Prokaryotes

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 proteins . Furthermore, if one is to inhibit multidrug transporters in human cells, one should know more about their physiological substrates and functions . This review will summarize important new insights into the role that multidrug transporters in general, and P-glycoprotein and its bacterial homologue LmrA in particular, play in the physiology of the cell . In addition, the molecular basis of drug transport by these proteins will be discussed.

Annu Rev Biochem, 2001, 70, 755 - 75
The signal recognition particle; Keenan RJ et al.; The signal recognition particle (SRP) and its membrane-associated receptor (SR) catalyze targeting of nascent secretory and membrane proteins to the protein translocation apparatus of the cell . Components of the SRP pathway and salient features of the molecular mechanism of SRP-dependent protein targeting are conserved in all three kingdoms of life . Recent advances in the structure determination of a number of key components in the eukaryotic and prokaryotic SRP pathway provide new insight into the molecular basis of SRP function, and they set the stage for future work toward an integrated picture that takes into account the dynamic and contextual properties of this remarkable cellular machine.

Annu Rev Biochem, 2001, 70, 603 - 47
Folding of newly translated proteins in vivo: the role of molecular chaperones; Frydman J; Recent years have witnessed dramatic advances in our understanding of how newly translated proteins fold in the cell and the contribution of molecular chaperones to this process . Folding in the cell must be achieved in a highly crowded macromolecular environment, in which release of nonnative polypeptides into the cytosolic solution might lead to formation of potentially toxic aggregates . Here I review the cellular mechanisms that ensure efficient folding of newly translated proteins in vivo . De novo protein folding appears to occur in a protected environment created by a highly processive chaperone machinery that is directly coupled to translation . Genetic and biochemical analysis shows that several distinct chaperone systems, including Hsp70 and the cylindrical chaperonins, assist the folding of proteins upon translation in the cytosol of both prokaryotic and eukaryotic cells . The cellular chaperone machinery is specifically recruited to bind to ribosomes and protects nascent chains and folding intermediates from nonproductive interactions . In addition, initiation of folding during translation appears to be important for efficient folding of multidomain proteins.

Annu Rev Biochem, 2001, 70, 369 - 413
DNA topoisomerases: structure, function, and mechanism; Champoux JJ; DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA . In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious . In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved . These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses . Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes . The type IB enzymes are structurally distinct from all other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases . The structural themes common to all topoisomerases include hinged clamps that open and close to bind DNA, the presence of DNA binding cavities for temporary storage of DNA segments, and the coupling of protein conformational changes to DNA rotation or DNA movement . For the type II topoisomerases, the binding and hydrolysis of ATP further modulate conformational changes in the enzymes to effect changes in DNA topology.

Annu Rev Biochem, 2001, 70, 181 - 208
Replisome-mediated DNA replication; Benkovic SJ et al.; The elaborate process of genomic replication requires a large collection of proteins properly assembled at a DNA replication fork . Several decades of research on the bacterium Escherichia coli and its bacteriophages T4 and T7 have defined the roles of many proteins central to DNA replication . These three different prokaryotic replication systems use the same fundamental components for synthesis at a moving DNA replication fork even though the number and nature of some individual proteins are different and many lack extensive sequence homology . The components of the replication complex can be grouped into functional categories as follows: DNA polymerase, helix destabilizing protein, polymerase accessory factors, and primosome (DNA helicase and DNA primase activities) . The replication of DNA derives from a multistep enzymatic pathway that features the assembly of accessory factors and polymerases into a functional holoenzyme; the separation of the double-stranded template DNA by helicase activity and its coupling to the primase synthesis of RNA primers to initiate Okazaki fragment synthesis; and the continuous and discontinuous synthesis of the leading and lagging daughter strands by the polymerases . This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.

Annu Rev Biochem, 2001, 70, 39 - 80
DNA primases; Frick DN et al.; DNA primases are enzymes whose continual activity is required at the DNA replication fork . They catalyze the synthesis of short RNA molecules used as primers for DNA polymerases . Primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long . Most DNA primases can be divided into two classes . The first class contains bacterial and bacteriophage enzymes found associated with replicative DNA helicases . These prokaryotic primases contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either is itself a DNA helicase or interacts with a DNA helicase . The second major primase class comprises heterodimeric eukaryotic primases that form a complex with DNA polymerase alpha and its accessory B subunit . The small eukaryotic primase subunit contains the active site for RNA synthesis, and its activity correlates with DNA replication during the cell cycle.

Antonie Van Leeuwenhoek, 2001 Jan, 79(1), 17 - 31
Signal recognition particle mediated protein targeting in Escherichia coli; Valent QA; The signal recognition particle (SRP) is a conserved ribonucleoprotein complex that binds to targeting sequences in nascent secretory and membrane proteins . The SRP guides these proteins to the cytoplasmic membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes via an interaction with its cognate receptor . The E . coli SRP is relatively small and is currently used as a model for fundamental and applied studies on translation-linked protein targeting . In this review recent advances in our understanding of the structure and function of the E . coli SRP and its receptor are discussed . In particular, the interplay between the SRP pathway and other targeting routes, the role of guanine nucleotides in cycling of the SRP and the substrate specificity of the SRP are highlighted.

Microbiology, 2001 Jun, 147(Pt 6), 1641 - 50
A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect; Angelova M et al.; The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn SOD . To improve its yield, the effect of an increased concentration of dissolved oxygen (DO) on growth and enzyme biosynthesis by the producer, cultivated in a 3 l bioreactor, was examined . Exposure to a 20% DO level caused a 1.7-fold increase of SOD activity compared to the DO-uncontrolled culture . Maximum enzyme productivity of SOD was approximately 300 x 10(3) U (kg wet biomass)(-1) . The novel enzyme was purified to electrophoretic homogeneity . The presence of Cu and Zn were confirmed by atomic absorption spectrometry . The molecular mass of H . lutea Cu/Zn SOD was calculated to be 31870 Da for the whole molecule and 15936 Da for the structural subunits . The N-terminal sequence revealed a high degree of structural homology with Cu/Zn SOD from other prokaryotic and eukaryotic sources . H . lutea Cu/Zn SOD was used in an in vivo model for the demonstration of its protective effect against myeloid Graffi tumour in hamsters . Comparative studies revealed that the enzyme (i) elongated the latent time for tumour appearance, (ii) inhibited tumour growth in the early stage of tumour progression (73-75% at day 10) and (iii) increased the mean survival time of Graffi-tumour-bearing hamsters . Moreover, the fungal Cu/Zn SOD exhibited a strong protective effect on experimental influenza virus infection in mice . The survival rate increased markedly, the time of survival rose by 5.2 d and the protective index reached 86% . The H . lutea SOD protected mice from mortality more efficiently compared to the selective antiviral drug ribavirin and to commercial bovine SOD . In conclusion, our results suggest that appropriate use of the novel fungal SOD, applied as such or in combination with selective inhibitors, could outline a promising strategy for the treatment of myeloid Graffi tumour and influenza virus infection.

Trends Microbiol, 2001 Jun, 9(6), 267 - 73
Intracellular survival strategies of mutualistic and parasitic prokaryotes; Goebel W et al.; Endosymbiotic bacteria closely related to mammalian pathogens are widespread in invertebrates . Mutualistic and parasitic bacteria-host interactions on the various evolutionary levels apparently involve similar factors, indicating that relevant genetic information developed early in evolution . The detailed characterization of symbiotic interactions of bacteria with non-mammalian hosts should provide profound insights into the basic mechanisms of bacteria-host interactions and their evolution.

J Cell Biochem Suppl, 2000, Suppl 35, 3 - 22
Fatal connections: when DNA ends meet on the nuclear matrix; Bode J et al.; A damaged nucleus has long been regarded simply as a "bag of broken chromosomes," with the DNA free ends moving around and forming connections with randomly encountered partners . Recent evidence shows this picture to be fundamentally wrong . Chromosomes occupy specific nuclear domains within which only limited movement is possible . In a human diploid nucleus, 6.6 x 10(9) base pairs (bp) of DNA are compartmentalized into chromosomes in a way that allows stringent control of replication, differential gene expression, recombination and repair . Most of the chromatin is further organized into looped domains by the dynamic binding of tethered bases to a network of intranuclear proteins, the so-called nuclear scaffold or matrix . Thus, DNA movement is severely curtailed, which limits the number of sites where interchanges can occur . This intricate organizational arrangement may render the genome vulnerable to processes that interfere with DNA repair . Both lower and higher eukaryotic cells perform homologous recombination (HR) and illegitimate recombination (IR) as part of their survival strategies . The repair processes comprising IR must be understood in the context of DNA structural organization, which is fundamentally different in prokaryotic and eukaryotic genomes . In this paper we first review important cellular processes including recombination, DNA repair, and apoptosis, and describe the central elements involved . Then we review the different DNA targets of recombination, and present recent evidence implicating the nuclear matrix in processes which can induce either repair, translocation, deletion, or apoptosis . J . Cell . Biochem . Suppl . 35:3-22, 2000 .

Protein Expr Purif, 2001 Jun, 22(1), 135 - 40
Intein-mediated rapid purification of Cre recombinase; Cantor EJ et al.; Cre recombinase produced by bacteriophage P1 catalyzes site-specific recombination of DNA between loxP recognition sites in both prokaryotic and eukaryotic cells and has been widely used for genome engineering and in vitro cloning . Recombinant Cre has been overproduced in Escherichia coli and its purification involves multiple steps . In this report, we used an "intein" fusion system to express Cre as a C-terminal fusion to a modified protein splicing element, i.e., intein . The modified intein contained a Bacillus circulans chitin-binding domain which allowed binding of the fusion protein on a chitin column and could be induced to undergo in vitro peptide bond cleavage which specifically released Cre from the column . Using the intein system, we have obtained highly pure nontagged Cre after just a single chromatographic step, which corresponded to approximately 80% recovery and 27-fold purification . The activity of the purified Cre was determined in an in vitro assay system and was found to remain stable over a period of more than 6 months .

Am J Trop Med Hyg, 2000 Sep-Oct, 63(3-4), 121 - 7
Therapy for human gastrointestinal microsporidiosis; Conteas CN et al.; Gastrointestinal microsporidiosis is a major cause of diarrhea and wasting in persons with acquired immune deficiency syndrome (AIDS) . Microsporidia demonstrate properties of both true eukaryotes and prokaryotes . The biology of microsporidia makes its elimination from the gastrointestinal tract therapeutically challenging . This organism depends greatly on the host for its energy needs and reproduction; microsporidial spores are impervious to the elements . Microsporidial infection of the gastrointestinal tract, principally with Enterocytozoon bieneusi and Encephalitozoon intestinalis in patients with AIDS has been treated with different medical regimens with variable success . The less common pathogen, E . intestinalis, responds well to albendazole, making it excellent first-line therapy, but such is not the case for E . bieneusi . None of the benzimidazoles has been demonstrated to be efficacious for E . bieneusi . On the other hand, E . bieneusi has shown excellent clinical therapeutic response to either direct action with fumagillin or its analogue, TNP-470, or indirectly by immune enhancement by suppression of the HIV virus with more aggressive, highly effective antiretroviral therapy . Further work is necessary to fully establish proper therapeutic protocols and manage side effects of the treatments . Other promising forms of therapy such as polyamine inhibitors and thalidomide demonstrate certain effectiveness in treatment of microsporidian in vitro (polyamine inhibitors) and in selected cases in vivo (thalidomide) . Lack of either sufficiently suggestive or definitive human studies prevents the endorsement of these modes of therapy for treatment of gastrointestinal microsporidiosis at this time.

Mol Genet Metab, 2001 Jun, 73(2), 173 - 8
Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes produced in a prokaryotic expression system; Leandro P et al.; Chemical chaperones are low molecular weight compounds known to stabilize proteins in vitro . Recently it was shown that, in transfected cells, these molecules can also correct the defective folding of some mutant proteins . Hyperphenylalaninemia (HPA) has been proposed to be classified as a "conformational disease," since it has been shown that the majority of the PAH mutations affect protein folding, thereby causing an increasing tendency toward aggregation and proteolytic degradation . Based on these observations, the effect of glycerol as a stabilizer agent of recombinant mutant forms of human phenylalanine hydroxylase enzymes (hPAH) produced in a prokaryotic expression system was investigated . The wild-type and two mutant forms of the hPAH protein (R270K and V388M) were expressed in the presence of glycerol in the culture medium . The yield, specific enzymatic activities, and kinetic properties of the recombinant proteins were determined and compared with the data obtained under normal growth conditions . The results obtained demonstrate that glycerol not only improved the yield of the soluble hPAH proteins (2- to 3-fold depending on the mutant enzyme) produced but also increased the specific activity of the purified recombinant enzymes . We speculate that correction of protein folding abnormalities by chemical chaperones may be a possible therapeutic approach to correct conformational diseases .

Vopr Med Khim, 2001 Jan-Feb, 47(1), 3 - 19
{Energy-dependent selective intracellular proteolysis . Structure, active sites and specificity of ATP-dependent proteinases}; Rotanova TV; The enzymatic systems of selective proteolysis serving for the maintenance of cell homeostasis and functioning in prokaryotic and eukaryotic cells are characterized . The data on structure, active sites and specificity towards protein targets are given.

Bioessays, 2001 Jun, 23(6), 534 - 42
Glycerol: a neglected variable in metabolic processes?
Brisson D, Vohl MC, St-Pierre J, Hudson TJ, Gaudet D.
Glycerol is a small and simple molecule produced in the breakdown of glucose, proteins, pyruvate, triacylglycerols and other glycerolipid, as well as release from dietary fats . An increasing number of observations show that glycerol is probably involved in a surprising variety of physiopathologic mechanisms . Glycerol has long been known to play fundamental roles in several vital physiological processes, in prokaryotes and eukaryotes, and is an important intermediate of energy metabolism . Despite some differences in the details of their operation, many of these mechanisms have been preserved throughout evolution, demonstrating their fundamental importance . In particular, glycerol can control osmotic activity and crystal formation and then act as a cryoprotective agent . Furthermore, its properties make it useful in numerous industrial, therapeutic and diagnostic applications . Few studies have focussed directly on glycerol, however, and while its metabolism is increasingly well documented, much of the details remain unknown . Considering the importance of glycerol in multiple vital physiological processes, its study could help unlock important physiopathological mechanisms .

Dis Aquat Organ, 2001 Apr 10, 44(3), 203 - 16
Characterization of intracytoplasmic prokaryote infections in Dreissena sp . (Bivalvia: Dreissenidae); Molloy DP et al.; This study characterizes intracytoplasmic infections with prokaryote microorganisms in Dreissena sp . (near Dreissena polymorpha) from northeastern Greece and represents the first report of such infections in freshwater bivalves . Light microscope observations of stained tissues revealed basophilic, cytoplasmic inclusion bodies in 87.5% (28/32) of the mussels sectioned . Inclusions in epithelial cells and connective tissues were noted, respectively, in 34.4 and 71.9% of the sample, with 5 mussels (15.6%) having both tissue types infected . Epithelial cell infections were observed in histological sections only in digestive gland tubules and ducts; within tubules, inclusions were present more often in secretory than digestive cells . Connective tissue infections, however, were systemic; among the 32 mussels sectioned, inclusions were found in the gills (65.6%), foot (12.5%), mantle (9.4%), labial palps (6.3%), digestive gland (6.3%), stomach (6.3%), and gonads (3.1%) . Cytoplasmic inclusions (maximum dimension, 138 microm) were prominent enough in the gills to be visible in 17.0% of the 247 mussels dissected . Ultrastructurally, prokaryote cells in gill connective tissues were clearly characteristic of Chlamydiales-like organisms, with each intracytoplasmic inclusion containing a loosely packed mixture of elementary, reticulate, intermediate bodies, and blebs . Prokaryote colonies in digestive gland epithelial cells exclusively contained 1 of 4 morphological cell types and were considered Rickettsiales-like . Hexagonal, virus-like particles were present in the cytoplasm of the largest of these Rickettsiales-like prokaryotes . Although host stress was evident from localized cell necrosis and dense hemocyte infiltration, overall infection was fairly benign, with no major, adverse impact on body condition evident among sectioned or dissected mussels . A possible negative effect was partial constriction of gill water tubes, but at the infection intensity observed (typical range 1 to 7 inclusion bodies per section), significant interference with respiration and other metabolic functions of the gills was highly unlikely.

Arch Microbiol, 2001 Apr, 175(4), 241 - 9
The gene transfer agent of Rhodobacter capsulatus and "constitutive transduction" in prokaryotes; Lang AS et al.; Transduction, bacteriophage-mediated gene transfer, is thought to play an important role in the evolution of prokaryote genomes . Several gene transfer agents that resemble transducing phages have been found in diverse prokaryotes . This mini-review discusses these interesting agents of genetic exchange with a focus on the gene transfer agent (GTA) of Rhodobacter capsulatus, at present the only member of this group for which genetic information exists about the production of transducing particles . Production of GTA results from expression of genes that are similar to phage genes, yet transcription of these genes is dependent upon cellular (two-component) signaling proteins . The significance of these relationships, as well as the finding of GTA gene homologues in the bacterium Rhodopseudomonas palustris, is discussed.

Huan Jing Ke Xue, 2001 Jan, 22(1), 19 - 22
{The bindings of typical aldehydes pollutants with cell DNA}; Xi Z et al.; The bindings of 3 kinds of aidehyde pollutants, formaldehyde, acetaldehyde, acrolein, with both prokaryotic and eukaryotic DNA, and their genotoxic effect and mechanism were conducted by the shifts of maximum UV absorption peak to determine binding effects and by HPLC to determine binding sites in vitro model system . The shifts of maximum UV absorption peak of prokaryotic DNA contaminated by 3 kinds of aldehyde pollutants are not significant; but after the DNA extracted from prokaryotic bacteria reacting with formaldehyde in tube, the shifts of maximum UV absorption peak of DNA are significant; the shifts of maximum UV absorption peak of eukaryotic DNA contaminated by 3 kinds of aldehyde pollutants are significant also . The reacition of acetaldehyde with dG reduced by NaBH4 was separated and detected by HPLC, the product was determined qualitatively as N2-ethaldeoxyguanosine adduct . The 3 kinds of aldehyde pollutants could bind with cellular DNA to express genotoxic effects; and the N2 site of deoxyguanosine is the possible covalent binding site.

Nat Genet, 2001 Jun, 28(2), 188 - 91
Identification of the gene that, when mutated, causes the human obesity syndrome BBS4; Mykytyn K et al.; Bardet-Biedl syndrome (BBS, MIM 209900) is a heterogeneous autosomal recessive disorder characterized by obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism . The disorder is also associated with diabetes mellitus, hypertension, and congenital heart disease . Six distinct BBS loci map to 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22.3-q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6) . Although BBS is rare in the general population (<1/100,000), there is considerable interest in identifying the genes causing BBS because components of the phenotype, such as obesity and diabetes, are common . We and others have demonstrated that BBS6 is caused by mutations in the gene MKKS (refs . 12,13), mutation of which also causes McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly, and congenital heart defects) . MKKS has sequence homology to the alpha subunit of a prokaryotic chaperonin in the thermosome Thermoplasma acidophilum . We recently identified a novel gene that causes BBS2 . The BBS2 protein has no significant similarity to other chaperonins or known proteins . Here we report the positional cloning and identification of mutations in BBS patients in a novel gene designated BBS4.

Curr Opin Microbiol, 2001 Jun, 4(3), 301 - 6
Carbon cycling: the prokaryotic contribution; Shively JM et al.; Although the debate continues, the concept of global warming as a consequence of the increased production of 'greenhouse gases' via human activities is now widely accepted . The role of microbes, especially the prokaryotes, in the formation, trapping and retention of 'greenhouse gases' has, for the most part, been overlooked . The future requires that we pay close attention to these organisms for possible solutions to adverse global changes.

Curr Opin Microbiol, 2001 Jun, 4(3), 285 - 9
Microbial genomes: dealing with diversity; Boucher Y et al.; We have now complete genome sequences of several pairs of closely related prokaryotes (conspecific strains or congeneric species) . Surprisingly, even strains of the same species can differ by as much as 20% in gene content . Conceptual and methodological approaches for dealing with such diversity are now being developed, and should transform microbial genomics.

Int J Biochem Cell Biol, 2001 Jun, 33(6), 541 - 53
Cotranslational folding--omnia mea mecum porto?
Kramer G, Ramachandiran V, Hardesty B.
Evidence for cotranslational folding on both prokaryotic and eukaryotic ribosomes is reviewed . Molecular chaperones appear to assist only a small fraction of newly synthesized proteins in folding into their native conformation . The recently published crystal structure of the large ribosomal subunit at 2.5 A resolution has provided the basis for understanding where and how peptide synthesis takes place on the ribosome . The nascent peptide is concluded to pass through a tunnel that extends about 100 A between the peptidyl transferase center and its exit site . The minimum diameter of the tunnel and the apparent physical and chemical properties of its walls appear to preclude complex folding of the nascent peptide within most of the length of the tunnel . However, results indicate that nascent peptides that are protected within the ribosomes vary in length from about 30 to 72 amino acid residues . This suggests that nascent peptides have different conformations . It is hypothesized that folding of the nascent polypeptide into its native conformation starts in the distal portion of the tunnel, and proceeds at the surface of the ribosomal subunit in a depression or bay near the exit opening of the tunnel.

Bioorg Med Chem Lett, 2001 Jun 4, 11(11), 1355 - 8
Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition; Thorarensen A et al.; Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis . Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range . Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC) . In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues . This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF . In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.

Gene, 2001 May 16, 269(1-2), 195 - 204
Identification of putative exported/secreted proteins in prokaryotic proteomes; Saleh MT et al.; The increasing number of bacterial genomes being sequenced fuels an equal demand for methods to rapidly analyze the proteomes of these organisms . One group of proteins of pressing importance is the exported/secreted proteins, given their dominant immunogenicity and role in pathogenesis . With this in mind, a weight matrix algorithm and two artificial neural networks, one based on amino acid position within the N-terminus and the other on amino acid frequency, were developed for identification of such proteins . The neural networks and a hybrid method, combining the weight matrix algorithm and the amino acid frequency neural network, were tested independently against a standard data set of secreted and cytoplasmic proteins to determine their accuracy in predicting secreted prokaryotic proteins . The results of these analyses demonstrated that the amino acid position neural network provided the highest accuracy (Mathews correlation coefficient of 0.93) in predicting secreted proteins of Gram-negative bacteria, whereas the hybrid method was best (Mathews correlation coefficient of 0.97) for prediction of Gram-positive secreted proteins . These two methods were integrated into a single program (ExProt) designed to analyze whole proteomes . In addition to protein localization, ExProt also contains a neural network trained to identify the most probable signal peptidase I cleavage site of secreted proteins . When tested against the standard protein data set ExProt correctly predicted 73.5 and 84.5% of the cleavage sites in Gram-positive and Gram-negative secreted proteins, respectively . Comparative analysis of Gram-negative, Gram-positive, Mycobacterium tuberculosis, and Archaea proteomes with ExProt revealed that the fraction of putative exported/secreted proteins encoded by bacterial genomes ranged from 8% for Methanococcus jannaschii to 37% for Mycoplasma pneumoniae.

J Biol Chem, 2001 Jul 27, 276(30), 28516 - 24 Epub 2001 May 25.
Bacteriophage T4 RNase H removes both RNA primers and adjacent DNA from the 5' end of lagging strand fragments; Bhagwat M et al.; Bacteriophage T4 RNase H belongs to a family of prokaryotic and eukaryotic nucleases that remove RNA primers from lagging strand fragments during DNA replication . Each enzyme has a flap endonuclease activity, cutting at or near the junction between single- and double-stranded DNA, and a 5'- to 3'-exonuclease, degrading both RNA.DNA and DNA.DNA duplexes . On model substrates for lagging strand synthesis, T4 RNase H functions as an exonuclease removing short oligonucleotides, rather than as an endonuclease removing longer flaps created by the advancing polymerase . The combined length of the DNA oligonucleotides released from each fragment ranges from 3 to 30 nucleotides, which corresponds to one round of processive degradation by T4 RNase H with 32 single-stranded DNA-binding protein present . Approximately 30 nucleotides are removed from each fragment during coupled leading and lagging strand synthesis with the complete T4 replication system . We conclude that the presence of 32 protein on the single-stranded DNA between lagging strand fragments guarantees that the nuclease will degrade processively, removing adjacent DNA as well as the RNA primers, and that the difference in the relative rates of synthesis and hydrolysis ensures that there is usually only a single round of degradation during each lagging strand cycle.

J Biol Chem, 2001 Jul 27, 276(30), 27981 - 8 Epub 2001 May 25.
Subcellular distribution and membrane topology of the mammalian concentrative Na+-nucleoside cotransporter rCNT1; Hamilton SR et al.; The rat transporter rCNT1 is the archetype of a family of concentrative nucleoside transporters (CNTs) found both in eukaryotes and in prokaryotes . In the present study we have used antibodies to investigate the subcellular distribution and membrane topology of this protein . rCNT1 was found to be expressed predominantly in the brush-border membranes of the polarized epithelial cells of rat jejunum and renal cortical tubules and in the bile canalicular membranes of liver parenchymal cells, consistent with roles in the absorption of dietary nucleosides, of nucleosides in the glomerular filtrate, or of nucleosides arising from the action of extracellular nucleotidases, respectively . The effect of endoglycosidase F treatment on wild-type and mutant rCNT1 expressed in Xenopus oocytes revealed that the recombinant transporter could be glycosylated at either or both of Asn605 and Asn643, indicating that its C terminus is extracellular . In contrast, potential N-glycosylation sites introduced near the N terminus, or between putative transmembrane (TM) helices 4 and 5, were not glycosylated . The deduced orientation of the N terminus in the cytoplasm was confirmed by immunocytochemistry on intact and saponin-permeabilized Chinese hamster ovary cells expressing recombinant rCNT1 . These results, in conjunction with extensive analyses of CNT family protein sequences using predictive algorithms, lead us to propose a revised topological model, in which rCNT1 possesses 13 TM helices with the hydrophilic N-terminal and C-terminal domains on the cytoplasmic and extracellular sides of the membrane, respectively . Furthermore, we show that the first three TM helices, which are absent from prokaryote CNTs, are not essential for transporter function; truncated proteins lacking these helices, derived either from rCNT1 or from its human homolog hCNT1, were found to retain significant sodium-dependent uridine transport activity when expressed in oocytes.

EMBO Rep, 2001 May, 2(5), 409 - 14
New insights into the activation of o-xylene biodegradation in Pseudomonas stutzeri OX1 by pathway substrates; Arenghi FL et al.; The regulation of the tou operon of Pseudomonas stutzeri OX1, for degradation of toluene and o-xylene via phenolic intermediates, has been faithfully reconstructed in vitro with purified proteins . The set-up included the prokaryotic enhancer-binding protein TouR, the sigma54-dependent PToMO promoter and the sigma54-containing RNA polymerase . With this system we prove that direct binding of 2-methylphenol (o-cresol) to TouR is the only regulatory step for activation of PToMO in response to aromatic effectors, thereby ruling out the involvement of other factors or a need for protein processing . In addition, we found that while TouR failed entirely to activate PToMO in the absence of inducers, the protein had per se a very significant ATPase activity, which was only moderately increased by o-cresol addition . The results presented here support the view that TouR-like proteins are particularly suitable as evolutionary assets to endow recently evolved pathways for the degradation of environmental pollutants with an optimal degree of transcriptional regulation.

Genomics, 2001 May 15, 74(1), 109 - 14
Cloning and characterization of human and mouse mitochondrial elongation factor G, GFM and Gfm, and mapping of GFM to human chromosome 3q25.1-q26.2; Gao J et al.; Similar to the translational system in the cell cytoplasm, the initiation, elongation, and termination of protein synthesis in the mitochondria of eukaryotes are catalyzed by several protein factors . These factors, from the viewpoint of evolution, are more closely related to the corresponding prokaryotic factors than to those in the eukaryotic cytoplasm . In this paper, we isolated two cDNAs coding for human and mouse mitochondrial elongation factor G (GFM and Gfm, respectively) . The GFM cDNA, which is 3481 bp in length, predicts a protein of 751 amino acids sharing 84 and 42% identity and 88 and 62% similarity to rat EF-G(mt) and Escherichia coli EF-G, respectively, and 24% identity and 39% similarity to human EF-2, the equivalent of EF-G in the cytoplasm . The mouse Gfm cDNA is 2564 bp and contains an intact open reading frame that encodes 751 amino acids showing 89% sequence identity and 94% similarity to human GFM . Northern blot analysis of human GFM revealed three transcripts of 3.8, 3.4, and 2.9 kb . The first two were expressed at high levels in heart, skeletal muscle, and testis, at moderate levels in liver and kidney, and at low levels in other tissues including brain, placenta, and lung, while the last transcript was expressed only in testis . The relative abundance of GFM was consistent with the observations for human EF-Tu(mt) and EF-Ts(mt), the other two mitochondrial elongation factors, indicating that the three factors were expressed at corresponding levels . The expression pattern of mouse Gfm was also determined, which showed that Gfm was expressed as a 3.0-kb transcript, abundantly in heart, skeletal muscle, kidney, and testis . In addition, GFM was assigned to human chromosome 3q25.1-q26.2 by the radiation hybrid mapping method . The genomic organization of GFM was also analyzed by comparing this cDNA with a genomic DNA sequence (Accession No . AC010936), which showed that GFM contained 18 exons and spanned at least 40 kb .

Mol Biol Evol, 2001 Jun, 18(6), 1057 - 69
Is the 16S-23S rRNA internal transcribed spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria; Boyer SL et al.; We amplified, TA-cloned, and sequenced the 16S-23S internal transcribed spacer (ITS) regions from single isolates of several cyanobacterial species, Calothrix parietina, Scytonema hyalinum, Coelodesmium wrangelii, Tolypothrix distorta, and a putative new genus (isolates SRS6 and SRS70), to investigate the potential of this DNA sequence for phylogenetic and population genetic studies . All isolates carried ITS regions containing the sequences coding for two tRNA molecules (tRNA and tRNA) . We retrieved additional sequences without tRNA features from both C . parietina and S . hyalinum . Furthermore, in S . hyalinum, we found two of these non-tRNA-encoding regions to be identical in length but different in sequence . This is the first report of ITS regions from a single cyanobacterial isolate not only different in configuration, but also, within one configuration, different in sequence . The potential of the ITS region as a tool for studying molecular systematics and population genetics is significant, but the presence of multiple nonidentical rRNA operons poses problems . Multiple nonidentical rRNA operons may impact both studies that depend on comparisons of phylogenetically homologous sequences and those that employ restriction enzyme digests of PCR products . We review current knowledge of the numbers and kinds of 16S-23S ITS regions present across bacterial groups and plastids, and we discuss broad patterns congruent with higher-level systematics of prokaryotes.

J Biol Chem, 2001 Jul 13, 276(28), 26724 - 31 Epub 2001 May 22.
Stage-dependent localization of a novel gene product of the malaria parasite, Plasmodium falciparum; Nguyen TV et al.; A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P . falciparum-infected and irradiated mosquitoes . The single-exon, single-copy MB2 gene is predicted to encode a protein with an M(r) of 187,000 . The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2 . MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes . The MB2 protein is distributed as a approximately 120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a approximately 66-kDa species . Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein . This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target.

Proc R Soc Lond B Biol Sci, 2001 May 7, 268(1470), 961 - 5
Cooperation in the dark: signalling and collective action in quorum-sensing bacteria; Brown SP et al.; The study of quorum-sensing bacteria has revealed a widespread mechanism of coordinating bacterial gene expression with cell density . By monitoring a constitutively produced signal molecule, individual bacteria can limit their expression of group-beneficial phenotypes to cell densities that guarantee an effective group outcome . In this paper, we attempt to move away from a commonly expressed view that these impressive feats of coordination are examples of multicellularity in prokaryotic populations . Here, we look more closely at the individual conflict underlying this cooperation, illustrating that, even under significant levels of genetic conflict, signalling and resultant cooperative behaviour can stably exist . A predictive two-trait model of signal strength and of the extent of cooperation is developed as a function of relatedness (reflecting multiplicity of infection) and basic population demographic parameters . The model predicts that the strength of quorum signalling will increase as conflict (multiplicity of infecting strains) increases, as individuals attempt to coax more cooperative contributions from their competitors, leading to a devaluation of the signal as an indicator of density . Conversely, as genetic conflict increases, the model predicts that the threshold density for cooperation will increase and the subsequent strength of group cooperation will be depressed.

Leukemia, 2001 Apr, 15(4), 601 - 12
Cloning and expression of human HBP1, a high mobility group protein that enhances myeloperoxidase (MPO) promoter activity; Lin KM et al.; Factors which regulate transcription in immature myeloid cells are of great current interest for the light they may shed upon myeloid differentiation . In the course of screening for transcription factors which interact with the human myeloperoxidase (MPO) promoter we, for the first time, identified and cloned the cDNA and genomic DNA for human HBP1 (HMG-Box containing protein 1), a member of the high mobility group of non-histone chromosomal proteins . HBP1 cDNA was initially cloned from rat brain in 1994, but its presence in human cells or in myeloid tissue had not been described previously . The sequence of human HBP1 cDNA shows 84% overall homology with the rat HBP1 cDNA sequence . We have subsequently cloned the gene, which is present as a single copy, 25 kbp in length . Northern blotting reveals a single 2.6 kb mRNA transcript which is expressed at higher levels in human myeloid and B lymphoid cell lines than in T cell lines tested and is present in several non-myeloid human cell lines . Comparison of the mRNA and genomic sequences reveals the gene to contain 10 exons and 9 introns . The sequence of human HBP1 mRNA contains a single open reading frame, which codes for a protein 514 amino acids in length . The amino acid sequence specified by the coding region shows 95% homology with the rat HBP1 protein . The human protein sequence exhibits a putative DNA-binding domain similar to that seen in rat HBP1 and shows homology with the activation and repressor domains previously demonstrated in the rat protein . We have expressed human HBP1 protein both in vitro and in prokaryotic and eukaryotic cells . The expressed fusion protein binds to a sequence in a functionally important region within the basal human MPO promoter . In transient co-transfection experiments HBP1 enhances MPO promoter activity . Human HBP1 appears to be a novel transcription factor which is likely to play an important role in regulating transcription in developing myeloid cells.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1998 Jun, 20(3), 168 - 72
{Cloning of human papillomavirus type 16 E7 gene and expression in E . coli}; Liu C et al.; OBJECTIVE: To study the biological activity of the E7 gene of human papillomavirus 16 (HPV16) . METHODS: E7 gene from the cervical carcinoma of Chinese women was amplified by the PCR . The E7 DNA was sequenced and compared with the prototype E7 gene of HPV 16 . RESULTS: It showed no mutations . However when E7 gene was inserted into the prokaryotic expression vector pGEX-2T and the fused E7 protein was efficiently expressed in E . Coli (DH5 alpha) . The E7 antibody could combine specifically with this fused protein by Western blot technique . CONCLUSIONS: The result might aid for epidemiological investigation and preparation of the vaccine.

Mol Genet Genomics, 2001 Apr, 265(2), 293 - 301
A functional OGG1 homologue from Arabidopsis thaliana; Dany AL et al.; One of the major mutagenic base lesions in DNA caused by exposure to reactive oxygen species is 7,8-dihydro-8-oxoguanine (8-oxoG) . Genes coding for DNA repair enzymes that recognise 8-oxoG have been reported in bacteria, yeast, mammals and plants . The prokaryotic and eukaryotic genes are functional homologues but differ in their primary sequence . We have cloned, sequenced, and expressed a new Arabidopsis thaliana cDNA that shows sequence homology to the eukaryotic genes coding for 8-oxoG DNA N-glycosylases (OGG1) . The 40.3-kDa enzyme it encodes (AtOGG1) introduces a chain break in a double-stranded oligonucleotide specifically at an 8-oxoG residue . In addition, AtOGG1 can form a Schiff base with 8-oxoG in the presence of NaBH4, suggesting that it is a bifunctional DNA N-glycosylase . Furthermore, expression of AtOGG1 in an Escherichia coli strain that is deficient in the repair of 8-oxoG in DNA suppresses its spontaneous-mutator phenotype . Thus, we have demonstrated that AtOGG1 is not only a structural but also a functional eukaryotic OGG1 homologue.

J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 491 - 7
Expression of growth factors in Dictyostelium discoideum; Asgari S et al.; Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors . Many growth factors require post-translational modifications making them unsuitable for production in Escherichia coli or other prokaryotes . Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins . Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring.

J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 457 - 9
Prokaryotic calmodulins: recent developments and evolutionary implications; Yang K; Ca2+ is a common intracellular second-messenger molecule in eukaryotic cells and regulates a myriad of cellular processes . Many effects of Ca2+ are mediated by calcium-binding regulatory proteins of the calmodulin superfamily . We propose the idea that calcium-binding proteins originated in high G+C Gram-positive bacteria and were later acquired by eukaryotes.

Mol Microbiol, 2001 May, 40(3), 509 - 19
A guided tour: small RNA function in Archaea; Dennis PP et al.; In eukaryotes, the C/D box family of small nucleolar (sno)RNAs contain complementary guide regions that are used to direct 2'-O-ribose methylation to specific nucleotide positions within rRNA during the early stages of ribosome biogenesis . Direct cDNA cloning and computational genome searches have revealed homologues of C/D box snoRNAs (called sRNAs) in prokaryotic Archaea that grow at high temperature . The guide sequences within the sRNAs indicate that they are used to direct methylation to nucleotides in both rRNAs and tRNAs . The number of sRNA genes that are detectable within currently sequenced genomes correlates with the optimal growth temperature . We suggest that archaeal sRNAs may have two functions: to guide the deposition of methyl groups at the 2'-O position of ribose, which is an important determinant in RNA structural stability, and to serve as a molecular chaperones to help orchestrate the folding of rRNAs and tRNAs at high temperature.

Science, 2001 Jun 8, 292(5523), 1903 - 6 Epub 2001 May 17.
Microbial genes in the human genome: lateral transfer or gene loss?
Salzberg SL, White O, Peterson J, Eisen JA.
The human genome was analyzed for evidence that genes had been laterally transferred into the genome from prokaryotic organisms . Protein sequence comparisons of the proteomes of human, fruit fly, nematode worm, yeast, mustard weed, eukaryotic parasites, and all completed prokaryote genomes were performed, and all genes shared between human and each of the other groups of organisms were collected . About 40 genes were found to be exclusively shared by humans and bacteria and are candidate examples of horizontal transfer from bacteria to vertebrates . Gene loss combined with sample size effects and evolutionary rate variation provide an alternative, more biologically plausible explanation.

Eur J Biochem, 2001 May, 268(10), 2896 - 904
The polypeptide chain release factor eRF1 specifically contacts the s(4)UGA stop codon located in the A site of eukaryotic ribosomes; Chavatte L et al.; It has been shown previously {Brown, C.M . & Tate, W.P . (1994) J . Biol . Chem . 269, 33164-33170.} that the polypeptide chain release factor RF2 involved in translation termination in prokaryotes was able to photocrossreact with mini-messenger RNAs containing stop signals in which U was replaced by 4-thiouridine (s4U) . Here, using the same strategy we have monitored photocrosslinking to eukaryotic ribosomal components of 14-mer mRNA in the presence of tRNA(f)(Met), and 42-mer mRNA in the presence of tRNA(Asp) (tRNA(Asp) gene transcript) . We show that: (a) both 14-mer and 42-mer mRNAs crossreact with ribosomal RNA and ribosomal proteins . The patterns of the crosslinked ribosomal proteins are similar with both mRNAs and sensitive to ionic conditions; (b) the crosslinking patterns obtained with 42-mer mRNAs show characteristic modification upon addition of tRNA(Asp) providing evidence for appropriate mRNA phasing onto the ribosome . Similar changes are not detected with the 14-mer mRNA.tRNA(f)(Met) pairs; (c) when eukaryotic polypeptide chain release factor 1 (eRF1) is added to the ribosome.tRNA(Asp) complex it crossreacts with the 42-mer mRNA containing the s(4)UGA stop codon located in the A site, but not with the s(4)UCA sense codon; this crosslink involves the N-terminal and middle domains of eRF1 but not the C domain which interacts with eukaryotic polypeptide chain release factor 3 (eRF3); (d) addition of eRF3 has no effect on the yield of eRF1-42-mer mRNA crosslinking and eRF3 does not crossreact with 42-mer mRNA . These experiments delineate the in vitro conditions allowing optimal phasing of mRNA on the eukaryotic ribosome and demonstrate a direct and specific contact of 'core' eRF1 and s(4)UGA stop codon within the ribosomal A site.

Prikl Biokhim Mikrobiol, 2001 Mar-Apr, 37(2), 141 - 55
{The fungi kingdom: heterogeneity of physiological-biochemical properties and closeness to plants, animals, and prokaryotes}; Feofilova EP; This review analyzes the current taxonomy of fungi whose criteria are based on a range of episemantic molecules . In this context, data on the chemical composition of fungal cells (membrane lipids, cytosolic carbohydrates, etc.) are considered in comparison with their counterparts found in higher eukaryotes and prokaryotes . Modern theories explaining the origin of fungi and their similarity to plants, animals, and bacteria are discussed . The biochemical criteria used in this work supported the division of fungi into Eumycota and Neomycota . The latter division, especially Basidiomycota, are more closely related to plants . The heterogeneity of the kingdom Fungi is underlined, the existence of Oomycota as a separate entity is supported, and the theory of the primitive nature of fungal cells is criticized from the viewpoint of biochemical adaptation.

Izv Akad Nauk Ser Biol, 2001 Mar-Apr, (2), 220 - 6
{Significance of the energy of symbiotic bacteria in metabolism of hydrothermal and other chemotrophic biota of the ocean}; Kuznetsov AP et al.; The bacterial origin of eukaryotic mitochondria, specifically in Metazoa, as a mechanism of their basic (aerobic) respiration and the role of symbiotic bacteria during the supply of energy to the metazoan host is proved for the first time from the viewpoint of the monophyletic development of the organic world and the origin of eukaryotes as descendants of prokaryotes Representatives of the hydrothermal bacteriochemosymbiotrophic bottom fauna of the open sea were used as examples.

J Biol Chem, 2001 Jul 13, 276(28), 25982 - 9 Epub 2001 May 15.
FtsY binds to the Escherichia coli inner membrane via interactions with phosphatidylethanolamine and membrane proteins; Millman JS et al.; Targeting of many polytopic proteins to the inner membrane of prokaryotes occurs via an essential signal recognition particle-like pathway . FtsY, the Escherichia coli homolog of the eukaryotic signal recognition particle receptor alpha-subunit, binds to membranes via its amino-terminal AN domain . We demonstrate that FtsY assembles on membranes via interactions with phosphatidylethanolamine and with a trypsin-sensitive component . Both interactions are mediated by the AN domain of FtsY . In the absence of phosphatidylethanolamine, the trypsin-sensitive component is sufficient for binding and function of FtsY in the targeting of membrane proteins . We propose a two-step mechanism for the assembly of FtsY on the membrane similar to that of SecA on the E . coli inner membrane.

Antimicrob Agents Chemother, 2001 Jun, 45(6), 1743 - 5
Trifluoromethionine, a prodrug designed against methionine gamma-lyase-containing pathogens, has efficacy in vitro and in vivo against Trichomonas vaginalis; Coombs GH et al.; Methionine gamma-lyase, the enzyme which catalyzes the single-step conversion of methionine to alpha-ketobutyrate, ammonia, and methanethiol, is highly active in many anaerobic pathogenic microorganisms but has no counterpart in mammals . This study tested the hypothesis that this pathogen-specific enzyme can be exploited as a drug target by prodrugs that are exclusively activated by it . Trifluoromethionine was confirmed as such a prodrug and shown to be highly toxic in vitro to the anaerobic protozoan parasite Trichomonas vaginalis, to anaerobic bacteria containing methionine gamma-lyase, and to Escherichia coli expressing the trichomonad gene . The compound also has exceptional activity against the parasite growing in vivo, with a single dose preventing lesion formation in five of the six mice challenged . These findings suggest that trifluoromethionine represents a lead compound for a novel class of anti-infective drugs with potential as chemotherapeutic agents against a range of prokaryotic and eukaryotic anaerobic pathogens.

Biochemistry, 2001 May 22, 40(20), 6155 - 63
A model for ligand binding to hexacoordinate hemoglobins; Trent JT 3rd et al.; Hexacoordinate hemoglobins are heme proteins capable of reversible intramolecular coordination of the ligand binding site by an amino acid side chain from within the heme pocket . Examples of these proteins are found in many living organisms ranging from prokaryotes to humans . The nonsymbiotic hemoglobins (nsHbs) are a class of hexacoordinate heme proteins present in all plants . The nsHb from rice (rHb1) has been used as a model system to develop methods for determining rate constants characterizing binding and dissociation of the His residue responsible for hexacoordination . Measurement of these reactions exploits laser flash photolysis to initiate the reaction from the unligated, pentacoordinate form of the heme protein . A model for ligand binding is presented that incorporates the reaction following rapid mixing with the reaction starting from the pentacoordinate hemoglobin (Hb) . This model is based on results indicating that ligand binding to hexacoordinate Hbs is not a simple combination of competing first order (hexacoordination) and second order (exogenous ligand binding) reactions . Ligand binding following rapid mixing is a multiphasic reaction displaying time courses ranging from milliseconds to minutes . The new model incorporates a "closed", slow reacting form of the protein that is not at rapid equilibrium with the reactive conformation . It is also demonstrated that formation of the closed protein species is not dependent on hexacoordination.

J Mol Biol, 2001 May 18, 308(5), 823 - 37
Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection; Patel PH et al.; Accurate transmission of DNA material from one generation to the next is crucial for prolonged cell survival . Following the discovery of DNA polymerse I in Escherichia coli, the DNA polymerase I class of enzymes has served as the prototype for studies on structural and biochemical mechanisms of DNA replication . Recently, a series of genomic, mutagenesis and structural investigations have provided key insights into how Pol I class of enzymes function and evolve . X-ray crystal structures of at least three Pol I class of enzymes have been solved in the presence of DNA and dNTP, thus allowing a detailed description of a productive replication complex . Rapid-quench stop-flow studies have helped define individual steps during nucleotide incorporation and conformational changes that are rate limiting during catalysis . Studies in our laboratory have generated large libraries of active mutant enzymes (8000) containing a variety of substitutions within the active site, some of which exhibit altered biochemical properties . Extensive genomic information of Pol I has recently become available, as over 50 polA genes from different prokaryotic species have been sequenced . In light of these advancements, we review here the structure-function relationships of Pol I, and we highlight those interactions that are responsible for the high fidelity of DNA synthesis . We present a mechanism for "flipping" of the complementary template base to enhance interactions with the incoming nucleotide substrate during DNA synthesis .

EMBO J, 2001 May 15, 20(10), 2587 - 95
Replication slippage involves DNA polymerase pausing and dissociation; Viguera E et al.; Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination . The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases . We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis . We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA . We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat . Resumption of DNA replication then completes the slippage process.

J Mol Biol, 2001 May 11, 308(4), 597 - 608
Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein; Schepers A et al.; The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication . Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins . These proteins share a number of conserved regions of homology and a common three-dimensional architecture . Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo . Here, we analyse mutants in the other conserved sequence motifs . Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature . In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine . Overexpression of this mutant protein is lethal . This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function .

J Biol Chem, 2001 Aug 10, 276(32), 30167 - 77 Epub 2001 May 10.
Contacts between the 5' nuclease of DNA polymerase I and its DNA substrate; Xu Y et al.; The 5' nuclease of DNA polymerase I (Pol I) of Escherichia coli is a member of an important class of prokaryotic and eukaryotic nucleases, involved in DNA replication and repair, with specificity for the junction between single-stranded and duplex DNA . We have investigated the interaction of the 5' nuclease domain with DNA substrates from the standpoint of both the protein and the DNA . Phosphate ethylation interference showed that the nuclease binds to the nucleotides immediately surrounding the cleavage site and also contacts the complementary strand one-half turn away, indicating that contacts are made to one face only of the duplex portion of the DNA substrate . Phosphodiester contacts were investigated further using DNA substrates carrying unique methylphosphonate substitutions, together with mutations in the 5' nuclease . These experiments suggested that two highly conserved basic residues, Lys(78) and Arg(81), are close to the phosphodiester immediately 5' to the cleavage site, while a third highly conserved residue, Arg(20), may interact with the phosphodiester 3' to the cleavage site . Our results provide strong support for a DNA binding model proposed for the related exonuclease from bacteriophage T5, in which the conserved basic residues mentioned above define the two ends of a helical arch that forms part of the single-stranded DNA-binding region . The nine highly conserved carboxylates in the active site region appear to play a relatively minor role in substrate binding, although they are crucial for catalysis . In addition to binding the DNA backbone around the cleavage point, the 5' nuclease also has a binding site for one or two frayed bases at the 3' end of an upstream primer strand . In agreement with work in related systems, 5' nuclease cleavage is blocked by duplex DNA in the 5' tail, but the enzyme is quite tolerant of abasic DNA or polarity reversal within the 5' tail.

Infect Immun, 2001 Jun, 69(6), 3658 - 62
Secreted variant of nucleoside diphosphate kinase from the intracellular parasitic nematode Trichinella spiralis; Gounaris K et al.; The molecular components involved in the survival of the parasitic nematode Trichinella spiralis in an intracellular environment are poorly characterized . Here we demonstrate that infective larvae secrete a nucleoside diphosphate kinase when maintained in vitro . The secreted enzyme forms a phosphohistidine intermediate and shows broad specificity in that it readily accepts gamma-phosphate from both ATP and GTP and donates it to all nucleoside and deoxynucleoside diphosphate acceptors tested . The enzyme was partially purified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by autophosphorylation and reactivity with an antibody to a plant-derived homologue . Secreted nucleoside diphosphate kinases have previously been identified only in prokaryotic organisms, all of them bacterial pathogens . The identification of a secreted variant of this enzyme from a multicellular eukaryote is very unusual and is suggestive of a role in modulating host cell function.

Folia Microbiol (Praha), 2000, 45(5), 407 - 13
WD-repeat protein encoding genes among prokaryotes of the Streptomyces genus; Stoytcheva Z et al.; Southern hybridization with probes designed for detection of WD-repeats coding sequences gave positive results in 21 streptomycete strains indicating that WD-repeats encoding genes are massively spread among streptomycetes . One of them, the wdlA gene of Streptomyces lincolnensis, codes for a 971 amino acid protein with seven WD-repeats in its C-terminus, two transmembrane domains and an ATP/GTP binding site upstream of the WD-repeat region.

Protein Sci, 2001 Mar, 10(3), 471 - 81
Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach; Koc EC et al.; Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis . Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion . The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry . Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico . The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases . Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified . Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes . These proteins are homologs to Escherichia coli S9 and S5 proteins . The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.

Biochim Biophys Acta, 2001 May 5, 1547(1), 104 - 16
Molecular cloning and functional characterization of a unique multipotent polyphenol oxidase from Marinomonas mediterranea; Sanchez-Amat A et al.; Marinomonas mediterranea is a recently isolated melanogenic marine bacterium containing laccase and tyrosinase activities . These activities are due to the expression of two polyphenol oxidases (PPOs), a blue multicopper laccase and an SDS-activated tyrosinase . The gene encoding the first one, herein denominated M . mediterranea PpoA, has been isolated by transposon mutagenesis, cloned and expressed in Escherichia coli . Its predicted amino acid sequence shows the existence of a signal peptide and four copper-binding sites characteristic of the blue multicopper proteins, including all fungal laccases . In addition, two additional putative copper-binding sites near its N-terminus are also present . Recombinant expression in E . coli of this protein clearly demonstrates its multipotent capability, showing both laccase-like and tyrosinase-like activities . This is the first prokaryotic laccase sequenced and the first PPO showing such multipotent catalytic activity . The expression of several truncated products indicates that the four copper-binding sites typical of blue multicopper proteins are essential for the laccase activity of this enzyme . However, the last two of these sites are not necessary for tyrosine hydroxylase activity as this activity is retained in a truncated product containing the first two sites as well as the extra histidine-rich clusters close to the N-terminus of the protein.

Biochim Biophys Acta, 2001 Feb 16, 1517(3), 376 - 83
Identification of human and mouse HIRA-interacting protein-5 (HIRIP5), two mammalian representatives in a family of phylogenetically conserved proteins with a role in the biogenesis of Fe/S proteins; Lorain S et al.; The human HIRA protein is encoded from a region of chromosome 22q that is critical for the DiGeorge syndrome and the velocardiofacial syndrome . We have previously reported that it directly interacts with core histones, with a novel histone-binding protein, HIRIP3, and during mouse embryogenesis, with the developmentally regulated homeodomain protein Pax3, suggesting a promoter-targeted function at the chromatin level . We here report on HIRA-interacting protein 5 (HIRIP5), a small acidic protein that interacted with HIRA in a double-hybrid screen performed in yeast and in in vitro protein interaction experiments . HIRIP5 has highly conserved homologs in both prokaryotes and eukaryotes, including the NFU1 gene product which has been implicated in iron metabolism in mitochondria of the yeast Saccharomyces cerevisiae . By radioactive in situ hybridization, the HIRIP5 gene was mapped to the 2p13-p15 chromosomal region, separate from any region previously associated with DiGeorge syndrome.

Biochim Biophys Acta, 2001 Feb 9, 1545(1-2), 367 - 71
Nucleotide and deduced amino acid sequences of an alkaline pullulanase from the alkaliphilic bacterium Bacillus sp . KSM-1876; Hatada Y et al.; The nucleotide sequence of an alkaline pullulanase-encoding gene from alkaliphilic Bacillus sp . strain KSM-1876 was determined . The open reading frame of the gene encoded 1142 amino acids with a calculated molecular mass of 128739 Da . The alkaline pullulanase showed very limited homology (<32% identity) to previously reported debranching enzymes from prokaryotes and eukaryotes . It contained unique tandem repeats in both the N-terminal and the C-terminal regions.

Mutat Res, 2001 Feb 25, 485(1), 23 - 36
Nucleotide excision repair "a legacy of creativity"; Cleaver JE et al.; The first half of the 20th century has seen an enormous growth in our knowledge of DNA repair, in no small part due to the work of Dirk Bootsma, Philip Hanawalt and Bryn Bridges; those honored by this issue . For the new millennium, we have asked three general questions: (A) Do we know all possible strategies of nucleotide excision repair (NER) in all organisms? (B) How is NER integrated and regulated in cells and tissues? (C) Does DNA replication represent a new frontier in the roles of DNA repair? We make some suggestions for the kinds of answers the next generation may provide . The kingdom of archea represents an untapped field for investigation of DNA repair in organisms with extreme lifestyles . NER appears to involve a similar strategy to the other kingdoms of prokaryotes and eukaryotes, but subtle differences suggest that individual components of the system may differ . NER appears to be regulated by several major factors, especially p53 and Rb which interact with transcription coupled repair and global genomic repair, respectively . Examples can be found of major regulatory changes in repair in testicular tissue and melanoma cells . Our understanding of replication of damaged DNA has undergone a revolution in recent years, with the discovery of multiple low-fidelity DNA polymerases that facilitate replicative bypass . A secondary mechanism of replication in the absence of NER or of one or more of these polymerases involves sister chromatid exchange and recombination (hMre11/hRad50/Nbs1) . The relative importance of bypass and recombination is determined by the action of p53 . We hypothesise that these polymerases may be involved in resolution of complex DNA structures during completion of replication and sister chromatid resolution . With these fascinating problems to investigate, the field of DNA repair will surely not disappoint the next generation.

Biochim Biophys Acta, 2001 Jan 15, 1530(1), 23 - 31
Identification of novel membrane-bound phospholipase D from Streptoverticillium cinnamoneum, possessing only hydrolytic activity; Ogino C et al.; A membrane-bound phospholipase D (PLD) has been identified and isolated in a soluble form from an actinomycete, Streptoverticillium cinnamoneum . The enzyme has a monomeric structure with a molecular size of about 37 kDa, being the smallest among the enzymes so far reported . The enzyme catalyzes the hydrolysis of phosphatidylethanolamine and phosphatidylserine as preferred substrates, but not the transphosphatidylation reaction of their phospholipid groups to ethanol . Together with the absence of immunochemical cross-reactivity, these enzymatic properties demonstrate that the membrane-bound enzyme is distinct from the extracellular enzyme recently characterized and cloned from the same bacterial strain {C . Ogino et al., J . Biochem . 125 (1999) 263-269} and is therefore regarded as a novel prokaryotic PLD.

Biochemistry, 2001 May 15, 40(19), 5615 - 21
Alanine-scanning mutagenesis of the small-subunit beta A-beta B loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: substitution at Arg-71 affects thermal stability and CO2/O2 specificity; Spreitzer RJ et al.; Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) enzymes from different species differ with respect to carboxylation catalytic efficiency and CO2/O2 specificity, but the structural basis for these differences is not known . Whereas much is known about the chloroplast-encoded large subunit, which contains the alpha/beta-barrel active site, much less is known about the role of the nuclear-encoded small subunit in Rubisco structure and function . In particular, a loop between beta-strands A and B contains 21 or more residues in plants and green algae, but only 10 residues in prokaryotes and nongreen algae . To determine the significance of these additional residues, a mutant of the green alga Chlamydomonas reinhardtii, which lacks both small-subunit genes, was used as a host for transformation with directed-mutant genes . Although previous studies had indicated that the betaA-betaB loop was essential for holoenzyme assembly, Ala substitutions at residues conserved among land plants and algae (Arg-59, Tyr-67, Tyr-68, Asp-69, and Arg-71) failed to block assembly or eliminate function . Only the Arg-71 --> Ala substitution causes a substantial decrease in holoenzyme thermal stability . Tyr-68 --> Ala and Asp-69 --> Ala enzymes have lower K(m)(CO2) values, but these improvements are offset by decreases in carboxylation V(max) values . The Arg-71 --> Ala enzyme has a decreased carboxylation V(max) and increased K(m)(CO2) and K(m)(O2) values, which account for an observed 8% decrease in CO2/O2 specificity . Despite the fact that Arg-71 is more than 20 A from the large-subunit active site, it is apparent that the small-subunit betaA-betaB loop region can influence catalytic efficiency and CO2/O2 specificity.

Exp Cell Res, 2001 May 15, 266(1), 11 - 20
Bacterial heat shock protein-60 increases epithelial cell proliferation through the ERK1/2 MAP kinases; Zhang L et al.; Heat shock proteins (hsp) have important roles in the regulation and protection of both prokaryotic and eukaryotic cells, especially during environmental stress . Hsps are also important bacterial virulence factors . We investigated whether bacterial hsp60 can alter epithelial cell mitogen-activated protein kinase (MAPK) signaling and cell proliferation . Human skin keratinocytes (HaCaT cell line) were cultured in the presence of hsp60 purified from Actinobacillus actinomycetemcomitans, an important oral pathogen . Protein kinases in the ERK1/2 and p38 MAPK signaling pathways were probed with kinase-specific and phosphorylation-site-specific antibodies on Western blots . In quiescent cultures, hsp60 increased ERK1/2 phosphorylation in a sustained manner and p38 phosphorylation transiently . Hsp60 also increased epithelial cell proliferation by about 30% . Inhibition of the ERK1/2 pathway by PD 98059 (a MEK1 inhibitor) reversed partially ERK1/2 phosphorylation and totally cell proliferation indicating that the ERK1/2 MAPK pathway is involved in the hsp60-induced cell growth . This was supported by findings that hsp60 stimulated phosphorylation of RSK1/2 and cyclic AMP response element-binding protein and increased expression of transcription factors c-Jun and c-Fos . Recombinant human hsp60 did not alter ERK1/2 or p38 phosphorylation and had no effect on epithelial cell proliferation . Inhibition of p38 MAPK pathway by SB 203580 increased both ERK1/2 phosphorylation and cell proliferation demonstrating that the inhibitor can either directly or indirectly activate the ERK1/2 MAPK pathway . The results show that exogenous bacterial hsp60 is able to activate ERK1/2 phosphorylation and thereby cause increased epithelial proliferation . In case of mucosal infection this effect may either lead to increased wound repair or participate in the pathological mechanism of some bacterial diseases that involve increased epithelial proliferation .

Mikrobiologiia, 2001 Jan-Feb, 70(1), 86 - 91
{A system of oligonucleotide primers for amplifying nifH genes from various taxonomic groups of prokaryotes}; Marusina AI et al.; Based on the analysis of the nifH gene nucleotide sequences from GenBank, a system of primers was developed that makes it possible to obtain 370- and 470-bp PCR fragments of the nifH gene of nitrogen-fixing bacteria and archaea . The effectiveness of the proposed system for revealing the presence of nifH genes was demonstrated by PCR on the DNA isolated from nitrogen-fixing prokaryotes for which the primary structure of these genes is known and which belong to different taxonomic groups . nifH sequences of nitrogen-fixing prokaryotes of the genera Xanthobacter, Beijerinckia, and Methanosarcina, for which the capacity for nitrogen fixation was demonstrated earlier, but no data existed on the nucleotide composition of these genes, were determined and deposited in GenBank.

Photodermatol Photoimmunol Photomed, 2001 Apr, 17(2), 47 - 54
Xeroderma pigmentosum--bridging a gap between clinic and laboratory; Moriwaki S et al.; Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of UV-related skin cancers associated with impaired ability to repair UV-induced DNA damage . There are seven nucleotide excision repair (NER) complementation groups (A through G) and an NER proficient form (XP variant) . XPA, B, D and G patients may also develop XP neurological disease . The laboratory diagnosis of XP can be performed by autoradiography . Recently, the isolation and characterization of the genes responsible for XP have made it possible to use molecular biological techniques to diagnose XP patients, for carrier detection and for prenatal diagnosis, especially in Japanese XPA patients . These techniques include polymerase chain reaction (PCR) and plasmid host cell reactivation assays with cloned XP genes . DNA damage is not repaired by the NER system equally throughout the genome . There are two DNA repair pathways: 1) transcription-coupled repair, and 2) global genome repair . Many factors involved in these pathways are related to the pathogenesis of XP and a related photosensitive disease, Cockayne syndrome . Clinical management consists of early diagnosis followed by a rigorous program of sun protection including avoidance of unnecessary UV exposure, wearing UV blocking clothing, and use of sunblocks on the skin . Although there is no cure for XP, the efficacy of oral retinoids for the prevention of new skin cancers, local injection of interferon, and the external use of a prokaryotic DNA repair enzyme have been reported.

Annu Rev Plant Physiol Plant Mol Biol, 2001 Jun, 52, 315 - 333
THE PLASTID DIVISION MACHINE; Osteryoung KW et al.; Plastid division is essential for the maintenance of plastid populations in cells undergoing division and for the accumulation of large chloroplast numbers in photosynthetic tissues . Although the mechanisms mediating plastid division are poorly understood, ultrastructural studies imply this process is accomplished by a dynamic macromolecular machine organized into ring structures at the plastid midpoint . A key component of the engine that powers this machine is the motor-like protein FtsZ, a cytoskeletal GTPase of endosymbiotic origin that forms a ring at the plastid division site, similar to the function of its prokaryotic relatives in bacterial cytokinesis . This review considers the phylogenetic distribution and structural properties of two recently identified plant FtsZ protein families in the context of their distinct roles in plastid division and describes current evidence regarding factors that govern their placement at the division site . Because of their evolutionary and mechanistic relationship, the process of bacterial cell division provides a valuable, though incomplete, paradigm for understanding plastid division in plants.

Comp Biochem Physiol B Biochem Mol Biol, 2001 May, 129(1), 17 - 28
Phylogeny of ion channels: clues to structure and function; Anderson PA et al.; Voltage-gated ion channels are responsible for the electrical activity in a variety of cell types in modern-day animals . However, they represent the result of many millions of years of evolution of a family of ion channel proteins that are also found in prokaryotes and diverse eukaryotes, and probably exist in all life forms . This review traces the evolution of ion channels, with particular emphasis on the factors and evolutionary pathways that may have given rise to voltage-gated potassium (K+), calcium (Ca2+), and sodium (Na+) channels . The review also highlights the utility of comparing phylogenetically distinct versions of the same protein as a means to better understand the structure and function of proteins.

Pharmacol Ther, 2000 Dec, 88(3), 349 - 425
Purine nucleoside phosphorylases: properties, functions, and clinical aspects; Bzowska A et al.; The ubiquitous purine nucleoside phosphorylases (PNPs) play a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function . This review updates the properties of the enzymes from eukaryotes and a wide range of prokaryotes, including a tentative classification of the enzymes from various sources, based on three-dimensional structures in the solid state, subunit composition, amino acid sequences, and substrate specificities . Attention is drawn to the compelling need of quantitative experimental data on subunit composition in solution, binding constants, and stoichiometry of binding; order of ligand binding and release; and its possible relevance to the complex kinetics exhibited with some substrates . Mutations responsible for PNP deficiency are described, as well as clinical methods, including gene therapy, for corrections of this usually fatal disease . Substrate discrimination between enzymes from different sources is also being profited from for development of tumour-directed gene therapy . Detailed accounts are presented of design of potent inhibitors, largely nucleosides and acyclonucleosides, their phosphates and phosphonates, particularly of the human erythrocyte enzyme, some with Ki values in nanomolar and picomolar range, intended for induction of the immunodeficient state for clinical applications, such as prevention of host-versus-graft response in organ transplantations . Methods of assay of PNP activity are reviewed . Also described are applications of PNP from various sources as tools for the enzymatic synthesis of otherwise inaccessible therapeutic nucleoside analogues, as coupling enzymes for assays of orthophosphate in biological systems in the micromolar and submicromolar ranges, and for coupled assays of other enzyme systems.

Virology, 2001 May 10, 283(2), 343 - 52
Membrane relocation but not tight binding of human immunodeficiency virus type 1 Gag particles myristoylated in Escherichia coli; Morikawa Y et al.; Expression of human immunodeficiency virus Gag protein and the N-terminal matrix (MA) domain in Escherichia coli yielded spherical structures in the cytoplasm . When human N-myristoyltransferase was coexpressed, both Gag and MA were fully myristoylated and spherical structures were relocated in close proximity to the cytoplasmic membrane . However, neither myristoylated Gag nor MA exhibited tight binding to E . coli membrane, suggesting that myristoylation in E . coli did not confer membrane affinity on Gag despite the relocation . Our data also suggest that the morphogenetic pathway of Gag particles in prokaryotic cells differs from that in eukaryotic cells despite biochemical similarities of in the form of Gag expressed .

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2001 Mar, 18(1), 68 - 71
{Expression of human brain derived neurotrophic factor gene in E . coli}; Liu Z et al.; The primers specific for the full-length BDNF coding sequence was designed and synthesized . The BDNF coding sequence was directly amplified from human genomic DNA by using PCR and inserted into vector pGEM-3Zf(+) . The recombinant DNA was transformed into the host cells JM109 to obtain the positive clone pGEMBF18 . The restriction enzyme analysis and DNA sequence detection confirmed that the inserted fragment of clone pGEMBF18 is the full-length BDNF coding sequence . The hBDNF DNA fragment was recovered from the clone pGEMBF18 and ligated with prokaryotic expression vector pGEX-5T to construct the recombinant expression plasmid p5TBF34 . The E . coli JM109 transformed with p5TBF34 was induced with IPTG . A new protein band with apparent molecular weight 43 kDa was detected in the lysate of the transformed cell by using SDS-PAGE . The result of western hybridization showed that this fusion protein reacted specifically to the antibodies to human BDNF . The amount of the soluble fusion protein was about 503.04 mg/L lysate, 7.53% of total bacterial soluble protein of transformed cells, estimated by absorbance scanning of SDS-PAGE and protein quantitation.

Yi Chuan Xue Bao, 2001, 28(4), 306 - 12
{Prokaryotic expression of recombinant grass carp growth hormone}; Wang W et al.; The recombinant plasmid containing a synthetic grass carp growth hormone cDNA was transformed into Escherichia coli BL21 (DE3) and expressed efficiently, induced by 1 mmol/L IPTG . 12.5% SDS-PAGE showed the molecular weight of new added protein in Escherichia coli BL21 (DE3) is consistent with that of grass carp growth hormone . The recombinant protein was composed of approximately 40% of the total bacterial proteins . The target protein was purified with Ni-NTA affinity column . Western-blotting conformed that recombinant protein could specifically react with antibody against grass carp growth hormone . ELISA-receptor assay showed the bioactivity of recombinant protein is consistent with that of nature grass carp growth hormone.

Biochemistry, 2001 Feb 20, 40(7), 1930 - 6
Covariation of a specificity-determining structural motif in an aminoacyl-tRNA synthetase and a tRNA identity element; Hawko SA et al.; Transfer RNA (tRNA) identity determinants help preserve the specificity of aminoacylation in vivo, and prevent cross-species interactions . Here, we investigate covariation between the discriminator base (N73) element in histidine tRNAs and residues in the histidyl-tRNA synthetase (HisRS) motif 2 loop . A model of the Escherichia coli HisRS--tRNA(His) complex predicts an interaction between the prokaryotic conserved glutamine 118 of the motif 2 loop and cytosine 73 . The substitution of Gln 118 in motif 2 with glutamate decreased discrimination between cytosine and uracil some 50-fold, but left overall rates of adenylation and aminoacylation unaffected . By contrast, substitutions at neighboring Glu 115 and Arg 121 affected both adenylation and aminoacylation, consistent with their predicted involvement in both half-reactions . Additional evidence for the involvement of the motif 2 loop was provided by functional analysis of a hybrid Saccharomyces cerevisiae-- E . coli HisRS possessing the 11 amino acid motif 2 loop of the yeast enzyme . Despite an overall decreased activity of nearly 1000-fold relative to the E . coli enzyme, the chimera nevertheless exhibited a modest preference for the yeast tRNA(His) over the E . coli tRNA, and preferred wild-type yeast tRNA(His) to a variant with C at the discriminator position . These experiments suggest that part of, but not all of, the specificity is provided by the motif 2 loop . The close interaction between enzyme loop and RNA sequence elements suggested by these experiments reflects a covariation between enzyme and tRNA that may have acted to preserve aminoacylation fidelity over evolutionary time.

J Antimicrob Chemother, 2001 May, 47(5), 671 - 4
Activities of cholic acid-derived antimicrobial agents against multidrug-resistant bacteria; Schmidt EJ et al.; Cationic cholic acid derivatives displayed potent and broad-spectrum activity against multidrug-resistant Gram-negative and -positive bacteria . Specific examples were effective permeabilizers of the outer membranes of many strains of multidrug-resistant Gram-negative bacteria and sensitized these to hydrophobic antibiotics . We also prepared a new cholic acid derivative with improved apparent selectivity for prokaryote membranes.

Helicobacter, 2001 Mar, 6(1), 55 - 9
A possible relation of the Helicobacter pylori pfr gene to iron deficiency anemia?
Choe YH, Hwang TS, Kim HJ, Shin SH, Song SU, Choi MS.
BACKGROUND: H . pylori infection is thought to contribute to iron-deficiency anemia, especially during puberty . The ferritin protein Pfr of H . pylori is homologous to eukaryotic and prokaryotic ferritins . The purpose of this study was to analyze the H . pylori pfr status in gastric biopsy specimens according to clinical data, including antral gastritis with or without iron-deficiency anemia . METHODS: A total of 26 H . pylori-positive patients aged from 10-18 years were categorized into subgroups based on the presence or absence of iron-deficiency anemia . All of them had antral gastritis . Sixteen patients were proved to have iron-deficiency anemia by hematological study, two of which had a duodenal ulcer . The other 10 patients showed normal hematological findings . DNA isolation was performed from each of the gastric biopsy specimens . PCR amplification of the pfr gene coding was done using two sets of primers . The pfr region, 501 bp, was generated by linking the sequences of the two PCR products . The nucleotide and protein sequences were compared between the pfr regions from Korean H . pylori strains and the NCTC 11638 strain, which was obtained from the Genbank . Sequence comparisons were also performed for the pfr regions between the iron-deficiency anemia (+) and (-) groups . RESULTS: Analysis of the complete coding region of the pfr gene revealed three sites of mutation . The Ser39Ala mutation was found in 100% (26/26), Gly111Asn in 26.9% (7/26), and Gly82Ser in 11.5% (3/26) . There were no significant differences in the mutations of the pfr regions between the iron deficiency anemia (+) and (-) groups . CONCLUSION: The mutation in the pfr gene did not relate with the clinical phenotype, iron deficiency anemia . Further studies are needed on the aspects of host side or other complex factors to elucidate the mechanisms by which the H . pylori infection might lead to iron deficiency anemia.

J Biol Chem, 2001 Jul 6, 276(27), 24690 - 6 Epub 2001 Apr 26.
The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1; Urano T et al.; In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1) . Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B . subtilis (natto) . Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments . Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)) . rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm) . Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm) . The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT . The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1 . This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.

J Bacteriol, 2001 May, 183(10), 2995 - 3003
Forespore-specific transcription of the lonB gene during sporulation in Bacillus subtilis; Serrano M et al.; The Bacillus subtilis genome encodes two members of the Lon family of prokaryotic ATP-dependent proteases . One, LonA, is produced in response to temperature, osmotic, and oxidative stress and has also been implicated in preventing sigma(G) activity under nonsporulation conditions . The second is encoded by the lonB gene, which resides immediately upstream from lonA . Here we report that transcription of lonB occurs during sporulation under sigma(F) control and thus is restricted to the prespore compartment of sporulating cells . First, expression of a lonB-lacZ transcriptional fusion was abolished in strains unable to produce sigma(F) but remained unaffected upon disruption of the genes encoding the early and late mother cell regulators sigma(E) and sigma(K) or the late forespore regulator sigma(G) . Second, the fluorescence of strains harboring a lonB-gfp fusion was confined to the prespore compartment and depended on sigma(F) production . Last, primer extension analysis of the lonB transcript revealed -10 and -35 sequences resembling the consensus sequence recognized by sigma(F)-containing RNA polymerase . We further show that the lonB message accumulated as a single monocistronic transcript during sporulation, synthesis of which required sigma(F) activity . Disruption of the lonB gene did not confer any discernible sporulation phenotype to otherwise wild-type cells, nor did expression of lonB from a multicopy plasmid . In contrast, expression of a fusion of the lonB promoter to the lonA gene severely reduced expression of the sigma(G)-dependent sspE gene and the frequency of sporulation . In confirmation of earlier observations, we found elevated levels of sigma(F)-dependent activity in a spoIIIE47 mutant, in which the lonB region of the chromosome is not translocated into the prespore . Expression of either lonB or the P(lonB)-lonA fusion from a plasmid in the spoIIIE47 mutant reduced sigma(F) -dependent activity to wild-type levels . The results suggest that both LonA and LonB can prevent abnormally high sigma(F) activity but that only LonA can negatively regulate sigma(G).

Biophys J, 2001 May, 80(5), 2074 - 81
Structural determinants of MscL gating studied by molecular dynamics simulations; Gullingsrud J et al.; The mechanosensitive channel of large conductance (MscL) in prokaryotes plays a crucial role in exocytosis as well as in the response to osmotic downshock . The channel can be gated by tension in the membrane bilayer . The determination of functionally important residues in MscL, patch-clamp studies of pressure-conductance relationships, and the recently elucidated crystal structure of MscL from Mycobacterium tuberculosis have guided the search for the mechanism of MscL gating . Here, we present a molecular dynamics study of the MscL protein embedded in a fully hydrated POPC bilayer . Simulations totaling 3 ns in length were carried out under conditions of constant temperature and pressure using periodic boundary conditions and full electrostatics . The protein remained in the closed state corresponding to the crystal structure, as evidenced by its impermeability to water . Analysis of equilibrium fluctuations showed that the protein was least mobile in the narrowest part of the channel . The gating process was investigated through simulations of the bare protein under conditions of constant surface tension . Under a range of conditions, the transmembrane helices flattened as the pore widened . Implications for the gating mechanism in light of these and experimental results are discussed.

Biochim Biophys Acta, 2001 Apr 30, 1531(3), 165 - 8
Characterization of mevalonate kinase V377I, a mutant implicated in defective isoprenoid biosynthesis and HIDS/periodic fever syndrome; Rios SE et al.; The list of diseases linked to defects in lipid metabolism has recently been augmented by the addition of hyperimmunoglobulinemia D and periodic fever syndrome (HIDS: MIM 260920), which are correlated with depressed levels of mevalonate kinase activity {1,2} and protein {1} . More specifically, a V377I substitution has been proposed to account for this disease . We observed that V377 appears to be far from invariant in eukaryotic mevalonate kinases . Prokaryotic mevalonate kinases are lower in molecular weight and several terminate prior to residue 377 of the eukaryotic proteins . These observations prompted our direct test of the impact of V377 on activity and protein stability by engineering a V377I mutation in a recombinant human mevalonate kinase . The mutant protein has been isolated and kinetically characterized . In comparison with wild-type enzyme, V377I exhibits only modest differences (notably > or = 6-fold inflation of K(m(MVA))) that do not account for the diminished mevalonate kinase activity assayed in HIDS cell extracts . Moreover, thermal inactivation (50 degrees C) of isolated wild-type and V377I enzymes demonstrates little difference in stability between these proteins . We conclude that a single V377I substitution is unlikely to explain the observation of depressed mevalonate kinase stability and catalytic activity in HIDS.

FEMS Microbiol Lett, 2001 Apr 20, 198(1), 1 - 7
Structure, function, and assembly of the terminal organelle of Mycoplasma pneumoniae; Krause DC et al.; Mycoplasmas are cell wall-less bacteria at the low extreme in genome size in the known prokaryote world, and the minimal nature of their genomes is clearly reflected in their metabolic and regulatory austerity . Despite this apparent simplicity, certain species such as Mycoplasma pneumoniae possess a complex terminal organelle that functions in cytadherence, gliding motility, and cell division . The attachment organelle is a membrane-bound extension of the cell and is characterized by an electron-dense core that is part of the mycoplasma cytoskeleton, defined here for working purposes as the protein fraction that remains after extraction with the detergent Triton X-100 . This review focuses on the architecture and assembly of the terminal organelle of M . pneumoniae . Characterizing the downstream consequences of defects involving attachment organelle components has made it possible to begin to elucidate the probable sequence of certain events in the biogenesis of this structure.

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 193 - 200
Prokaryote multidrug efflux proteins of the major facilitator superfamily: amplified expression, purification and characterisation; Ward A et al.; In bacterial genomes 3-12% of open reading frames are predicted to encode membrane transport proteins . These proteins can be vital for antibiotic efflux, protein/ toxin secretion, cell nutrition, environmental sensing, ATP synthesis, and other functions . Some, such as the multidrug efflux proteins, are potential targets for the development of new antibacterials and also for applications in biotechnology . In general membrane transport proteins are poorly understood, because of the technical difficulties involved in isolating sufficient protein for elucidation of their structure-activity relationships . We describe a general strategy for the amplified expression, purification and characterisation of prokaryotic multidrug efflux proteins of the 'Major facilitator superfamily' of transport proteins, using the Bacillus subtilis multidrug resistance protein, 'Bmr', as example.

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 185 - 92
Molecular basis of multidrug transport by ATP-binding cassette transporters: a proposed two-cylinder engine model; van Veen HW et al.; ATP-binding cassette multidrug transporters are probably present in all living cells, and are able to export a variety of structurally unrelated compounds at the expense of ATP hydrolysis . The elevated expression of these proteins in multidrug resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms . Multidrug transporters interact directly with the drug substrates . Insights into the structural elements in drug molecules and transport proteins that are required for this interaction are now beginning to emerge . However, much remains to be learned about the nature and number of drug binding sites in the transporters, and the mechanism(s) by which ATP hydrolysis is coupled to changes in affinity and/or accessibility of drug binding sites . This review summarizes recent advances in answering these questions for the human multidrug resistance P-glycoprotein and its prokaryotic homolog LmrA . The relevance of these findings for other ATP-binding cassette transporters will be discussed.

Environ Microbiol, 2001 Mar, 3(3), 168 - 75
Differential expression of antenna and core genes in Prochlorococcus PCC 9511 (Oxyphotobacteria) grown under a modulated light-dark cycle; Garczarek L et al.; The continuous changes in incident solar light occurring during the day oblige oxyphototrophs, such as the marine prokaryote Prochlorococcus, to modulate the synthesis and degradation rates of their photosynthetic components finely . How this natural phenomenon influences the diel expression of photosynthetic genes has never been studied in this ecologically important oxyphotobacterium . Here, the high light-adapted strain Prochlorococcus sp . PCC 9511 was grown in large-volume continuous culture under a modulated 12 h-12 h light-dark cycle mimicking the conditions found in the upper layer of equatorial oceans . The pcbA gene encoding the major light-harvesting complex showed strong diel variations in transcript levels with two maxima, one before the onset of illumination and the other near the end of the photoperiod . In contrast, the mRNA level of psbA (encoding the reaction centre II subunit D1), the monocistronic transcript of psbD (encoding D2) and the dicistronic transcript of psbDC were all tightly correlated with light irradiance, with a minimum at night and a maximum at noon . The occurrence of a second peak during the dark period for the monocistronic transcript of psbC (encoding one of the PS II core Chl a antenna proteins) suggested the involvement of post-transcriptional regulation . Differential expression of the external antenna and core genes may constitute a mechanism of regulation of the antenna size to cope with the excess photon fluxes that Prochlorococcus cells experience in the upper layer of oceans around midday . The 5' ends of all transcripts were mapped, and a conserved motif, 5'-TTGATGA-3', was identified within the putative psbA and pcbA promoters.

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 667 - 78
Relationship of 16S rRNA sequence similarity to DNA hybridization in prokaryotes; Keswani J et al.; The relationship between 16S rRNA sequence similarity (S) and the extent of DNA hybridization (D) was well described by the equation In(-InD) = 0.53 {In(-InS)}+2.201 when D was determined by either the S1 nuclease or membrane filter methods . When the presence of nonultrametric rRNA sequences and differences between genera or families were controlled, this relationship accounted for 78% of the variability of D given S, and it was possible to estimate the distribution of D from S with a known precision . Thus, D<0.70 was expected to occur 50, 95 and 99% of the time when S was 0.998, 0.992 and 0.986, respectively . The relationship between D and S varied between prokaryotic taxa even within the same subphylum, and more precise estimates of D could be made when the relationship for a particular taxon was known . The relationship between D and S was not significantly different between the prokaryotic domains, and S appeared to be a quasi-molecular clock of approximately constant rate when averaging effects and stochastic factors were taken into account . The relationship between logD and logS was nonlinear, and D provided a very poor measure of relatedness for distantly related organisms . For instance, within the range 1.0 >S>> 0.95, D decreased from 1.0 to 0.15; and within the range 0.95 >S> 0.90, D decreased from 0.15 to 0.06 . Lastly, at least some of the rRNA sequences from about one-third of the taxa examined had nonultrametric properties where S was much lower than expected from the value of D . For these taxa, S was a poor indicator of relatedness for closely related strains . Thus, the ultrametric properties of rRNA sequences should be tested before making taxonomic or phylogenetic conclusions based upon S.

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 385 - 91
A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data; Dalevi D et al.; The 16S rRNA gene (16S rDNA) is currently the most widely used gene for estimating the evolutionary history of prokaryotes . To date, there are more than 30,000 16S rDNA sequences available from the core databases, GenBank, EMBL and DDBJ . This great number may cause a dilemma when composing datasets for phylogenetic analysis, since the choice and number of reference organisms are known to affect the resulting tree topology . A group of sequences appearing monophyletic in one dataset may not be so in another . This can be especially problematic when establishing the relationships of distantly related sequences at the division (phylum) level . In this study, a multiple-outgroup approach to resolving division-level phylogenetic relationships is suggested using 16S rDNA data . The approach is illustrated by two case studies concerning the monophyly of two recently proposed bacterial divisions, OP9 and OP10.

Microbiology, 2001 May, 147(Pt 5), 1235 - 41
Cyclic heptapeptide microcystin biosynthesis requires the glutamate racemase gene; Nishizawa T et al.; It was demonstrated previously that the operon consisting of the non-ribosomal peptide synthetase (NRPS) gene coupled with the polyketide synthase (PKS) gene involved in cyclic heptapeptide microcystin synthesis includes two different D-amino acid synthetase genes, an epimerization domain at the 3' end of module 2, and the racemase gene mcyF . To determine the role of mcyF in microcystin synthesis, gene-disruption and complementation analyses were carried out . Insertional mutagenesis in the mcyF gene, generated by homologous recombination, abolished only microcystin synthesis, but did not influence cell growth . Furthermore, McyF supported D-Glu-independent growth of a strain of Escherichia coli defective in D-Glu synthesis . It is concluded that mcyF is the glutamic acid racemase gene involved in the synthesis of D-Glu residues in the microcystin molecule . This is the first report of the racemase in prokaryotic NRPS.

Mol Ther, 2001 Apr, 3(4), 591 - 601
Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome; Saeki Y et al.; Herpes simplex virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal . In the presence of HSV-1 "helper" functions, amplicons are replicated and packaged into HSV-1 virions . Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6) . To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent helper virus (<1 helper virus per 10(8) TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration . Elimination of replication-competent helper virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

J Biol Chem, 2001 Jun 29, 276(26), 23639 - 44 Epub 2001 Apr 20.
DnaA box sequences as the site for helicase delivery during plasmid RK2 replication initiation in Escherichia coli; Pacek M et al.; DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements . Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site . For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D . R., Pacek, M., and Konieczny, I . (2000) J . Biol . Chem . 275, 18454-18461) . Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized approximately 200 base pairs upstream from the region of origin opening and a potential site for helicase entry . The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates . A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed . In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region . This interaction is facilitated via direct contact between TrfA and DnaB proteins.

Arch Virol, 2001, 146(2), 303 - 26
Identification, transcription and sequence analysis of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase gene; Huang J et al.; Sequence analysis of a 6.4 kb DNA region from the Spodoptera littoralis multinucleocapsid nucleopolyhedrovirus (SpliNPV) revealed a large open reading frame (ORF) encoding a predicted polypeptide of 998 amino acid (aa) residues with a molecular mass of 114.93 kDa, located between 47.2-52.3 m.u . on the SpliNPV genome . Comparative sequence analyses demonstrated that the ORF encodes a DNA polymerase gene (dnapol) that contains conserved exonuclease domains and DNA polymerase motifs found in many prokaryotic, eukaryotic, and viral replicative DNA polymerases . A second ORF, ORF138, located between the lef-3 and dnapol, encodes a 138 aa polypetide that is homologous to ORF66 of the Autographa californica MNPV (AcMNPV) . SpliNPV DNA polymerase shares an overall aa sequence identity of 39% with that of AcMNPV . A 3.0 kb SpliNPV dnapol-specific transcript was detected initially at 2 hpi and became abundant 48 hpi by Northern blot analysis . The transcription initiation site was mapped to an NPV early promoter element, ACGT . 3' RACE demonstrated that the SpliNPV dnapol transcript terminated at the polyadenylation signal AATAAA . Sequence analysis suggested that the SpliNPV dnapol and the dnapol of the NPV of S . litura (SpltNPV) are closely related.

Mol Microbiol, 2001 Apr, 40(2), 476 - 84
Regulation of translation elongation in cyanobacteria: membrane targeting of the ribosome nascent-chain complexes controls the synthesis of D1 protein; Tyystjarvi T et al.; The photosystem II reaction centre protein D1 is encoded by the psbA gene . The D1 protein is stable in darkness but undergoes rapid turnover in the light . Here, we show that, in cyanobacterium Synechocystis sp . PCC6803, the synthesis of the D1 protein is regulated at the level of translation elongation in addition to the previously known transcriptional regulation . When Synechocystis sp . PCC6803 cells were transferred from light to darkness, the psbA mRNA remained abundant for hours . Cytosolic ribosomes were attached to psbA transcripts in the dark, and translation continued up to a distinct pausing site . However, ribosome nascent D1 chain complexes were not targeted to the thylakoid membrane, and no full-length D1 protein was produced in darkness . The arrest in translation elongation was released in the light, concomitantly with targeting of ribosome D1 nascent-chain complexes to the thylakoid membrane, allowing the synthesis of the full-length D1 protein . Downregulation of membrane targeting of ribosome complexes was also observed in the light if damage to the D1 protein was slow . This novel type of regulation of prokaryotic translation functions to balance the synthesis and degradation of the rapidly turning over photosystem II D1 protein in Synechocystis sp . PCC6803.

Biol Chem, 2001 Feb, 382(2), 179 - 86
Mitochondrial single-stranded DNA-binding proteins: in search for new functions; Tomaska L et al.; During the evolution of the eukaryotic cell, genes encoding proteins involved in the metabolism of mitochondrial DNA (mtDNA) have been transferred from the endosymbiont into the host genome . Mitochondrial single-stranded DNA-binding (mtSSB) proteins serve as an excellent argument supporting this aspect of the endosymbiotic theory . The crystal structure of the human mtSSB, together with an abundance of biochemical and genetic data, revealed several exciting features of mtSSB proteins and enabled a detailed comparison with their prokaryotic counterparts . Moreover, identification of a novel member of the mtSSB family, mitochondrial telomere-binding protein of the yeast Candida parapsilosis, has raised interesting questions regarding mtDNA metabolism and evolution.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 955 - 62
Binding of pyridine coenzymes to the beta-subunit of the voltage sensitive potassium channels; Liu SQ et al.; The beta-subunit of the voltage-sensitive K(+) channels shares 15-30% amino acid identity with the sequences of aldo-keto reductases (AKR) genes . However, the AKR properties of the protein remain unknown . To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro . The cDNA of K(v)beta2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli . The purified protein was tetrameric in solution as determined by size exclusion chromatography . The protein displayed high affinity binding to NADPH as determined by fluorometric titration . The K(D) values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1+/-0.007 and 0.05+/-0.006 M, respectively - indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the alpha-subunit . The protein displayed poor affinity for other coenzymes and the corresponding values of the K(D) for NADH and NAD were between 1-3 microM whereas the K(D) for FAD was >10 microM . However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2' phosphate at the binding site . The selectivity of K(v)beta2.1 for NADPH over NADP may be significant in regulating the K(+) channels as a function of the cellular redox state.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 815 - 24
Metabolic activation of polycyclic aromatic hydrocarbon trans-dihydrodiols by ubiquitously expressed aldehyde reductase (AKR1A1); Palackal NT et al.; Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by CYP1A1 and epoxide hydrolase (EH) . CYP1A1 or aldo-keto reductases (AKRs) from the 1C subfamily can further activate the trans-dihydrodiols by forming either anti-diol-epoxides or reactive and redox active o-quinones, respectively . To determine whether other AKR superfamily members can divert trans-dihydrodiols to o-quinones, the cDNA encoding human aldehyde reductase (AKR1A1) was isolated from hepatoma HepG2 cells using RT-PCR, subcloned into a prokaryotic expression vector, overexpressed in E . coli and purified to homogeneity in milligram amounts . Studies revealed that AKR1A1 preferentially oxidized the metabolically relevant (-)-{3R,4R}-dihydroxy-3,4-dihydrobenz{a}anthracene . AKR1A1 also displayed high utilization ratios (V(max)/K(m)) for the following PAH trans-dihydrodiols: (+/-)trans-3,4-dihydroxy-3,4-dihydro-7-methylbenz{a}anthracene, (+/-)trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz{a}anthracene and (+/-)trans-7,8-dihydroxy-7,8-dihydro-5-methylchrysene . Multiple tissue expression (MTE) arrays were used to measure the co-expressed of CYP1A1, EH and AKR1A1 . All the three enzymes co-expressed to sites of PAH activation . The high catalytic efficiency of AKR1A1 for potent proximate carcinogen trans-dihydrodiols and its presence in tissues that contain CYP1A1 and EH suggests that it plays an important role in this alternative pathway of PAH activation (supported by CA39504).

J Biol Chem, 2001 Jul 13, 276(28), 26430 - 40 Epub 2001 Apr 13.
Galactan biosynthesis in Mycobacterium tuberculosis . Identification of a bifunctional UDP-galactofuranosyltransferase; Kremer L et al.; The cell wall of Mycobacterium tuberculosis and related genera is unique among prokaryotes, consisting of a covalently bound complex of mycolic acids, D-arabinan and D-galactan, which is linked to peptidoglycan via a special linkage unit consisting of Rhap-(1-->3)-GlcNAc-P . Information concerning the biosynthesis of this entire polymer is now emerging with the promise of new drug targets against tuberculosis . Accordingly, we have developed a galactosyltransferase assay that utilizes the disaccharide neoglycolipid acceptors beta-d-Galf-(1-->5)-beta-D-Galf-O-C(10:1) and beta-D-Galf-(1-->6)-beta-D-Galf-O-C(10:1), with UDP-Gal in conjunction with isolated membranes . Chemical analysis of the subsequent reaction products established that the enzymatically synthesized products contained both beta-D-Galf linkages ((1-->5) and (1-->6)) found within the mycobacterial cell, as well as in an alternating (1-->5) and (1-->6) fashion consistent with the established structure of the cell wall . Furthermore, through a detailed examination of the M . tuberculosis genome, we have shown that the gene product of Rv3808c, now termed glfT, is a novel UDP-galactofuranosyltransferase . This enzyme possesses dual functionality in performing both (1-->5) and (1-->6) galactofuranosyltransferase reactions with the above neoglycolipid acceptors, using membranes isolated from the heterologous host Escherichia coli expressing Rv3808c . Thus, at a biochemical and genetic level, the polymerization of the galactan region of the mycolyl-arabinogalactan complex has been defined, allowing the possibility of further studies toward substrate recognition and catalysis and assay development . Ultimately, this may also lead to a more rational approach to drug design to be explored in the context of mycobacterial infections.

Curr Biol, 2001 Mar 20, 11(6), R222 - 5
Bacterial cell cycle: seeing the big picture with microarrays; Stephens C; Global assays of gene expression and protein stability during the Caulobacter crescentus cell cycle reveal that a surprisingly large fraction of the genome and proteome is affected as cells grow and divide . These studies are an important step toward understanding how the cell cycle is controlled in prokaryotes.

Neuron, 2001 Mar, 29(3), 669 - 80
Coupling Gbetagamma-dependent activation to channel opening via pore elements in inwardly rectifying potassium channels; Sadja R et al.; G protein-coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gbetagamma subunits of the G protein . The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gbetagamma . Mutations in TM2 were found that mimicked the Gbetagamma-activated state . The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gbetagamma subunits but depended on PtdIns(4,5)P(2) . The results suggest that the TM2 region plays a key role in channel gating following Gbetagamma binding in a phospholipid-dependent manner . This mechanism of gating in inwardly rectifying K+ channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.

Neuron, 2001 Mar, 29(3), 593 - 601
Structure of the RCK domain from the E . coli K+ channel and demonstration of its presence in the human BK channel; Jiang Y et al.; The intracellular C-terminal domain structure of a six-transmembrane K+ channel from Escherichia coli has been solved by X-ray crystallography at 2.4 A resolution . The structure is representative of a broad class of domains/proteins that regulate the conductance of K+ (here referred to as RCK domains) in prokaryotic K+ transporters and K+ channels . The RCK domain has a Rossmann-fold topology with unique positions, not commonly conserved among Rossmann-fold proteins, composing a well-conserved salt bridge and a hydrophobic dimer interface . Structure-based amino acid sequence alignments and mutational analysis are used to demonstrate that an RCK domain is also present and is an important component of the gating machinery in eukaryotic large-conductance Ca2+ activated K+ channels.

Mol Microbiol, 2001 Apr, 40(1), 169 - 78
Two-hybrid analysis of domain interactions involving NtrB and NtrC two-component regulators; Martinez-Argudo I et al.; Signal transduction by two-component regulatory systems involves phosphorylation of the receiver domain of a response regulator by the transmitter domain of the cognate histidine kinase . In the NtrBC system, phosphorylation of NtrC by NtrB results in transcriptional activation of nitrogen-regulated genes . We have used the yeast two-hybrid system to probe interactions between domains of the NtrB and NtrC proteins from Klebsiella pneumoniae . We constructed fusions from each of a series of proteins or protein domains to the activation and the DNA-binding domains of GAL4 and analysed expression of GAL1:lacZ and GAL1:HIS3 reporters in yeast . The DNA-binding domain of NtrC and the so-called sensor domain of NtrB appeared to provide the major determinants for dimerization of the fusion proteins . A strong and specific interaction was also shown between NtrB and NtrC, localized to the HN region of the NtrB transmitter module and to the NtrC receiver domain, whereas other domains of these proteins do not appear to contribute to the recognition specificity . The results presented here indicate that communication between two-component partners also involves protein-protein interactions that can be detected in vivo, suggesting that the yeast two-hybrid system is a powerful genetic tool for identifying functional partners of prokaryotic signal transduction pathways.

Mol Microbiol, 2001 Apr, 40(1), 20 - 36
Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS; Hommais F et al.; Despite many years of intense work investigating the function of nucleoid-associated proteins in prokaryotes, their role in bacterial physiology remains largely unknown . The two-dimensional protein patterns were compared and expression profiling was carried out on H-NS-deficient and wild-type strains of Escherichia coli K-12 . The expression of approximately 5% of the genes and/or the accumulation of their protein was directly or indirectly altered in the hns mutant strain . About one-fifth of these genes encode proteins that are involved in transcription or translation and one-third are known to or were in silico predicted to encode cell envelope components or proteins that are usually involved in bacterial adaptation to changes in environmental conditions . The increased expression of several genes in the mutant resulted in a better ability of this strain to survive at low pH and high osmolarity than the wild-type strain . In particular, the putative regulator, YhiX, plays a central role in the H-NS control of genes required in the glutamate-dependent acid stress response . These results suggest that there is a strong relationship between the H-NS regulon and the maintenance of intracellular homeostasis.

FEBS Lett, 2001 Apr 6, 494(1-2), 34 - 7
Substitution of a conserved aspartate allows cation-induced polymerization of FtsZ; Scheffers DJ et al.; The prokaryotic tubulin homologue FtsZ polymerizes in vitro in a nucleotide dependent fashion . Here we report that replacement of the strictly conserved Asp212 residue of Escherichia coli FtsZ by a Cys or Asn, but not by a Glu residue results in FtsZ that polymerizes with divalent cations in the absence of added GTP . FtsZ D212C and D212N mutants co-purify with GTP as bound nucleotide, providing an explanation for the unusual phenotype . We conclude that D212 plays a critical role in the coordination of a metal ion and the nucleotide at the interface of two FtsZ monomers.

Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 5240 - 5 Epub 2001 Apr 10.
Predicted highly expressed and putative alien genes of Deinococcus radiodurans and implications for resistance to ionizing radiation damage; Karlin S et al.; Predicted highly expressed (PHX) and putative alien genes determined by codon usages are characterized in the genome of Deinococcus radiodurans (strain R1) . Deinococcus radiodurans (DEIRA) can survive very high doses of ionizing radiation that are lethal to virtually all other organisms . It has been argued that DEIRA is endowed with enhanced repair systems that provide protection and stability . However, predicted expression levels of DNA repair proteins with the exception of RecA tend to be low and do not distinguish DEIRA from other prokaryotes . In this paper, the capability of DEIRA to resist extreme doses of ionizing and UV radiation is attributed to an unusually high number of PHX chaperone/degradation, protease, and detoxification genes . Explicitly, compared with all current complete prokaryotic genomes, DEIRA contains the greatest number of PHX detoxification and protease proteins . Other sources of environmental protection against severe conditions of UV radiation, desiccation, and thermal effects for DEIRA are the several S-layer (surface structure) PHX proteins . The top PHX gene of DEIRA is the multifunctional tricarboxylic acid (TCA) gene aconitase, which, apart from its role in respiration, also alerts the cell to oxidative damage.

Nucleic Acids Res, 2001 Apr 15, 29(8), 1765 - 71
Substrate-specific inhibition of RecQ helicase; Wu X et al.; The RecQ helicases constitute a small but highly conserved helicase family . Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes . Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs) . We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation . RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays . The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (K(i)) for NMM inhibition of G4 DNA unwinding is 1.7 microM, approximately two orders of magnitude below the K(i) for inhibition of duplex DNA unwinding (>100 microM) . NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.

J Mol Biol, 2001 Apr 13, 307(5), 1293 - 303
The C-terminal half of the colicin A pore-forming domain is active in vivo and in vitro; Nardi A et al.; The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of Escherichia coli from the periplasmic side and forms a functional channel . The soluble structure of pfColA consists of a ten-helix bundle containing a hydrophobic helical hairpin . Here, we generated a series of mutants in which an increasing number of sp-pfColA alpha-helices was deleted . These peptides were tested for their ability to form ion channels in vivo and in vitro . We found that the shortest sp-pfColA mutant protein that killed Escherichia coli was composed of the five last alpha-helices of sp-pfColA, whereas the shortest peptide that formed a channel in planar lipid bilayer membranes similar to that of intact pfColA was the protein composed of the last six alpha-helices . The peptide composed of the last five alpha-helices of pfColA generated a voltage-independent conductance in planar lipid bilayer with properties very different from that of intact pfColA . Thus, helices 1 to 4 are unnecessary for channel formation, while helix 5, or some part of it, is important but not absolutely necessary . Voltage-dependence of colicin is evidently controlled by the first four alpha-helices of pfColA .

J Mol Biol, 2001 Apr 13, 307(5), 1271 - 92
Regulatory potential, phyletic distribution and evolution of ancient, intracellular small-molecule-binding domains; Anantharaman V et al.; Central cellular functions such as metabolism, solute transport and signal transduction are regulated, in part, via binding of small molecules by specialized domains . Using sensitive methods for sequence profile analysis and protein structure comparison, we exhaustively surveyed the protein sets from completely sequenced genomes for all occurrences of 21 intracellular small-molecule-binding domains (SMBDs) that are represented in at least two of the three major divisions of life (bacteria, archaea and eukaryotes) . These included previously characterized domains such as PAS, GAF, ACT and ferredoxins, as well as three newly predicted SMBDs, namely the 4-vinyl reductase (4VR) domain, the NIFX domain and the 3-histidines (3H) domain . Although there are only a limited number of different superfamilies of these ancient SMBDs, they are present in numerous distinct proteins combined with various enzymatic, transport and signal-transducing domains . Most of the SMBDs show considerable evolutionary mobility and are involved in the generation of many lineage-specific domain architectures . Frequent re-invention of analogous architectures involving functionally related, but not homologous, domains was detected, such as, fusion of different SMBDs to several types of DNA-binding domains to form diverse transcription regulators in prokaryotes and eukaryotes . This is suggestive of similar selective forces affecting the diverse SMBDs and resulting in the formation of multidomain proteins that fit a limited number of functional stereotypes . Using the "guilt by association approach", the identification of SMBDs allowed prediction of functions and mode of regulation for a variety of previously uncharacterized proteins .

J Mol Biol, 2001 Apr 13, 307(5), 1161 - 70
Selective recognition of pyrimidine motif triplexes by a protein encoded by the bacterial transposon Tn7; Rao JE et al.; The bacterial transposon Tn7 is distinguished among mobile genetic elements by its targeting abilities . Recently, we reported that Tn7 is able to selectively insert adjacent to triple-helical DNA . The binding of TnsC, a Tn7-encoded protein, to the triplex DNA target leads to the specific transposition of Tn7 adjacent to both inter- and intramolecular pyrimidine motif triplexes . Here, we further probe how Tn7 targets triplex DNA . We report that TnsC discriminates between different types of triplexes, showing binding preference for pyrimidine but not for purine motif intermolecular triplex DNA . The binding preferences of TnsC and the Tn7 insertion profiles were obtained using psoralenated, triplex- forming oligonucleotides annealed to plasmid DNAs . Although the presence of psoralen is not required for targeting nor is it alone able to attract TnsC, we show that the location of psoralen within the pyrimidine motif triplex does alter the position of Tn7 insertion relative to the triplex . Comparison between the triplex-targeting pathway and the highly site-specific targeting pathway mediated by the binding of the Tn7-encoded protein, TnsD, to the unique site attTn7, suggests that similar structural features within each target DNA are recognized by TnsC, leading to site-specific transposition . This work demonstrates that a prokaryotic protein involved in the targeting and regulation of Tn7 translocation, TnsC, can selectively recognize pyrimidine motif triplexes .

Plant Mol Biol, 2001 Feb, 45(3), 327 - 40
The stress- and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms; Shen Q et al.; Abscisic acid (ABA) induces the expression of a battery of genes in mediating plant responses to environmental stresses . Here we report one of the early ABA-inducible genes in barley (Hordeum vulgare L.), HVA22, which shares little homology with other ABA-responsive genes such as LEA (late embryogenesis-abundant) and RAB (responsive to ABA) genes . In grains, the expression of HVA22 gene appears to be correlated with the dormancy status . The level of HVA22 mRNA increases during grain development, and declines to an undetectable level within 12 h after imbibition of non-dormant grains . In contrast, the HVA22 mRNA level remains high in dormant grains even after five days of imbibition . Treatment of dormant grains with gibberellin (GA) effectively breaks dormancy with a concomitant decline of the level of HVA22 mRNA . The expression of HVA22 appears to be tissue-specific with the level of its mRNA readily detectable in aleurone layers and embryos, yet undetectable in the starchy endosperm . The expression of HVA22 in vegetative tissues can be induced by ABA and environmental stresses, such as cold and drought . Apparent homologues of this barley gene are found in phylogenetically divergent eukaryotic organisms, including cereals, Arabidopsis, Caenorhabditis elegans, man, mouse and yeast, but not in any prokaryotes . Interestingly, similar to barley HVA22, the yeast homologue is also stress-inducible . These observations suggest that the HVA22 and its homologues encode a highly conserved stress-inducible protein which may play an important role in protecting cells from damage under stress conditions in many eukaryotic organisms.

Environ Pollut, 2001, 112(3), 427 - 34
Differential responses of benthic microbes and meiofauna to fish-farm disturbance in coastal sediments; La Rosa T et al.; Bacterial and meiofaunal abundance and biomass and their response to the disturbance induced by fish-farm biodeposition were investigated from March to October 1997 on a monthly basis at two stations of the Gaeta Gulf (Tyrrhenian Sea, Mediterranean Sea) . The biopolymeric fraction of the organic matter was characterized by high concentrations which was similar at both fish-farming-impacted and control stations . Similarly, bacteria accounted for a small fraction of the biopolymeric organic carbon (< 1%), while the contribution due to auto-fluorescent cell biomass (i.e . prokaryotic and eukaryotic cells displaying auto-fluorescence) to the total biopolymeric carbon was quantitatively negligible (< 0.1%) . Benthic bacteria appear to be sensitive to organic enrichment as their abundance increased significantly beneath the cage, whilst numbers of meiofauna was lower than in the control . Changes occurred also in terms of individual nematode biomass that increased as result of the biodeposition . A particularly useful tool appeared to be represented by the ratio of benthic auto-fluorescent cells to bacterial abundance, bacteria to meiofaunal biomass and auto-fluorescent cells to meiofauna biomass . All these parameters described well the impact due to biodeposition on the benthic environment as their ratios displayed significantly higher values in farm sediments, but recovered rapidly (15 days) to values observed in the control (i.e . undisturbed conditions) immediately after cage removal . Changes observed in the present study highlight that the increased organic loading determined a shift of the relative contribution of the different benthic components to the total biopolymeric carbon, so that in highly impacted systems total benthic biomass becomes increasingly dominated by microbial components.

C R Acad Sci III, 2001 Mar, 324(3), 201 - 8
Composition strand asymmetries in prokaryotic genomes: mutational bias and biased gene orientation; Lopez P et al.; Most prokaryotic genomes display strand compositional asymmetries, but the reasons for these biases remain unclear . When the distribution of gene orientation is biased, as it often is, this may induce a bias in composition, as codon frequencies are not identical . We show here that this effect can be estimated and removed, and that the residual base skews are the highest at third base codon positions and lower at first and second positions . This strongly suggests that compositional asymmetries result from 1) a replication-related mutational bias that is filtered through selective pressure and/or from 2) an uneven distribution of gene orientation . In most cases, the mutational bias alters the codon usage and amino acid frequencies of the leading and the lagging strand . However, these features are not ubiquitous amongst prokaryotes, and the biological reasons for them remain to be found.

Genetics, 2001 Apr, 157(4), 1711 - 21
Nuclear gene dosage effects upon the expression of maize mitochondrial genes; Auger DL et al.; Each mitochondrion possesses a genome that encodes some of its own components . The nucleus encodes most of the mitochondrial proteins, including the polymerases and factors that regulate the expression of mitochondrial genes . Little is known about the number or location of these nuclear factors . B-A translocations were used to create dosage series for 14 different chromosome arms in maize plants with normal cytoplasm . The presence of one or more regulatory factors on a chromosome arm was indicated when variation of its dosage resulted in the alteration in the amount of a mitochondrial transcript . We used quantitative Northern analysis to assay the transcript levels of three mitochondrially encoded components of the cytochrome c oxidase complex (cox1, cox2, and cox3) . Data for a nuclearly encoded component (cox5b) and for two mitochondrial genes that are unrelated to cytochrome c oxidase, ATP synthase alpha-subunit and 18S rRNA, were also determined . Two tissues, embryo and endosperm, were compared and most effects were found to be tissue specific . Significantly, the array of dosage effects upon mitochondrial genes was similar to what had been previously found for nuclear genes . These results support the concept that although mitochondrial genes are prokaryotic in origin, their regulation has been extensively integrated into the eukaryotic cell.

Cell Mol Life Sci, 2001 Feb, 58(2), 165 - 78
Functional, biochemical and genetic diversity of prokaryotic nitrate reductases; Richardson DJ et al.; Prokaryotic nitrate reduction can serve a number of physiological roles and can be catalysed by a number of biochemically distinct nitrate reductases . Three distinct nitrate reductase classes can be indentified in prokaryotes, NAS, NAR and NAP . NAS is located in the cytoplasmic compartment and participates in nitrogen assimilation . NAR is usually a three-subunit complex anchored to the cytoplasmic face of the membrane with its active site located in the cytoplasmic compartment and is involved in anaerobic nitrate respiration . NAP is a two-subunit complex, located in the periplasmic compartment, that is coupled to quinol oxidation via a membrane anchored tetraheme cytochrome . It shows considerable functional flexibility by participating in anaerobic respiration or redox energy dissipation depending on the organism in which it is found . The members of all three classes of enzymes bind the bis-molybdopterin guanine dinucleotide cofactor at the active site, but they differ markedly in the number and nature of cofactors used to transfer electrons to this site . Analysis of prokaryotic genome sequences available at the time of writing reveals that the different nitrate reductases are phylogenetically widespread.

Curr Opin Biotechnol, 2001 Apr, 12(2), 126 - 30
Computational methods for gene annotation: the Arabidopsis genome; Cho Y et al.; Since the structure of the DNA molecule was identified half a century ago, the complete genome sequence has been determined for 37 prokaryotes and several eukaryotes . With the exponential growth of genetic information, bioinformatics has attempted to predict gene locations and functions in cyberspace prior to experimental confirmation at the bench.

Biochim Biophys Acta, 2001 Apr 2, 1511(2), 297 - 308
Thermal transitions of DMPG bilayers in aqueous solution: SAXS structural studies; Riske KA et al.; Dimyristoylphosphatidylglycerol (DMPG) has been extensively studied as a model for biological membranes, since phosphatidylglycerol is the most abundant anionic phospholipid in prokaryotic cells . At low ionic strengths, this lipid presents a peculiar thermal behavior, with two sharp changes in the light scattering profile, at temperatures named here T(on)(m) and T(off)(m) . Structural changes involved in the DMPG thermal transitions are here investigated by small angle X-ray scattering (SAXS), and compared to the results yielded by differential scanning calorimetry (DSC) and electron spin resonance (ESR) . The SAXS results show a broad peak, indicating that DMPG is organized in single bilayers, for the range of temperature studied (10-45 degrees C) . SAXS intensity shows an unusual effect, starting to decrease at T(on)(m), and presenting a sharp increase at T(off)(m) . The bilayer electron density profiles, obtained from modeling the SAXS curves, show a gradual decrease in electron density contrast (attributed to separation between charged head groups) and in bilayer thickness between T(on)(m) and T(off)(m) . Results yielded by SAXS, DSC and ESR indicate that a chain melting process starts at T(on)(m), but a complete fluid phase exists only for temperatures above T(off)(m), with structural changes occurring at the bilayer level in the intermediate region.

Trends Plant Sci, 2001 Apr, 6(4), 151 - 5
Was the evolution of plastid genetic machinery discontinuous?
Sato N.
The plastid nucleoid consists of plastid DNA and various, mostly uncharacterized, DNA-binding proteins . The plastid DNA undoubtedly originated from an ancestral cyanobacterial genome, but the origin of the nucleoid proteins appears complex . Initial biochemical analysis of these proteins, as well as comparative genome informatics, suggest that proteins of eukaryotic origin replaced most of the original prokaryotic proteins during the evolution of plastids in the lineage of green plants.

Trends Microbiol, 2001 Apr, 9(4), 169 - 75
Ureaplasma urealyticum: an opportunity for combinatorial genomics; Pollack JD; Examination of genomic or enzymatic activity data alone neither provides a complete picture of metabolic function or potential nor confidently reveals sites amenable to inhibition . Furthermore, in some cases, gene annotation and in aqua assays disagree by describing gene annotation without enzyme activity and enzyme activity without homologous annotation . The newly sequenced genome of Ureaplasma urealyticum (parvum) is another prokaryote example of the class Mollicutes where such confounding differences are observed . The little-considered role of some proteins as multifunctional enzymes - substitutes for 'missing' genes - could both partially explain the apparent anomalies and relate to any inaccurate deductions of inhibitor function . A combinatorial analysis involving available evidence of genomic sequence, transcription, translational phenomena, structure and enzymatic activity gives the best picture of the organism's vital metabolic alternatives.

J Invest Dermatol, 2001 Apr, 116(4), 610 - 3
Homozygous variegate porphyria: 20 y follow-up and characterization of molecular defect; Kauppinen R et al.; The long-term follow-up of a homozygous variegate porphyria patient revealed severe photosensitivity accompanied by mild sensory neuropathy and IgA nephropathy . A 35T to C transition in exon 2 (I12T) and a 767C to G transversion in exon 7 (P256R) of the protoporphyrinogen oxidase gene were identified from both alleles of the patient's cDNA and genomic DNA samples . Both prokaryotic and eukaryotic expression studies showed that the first mutation in the evolutionary conserved region resulted in a decrease in the protoporphyrinogen oxidase activity in contrast to the polymorphic substitution in exon 7, which affected the function of the enzyme assayed in Escherichia coli but not COS-1 cells.

J Cell Biol, 2001 Apr 2, 153(1), 111 - 20
FtsZ ring formation at the chloroplast division site in plants; Vitha S et al.; Among the events that accompanied the evolution of chloroplasts from their endosymbiotic ancestors was the host cell recruitment of the prokaryotic cell division protein FtsZ to function in chloroplast division . FtsZ, a structural homologue of tubulin, mediates cell division in bacteria by assembling into a ring at the midcell division site . In higher plants, two nuclear-encoded forms of FtsZ, FtsZ1 and FtsZ2, play essential and functionally distinct roles in chloroplast division, but whether this involves ring formation at the division site has not been determined previously . Using immunofluorescence microscopy and expression of green fluorescent protein fusion proteins in Arabidopsis thaliana, we demonstrate here that FtsZ1 and FtsZ2 localize to coaligned rings at the chloroplast midpoint . Antibodies specific for recognition of FtsZ1 or FtsZ2 proteins in Arabidopsis also recognize related polypeptides and detect midplastid rings in pea and tobacco, suggesting that midplastid ring formation by FtsZ1 and FtsZ2 is universal among flowering plants . Perturbation in the level of either protein in transgenic plants is accompanied by plastid division defects and assembly of FtsZ1 and FtsZ2 into filaments and filament networks not observed in wild-type, suggesting that previously described FtsZ-containing cytoskeletal-like networks in chloroplasts may be artifacts of FtsZ overexpression.

EMBO J, 2001 Apr 2, 20(7), 1681 - 91
Molecular basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis; Aguilar PS et al.; Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins . Although a large body of information concerning cold shock-induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures . Here we identified a two-component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Delta5-lipid desaturase from Bacillus subtilis . We found that DesR binds to a DNA sequence extending from position -28 to -77 relative to the start site of the temperature-regulated des gene . We show further that unsaturated fatty acids (UFAs), the products of the Delta5-desaturase, act as negative signalling molecules of des transcription . Thus, a regulatory loop composed of the DesK-DesR two-component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures.

EMBO J, 2001 Apr 2, 20(7), 1555 - 62
Turning a disulfide isomerase into an oxidase: DsbC mutants that imitate DsbA; Bader MW et al.; There are two distinct pathways for disulfide formation in prokaryotes . The DsbA-DsbB pathway introduces disulfide bonds de novo, while the DsbC-DsbD pathway functions to isomerize disulfides . One of the key questions in disulfide biology is how the isomerase pathway is kept separate from the oxidase pathway in vivo . Cross-talk between these two systems would be mutually destructive . To force communication between these two systems we have selected dsbC mutants that complement a dsbA null mutation . In these mutants, DsbC is present as a monomer as compared with dimeric wild-type DsbC . Based on these findings we rationally designed DsbC mutants in the dimerization domain . All of these mutants are able to rescue the dsbA null phenotype . Rescue depends on the presence of DsbB, the native re-oxidant of DsbA, both in vivo and in vitro . Our results suggest that dimerization acts to protect DsbC's active sites from DsbB-mediated oxidation . These results explain how oxidative and reductive pathways can co-exist in the periplasm of Escherichia coli.

Science, 2001 May 11, 292(5519), 1127 - 31 Epub 2001 Mar 29.
14C-dead living biomass: evidence for microbial assimilation of ancient organic carbon during shale weathering; Petsch ST et al.; Prokaryotes have been cultured from a modern weathering profile developed on a approximately 365-million-year-old black shale that use macromolecular shale organic matter as their sole organic carbon source . Using natural-abundance carbon-14 analysis of membrane lipids, we show that 74 to 94% of lipid carbon in these cultures derives from assimilation of carbon-14-free organic carbon from the shale . These results reveal that microorganisms enriched from shale weathering profiles are able to use a macromolecular and putatively refractory pool of ancient organic matter . This activity may facilitate the oxidation of sedimentary organic matter to inorganic carbon when sedimentary rocks are exposed by erosion . Thus, microorganisms may play a more active role in the geochemical carbon cycle than previously recognized, with profound implications for controls on the abundance of oxygen and carbon dioxide in Earth's atmosphere over geologic time.

Plant Cell, 2001 Apr, 13(4), 853 - 71
The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium; Debeaujon I et al.; Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination . We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 (tt12), from a reduced seed dormancy screen . Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells . Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway . The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family . TT12 is expressed specifically in ovules and developing seeds . In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation . The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium.

Genome Res, 2001 Apr, 11(4), 555 - 65
Lineage-specific gene expansions in bacterial and archaeal genomes; Jordan IK et al.; Gene duplication is an important mechanistic antecedent to the evolution of new genes and novel biochemical functions . In an attempt to assess the contribution of gene duplication to genome evolution in archaea and bacteria, clusters of related genes that appear to have expanded subsequent to the diversification of the major prokaryotic lineages (lineage-specific expansions) were analyzed . Analysis of 21 completely sequenced prokaryotic genomes shows that lineage-specific expansions comprise a substantial fraction (approximately 5%-33%) of their coding capacities . A positive correlation exists between the fraction of the genes taken up by lineage-specific expansions and the total number of genes in a genome . Consistent with the notion that lineage-specific expansions are made up of relatively recently duplicated genes, >90% of the detected clusters consists of only two to four genes . The more common smaller clusters tend to include genes with higher pairwise similarity (as reflected by average score density) than larger clusters . Regardless of size, cluster members tend to be located more closely on bacterial chromosomes than expected by chance, which could reflect a history of tandem gene duplication . In addition to the small clusters, almost all genomes also contain rare large clusters of size > or =20 . Several examples of the potential adaptive significance of these large clusters are explored . The presence or absence of clusters and their related genes was used as the basis for the construction of a similarity graph for completely sequenced prokaryotic genomes . The topology of the resulting graph seems to reflect a combined effect of common ancestry, horizontal transfer, and lineage-specific gene loss.

Comb Chem High Throughput Screen, 2001 Apr, 4(2), 135 - 43
Shotgun phage display cloning; Jacobsson K et al.; Shotgun phage display cloning is a useful tool for studying interactions between bacterial and host proteins . Libraries are constructed by cloning randomly fragmented prokaryotic DNA into phage mid-vectors . Theoretically, these libraries will consist of phages that together display all proteins encoded by the bacterial genome . Selecting a gene III-based library, made from Staphylococcus aureus DNA, against IgG and fibronectin resulted in 20-40% positive clones after two pannings . Increasing the number of fusion proteins per phage particle by using gene VIII-based display, increased the frequency of correct clones to 75-100%.

Mol Cell Biol Res Commun, 2000 Sep, 4(3), 172 - 3
Using neural networks for prediction of subcellular location of prokaryotic and eukaryotic proteins; Cai YD et al.; T . Kohonen's self-organization model, a typical neural network model, was applied to predict the subcellular location of proteins from their amino acid composition . The Reinhardt and Hubbard database was used to examine the performance of the neural network method . The rates of correct prediction for the three possible subcellular location of prokaryotic proteins were 96.1% by the self-consistency test and 84.4% by the jackknife test . The rates of correct prediction for the four possible subcellular location of eukaryotic proteins were 95.6% by the self-consistency test and 70.6% by the jackknife test .

Protein Expr Purif, 2001 Apr, 21(3), 470 - 84
Identification, cloning, and expression of a functional phenylalanyl-tRNA synthetase (pheRS) from Staphylococcus aureus; Savopoulos JW et al.; Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthetases in that it is a heterotetrameric enzyme composed of two alpha-subunits and two larger beta-subunits . In prokaryotes, the alpha- and beta-subunits of pheRS are encoded by the genes pheS and pheT, respectively . In this report we describe the isolation of a DNA fragment (3.52 kb) containing the pheS and pheT genes from a Staphylococcus aureus (WCUH29) genomic DNA library . Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarity to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta- subunits . We describe the high-level overexpression and purification of recombinant S . aureus pheRS using pheS and pheT genes as part of an artificial operon in Escherichia coli . For comparative analysis we also report a procedure for the purification of native pheRS from S . aureus (Oxford Strain) and demonstrate that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable .

Res Microbiol, 2001 Jan-Feb, 152(1), 3 - 10
Cell division protein FtsZ: running rings around bacteria, chloroplasts and mitochondria; Gilson PR et al.; Of all the proteins involved in prokaryotic cell division FtsZ is one of the earliest acting and most widely distributed, being found in all but a few species . We discuss several recent discoveries of FtsZ in eukaryotic cells and the protein's role in the division of chloroplasts and mitochondria, organelles that are of bacterial origin.

Free Radic Biol Med, 2000 Jan 15, 28(2), 281 - 8
Polynucleotide degradation during early stage response to oxidative stress is specific to mitochondria; Abramova NE et al.; Oxidative stress is known to modulate RNA expression in both prokaryotic and eukaryotic cells . We have previously determined that a preferential and calcium-dependent downregulation of mitochondrial RNA occurs in HA-1 hamster fibroblasts in response to hydrogen peroxide, and that this is accompanied by the degradation of mitochondrial genomic DNA . Here we extended these studies to determine whether downregulation is specific to transcripts derived from mitochondrial-encoded genes; to determine whether genomic DNA degradation occurs in the nucleus; and to compare overall polynucleotide stress response with cellular growth arrest and apoptosis . We observed that nuclear genome-encoded mRNAs whose protein products are targeted for the electron transport chain of mitochondria were not degraded . Furthermore, early stage degradation of genomic DNA, assessed within the first 5 h of peroxide exposure, was specific to mitochondria, as nuclear genomic DNA was not degraded under the same treatment conditions . These differential degradations occurred under conditions where extensive growth-arrest and moderate apoptosis were observed, and were accompanied by significant induction of the growth arrest mRNAs gadd45, gadd153, and adapt15/gadd7 . Combined, these results indicate that there is a general degradation of mitochondrial- but not nuclear-polynucleotides during early stage response of HA-1 fibroblasts to oxidative stress.

J Biol Chem, 2001 Jun 1, 276(22), 19363 - 74 Epub 2001 Mar 02.
The small subunit of the mammalian mitochondrial ribosome . Identification of the full complement of ribosomal proteins present; Cavdar Koc E et al.; Identification of all the protein components of the small subunit (28 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS) . Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled . The human mitochondrial ribosome has 29 distinct proteins in the small subunit . Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21 . All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes . Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D . melanogaster mitochondrial ribosomes while C . elegans has two S18 homologs . The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s . No mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases . The remaining 15 proteins present in mammalian mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to mitochondrial ribosomes . Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes . All but two of these proteins have a clear homolog in D . melanogaster while all but three can be found in the genome of C . elegans . Five of the mitochondrial specific ribosomal proteins have homologs in S . cerevisiae.

J Biol Chem, 2001 Jun 1, 276(22), 19141 - 9 Epub 2001 Mar 15.
Circular permutation of 5-aminolevulinate synthase . Mapping the polypeptide chain to its function; Cheltsov AV et al.; 5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes . The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor . Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function . To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library . One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase . Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain . The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus . This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits . Furthermore, the order of two identified functional elements (i.e . the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme . Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment . Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.

J Biol Chem, 2001 May 11, 276(19), 16257 - 64 Epub 2001 Jan 30.
New insights into host factor requirements for prokaryotic beta-recombinase-mediated reactions in mammalian cells; Diaz V et al.; The prokaryotic beta-recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in mammalian cells, both on episomic and chromatin-integrated substrates . Using a specific recombination activated gene expression system, we report the site-specific recombination activity of an enhanced green fluorescent protein (EGFP) fused version of beta-recombinase (beta-EGFP) . This allows expression of active beta-recombinase detectable in vivo and in fixed cells by fluorescence microscopy . In addition, cellular viability is compatible with a substantial level of expression of the beta-EGFP protein . Using fluorescence-activated cell sorting, we have been able to enrich cell populations expressing this fusion protein . Application of this strategy has allowed us to study in more depth the host factor requirements for this system . Previous work showed that eukaryotic HMG1 protein was necessary and sufficient to help beta-recombinase activity in vitro . The influence of ectopic expression of HMG1 protein in the recombination process has been analyzed, indicating that HMG1 overexpression does not lead to a significant increase on the efficiency of beta-recombinase-mediated recombination both on episomal substrates and chromatin-associated targets . In addition, beta-recombinase-mediated recombination has been demonstrated in HMG1 deficient cells at the same levels as in wild type cells . These data demonstrate the existence of cellular factors different from HMG-1 that can act as helpers for beta-recombinase activity in the eukaryotic environment.

J Biol Chem, 2001 May 4, 276(18), 15275 - 83 Epub 2001 Feb 02.
Overlapping destinations for two dual targeted glycyl-tRNA synthetases in Arabidopsis thaliana and Phaseolus vulgaris; Duchene AM et al.; In plant mitochondria, some of the tRNAs are encoded by the mitochondrial genome and resemble their prokaryotic counterparts, whereas the remaining tRNAs are encoded by the nuclear genome and imported from the cytosol . Generally, mitochondrial isoacceptor tRNAs all have the same genetic origin . One known exception to this rule is the group of tRNA(Gly) isoacceptors in dicotyledonous plants . A mitochondrion-encoded tRNA(Gly) and at least one nucleus-encoded tRNA(Gly) coexist in the mitochondria of these plants, and both are required to allow translation of all four GGN glycine codons . We have taken advantage of this atypical situation to address the problem of tRNA/aminoacyl-tRNA synthetase coevolution in plants . In this work, we show that two different nucleus-encoded glycyl-tRNA synthetases (GlyRSs) are imported into Arabidopsis thaliana and Phaseolus vulgaris mitochondria . The first one, GlyRS-1, is similar to human or yeast glycyl-tRNA synthetase, whereas the second, GlyRS-2, is similar to Escherichia coli glycyl-tRNA synthetase . Both enzymes are dual targeted, GlyRS-1 to mitochondria and to the cytosol and GlyRS-2 to mitochondria and chloroplasts . Unexpectedly, GlyRS-1 seems to be active in the cytosol but inactive in mitochondrial fractions, whereas GlyRS-2 is likely to glycylate both the organelle-encoded tRNA(Gly) and the imported tRNA(Gly) present in mitochondria.

J Biol Chem, 2001 May 18, 276(20), 16786 - 96 Epub 2001 Feb 13.
Cyclobutane pyrimidine dimers and bulky chemical DNA adducts are efficiently repaired in both strands of either a transcriptionally active or promoter-deleted APRT gene; Zheng Y et al.; Both prokaryotic and eukaryotic cells have the capacity to repair DNA damage preferentially in the transcribed strand of actively expressed genes . However, we have found that several types of DNA damage, including cyclobutane pyrimidine dimers (CPDs) are repaired with equal efficiency in both the transcribed and nontranscribed strands of the adenine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells . We further found that, in two mutant cell lines in which the entire APRT promoter region has been deleted, CPDs are still efficiently repaired in both strands of the promoterless APRT gene, even though neither strand appears to be transcribed . These results suggest that efficient repair of both strands at this locus does not require transcription of the APRT gene . We have also mapped CPD repair in exon 3 of the APRT gene in each cell line at single nucleotide resolution . Again, we found similar rates of CPD repair in both strands of the APRT gene domain in both APRT promoter-deletion mutants and their parental cell line . Our findings suggest that current models of transcription-coupled repair and global genomic repair may underestimate the importance of factors other than transcription in governing the efficiency of nucleotide excision repair.

J Biol Chem, 2001 May 4, 276(18), 14744 - 51 Epub 2001 Feb 08.
Identification of the active oligomeric state of an essential adenine DNA methyltransferase from Caulobacter crescentus; Shier VK et al.; Caulobacter crescentus contains one of the two known prokaryotic DNA methyltransferases that lacks a cognate endonuclease . This endogenous cell cycle regulated adenine DNA methyltransferase (CcrM) is essential for C . crescentus cellular viability . DNA methylation catalyzed by CcrM provides an obligatory signal for the proper progression through the cell cycle . To further our understanding of the regulatory role played by CcrM, we sought to investigate its biophysical properties . In this paper we employed equilibrium ultracentrifugation, velocity ultracentrifugation, and chemical cross-linking to show that CcrM is dimeric at physiological concentrations . However, surface plasmon resonance experiments in the presence of S-adenosyl-homocysteine evince that CcrM binds as a monomer to a defined hemi-methylated DNA substrate containing the canonical methylation sequence, GANTC . Initial velocity experiments demonstrate that dimerization of CcrM does not affect DNA methylation . Collectively, these findings suggest that CcrM is active as a monomer and provides a possible in vivo role for dimerization as a means to stabilize CcrM from premature catabolism.

Biochimie, 2001 Feb, 83(2), 187 - 92
Chromosome separation and segregation in dinoflagellates and bacteria may depend on liquid crystalline states; Bouligand Y et al.; The patterns characteristic of certain liquid crystals called 'twisted nematics' or 'cholesterics' have been observed in thin sections of both dinoflagellates and bacterial chromosomes . These liquid crystals have also been obtained in vitro in concentrated DNA solutions . A large part of DNA in prokaryotic chromosomes forms such a twisted liquid crystal, whilst the remainder consists of lateral loops and is less concentrated . These semi-ordered phases could help chromosome separation to occur during and after DNA replication . We suggest that, owing to chemical differences, one of the two replicated filaments is immiscible with the rest of DNA in this chromosome . This immiscibility occurs in the context of an ordered liquid, with the DNA closely layered by a regular twist, a situation proposed to strongly minimize entangling after replication and hence to facilitate segregation.

Bull Math Biol, 2001 Mar, 63(2), 329 - 51
A mathematical model for prokaryotic protein synthesis; Drew DA; A kinetic model for the synthesis of proteins in prokaryotes is presented and analysed . This model is based on a Markov model for the state of the DNA strand encoding the protein . The states that the DNA strand can occupy are: ready, repressed, or having a mRNA chain of length i in the process of being completed . The case i = 0 corresponds to the RNA polymerase attached, but no nucleotides attached to the chain . The Markov model consists of differential equations for the rates of change of the probabilities . The rate of production of the mRNA molecules is equal to the probability that the chain is assembled to the penultimate nucleotide, times the rate at which that nucleotide is attached . Similarly, the mRNA molecules can also be in different states, including: ready and having an amino acid chain of length j attached . The rate of protein synthesis is the rate at which the chain is completed . A Michaelis-Menten type of analysis is done, assuming that the rate of protein degradation determines the 'slow' time, and that all the other kinetic rates are 'fast' . In the self-regulated case, this results in a single ordinary differential equation for the protein concentration.

Cell Physiol Biochem, 2001, 11(2), 61 - 76
Mechanosensitive channels in prokaryotes; Martinac B; Compared to voltage-dependent or ligand-gated ion channels that have extensively been studied over the last twenty years, there is little knowledge available on structure and function of mechanosensitive (MS) channels that constitute the third major group of ion channels classified according to their gating mechanism . The main purpose of this review is to summarize an area of the MS channel research where major progress has occurred . Cloning of MscL and MscS, the MS channels of Large and Small conductance found in Bacteria and elucidation of the 3D crystal structure of MscL are discussed in conjunction with the physiological role of the MS channels in bacterial osmoregulation . Furthermore, cloning and molecular characterization of MS channels in Archaea Methanococcus jannashii and Thermoplasma acidophilum are described . They present examples of the recent promising developments, which may ultimately lead to the understanding of the biophysical principles and evolutionary origins of mechanosensory transduction . Although they conduct ions and are usually characterized by their ionic conductance and selectivity, the MS channels in prokaryotes may primarily serve to transport osmoticants other than ions . Therefore, throughout this review prokaryotic MS ion channels are referred to as MS channels rather than MS ion channels.

Mol Immunol, 2000 Aug-Sep, 37(12-13), 789 - 98
Unique and shared IgE epitopes of Hev b 1 and Hev b 3 in latex allergy; Banerjee B et al.; Of the several latex proteins cloned and expressed, the rubber elongation factor, Hev b 1, and the closely related Hev b 3, represent two major allergens associated with latex allergy . Although both allergens demonstrated IgE binding with sera from latex allergic patients, it was not known whether these two molecules shared any epitopes . Hence, in the present study using health care workers (HCW) and spina bifida (SB) patients with latex allergy, we investigated the IgE binding epitopes in Hev b 1 and Hev b 3 . Recombinant Hev b 1 and Hev b 3 were expressed in a prokaryotic expression system, while overlapping decapeptides of Hev b 1 and Hev b 3 were synthesized on derivatized cellulose membrane . Eight IgE binding epitopes for Hev b 1 and eleven for Hev b 3 were identified using sera from latex allergic patients with SB . On further analysis of synthetic peptides encompassing these epitopes, similar IgE antibody reactivity was demonstrated with three Hev b 1 epitopes b1E3, b1E5, b1E6 and two Hev b 3 epitopes; b3E10 and b3E 11 . For Hev b 1, a unique IgE binding epitope was identified in the region of amino acid residues 16-25 . In competitive ELISA, peptides bIE2 and bIE4 together inhibited 58% of IgE binding of Hev b 1, while b3E5 showed 22% inhibition in the IgE binding of Hev b 3 . The results of the present study suggest that the understanding of linear and conformational IgE epitopes in the major latex allergens may provide better insight into the structure-function relationship of the allergens, and may lead to the development of better patient care and management strategies in latex allergy.

Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 4095 - 100 Epub 2001 Mar 13.
Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle; Jacobs C et al.; We present evidence that a bacterial signal transduction cascade that couples morphogenesis with cell cycle progression is regulated by dynamic localization of its components . Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus . Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell . The membrane-bound DivJ and PleC histidine kinases, which are asymmetrically localized at the opposite poles of the predivisional cell, control the temporal and spatial localization of DivK . DivJ mediates DivK targeting to the poles whereas PleC controls its release from one of the poles at times and places that are consistent with the activities and location of DivJ and PleC in the late predivisional cell . Thus, dynamic changes in subcellular location of multiple components of a signal transduction cascade may constitute a novel mode of prokaryotic regulation to generate and maintain cellular asymmetry.

J Biol Chem, 2001 Jun 15, 276(24), 21070 - 6 Epub 2001 Mar 26.
Structural compatibility between the putative voltage sensor of voltage-gated K+ channels and the prokaryotic KcsA channel; Caprini M et al.; Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K(+) channel (KcsA), support the tenet that voltage-gated K(+) channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward . To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel . Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane . Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used . Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity . Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels . Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels . Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.

J Biol Chem, 2001 Jun 29, 276(26), 23785 - 9 Epub 2001 Mar 26.
A repressor with similarities to prokaryotic and eukaryotic DNA helicases controls the assembly of the CAAT box binding complex at a photosynthesis gene promoter; Bezhani S et al.; A single nucleotide exchange in a promoter region located immediately upstream of the CAAT box of the spinach photosynthesis gene AtpC (gene product is subunit gamma of the chloroplast ATP synthase) prevents the formation of a secondary structure and causes an unregulated, constitutive high level of expression (Kusnetsov, V., Landsberger, M., Oelmuller, R . (1999) J . Biol . Chem . 274, 36009-36014) . We have isolated cDNAs for ATPC-2, a new polypeptide with homologies to pro- and eukaryotic helicases, which specifically binds to this promoter region . Binding of ATPC-2 competes strongly with that of a CAAT box binding factor (CBF), consistent with the idea that both complexes cannot be formed simultaneously because of sterical reasons . In gel mobility shift assays, high binding activities of ATPC-2 and low binding activities of CBF were observed with nuclear extracts from tissue with low AtpC expression levels, and the opposite was observed with extracts from tissues with high AtpC expression levels . Binding of ATPC-2 to the mutant sequence, which directs a constitutively high level expression in vivo and prevents the formation of a secondary structure in vitro, is significantly weaker than binding to the wild-type sequence . Again, the opposite results were obtained for the CBF . Thus, we conclude that the assembly of the CBF.DNA complex stimulates transcription of AtpC and that CBF binding is prevented if ATPC-2 is bound to the promoter region . The novel mechanism of gene regulation and the role of the helicase-like protein ATPC-2 as a potential transcriptional repressor is discussed in relation to its modular structure.

Folia Microbiol (Praha), 2000, 45(3), 239 - 42
Immuno-electron localization of DNA in chondriolites of Saccharomyces cerevisiae mitochondria; Vorisek J et al.; Under electron microscope, the matrix of sectioned mitochondria exhibits ribosomes and an oval, electron-transparent zone which is devoid of ribosomes and is named chondriolite . Fine fibers or clumps of an electron-dense material appeared in this zone after several fixation and contrasting steps and were identified with mitochondrial DNA by cytologists . To verify this assumption, we labeled DNA by a monoclonal antibody and a secondary antibody coupled to immunogold . The label was observed in the nucleus and in the chondriolite zone of sectioned mitochondria . Because the ultrastructure of chondriolites resembles that of nucleoids of prokaryotes, we suggest the term mitochondrial nucleoid for the zone of mitochondrial matrix devoid of ribosomes and containing DNA.

EMBO Rep, 2000 Oct, 1(4), 340 - 6
Efficient membrane assembly of the KcsA potassium channel in Escherichia coli requires the protonmotive force; van Dalen A et al.; Very little is known about the biogenesis and assembly of oligomeric membrane proteins . In this study, the biogenesis of KcsA, a prokaryotic homotetrameric potassium channel, is investigated . Using in vivo pulse-chase experiments, both the monomeric and tetrameric form could be identified . The conversion of monomers into a tetramer is found to be a highly efficient process that occurs in the Escherichia coli inner membrane . KcsA does not require ATP hydrolysis by SecA for insertion or tetramerization . The presence of the proton-motive force (pmf) is not necessary for transmembrane insertion of KcsA; however, the pmf proved to be essential for the efficiency of oligomerization . From in vivo and in vitro experiments it is concluded that the electrical component, deltapsi, is the main determinant for this effect . These results demonstrate a new role of the pmf in membrane protein biogenesis.

Bioessays, 2001 Apr, 23(4), 344 - 51
Innate immunity and its evasion and suppression by hymenopteran endoparasitoids; Schmidt O et al.; Recent studies suggest that insects use pattern recognition molecules to distinguish prokaryotic pathogens and fungi from "self" structures . Less understood is how the innate immune system of insects recognizes endoparasitic Hymenoptera and other eukaryotic invaders as foreign . Here we discuss candidate recognition factors and the strategies used by parasitoids to overcome host defense responses . We suggest that host-parasitoid systems are important experimental models for studying how the innate immune system of insects recognizes foreign invaders that are phylogenetically more closely related to their hosts . The strategies used by parasitoids suggest that insects may employ "hidden-self" recognition molecules for attacking foreign objects intruding the open circulatory system . BioEssays 23:344-351, 2001 .

Bioessays, 2001 Apr, 23(4), 327 - 32
Sound silencing: the Sir2 protein and cellular senescence; Defossez PA et al.; The model organism Saccharomyces cerevisiae is providing new insights into the molecular and cellular changes that are related to aging . The yeast protein Sir2p (Silent Information Regulator 2) is a histone deacetylase involved in transcriptional silencing and the control of genomic stability . Recent results have led to the identification of Sir2p as a crucial determinant of yeast life span . Dosage, intracellular localization, and activity of Sir2p all have important effects on yeast longevity . For instance, calorie restriction apparently increases yeast life span by increasing Sir2p activity . Since Sir2p-related proteins have been identified in many prokaryotic and eukaryotic organisms, the fundamental principles derived from the studies in yeast may prove valuable in directing our future research toward an understanding of the mechanisms of aging in higher eukaryotes . BioEssays 23:327-332, 2001 .

FEMS Microbiol Lett, 2001 Mar 15, 196(2), 93 - 8
Characterization of the alanine racemases from two mycobacteria; Strych U et al.; D-Alanine is a necessary precursor in the biosynthesis of the bacterial peptidoglycan . The naturally occurring L-alanine isomer is racemized to its D-form through the action of a class of enzymes called alanine racemases . These enzymes are ubiquitous among prokaryotes, and with very few exceptions are absent in eukaryotes, making them a logical target for the development of novel antibiotics . The alanine racemase gene from both Mycobacterium tuberculosis and M . avium was amplified by PCR and cloned in Escherichia coli . Overexpression of the proteins in the E . coli BL21 system, both as native and as His-tagged recombinant products, has been achieved . The proteins have been purified to electrophoretic homogeneity and analyzed biochemically . A D-alanine requiring double knock-out mutant of E . coli (alr, dadX) was constructed and the cloned genes were able to complement its deficiencies.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1491 - 506
DNA methyltransferases of the cyanobacterium Anabaena PCC 7120; Matveyev AV et al.; From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium ANABAENA PCC 7120 has been deduced . ANABAENA has nine DNA MTases . Four are associated with Type II restriction enzymes (AVAI, AVAII, AVAIII and the newly recognized inactive AVAIV), and five are not . Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system . The group is defined here based on biochemical and genetic characteristics . The four solitary MTases, DmtA/M.AVAVI, DmtB/M.AVAVII, DmtC/M.AVAVIII and DmtD/M.AVAIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively . DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former . The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems . One Mtase, M.AVAV, cannot reliably be classified as either a solitary or restriction MTase . It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.

EMBO Rep, 2000 Aug, 1(2), 158 - 63
Decoding apparatus for eukaryotic selenocysteine insertion; Tujebajeva RM et al.; Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements . Eukaryotic SECIS elements are found in the 3' untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA . Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms . We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor . This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells . Expression of the two functional domains of the bacterial elongation factor-SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA-elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.

Acta Crystallogr D Biol Crystallogr, 2001 Apr, 57(Pt 4), 586 - 8
Expression, purification and preliminary crystallographic studies of human ketohexokinase; Kozak M et al.; Ketohexokinase (KHK; E.C . 2.7.1.3) catalyses the (reversible) phosphorylation of fructose to fructose-1-phosphate . KHK is the first enzyme in a specialized catabolic pathway metabolizing dietary fructose to the glycolytic intermediate glyceraldehyde-3-phosphate . Mutations inactivating KHK underlie the metabolic disorder essential fructosuria . The primary structure of KHK shows no significant homology to other mammalian hexokinases . It is most similar to prokaryotic ribokinases, but catalyses a distinct phosphorylation reaction . Recombinant human KHK has been crystallized in the orthorhombic form (space group P2(1)2(1)2 or P2(1)2(1)2(1)) . Single crystals of this polymorph suitable for X-ray diffraction have been obtained by vapour diffusion using 2-propanol and MPD as precipitants (pH 7.5) . The crystals have unit-cell parameters a = 93.4, b = 121.5, c = 108.4 A . Diffraction data were collected to 4.3 A resolution . The asymmetric unit contains four protein molecules.

Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3768 - 72 Epub 2001 Mar 20.
ClpA mediates directional translocation of substrate proteins into the ClpP protease; Reid BG et al.; The intracellular degradation of many proteins is mediated in an ATP-dependent manner by large assemblies comprising a chaperone ring complex associated coaxially with a proteolytic cylinder, e.g., ClpAP, ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes . Recent studies of the chaperone ClpA indicate that it mediates ATP-dependent unfolding of substrate proteins and directs their ATP-dependent translocation into the ClpP protease . Because the axial passageway into the proteolytic chamber is narrow, it seems likely that unfolded substrate proteins are threaded from the chaperone into the protease, suggesting that translocation could be directional . We have investigated directionality in the ClpA/ClpP-mediated reaction by using two substrate proteins bearing the COOH-terminal ssrA recognition element, each labeled near the NH(2) or COOH terminus with fluorescent probes . Time-dependent changes in both fluorescence anisotropy and fluorescence resonance energy transfer between donor fluorophores in the ClpP cavity and the substrate probes as acceptors were measured to monitor translocation of the substrates from ClpA into ClpP . We observed for both substrates that energy transfer occurs 2--4 s sooner with the COOH-terminally labeled molecules than with the NH(2)-terminally labeled ones, indicating that translocation is indeed directional, with the COOH terminus of the substrate protein entering ClpP first.

Virology, 2001 Mar 30, 282(1), 102 - 12
Binding properties, cell delivery, and gene transfer of adenoviral penton base displaying bacteriophage; Di Giovine M et al.; The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalisation, properties that can be exploited to reengineer prokaryotic viruses for the infection of mammalian cells . We report that filamentous phage displaying either the full-length penton base gene or a central region of 107 amino acids on their surface were able to bind, internalise, and transduce mammalian cells expressing integrin receptors . Both phage bound alphavbeta3, alphavbeta5, alpha3beta1, and alpha5beta1 integrin subtypes . Cell-binding was shown by electron microscopy; internalisation was investigated by immunofluorescence and confirmed by micropanning . As it has been described for adenovirus, pharmacologic disruption of phosphoinositide-30H kinase, but not of myosin light-chain kinase, inhibited phage internalisation . Recombinant phage encoding an eukaryotic expression cassette was able to mediate gene expression in mammalian cells . Taken together, these data open insights for the exploit of recombinant phage for integrin-targeted gene delivery .

Biochemistry, 2001 Mar 13, 40(10), 3222 - 31
Proposed secondary structure of eukaryote specific expansion segment 15 in 28S rRNA from mice, rats, and rabbits; Larsson SL et al.; The expansion segments in eukaryotic ribosomal RNAs are additional RNA sequences not found in the RNA core common to both prokaryotes and eukaryotes . These regions show large species-dependent variations in sequence and size . This makes it difficult to create secondary structure models for the expansion segments exclusively based on phylogenetic sequence comparison . Here we have used a combination of experimental data and computational methods to generate secondary structure models for expansion segment 15 in 28S rRNA in mice, rats, and rabbits . The experimental data were collected using the structure sensitive reagents DMS, CMCT, kethoxal, micrococcal nuclease, RNase T(1), RNase CL3, RNase V(1), and lead(II) acetate . ES15 was folded with the computer program RNAStructure 3.5 using modification data and phylogenetic similarities between different ES15 sequences . This program uses energy minimization to find the most stable secondary structure of an RNA sequence . The presented secondary structure models include several common structural motifs, but they also have characteristics unique to each organism . Overall, the secondary structure models showed indications of an energetically stable but dynamic structure, easily accessible from the solution by the modification reagents, suggesting that the expansion segment is located on the ribosomal surface.

J Mol Biol, 2001 Mar 23, 307(2), 541 - 56
Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication; Chapados BR et al.; DNA replication and cellular survival requires efficient removal of RNA primers during lagging strand DNA synthesis . In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex . This conserved type 2 RNase H family of replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic properties . Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis . The two-domain architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic domain . This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif, to substrate-discrimination, by a tyrosine finger motif, thereby providing substrate-specific catalytic activity . Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode . These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays . In combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity .

J Mol Biol, 2001 Mar 23, 307(2), 481 - 6
Non-clonability correlates with genomic instability: a case study of a unique DNA region; Razin SV et al.; Instability of eukaryotic DNA in constructs propagated in prokaryotic hosts is a frequently observed phenomenon . With the exception of a very high A+T-content and the presence of multiple repetitions, no general rule at the basis of this phenomenon is actually known . The intergenic spacer located between the pi and alpha(D) chicken alpha-type globin genes is frequently deleted from recombinant phages and plasmids . Here we have cloned this DNA fragment using a specially designed bacterial strain (SURE competent cells, Stratogene) . Comparative analysis of DNA of recombinant clones bearing deletions and clones containing the intact genomic DNA fragment has revealed two important DNA sequence motifs that contribute to the unclonability of eukaryotic DNA in prokaryotic cells . First, the similarity to bacterial transposons (i.e . the presence of repeats flanking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombinant DNA in E . coli . Second, a high content of rotationally correlated kinkable elements (TG*CA steps) may result in non-clonability of the DNA sequence . Interestingly, the latter type of "unclonable" DNA sequence motifs identified in the globin gene domain is unstable (frequently rearranged) also in the eukaryotic chromosome resulting in a local polymorphism . In the chicken domain of alpha globin genes this unstable DNA sequence seems to be partially protected by interaction with nuclear matrix proteins .

Mol Gen Genet, 2001 Feb, 264(6), 809 - 18
Functional analysis of the Bacillus subtilis y shD gene, a mutS paralogue; Rossolillo P et al.; In the course of the Bacillus subtilis genome sequencing project, an ORF called yshD was identified, and its product was classified as a mismatch repair protein . Further analysis of the YshD primary sequence showed that the protein belongs to the MutS2 protein family, sharing a high degree of identity with the Thermootoga Inaritima protein TM1278 (34%) and with the so-called MutS2 protein sl11772 of Synechocystis (32%) . The COG1193 family of MutS-like proteins is made up of polypeptides that have been predicted from genomic sequencing data from various prokaryotes, but their biological role has not yet been analysed . The functional study of yshD revealed that the gene is constitutively transcribed during the life cycle of B . subtilis, and in minimal medium expression remains at appreciable levels until very late in stationary phase . Fluctuation tests with yshD knock-out mutants did not indicate any role for the protein in preventing the accumulation of spontaneous forward mutations to RifR, nor was any functional interaction with MutS or MutL suggested in fluctuation experiments with mutants lacking combinations of the three genes . Nevertheless, the mutation spectrum observed in the rpoB gene in the deltayshD strain has some characteristic features . The gene does not seem to be involved in the prevention of interspecific recombination in transformation-competent cells.

Nat Rev Mol Cell Biol, 2000 Nov, 1(2), 101 - 9
The expanding polymerase universe; Goodman MF et al.; Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded . Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread . The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

J Antimicrob Chemother, 1999 Feb, 43(2), 219 - 26
Antimycobacterial activity of cerulenin and its effects on lipid biosynthesis; Parrish NM et al.; Cerulenin is a potent inhibitor of fatty acid synthase (FAS) in a variety of prokaryotic and eukaryotic cells . Using a standardized mycobacterial susceptibility test, we have observed that cerulenin inhibits the growth of several species of mycobacteria, including tuberculous species such as Mycobacterium tuberculosis (H37Rv and clinical isolates) and Mycobacterium bovis BCG (hereafter called BCG), as well as several non-tuberculous species: Mycobacterium smegmatis, the Mycobacterium avium-intracellulare complex (MAC), Mycobacterium kansasii and others . All species and strains tested, including multi-drug resistant isolates of M . tuberculosis, were susceptible to cerulenin with MICs ranging from 1.5 to 12.5 mg/L . Two-dimensional thin-layer chromatography revealed different inhibition patterns of lipid synthesis between tuberculous and non-tuberculous mycobacteria . Cerulenin treatment resulted in a relative increase in phospholipids and mycolic acids in MAC and M . smegmatis, whereas in cerulenin-treated BCG, phospholipids and mycolic acids diminished relative to controls . In addition, long-chain extractable lipids (intermediate in polarity), triglycerides and glycopeptidolipids decreased with cerulenin treatment in all three species of mycobacteria tested . Qualitative changes in several of these lipid classes indicate inhibition in the synthesis of intermediate and long-chain fatty acids . Our results suggest that cerulenin's primary effect may be inhibition of intermediate and long-chain lipid synthesis, with little effect on the synthesis of other lipid classes . In addition, the BCG-specific reduction in phospholipids and mycolic acids suggests the presence of a unique cerulenin-sensitive FAS system in tuberculous mycobacteria . Since pathogenic mycobacteria produce novel long-chain fatty acids, inhibition of fatty acid synthesis offers a potential target for the development of antimycobacterial drugs.

Mol Microbiol, 2001 Feb, 39(4), 1061 - 8
Evidence for non-enzymatic glycosylation in Escherichia coli; Mironova R et al.; Non-enzymatic glycosylation (glycation) is a chain of chemical reactions affecting free amino groups in proteins of long-living eukaryotes . It proceeds in several steps leading to the consecutive formation of Schiff bases, Amadori products and advanced glycation end-products (AGEs) . To our knowledge, this process has not been observed in prokaryotes so far . However, the present study provides clear-cut evidence that glycation takes place in bacteria despite their short life span . We have detected AGEs in recombinant human interferon gamma (rhIFN-gamma) produced in Escherichia coli as well as in total protein of the same bacterium using three different approaches: (i) Western blotting using two monoclonal antibodies raised against AGEs; (ii) fluorescent spectroscopy; and (iii) investigation of the effect of known AGE inhibitors (such as acetyl salicylic acid and thiamine) on the glycation reaction . Our study shows that non-enzymatic glycosylation is initiated during the normal growth of E . coli and results in AGE formation even after isolation of proteins . This process seems to be tightly associated with some post-translational modifications observed in the cysteineless rhIFN-gamma, such as covalent dimerization and truncation.

Int J Biol Macromol, 2001 Mar 14, 28(3), 255 - 61
Prediction of the subcellular location of prokaryotic proteins based on the hydrophobicity index of amino acids; Feng ZP et al.; An algorithm of predicting the subcellular location of prokaryotic proteins is proposed in this paper . In addition to the amino acid composition, the auto-correlation functions based on the hydrophobicity profile of amino acids along the primary sequence of the query protein have been used . Consequently, the best predictive accuracy to date has been achieved . Of the 997 prokaryotic proteins in the database used here, 688 cytoplasmic, 107 extracellular and 202 periplasmic proteins, the overall predictive accuracies are as high as 97.7 and 90.4% in the resubstitution and jackknife tests, respectively, using the hydrophilicity value of Hopp and Woods . The underlying mechanism of the improvement is also discussed . This work would be useful for a systematic analysis of the great amounts of prokaryotic genome sequences . The computer programs used in this paper are available on request via email.

Endocrinology, 2001 Apr, 142(4), 1517 - 24
High-level expression of a functional single-chain human chorionic gonadotropin-luteinizing hormone receptor ectodomain complex in insect cells; Fralish GB et al.; Reproductive capacity in primates is dependent on the high-affinity binding of the glycoprotein hormones LH and human (h)CG to the large ectodomain (ECD) of their common receptor (LHR) . Our understanding of the precise molecular determinants of hormone binding is limited, because there are no structural data for any of the glycoprotein hormone receptors . Overexpression of the ECD of the receptor has been attempted in various expression systems . Prokaryotic expression does not yield properly folded ECD . Eukaryotic expression, on the other hand, results in mostly heterogeneous, intracellularly trapped protein, but the secreted ECD is completely folded . Accordingly, we have tethered the single-chain hormone, yoked hCG, to the N terminus of LHR-ECD (yoked hormone-extracellular domain) . Yoked hCG is secreted at high levels; binds LHR with high affinity; and, when tethered to the N terminus of full-length LHR, it binds and constitutively activates the receptor . Using recombinant baculovirus, yoked hormone-extracellular domain is secreted from insect cells at levels greater than 1 microg/ml, nearly 20-fold higher than that previously reported in eukaryotic expression systems . The protein was purified and binds exogenous (125)I-hCG with high affinity but, significantly, only after protease treatment to remove the tethered hormone . Thus, the fusion protein seems to form a functional hormone-receptor complex that is expressed at levels sufficient for its biophysical characterization.

Structure (Camb), 2001 Feb 7, 9(2), 115 - 24
Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens betagamma-crystallin domain fold; Clout NJ et al.; BACKGROUND: The betagamma-crystallins belong to a superfamily of two-domain proteins found in vertebrate eye lenses, with distant relatives occurring in microorganisms . It has been considered that an eukaryotic stress protein, spherulin 3a, from the slime mold Physarum polycephalum shares a common one-domain ancestor with crystallins, similar to the one-domain 3-D structure determined by NMR . RESULTS: The X-ray structure of spherulin 3a shows it to be a tight homodimer, which is consistent with ultracentrifugation studies . The (two-motif) domain fold contains a pair of calcium binding sites very similar to those found in a two-domain prokaryotic betagamma-crystallin fold family member, Protein S . Domain pairing in the spherulin 3a dimer is two-fold symmetric, but quite different in character from the pseudo-two-fold pairing of domains in betagamma-crystallins . There is no evidence that the spherulin 3a single domain can fold independently of its partner domain, a feature that may be related to the absence of a tyrosine corner . CONCLUSION: Although it is accepted that the vertebrate two-domain betagamma-crystallins evolved from a common one-domain ancestor, the mycetezoan single-domain spherulin 3a, with its unique mode of domain pairing, is likely to be an evolutionary offshoot, perhaps from as far back as the one-motif ancestral stage . The spherulin 3a protomer stability appears to be dependent on domain pairing . Spherulin-like domain sequences that are found within bacterial proteins associated with virulence are likely to bind calcium.

Prog Neurobiol, 2001 May, 64(1), 1 - 33
Cooperation of the basal ganglia, cerebellum, sensory cerebrum and hippocampus: possible implications for cognition, consciousness, intelligence and creativity; Cotterill RM; It is suggested that the anatomical structures which mediate consciousness evolved as decisive embellishments to a (non-conscious) design strategy present even in the simplest unicellular organisms . Consciousness is thus not the pinnacle of a hierarchy whose base is the primitive reflex, because reflexes require a nervous system, which the single-celled creature does not possess . By postulating that consciousness is intimately connected to self-paced probing of the environment, also prominent in prokaryotic behavior, one can make mammalian neuroanatomy amenable to dramatically straightforward rationalization . Muscular contraction is the nervous system's only externally directed product, and the signaling routes which pass through the various brain components must ultimately converge on the motor areas . The function of several components is still debatable, so it might seem premature to analyze the global operation of the circuit these routes constitute . But such analysis produces a remarkably simple picture, and it sheds new light on the roles of the individual components . The underlying principle is conditionally permitted movement, some components being able to veto muscular contraction by denying the motor areas sufficient activation . This is true of the basal ganglia (BG) and the cerebellum (Cb), which act in tandem with the sensory cerebrum, and which can prevent the latter's signals to the motor areas from exceeding the threshold for overt movement . It is also true of the anterior cingulate, which appears to play a major role in directing attention . In mammals, the result can be mere thought, provided that a second lower threshold is exceeded . The veto functions of the BG and the Cb stem from inhibition, but the countermanding disinhibition develops at markedly different rates in those two key components . It develops rapidly in the BG, control being exercised by the amygdala, which itself is governed by various other brain regions . It develops over time in the Cb, thereby permitting previously executed movements that have proved advantageous . If cognition is linked to overt or covert movement, intelligence becomes the ability to consolidate individual motor elements into more complex patterns, and creativity is the outcome of a race-to-threshold process which centers on the motor areas . Amongst the ramifications of these ideas are aspects of cortical oscillations, phantom limb sensations, amyotrophic lateral sclerosis (ALS) the difficulty of self-tickling and mirror neurons.

FEMS Microbiol Rev, 2001 Apr, 25(2), 147 - 74
The archaeal flagellum: a different kind of prokaryotic motility structure; Thomas NA et al.; The archaeal flagellum is a unique motility apparatus distinct in composition and likely in assembly from the bacterial flagellum . Gene families comprised of multiple flagellin genes co-transcribed with a number of conserved, archaeal-specific accessory genes have been identified in several archaea . However, no homologues of any bacterial genes involved in flagella structure have yet been identified in any archaeon, including those archaea in which the complete genome sequence has been published . Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins . Also unlike bacterial flagellins but similar to type IV pilins, archaeal flagellins are initially synthesized with a short leader peptide that is cleaved by a membrane-located peptidase . With recent advances in genetic transfer systems in archaea, knockouts have been reported in several genes involved in flagellation in different archaea . In addition, techniques to isolate flagella with attached hook and anchoring structures have been developed . Analysis of these preparations is under way to identify minor structural components of archaeal flagella . This and the continued isolation and characterization of flagella mutants should lead to significant advances in our knowledge of the composition and assembly of archaeal flagella.

Eur J Biochem, 2001 Mar, 268(6), 1869 - 75
Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp . strain PCC 7120; Gonzalez L et al.; Eukaryotic-like protein Ser/Thr and Tyr kinases have only recently been discovered in prokaryotes . In most cases, their biochemical properties have been poorly characterized . The nitrogen-fixing and heterocyst-forming cyanobacterium Anabaena sp . strain PCC 7120 houses a family of eukaryotic-like Ser/Thr kinases . Some of these enzymes are required for cell growth or development under certain conditions . None of them, however, has been shown experimentally to possess Ser/Thr kinase activity . A gene, pknC, encoding a novel putative Ser/Thr kinase was isolated from Anabaena sp . PCC 7120 . The recombinant PknC was shown to be phosphorylated on a Thr residue . This phosphorylation was probably due to the autophosphorylation activity of PknC itself because mutation of two amino acid residues within the subdomain II of its catalytic domain eliminated the phosphorylation of PknC . PknC displayed also a Ser kinase activity towards several nonspecific substrates, and the two residues needed for PknC autophosphorylation was equally required for the phosphorylation of other substrates . PknC is thus a Ser/Thr kinase with broad substrate specificity . The activity of PknC is likely to be regulated in vivo in order to limit the spectrum of its substrate specificity.

FEMS Microbiol Ecol, 2001 Mar, 35(1), 27 - 36
Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits; Brauman A et al.; Termites are an important group of terrestrial insects that harbor an abundant gut microbiota, many of which contribute to digestion, termite nutrition and gas (CH(4), CO(2) and H(2)) emission . With 2200 described species, termites also provide a good model to study relationships between host diet and gut microbial community structure and function . We examined the relationship between diet and gut prokaryotic community profiles in 24 taxonomically and nutritionally diverse species of termites by using nucleic acid probes targeting 16S-like ribosomal RNAs . The relative abundance of domain-specific 16S-like rRNAs recovered from gut extracts varied considerably (ranges: Archaea (0-3%); Bacteria (15-118%)) . Although Bacteria were always detectable and the most abundant, differences in domain-level profiles were correlated with termite diet, as evidenced by higher relative abundances of Archaea in guts of soil-feeding termites, compared to those of wood-feeding species in the same family . The oligonucleotide probes also readily distinguished gut communities of wood-feeding taxa in the family Termitidae (higher termites) from those of other wood-feeding termite families (lower termites) . The relative abundances of 16S-like archaeal rRNA in guts were positively correlated with rates of methane emission by live termites, and were consistent with previous work linking high relative rates of methanogenesis with the soil (humus)-feeding habit . Probes for methanogenic Archaea detected members of only two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these typically accounted for 60% of the all archaeal probe signal . In four species of termites, Methanosarcinaceae were dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogen family profiles and termite diet or taxonomy.

Biochim Biophys Acta, 2001 May 1, 1505(1), 131 - 43
Towards the molecular mechanism of Na(+)/solute symport in prokaryotes; Jung H; The Na(+)/solute symporter family (SSF, TC No . 2.A.21) contains more than 40 members of pro- and eukaryotic origin . Besides their sequence similarity, the transporters share the capability to utilize the free energy stored in electrochemical Na(+) gradients for the accumulation of solutes . As part of catabolic pathways most of the transporters are most probably involved in the acquisition of nutrients . Some transporters play a role in osmoadaptation . With a high resolution structure still missing, a combination of genetic, protein chemical and spectroscopic methods has been used to gain new insights into the structure and molecular mechanism of action of the transport proteins . The studies suggest a common 13-helix motif for all members of the SSF according to which the N-terminus is located in the periplasm and the C-terminus is directed into the cytoplasm (except for proteins containing a N- or C-terminal extension) . Furthermore, an amino acid substitution analysis of the Na(+)/proline transporter (PutP) of Escherichia coli, a member of the SSF, has identified regions of particular functional importance . For example, amino acids of TM II of PutP proved to be critical for high affinity binding of Na(+) and proline . In addition, it was shown that ligand binding induces widespread conformational alterations in the transport protein . Taken together, the studies substantiate the common idea that Na(+)/solute symport is the result of a series of ligand-induced structural changes.

Traffic, 2001 Feb, 2(2), 99 - 104
Strategies for prokaryotic expression of eukaryotic membrane proteins; Laage R et al.; High-level heterologous expression of integral membrane proteins at full-length is a useful tool for their structural and functional characterization . Here, systems that have previously been used for efficient bacterial expression of eukaryotic membrane proteins are reviewed and novel vectors consisting of a modular fusion moiety based on nuclease A from Staphylococcus aureus are presented.

Plant Physiol, 2001 Mar, 125(3), 1206 - 15
Aquaporins constitute a large and highly divergent protein family in maize; Chaumont F et al.; Aquaporins (AQPs) are an ancient family of channel proteins that transport water and neutral solutes through a pore and are found in all eukaryotes and most prokaryotes . A comparison of the amino acid sequences and phylogenetic analysis of 31 full-length cDNAs of maize (Zea mays) AQPs shows that they comprise four different groups of highly divergent proteins . We have classified them as plasma membrane intinsic proteins (PIPs), tonoplast intrinsic proteins, Nod26-like intrinsic proteins, and small and basic intrinsic proteins . Amino acid sequence identities vary from 16% to 100%, but all sequences share structural motifs and conserved amino acids necessary to stabilize the two loops that form the aqueous pore . Most divergent are the small and basic integral proteins in which the first of the two highly conserved Asn-Pro-Ala motifs of the pore is not conserved, but is represented by alanine-proline-threonine or alanine-proline-serine . We present a model of ZmPIP1-2 based on the three-dimensional structure of mammalian AQP1 . Tabulation of the number of times that the AQP sequences are found in a collection of databases that comprises about 470,000 maize cDNAs indicates that a few of the maize AQPs are very highly expressed and many are not abundantly expressed . The phylogenetic analysis supports the interpretation that the divergence of PIPs through gene duplication occurred more recently than the divergence of the members of the other three subfamilies . This study opens the way to analyze the function of the proteins in Xenopus laevis oocytes, determine the tissue specific expression of the genes, recover insertion mutants, and determine the in planta function.

J Bacteriol, 2001 Apr, 183(7), 2187 - 97
Physiological basis for conservation of the signal recognition particle targeting pathway in Escherichia coli; Bernstein HD et al.; The Escherichia coli signal recognition particle (SRP) is a ribonucleoprotein complex that targets nascent inner membrane proteins (IMPs) to transport sites in the inner membrane (IM) . Since SRP depletion only partially inhibits IMP insertion under some growth conditions, however, it is not clear why the particle is absolutely essential for viability . Insights into this question emerged from experiments in which we analyzed the physiological consequences of reducing the intracellular concentration of SRP below the wild-type level . We found that even moderate SRP deficiencies that have little effect on cell growth led to the induction of a heat shock response . Genetic manipulations that suppress the heat shock response were lethal in SRP-deficient cells, indicating that the elevated synthesis of heat shock proteins plays an important role in maintaining cell viability . Although it is conceivable that the heat shock response serves to increase the capacity of cells to target IMPs via chaperone-based mechanisms, SRP-deficient cells did not show an increased dependence on either GroEL or DnaK . By contrast, the heat shock-regulated proteases Lon and ClpQ became essential for viability when SRP levels were reduced . These results suggest that the heat shock response protects SRP-deficient cells by increasing their capacity to degrade mislocalized IMPs . Consistent with this notion, a model IMP that was mislocalized in the cytoplasm as the result of SRP depletion appeared to be more stable in a Deltalon DeltaclpQ strain than in control cells . Taken together, the data provide direct evidence that SRP is essential in E . coli and possibly conserved throughout prokaryotic evolution as well partly because efficient IMP targeting prevents a toxic accumulation of aggregated proteins in the cytoplasm.

Biochem Biophys Res Commun, 2001 Mar, 281(5), 1261 - 5
Molecular cloning of actinohivin, a novel anti-HIV protein from an actinomycete, and its expression in Escherichia coli; Inokoshi J et al.; Syncytium-inducing variants of the HIV-1 virus are correlated with poor diagnosis and rapid disease progression . We have recently discovered a novel anti-HIV protein, referred to as actinohivin, that inhibits syncytium formation . Here we describe the cloning and sequencing of the gene encoding actinohivin from the actinomycete strain K97-0003, and its expression in Escherichia coli . The actinohivin gene was located on a 0.8-kb BamHI fragment of genomic DNA . The fragment contained an open reading frame of 480 bp, which encoded a protein of 160 amino acids with calculated molecular weight of 17492.7 . The N-terminal region was found to be a typical signal peptide of prokaryotes, and actinohivin was located at amino acid positions 46-160 . The actinohivin gene could be expressed in E . coli using a pET30Xa/LIC expression vector and the purified recombinant actinohivin was found to inhibit syncytium formation to a similar extent as actinohivin from its natural source .

Environ Microbiol, 2000 Feb, 2(1), 11 - 26
Cyanobacterial-bacterial mat consortia: examining the functional unit of microbial survival and growth in extreme environments; Paerl HW et al.; Cyanobacterial-bacterial consortial associations are taxonomically complex, metabolically interactive, self-sustaining prokaryotic communities representing pioneer and often the only biota inhabiting extreme aquatic and terrestrial environments . Laminated mats and aggregates exemplify such communities . The fossil record indicates that these associations represent the earliest extant inhabitants and modifiers (i.e . anoxic to oxic conditions) of the Earth's biosphere . Present-day consortia flourish in physically and chemically stressed environments, including nutrient-deplete, hypersaline, calcified, desiccated and high-irradiance ecosystems ranging from the tropics to polar regions . Consortial members exhibit extensive metabolic diversification, but have remained structurally simple . Structural simplicity, while advantageous in countering environmental extremes, presents a 'packaging problem' with regard to compartmentalizing potentially cross-inhibitory aerobic versus anaerobic growth processes . To circumvent these metabolic constraints, phototrophic cyanobacteria and microheterotrophs orient along microscale chemical (i.e . O2, pH, Eh) gradients to meet and optimize the biogeochemical processes (C, N, S cycling) essential for survival, growth and the maintenance of genetic diversity, needed to sustain life . Microscale ecophysiological, analytical, molecular (immunological and nucleic acid) techniques have helped to develop a mechanistic basis for understanding consortial growth and survival under extreme environmental conditions on Earth . Consortia are ideal model systems for developing a process-based understanding of the structural and functional requirements for life in extreme environments representative of the Earth's earliest biosphere and possibly other planets.

Zhonghua Xue Ye Xue Za Zhi, 1998 Mar, 19(3), 146 - 8
{Expression and purification of recombinant hirudin}; Guo J et al.; OBJECTIVE: To express the recombinant hirudin variant 1 (rHV1) with biological activity in prokaryotic cells and then isolate and purify the expressed products . METHODS: The hirudin variant 1 gene in plasmid vector pBV220 was expressed in E . coli strain DH5 alpha . The biological activity of rHV1 was determined with chromogenic substrate method . The expressed product was purified by ultrafiltration, DEAE-Sephadex A-50 filtration and thrombin-Sepharose 4B affinity chromatography . RESULTS: The expressed rHV1 accounted for approximately 16.9% of E . coli cell proteins with an activity of 20-30 ATU (antithrombin unit) per milliliter culture . The purified rHV1 showed a homogeneous band on SDS-PAGE . CONCLUSIONS: This is the first report in China on successful expression of hirudin variant 1 gene in E . coli and purification of the expressed rHV1.

Br J Nutr, 2000 Nov, 84 Suppl 1, S55 - 8
Polyamines in human and animal milk; Loser C; Polyamines are highly regulated polycations which are essentially involved in cell growth and differentiation . Polyamines in food significantly contribute to the polyamine body pool . Dietary polyamines exert various direct and indirect trophic effects on the rat's immature intestine and play an important role during intestinal maturation . Human milk and that of other mammalians contain relatively high levels of polyamines which are essential luminal growth and maturation factors . The polyamines spermidine and spermine as well as their diamine precursor putrescine are ubiquitous normal constituents of all prokaryotic and eukaryotic cells and are essentially involved in various processes of cell growth and differentiation (Pegg & McCann, 1982; Tabor & Tabor, 1984; Seiler, 1990).

Zhonghua Gan Zang Bing Za Zhi, 2001 Feb, 9(1), 22 - 4
Phosphorylation of hepatic stimulator substance on mitogen-activated protein kinase in BEL-7402 hepatoma cells; Chen L et al.; OBJECTIVE: To obtain more insight into information of signal transduction of EGF-receptor-mediated pathway response to the stimulation of hepatic stimulator substance (HSS) . METHODS: HSS was extracted from weanling rat liver and partially purified . Bioactivity of HSS was confirmed with its ability to proliferate hepatoma cell in vitro . Meanwhile, a rat recombinant HSS vector was constructed and expressed in BL-21 E . Coli . Mitogen-activated protein kinase (MAPK) activation marked by phosphorylation at Thr202/Tyr204 was determined by Western blot . RESULTS: The molecular weight of the biochemically purified HSS was found identically to that of the recombined HSS as expressed in the prokaryotic cells . After the treatment of HSS, cellular MAPK phosphorylation was initiated obviously at 15 min and maintained to 30 min . In comparison with EGF, MAPK phosphorylation as stimulated by HSS appeared less intensive and later time-kinetics as well . The HSS induction on cellular MAPK phosphorylation was gradually inhibited by PD98059, a specific inhibitor of MAPK kinase (MEK) . A complete blockade was seen at 100 micromol/L of PD98059 . CONCLUSIONS: The involvement of HSS on MAPK activation implies that this liver-specific growth factor might take part in, either individually or as combined with other growth factors, the regulation of TPK signaling cascade during hepatocyte proliferation.

J Exp Zool, 2001 Apr 1, 289(4), 232 - 44
Biology of the 2Na+/1H+ antiporter in invertebrates; Ahearn GA et al.; The functional expression of membrane transport proteins that are responsible for exchanging sodium and protons is a ubiquitous phenomenon . Among vertebrates the Na+/H+ antiporter occurs in plasma membranes of polarized epithelial cells and non-polarized cells such as red blood cells, muscle cells, and neurons, and in each cell type the transporter exchanges one sodium for one hydrogen ion, is inhibited by amiloride, and regulates intracellular pH and sodium concentration within tight limitations . In polarized epithelial cells this transporter occurs in two isoforms, each of which is restricted to either the brush border or basolateral cell membrane, and perform somewhat different tasks in the two locations . In prokaryotic cells, sodium/proton exchange occurs by an electrogenic 1Na+/2H+ antiporter that is coupled to a primary active proton pump and together these two proteins are capable of tightly regulating the intracellular concentrations of these cations in cells that may occur in environments of 4 M NaCl or pH 10-12 . Invertebrate epithelial cells from the gills, gut, and kidney also exhibit electrogenic sodium/proton exchange, but in this instance the transport stoichiometry is 2Na+/1H+ . As with vertebrate electroneutral Na+/H+ exchange, the invertebrate transporter is inhibited by amiloride, but because of the occurrence of two external monovalent cation binding sites, divalent cations are able to replace external sodium and also be transported by this system . As a result, both calcium and divalent heavy metals, such as zinc and cadmium, are transported across epithelial brush border membranes in these animals and subsequently undergo a variety of biological activities once accumulated within these cells . Absorbed epithelial calcium in the crustacean hepatopancreas may participate in organismic calcium balance during the molt cycle and accumulated heavy metals may undergo complexation reactions with intracellular anions as a detoxification mechanism . Therefore, while the basic process of sodium/proton exchange may occur in invertebrate cells, the presence of the electrogenic 2Na+/1H+ antiporter in these cells allows them to perform a wide array of functions without the need to develop and express additional specialized transport proteins . J . Exp . Zool . 289:232-244, 2001 .

Biopolymers, 2001 Apr 15, 58(5), 491 - 9
Prediction of the subcellular location of prokaryotic proteins based on a new representation of the amino acid composition; Feng ZP; A new representation of protein sequence is devoted in this paper, in which each protein can be represented by a 20-dimensional (20D) vector of unit length . Inspired by the principle of superposition of state in quantum mechanics, the squares of the 20 components of the vector correspond to the amino acid composition . Using the new representation of the primary sequence and Bayes Discriminant Algorithm, the subcellular location of prokaryotic proteins was predicted . The overall predictive accuracy in the jackknife test can be 3% higher than the result of using amino acid composition directly for the database of sequence identity is less than 90%, but 5% higher when sequence identity is less than 80% . The higher predictive accuracy indicates that the current measure of extracting the information from the primary sequence is efficient . Since the subcellular location restricting a protein's possible function, the present method should also be a useful measure for the systematic analysis of genome data . The program used in this paper is available on request.

Parasite Immunol, 2001 Mar, 23(3), 141 - 52
A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins; Kasper G et al.; Calreticulin was recently identified as a hookworm (Necator americanus) allergen, implying secretion, and contact with cells of the immune system, or significant worm attrition in the tissues of the host . As human calreticulin has been shown to bind to and neutralize the haemolytic activity of the complement component C1q, and to be putatively involved in integrin-mediated intracellular signalling events in platelets, it was of interest to determine whether a calreticulin from a successful nematode parasite of humans, with known immune modulatory and antihaemostatic properties, exhibited a capacity to interfere with complement activation and to interact with integrin domains associated with cell signalling in platelets and other leucocytes . We can now report that recombinant calreticulin failed to demonstrate significant calcium binding capacity, which is a hallmark of calreticulins in general and may indicate inappropriate folding following expression in a prokaryote . Nevertheless, recombinant calreticulin retained sufficient molecular architecture to bind to, and inhibit the haemolytic capacity of, human C1q . Furthermore, recombinant calreticulin reacted in surface plasmon resonance analysis (SPR) with peptides corresponding to cytoplasmic signalling domains of the integrins alphaIIb and alpha5, in a calcium independent manner . SPR was also used to ratify the specificity of a polyclonal antibody to hookworm calreticulin, which was then used to assess the stage specificity of expression of the native molecule (in comparison with reverse transcriptase-polymerase chain reaction), to indicate its apparent secretion, and to purify native calreticulin from worm extracts by affinity chromatography . This development will allow the functional tests described above to be repeated for native calreticulin, to ascertain its role in the host-parasite relationship.

J Microbiol Methods, 2001 Apr, 44(3), 217 - 23
Identification of novel Mycoplasma hyorhinis gene fragments by differential display analysis of co-cultures; Taxman DJ et al.; Mycoplasmas are a diverse group of wall-less prokaryotes that have evolved an unusually small genome by adopting a parasitic mode of life . Recently, intense efforts have been made to sequence mycoplasma genomes and to define a minimal genome using mycoplasma as a model . Due to their parasitic nature, mycoplasma species are often difficult to cultivate, making it challenging to identify and sequence mycoplasma genes . In this report, we describe a method for identifying mycoplasma gene fragments from co-cultures using differential display analysis . Using this technique, we have identified fragments of seven putative genes from Mycoplasma hyorhinis . Sequence similarities suggest that four of these genes are members of the proposed minimal mycoplasma genome . The application of differential display analysis to co-cultures should be useful in the identification of genes from a variety of pathogenic organisms that are difficult to cultivate without a host.

Microbiol Mol Biol Rev, 2001 Mar, 65(1), 80 - 105
P(II) signal transduction proteins, pivotal players in microbial nitrogen control; Arcondeguy T et al.; The P(II) family of signal transduction proteins are among the most widely distributed signal proteins in the bacterial world . First identified in 1969 as a component of the glutamine synthetase regulatory apparatus, P(II) proteins have since been recognized as playing a pivotal role in control of prokaryotic nitrogen metabolism . More recently, members of the family have been found in higher plants, where they also potentially play a role in nitrogen control . The P(II) proteins can function in the regulation of both gene transcription, by modulating the activity of regulatory proteins, and the catalytic activity of enzymes involved in nitrogen metabolism . There is also emerging evidence that they may regulate the activity of proteins required for transport of nitrogen compounds into the cell . In this review we discuss the history of the P(II) proteins, their structures and biochemistry, and their distribution and functions in prokaryotes . We survey data emerging from bacterial genome sequences and consider other likely or potential targets for control by P(II) proteins.

Mol Cell Biol, 2001 Mar, 21(5), 1491 - 8
Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2; Copeland PR et al.; The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec . The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3' untranslated region as well as the SECIS binding protein SBP2 . Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis . We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1 . We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding . Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding . Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity . This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA . SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

Biochem J, 2001 Mar 15, 354(Pt 3), 511 - 9
Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter; Goswami T et al.; In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases . Nramp2 is associated with anaemia . Both belong to a highly conserved eukaryote/prokaryote protein family . We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter . Strikingly, however, where Nramp2 is a symporter of H+ and metal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient . At pH 9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1(G169); P=3.22x10(-6)) and human NRAMP1 (P=3.87x10(-5)) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls . At pH 5.5, Nramp1(G169) (P=1.34x10(-13)) and NRAMP1 (P=1.09x10(-6)) oocytes showed significant efflux of Zn2+ . Zn2+ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone . Using pre-acidified oocytes, currents of 130+/-57 nA were evoked by 100 microM Zn2+ at pH 7.5, and 139+/-47 nA by 100 microM Fe2+ at pH 7.0, in Nramp1(G169) oocytes; currents of 254+/-49 nA and 242+/-26 nA were evoked, respectively, in NRAMP1 oocytes . Steady-state currents evoked by increasing concentrations of Zn2+ were saturable, with apparent affinity constants of approx . 614 nM for Nramp1(G169) and approx . 562 nM for NRAMP1 oocytes, and a curvilinear voltage dependence of transporter activity (i.e . the data points approximate to a curve that approaches a linear asymptote) . In the present study we propose a new model for metal ion homoeostasis in macrophages . Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol . Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into this acidic compartment where they may directly affect antimicrobial activity.

Mol Ther, 2001 Feb, 3(2), 178 - 85
Targeted correction of a defective selectable marker gene in human epithelial cells by small DNA fragments; Colosimo A et al.; A novel gene targeting strategy, small fragment homologous replacement (SFHR), has been used to correct specific genomic lesions in human epithelial cells . The frequency of targeting was estimated to be 1-10% . However, given the genomic target, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, it is difficult to accurately quantify targeting frequency . As an alternative to targeting CFTR, targeted correction of a mutant selectable marker or reporter gene would be more amenable to accurate and rapid quantification of gene targeting efficiency . The present study evaluates the conditions that modulate SFHR-mediated correction of a defective Zeocin antibiotic resistance (Zeo(r)) gene that has been inactivated by a 4-bp insertion . The conditions include delivery systems, plasmid-to-fragment ratio, fragment length, and fragment strandedness (single- or double-stranded DNA) . Targeting fragments comprise the wild-type Zeo(r) gene sequence and were either 410 (Zeo1) or 458 bp (Zeo3) . Expression vectors containing the corrected Zeo(r) gene were isolated as episomal plasmids or were allowed to stably integrate into cultured human airway epithelial cells . Correction of the Zeo(r) gene was phenotypically defined as restoration of resistance to Zeocin in either bacteria or epithelial cell clones . Extrachromosomal gene correction was assayed using polymerase chain reaction amplification, restriction enzyme digestion, DNA sequencing, and Southern blot hybridization analysis of DNA from isolated prokaryotic and eukaryotic clones . Neither random sequence alteration in the target episomal gene nor random integration of the small fragments was detected . Targeted correction efficiencies of up to 4% were attained . These studies provide insight into parameters that can be modulated for the optimization of SFHR-mediated targeting.

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