J Mol Biol, 2001 Aug 3, 311(1), 87 - 100 X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 A resolution; Li de la Sierra I et al.; The X-ray structure of Mycobacterium tuberculosis TMP kinase at 1.95 A resolution is described as a binary complex with its natural substrate TMP . Its main features involve: (i) a clear magnesium-binding site; (ii) an alpha-helical conformation for the so-called LID region; and (iii) a high density of positive charges in the active site . There is a network of interactions involving highly conserved side-chains of the protein, the magnesium ion, a sulphate ion mimicking the beta phosphate group of ATP and the TMP molecule itself . All these interactions conspire in stabilizing what appears to be the closed form of the enzyme . A complete multialignment of all (32) known sequences of TMP kinases is presented . Subtle differences in the TMP binding site were noted, as compared to the Escherichia coli, yeast and human enzyme structures, which have been reported recently . These differences could be used to design specific inhibitors of this essential enzyme of nucleotide metabolism . Two cases of compensatory mutations were detected in the TMP binding site of eukaryotic and prokaryotic enzymes . In addition, an intriguing high value of the electric field is reported in the vicinity of the phosphate group of TMP and the putative binding site of the gamma phosphate group of ATP . Arch Biochem Biophys, 2001 Aug 1, 392(1), 71 - 8 Structure--function relationships of rat hepatic tryptophan 2,3-dioxygenase: identification of the putative heme-ligating histidine residues; Dick R et al.; The liver cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidation of L-tryptophan to formylkynurenine and controls the physiological flux of tryptophan into both the serotonergic and kynureninic pathways . This hemoprotein enzyme is composed of four noncovalently bound subunits of equivalent mass and contains two heme moieties per molecule . Electron paramagnetic resonance analyses have indicated that a histidyl nitrogen is involved in heme ligation {Henry et al., (1976) J . Biol . Chem . 251, 1578}, but the identity of the His residue(s) is unknown . In an attempt to characterize the active site of the enzyme we have substituted each of the 12 His residues in the rat TDO subunit with Ala, to determine their relative importance in heme binding . Sequence alignment of the rat liver protein with that of known or putative TDO sequences from other organisms reveals that four of the His residues are conserved in eukaryotes, two of which are also conserved in prokaryotes . Our findings indicate that replacement of the evolutionarily conserved His 76 and 328 residues resulted in a dramatic reduction of TDO activity, whereas that of the eukaryotically conserved His70 resulted in a significant reduction relative to that of the wild-type enzyme . On the other hand, replacement of the other eukaryotically conserved His273 residue, while affecting the relative expression of the enzyme, had little effect on its specific activity . Size-exclusion analyses revealed that the His76Ala and His328Ala mutants retained little or no heme, suggesting that these may be key residues in ligating the prosthetic heme moieties . Whether these His residues are both provided by the same TDO subunit or a different TDO subunit remains to be determined . Biochem Biophys Res Commun, 2001 Jul 27, 285(4), 976 - 80 An investigation into the membrane-interactive potential of the Escherichia coli KpsE C-terminus; Phoenix DA et al.; Membrane binding via C-terminal amphiphilic alpha-helical structure is a novel anchoring mechanism, which has been characterised in a number of prokaryotic carboxypeptidases . Here, we have used graphical and DWIH analyses to ascertain if a similar anchoring mechanism may be utilised by the Escherichia coli KpsE protein in its binding to the periplasmic face of the inner membrane . The results of these analyses have been compared to those obtained for similar analyses of the C-terminal sequences of E . coli penicillin-binding proteins (PBPs) PBP5 and PBP6 which, are known to function as amphiphilic alpha-helical membrane anchors, and of melittin, a known membrane-interactive toxin . We have also used FTIR spectroscopy and lipid phase transition temperature analysis to investigate the interaction of a peptide homologue of KpsE C-terminal region with membrane lipid . Our results suggest that the KpsE C-terminal sequence has the potential to form an amphiphilic alpha-helix and that this alpha-helix could feature in the membrane binding of the protein . J Bacteriol, 2001 Aug, 183(16), 4752 - 60 Membrane topology of the Streptomyces lividans type I signal peptidases; Geukens N et al.; Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins . For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described . In this communication, we report the experimental determination of the membrane topology of these SPases . A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY . SipX and SipZ have a predicted topology similar to that of SipY . These three S . lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases . In contrast, S . lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria . Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo . Moreover, for the S . lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell . SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell . Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found. Semin Cell Dev Biol, 2001 Aug, 12(4), 271 - 8 Circadian programming in cyanobacteria; Mori T et al.; Prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a timing mechanism that is independent of the cell division cycle . This biological clock orchestrates global regulation of gene expression . Competition experiments demonstrate that fitness is enhanced when the circadian period is consonant with the period of the environmental cycle . Mutational analyses have identified three clock genes in the organism, one of which is related to DNA recombinases and helicases . We propose a new model for the core 'clockwork' that implicates rhythmic changes in the status of the chromosome that underly the rhythms of gene expression . J Bioenerg Biomembr, 2001 Feb, 33(1), 9 - 26 A comprehensive phylogenetic analysis of Rieske and Rieske-type iron-sulfur proteins; Schmidt CL et al.; The Rieske iron-sulfur center consists of a {2Fe-2S} cluster liganded to a protein via two histidine and two cysteine residues present in conserved sequences called Rieske motifs . Two protein families possessing Rieske centers have been defined . The Rieske proteins occur as subunits in the cytochrome bc1 and cytochrome b6f complexes of prokaryotes and eukaryotes or form components of archaeal electron transport systems . The Rieske-type proteins encompass a group of bacterial oxygenases and ferredoxins . Recent studies have uncovered several new proteins containing Rieske centers, including archaeal Rieske proteins, bacterial oxygenases, bacterial ferredoxins, and, intriguingly, eukaryotic Rieske oxygenases . Since all these proteins contain a Rieske motif, they probably form a superfamily with one common ancestor . Phylogenetic analyses have, however, been generally limited to similar sequences, providing little information about relationships within the whole group of these proteins . The aim of this work is, therefore, to construct a dendrogram including representatives from all Rieske and Rieske-type protein classes in order to gain insight into their evolutionary relationships and to further define the phylogenetic niches occupied by the recently discovered proteins mentioned above. Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8376 - 80 Interaction of the beta sliding clamp with MutS, ligase, and DNA polymerase I; Lopez de Saro FJ et al.; The beta and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication . Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone . Indeed, the Escherichia coli beta clamp is known to function with DNA polymerases II and V, indicating that beta also interacts with more than just the chromosomal replicase, DNA polymerase III . This report demonstrates three previously undetected protein-protein interactions with the beta clamp . Thus, beta interacts with MutS, DNA ligase, and DNA polymerase I . Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of beta interactive proteins suggests that the prokaryotic beta ring participates in a wide variety of reactions beyond its role in chromosomal replication. Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8342 - 9 Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination; Sutton MD et al.; Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years . Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates . This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life . Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases . Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression . In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes. Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8283 - 9 A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability; Hishida T et al.; Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases . If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability . Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans . Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA(+) class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein . The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes . The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities . An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability . The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability . In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant . Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability. Proc Natl Acad Sci U S A, 2001 Jul 31, 98(16), 8991 - 6 Epub 2001 Jul 17. PAS kinase: an evolutionarily conserved PAS domain-regulated serine/threonine kinase; Rutter J et al.; PAS domains regulate the function of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli . PAS domain-regulated histidine kinases are common in prokaryotes and control a wide range of fundamental physiological processes . Similarly regulated kinases are rare in eukaryotes and are to date completely absent in mammals . PAS kinase (PASK) is an evolutionarily conserved gene product present in yeast, flies, and mammals . The amino acid sequence of PASK specifies two PAS domains followed by a canonical serine/threonine kinase domain, indicating that it might represent the first mammalian PAS-regulated protein kinase . We present evidence that the activity of PASK is regulated by two mechanisms . Autophosphorylation at two threonine residues located within the activation loop significantly increases catalytic activity . We further demonstrate that the N-terminal PAS domain is a cis regulator of PASK catalytic activity . When the PAS domain-containing region is removed, enzyme activity is significantly increased, and supplementation of the purified PAS-A domain in trans selectively inhibits PASK catalytic activity . These studies define a eukaryotic signaling pathway suitable for studies of PAS domains in a purified in vitro setting. Mutat Res, 2001 Aug 9, 486(3), 167 - 84 DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae; Broomfield S et al.; DNA postreplication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions . In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis . Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes . In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities . PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown . This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes . Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed . Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle . The PRR processes appear to be highly conserved within eukaryotes, from yeast to human. Mol Genet Genomics, 2001 Jun, 265(4), 711 - 20 Fish retroposons related to the Penelope element of Drosophila virilis define a new group of retrotransposable elements; Volff JN et al.; Poseidon and Neptune are two ancient lineages of retroposons related to the Penelope element from Drosophila virilis . They have been identified in various teleost fish species, including the medakafish (Oryzias latipes), and the pufferfishes Fugu rubripes and Tetraodon nigroviridis, whose genomes are currently being sequenced . Some of these elements are highly reiterated in fish genomes . Penelope-related elements were also identified in blood fluke, shrimp, sea urchin, cichlid fish and frog, showing that they are widespread in animals . Penelope-related retroposons were not detected among sequences from the Drosophila melanogaster and human genome projects, suggesting that they have been lost from certain animal lineages . A sequence encoding a putative Uri (also called GIY-YIG) endonuclease domain was detected downstream from the gene for reverse transcriptase . To the best of our knowledge, this type of endonuclease sequence has previously been identified in group I introns and in genes for prokaryotic excinucleases but not in retrotransposable elements . Penelope-related elements are frequently truncated at their 5' ends and can also be flanked by long terminal repeat-like structures . Phylogenetic analysis of the reverse transcriptase domain failed to assign Penelope-related retroposons to one of the major groups of retroelements . Overall, therefore, the evidence strongly suggests that these sequences represent a new group of retrotransposable elements. Biochemistry, 2001 Jul 24, 40(29), 8438 - 43 A ParE-ParC fusion protein is a functional topoisomerase; Lavasani LS et al.; Type II topoisomerases are responsible for DNA unlinking during DNA replication and chromosome segregation . Although eukaryotic enzymes are homodimers and prokaryotic enzymes are heterotetramers, both prokaryotic and eukaryotic type II topoisomerases belong to a single protein family . The amino- and carboxyl-terminal domains of eukaryotic enzymes are homologous to the ATP-binding and catalytic subunits of prokaryotic enzymes, respectively . Topoisomerase IV, a prokaryotic type II topoisomerase, consists of the ATP-binding subunit, ParE, and the catalytic subunit, ParC . We have joined the coding regions of parE and parC in frame and constructed a fusion protein of the two subunits of topoisomerase IV . This fusion protein, ParEC, can catalyze both decatenation and relaxation reactions . The ParEC protein is also capable of decatenating replicating daughter DNA molecules during oriC DNA replication in vitro . Furthermore, the fusion gene, parEC, complements the temperature-sensitive growth of both parC and parE strains, indicating that the ParEC protein can substitute for topoisomerase IV in vivo . These results demonstrate that a fusion protein of the two subunits of topoisomerase IV is a functional topoisomerase . Thus, a heterotetrameric type II topoisomerase can be converted into a homodimeric type II topoisomerase by gene fusion. Fungal Genet Biol, 2001 Jul, 33(2), 115 - 25 Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans; Tavoularis S et al.; The PrnB protein is a highly specific proline transporter that belongs to an amino acid transporter family conserved in both prokaryotes and eukaryotes . In this work, we detected and analyzed the cellular localization of PrnB in vivo by means of green fluorescent protein (GFP) fusions . Several prnB-gfp gene fusions, driven by prnB native promoter sequences, were constructed and targeted to the genomic locus of a prnB null mutant . Chimeric proteins containing GFP fused to the C terminus of PrnB through a linker of two, four, or eight amino acids, with low potential to form secondary structure elements, were shown to be functional in vivo . A two-linker fusion results in partial complementation at both 25 and 37 degrees C . A four-linker fusion affords almost full complementation at 25 degrees C but partial complementation at 37 degrees C, whereas the eight-linker fusion results in partial complementation at both temperatures but in no GFP fluorescence . These results show that the number of linker amino acids is critical for the correct expression and/or translocation of PrnB-GFP fused proteins to the plasma membrane and for the fluorescence of the GFP . The expression of the four-linker PrnB-GFP transporter was detected and analyzed in vivo by both conventional fluorescence and confocal laser microscopy . This chimeric protein is localized in the plasma membrane, secondarily in large vacuoles found in the swollen conidial end of the germlings, and in other small intracellular structures observed as fluorescent granules . A strong correlation between known patterns of PrnB expression and intensity of PrnB-GFP fluorescence was observed . This work also demonstrates that the GFP fusion technology is a unique tool with which to study the expression and cellular localization of low-abundance transmembrane transporters expressed from their native promoters . J Cell Biochem Suppl, 2001, Suppl 36, 117 - 28 Green fluorescent protein variants fold differentially in prokaryotic and eukaryotic cells; Sacchetti A et al.; Better-folding Green Fluorescent Protein (GFP) mutants selected from bacterial screenings are commonly used in widely different cellular environments . However, it is unclear if the folding efficiency of GFPs is invariant in different cell types . In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells . Remarkably, S65T was found to fold at comparable levels with the wild type GFP in bacteria, but at 10-fold lower levels in mammalian cells . On the other hand, Bex1 folded 3-4 times better than the wtGFP or S65T in E . coli, and 10-20-fold or more than 95-fold better, respectively, in mammalian cells . The Vex1 mutant demonstrated similar properties to Bex1 . No evidence of differential GFP unfolding in vivo or of preferential degradation of unfolded GFP molecules was found . Moreover, no relationship between GFP folding efficiency and expression levels, or protein stability was detected . Trivial Aconfounding factors, like GFP unfolding caused by different pH or fluorescence quenching due to molecular crowding, were also excluded . In summary, our results demonstrate that specific GFP variants follow different folding trajectories in mammalian versus bacterial cells . The specificity of this differential folding supports a role of chaperones in guiding the folding of GFP in vivo . J . Cell . Biochem . Suppl . 36: 117-128, 2001 . Genetics, 2001 Jul, 158(3), 1081 - 8 Tc8, a Tourist-like transposon in Caenorhabditis elegans; Le QH et al.; Members of the Tourist family of miniature inverted-repeat transposable elements (MITEs) are very abundant among a wide variety of plants, are frequently found associated with normal plant genes, and thus are thought to be important players in the organization and evolution of plant genomes . In Arabidopsis, the recent discovery of a Tourist member harboring a putative transposase has shed new light on the mobility and evolution of MITEs . Here, we analyze a family of Tourist transposons endogenous to the genome of the nematode Caenorhabditis elegans (Bristol N2) . One member of this large family is 7568 bp in length, harbors an ORF similar to the putative Tourist transposase from Arabidopsis, and is related to the IS5 family of bacterial insertion sequences (IS) . Using database searches, we found expressed sequence tags (ESTs) similar to the putative Tourist transposases in plants, insects, and vertebrates . Taken together, our data suggest that Tourist-like and IS5-like transposons form a superfamily of potentially active elements ubiquitous to prokaryotic and eukaryotic genomes. Mol Microbiol, 2001 Jul, 41(1), 279 - 87 The cytosolic HinT protein of Mycoplasma hominis interacts with two membrane proteins; Kitzerow A et al.; Histidine triad nucleotide-binding (HinT) proteins are dimeric proteins that bind to purines and are found in all three kingdoms: the eukarya, bacteria and archaea . In eukaryotes, HinT proteins have been detected intracellularly, but their function is unknown . Until now, knowledge about HinT proteins in prokaryotes was restricted to sequence similarities and nucleotide-binding studies . In this study, we provide evidence that, in the cell wall-less prokaryote, Mycoplasma hominis, the gene encoding the HinT protein forms an operon with two other genes . These genes encode the species-specific membrane proteins, P60 and P80, which are associated within the mycoplasma membrane . The finding that HinT interacts with this complex by binding to P80 provides novel insight into the organization of bacterial HinT proteins. Mol Microbiol, 2001 Jul, 41(1), 217 - 27 The genetic organization of Desulfovibrio desulphuricans ATCC 27774 bacterioferritin and rubredoxin-2 genes: involvement of rubredoxin in iron metabolism; da Costa PN et al.; The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di-iron centre and having iron-coproporphyrin III as its haem cofactor, as well as a type 2 rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines . The genes encoding for these two proteins were cloned and sequenced . The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family . Most interestingly, the bacterioferritin and rubredoxin-2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins . Indeed, bacterioferritin and rubredoxin-2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of rubredoxin-2 tryptophan(s) when mixed with bacterioferritin . In addition, rubredoxin-2 donates electrons to bacterioferritin . This is the first identification of an electron donor to a bacterioferritin and shows the involvement of rubredoxin-2 in iron metabolism . Furthermore, analysis of the genomic data for anaerobes suggests that rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes. Science, 2001 Jul 13, 293(5528), 290 - 3 Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes; Metz JG et al.; Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and are the precursors of many lipid-derived signaling molecules . Here, pathways for PUFA synthesis are described that do not require desaturation and elongation of saturated fatty acids . These pathways are catalyzed by polyketide synthases (PKSs) that are distinct from previously recognized PKSs in both structure and mechanism . Generation of cis double bonds probably involves position-specific isomerases; such enzymes might be useful in the production of new families of antibiotics . It is likely that PUFA synthesis in cold marine ecosystems is accomplished in part by these PKS enzymes. FEMS Microbiol Ecol, 2001 Jul, 36(2-3), 193 - 202 Diversity of free-living prokaryotes from a deep-sea site at the Antarctic Polar Front; Lopez-Garcia P et al.; To contribute to the understanding of deep-sea planktonic communities, we explored the prokaryotic diversity of a 3000 m deep site at the Antarctic Polar Front using molecular methods . Bacterial 16S rDNA-amplified sequences corresponded to the as yet uncultivated groups SAR11, within the alpha-Proteobacteria, and SAR324, within the delta-Proteobacteria, as well as to the gamma-Proteobacteria, Cytophagales, Planctomyces, Gram-positives, and the group of environmental sequences SAR406 . Among them, gamma-proteobacterial sequences were the most abundant and diverse . Within Archaea, and using six different primer sets for 16S rDNA amplification, only euryarchaeotal sequences were retrieved . Most of them clustered with the Thermoplasma-related marine groups II and III, but some corresponded to a recently described group of marine sequences emerging at the base of haloarchaea . Our data suggest that gamma-Proteobacteria and Euryarchaeota may be dominant elements in terms of genetic diversity of the two prokaryotic domains in this deep-sea pelagic area. Mikrobiologiia, 2001 May-Jun, 70(3), 412 - 20 {Micromycetes and actinobacteria under conditions of many years of natural cryopreservation}; Kochkina GA et al.; Almost all of the investigated samples of the Arctic and Antarctic permafrost sediments of different genesis with ages from 5-10 thousand to 2-3 million years were found to contain viable micromycete and bacterial cells . The maximum amounts of viable cells of fungi (up to 10(4) CFU/g air-dried sample) and bacteria (up to 10(7)-10(9) CFU/g air-dried sample) were present in fine peaty sediment samples taken from different depths . The identified micromycetes belonged to more than 20 genera of the divisions Basidiomycota, Ascomycota, and Zygomycota, and some represented mitosporic fungi . Thawing the samples at 35 and 52 degrees C allowed the number of detected fungal genera to be increased by more than 30% . Aerobic heterotrophic prokaryotes were dominated by coryneform, nocardioform, and spore-forming microorganisms of the order Actinomycetales . Analysis of the isolated fungi and actinomycetes showed that most of them originated from the microbial communities of ancient terrestrial biocenoses. Mikrobiologiia, 2001 May-Jun, 70(3), 374 - 83 {Features of microorganism distribution in the Al-Fe-humus podzol of the northern-taiga spruce forests: natural and technogenic aspects}; Nikonov VV et al.; Natural and anthropogenically induced seasonal variations in the abundance and biomass of various groups of microorganisms in the Al-Fe-humus podzols of boreal spruce forests were analyzed . The fungal biomass in these soils was found to be considerably higher than the bacterial biomass . Microbial population was mainly concentrated in a thin surface layer (10-15 cm in thickness), which included the forest litter and the upper mineral root-inhabited soil horizon and greatly differed from other soil horizons in morphology and other properties . This layer was found to be optimal with respect to hydrothermal and nutritional conditions and was characterized by profound seasonal variations in the abundance and biomass of microbiota . The high acidity, typical of the Al-Fe-humus podzols, resulted from the metabolism of their microbial communities . In the polluted podzols, the population of prokaryotes increased and that of eukaryotes decreased. Adv Microb Physiol, 2001, 45, 157 - 98 The superfamily of chemotaxis transducers: from physiology to genomics and back; Zhulin IB; Chemotaxis transducers are specialized receptors that microorganisms use in order to sense the environment in directing their motility to favorable niches . The Escherichia coli transducers are models for studying the sensory and signaling events at the molecular level . Extensive studies in other organisms and the arrival of genomics has resulted in the accumulation of sequences of many transducer genes, but they are not fully understood . In silico analysis provides some assistance in classification of various transducers from different species and in predicting their function . All transducers contain two structural modules: a conserved C-terminal multidomain module, which is a signature element of the transducer superfamily, and a variable N-terminal module, which is responsible for the diversity within the superfamily . These structural modules have two distinct functions: the conserved C-terminal module is involved in signaling and adaptation, and the N-terminal module is involved in sensing various stimuli . Both C-terminal and N-terminal modules appear to be mobile genetic elements and subjects of duplication and lateral transfer . Although chemotaxis transducers are found exclusively in prokaryotic organisms that have some type of motility (flagellar, gliding or pili-based), several types of domains that are found in their N-terminal modules are also present in signal transduction proteins from eukaryotes, including humans . This indicates that basic principles of sensory transduction are conserved throughout the phylogenetic tree and that the chemotaxis transducer superfamily is a valuable source of novel sensory elements yet to be discovered. Antisense Nucleic Acid Drug Dev, 2001 Jun, 11(3), 181 - 8 From bugs to drugs: therapeutic immunomodulation with oligodeoxynucleotides containing CpG sequences from bacterial DNA; Krieg AM; Several types of immune cells possess pattern recognition receptors (PRR) that can distinguish prokaryotic DNA from vertebrate DNA by detecting unmethylated CpG dinucleotides in particular base contexts (CpG motifs) . Bacterial DNA or synthetic oligodeoxynucleotides containing these CpG motifs activate both innate and acquired immune responses that have evolved to protect against intracellular infections . These T helper 1 (Th1)-like immune responses include activation of B cells, dendritic cells, macrophages, and natural killer (NK) cells . CpG DNA-induced immune activation can protect against infection either alone or in combination with a vaccine and is effective in the immunotherapy of allergic diseases and cancer . Human clinical trials using such CpG DNA are currently underway. Genes Dev, 2001 Jul 1, 15(13), 1637 - 51 Identification of novel small RNAs using comparative genomics and microarrays; Wassarman KM et al.; A burgeoning list of small RNAs with a variety of regulatory functions has been identified in both prokaryotic and eukaryotic cells . However, it remains difficult to identify small RNAs by sequence inspection . We used the high conservation of small RNAs among closely related bacterial species, as well as analysis of transcripts detected by high-density oligonucleotide probe arrays, to predict the presence of novel small RNA genes in the intergenic regions of the Escherichia coli genome . The existence of 23 distinct new RNA species was confirmed by Northern analysis . Of these, six are predicted to encode short ORFs, whereas 17 are likely to be novel functional small RNAs . We discovered that many of these small RNAs interact with the RNA-binding protein Hfq, pointing to a global role of the Hfq protein in facilitating small RNA function . The approaches used here should allow identification of small RNAs in other organisms. FEBS Lett, 2001 Jul 6, 500(3), 153 - 6 Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition; Colnaghi R et al.; The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family . E . coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238) . In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese) . The biochemical characterization of E . coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate . Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding . On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate. Mol Biol (Mosk), 2001 May-Jun, 35(3), 492 - 9 {Comparative study of the morphology and antigenic properties of recombinant analogs of a Marburg virus nucleoprotein}; Kachko AV et al.; The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the promoter for T7 RNA polymerase . Recombinant NP was synthesized in Escherichia coli and in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase . On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E . coli and of a single type in cell line 293 . ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP. News Physiol Sci, 2001 Jun, 16, 123 - 6 A nonconventional role of molecular chaperones: involvement in the cytoarchitecture; Csermely P; A hallmark of chaperone action is assistance in protein folding . Indeed, folding of nascent prokaryotic proteins proceeds mostly as a chaperone-assisted, posttranslational event . On the contrary, in nonstressed eukaryotic cells folding-related tasks of eukaryotic chaperones are restricted to a subset of proteins, and "jobless" chaperones may form an extension of the cytoarchitecture, facilitating intracellular traffic of proteins and other macromolecules. J Bacteriol, 2001 Aug, 183(15), 4413 - 20 Molecular characterization of Desulfovibrio gigas neelaredoxin, a protein involved in oxygen detoxification in anaerobes; Silva G et al.; Desulfovibrio gigas neelaredoxin is an iron-containing protein of 15 kDa, having a single iron site with a His(4)Cys coordination . Neelaredoxins and homologous proteins are widespread in anaerobic prokaryotes and have superoxide-scavenging activity . To further understand its role in anaerobes, its genomic organization and expression in D . gigas were studied and its ability to complement Escherichia coli superoxide dismutase deletion mutant was assessed . In D . gigas, neelaredoxin is transcribed as a monocistronic mRNA of 500 bases as revealed by Northern analysis . Putative promoter elements resembling sigma(70) recognition sequences were identified . Neelaredoxin is abundantly and constitutively expressed, and its expression is not further induced during treatment with O(2) or H(2)O(2) . The neelaredoxin gene was cloned by PCR and expressed in E . coli, and the protein was purified to homogeneity . The recombinant neelaredoxin has spectroscopic properties identical to those observed for the native one . Mutations of Cys-115, one of the iron ligands, show that this ligand is essential for the activity of neelaredoxin . In an attempt to elucidate the function of neelaredoxin within the cell, it was expressed in an E . coli mutant deficient in cytoplasmic superoxide dismutases (sodA sodB) . Neelaredoxin suppresses the deleterious effects produced by superoxide, indicating that it is involved in oxygen detoxification in the anaerobe D . gigas. Int J Obes Relat Metab Disord, 2001 Jun, 25(6), 770 - 6 A new gene related to human obesity identified by suppression subtractive hybridization; Larose M et al.; OBJECTIVE: The aim of the research was to identify genes specially expressed in the obese state and potentially involved in the pathogenesis of obesity . DESIGN AND SUBJECTS: We used the technique of suppression subtractive hybridization (SSH), which combines subtractive hybridization with PCR, to generate a population of PCR fragments enriched for transcripts of high or low abundance from differentially expressed genes . PolyA+ mRNA was isolated from subcutaneous abdominal adipose tissue of five massively obese (>35 kg/m(2)) and five normal-weight (<25 kg/m(2)) women . cDNA generated from RNA pooled from the obese subjects was contrasted by SSH with an excess of pooled cDNA from the normal-weight women . RESULTS: Seventy-nine clones were obtained among which one showed by RT-PCR a higher expression in obese than in normal-weight subjects . This gene was shown to be predominantly expressed in adipose tissue in contrast to brain, liver, kidney, heart and skeletal muscle, and was called "Adipogene" . No expression was detected in lung, pancreas and placenta . The cDNA was 1.5 kb long with an open reading frame of 1004 nucleotides encoding a protein of 334 amino acids (37 kDa) . No significant sequence similarity was found in databanks, except for weak amino acid homologies with prokaryotic AraC/XylS transcriptional regulator family . Adipogene is encoded on chromosome 8, less than 1 centiMorgan (cM) from the beta3 adrenergic receptor (ADRB3) locus . Weak linkages were observed with body mass index (BMI) and three microsatellite markers located within 10 cM of Adipogene, whereas no linkage was observed with Trp64Arg ADRB3 polymorphism using the Quebec Family Study database . CONCLUSION: Using the SSH technique, we have identified a new gene, called Adipogene, which is overexpressed in the adipose tissue of the obese individuals and could be involved in obesity. J Mol Biol, 2001 Jul 13, 310(3), 617 - 34 Crystal structures of YBHB and YBCL from Escherichia coli, two bacterial homologues to a Raf kinase inhibitor protein; Serre L et al.; In rat and human cells, RKIP (previously known as PEBP) was characterized as an inhibitor of the MEK phosphorylation by Raf-1 . In Escherichia coli, the genes ybhb and ybcl possibly encode two RKIP homologues while in the genomes of other bacteria and archaebacteria other homologous genes of RKIP have been found . The parallel between the cellular signaling mechanisms in eukaryotes and prokaryotes suggests that these bacterial proteins could be involved in the regulation of protein phosphorylation by kinases as well . We first showed that the proteins YBHB and YBCL were present in the cytoplasm and periplasm of E . coli, respectively, after which we determined their crystallographic structures . These structures verify that YBHB and YBCL belong to the same structural family as mammalian RKIP/PEBP proteins . The general fold and the anion binding site of these proteins are extremely well conserved between mammals and bacteria and suggest functional similarities . However, the bacterial proteins also exhibit some specific structural features, like a substrate binding pocket formed by the dimerization interface and the absence of cis peptide bonds . This structural variety should correspond to the recognition of multiple cellular partners . J Mol Biol, 2001 Jul 13, 310(3), 563 - 75 Analysis of the NF-kappaB p50 dimer interface by diversity screening; Hart DJ et al.; An in vivo screen has been devised for NF-kappaB p50 activity in Escherichia coli exploiting the ability of the mammalian transcription factor to emulate a prokaryotic repressor . Active intracellular p50 was shown to repress the expression of a green fluorescent protein reporter gene allowing for visual screening of colonies expressing active p50 on agar plates . A library of mutants was constructed in which the residues Y267, L269, A308 and V310 of the dimer interface were simultaneously randomised and twenty-five novel functional interfaces were selected which repressed the reporter gene to similar levels as the wild-type protein . The leucine-269 alanine-308 core was repeatedly, but not exclusively, selected from the library whilst a diversity of predominantly non-polar residues were selected at positions 267 and 310 . These results indicate that L269 and A308 may form a hot spot of interaction and allow an insight into the processes of dimer selectivity and evolution within this family of transcription factors . Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8513 - 8 Epub 2001 Jul 03. Biosynthesis of the thiazole moiety of thiamin in Escherichia coli: identification of an acyldisulfide-linked protein--protein conjugate that is functionally analogous to the ubiquitin/E1 complex; Xi J et al.; A covalently linked protein--protein conjugate between ThiF and ThiS thiocarboxylate was found in a partially purified coexpressed ThiF/ThiS protein mixture by using Fourier transform mass spectrometry . The Cys-184 of ThiF and the C terminus of ThiS thiocarboxylate were identified to be involved in the formation of this complex by using both mutagenesis and chemical modification methods . A complementation study of Escherichia coli thiF(-) using thiF(C184S) suggests that this conjugate is an essential intermediate involved in the biosynthesis of the thiazole moiety of thiamin . This ThiF/ThiS conjugate is the first characterized example of a unique acyldisulfide intermediate in a biosynthetic system . This protein conjugate is also an example of an ubiquitin-E1 like protein-protein conjugate in prokaryotes and supports a strong evolutionary link between thiamin biosynthesis and the ubiquitin conjugating system. Virology, 2001 Jul 5, 285(2), 270 - 7 A plasmid of phytoplasma encodes a unique replication protein having both plasmid- and virus-like domains: clue to viral ancestry or result of virus/plasmid recombination? Oshima K, Kakizawa S, Nishigawa H, Kuboyama T, Miyata S, Ugaki M, Namba S. The genomes of most prokaryotic and eukaryotic single-stranded (ss) DNA viruses, and some prokaryotic plasmids such as pLS1, commonly replicate via a rolling circle replication (RCR) strategy, and thus the viruses are hypothesized to have evolved from the plasmids, although evidence for this view is sparse . We have sequenced a circular plasmid of 3933 nt, pOYW, obtained from onion yellows phytoplasma (OY-W), a cell-wall-less, unculturable prokaryote that inhabits the cytoplasm of both plant and insect cells . pOYW contains five open reading frames (ORFs) on the same strand and apparently replicates by an RCR mechanism . Its rep gene (ORF5) encodes a unique protein, pOYW-Rep, with an unprecedented structure . The N-terminal region of pOYW-Rep has similarities to the RCR initiator protein (Rep) of pLS1 family plasmids but, unlike the Rep of other plasmids, its C-terminal region was unexpectedly similar to the helicase domain of the replication-associated proteins (Rap) of eukaryotic viruses, especially circoviruses (ssDNA viruses of vertebrates) . The pOYW-Rep was specifically detected in OY-W-infected plant phloem cells, suggesting that it is a functional protein . We suggest that an ancestral phytoplasma plasmid pOYW may have acquired a helicase domain from host phytoplasmal DNA, entered the surrounding eukaryotic cytoplasm, and subsequently evolved into an ancestral eukaryotic ssDNA virus . Alternatively, a pOYW ancestor could have obtained the helicase domain by recombination with a virus: this would be the first example of recombination between plasmids and viruses . Int Arch Allergy Immunol, 2001 Jun, 125(2), 120 - 7 Identification of a Hevea brasiliensis latex manganese superoxide dismutase (Hev b 10) as a cross-reactive allergen; Wagner S et al.; BACKGROUND: Cross-reactive allergens play an increasingly important role in latex allergy in complicating both the diagnosis and time course of allergic symptoms . Manganese superoxide dismutase (MnSOD), a ubiquitous protein of prokaryotic and eukaryotic organisms, was described as a cross-reactive allergen in Aspergillus fumigatus . Little information is available on the importance of this pan-allergen in Hevea brasiliensis latex . The aim of this study was to clone and express MnSOD from H . brasiliensis latex, and to obtain the soluble and immunologically active recombinant allergen for diagnosis of latex allergy and to investigate possible cross-reactivities with the structurally related A . fumigatus and human MnSODs . METHODS: A complementary DNA coding for Hevea latex MnSOD was amplified by PCR . The recombinant protein was produced in Escherichia coli with an N-terminal hexahistidyl tag . Enzymatic activity of the recombinant protein was determined using an enzyme assay for SODs . IgE immunoblotting and IgE inhibition assays were performed to characterize the recombinant allergen and its cross-reactivity . RESULTS: A Hevea latex MnSOD consisting of 206 amino acid residues was cloned and expressed in E . coli . The allergen was designated Hev b 10 . The recombinant protein was enzymatically active, indicating the correct folding of the protein . In immunoblots, latex- as well as A . fumigatus-allergic patients revealed IgE binding to recombinant (r)Hev b 10 . Cross-reactivity to Asp f 6, the MnSOD from A . fumigatus, and human MnSOD was determined by inhibition of IgE binding to these MnSODs by rHev b 10 . CONCLUSIONS: Hev b 10 is a new cross-reactive allergen of H . brasiliensis which belongs to the 'latex-mold' group of latex allergens . Furthermore, it is a candidate for primary sensitization in patients allergic to the pan-allergen MnSOD . Comp Biochem Physiol B Biochem Mol Biol, 2001 Jul, 129(4), 711 - 23 Identifying 'prime suspects': symbioses and the evolution of multicellularity; McFall-Ngai MJ; The possible involvement of symbioses in the evolution of multicellularity is explored . Evidence is drawn principally from the biology of present day associations of plants and animals with prokaryotes . A particular emphasis is placed on future research opportunities in this area of biology that have been provided by the advent of specific molecular techniques and new model systems . With the application of new approaches that result from these advances, a more holistic understanding of the biology of the coevolved communities, composed of animals or plants and their associated prokaryotes, is within the reach of biologists over the next few decades. Int J Hyg Environ Health, 2001 May, 203(4), 327 - 34 Biochemical and pharmacological investigations of selected cyanobacteria; Mundt S et al.; Cyanobacteria are a very old group of prokaryotic organisms that produce a variety of secondary metabolites with antibiotic, algicide, cytotoxic, immunosuppressive and enzyme inhibiting activities . In the last decades structures of pure compounds have been determined as phenols, peptides, alkaloids or terpenoids (Falch, 1996) . Screening of lipophilic and hydrophilic extracts from cultured cyanobacteria or waterbloom material, isolated from German lakes and the Baltic sea for antiviral, antibiotic, immunomodulating and enzyme inhibiting activity in different in vitro systems revealed strains with interesting effects . These strains were cultivated in 45 litre photobioreactors to produce enough biomass for bioassay-guided isolation of the active substances . First results characterising active substances are reported. Orig Life Evol Biosph, 2001 Jun, 31(3), 257 - 69 On the relative content of G,C bases in codons of amino acids corresponding to class I and II aminoacyl-tRNA synthetases; Cavalcanti AR et al.; We have analyzed the relative G,C content from protein coding regions of 530 organisms and found that the ratio of the G,C content of the codons of the amino acids corresponding to Class II and Class I aminoacyl-tRNA synthetases decreases in a statistically significant way from prokaryotes to animals . This can be interpreted assuming that an initial asymmetry between the G,C content of codons of Class I and II amino acids existed and has decreased in the course of evolution. Nucleic Acids Res, 2001 Jul 1, 29(13), 2822 - 8 Developmentally-regulated packaging of mitochondrial DNA by the HMG-box protein mtTFA during Xenopus oogenesis; Shen EL et al.; Mature Xenopus oocytes are highly enriched for mitochondria . The organelles are stored and partitioned to newly-arising cells during embryogenesis, when there is little mitochondrial DNA (mtDNA) replication or transcription . A previously described member of the high mobility group (HMG) family of proteins, mtTFA, has been suggested to play a role in control of mtDNA copy number . mtTFA serves as a mitochondrial transcription factor in humans and Xenopus and as an abundant mtDNA packaging protein in yeast, like its prokaryotic histone-like counterpart, HU protein . Northern blot analysis demonstrated that expression of the gene was regulated during Xenopus oogenesis and specifically peaked at stage II . Western and Southern blotting were used to quantify amounts of the protein and mtDNA, respectively, in each stage of oogenesis . mtTFA:mtDNA ratios were found to be relatively low in previtellogenic oocytes while the ratios increased markedly in mature oocytes. J Biochem (Tokyo), 2001 Jul, 130(1), 57 - 61 Detection of the protein-protein interaction between cyclic AMP receptor protein and RNA polymerase, by (13)C-carbonyl NMR; Lee TW et al.; Cyclic AMP receptor protein (CRP) plays a key role in the transcription regulation of many prokaryotic genes . Upon the binding of cyclic AMP, CRP is allosterically activated, binds to target DNA sites, and interacts with RNA polymerase . Although the protein-protein interaction between CRP and RNA polymerase is known to be important for the transcription initiation of the target genes, its structural understanding is still lacking, particularly due to the high molecular mass (approximately 120 kDa) of the protein complex . We assigned all of the (13)C-carbonyl resonances of methionine residues in CRP by using the double labeling and the enzyme digestion techniques . The result of (13)C-carbonyl NMR experiment on {(13)C'-Met}-CRP in the presence of both cyclic AMP and RNA polymerase alpha subunit showed that the two proteins interact with each other in solution in the absence of DNA via the region around the residues from Met 157 to Met 163 in CRP . The results also showed the effectiveness of the selective labeling and (13)C-carbonyl NMR spectroscopy in the specific detection of the protein-protein interaction between large molecules. Eur J Biochem, 2001 Jul, 268(13), 3807 - 15 The variant tuf3 gene of Streptomyces coelicolor A3(2) encodes a real elongation factor Tu, as shown in a novel Streptomyces in vitro translation system; Olsthoorn-Tieleman LN et al.; In Streptomyces coelicolor, the regular and abundant elongation factor (EF)-Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known . Expression of the latter could so far only be detected on the transcriptional level under stress conditions . In this paper we demonstrate the presence of low levels of EF-Tu3 in strains of the J1501 lineage . Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome-bound ppGpp synthetase, respectively . To assess the putative translational capacities of EF-Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni2+ affinity adsorption of chromosomally encoded (His)6-tagged EF-Tu1 from a S . coelicolor cell-free extract . Translational activity of this system is totally dependent on the addition of purified EF-Tu species or on the presence of an additional elongation factor Tu in the extract, e.g . encoded by a plasmid-borne tuf gene . Using this EF-Tu-dependent translation system, we have established that S . coelicolor EF-Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF-Tu sequence at positions involved in either aminoacyl-tRNA binding or interaction with the guanine-nucleotide exchange factor EF-Ts. Eur J Biochem, 2001 Jul, 268(13), 3620 - 39 The drug/metabolite transporter superfamily; Jack DL et al.; Previous work defined several families of secondary active transporters, including the prokaryotic small multidrug resistance (SMR) and rhamnose transporter (RhaT) families as well as the eukaryotic organellar triose phosphate transporter (TPT) and nucleotide-sugar transporter (NST) families . We show that these families as well as several other previously unrecognized families of established or putative secondary active transporters comprise a large ubiquitous superfamily found in bacteria, archaea and eukaryotes . We have designated it the drug/metabolite transporter (DMT) superfamily (transporter classification number 2.A.7) and have shown that it consists of 14 phylogenetic families, five of which include no functionally well-characterized members . The largest family in the DMT superfamily, the drug/metabolite exporter (DME) family, consists of over 100 sequenced members, several of which have been implicated in metabolite export . Each DMT family consists of proteins with a distinctive topology: four, five, nine or 10 putative transmembrane alpha helical spanners (TMSs) per polypeptide chain . The five TMS proteins include an N-terminal TMS lacking the four TMS proteins . The full-length proteins of 10 putative TMSs apparently arose by intragenic duplication of an element encoding a primordial five-TMS polypeptide . Sequenced members of the 14 families are tabulated and phylogenetic trees for all the families are presented . Sequence and topological analyses allow structural and functional predictions. FEMS Microbiol Lett, 2001 May 1, 198(2), 99 - 103 Effects of lipids on n-alkane attenuation in media supporting oil-utilizing microorganisms from the oily Arabian Gulf coasts; Radwan SS et al.; The Arabian Gulf is one of the most extensively oil-polluted areas of the world . The major objectives of this work were to study whether hydrocarbon-utilizing microorganisms indigenous to that area would readily accumulate added lipids, and whether this might affect their hydrocarbon consumption potential . Two prokaryotes, Arthrobacter nicotianae KCC B35 and the unidentified organisms KCC B6, as well as one eukaryote, Candida parapsilosis KCC Y1, were selected for this study . Biomass samples of the test organisms were incubated in an inorganic medium containing various concentrations of cholesterol, stearic acid, triolein or egg-phospholipids . The results revealed that all lipid classes were readily accumulated by the three test organisms . In addition, biomass samples were incubated for 6 h in an inorganic medium containing mixtures of individual lipid classes and either n-octadecane or n-docosane . The cells were removed and the residual alkanes in the medium were quantitatively recovered and analyzed by GLC . The results showed that out of the tested lipid classes only stearic acid exhibited a common stimulatory effect on the consumption of both n-alkanes by all test organisms . Other lipid classes were either inhibitory or had less pronounced effects than stearic acid. Proc R Soc Lond B Biol Sci, 2001 Jul 7, 268(1474), 1417 - 22 The unique features of starch metabolism in red algae; Viola R et al.; Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules outside of their plastids . The starch granules from red algae (floridean starch) show structural similarities with higher plant starch granules but lack amylose . Recent studies have indicated that the extra-plastidic starch synthesis in red algae proceeds via a UDP glucose-selective alpha-glucan synthase, in analogy with the cytosolic pathway of glycogen synthesis in other eukaryotes . On the other hand, plastidic starch synthesis in green cells occurs selectively via ADP glucose in analogy with the pathway of glycogen synthesis in prokaryotes from which plastids have evolved . Given the emerging consensus of a monophyletic origin of plastids, it would appear that the capacity for starch synthesis selectively evolved from the alpha-glucan synthesizing machinery of the host ancestor and its endosymbiont in red algae and green algae, respectively . This implies the evolution of fundamentally different functional relationships between the different subcellular compartments with regard to photosynthetic carbon metabolism in these organisms . It is suggested that the biochemical and molecular elucidation of floridean starch synthesis may offer new insights into the metabolic strategies of photosynthetic eukaryotes. Semin Cell Dev Biol, 2001 Jun, 12(3), 239 - 45 Towards the molecular mechanism of prokaryotic and eukaryotic multidrug transporters; van Veen HW; Due to their ability to extrude structurally dissimilar cytotoxic drugs out of the cell, multidrug transporters are able to reduce the cytoplasmic drug concentration, and, hence, are able to confer drug resistance on human cancer cells and pathogenic microorganisms . This review will focus on the molecular properties of two bacterial multidrug transporters, the ATP-binding cassette transporter LmrA and the proton motive force-dependent major facilitator superfamily transporter LmrP, which each represent a major class of multidrug transport proteins encountered in pro- and eukaryotic cells . In spite of the structural differences between LmrA and LmrP, the molecular bases of their drug transport activity may turn out to be more similar than might currently appear . J Mol Biol, 2001 Jul 6, 310(2), 419 - 31 The crystal structure of Escherichia coli MoeA, a protein from the molybdopterin synthesis pathway; Schrag JD et al.; MoeA is involved in synthesis of the molybdopterin cofactor, although its function is not yet clearly defined . The three-dimensional structure of the Escherichia coli protein was solved at 2.2 A resolution . The locations of highly conserved residues among the prokaryotic and eukaryotic MoeA homologs identifies a cleft in the dimer interface as the likely functional site . Of the four domains of MoeA, domain 2 displays a novel fold and domains 1 and 4 each have only one known structural homolog . Domain 3, in contrast, is structurally similar to many other proteins . The protein that resembles domain 3 most closely is MogA, another protein required for molybdopterin cofactor synthesis . The overall similarity between MoeA and MogA, and the similarities in a constellation of residues that are strongly conserved in MoeA, suggests that these proteins bind similar ligands or substrates and may have similar functions . J Mol Evol, 2001 Mar, 52(3), 275 - 80 Synonymous codon usage, accuracy of translation, and gene length in Caenorhabditis elegans; Marais G et al.; In many unicellular organisms, invertebrates, and plants, synonymous codon usage biases result from a coadaptation between codon usage and tRNAs abundance to optimize the efficiency of protein synthesis . However, it remains unclear whether natural selection acts at the level of the speed or the accuracy of mRNAs translation . Here we show that codon usage can improve the fidelity of protein synthesis in multicellular species . As predicted by the model of selection for translational accuracy, we find that the frequency of codons optimal for translation is significantly higher at codons encoding for conserved amino acids than at codons encoding for nonconserved amino acids in 548 genes compared between Caenorhabditis elegans and Homo sapiens . Although this model predicts that codon bias correlates positively with gene length, a negative correlation between codon bias and gene length has been observed in eukaryotes . This suggests that selection for fidelity of protein synthesis is not the main factor responsible for codon biases . The relationship between codon bias and gene length remains unexplained . Exploring the differences in gene expression process in eukaryotes and prokaryotes should provide new insights to understand this key question of codon usage. Nat Struct Biol, 2001 Jul, 8(7), 641 - 8 Structure of 4-diphosphocytidyl-2-C- methylerythritol synthetase involved in mevalonate- independent isoprenoid biosynthesis; Richard SB et al.; The YgbP protein of Escherichia coli encodes the enzyme 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) synthetase, a member of the cytidyltransferase family of enzymes . CDP-ME is an intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis in a number of prokaryotic organisms, algae, the plant plastids and the malaria parasite . Because vertebrates synthesize isoprenoid precursors using a mevalonate pathway, CDP-ME synthetase and other enzymes of the mevalonate-independent pathway for isoprenoid production represent attractive targets for the structure-based design of selective antibacterial, herbicidal and antimalarial drugs . The high-resolution structures of E . coli CDP-ME synthetase in the apo form and complexed with both CTP-Mg2+ and CDP-ME-Mg2+ reveal the stereochemical principles underlying both substrate and product recognition as well as catalysis in CDP-ME synthetase . Moreover, these complexes represent the first experimental structures for any cytidyltransferase with both substrates and products bound. Proc Natl Acad Sci U S A, 2001 Jul 3, 98(14), 7928 - 33 Epub 2001 Jun 26. Genetic fidelity under harsh conditions: analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius; Grogan DW et al.; Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046) . However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions . In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes . Both of these conditions are met by the extreme thermoacidophile Sulfolobus acidocaldarius . We determined the genomic mutation rate for this species when growing at pH 3.5 and 75 degrees C based on the rate of forward mutation at the pyrE gene and the nucleotide changes identified in 101 independent mutants . The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA . The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the S . acidocaldarius spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs . The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of Sulfolobus spp . It suggests, however, that molecular evolution per genome replication may proceed more slowly in S . acidocaldarius than in other DNA-based organisms examined to date. Mutat Res, 2001 Jul 12, 486(2), 125 - 36 Spontaneous germline amplification and translocation of a transgene array; Kearns M et al.; The majority of the mammalian genome is thought to be relatively stable throughout and between generations . There are no developmentally programmed gene amplifications as seen in lower eukaryotes and prokaryotes, however a number of unscheduled gene amplifications have been documented . Apart from expansion of trinucleotide repeats and minisatellite DNA, which involve small DNA elements, other cases of gene or DNA amplifications in mammalian systems have been reported in tumor samples or permanent cell lines . The mechanisms underlying these amplifications remain unknown . Here, we report a spontaneous transgene amplification through the male germline which resulted in silencing of transgene expression . During routine screening one mouse, phenotypically negative for transgene expression, was found to have a transgene copy number much greater than that of the transgenic parent . Analysis of the transgene expansion revealed that the amplification in the new high copy transgenic line resulted in a copy number approximately 40-60 times the primary transgenic line copy number of 5-8 copies per haploid genome . Genetic breeding analysis suggested that this amplification was the result of insertion at only one integration site, that it was stable for at least two generations and that the site of insertion was different from the site at which the original 5-8 copy array had integrated . FISH analysis revealed that the new high copy array was on chromosome 7 F3/4 whereas the original low copy transgene array had been localised to chromosome 3E3 . DNA methylation analysis revealed that the high copy transgene array was heavily methylated . The amplification of transgenes, although a rare event, may give insight into amplification of endogenous genes which can be associated with human disease. J Biol Chem, 2001 Sep 7, 276(36), 33319 - 27 Epub 2001 Jun 22. MAP kinase phosphatase-1 gene transcription in rat neuroendocrine cells is modulated by a calcium-sensitive block to elongation in the first exon; Ryser S et al.; Transcriptional elongation of many eukaryotic, prokaryotic, and viral genes is tightly controlled, which contributes to gene regulation . Here we describe this phenomenon for the MAP kinase phosphatase 1 (MKP-1) immediate early gene . In rat GH4C1 pituitary cells, MKP-1 mRNA is rapidly and transiently induced by the thyrotropin-releasing hormone (TRH) and the epidermal growth factor EGF via transcriptional activation of the gene . Ca(2+) signals are necessary for the induction of MKP-1 in response to TRH but not to EGF . Reporter gene analysis with the newly cloned rat promoter sequence shows only limited induction in response to various stimuli, including TRH or EGF . By nuclear run-on assays we demonstrate that in basal conditions, a strong block to elongation in the first exon regulates the MKP-1 gene and that stimulation with either TRH or EGF overcomes the block . Ca(2+) signals are important to release the MKP-1 elongation block in a manner similar to the c-fos oncogene . These results suggest that a common mechanism of intragenic regulation may be conserved between MKP-1 and c-fos in mammalian cells. J Biol Chem, 2001 Sep 7, 276(36), 33952 - 63 Epub 2001 Jun 21. A non-Golgi alpha 1,2-fucosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium; van Der Wel H et al.; Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation . In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core . To address how the third sugar, fucose alpha1,2-linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDP-Fuc:Skp1 alpha 1,2-fucosyltransferase . Tryptic-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry . Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker polymerase chain reaction method to yield an intronless open reading frame of 768 amino acids . Disruption of the FT85 gene by homologous recombination resulted in viable cells, which had altered light scattering properties as revealed by flow cytometry . FT85 was necessary and sufficient for Skp1 fucosylation, based on biochemical analysis of FT85 mutant cells and Escherichia coli that express FT85 recombinantly . FT85 lacks sequence motifs that characterize all other known alpha 1,2-fucosyltransferases and lacks the signal-anchor sequence that targets them to the secretory pathway . The C-terminal region of FT85 harbors motifs found in inverting Family 2 glycosyltransferase domains, and its expression in FT85 mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate . Whereas most prokaryote and eukaryote Family 2 glycosyltransferases are membrane-bound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1-fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus. Genome Biol . 2001;2(6):RESEARCH0020 . Epub 2001 Jun 01. Evolution of gene order conservation in prokaryotes; Tamames J; BACKGROUND: As more complete genomes are sequenced, conservation of gene order between different organisms is emerging as an informative property of the genomes . Conservation of gene order has been used for predicting function and functional interactions of proteins, as well as for studying the evolutionary relationships between genomes . The reasons for the maintenance of gene order are still not well understood, as the organization of the prokaryote genome into operons and lateral gene transfer cannot possibly account for all the instances of conservation found . Comprehensive studies of gene order are one way of elucidating the nature of these maintaining forces . RESULTS: Gene order is extensively conserved between closely related species, but rapidly becomes less conserved among more distantly related organisms, probably in a cooperative fashion . This trend could be universal in prokaryotic genomes, as archaeal genomes are likely to behave similarly to bacterial genomes . Gene order conservation could therefore be used as a valid phylogenetic measure to study relationships between species . Even between very distant species, remnants of gene order conservation exist in the form of highly conserved clusters of genes . This suggests the existence of selective processes that maintain the organization of these regions . Because the clusters often span more than one operon, common regulation probably cannot be invoked as the cause of the maintenance of gene order . CONCLUSIONS: Gene order conservation is a genomic measure that can be useful for studying relationships between prokaryotes and the evolutionary forces shaping their genomes . Gene organization is extensively conserved in some genomic regions, and further studies are needed to elucidate the reason for this conservation. Oncogene, 2001 May 28, 20(24), 3039 - 46 Glucocorticoid receptor-mediated chromatin remodeling in vivo; Deroo BJ et al.; The compaction of DNA into chromatin provides an additional level of gene regulation in eukaryotes that may not be available to prokaryotes . When packaged as chromatin, most promoters are transcriptionally repressed, and transcription factors have reduced access to their binding sites . The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates the activity of genes involved in many physiological processes . To regulate eukaryotic genes, the GR binds to target sites within promoter regions of genes assembled as chromatin . This interaction alters the nucleosomal architecture to allow binding of other transcription factors, and formation of the preinitiation complex . The mouse mammary tumor virus (MMTV) promoter has been used extensively as a model to explore the processes by which the GR remodels chromatin and activates transcription . Significant progress has been made in our understanding of the mechanisms used by the GR to modify chromatin structure, and the limits placed on the GR by post-translational modifications of histones . We will describe recent developments in the processes used by the GR to activate transcription in vivo via chromatin remodeling complexes, histone H1 phosphorylation, and recruitment of diverse coactivators. Avian Dis, 2001 Apr-Jun, 45(2), 389 - 99 The in vivo and in vitro effects of chicken interferon alpha on infectious bursal disease virus and Newcastle disease virus infection; Mo CW et al.; The in vitro and in vivo effects of chicken interferon alpha on infectious bursal disease virus (IBDV) infection were investigated in this study . A cDNA of interferon alpha was first cloned from a Chinese strain chicken Shiqi by reverse transcription-polymerase chain reaction . The deduced amino acid sequence has one amino acid substitution with chicken interferon alpha 1 at residue 65 (N to S) and two amino acid substitutions with chicken interferon alpha 2 at residues 50 (N to S) and 58 (P to L), respectively . A prokaryotic expression system was employed to produce a large quantity of recombinant protein . Recombinant interferon was purified in a one-step process, and an optimal refolding process was devised . About 51% recombinant protein from inclusion bodies was refolded, and the final yield of the recombinant interferon reached 24.66 mg/liter culture . The recombinant interferon suppressed IBDV plaque formation in a dose-dependent manner and ameliorated IBDV and Newcastle disease virus infection in both specific-pathogen-free (SPF) and commercial chickens . The antiviral effect of interferon alpha is more significant in commercial chickens than in SPF chickens, and the route of administration affects the efficacy of interferon therapy . This is the first reported study of the effects of interferon alpha on IBDV infection. Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7018 - 24 Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome; Keene JD; Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs) . The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure . The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome . The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs . Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins . Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons . mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them . This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions . Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms . Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo. J Gen Virol, 2001 Jul, 82(Pt 7), 1767 - 76 Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication; Huang J et al.; The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems . While less protein was obtained from the E . coli expression system, SpliNPV DNAPOL purified from E . coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system . Biochemical analyses suggested that the DNA polymerase and the 3'-5' exonuclease activities are intrinsic to the protein . Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme . Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive . Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment . Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay. J Virol, 2001 Jul, 75(14), 6392 - 401 In the simian virus 40 in vitro replication system, start site selection by the polymerase alpha-primase complex is not significantly altered by changes in the concentration of ribonucleotides; Purviance JD et al.; The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) alpha-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences . However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis . Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated . Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites . Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases . It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin . These studies provide additional evidence that the Pol alpha-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated. Prog Lipid Res, 2001 Jul, 40(4), 299 - 324 Do sterols reduce proton and sodium leaks through lipid bilayers? Haines TH. Proton and/or sodium electrochemical gradients are critical to energy handling at the plasma membranes of all living cells . Sodium gradients are used for animal plasma membranes, all other living organisms use proton gradients . These chemical and electrical gradients are either created by a cation pumping ATPase or are created by photons or redox, used to make ATP . It has been established that both hydrogen and sodium ions leak through lipid bilayers at approximately the same rate at the concentration they occur in living organisms . Although the gradients are achieved by pumping the cations out of the cell, the plasma membrane potential enhances the leakage rate of these cations into the cell because of the orientation of the potential . This review proposes that cells use certain lipids to inhibit cation leakage through the membrane bilayers . It assumes that Na(+) leaks through the bilayer by a defect mechanism . For Na(+) leakage in animal plasma membranes, the evidence suggests that cholesterol is a key inhibitor of Na(+) leakage . Here I put forth a novel mechanism for proton leakage through lipid bilayers . The mechanism assumes water forms protonated and deprotonated clusters in the lipid bilayer . The model suggests how two features of lipid structures may inhibit H(+) leakage . One feature is the fused ring structure of sterols, hopanoids and tetrahymenol which extrude water and therefore clusters from the bilayer . The second feature is lipid structures that crowd the center of the bilayer with hydrocarbon . This can be accomplished either by separating the two monolayers with hydrocarbons such as isoprenes or isopranes in the bilayer's cleavage plane or by branching the lipid chains in the center of the bilayers with hydrocarbon . The natural distribution of lipids that contain these features are examined . Data in the literature shows that plasma membranes exposed to extreme concentrations of cations are particularly rich in the lipids containing the predicted qualities . Prokaryote plasma membranes that reside in extreme acids (acidophiles) contain both hopanoids and iso/anteiso- terminal lipid branching . Plasma membranes that reside in extreme base (alkaliphiles) contain both squalene and iso/anteiso- lipids . The mole fraction of squalene in alkaliphile bilayers increases, as they are cultured at higher pH . In eukaryotes, cation leak inhibition is here attributed to sterols and certain isoprenes, dolichol for lysosomes and peroxysomes, ubiquinone for these in addition to mitochondrion, and plastoquinone for the chloroplast . Phytosterols differ from cholesterol because they contain methyl and ethyl branches on the side chain . The proposal provides a structure-function rationale for distinguishing the structures of the phytosterols as inhibitors of proton leaks from that of cholesterol which is proposed to inhibit leaks of Na(+) . The most extensively studied of sterols, cholesterol, occurs only in animal cells where there is a sodium gradient across the plasma membrane . In mammals, nearly 100 proteins participate in cholesterol's biosynthetic and degradation pathway, its regulatory mechanisms and cell-delivery system . Although a fat, cholesterol yields no energy on degradation . Experiments have shown that it reduces Na(+) and K(+) leakage through lipid bilayers to approximately one third of bilayers that lack the sterol . If sterols significantly inhibit cation leakage through the lipids of the plasma membrane, then the general role of all sterols is to save metabolic ATP energy, which is the penalty for cation leaks into the cytosol . The regulation of cholesterol's appearance in the plasma membrane and the evolution of sterols is discussed in light of this proposed role. Biochemistry, 2001 Jun 26, 40(25), 7491 - 7 Fast deuterium access to the buried magnesium/manganese site in cytochrome c oxidase; Florens L et al.; The recently determined crystal structures of bacterial and bovine cytochrome c oxidases show an area of organized water within the protein immediately above the active site where oxygen chemistry occurs . A pathway for exit of protons or water produced during turnover is suggested by possible connections of this aqueous region to the exterior surface . A non-redox-active Mg(2+) site is located in the interior of this region, and our previous studies {Florens, L., Hoganson, C., McCracken, J., Fetter, J., Mills, D., Babcock, G . T., and Ferguson-Miller, S . (1998) in Phototropic Prokaryotes (Peschek, G . A., Loeffelhard, W., and Schmetterer, G., Eds.) Kluwer Academic/Plenum, New York} have shown that the protons of water molecules that coordinate the metal can be exchanged within minutes of mixing with (2)H(2)O . Here we examine the extent and rate of deuterium exchange, using a combination of rapid freeze-quench and electron spin echo envelope modulation (ESEEM) analysis of Mn(2+)-substituted cytochrome c oxidase, which retains full activity . In the oxidized enzyme at room temperature, deuterium exchange at the Mn(2+) site occurs in less than 11 ms, which corresponds to an apparent rate constant higher than 3000 s(-1) . The extent of deuterium substitution is dependent on the concentration of (2)H(2)O in the sample, indicative of rapid equilibrium, with three inner sphere (2)H(2)O exchanged per Mn(2+) . This indicates that the water ligands of the Mn(2+)/Mg(2+) site, or the protons of these waters, can exchange with bulk solvent at a rate consistent with a role for this region in product release during turnover. Sheng Wu Gong Cheng Xue Bao, 2001 Mar, 17(2), 140 - 4 {Cloning of VEGF receptor KDR and its expression in insect cells}; Zeng GF et al.; The cDNA fragment of the first 3 loops of VEGF receptor, KDR, was cloned by PCR and inserted into a baculovirus expression plasmid pFASTBACI . The competent E . coli DH10BAC cell, which contain another plasmid with baculovirus genome in it, was transformed with pFASTBACI-KDRn3 . Homologous recombination in the prokaryotic cells resulted in a recombinant plasmid containing KDRn3 in baculovirus genome . Transfection of the insect cell SF-9 with above plasmid generated a recombinant baculorvirus contain target gene fragment . SDS-PAGE and Western blot analysis of the supernatant of the infected SF-9 cell showed that KDRn3 was secreted in the medium . The recombinant protein was verified with Western blot and tested for their binding activity with VEGF . Its anti-angiogenic activity was assayed on chorionic allantoic membrane(CAM) of fertilized egg . The results showed that the recombinant protein could inhibit new vessel formation on CAM of fertilized eggs. Nucleic Acids Res, 2001 Jun 15, 29(12), 2607 - 18 GeneMarkS: a self-training method for prediction of gene starts in microbial genomes . Implications for finding sequence motifs in regulatory regions; Besemer J et al.; Improving the accuracy of prediction of gene starts is one of a few remaining open problems in computer prediction of prokaryotic genes . Its difficulty is caused by the absence of relatively strong sequence patterns identifying true translation initiation sites . In the current paper we show that the accuracy of gene start prediction can be improved by combining models of protein-coding and non-coding regions and models of regulatory sites near gene start within an iterative Hidden Markov model based algorithm . The new gene prediction method, called GeneMarkS, utilizes a non-supervised training procedure and can be used for a newly sequenced prokaryotic genome with no prior knowledge of any protein or rRNA genes . The GeneMarkS implementation uses an improved version of the gene finding program GeneMark.hmm, heuristic Markov models of coding and non-coding regions and the Gibbs sampling multiple alignment program . GeneMarkS predicted precisely 83.2% of the translation starts of GenBank annotated Bacillus subtilis genes and 94.4% of translation starts in an experimentally validated set of Escherichia coli genes . We have also observed that GeneMarkS detects prokaryotic genes, in terms of identifying open reading frames containing real genes, with an accuracy matching the level of the best currently used gene detection methods . Accurate translation start prediction, in addition to the refinement of protein sequence N-terminal data, provides the benefit of precise positioning of the sequence region situated upstream to a gene start . Therefore, sequence motifs related to transcription and translation regulatory sites can be revealed and analyzed with higher precision . These motifs were shown to possess a significant variability, the functional and evolutionary connections of which are discussed. FEMS Microbiol Lett, 2001 Jun 12, 200(1), 79 - 84 On the origin of Ser/Thr kinases in a prokaryote; Han G et al.; The family of Ser/Thr and/or Tyr kinases and that of His kinases play essential roles in signal transduction . For a long time, the former has been found in eukaryotes, the latter in prokaryotes . Studies in the last decade have shown, however, that most bacteria possess from one to more than 10 genes encoding Ser/Thr kinases . This observation raises an important question concerning the evolutionary origin of Ser/Thr kinases found in bacteria . To answer this question, we have analyzed a family of 11 genes encoding Ser/Thr kinases in the cyanobacterium Synechocystis sp . PCC 6803 . This bacterium contains the largest number of Ser/Thr kinases among all bacteria whose genomic sequences have been released so far . In this study, we have developed a user-friendly computer program for statistical analysis of codon usages and GC content . The results demonstrate that Ser/Thr kinases have similar codon usages and GC contents as the average of all possible open reading frames (ORFs) deduced from the genome . In contrast, ORFs encoding transposases, as a control in our analysis, display a disparity in both codon usage and GC content, confirming their multiple origin and genetic promiscuity . In light of our results, we propose that Ser/Thr kinases existed before the divergence between prokaryotes and eukaryotes during evolution, or were laterally transferred into prokaryotes at the early stages of bacterial evolution . If Ser/Thr kinases have persisted ever since in prokaryotes under evolutionary pressure, it is then expected that they play important, possibly even essential roles in regulating bacterial activities as do their counterparts in eukaryotes. Mutat Res, 2001 Jul 1, 478(1-2), 153 - 8 DNA breakage due to metronidazole treatment; Menendez D et al.; The mutagenicity of metronidazole {1-(hidroxyethyl)-2-methyl-5-nitroimidazole} (MTZ) has been shown in different prokaryotic systems . However, data on human cells are still contradictory . In this study DNA damage was determined by the single cell gel electrophoresis (SCGE) assay, in lymphocytes from 10 healthy subjects treated with therapeutic doses of this drug . Samples were obtained before treatment, as well as 1 and 15 days after ending treatment . Results showed a significant increase of DNA strand breaks 1 day after ending treatment, although, an inverse correlation between the amount of DNA damage and plasma concentrations of MTZ was obtained . Thus, the observed damage may be induced by some MTZ metabolite rather than by the parent drug . Interestingly, the amount of DNA damage returned to basal levels 15 days after ending treatment, except in two individuals . This persistent damage should be further investigated. Biochemistry (Mosc), 2001 May, 66(5), 476 - 89 Oxidative stress and mechanisms of protection against it in bacteria; Lushchak VI; In the review contemporary data on the effects of oxidative stresses of various kinds in bacteria are summarized . A general theory of oxidative stress, peculiarities of oxidative stress in eukaryotes and prokaryotes, and natural and induced oxidative stresses are described . Data on the mechanisms of protection against oxidative stress are given, including prevention of the generation of oxidative stress, prevention of propagation of free radical chain reactions, and the mechanisms of repair of damaged DNA . The regulation of effector genes via redox-sensitive iron-containing proteins is analyzed . Special attention is given to the expression of so-called antioxidant and associated enzymes as protection mechanisms and to the space-time organization of the response of bacteria to oxidative stress. Biol Chem, 2001 Apr, 382(4), 661 - 8 Chemical accessibility of 18S rRNA in native ribosomal complexes: interaction sites of mRNA, tRNA and translation factors; Sloma MS et al.; During protein synthesis the ribosome interacts with ligands such as mRNA, tRNA and translation factors . We have studied the effect of ribosome-ligand interaction on the accessibility of 18S rRNA for single strand-specific modification in ribosomal complexes that have been assembled in vivo, i . e . native polysomes . A comparison of the modification patterns derived from programmed and non-programmed ribosomes showed that bases in the 630- and 1060-loops (530- and 790-loops in E . coli) together with two nucleotides in helices 33 and 34 were protected from chemical modification . The majority of the protected sites were homologous to sites previously suggested to be involved in mRNA and/or tRNA binding in prokaryotes and eukaryotes, implying that the interaction sites for these ligands are similar, if not identical, in naturally occurring programmed ribosomes and in in vitro assembled ribosomal complexes . Additional differences between programmed and non-programmed ribosomes were found in hairpin 8 . The bases in helix 8 showed increased exposure to chemical modification in the programmed ribosomes . In addition, structural differences in helices 36 and 37 were observed between native 80S run-off ribosomes and 80S ribosomes assembled from isolated 40S and 60S subunits. Biol Chem, 2001 Apr, 382(4), 645 - 54 Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families; Cukovic D et al.; The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in general phenylpropanoid metabolism . 4CL is related to a larger class of prokaryotic and eukaryotic adenylate-forming enzymes and shares several conserved peptide motifs with these enzymes . In order to better characterize the nature of 4CL gene families in poplar, parsley, and tobacco, we used degenerate primers to amplify 4CL sequences from these species . In each species additional, divergent 4CL genes were found . Complete cDNA clones for the two new poplar 4CL genes were obtained, allowing examination of their expression patterns and determination of the substrate utilization profile of a xylem-specific isoform . Phylogenetic analysis of these genes and gene fragments confirmed previous results showing that 4CL proteins fall into two evolutionarily ancient subgroups . A comparative phylogenetic analysis of enzymes in the adenylate-forming superfamily showed that 4CLs, luciferases, and acetate CoA ligases each form distinct clades within the superfamily . According to this analysis, four Arabidopsis 4CL-like genes identified from the Arabidopsis Genome Project are only distantly related to bona fide 4CLs or are more closely related to fatty acid CoA ligases, suggesting that the three Arabidopsis 4CL genes previously characterized represent the extent of the 4CL gene family in this species. Gene, 2001 May 30, 270(1-2), 191 - 200 Analysis of the enzymatic domains in the modular portion of the coronafacic acid polyketide synthase; Jiralerspong S et al.; Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen Pseudomonas syringae . The CFA polyketide synthase (PKS) consists of two open reading frames (ORFs) that encode type I multifunctional proteins and several ORFs that encode monofunctional proteins . Sequence comparisons of the modular portions of the CFA PKS with other prokaryotic, modular PKSs elucidated the boundaries of the domains that are involved in the individual stages of polyketide assembly . The two beta-ketoacyl:acyl carrier protein synthase (KS) domains in the modular portion of the CFA PKS exhibit a high degree of similarity to each other (53%), but are even more similar to the KS domains of DEBS, RAPS, and RIF . Cfa6 possesses two acyltransferases- AT0, which is associated with a loading domain, and AT1, which uses ethylmalonyl-CoA (eMCoA) as a substrate for chain extension . Cfa7 contains an AT that uses malonyl-CoA as a substrate for chain extension . The Cfa6 AT0 shows 35 and 32% similarity to the DEBS1 and NidA1 AT0s, respectively, and 32 and 36% similarity to the Cfa6 and Cfa7 AT1s . Sequence motifs have previously been identified that correlate with AT substrates . The motifs in Cfa6 AT1 were found to correlate reasonably well with those predicted for methylmalonyl-CoA (mMCoA) ATs . The motifs in the AT of Cfa7 correlated more poorly with those predicted for MCoA ATs . Three ACP domains occur in the modular proteins of the COR PKS . The loading domain-associated ACP0 showed 38% similarity to the loading domain ACP0s of DEBS1 and NidA1 and 32-36% similarity to the two module-associated ACPs of the COR PKS . It exhibited a higher degree of similarity to the module-associated ACPs of RAPS . The two module-associated ACPs show 39% similarity to each other, but appear more closely related to module-associated ACP domains in RAPS and RIFS . Furthermore, the DH and KR domains of Cfa6 and Cfa7 show greater similarity to DH and KR domains in RAPS and RIFS than to each other . The CFA PKS includes a thioesterase domain (TE I) that resides at the C-terminus of Cfa7 and a second thioesterase, which exists as a separate ORF (Cfa9, a TE II) . Analysis of a Cfa7 thioesterase mutant demonstrated that the TE domain is required for the production of CFA . The co-existence of TE domains within modular PKSs along with physically separated, monofunctional TEs (TE IIs) has been reported for a number of modular polyketide and non-ribosomal peptide synthases (NRPS) . An analysis of the two types of thioesterases using Clustal X yielded a dendrogram showing that TE IIs from PKSs and NRPSs are more closely related to each other than to domain TEs from either PKSs or NRPSs . Furthermore, the dendrogram indicates that both types of TE IIs are more closely related to TE domains associated with PKSs than to TE domains in NRPSs . Finally, the overall % G+C content and the % G+C content at the third codon for all of the PKS genes in the COR cluster suggest that these genes may have been recruited from a gram-positive bacterium. J Biol Chem, 2001 Aug 17, 276(33), 30779 - 85 Epub 2001 Jun 14. Species-specific differences in amino acid editing by class II prolyl-tRNA synthetase; Beuning PJ et al.; Aminoacyl-tRNA synthetases are a family of enzymes responsible for ensuring the accuracy of the genetic code by specifically attaching a particular amino acid to their cognate tRNA substrates . Through primary sequence alignments, prolyl-tRNA synthetases (ProRSs) have been divided into two phylogenetically divergent groups . We have been interested in understanding whether the unusual evolutionary pattern of ProRSs corresponds to functional differences as well . Previously, we showed that some features of tRNA recognition and aminoacylation are indeed group-specific . Here, we examine the species-specific differences in another enzymatic activity, namely amino acid editing . Proofreading or editing provides a mechanism by which incorrectly activated amino acids are hydrolyzed and thus prevented from misincorporation into proteins . "Prokaryotic-like" Escherichia coli ProRS has recently been shown to be capable of misactivating alanine and possesses both pretransfer and post-transfer hydrolytic editing activity against this noncognate amino acid . We now find that two ProRSs belonging to the "eukaryotic-like" group exhibit differences in their hydrolytic editing activity . Whereas ProRS from Methanococcus jannaschii is similar to E . coli in its ability to hydrolyze misactivated alanine via both pretransfer and post-transfer editing pathways, human ProRS lacks these activities . These results have implications for the selection or design of antibiotics that specifically target the editing active site of the prokaryotic-like group of ProRSs. Curr Microbiol, 2001 Sep, 43(3), 163 - 9 Effects of mycoplasmal LAMPs on receptor responses to steroid hormones in mammalian cells; Iyama K et al.; Many individuals are chronically infected or parasitically colonized with mycoplasmas in their respiratory or urogenital tracts without apparent clinical significance . However, prolonged close interaction between prokaryotic agents and eukaryotic host cells may gradually and significantly alter normal biological or physiological properties of infected hosts . Steroid hormones are associated with rates of cancer formation in human . The purpose of this study is to establish a sensitive reporting system to examine whether mycoplasmal infections affect biological responses to steroid hormones in mammalian cells . We established pMTV-CAT stably transfected cell lines to test the effect of mycoplasmal lipid-associated membrane proteins (LAMPs) . Results showed that LAMPs (1 microg/ml) from seven different species of human mycoplasmas-M . penetrans, M . fermentans, M . genitalium, M . salivarium, M . pneumoniae, M . orale, and M . hominis-had an inhibitory effect on androgen receptor (AR) response to 5alpha-dihydrotestosterone (DHT) in the E82 transfectants . The inhibitory effect of mycoplasmal LAMPs appeared to be dose dependent . LAMPs from M . penetrans, M . genitalium, M . salivarium, M . pneumoniae, and M . orale also had an inhibitory effect on glucocorticoid receptor (GR) response to hormone dexamethasone (Dex) in TSU transfectants . In contrast, LAMPs from M . fermentans and M . hominis showed a stimulatory effect on the GR response to Dex in these TSU cells . The results suggest that colonization or chronic infection by mycoplasmas may significantly affect the responses of mammalian host cells to various steroid hormones, potentially affecting rates of cancer formation. J Biol Chem, 2001 Jun 15, 276(24), 20890 - 7 Epub 2001 Apr 03. Characterization of clutathione amide reductase from Chromatium gracile . Identification of a novel thiol peroxidase (Prx/Grx) fueled by glutathione amide redox cycling; Vergauwen B et al.; Among the Chromatiaceae, the glutathione derivative gamma-l-glutamyl-l-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species . The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined . The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus . The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase . Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH . By establishing Michaelis constants of 97 and 13.2 microm for glutathione amide disulfide and NADH, respectively (in contrast to K(m) values of 6.9 mm for glutathione disulfide and 1.98 mm for NADPH), the exclusive substrate specificities of GAR have been documented . Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21 . On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2'-phosphate group, by Leu-197, Glu-198, and Phe-203 . We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases. Hematology, 1999, 4(1), 59 - 66 Molecular Medicine and Molecular Pathology for Haematologists: I . Gene Speak made Easy: An Overview of Genes and Gene Expression; Lawler M; Molecular Medicine and Molecular Pathology are integral parts of Haematology as we enter the new millennium . Their origins can be linked to fundamental developments in the basic sciences, particularly genetics, chemistry and biochemistry . The structure of DNA and the genetic code that it encrypts are the critical starting points to our understanding of these new disciplines . The genetic alphabet is a simple one, consisting of just 4 letters, buts its influence is crucial to human development and differentiation . The concept of a gene is not a new one but the Human Genome Project (a joint world-wide effort to characterise our entire genetic make-up) is providing an invaluable understanding of how genes function in normal cellular processes and pinpointing how disruption of these processes can lead to disease . Transcription and translation are the key events by which our genotype is converted to our phenotype (via a messenger RNA intermediate), producing the myriad proteins and enzymes which populate the cellular factory of our body . Unlike the bacterial or prokaryotic genome, the human genome contains a large amount of non coding DNA (less than 1% of our genome codes for proteins), and our genes are interrupted, with the coding regions or exons separated by non coding introns . Precise removal of the intronic material after transcription (though a process called splicing) is critical for efficient translation to occur . Incorrect splicing can lead to the generation of mutant proteins, which can have a dilaterious effect on the phenotype of the individual . Thus the 100,000-200,000 genes which are present in each cell in our body have a defined control mechanism permitting efficient and appropriate expression of proteins and enzymes and yet a single base change in just one of those genes can lead to diseases such as haemophilia or fanconis anaemia. Mol Microbiol, 2001 May, 40(4), 804 - 14 A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2); Kelemen GH et al.; Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony . sigmaH was identified previously as one of three paralogous sigma factors associated with stress responses in S . coelicolor . Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sigmaH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2 . While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation . Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2-egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S . coelicolor . However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sigmaH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2-egfp fusion was expressed ectopically in the substrate mycelium in the bldD background . Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S . coelicolor and shown to be BldD . The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting . These data show that expression of sigmaH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis. Chromosoma, 2001 Apr, 110(1), 1 - 9 The hAT family: a versatile transposon group common to plants, fungi, animals, and man; Kempken F et al.; Transposons are ubiquitous mobile genetic elements found in all eu- and prokaryotic cells . The first transposon identified, the maize Activator element, belongs to the hAT family . hAT transposons have been identified in most eukaryotic lineages, including plants, fungi, animals and even man . The basic structural and functional features of this transposon family and its phylogenetic roots are discussed in detail, including a phylogenetic tree deduced from the amino acid sequence of the most conserved part of the transposon-encoded transposase . Emphasis is given to the use of hAT transposons as tools for gene tagging and insect transformation as well as to their biological function, i.e . are they selfish DNA, beneficial companions, or even both? J Neuropathol Exp Neurol, 2001 Jun, 60(6), 613 - 20 Spiroplasma sp . 16S rDNA in Creutzfeldt-Jakob disease and scrapie as shown by PCR and DNA sequence analysis; Bastian FO et al.; The pathogenesis of the transmissible spongiform encephalopathies (TSE), which include Creutzfeldt-Jakob disease (CJD) in humans and scrapie in sheep, remains an enigma . In this paper we present evidence for the association of Spiroplasma sp., a wall-less prokaryote, with TSE . We have shown PCR amplification of Spiroplasma 16S rDNA in TSE-infected brain tissues (13 of 13 CJD cases and 5 of 9 scrapie cases) and not in control brains (0 of 50) . Direct sequencing of the amplified PCR products has confirmed the presence of Spiroplasma-like DNA in all 5 of the TSE brains tested . Our evidence is not necessarily in conflict with involvement of a PrPres--a protease-resistant host-derived protein referred to as the prion--in the pathogenesis of TSE, since there is evidence that another factor is involved . We propose a bacterium, namely Spiroplasma, as this associated factor although the role of Spiroplasma in TSE cannot be determined from these experiments . The presence of the nucleic acid sequence of this microbe in all cases of TSE in our laboratory and not in controls provides direct evidence of the association of Spiroplasma sp . with TSE. J Mol Biol, 2001 Jun 8, 309(3), 727 - 35 Structural studies of the tRNA domain of tmRNA; Stagg SM et al.; tmRNA is a small, stable prokaryotic RNA . It rescues ribosomes that have become stalled during the translation of mRNA fragments lacking stop codons, or during periods of tRNA scarcity . It derives its name from the presence of two separate domains, one that functions as a tRNA, and another that serves as an mRNA . We have carried out modeling and transient electric birefringence studies to determine the angle between the acceptor stem and anticodon stem of the tRNA domain of Eschericia coli tmRNA . The results of the modeling studies yielded an interstem angle of 110 degrees, in agreement with the lower end of the range of angles (111 degrees -137 degrees ) determined experimentally for various solution conditions . The range of experimental angles is greater than the angles observed for any of the tRNA crystal structures, in line with the presence of a shortened D stem . The secondary structure of the tRNA domain is conserved for all known tmRNA sequences, so we propose that the angle is also conserved . These results also suggest that the region of tmRNA between P2a and P2b may interact with the decoding site of the ribosome . Mol Membr Biol, 2001 Jan-Mar, 18(1), 97 - 103 Multidrug transporters in prokaryotic and eukaryotic cells: physiological functions and transport mechanisms; Blackmore CG et al.; Multidrug transporters mediate the extrusion of structurally unrelated drugs from prokaryotic and eukaryotic cells . As a result of this efflux activity, the cytoplasmic drug concentration in the cell is lowered to subtoxic levels and, hence, cells become multidrug resistant . The activity of multidrug transporters interferes with the drug-based control of tumours and infectious pathogenic microorganisms . There is an urgent need to understand the structure-function relationships in multidrug transporters that underlie their drug specificity and transport mechanism . Knowledge about the architecture of drug and modulator binding sites and the link between energy-generating and drug translocating functions of multidrug transporters may allow one to rationally design new drugs that can poison or circumvent the activity of these transport