile nature of the compartment, and therein the enzymes for posttranslational assembly of heme with the translocated protein. Protein Expr Purif, 2001 Feb, 21(1), 235 - 42 Bacterial expression, purification, and characterization of rat hydroxysteroid sulfotransferase STa; Sheng JJ et al.; Hydroxysteroid (alcohol) sulfotransferase catalyzes numerous reactions that are important to our understanding of the metabolism of both endogenous steroids and exogenous alcohols . Here we report a method for prokaryotic expression and rapid purification of the recombinant hydroxysteroid sulfotransferase STa, a major isoform of hydroxysteroid sulfotransferase in the rat . The cDNA encoding STa was cloned into a pET-3c vector and expressed in Escherichia coli BL21 cells . After disruption of the cells by sonication, the enzyme was purified in one step by affinity chromatography on adenosine 3',5'-diphosphate-agarose . The purified recombinant STa had a relative molecular mass on SDS-PAGE that was identical with the native hepatic STa in rat liver . The expressed enzyme displayed similar substrate inhibition characteristics with dehydroepiandrosterone as have been noted previously with the native enzyme purified from rat liver . Furthermore, the catalytic efficiency in sulfation of 7-hydroxymethyl-12-methylbenz{a}anthracene, as well as the stereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent with characteristics of the STa isolated from rat liver . J Mol Biol, 2001 Jan 26, 305(4), 715 - 27 Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA; Velichutina IV et al.; Functional and structural similarities between tRNA and eukaryotic class 1 release factors (eRF1) described previously, provide evidence for the molecular mimicry concept . This concept is supported here by the demonstration of a genetic interaction between eRF1 and the decoding region of the ribosomal RNA, the site of tRNA-mRNA interaction . We show that the conditional lethality caused by a mutation in domain 1 of yeast eRF1 (P86A), that mimics the tRNA anticodon stem-loop, is rescued by compensatory mutations A1491G (rdn15) and U1495C (hyg1) in helix 44 of the decoding region and by U912C (rdn4) and G886A (rdn8) mutations in helix 27 of the 18 S rRNA . The rdn15 mutation creates a C1409-G1491 base-pair in yeast rRNA that is analogous to that in prokaryotic rRNA known to be important for high-affinity paromomycin binding to the ribosome . Indeed, rdn15 makes yeast cells extremely sensitive to paromomycin, indicating that the natural high resistance of the yeast ribosome to paromomycin is, in large part, due to the absence of the 1409-1491 base-pair . The rdn15 and hyg1 mutations also partially compensate for inactivation of the eukaryotic release factor 3 (eRF3) resulting from the formation of the {PSI+} prion, a self-reproducible termination-deficient conformation of eRF3 . However, rdn15, but not hyg1, rescues the conditional cell lethality caused by a GTPase domain mutation (R419G) in eRF3 . Other antisuppressor rRNA mutations, rdn2(G517A), rdn1T(C1054T) and rdn12A(C526A), strongly inhibit {PSI+}-mediated stop codon read-through but do not cure cells of the {PSI+} prion . Interestingly, cells bearing hyg1 seem to enable {PSI+} strains to accumulate larger Sup35p aggregates upon Sup35p overproduction, suggesting a lower toxicity of overproduced Sup35p when the termination defect, caused by {PSI+}, is partly relieved . Plant Physiol, 2001 Feb, 125(2), 1001 - 11 Characterization of plant beta-ureidopropionase and functional overexpression in Escherichia coli; Walsh TA et al.; Pyrimidine bases are rapidly catabolized in growing plant tissues . The final enzyme of the catabolic pathway, beta-ureidopropionase (beta-UP; EC 3.5.1.6), was partially purified from the shoots of etiolated maize (Zea mays) seedlings . The enzyme had a K(m) for beta-ureidopropionate (the substrate derived from uracil) of 11 microM . Only one enantiomer of racemic beta-ureidoisobutyrate (derived from thymine) was processed with a K(m) of 6 microM . The enzyme was inactivated by dialysis against 1,10-phenanthroline and activity could be partially restored by addition of Zn(2+) . Maize beta-UP was very sensitive to inactivation by iodoacetamide . This could be prevented by addition of substrate, indicating the presence of an active site Cys . The enzyme was strongly inhibited by short chain aliphatic acids and aryl propionates, the most potent inhibitor of which was 2-(2, 6-dinitrophenoxy)-propionate (I(50) = 0.5 microM) . A gene for Arabidopsis beta-UP encodes a polypeptide of 405 amino acids and has about 55% homology with the enzymes from other eukaryotic organisms . Several highly conserved residues link the plant beta-UP with a larger class of prokaryotic and eukaryotic amidohydrolases . An Arabidopsis cDNA truncated at the N terminus by 14 residues was cloned and overexpressed in Escherichia coli . The recombinant enzyme (43.7 kD) was soluble, functional, and purified to homogeneity with yields of 15 to 20 mg per 30 g fresh weight of E . coli cells . The recombinant enzyme from Arabidopsis and the native enzyme from maize had molecular masses of approximately 440 kD, indicating the enzyme is a decamer at pH 7. Nucleic Acids Res, 2001 Feb 1, 29(3), 753 - 8 Pseudoknots in prion protein mRNAs confirmed by comparative sequence analysis and pattern searching; Barrette I et al.; The human prion gene contains five copies of a 24 nt repeat that is highly conserved among species . An analysis of folding free energies of the human prion mRNA, in particular in the repeat region, suggested biased codon selection and the presence of RNA patterns . In particular, pseudoknots, similar to the one predicted by Wills in the human prion mRNA, were identified in the repeat region of all available prion mRNAs available in GenBank, but not those of birds and the red slider turtle . An alignment of these mRNAs, which share low sequence homology, shows several co-variations that maintain the pseudoknot pattern . The presence of pseudoknots in yeast Sup35p and Rnq1 suggests acquisition in the prokaryotic era . Computer generated three-dimensional structures of the human prion pseudoknot highlight protein and RNA interaction domains, which suggest a possible effect in prion protein translation . The role of pseudoknots in prion diseases is discussed as individuals with extra copies of the 24 nt repeat develop the familial form of Creutzfeldt-Jakob disease. Biophys J, 2001 Jan, 80(1), 229 - 40 Molecular identification of a mechanosensitive channel in archaea; Kloda A et al.; The TM1 domain of the large conductance mechanosensitive (MS) channel of Escherichia coli was used as a genetic probe to search the genomic database of the archaeon Methanoccoccus jannashii for MscL homologs . We report that the hypothetical protein MJ0170 of M . jannashii exhibited 38.5% sequence identity with the TM1 domain of Eco-MscL . Moreover, MJ0170 was found to be a conserved homolog of MscS, the second type of E . coli MS channel encoded by the yggB gene . Furthermore, we identified a cluster of charged residues KIKEE in the C-terminus of MJ0170 that strikingly resembled the charged C-terminal amino acid cluster present in Eco-MscL (RKKEE) . We cloned and expressed MJ0170 in E . coli, which when reconstituted into liposomes or expressed in the cell membrane of giant E . coli spheroplasts, exhibited similar activity to the bacterial MS channels . Our study suggests that the M . jannashii MS channel and its homologs evolved as a result of gene duplication of the ancestral MscL-like molecule with the TM1 domain remaining the most conserved structural motif among prokaryotic MS channels. Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 926 - 31 Epub 2001 Jan 23. Characterization of a quinone reductase activity for the mitomycin C binding protein (MRD): Functional switching from a drug-activating enzyme to a drug-binding protein; He M et al.; Self-protection in the mitomycin C (MC)-producing microorganism Streptomyces lavendulae includes MRD, a protein that binds MC in the presence of NADH and functions as a component of a unique drug binding-export system . Characterization of MRD revealed that it reductively transforms MC into 1,2-cis-1-hydroxy-2,7-diaminomitosene, a compound that is produced in the reductive MC activation cascade . However, the reductive reaction catalyzed by native MRD is slow, and both MC and the reduced product are bound to MRD for a relatively prolonged period . Gene shuffling experiments generated a mutant protein (MRD(E55G)) that conferred a 2-fold increase in MC resistance when expressed in Escherichia coli . Purified MRD(E55G) reduces MC twice as fast as native MRD, generating three compounds that are identical to those produced in the reductive activation of MC . Detailed amino acid sequence analysis revealed that the region around E55 in MRD strongly resembles the second active site of prokaryotic catalase-peroxidases . However, native MRD has an aspartic acid (D52) and a glutamic acid (E55) residue at the positions corresponding to the catalytic histidine and a nearby glycine residue in the catalase-peroxidases . Mutational analysis demonstrated that MRD(D52H) and MRD(D52H/E55G) conferred only marginal resistance to MC in E . coli . These findings suggest that MRD has descended from a previously unidentified quinone reductase, and mutations at the active site of MRD have greatly attenuated its catalytic activity while preserving substrate-binding capability . This presumed evolutionary process might have switched MRD from a potential drug-activating enzyme into the drug-binding component of the MC export system. J Gen Physiol, 2001 Feb, 117(2), 165 - 80 Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating; Cortes DM et al.; The molecular architecture of the NH(2) and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy . Cysteine mutants were generated (residues 5-24 and 121-160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature . Data on probe mobility (DeltaHo(-1)), accessibility parameters (PiO(2) and PiNiEdda), and inter-subunit spin-spin interaction (Omega) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs . 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs . Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed . In this model, the NH(2) terminus of KcsA forms an alpha-helix anchored at the membrane-water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40-50 A towards the cytoplasm . Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism. Appl Environ Microbiol, 2001 Feb, 67(2), 888 - 94 Diversity of sulfur isotope fractionations by sulfate-reducing prokaryotes; Detmers J et al.; Batch culture experiments were performed with 32 different sulfate-reducing prokaryotes to explore the diversity in sulfur isotope fractionation during dissimilatory sulfate reduction by pure cultures . The selected strains reflect the phylogenetic and physiologic diversity of presently known sulfate reducers and cover a broad range of natural marine and freshwater habitats . Experimental conditions were designed to achieve optimum growth conditions with respect to electron donors, salinity, temperature, and pH . Under these optimized conditions, experimental fractionation factors ranged from 2.0 to 42.0 per thousand . Salinity, incubation temperature, pH, and phylogeny had no systematic effect on the sulfur isotope fractionation . There was no correlation between isotope fractionation and sulfate reduction rate . The type of dissimilatory bisulfite reductase also had no effect on fractionation . Sulfate reducers that oxidized the carbon source completely to CO2 showed greater fractionations than sulfate reducers that released acetate as the final product of carbon oxidation . Different metabolic pathways and variable regulation of sulfate transport across the cell membrane all potentially affect isotope fractionation . Previous models that explained fractionation only in terms of sulfate reduction rates appear to be oversimplified . The species-specific physiology of each sulfate reducer thus needs to be taken into account to understand the regulation of sulfur isotope fractionation during dissimilatory sulfate reduction. Appl Environ Microbiol, 2001 Feb, 67(2), 782 - 90 Cell cycle regulation by light in Prochlorococcus strains; Jacquet S et al.; The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry . When exposed to a light-dark (L-D) cycle with an irradiance of 25 micromol of quanta x m(-2) x s(-1), the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511 . Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the "light on" signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles . Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift . Complete darkness arrested most of the cells in the G1 phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division . However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp . are not as strict as in marine Synechococcus spp . Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G2 phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate . These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp. Trends Microbiol, 2000 Apr, 8(4), 180 - 5 The question of DNA repair in hyperthermophilic archaea; Grogan DW; Hyperthermophilic archaea grow at temperatures that destabilize the primary structure of DNA and in evolutionary terms they are highly divergent from other well studied microorganisms . These prokaryotes should therefore require DNA damage repair to be unusually effective, and could employ novel mechanisms for this repair . Recent genome sequence analyses and biochemical and genetic assays suggest a distribution of DNA repair strategies that raises intriguing questions for future study. J Mol Evol, 2000 Mar, 50(3), 249 - 57 The contributions of replication orientation, gene direction, and signal sequences to base-composition asymmetries in bacterial genomes; Tillier ER et al.; Asymmetries in base composition between the leading and the lagging strands have been observed previously in many prokaryotic genomes . Since a majority of genes is encoded on the leading strand in these genomes, previous analyses have not been able to determine the relative contribution to the base composition skews of replication processes and transcriptional and/or translational forces . Using qualitative graphical presentations and quantitative statistical analyses (analysis of variance), we have found that a significant proportion of the GC and AT skews can be attributed to replication orientation, i.e., the sequence of a gene is influenced by whether it is encoded on the leading or lagging strand . This effect of replication orientation on skews is independent of, and can be opposite in sign to, the effects of transcriptional or translational processes, such as selection for codon usage, amino acid preferences, expression levels (inferred from codon adaptation index), or potential short signal sequences (e.g., chi sequences) . Mutational differences between the leading and the lagging strands are the most likely explanation for a significant proportion of the base composition skew in these bacterial genomes . The finding that base composition skews due to replication orientation are independent of those due to selection for function of the encoded protein may complicate the interpretation of phylogenetic relationships, conserved positions in nucleotide or amino acid sequence alignments, and codon usage patterns. Curr Biol, 2000 Apr 6, 10(7), R272 - 5 Microbiology: intimate strangers; Stephens C; A more robust view of the diversity of prokaryotes has come from sequencing rRNAs amplified directly from environmental samples . This approach has now been used to examine microbial communities in the human body, revealing populations rich in undescribed species whose impact on humans remains to be determined. J Biol Chem, 2001 Apr 13, 276(15), 11743 - 53 Epub 2001 Jan 04. Polymerization of Ftsz, a bacterial homolog of tubulin . is assembly cooperative? Romberg L, Simon M, Erickson HP. FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis . In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers . Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or "isodesmic") assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration . We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme . The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates . It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolar K(D). J Biol Chem, 2001 Apr 13, 276(15), 12222 - 7 Epub 2001 Jan 08. Signal peptides bind and aggregate RNA . An alternative explanation for GTPase inhibition in the signal recognition particle; Swain JF et al.; N-terminal signal sequences can direct nascent protein chains to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes by interacting with the signal recognition particle . In this study, we show that isolated peptides corresponding to several bacterial signal sequences inhibit the GTPase activity of the Escherichia coli signal recognition particle, as previously reported (Miller, J . D., Bernstein, H . D., and Walter, P . (1994) Nature 367, 657-659), but not by the direct mechanism proposed . Instead, isolated signal peptides bind nonspecifically to the RNA component and aggregate the entire signal recognition particle, leading to a loss of its intrinsic GTPase activity . Surprisingly, only "functional" peptide sequences aggregate RNA; the peptides in general use as "nonfunctional" negative controls (e.g . those with deletions or charged substitutions within the hydrophobic core), are sufficiently different in physical character that they do not aggregate RNA and thus have no effect on the GTPase activity of the signal recognition particle . We propose that the reported effect of functional signal peptides on the GTPase activity of the signal recognition particle is an artifact of the high peptide concentrations and low salt conditions used in these in vitro studies and that signal sequences at the N terminus of nascent chains in vivo do not exhibit this activity. FEMS Microbiol Rev, 2001 Jan, 25(1), 39 - 67 The species concept for prokaryotes; Rossello-Mora R et al.; The species concept is a recurrent controversial issue that preoccupies philosophers as well as biologists of all disciplines . Prokaryotic species concept has its own history and results from a series of empirical improvements parallel to the development of the techniques of analysis . Among the microbial taxonomists, there is general agreement that the species concept currently in use is useful, pragmatic and universally applicable within the prokaryotic world . However, this empirically designed concept is not encompassed by any of the, at least, 22 concepts described for eukaryotes . The species could be described as 'a monophyletic and genomically coherent cluster of individual organisms that show a high degree of overall similarity in many independent characteristics, and is diagnosable by a discriminative phenotypic property' . We suggest to refer it as a phylo-phenetic species concept . Here, we discuss the validity of the concept in use which we believe is more pragmatic in comparison with those concepts described for eukaryotes. FEMS Microbiol Lett, 2001 Jan 1, 194(1), 7 - 11 Measurement of the glycogen synthetic pathway in permeabilized cells of cyanobacteria; Gomez Casati DF et al.; A simple, rapid and reliable procedure for permeabilizing cyanobacterial cells and measuring the glycogen synthetic pathway in situ, is presented . Cells from Anabaena sp . strain PCC 7120 were permeabilized with a mixture of toluene:ethanol (1:4 v/v) . Fluorescence microscopy of cells incubated with fluorescein diacetate showed Anabaena non-permeabilized cells as green fluorescents, whereas permeabilized (viable) cells exhibited the intrinsic red fluorescence . Labelled alpha-1,4-glucan was recovered when permeabilized cells were incubated with the substrates of ADP-glucose pyrophosphorylase or glycogen synthase . The kinetic and regulatory properties of both enzymes could be reproduced in situ . The simplicity of the procedure and the ability to measure in situ glucan fluxes show the methodology as useful for studying the intracellular regulation of storage polysaccharides in a photosynthetic prokaryote. Biochim Biophys Acta, 2000 Dec 29, 1543(2), 417 - 433 Approaches for deciphering the structural basis of low temperature enzyme activity; Sheridan PP et al.; An increasing number of enzymes active at low temperature are being studied to help determine the structural features important for cold-activity . This review examines the diversity of prokaryotic cold-active enzymes and the features proposed to account for low temperature activity . We then consider the difficulty of identifying the key structural features needed for cold-activity and the need to compare enzymes having different temperature optima from phylogenetically related organisms to determine features responsible for low temperature activity . In addition to studying naturally occurring enzymes, directed evolution experiments are discussed as methods for examining the proposed mechanisms influencing the thermal dependence of activity. Plant Cell Physiol, 2000 Oct, 41(10), 1157 - 63 Mitochondrial localization of AtOXA1, an arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane; Sakamoto W et al.; Components of some protein complexes present in the inner membrane of mitochondria are encoded in both nuclear and mitochondrial genomes, and correct sorting and assembly of these proteins is necessary for proper respiratory function . Recent studies in yeast suggest that Oxa1p, a protein conserved between prokaryotes and eukaryotes, is an essential factor for protein sorting and assembly into membranes . We previously identified AtOXA1, an Arabidopsis homologue of OXA1 by functional complementation of a yeast oxa1- mutant . In this study, we investigated the genomic organization of AtOXA1 and localization of the AtOXA1 protein . Characterization of the AtOXA1 genomic region indicated that the gene consists of 10 exons and is located on chromosome V . A database search also revealed another gene coding for a putative protein homologous to AtOXA1 on chromosome II . Transient expression of a green fluorescent protein (GFP) fusion in suspension-cultured tobacco cells showed that AtOXA1 is targeted into mitochondria by its N-terminal presequence . Antibodies raised against AtOXA1 recognized a 38-kDa intrinsic protein of the inner mitochondrial membrane . Thus, localization of AtOXA1 in the mitochondrial inner membrane, together with our previous complementation experiment in yeast, suggested that it is a functional homologue of Oxa1p. Arch Biochem Biophys, 2000 Dec 1, 384(1), 116 - 22 Production of recombinant human apoferritin heteromers; Grace JE Jr et al.; We are interested in learning how iron is safely inserted and stored in ferritin . Recombinant DNA technology has considerable potential in determining the functional roles of the two ferritin subunits (H and L) . In previous studies, we have observed that recombinant rat H ferritin was repressive to cell growth in both prokaryotic and eukaryotic expression systems (Guo et al., Biochem . Biophys . Res . Commun . 242, 39-45 (1998)) . This results in the protein being expressed at very low levels . This problem was partially bypassed by the use of an inducible expression system, which utilizes T7 RNA polymerase dependent expression of the gene, induced by isopropyl beta-D-thiogalactopyranoside (IPTG) . Simultaneously expressing the H and L ferritin genes in this system resulted in only a narrow range of ferritin heteromers, which predominantly consisted of the L subunit . Addition of rifampicin to cultures, 1 h following the induction of protein synthesis by IPTG, increased the production of the H subunit and thus increased the range of ferritin H:L subunit ratios . Simultaneous expression of the H and L ferritin genes in Escherichia coli grown in a deficient medium with minimal iron and with the addition of rifampicin resulted in the production of a range of recombinant human apoferritin heteromers that could be separated based on their subunit composition. Virology, 2001 Jan 5, 279(1), 292 - 301 A C-terminal deletion mutant of pokeweed antiviral protein inhibits programmed +1 ribosomal frameshifting and Ty1 retrotransposition without depurinating the sarcin/ricin loop of rRNA; Hudak KA et al.; Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes . Here, a series of PAP mutants were used to examine the relationship between depurination of the S/R loop and inhibition of +1 programmed ribosomal frameshifting (PRF) and to define PAP sequences critical for inhibition of +1 PRF and Ty1 retrotransposition in the yeast Saccharomyces cerevisiae . Using three different classes of mutants we present evidence that strong binding of a C-terminal PAP mutant (PAPc) to ribosomes is sufficient to inhibit +1 PRF and Ty1 retrotransposition in the absence of S/R loop depurination . PAPc did not affect the totivirus ScV-L-A and HIV-1-directed -1 PRF efficiencies or the ability of cells to maintain the M(1)-dependent killer phenotype, demonstrating the specificity of the effect of PAPc on +1 PRF . Semin Cell Dev Biol, 2000 Dec, 11(6), 467 - 73 Phytochromes as light-modulated protein kinases; Fankhauser C; Many phytochrome responses in plants are induced by red light and inhibited by far-red light . To explain the biochemical basis of these observations, it was speculated that plant phytochromes are light-regulated enzymes more than 40 years ago . The search for such an enzymatic activity has a long and rather tumultuous history . Biochemical data in the late 1980s had suggested that oat phytochrome might be a light-regulated protein kinase . The topic was the subject of intense debate, but solid experimental data backing the kinase model has been published recently . Two lines of research played a key role in this finding: the production of biologically active highly purified recombinant phytochrome and the discovery of phytochromes in prokaryotes . This review discusses the key steps of this discovery, and suggests some hypotheses for the role of protein kinase activity in photomorphogenesis . Ned Tijdschr Geneeskd, 2000 Dec 9, 144(50), 2384 - 91 {Bile formation and cholestasis}; Jansen PL et al.; Transport proteins in hepatocytes and bile duct epithelium mediate uptake and secretion of cholephilic compounds in the liver and are involved in bile formation . Many of these proteins have recently been cloned and characterized and appear to belong to large gene families . Apart from the liver these proteins are expressed in the blood-brain barrier, placenta, kidneys, lungs, intestine and seminiferous tubules . Prokaryotes and yeasts contain similar proteins . In cancer cells they are involved in multidrug resistance . Some genetic cholestatic liver diseases, including progressive familial intrahepatic cholestasis, Dubin-Johnson syndrome, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy result from mutations in transport protein genes . These proteins also play a role in drug-induced liver disease and in primary biliary cirrhosis . Cyclosporine and oestradiol (glucuronide) for instance inhibit bile salt export protein (BSEP). J Biotechnol, 2000 Sep, 74(3), 233 - 54 S-layer-supported lipid membranes; Schuster B et al.; Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component . S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution . Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations . Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies . Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g . silicon wafers) . Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes . Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice . Electrostatic interactions are the most prevalent forces . The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed . Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer . Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes . The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes. Hum Immunol, 2000 Nov, 61(11), 1113 - 7 HLA-G in reproduction: studies on the maternal-fetal interface; Hunt JS et al.; For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G, are present at the maternal-fetal interface . Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute . Recent studies in our laboratory raised the question of whether soluble isoforms might reach the maternal and/or fetal blood circulation . A capture enzyme-linked immunoabsorbent assay (ELISA) identified soluble HLA-G (sHLA-G) in maternal blood throughout pregnancy but failed to detect sHLA-G in cord sera . Further studies suggested that the circulating proteins may be either free heavy chain (sHLA-G1 and/or sHLA-G2) or exclusively sHLA-G2 . To study the potential function(s) of the soluble isoforms to modulate local or systemic immunity in mothers, we generated recombinant sHLA-G1 and -G2 in both prokaryotic and eukaryotic systems . Preliminary experiments conducted using DNA microarray analysis suggest that sHLA-G is capable of modulating gene expression in blood mononuclear leukocytes . Potential local targets were also identified; decidual and placental macrophages but not trophoblast cells contained mRNA encoding two of the known receptors for HLA-G, ILT2 and ILT4 . Collectively, the studies are consistent with the hypothesis that sHLA-G produced at the maternal-fetal interface targets to the cells of the monocyte/macrophage lineage and modulates their functions for the benefit of pregnancy. FEMS Microbiol Ecol, 2001 Jan, 34(3), 197 - 206 A new look at microbial leaching patterns on sulfide minerals; Edwards KJ et al.; Leaching patterns on sulfide minerals were investigated by high-resolution scanning electron microscopy (SEM) . Our goal was to evaluate the relative contributions of inorganic surface reactions and reactions localized by attached cells to surface morphology evolution . Experiments utilized pyrite (FeS(2)), marcasi