Prokaryotes

DNA Cell Biol, 2002 Mar, 21(3), 163 - 88
Interaction in vitro of type III intermediate filament proteins with triplex DNA; Li G et al.; As previously shown, type III intermediate filaments (IFs) select from a mixture of linear mouse genomic DNA fragments mobile and repetitive, recombinogenic sequences that have also been identified in SDS-stable crosslinkage products of vimentin and DNA isolated from intact fibroblasts . Because these sequences also included homopurine.homopyrimidine (Pu.Py) tracts known to adopt triple-helical conformation under superhelical tension, and because IF proteins are single-stranded (ss) and supercoiled DNA-binding proteins, it was of interest whether they have a particular affinity for triplex DNA . To substantiate this, IF-selected DNA fragments harboring a (Pu.Py) segment and synthetic d(GA)(n) microsatellites were inserted into a vector plasmid and the constructs analyzed for their capacity to interact with IF proteins . Band shift assays revealed a substantially higher affinity of the IF proteins for the insert-containing plasmids than for the empty vector, with an activity decreasing in the order of vimentin > glial fibrillary acidic protein > desmin . In addition, footprint analyses performed with S1 nuclease, KMnO(4), and OsO(4)/bipyridine showed that the (Pu.Py) inserts had adopted triplex conformation under the superhelical strain of the plasmids, and that the IF proteins protected the triple-helical insert sequences from nucleolytic cleavage and chemical modification . All these activities were largely reduced in extent when analyzed on linearized plasmid DNAs . Because intramolecular triplexes (H-DNA) expose single-stranded loops, and the prokaryotic ssDNA-binding proteins g5p and g32p also protected at least the Pu-strand of the (Pu.Py) inserts from nucleolytic degradation, it seemed likely that the IF proteins take advantage of their ssDNA-binding activity in interacting with H-DNA . However, in contrast to g5p and E . coli SSB, they produced no clear band shifts with single-stranded d(GA)(20) and d(TC)(20), so that the interactions rather appear to occur via the duplex-triplex and triplex-loop junctions of H-DNA . On the other hand, the IF proteins, and also g32p, promoted the formation of intermolecular triplexes from the duplex d{A(GA)(20).(TC)(20)T} and d(GA)(20) and d(TC)(20) single strands, with preference of the Py (Pu.Py) triplex motif, substantiating an affinity of the proteins for the triplex structure as such . This triplex-stabilizing effect of IF proteins also applies to the H-DNA of (Pu.Py) insert-containing plasmids, as demonstrated by the preservation of intramolecular triplex-vimentin complexes upon linearization of their constituent supercoiled DNAs, in contrast to poor complex formation from free, linearized plasmid DNA and vimentin . Considering that (Pu.Py) sequences are found near MAR/replication origins, in upstream enhancer and promoter regions of genes, and in recombination hot spots, these results might point to roles of IF proteins in DNA replication, transcription, recombination, and repair.

J Comput Biol, 2002, 9(2), 225 - 42
Finding motifs using random projections; Buhler J et al.; The DNA motif discovery problem abstracts the task of discovering short, conserved sites in genomic DNA . Pevzner and Sze recently described a precise combinatorial formulation of motif discovery that motivates the following algorithmic challenge: find twenty planted occurrences of a motif of length fifteen in roughly twelve kilobases of genomic sequence, where each occurrence of the motif differs from its consensus in four randomly chosen positions . Such "subtle" motifs, though statistically highly significant, expose a weakness in existing motif-finding algorithms, which typically fail to discover them . Pevzner and Sze introduced new algorithms to solve their (15,4)-motif challenge, but these methods do not scale efficiently to more difficult problems in the same family, such as the (14,4)-, (16,5)-, and (18,6)-motif problems . We introduce a novel motif-discovery algorithm, PROJECTION, designed to enhance the performance of existing motif finders using random projections of the input's substrings . Experiments on synthetic data demonstrate that PROJECTION remedies the weakness observed in existing algorithms, typically solving the difficult (14,4)-, (16,5)-, and (18,6)-motif problems . Our algorithm is robust to nonuniform background sequence distributions and scales to larger amounts of sequence than that specified in the original challenge . A probabilistic estimate suggests that related motif-finding problems that PROJECTION fails to solve are in all likelihood inherently intractable . We also test the performance of our algorithm on realistic biological examples, including transcription factor binding sites in eukaryotes and ribosome binding sites in prokaryotes.

Biotechniques, 2002 May, 32(5), 1178, 1180, 1182 - 7
BiZyme: a novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance; Hansen SG et al.; Recombinant vaccinia virus is a useful and powerful tool for the expression and study of foreign genes . Methods that are currently available for the selection of vaccinia virus recombinants include the restoration of viral plaque-forming phenotype, the replication of viral DNA in the presence of BUdR or mycophenolic acid, and the maturation and propagation of virus under antibiotic selection . Though effective, each of these methods requires several weeks of concerted effort to isolate, purify, and amplify a potential recombinant virus . Here we report the development of a bifunctional enzyme (BiZyme) to simplify and expedite the isolation and purification of vaccinia virus recombinants . This novel selection marker is composed of an in-frame fusion between the genes encoding gfp and the neomycin phosphotransferase enzyme (neo) . Remarkably, expression of the chimeric gfp-neo cassette in the presence of G418 confers both viability and fluorescence to transfected or recombinant virus-infected cells, indicating that both activities are retained within the fusion protein . Therfore, BiZyme was incorporated into a recombination plasmid (pGNR) to enable the concomitant insertion of a foreign gene of interest . Here we demonstrate that this selection/amplification process requires a minimum of 11 days to produce the desired vaccinia virus recombinants . Furthermore, recombinants produced in this fashion have been shown to express both biologically active enzymes and antigenically authenticforeign antigens . In addition to its use in the vaccinia virus vector system, the BiZyme bifunctional selection scheme should be applicable to other eukaryotic and prokaryotic expression systems, simply by coupling it to the appropriate host-specific transcription regulatory signals.

Biotechniques, 2002 May, 32(5), 1020, 1022, 1024 - 5 passim
Cyclic nucleotide-dependent screening of a lamda expression library for nucleic acid binding activities; Loflin P et al.; Here we describe a modified ligand screening strategy for the expression cloning of mammalian proteins that require the activation of protein kinase cascades to activate ligand binding activity . The manipulation of prokaryotic signaling pathways by the application of appropriate inhibitors or agonists to the nitrocellulose filter during functional screening of standard bacteriophage cDNA expression libraries can permit detection of activities that would not otherwise be found in their active state . We have applied this strategy to a A expression library to clone a novel renal cDNA that exhibits cAMP-dependent RNA binding activity when subsequently tested by ectopic expression in mammalian cells.

Proteins, 2002 Jul 1, 48(1), 134 - 40
Simple sequences are rare in the Protein Data Bank; Huntley MA et al.; A simple sequence is abundant in the proteins that have been sequenced to date . But unusual protein features, such as a simple sequence, are not present in the same high frequency within structural databases . A subset of these simple sequences, a group with a highly repetitive nature has been shown to be abundant in eukaryotes but not in prokaryotes . In this study, an examination of the eukaryotic proteins in the Protein Data Bank (PDB) has revealed a large deficiency of low complexity, highly repetitive protein repeats . Through simulated databases of similar samples of eukaryotic proteins taken from the National Center for Biotechnology Information (NCBI) database, it is shown that the PDB contains a significantly less highly repetitive, simple sequence than artificial databases of similar composition randomly derived from NCBI . When the structural data for those few PDB sequences that did contain a highly repetitive simple sequence is examined in detail, it is found that in most cases the tertiary structure is unknown for the regions consisting of a simple sequence . This lack of a simple sequence both in the PDB database and in the structural information suggests that this type of simple sequence may produce disordered structures that make structural characterization difficult .

Proteins, 2002 Jul 1, 48(1), 75 - 84
Expanding the nitrogen regulatory protein superfamily: Homology detection at below random sequence identity; Kinch LN et al.; Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots . PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation . Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold . Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages . The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups . Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function . Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them . Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information . For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions . Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction . In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect homology between highly diverse sequences and are aimed at understanding the most remote evolutionary connections in the protein world .

Biochim Biophys Acta, 2002 May 20, 1597(1), 107 - 14
Conformational change of the N-domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY; Shepotinovskaya IV et al.; The structural basis for the GTP-dependent co-translational targeting complex between the signal recognition particle (SRP) and its receptor is unknown . The complex has been shown to have unusual kinetics of formation, and association in vivo is likely to be dependent on catalysis by the SRP RNA . We have determined conditions for RNA-independent association of the 'NG' GTPase domains of the prokaryotic homologs of the SRP components, Ffh and FtsY, from Thermus aquaticus . Consistent with previous studies of the Escherichia coli proteins, the kinetics of association and dissociation are slow . The T . aquaticus FtsY is sensitive to an endogenous proteolytic activity that cleaves at two sites--the first in a lengthy linker peptide that spans the interface between the N and G domains, and the second near the N-terminus of the N domain of FtsY . Remarkably, this second cleavage occurs only on formation of the Ffh/FtsY complex . The change in protease sensitivity of this region, which is relatively unstructured in the FtsY but not in the Ffh NG domain, implies that it undergoes conformational change on formation of the complex between the two proteins . The N domain, therefore, participates in the interactions that mediate the GTP-dependent formation of the targeting complex.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 255 - 60
Recurrent intragenomic recombination leading to sequence homogenization during the evolution of the lipoyl-binding domain; Omelchenko MV et al.; The lipoyl-binding domain is often present, in one or several copies, in the E2 subunit and, less often, in the E1 and E3 subunits of 2-oxo acid dehydrogenase complexes . Phylogenetic analysis shows evidence of multiple, independent intragenomic recombination events between different versions of the lipoyl-binding domain in various bacteria and eukaryotic mitochondria, leading to homogenization of the sequences of the lipoyl-binding domain within the same enzymatic complex in several bacterial lineages . This appears to be the first case of sequence homogenization at the level of an individual domain in prokaryotes.

FEMS Microbiol Rev, 2002 Mar, 26(1), 49 - 71
Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence; Sleator RD et al.; Two general strategies exist for the growth and survival of prokaryotes in environments of elevated osmolarity . The 'salt in cytoplasm' approach, which requires extensive structural modifications, is restricted mainly to members of the Halobacteriaceae . All other species have convergently evolved to cope with environments of elevated osmolarity by the accumulation of a restricted range of low molecular mass molecules, termed compatible solutes owing to their compatibility with cellular processes at high internal concentrations . Herein we review the molecular mechanisms governing the accumulation of these compounds, both in Gram-positive and Gram-negative bacteria, focusing specifically on the regulation of their transport/synthesis systems and the ability of these systems to sense and respond to changes in the osmolarity of the extracellular environment . Finally, we examine the current knowledge on the role of these osmostress responsive systems in contributing to the virulence potential of a number of pathogenic bacteria.

Int J Biochem Cell Biol, 2002 Aug, 34(8), 983 - 91
Effects of removal of the N-terminal amino acid residues on the activity and conformation of firefly luciferase; Wang XC et al.; A series of deletion mutants were constructed using polymerase chain reaction (PCR) to investigate the roles of luciferase N-terminal residues . The coding sequences of the first 0 (Luc0), 6 (Luc6), 7 (Luc7), 8 (Luc8), 9 (Luc9), 10 (Luc10) and 20 (Luc20) amino acids of the N-terminus were deleted and inserted into the prokaryotic expression vector pBV220 . The results showed that the enzymes were completely inactivated when the first eight or more N-terminal amino acids were removed . The recombinant Luc0 and mutants Luc6 and Luc7 were purified to homogeneity by ammonium sulfate precipitation and liquid chromatography for determination of their activity and conformational changes . The activity assay showed that removal of the first six amino acids resulted in 29% loss of enzymatic activity while removal of the first seven amino acids resulted in nearly complete inactivation (with remaining activity <0.5% of the original activity) . Circular dichroism spectra showed no significant secondary structure changes . But the fluorescence emission maximum red-shift indicated some conformational changes . Luc6 and Luc7 were more sensitive to guanidine unfolding than Luc0 . The present result indicated the significant role of Ile7 to the luciferase stability.

Biochim Biophys Acta, 2002 Apr 29, 1596(2), 193 - 200
Novel anti-inflammatory compounds from Rubus sieboldii, triterpenoids, are inhibitors of mammalian DNA polymerases; Murakami C et al.; Two anti-inflammatory triterpenoids, tormentic acid (TA) and euscaphic acid (EA), were found from the plant Rubus sieboldii . These triterpenoids showed an inhibitory effect against enzymes involved in replication, such as calf DNA polymerase alpha (pol alpha) and rat DNA polymerase beta (pol beta) . The IC50 values of TA and EA were 37 and 61 microM for pol alpha and 46 and 108 microM for pol beta, respectively . However, TA and EA did not influence the activities of plant DNA polymerases, DNA primase, human immunodeficiency virus type-1 reverse transcriptase, terminal deoxynucleotidyl transferase, any of the prokaryotic DNA polymerases or DNA and RNA metabolic enzymes tested . TA and EA could prevent the growth of BALL-1 cancer cells, and the LD50 values were 11 and 48 microM, respectively . The cells were halted at G1 phase in the cell cycle . The mode of action of the triterpenoids against anti-inflammatory activity and their relationships to the DNA polymerase inhibitory activity and cell growth inhibition were discussed.

Chembiochem, 2002 May 3, 3(5), 384 - 97
Body shaping under water stress: osmosensing and osmoregulation of solute transport in bacteria; Morbach S et al.; Fluctuation of external osmolarity is one of the most common types of environmental stress factors for all kind of cells, both of prokaryotic and of eukaryotic origin . Cells try to keep their volume and/or turgor pressure constant; consequently, both a decrease (hypoosmotic stress) and an increase (hyperosmotic stress) of the solute concentration (correctly: increase or decrease in water activity) in the surrounding area, respectively, are challenges for cellular metabolism and survival . A common example from the prokaryotic world is the fate of a soil bacterium that, after a sunny day has dried out the soil (hyperosmotic stress), is suddenly exposed to a drop of distilled water from a rain cloud (hypoosmotic stress) . The immediate and inevitable passive response to the sudden osmotic shift in the surroundings is fast water efflux out of the cell in the former situation and water influx in the latter . In the worst case, these responses may lead to either loss of cell turgor and plasmolysis or to cell burst . In order to overcome such drastic consequences cells have developed effective mechanisms, namely osmoadaptation, to cope with the two different types of osmotic stress . For a graded reaction to osmotic shifts, cells must be able (1) to sense stimuli related to osmotic stress, (2) to transduce corresponding signals to those systems that properly respond (3) by activating transport or enzymatic functions or (4) by changing gene expression profiles . In this review, membrane proteins involved in the cell's active response to osmotic stress are described . Molecular details of structure, function, and regulation of mechanosensitive efflux channels from various organisms, as well as of osmoregulated uptake systems are discussed.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 138 - 42
Mouse restin inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis; Xu R et al.; Restin, a homologous protein of endostatin, was found by Ramchandran et al . It was the C-terminal fragment of type XV collagen . To analysis the inhibition activity of mouse restin on the proliferation of endothelial cells, the cDNA of restin was amplified from the total RNA of the mouse muscle and cloned into the prokaryotic expression plasmid pQE32 . The recombinant protein was expressed in inclusion body with a yield about 60%--70% of total protein . After refolding, the purified recombinant protein specifically inhibits bovine aortic endothelial (BAE) cell proliferation stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner, but the activity of restin was weaker than that of endostatin . Treatment of BAE cell with recombinant restin caused G(1) arrest and apoptosis in BAE cells.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 143 - 8
Cloning, expression and immunization of the new antigen gene Sj-Ts4 of Schistosoma japonicum; Zhou DM et al.; In order to explore the molecular mechanism of high immune protection against schistosomes infection in animals infected with Trichinella spiralis, and to provide several cross-protective antigen genes for developing anti-schistosomiasis vaccine, a Schistosoma japonicum adult worm cDNA library was immunoscreened using sera taken from mice infected with Trichinella spiralis . Nine positive clones were obtained after 3 rounds of immunoscreening . Among them, Sj-Ts4 represents a novel gene of S . japonicum, which coding for a novel protein of 210 amino acids . The protein has a deduced molecular mass of 23 kD and isoelectric point of 7.72 . Sj-Ts4 was expressed as a glutathione-S-transferase (GST) fusion protein by cloning into the prokaryotic expression vector pGEX-5X-3 . The recombinant Sj-Ts4 protein (rSj-Ts4) was purified and could be recognized by sera of mice infected with S.japonicum . Vaccination of several groups of mice with rSj-Ts4 or rSj-Ts4 incorporated into Freund's complete adjuvant induced high titers of specific IgG antibodies . Two vaccination groups all obtained significant reduction in worm burden (31.36%, 36.80%, P<0.01), compared with the control groups.

Science, 2002 May 10, 296(5570), 1127 - 9
Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment; Kaeberlein T et al.; The majority (>99%) of microorganisms from the environment resist cultivation in the laboratory . Ribosomal RNA analysis suggests that uncultivated organisms are found in nearly every prokaryotic group, and several divisions have no known cultivable representatives . We designed a diffusion chamber that allowed the growth of previously uncultivated microorganisms in a simulated natural environment . Colonies of representative marine organisms were isolated in pure culture . These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms . This observation may help explain the nature of microbial uncultivability.

Science, 2002 May 10, 296(5570), 1064 - 6
Prokaryotic diversity--magnitude, dynamics, and controlling factors; Torsvik V et al.; There are probably millions of species in the microorganismal domains Bacteria and Archaea (the prokaryotes), and we are only just beginning to work out the basic principles governing their distribution and abundance in natural environments . One characteristic that has become clear is that prokaryote diversity in aquatic environments is orders of magnitude less than in sediments and soils . Hypotheses and models explaining such differences are under development and are beginning to offer promising insights into the mechanisms governing prokaryote diversity and ecosystem function.

J Biol Chem, 2002 Jul 26, 277(30), 26779 - 87 Epub 2002 May 09.
IMP, GTP, and 6-phosphoryl-IMP complexes of recombinant mouse muscle adenylosuccinate synthetase; Iancu CV et al.; Prokaryotes have a single form of adenylosuccinate synthetase that controls the committed step of AMP biosynthesis, but vertebrates have two isozymes of the synthetase . The basic isozyme, which predominates in muscle, participates in the purine nucleotide cycle, has an active site conformation different from that of the Escherichia coli enzyme, and exhibits significant differences in ligand recognition . Crystalline complexes presented here of the recombinant basic isozyme from mouse show the following . GTP alone binds to the active site without inducing a conformational change . IMP in combination with an acetate anion induces major conformational changes and organizes the active site for catalysis . IMP, in the absence of GTP, binds to the GTP pocket of the synthetase . The combination of GTP and IMP results in the formation of a stable complex of 6-phosphoryl-IMP and GDP in the presence or absence of hadacidin . The response of the basic isozyme to GTP alone differs from that of synthetases from plants, and yet the conformation of the mouse basic and E . coli synthetases in their complexes with GDP, 6-phosphoryl-IMP, and hadacidin are nearly identical . Hence, reported differences in ligand recognition among synthetases probably arise from conformational variations observed in partially ligated enzymes.

J Biol Chem, 2002 Jul 26, 277(30), 26886 - 92 Epub 2002 May 09.
Reconstitution of a disulfide isomerization system; Collet JF et al.; Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides . In prokaryotes, the catalytic pathway responsible for disulfide isomerization involves thioredoxin, thioredoxin reductase, and the DsbC, DsbG, and DsbD proteins . To be active as isomerases, DsbC and DsbG must be kept reduced . This task is performed by the cytoplasmic membrane protein DsbD . DsbD in turn is reduced by the cytoplasmic thioredoxin and is composed of three domains . The beta domain is membrane-embedded, whereas the alpha and gamma domains are localized to the periplasm . It had been proposed that electrons are transferred within DsbD by a succession of disulfide exchange reactions between the three domains . To test this model using biochemical methods, we purified to homogeneity different polypeptides corresponding to the alpha, beta, gamma, and betagamma domains . Using these domains, we could reconstitute a DsbD activity and, for the first time, reconstitute in vitro the electron transport pathway from NADPH and thioredoxin to DsbC and DsbG . We showed that electrons are transferred from thioredoxin to the beta domain then successively to the gamma domain, the alpha domain, and finally on to DsbC or DsbG . We also determined the redox potential of the gamma domain to be -241 mV, and that of the alpha domain was found to be -229 mV . This shows that the direction of electron flow within DsbD is thermodynamically driven.

RNA, 2002 Mar, 8(3), 370 - 81
A conserved family of Saccharomyces cerevisiae synthases effects dihydrouridine modification of tRNA; Xing F et al.; Dihydrouridine modification of tRNA is widely observed in prokaryotes and eukaryotes, as well as in some archaea . In Saccharomyces cerevisiae every sequenced tRNA has at least one such modification, and all but one have two or more . We have used a biochemical genomics approach to identify the gene encoding dihydrouridine synthase 1 (Dus1, ORF YML080w), using yeast pre-tRNA(Phe) as a substrate . Dus1 is a member of a widespread family of conserved proteins, three other members of which are found in yeast: YNR015w, YLR405w, and YLR401c . We show that one of these proteins, Dus2, encoded by ORF YNR015w, has activity with two other substrates: yeast pre-tRNA(Tyr) and pre-tRNA(Leu) . Both Dus1 and Dus2 are active as a single subunit protein expressed and purified from Escherichia coli, and the activity of both is stimulated in the presence of flavin adenine dinucleotide . Dus1 modifies yeast pre-tRNA(Phe) in vitro at U17, one of the two positions that are known to bear this modification in vivo . Yeast extract from a dus1-A strain is completely defective in modification of yeast pre-tRNAPhe, and RNA isolated from dus1-delta and dus2-delta strains is significantly depleted in dihydrouridine content.

Crit Rev Microbiol, 2002, 28(1), 61 - 77
Control of the bacterial cell cycle by cytoplasmic growth; Koch AL; For free-living single-celled organisms, it can be assumed that it is their success in acquiring resources and converting them into cytoplasm that controls the timing of their cell cycles . Cytoplasm is the sink for the bulk of the environmental resources . It must be the case that this type of control must operate in dilute cultures under adequate nutrition in a constant environment . It follows that there ought to be mechanisms that measure or count the cell's biomass or some component of the cytoplasm to measure their growth success . Besides sensing their biomass, they need to know when a certain value of the cell size has been achieved . When this critical state has been achieved, the cell needs to have an all-or-none trigger that either initiates chromosome replication, the completion of cell replication, cell division, or the process of separating sister cells physiologically or physically . Any of these four different stages, in principle, may be the one triggered in response to cell growth in different species of microorganisms . Alternatively, multiple triggers at different cell sizes may be activated at different cell cycle stages . Although initiation of chromosome replication has been believed to be the event triggered in Escherichia coli, this probably is not generally the case and other control mechanisms may act in other prokaryotes . How the increase in cell biomass is self-assessed and used to carry out critical cell cycle events is not understood in any case . This deficiency in our knowledge of microbial cell physiology is grave . The factor that probably has prevented the elucidation of the mechanisms in any organism is that enzymatic processes deal with concentrations, and a cell cycle trigger must respond to the total amount of material present in a cell . This article discusses the theoretically possible classes of mechanisms for the cell to respond when it has achieved its appropriate critical size . These breakdown into three groups: those mechanisms that assess the total amount of biomass or some special subcellular component, and those that measure the ratio of one component to another component where their two syntheses are differently controlled by cell physiology and morphology, and a third group with some specialized mechanisms.

Proteins, 2002 Jun 1, 47(4), 481 - 8
M13 endopeptidases: New conserved motifs correlated with structure, and simultaneous phylogenetic occurrence of PHEX and the bony fish; Bianchetti L et al.; M13 endopeptidase alignments have focused mainly on mammalian sequences and on the active site region defining the catalytic sequence signatures . Aligning all available M13 from bacteria to human on a full-length basis, we have performed a sequence analysis . This enabled us to highlight the origin and function of the M13 PHEX subtype family endopeptidase (phosphate regulating gene with homologies to endopeptidases on the X chromosome) . New evolutionary conserved regions in both prokaryotes and eukaryotes have been detected and eukaryotic-specific regions clearly delineated . Using the recently solved neprilysin structure, we have observed that all new motifs, except one, localize in the spatial vicinity of the previously reported catalytic signatures . Interestingly, a highly hydrophobic pocket containing three newly reported motifs is centered by the C-terminal tryptophan residue . Extensive M13 searches in complete and in progress higher eukaryotic genomes have lead to the identification of Danio rerio as the simplest organism having PHEX . Finally, the human PHEX substrate, the parathyroid hormone-related peptide, PTHrP(107-139), is absent in bony fish: this suggests the existence of further PHEX substrates common to both bony fishes and higher vertebrates .

Nature . 2002 May 9;417(6885):137.
Microbiology: eukaryotic diversity in Spain's River of Fire; Amaral Zettler LA et al.; The Rio Tinto, known by the Phoenicians as 'Ur-yero', or 'River of Fire', because of its deep red colour and high acidity, flows through the world's largest pyritic belt in southwestern Spain . Surprisingly, eukaryotic microbes are the principal contributors of biomass in this hostile river, which has a pH of 2 and contains much higher concentrations of heavy metals than are typically found in fresh waters . Here we show that the Rio Tinto shows an unexpected degree of eukaryotic diversity and includes new lineages that we have identified by sequence analysis of genes encoding small-subunit ribosomal RNAs . The diversity of these eukaryotes is much greater than that of prokaryotes, whose metabolism is responsible for the extreme environment.

Nucleic Acids Res, 2002 May 15, 30(10), 2212 - 23
Connected gene neighborhoods in prokaryotic genomes; Rogozin IB et al.; A computational method was developed for delineating connected gene neighborhoods in bacterial and archaeal genomes . These gene neighborhoods are not typically present, in their entirety, in any single genome, but are held together by overlapping, partially conserved gene arrays . The procedure was applied to comparing the orders of orthologous genes, which were extracted from the database of Clusters of Orthologous Groups of proteins (COGs), in 31 prokaryotic genomes and resulted in the identification of 188 clusters of gene arrays, which included 1001 of 2890 COGs . These clusters were projected onto actual genomes to produce extended neighborhoods including additional genes, which are adjacent to the genes from the clusters and are transcribed in the same direction, which resulted in a total of 2387 COGs being included in the neighborhoods . Most of the neighborhoods consist predominantly of genes united by a coherent functional theme, but also include a minority of genes without an obvious functional connection to the main theme . We hypothesize that although some of the latter genes might have unsuspected roles, others are maintained within gene arrays because of the advantage of expression at a level that is typical of the given neighborhood . We designate this phenomenon 'genomic hitchhiking' . The largest neighborhood includes 79 genes (COGs) and consists of overlapping, rearranged ribosomal protein superoperons; apparent genome hitchhiking is particularly typical of this neighborhood and other neighborhoods that consist of genes coding for translation machinery components . Several neighborhoods involve previously undetected connections between genes, allowing new functional predictions . Gene neighborhoods appear to evolve via complex rearrangement, with different combinations of genes from a neighborhood fixed in different lineages.

Biol Cell, 2002 Feb, 94(1), 29 - 35
New magnet-sensitive structures in bacterial and archaeal cells; Vainshtein M et al.; The objects of the investigation were: distribution of intracellular magnet-sensitive structures among different taxonomic groups of prokaryotes, localisation and organisation of the magnet-sensitive inclusions (MsI) in cells . The MsI were discovered in representatives of both prokaryotic domains (Bacteria and Archaea), 2 kingdoms and 7 orders of bacteria . They were some amorphous or non-crystalline globules with the electron-transparent centre surrounded with an electron-dense homogenous matrix . The magnetic nature of the structures was shown by attraction with an applied magnet both for the cell suspensions and for the MsI isolated and separated from the destroyed cells . The MsI were studied with transparent electron microscopy and with X-ray analyses . When the cells were grown in the iron-containing nutrient medium, the matrix was enriched with iron . It was shown also that some bacteria grown with cobalt or with chromium contained the cobalt- or chromium-enriched magnetic inclusions.

Plant Mol Biol, 2002 Mar-Apr, 48(5-6), 805 - 20
Phylogenetic analysis of the acetyl-CoA carboxylase and 3-phosphoglycerate kinase loci in wheat and other grasses; Huang S et al.; We have applied a two-gene system based on the sequences of nuclear genes encoding multi-domain plastid acetyl-CoA carboxylase (ACCase) and plastid 3-phosphoglycerate kinase (PGK) to study grass evolution . Our analysis revealed that these genes are single-copy in most of the grass species studied, allowing the establishment of orthologous relationships between them . These relationships are consistent with the known facts of their evolution: the eukaryotic origin of the plastid ACCase, created by duplication of a gene encoding the cytosolic multi-domain ACCase gene early in grass evolution, and the prokaryotic (endosymbiont) origin of the plastid PGK . The major phylogenetic relationships among grasses deduced from the nucleotide sequence comparisons of ACCase and PGK genes are consistent with each other and with the milestones of grass evolution revealed by other methods . Nucleotide substitution rates were calculated based on multiple pairwise sequence comparisons . On a relative basis, with the divergence of the Pooideae and Panicoideae subfamilies set at 60 million years ago (MYA), events leading to the Triticum/Aegilops complex occurred at the following intervals: divergence of Lolium (Lolium rigidum) at 35 MYA, divergence of Hordeum (Hordeum vulgare) at 11 MYA and divergence of Secale (Secale cereale) at 7 MYA . On the same scale, gene duplication leading to the multi-domain plastid ACCase in grasses occurred at 129 MYA, divergence of grass and dicot plastid PGK genes at 137 MYA, and divergence of grass and dicot cytosolic PGK genes at 155 MYA . The ACCase and PGK genes provide a well-understood two-locus system to study grass phylogeny, evolution and systematics.

Mol Cell Biol, 2002 Jun, 22(11), 3707 - 17
tRNAs in Trypanosoma brucei: genomic organization, expression, and mitochondrial import; Tan TH et al.; The mitochondrial genome of Trypanosoma brucei does not encode tRNAs . Consequently, all mitochondrial tRNAs are imported from the cytosol and originate from nucleus-encoded genes . Analysis of all currently available T . brucei sequences revealed that its genome carries 50 tRNA genes representing 40 different isoacceptors . The identified set is expected to be nearly complete since all but four codons are accounted for . The number of tRNA genes in T . brucei is very low for a eukaryote and lower than those of many prokaryotes . Using quantitative Northern analysis we have determined the absolute abundance in the cell and the mitochondrion of a group of 15 tRNAs specific for 12 amino acids . Except for the initiator type tRNA(Met), which is cytosol specific, the cytosolic and the mitochondrial sets of tRNAs were qualitatively identical . However, the extent of mitochondrial localization was variable for the different tRNAs, ranging from 1 to 7.5% per cell . Finally, by using transgenic cell lines in combination with quantitative Northern analysis it was shown that import of tRNA(Leu)(CAA) is independent of its 5'-genomic context, suggesting that the in vivo import substrate corresponds to the mature, fully processed tRNA.

J Drug Target, 2002 Feb, 10(1), 47 - 54
Differential behaviour of fluid liposomes toward mammalian epithelial cells and bacteria: restriction of fusion to bacteria; Desjardins A et al.; Previous work demonstrated that fluid liposomes developed in our laboratory are able to fuse with bacterial outer membranes . This fusion improved the penetration and activity of liposome-encapsulated antibiotics and antisense oligonucleotides into the bacterial cells . Because it is anticipated that fluid liposome encapsulated antibiotics will be administered by aerosols to patients with chronic pulmonary infections or cystic fibrosis (CF), we conducted comparative studies in E . coli, P . aeruginosa and human lung epithelial cells using lipid-mixing assays to investigate the possibility that fluid liposomes might fuse with surrounding epithelial cells . After a 2 h incubation at 4 and 37 degrees C, no fusion between fluid liposomes and human lung epithelial cells was observed, whereas mean levels of 71 and 37% of fusion were observed at 37 degrees C with E . coli and P . aeruginosa cells, respectively . No fusion was observed at 4 degrees C in any cells . A kinetic study where temperature was gradually increased from 7 to 37 degrees C indicated that the fusion process in the two bacteria starts between 28 and 31 degrees C with a mean fusion rate of 0.60%/min at 31 degrees C to reach 1.18%/min at 37 degrees C . The present work suggests that it is unlikely that fluid liposomes fuse with host cells lining the human respiratory tract and further elucidates the fusogenic properties of fluid liposomes with respect to prokaryotes and eukaryotes.

J Immunol, 2002 May 15, 168(10), 4951 - 9
Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain; Riedl P et al.; Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes . When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity . HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable . Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity . HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells . The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag . Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag . Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency . Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity . Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine . Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.

Biochem J, 2002 Jul 1, 365(Pt 1), 13 - 8
Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase); Aghajanian S et al.; The final two enzymes in the CoA biosynthetic pathway, phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3) and dephospho-CoA kinase (DPCK; EC 2.7.1.24), are separate proteins in prokaryotes, but exist as a bifunctional enzyme in pig liver . In the present study we have obtained sequence information from purified pig-liver enzyme, and identified the corresponding cDNA in a number of species . The human gene localizes to chromosome 17q12-21 and contains regions with sequence similarity to the monofunctional Escherichia coli DPCK and PPAT . The recombinant 564-amino-acid human protein confirmed the associated transferase and kinase activities, and gave similar kinetic properties to the wild-type pig enzyme.

Front Biosci, 2002 May 01, 7, d1338 - 46
Mycoplasmosis and immunity of fish and reptiles; Brown DR; Advances in molecular phylogenetics have enabled reconstruction of the most likely chronology of events in prokaryotic evolution and correlation with the paleontologic record with increasing precision . Mycoplasmas probably evolved from clostridial ancestors by genome reduction leading to obligate parasitism of host cells . The vertebrate hosts present at the time of the origin of mycoplasmas about 400 million years ago were fish, and later amphibians and reptiles, whose descendants possess most elements of vertebrate innate and adaptive immunity . Successful colonization of those poikilothermous ("cold-blooded") hosts must have involved adaptation to those defenses, shaping mycoplasma-host interactions for more than 125 million years before the earliest emergence of mammals . That history illuminates one aspect of the potential significance of mycoplasmosis of poikilothermous vertebrates to health and disease of other hosts including humans.

Mol Biotechnol, 2002 May, 21(1), 19 - 37
Quantitative analysis of gene expression by reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection; Richards MP et al.; There has been a dramatic expansion of DNA sequence information compiled over the past several years for a variety of eukaryotic and prokaryotic genomes . Accompanying this increase in knowledge of genomic structure and organization has been a growing interest in studying the function of individual genes including regulation of their expression . A number of methods such as Northern blotting, ribonuclease protection assay, and hybridization arrays have been developed to analyze gene expression at the transcriptional (mRNA) level . Although quantitative estimates of mRNA transcripts can be obtained from each of these methods, oftentimes they lack sufficient sensitivity or the methodology is too costly or too labor-intensive to be applied to the analysis of a large number of samples . The most sensitive method for analyzing gene expression at the mRNA level involves the combination of reverse transcription and polymerase chain reaction (RT-PCR) . However, in order to provide accurate quantitative estimates of gene expression, a rapid and efficient method is required for separation and detection of the double-stranded DNA (dsDNA) products of RT-PCR . Recent advances in capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) have made this method suitable for the automated analysis of large numbers of RT-PCR samples . An overview of the application of CE/LIF to quantitative analysis of gene expression by RT-PCR is presented along with selected protocols and examples . Both relative-quantitative (RQ) and quantitative-competitive (QC) approaches to RT-PCR are discussed in conjunction with the use of CE/LIF for rapid and accurate quantitative analysis of PCR products.

Vestn Ross Akad Med Nauk, 2002, (3), 48 - 51
{Fundamental and applied aspects of neuroendocrine regulation of pro- and eukaryote interactions}; Stadnikov AA et al.; The paper presents the results of light, atomic-force, and electron microscopic studies of prokaryotes (various Staphylococcus aureus strains) and eukaryotes (Rat digestive and respiratory cells and tissues) in in vivo and in vivo interactions . It is concluded that hypothalamic nonapeptides play a modulating role in the persistence and symbiotic relations in the bacterium-host system and regulate cellular and tissue homeostasis through reparative histogenesis.

Microbiology, 2002 May, 148(Pt 5), 1389 - 96
Evidence of intermolecular recombination between extrachromosomal DNAs in phytoplasma: a trigger for the biological diversity of phytoplasma?
Nishigawa H, Oshima K, Kakizawa S, Jung HY, Kuboyama T, Miyata S, Ugaki M, Namba S.
Recombination among bacterial extrachromosomal DNAs (EC-DNAs) plays a major evolutionary role by creating genetic diversity, and provides the potential for rapid adaptation to new environmental conditions . Previously, a 7 kbp EC-DNA, EcOYW1, with a geminivirus-like rolling-circle-replication protein (Rep) gene was isolated and characterized from an original wild-type line (OY-W) of onion yellows (OY) phytoplasma, an endocellular cell-wall-less prokaryote that inhabits the cytoplasm of both plant and insect cells . EcOYW1, found in OY-W, was not present in a mild-symptom line (OY-M) derived from OY-W . A 4 kbp EC-DNA, pOYW, was also isolated and characterized from OY-W, and its pLS1-plasmid-like rep gene was expressed . This paper describes the isolation and sequencing of an EC-DNA of 5560 nt, EcOYW2, from OY-W, and its counterpart EC-DNA of 5025 nt, EcOYM, from OY-M . EcOYW2 and EcOYM contained seven and six ORFs, respectively . They both encoded a geminivirus-like Rep and a putative single-stranded-DNA-binding protein (SSB) . Southern blot analysis indicated that no more EC-DNAs with a rep gene exist in either OY-W or OY-M, which means that the complete set of EC-DNAs has been cloned from the OY-W and OY-M lines of OY phytoplasmas . Sequence analysis revealed that both EcOYW2 and EcOYM have chimeric structures of previously characterized EcOYW1 and pOYW, suggesting that they have a recombinational origin . This is the first evidence of intermolecular recombination between EC-DNAs in phytoplasma . The possible implications of these findings in increasing the biological diversity of phytoplasma are discussed.

Biochim Biophys Acta, 2002 Mar 19, 1561(1), 47 - 64
Structure and association of ATP-binding cassette transporter nucleotide-binding domains; Kerr ID; ATP-binding cassette transporters are responsible for the uptake and efflux of a multitude of substances across both eukaryotic and prokaryotic membranes . Members of this family of proteins are involved in diverse physiological processes including antigen presentation, drug efflux from cancer cells, bacterial nutrient uptake and cystic fibrosis . In order to understand more completely the role of these multidomain transporters an integrated approach combining structural, pharmacological and biochemical methods is being adopted . Recent structural data have been obtained on the cytoplasmic, nucleotide-binding domains of prokaryotic ABC transporters . This review evaluates both these data and the conflicting implications they have for domain communication in ABC transporters . Areas of biochemical research that attempt to resolve these conflicts will be discussed.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 69 - 73
{Establishment of a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase gene}; Gao C et al.; BACKGROUND: To establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene . METHODS: The full length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template, and inserted into the prokaryotic expression plasmid Pgex-2T . The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits . The antibody was labeled with biotin, and a sandwich ELISA technique with biotin streptavidin amplify system was established . Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro . The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay . RESULTS: SDS-PAGE displayed that the molecular weight of the expressed fusion protein was about 54,000 . The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells . Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method . CONCLUSIONS: The established method could sensitively reflect the activities of the upstream promoters, as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities . Therefore, it proposes a convenient assay for the studies using CAT as the reporter gene.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 31 - 2
{Preliminary evidence that a hepatitis E virus (HEV) ORF2 recombinant protein protects cynomolgus macaques against challenge with wild-type HEV}; Bi S et al.; BACKGROUND: To observe the protective effect of hepatitis E virus (HEV) ORF2 recombinant protein expressed in prokaryote cell cynomolgus macaques (cynos) against challenging with wild-type HEV . METHODS: Cynos were immunized with HEV ORF2 recombinant protein and then challenged with wild-type HEV, the unimmunized cynos were used as control . Blood samples were collected and tested to see if there were dynamic changes of ALT and antibody to HEV before and after challenge with wild-type HEV . RESULTS: All the five unimmunized cynos re-presented hepatitis 3 weeks after challenging with wild-type HEV . However, all the five immunized cynos showed no hepatitis and pathological changes . CONCLUSIONS: Cynos can be efficiently protected by immunization with HEV ORF2 recombinant protein against wild-type HEV . This protein can be a promising candidate for HEV vaccine.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2001 Dec, 15(4), 305 - 8
{Analysis and applications of a PrP antibody elicited by synthesized human PrP peptides}; Sun X et al.; BACKGROUND: To further study the immuno-reactivity and specificity of a PrP antibody elicited by synthesized human PrP peptides . METHODS: Construction of a plasmid which expresses a N-terminus deleted human PrP protein and purification of this truncated protein . The prepared PrP antibody was tested for reaction with various prokaryotic expressed human PrP proteins,including the full-length protein,the N-terminus-and C-terminus-truncated proteins with Western blot . The antibody was also used in the immunofluorescence assays to identify the PrP proteins expressed from the cell lines transfected with human PrP genes . Moreover,the brain tissues from scrapie agents-infected hamsters and a CJD patient were extracted and the proteinase K-resistant PrP-res were analyzed with the prepared antibody . RESULTS: The PrP-peptide elicited antibody could recognize both prokaryotic and eukaryotic expressed human PrP proteins, as well as the PrP-res proteins in the brain tissues both from the scrapie-infected hamsters and CJD patient . CONCLUSIONS: The tested antibody possesses eligible immuno-reactivity and specificity and can be used for diagnosis of CJD in the clinical work.

Protein Eng, 2002 Apr, 15(4), 337 - 45
Engineering a novel secretion signal for cross-host recombinant protein expression; Tan NS et al.; Protein secretion is conferred by a hydrophobic secretion signal usually located at the N-terminal of the polypeptide . We report here, the identification of a novel secretion signal (SS) that is capable of directing the secretion of recombinant proteins from both prokaryotes and eukaryotes . Secretion of fusion reporter proteins was demonstrated in Escherichia coli, Saccharomyces cerevisiae and six different eukaryotic cells . Estrogen-inducibility and secretion of fusion reporter protein was demonstrated in six common eukaryotic cell lines . The rate of protein secretion is rapid and its expression profile closely reflects its intracellular concentration of mRNA . In bacteria and yeast, protein secretion directed by SS is dependent on the growth culture condition and rate of induction . This secretion signal allows a flexible strategy for the production and secretion of recombinant proteins in numerous hosts, and to conveniently and rapidly study protein expression.

J Biol Chem, 2002 Jul 26, 277(30), 26879 - 85 Epub 2002 Apr 30.
Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide . Catalase-like activity, compound III formation, and enzyme inactivation; Hiner AN et al.; The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied . Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)) . The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h . The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1) . Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity . A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2) . A similar model is applicable to horseradish peroxidase . The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the {compound I.H(2)O(2)} complex . Inactivation does not occur from compound III . All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.

Dev Genes Evol, 2002 Apr, 212(3), 152 - 7 Epub 2002 Feb 28.
The Drosophila Pipsqueak protein defines a new family of helix-turn-helix DNA-binding proteins; Siegmund T et al.; Many prokaryotic and eukaryotic DNA-binding proteins use a helix-turn-helix (HTH) structure for DNA recognition . Here we describe a new family of eukaryotic HTH proteins, the Pipsqueak (Psq) family, which includes proteins from fungi, sea urchins, nematodes, insects, and vertebrates . Three subgroups of the Psq family can be distinguished . Like the HTH proteins of the prokaryotic resolvase family, members of the CENP-B/transposase subgroup catalyze site-specific recombination reactions . This functional conservation, together with a primary sequence similarity between the resolvase and Psq DNA-binding domains, suggests that the resolvase and Psq families are evolutionarily linked . More than half of the newly identified Drosophila Psq proteins contain a BTB protein-protein interaction domain . All proteins of this BTB subgroup belong to the conserved Tramtrack group of BTB-domain proteins . About half of the members of the Tramtrack group contain a Psq domain, while the other half is made up of proteins that contain a zinc finger domain . Thus, nearly all members of this group appear to be DNA-binding proteins . Among other developmental regulators, the Drosophila cell death protein E93 was found to contain a Psq motif and to define a third subgroup of Psq domain proteins . The high sequence conservation of the E93 Psq motif allowed the identification of E93 orthologs in humans and lower metazoans.

J Biol Chem, 2002 Jul 12, 277(28), 25692 - 6 Epub 2002 Apr 25.
The location of plastocyanin in vascular plant photosystem I; Ruffle SV et al.; We have studied the binding sites of the electron donor and acceptor proteins of vascular plant photosystem I by electron microscopy/crystallography . Previously, we identified the binding site for the electron acceptor (ferredoxin) . In this paper we complete these studies with the characterization of the electron donor (plastocyanin) binding site . After cross-linking, plastocyanin is detected using Fourier difference analysis of two dimensionally ordered arrays of photosystem I located at the periphery of chloroplast grana . Plastocyanin binds in a small cavity on the lumenal surface of photosystem I, close to the center and with a slight bias toward the PsaL subunit of the complex . The recent release of the full coordinates for the cyanobacterial photosystem I reaction center has allowed a detailed comparison between the structures of the eukaryotic and prokaryotic systems . This reveals a very close homology, which is particularly striking for the lumenal side of photosystem I.

Environ Microbiol, 2002 Feb, 4(2), 125 - 9
Effect of oil pollution on euendolithic cyanobacteria of the Arabian Gulf; Al-Thukair AA; Microbial euendoliths (true borer) cyanobacteria are carbonate-boring microorganisms found in modern and ancient marine environments . Modern euendoliths include a wide range of prokaryotes as well as eukaryotes, which have been reported world-wide . The importance of euendolithic cyanobacteria concerns their role in bio-erosion of calcium carbonate substrates and as ecological indicators of shallow, tropical and subtropical marine environments . Arabian Gulf ooids from four sites along the east coast of Saudi Arabia have been bored and inhabited by several species of euendolithic cyanobacteria . This assemblage of different species exists simultaneously within the same ooid grain . Comparisons of 1989 and 1992 data reveal a drastic reduction in active euendoliths, and the average numbers of colonies in these ooids . This study reveals the harmful effect of the 1991 oil spill on these unique microorganisms residing in these ooids.

J Biol Chem, 2002 Jul 12, 277(28), 25668 - 76 Epub 2002 Apr 22.
The conserved helix C region in the superfamily of interferon-gamma /interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK; Vandenbroeck K et al.; HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins . The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78 . DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the alpha-helical homodimeric secretory cytokine interferon-gamma (IFN-gamma) . We screened solid-phase peptide libraries from human and mouse IFN-gamma to identify DnaK-binding sites . Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN-gamma dimer interface . Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays . No DnaK-binding sites were found in the loops connecting the alpha-helices . The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-gamma, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs . These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain . We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

J Biol Chem, 2002 Jun 28, 277(26), 23882 - 7 Epub 2002 Apr 22.
Indolmycin resistance of Streptomyces coelicolor A3(2) by induced expression of one of its two tryptophanyl-tRNA synthetases; Kitabatake M et al.; Aminoacyl-tRNA synthetases, a family of enzymes essential for protein synthesis, are promising targets of antimicrobials . Indolmycin, a secondary metabolite of Streptomyces griseus and a selective inhibitor of prokaryotic tryptophanyl-tRNA synthetase (TrpRS), was used to explore the mechanism of inhibition and to explain the resistance of a naturally occurring strain . Streptomyces coelicolor A3(2), an indolmycin-resistant strain, contains two trpS genes encoding distinct TrpRS enzymes . We show that TrpRS1 is indolmycin-resistant in vitro and in vivo, whereas TrpRS2 is sensitive . The lysine (position 9) in the enzyme tryptophan binding site is essential for making TrpRS1 indolmycin-resistant . Replacement of lysine 9 by glutamine, which at this position is conserved in most bacterial TrpRS proteins, abolished the ability of the mutant trpS gene to confer indolmycin resistance in vivo . Molecular modeling suggests that lysine 9 sterically hinders indolmycin binding to the enzyme . Tryptophan recognition (assessed by k(cat)/K(M)) by TrpRS1 is 4-fold lower than that of TrpRS2 . Examination of the mRNA for the two enzymes revealed that only TrpRS2 mRNA is constitutively expressed, whereas mRNA for the indolmycin-resistant TrpRS1 enzyme is induced when the cells are exposed to indolmycin.

Exp Cell Res, 2002 May 1, 275(2), 200 - 14
Exogenous expression of heat shock protein 90kDa retards the cell cycle and impairs the heat shock response; Zhao C et al.; The 90-kDa heat shock protein, HSP90, is an abundant molecular chaperone which functions in cellular homeostasis in prokaryotes and eukaryotes . It is well known that HSP90 plays a critical and indispensable role in regulating cell growth through modulations of various signal transduction pathways, but its roles in cell cycle control are not so well known . We transferred human HSP90 (wild-type or mutated types) expression vectors into NIH-3T3 cells in order to study certain functions of HSP90 in the cell cycle and cell growth under physiological conditions . We found that the exogenous expression of HSP90 (wild-type) induced a decrease in cell growth via retardation of the G1/S transition . The inhibition of cell growth was caused by reduced expressions of cyclin D3 and cyclin A mRNA and protein . On the other hand, no stable transfectants with the three types of mutated HSP90 were obtained . Unexpectedly, exogenous HSP90 expression impaired the heat shock response by inhibiting both heat shock transcription factor 1(HSF1) activation and transportation of HSF1 into the nucleus . The HSF1 function was disrupted by the direct association between HSF1 and exogenous HSP90, which was present as a monomer . These results reveal important roles of HSP90 in cell cycle control and in the stress response of nontransformed cells.

Mol Biol (Mosk), 2002 Mar-Apr, 36(2), 277 - 85
{New insights into the molecular mechanisms of evolution: stress increases genetic diversity}; Vel'kov VV; The mechanisms of stress-induced mutagenesis in prokaryotes and realization of reserved (preaccumulated) genetic variation in eukaryotes are considered . In prokaryotes, replication becomes error-prone in stress because of the induction of the SOS response and the inactivation of the mismatch repair system; stress also increases the transposition rate and the efficiency of interspecific gene transfer . In eukaryotes, chaperone HSP90, which restores the native folding of mutant proteins (e.g., signal transduction and morphogenetic proteins) in normal conditions, fails to do so in stress, which leads to abrupt expression of multiple mutations earlier reserved in the corresponding genes . The role of these mechanisms in the evolution of prokaryotes and eukaryotes is discussed.

J Cell Biochem, 2002, 85(4), 678 - 88
Yeast methionine aminopeptidase type 1 is ribosome-associated and requires its N-terminal zinc finger domain for normal function in vivo; Vetro JA et al.; Methionine aminopeptidase type 1 (MetAP1) cotranslationally removes N-terminal methionine from nascent polypeptides, when the second residue in the primary structure is small and uncharged . Eukaryotic MetAP1 has an N-terminal zinc finger domain not found in prokaryotic MetAPs . We hypothesized that the zinc finger domain mediates the association of MetAP1 with the ribosomes and have reported genetic evidence that it is important for the normal function of MetAP1 in vivo . In this study, the intracellular role of the zinc finger domain in yeast MetAP1 function was examined . Wild-type MetAP1 expressed in a yeast map1 null strain removed 100% of N-terminal methionine from a reporter protein, while zinc finger mutants removed only 31-35% . Ribosome profiles of map1 null expressing wild-type MetAP1 or one of three zinc finger mutants were compared . Wild-type MetAP1 was found to be an 80S translational complex-associated protein that primarily associates with the 60S subunit . Deletion of the zinc finger domain did not significantly alter the ribosome profile distribution of MetAP1 . In contrast, single point mutations in the first or second zinc finger motif disrupted association of MetAP1 with the 60S subunit and the 80S translational complex . Together, these results indicate that the zinc finger domain is essential for the normal processing function of MetAP1 in vivo and suggest that it may be important for the proper functional alignment of MetAP1 on the ribosomes .

Protein Sci, 2002 May, 11(5), 1274 - 7
A model of the replication fork blocking protein Fob1p based on the catalytic core domain of retroviral integrases; Dlakic M; The replication fork blocks are common in both prokaryotes and eukaryotes . In most cases, these blocks are associated with increased levels of mitotic recombination . One of the best-characterized replication fork blocks in eukaryotes is found in ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae . It has been shown that the replication fork blocking protein Fob1p regulates the recombination rate and the number of rDNA copies in S . cerevisiae, but the mechanistic aspects of these events are still poorly understood . Sequence profile searches revealed that Fob1p is related to retroviral integrases . Subsequently, the catalytic domain of HIV-1 integrase was used as a template to build a reliable three-dimensional model of Fob1p . Structural insights from this study may be useful in explaining Fob1p-mediated formation of extrachromosomal rDNA circles that accelerate aging in yeast and recombination events that lead to expansion or contraction of rDNA.

J Virol, 2002 May, 76(10), 4836 - 47
Open reading frame UL26 of human cytomegalovirus encodes a novel tegument protein that contains a strong transcriptional activation domain; Stamminger T et al.; A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells . This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene . With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts . One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain . In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain . By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators . Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells . Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts . By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection . Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected . Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.

Mol Microbiol, 2002 Apr, 44(1), 1 - 8
Oxidative protein folding in bacteria; Collet JF et al.; Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously . Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides . Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB . DsbB generates disulphides de novo from oxidized quinones . These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport . Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing . Two isomerases exist in Escherichia coli, DsbC and DsbG . The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form . DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction.

Environ Microbiol, 2002 Jan, 4(1), 29 - 41
Deciphering the action of aromatic effectors on the prokaryotic enhancer-binding protein XylR: a structural model of its N-terminal domain; Devos D et al.; The prokaryotic enhancer-binding protein XylR is the central regulator of the toluene degradation pathway in Pseudomonas species . Copious genetic and biochemical data indicate that the N-terminal domain of the protein (domain A) interacts directly with m-xylene, which renders the protein competent as a transcriptional activator . Single-site and shuffling mutants of XylR or homologues have been reported to change or expand their effector profiles . Here, we follow a fold recognition approach to generate three-dimensional models of the domain A of XylR and DmpR with the purpose of deciphering the molecular activity of this protein family . The model is based on the crystallographic data of the rat catechol O-methyltransferase, a typical alpha/beta fold, consisting of eight alpha-helices and seven beta-strands . The fold identification is supported by physico-chemical properties of conserved amino acids, distribution of residues characteristic of the sequence families and confrontation with experimental data . The model not only provides a rationale for understanding published experimental data, but also suggests the molecular mechanism of the activation step and is a potentially useful conceptual tool for designing regulators with predefined inducer specificities.

Pharmacogenomics, 2002 Jan, 3(1), 19 - 30
The pharmacogenetics of NAT: structural aspects; Pompeo F et al.; Arylamine N-acetyltransferases (NATs) catalyze the transfer of an acetyl group from acetyl-CoA to arylhydrazines and to arylamine drugs and carcinogens or to their N-hydroxylated metabolites . NAT plays an important role in detoxification and metabolic activation of xenobiotics and was first identified as the enzyme responsible for inactivation of the antitubercular drug isoniazid, an arylhydrazine . The rate of inactivation was polymorphically distributed in the population: the first example of interindividual pharmacogenetic variation . Polymorphism in NAT activity is primarily due to single nucleotide polymorphisms (SNPs) in the coding region of NAT genes . NAT enzymes are widely distributed in eukaryotes and genome sequences have revealed many homologous members of this enzyme family in prokaryotes . The structures of S almonella typhimurium and Mycobacterium smegmatis NATs have been determined, revealing a unique fold in which a catalytic triad (Cys-His-Asp) forms the active site . Determination of prokaryotic and eukaryotic NAT structures could lead to a better understanding of their role in xenobiotics and endogenous metabolism.

Cell Death Differ, 2002 Apr, 9(4), 394 - 404
Origin and evolution of eukaryotic apoptosis: the bacterial connection; Koonin EV et al.; The availability of numerous complete genome sequences of prokaryotes and several eukaryotic genome sequences provides for new insights into the origin of unique functional systems of the eukaryotes . Several key enzymes of the apoptotic machinery, including the paracaspase and metacaspase families of the caspase-like protease superfamily, apoptotic ATPases and NACHT family NTPases, and mitochondrial HtrA-like proteases, have diverse homologs in bacteria, but not in archaea . Phylogenetic analysis strongly suggests a mitochondrial origin for metacaspases and the HtrA-like proteases, whereas acquisition from Actinomycetes appears to be the most likely scenario for AP-ATPases . The homologs of apoptotic proteins are particularly abundant and diverse in bacteria that undergo complex development, such as Actinomycetes, Cyanobacteria and alpha-proteobacteria, the latter being progenitors of the mitochondria . In these bacteria, the apoptosis-related domains typically form multidomain proteins, which are known or inferred to participate in signal transduction and regulation of gene expression . Some of these bacterial multidomain proteins contain fusions between apoptosis-related domains, such as AP-ATPase fused with a metacaspase or a TIR domain . Thus, bacterial homologs of eukaryotic apoptotic machinery components might functionally and physically interact with each other as parts of signaling pathways that remain to be investigated . An emerging scenario of the origin of the eukaryotic apoptotic system involves acquisition of several central apoptotic effectors as a consequence of mitochondrial endosymbiosis and probably also as a result of subsequent, additional horizontal gene transfer events, which was followed by recruitment of newly emerging eukaryotic domains as adaptors.

Curr Opin Infect Dis, 2000 Feb, 13(1), 37 - 45
Biology of trichomonosis; Lehker MW et al.; Trichomonas vaginalis is emerging as a major pathogen of men and women and is associated with serious health consequences . Advances in diagnosis and treatment are presented . The complexity of trichomonad pathogenesis is illustrated in the interaction of this parasite with human cells, tissues and the immune system . It is now becoming evident that the interaction of trichomonads with the host is frequently modulated by environmental signals . The molecular biology of trichomonads is still in its infancy, but analysis of genes, genomic structure and transcriptional mechanisms suggest that trichomonads combine both prokaryotic and eukaryotic features . Evidence for the ancient divergence of trichomonads from other eukaryotic lineages is discussed.

Curr Opin Allergy Clin Immunol, 2001 Dec, 1(6), 549 - 54
Can knowledge of the molecular structure of allergens improve immunotherapy?
Pomes A, Chapman MD.
Conventional immunotherapy may be associated with the development of adverse reactions, including anaphylaxis, due to the use of increasing doses of allergen . Standardization of extracts is necessary in order to assess the correct amount of allergen administered . In recent years, increased knowledge on the molecular structure of allergens has allowed the development of novel alternatives for immunotherapy . Initially, allergens were cloned and expressed as recombinant proteins in eukaryotic and prokaryotic systems . Crystallization of the purified proteins led to the elucidation of the tertiary structure of the allergen . Molecular biology techniques were used to construct modified allergens whose new IgE binding properties were studied . IgE antibody mapping combined with molecular modeling has allowed the recognition of IgE binding sites on the surface of the molecule . This information has been applied to the engineering of new modified allergens, with and without adjuvants, that retain immunogenicity but with reduced allergenicity . The use of these molecules for immunotherapy should allow the administration of greater doses of allergen, without the undesired side effects characteristic of conventional immunotherapy.

Biophys J, 2002 May, 82(5), 2627 - 34
First evidence for phototropin-related blue-light receptors in prokaryotes; Losi A et al.; A prokaryotic protein, YtvA from Bacillus subtilis, was found to possess a light, oxygen, voltage (LOV) domain sharing high homology with the photoactive, flavin mononucleotide (FMN)-binding LOV domains of phototropins (phot), blue-light photoreceptors for phototropism in higher plants . Computer-based three-dimensional modeling suggests that YtvA-LOV binds FMN in a similar pocket as phot-LOVs . Recombinant YtvA indeed exhibits the same spectroscopical features and blue-light-induced photochemistry as phot-LOVs, with the reversible formation of a blue-shifted photoproduct, assigned to an FMN-cysteine thiol adduct (Thio383) . By means of laser-flash photolysis and time-resolved optoacoustic experiments, we measured the quantum yield of formation for Thio383, Phi(Thio) = 0.49, and the enthalpy change, DeltaH(Thio) = 135 kJ/mol, with respect to the parent state . The formation of Thio383 is accompanied by a considerable volume contraction, DeltaV(Thio) = -13.5 ml/mol . Similar to phot-LOVs, Thio383 is formed from the decay of a red-shifted transient species, T650, within 2 micros . In both YtvA and free FMN, this transient has an enthalpy content of approximately 200 kJ/mol, and its formation is accompanied by a small contraction, DeltaV(T) approximately -1.5 ml/mol, supporting the assignment of T650 to the FMN triplet state, as suggested by spectroscopical evidences . These are the first studies indicating that phototropin-related, blue-light receptors may exist also in prokaryotes, besides constituting a steadily growing family in plants.

J Gen Virol, 2002 May, 83(Pt 5), 979 - 90
Cloning and sequencing of TT virus genotype 6 and expression of antigenic open reading frame 2 proteins; Kakkola L et al.; The near-full-length genome of a TT virus (TTV) (HEL32), closely related to the previously uncharacterized genotype 6, was cloned and sequenced . The genomic organization of HEL32 was compared to 41 published near-full-length TTV sequences representing 17 genotypes . In the majority of genomes, the open reading frame (ORF) 2 region was divided into two separate ORFs, 2a and 2b . The ORF2a sequence was conserved among all genotypes, while the ORF2b region showed more variability . The two corresponding putative proteins of HEL32 were expressed in prokaryotes and their antigenic potential was studied . IgM and IgG antibodies to the respective ORF2-encoded proteins, fp2a and fp2b, and the presence of TTV DNA were studied in the sera of 89 constitutionally healthy adults . By immunoblot using the small TTV proteins as antigens, strong IgM and IgG reactivities were found in 9 and 10% of subjects, respectively . Follow-up studies for 12-15 years of three subjects showed either a persistent coexistence of IgM and TTV DNA or the appearance of viral DNA regardless of pre-existing antibodies . The low prevalence of IgG could be due to the weak immunogenicity of these probably non-structural proteins or to a genotype-specific antibody response . By nested PCR of the conserved ORF2a region, the prevalence of TTV DNA was 85% . TTV genotype 6 sequences were found by specific PCR in 3 of 35 (8.6%) subjects . The low prevalence of TTV IgG compared to the high TTV DNA prevalence, the coexistence of antibodies and viral DNA and the appearance of TTV DNA regardless of pre-existing antibodies suggest that the B-cell immunity against these minor TTV proteins would not be cross protective.

Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 6428 - 33 Epub 2002 Apr 16.
Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants; Maser P et al.; Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins . These transporters contain four loop domains in one polypeptide with a proposed distant homology to K(+) channel selectivity filters . Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na(+)-coupled K(+) transport . Arabidopsis AtHKT1, however, transports only Na(+) in eukaryotic expression systems . To understand the molecular basis of this difference we constructed a series of AtHKT1/HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K(+) selectivity . A single-point mutation, Ser-68 to glycine, was sufficient to restore K(+) permeability to AtHKT1 . The reverse mutation in HKT1, Gly-91 to serine, abrogated K(+) permeability . This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K(+) channel selectivity filter GYG motif . The importance of such filter glycines for K(+) selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2 . Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na(+) transporters in plants and that Na(+)/K(+) symport in HKT proteins is associated with a glycine in the filter residue . These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K(+) channels.

FEMS Microbiol Lett, 2002 Feb 5, 207(2), 185 - 92
Cloning and characterization of a major surface protein (MspTL) of Treponema lecithinolyticum associated with rapidly progressive periodontitis; Park KK et al.; The gene encoding a major surface protein (MspTL) of Treponema lecithinolyticum, a periodontopathogen, was cloned and sequenced . The mspTL gene has a 1770-bp open reading frame (ORF) encoding a protein of 590 amino acids with a predicted molecular mass of 65 kDa which had a typical prokaryotic signal sequence (19 amino acids) . MspTL showed a high level of homology with major sheath protein (MspA) of Treponema maltophilum, phylogenetically the closest relative of T . lecithinolyticum . Southern blot analysis indicated that the mspTL gene exists in a single copy and Northern blot analysis showed that the mspTL transcript is monocistronic . Another ORF located downstream of mspTL was in the same orientation and encoded a putative protein, in which the first N-terminal 291 amino acids were identified . The homologous region of this protein is also a part on the T . maltophilum mspA locus.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jan, 34(1), 57 - 61
{Site-directed mutagenesis of the cysteines of human IL-18 and its effect on IL-18 activity}; Pei DS et al.; To study the structure-function relationships of human IL-18(hIL-18), site-directed mutagenesis was used to generate four hIL-18 cysteine mutants, C74S, C104S, C112S and C163S . The cDNAs of the four cysteine mutants were inserted into prokaryotic expression vector pJW2 and expressed as inclusion bodies in E . coli . The inclusion bodies were washed with 2 mol/L urea, dissolved in 8 mol/L urea, and purified by chromatography on Sephadex G-100 column . The purity of the purified mutants were greater than 90% as judged by SDS-PAGE . The activity of rhIL-18 C74S, C104S, C112S and C163S accounted for 5%, 81%, 58% and 11% of wild type, respectively . These results suggest that Cys74 and Cys163 play important roles in inducing IFN-gamma production in human peripheral blood mononuclear cells.

Funct Integr Genomics, 2002 Apr, 1(6), 357 - 66 Epub 2001 Dec 11.
Two-hybrid-based analysis of protein-protein interactions of the yeast multidrug resistance protein, Pdr5p; Subba Rao G et al.; The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes . The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood . In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae . Four of the major cytosolic loops that have been predicted for this protein {including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region} were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system . Results of these studies have revealed that the first cytosolic loop (CL1)--containing the first NBD domain--and also the C-terminal region of Pdr5p, interact with several candidate proteins . The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein.

Biochim Biophys Acta, 2002 Feb 20, 1574(1), 85 - 92
Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases; Murakami C et al.; Retinoic acids, vitamin A-related compounds, are known to be inhibitors of telomerase . We found that fucoxanthin from the sea alga Petalonia bingamiae is a potent inhibitor of mammalian replicative DNA polymerases (i.e., pol alpha, delta and epsilon) . Since fucoxanthin is a carotenoid (provitamin A-related) compound, we characterized the biochemical modes of vitamin A-related compounds including vitamin A and provitamin A in this report . Subsequently, we found that fucoxanthin, all-trans retinal (RAL, vitamin A aldehyde) and all-trans retinoic acid (RA, vitamin A acid) inhibited the activities of replicative DNA polymerases with IC(50) values of 18-190, 14-17 and 8-30 microM, respectively . On the other hand, all-trans retinol (vitamin A) did not influence any of the DNA polymerase activities . RA inhibited not only the activities of pol alpha, delta and epsilon with IC(50) values of 30, 28 and 8 microM, respectively, but of pol beta with an IC(50) value of 27 microM . The tested vitamin A-related compounds did not influence the activities of DNA polymerases from a higher plant, cauliflower, prokaryotic DNA polymerases, or DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I . RAL and RA should be called selective inhibitors of mammalian DNA polymerases including telomerase, and RAL was a specific inhibitor of mammalian replicative DNA polymerases . As expected from these results in vitro, some of them could prevent the growth of NUGC-3 human gastric cancer cells, and especially RAL was a potent antineoplastic agent with an LD(50) value of 19 microM . The cells were halted at G1 phase in the cell cycle by RAL.

Zhonghua Yi Xue Za Zhi, 2002 Mar, 82(5), 338 - 40
{Anti-apoptosis gene survivin promotes cell growth and transformation}; Zhu H et al.; OBJECTIVE: To investigate the role and molecular mechanism of surviving, an anti-apoptosis gene, in cell growth and transformation . METHODS: Coding sequence of surviving was amplified from Daudi cell mRNA by RT-PCR and then cloned into prokaryotic and eukaryotic vectors . The vectors with surviving were transfected into BL21 cells of Escherichia coli and human embryonic kidney 293 cells . The cells were cultured . Protein was extracted from the cells and examined by gel electrophoresis . Suspension of 293 cells was cultured and the number of cells was counted every other day, thus a growth curve was drawn . Another suspension of 293 cells was cultured in soft agar to observe the number of colonies . Cells transfected with plasmids void of surviving were used as controls . RESULTS: The anti-apoptosis gene surviving was well expressed in BL21 cells and 293 cells . The growth curve showed that the proliferation rate of 293 cells was slightly faster than that of control cells, however, without significant difference . Soft agar assay showed that the colonies formed by surviving-transfected 293 cells were of greater size and with greater number . Western blotting showed overexpression of cyclin D1 and c-myc, two important cancer proteins, in cells transfected with surviving . CONCLUSION: The anti-apoptosis gene surviving promotes cell transformation . These effects may depend on the functions of cyclin D1 and c-myc.

Mol Microbiol, 2002 Mar, 43(6), 1565 - 75
Identification of genes that are associated with DNA repeats in prokaryotes; Jansen R et al.; Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses . This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences . To appreciate their characteri-stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR) . In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300-500 b . The direct repeats and the leader sequences were conserved within a species, but dissimilar between species . The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements . Four CRISPR-associated (cas) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes . The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship . The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression . The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.

Plant Physiol, 2002 Apr, 128(4), 1470 - 9
Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii; Theiss C et al.; Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms . In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines . In synchronized C . reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels . Spermine, however, could not be detected in C . reinhardtii cells . Exogenous polyamines caused a decrease in ODC activity . Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase . Most of the cells had already doubled their cell mass after this growth period . The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine . The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period . Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.

Bioessays, 2002 Apr, 24(4), 293 - 6
Bacterial actins? An evolutionary perspective; Doolittle RF et al.; According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life . An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles . Two recent papers1, 2 present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin . Sequence comparisons reveal that eukaryotic actin and the bacterialhomolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories .

Biochemistry, 2002 Apr 16, 41(15), 4938 - 45
Inactivation of the PKR protein kinase and stimulation of mRNA translation by the cellular co-chaperone P58(IPK) does not require J domain function; Yan W et al.; P58(IPK) was discovered as an inhibitor of the interferon-induced, protein kinase, PKR . Upon virus infection, PKR can, as part of the host defense system, inhibit mRNA translation by phosphorylating the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2alpha) . We previously found that influenza virus recruits the cellular P58(IPK) co-chaperone to inhibit PKR activity and thus facilitate viral protein synthesis . P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain found in all DnaJ chaperone family members . To define the role of molecular chaperones in regulating cell growth in addition to PKR regulation, we performed a detailed analysis of the P58(IPK) J domain . Using growth rescue assays, we found that the P58(IPK) J domain substituted for the J domains of other DnaJ proteins, including DnaJ in Escherichia coli and Ydj1 in Saccharomyces cerevisiae . This is the first time a cellular J domain from a mammalian DnaJ family member was shown to be functional in both prokaryotic DnaJ and eukaryotic Ydj1 constructs . Furthermore, point mutations within the conserved HPD residue cluster of the P58(IPK) J domain disrupted P58(IPK) J function including stimulation of ATPase activity of Hsp70 . However, the P58(IPK) HPD mutants still inhibited PKR activity and thus supported cell growth in a yeast rescue assay . Overexpression of the HPD mutants of P58(IPK), similar to their wild-type counterpart, also stimulated mRNA translation in a mammalian cell system . Taken together, our data necessitate a model of P58(IPK) inhibition of PKR kinase activity and stimulation of mRNA translation, which does not require classical J domain function found in the DnaJ molecular chaperone family.

Am J Vet Res, 2002 Apr, 63(4), 551 - 8
Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants; Takafuji VA et al.; OBJECTIVES: To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture . SAMPLE POPULATION: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse . PROCEDURE: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified . Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml) . Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media . Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan . Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay . Data were collected at 48-hour intervals and normalized by DNA content . RESULTS: Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively . Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days . Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days . CONCLUSIONS AND CLINICAL RELEVANCE: The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro . These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.

Appl Microbiol Biotechnol, 2002 Mar, 58(3), 275 - 85 Epub 2001 Dec 20.
Microbial production of vitamin B12; Martens JH et al.; One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B12 (cobalamin), which was originally discovered as the anti pernicious anemia factor and whose enigmatic complex structure is matched only by the beguiling chemistry that it mediates . The biosynthesis of this essential nutrient is intricate, involved and, remarkably, confined to certain members of the prokaryotic world, seemingly never have to have made the eukaryotic transition . In humans, the vitamin is required in trace amounts (approximately 1 microg/day) to assist the actions of only two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase; yet commercially more than 10 t of B12 are produced each year from a number of bacterial species . The rich scientific history of vitamin B12 research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described . Current strategies for the improvement of vitamin B12 production using modern biotechnological techniques are outlined.

Science, 2002 Apr 5, 296(5565), 148 - 51
The interaction of Alba, a conserved archaeal chromatin protein, with Sir2 and its regulation by acetylation; Bell SD et al.; The conserved Sir2 family of proteins has protein deacetylase activity that is dependent on NAD (the oxidized form of nicotinamide adenine dinucleotide) . Although histones are one likely target for the enzymatic activity of eukaryotic Sir2 proteins, little is known about the substrates and roles of prokaryotic Sir2 homologs . We reveal that an archaeal Sir2 homolog interacts specifically with the major archaeal chromatin protein, Alba, and that Alba exists in acetylated and nonacetylated forms . Furthermore, we show that Sir2 can deacetylate Alba and mediate transcriptional repression in a reconstituted in vitro transcription system . These data provide a paradigm for how Sir2 family proteins influence transcription and suggest that modulation of chromatin structure by acetylation arose before the divergence of the archaeal and eukaryotic lineages.

Bioinformatics, 2002 Mar, 18(3), 470 - 81
STOCKS: STOChastic Kinetic Simulations of biochemical systems with Gillespie algorithm; Kierzek AM; MOTIVATION: The availability of a huge amount of molecular data concerning various biochemical reactions provoked numerous attempts to study the dynamics of cellular processes by means of kinetic models and computer simulations . Biochemical processes frequently involve small numbers of molecules (e.g . a few molecules of a transcriptional regulator binding to one 'molecule' of a DNA regulatory region) . Such reactions are subject to significant stochastic fluctuations . Monte Carlo methods must be employed to study the functional consequences of the fluctuations and simulate processes that cannot be modelled by continuous fluxes of matter . This provides the motivation to develop software dedicated to Monte Carlo simulations of cellular processes with the rigorously proven Gillespie algorithm . RESULTS: STOCKS, software for the stochastic kinetic simulation of biochemical processes is presented . The program uses a rigorously derived Gillespie algorithm that has been shown to be applicable to the study of prokaryotic gene expression . Features dedicated to the study of cellular processes are implemented, such as the possibility to study a process in the range of several cell generations with the application of a simple cell division model . Taking expression of Escherichia coli beta-galactosidase as an example, it is shown that the program is able to simulate systems composed of reactions varying in several orders of magnitude by means of reaction rates and the numbers of molecules involved . AVAILABILITY: The software is available at ftp://ibbrain.ibb.waw.pl/stocksand Supplementary information: Parameters of the model of prokaryotic gene expression are available in example files of software distribution.

Curr Opin Microbiol, 2002 Apr, 5(2), 135 - 41
Structure/function relationships in OmpR and other winged-helix transcription factors; Kenney LJ; Response regulators are the output component of two-component regulatory systems, the predominant form of signal transduction systems utilized by prokaryotes . The majority of response regulators function as transcription factors, yet detailed descriptions of their mechanisms of DNA binding and its consequences are lacking . Versatility in the modes of DNA binding is evident with winged helix-turn-helix proteins, raising doubts that mechanisms of DNA binding will be generalizable among members of the family . The current focus of some of the research efforts aimed at understanding activation and DNA binding by response regulators is highlighted in this review.

Chembiochem, 2002 Apr 2, 3(4), 274 - 93
Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases; Jeltsch A; DNA methyltransferases catalyze the transfer of a methyl group from S-adenosyl-L-methionine to cytosine or adenine bases in DNA . These enzymes challenge the Watson/Crick dogma in two instances: 1) They attach inheritable information to the DNA that is not encoded in the nucleotide sequence . This so-called epigenetic information has many important biological functions . In prokaryotes, DNA methylation is used to coordinate DNA replication and the cell cycle, to direct postreplicative mismatch repair, and to distinguish self and nonself DNA . In eukaryotes, DNA methylation contributes to the control of gene expression, the protection of the genome against selfish DNA, maintenance of genome integrity, parental imprinting, X-chromosome inactivation in mammals, and regulation of development . 2) The enzymatic mechanism of DNA methyltransferases is unusual, because these enzymes flip their target base out of the DNA helix and, thereby, locally disrupt the B-DNA helix . This review describes the biological functions of DNA methylation in bacteria, fungi, plants, and mammals . In addition, the structures and mechanisms of the DNA methyltransferases, which enable them to specifically recognize their DNA targets and to induce such large conformational changes of the DNA, are discussed.

Protein Eng, 2002 Mar, 15(3), 169 - 83
Knowledge-based selection of targets for structural genomics; Frishman D; The problem of rational target selection for protein structure determination in structural genomics projects on microbes is addressed . A flexible computational procedure is described that directly incorporates the whole body of annotation available in the PEDANT genome database into the sequence clustering and selection process in order to identify proteins that are likely to possess currently unknown structural domains . Filtering out gene products based on predicted structural features, such as known three-dimensional structures and transmembrane regions, allows one to reduce the complexity of neighbor relationships between sequences and all but eliminates the need for further partitioning of single-linkage clusters into disjoint protein groups corresponding to homologous families . The results of a large-scale computation experiment in which exemplary target selection for 32 prokaryotic genomes was conducted are presented.

Microbiology, 2002 Apr, 148(Pt 4), 1129 - 42
Redundancy, phylogeny and differential expression of Histoplasma capsulatum catalases; Johnson CH et al.; Histoplasma capsulatum produces an extracellular catalase termed M antigen, which is similar to catalase B of Aspergillus and Emericella species . Evidence is presented here for two additional catalase isozymes in H . capsulatum . Catalase A is highly similar to a large-subunit catalase in Aspergillus and Emericella species, while catalase P is a small-subunit catalase protein with greatest similarity to known peroxisomal catalases of animals and Saccharomycotina yeasts . Complete cDNAs for the CATA and CATP genes (encoding catalases A and P, respectively) were isolated . The transcriptional expression of the H . capsulatum CATA, CATB (M antigen) and CATP genes was assessed by Northern blot hybridizations on total RNA . Results at the transcript levels for these genes are shown for three conditions: cell morphology (mycelial versus yeast phase cells), oxidative stress (in response to a challenge with H(2)O(2)) and carbon source (glucose vs glycerol) . Collectively, these results demonstrated regulation of CATA by both cell morphology and oxidative stress, but not by carbon source, and regulation of CATB and CATP by carbon source but not cell morphology or oxidative stress . A phylogenetic analysis of presently available catalase sequences and intron residences was done . The results support a model for evolution of eukaryotic monofunctional catalase genes from prokaryotic genes.

J Biol Chem, 2002 Jun 14, 277(24), 21405 - 13 Epub 2002 Apr 03.
Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix; Baumann F et al.; The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria . Little is known about the intramitochondrial sorting of these proteins . We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space . Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane . The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane . The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria . A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.

Sheng Li Xue Bao, 2002 Feb 25, 54(1), 23 - 7
{Prokaryotic expression and purification of human hepatic stimulator substance}; Du HJ et al.; To explore the possibility of prokaryotic expression of human hepatic stimulator substance (hHSS), hHSS gene was inserted in the downstream of glutathion S-transferase (GST) in a pET-42a expression vector and recombinant GST-hHSS fusion protein was expressed under IPTG induction in BL-21(DE3) cells . The recombinant HSS was purified with His.Tag affinity chromatography, and its bioactivity was analyzed . The results showed that GST-hHSS fusion protein was expressed both as a soluble or a inclusive body in bacterial cytosol . The soluble GST-hHSS expression reached up to 30% of the whole soluble protein of bacteria as determined by densitometry . The cleavage of GST-hHSS fusion protein with Factor Xa produced two fragments of the protein, which sized 33 and 15 kD, respectively . The molecular weight of recombinant HSS protein was identical to theoretical deduction based on the DNA sequences . The protein homology of 15 kD hHSS could be efficiently eluted out after Factor Xa cleavage . It is further indicated that the recombinant hHSS is able to proliferate hepatoma cells of BEL-7402 in the preliminary experiments.

Neuroreport, 2002 Mar 25, 13(4), 437 - 41
HSP90 inhibitors alter capsaicin- and ATP-induced currents in rat dorsal root ganglion neurons; McDowell TS et al.; Heat shock proteins (HSPs) are major components of eukaryotic and prokaryotic cells with particularly high levels of expression in neurons . HSPs control protein folding, transport of proteins to and from the nucleus, incorporation of proteins into the cell membrane, and maintenance of the functional activity of several proteins involved in transcriptional control . In this study we demonstrate that inhibitors of HSP90 alter currents mediated by the ligand gated channels, P2X and VR1 . P2X and VR1 are membrane receptors activated by ATP and capsaicin, respectively, and are thought to be involved in inflammation-related nociception . The HSP90 inhibitors geldanamycin (GLD), radicicol (RAD) herbimycin A (HERB) potentiated ATP induced currents, whereas only GLD altered capsaicin-induced currents in isolated DRG neurons . At low (< 1 microM) concentrations, GLD potentiated the capsaicin-induced current, while at high concentrations (10-25 microM) it inhibited it . The results suggest a potential involvement of HSPs in nociception.

Cell Mol Biol (Noisy-le-grand), 2002 Feb, 48(1), 79 - 82
Functional studies of mutations in the human protoporphyrinogen oxidase gene in variegate porphyria; Morgan RR et al.; The autosomal dominant disorder, variegate porphyria (VP), results from mutations in the protoporphyrinogen oxidase (PPOX) gene . We have investigated the effects of 22 disease-associated missense mutations in this gene on enzyme activity . Mutants were generated in the expression plasmid pHPPOX by site-directed mutagenesis . They were screened for PPOX activity by complementation of the Escherischia coli strain SAS38X which lacks PPOX activity . Ten mutants (G40E, L85P, G232R, de1281H, V282D, L295P, V335G, S350P, L444P, G453V) had no detectable PPOX activity . PPOX activity of the remaining 12 mutants (L15F, R38P, L73P, V84G, D143V, R152C, L154P, V158M, R168H, A172V, V290L, G453R) ranged from less than 1% to 9.2% of wild-type activity . Our findings show that all 22 mutations substantially impair or abolish PPOX activity in a prokaryotic expression system and add to the evidence that they cause VP.

J Biosci, 2002 Feb, 27(1 Suppl 1), 7 - 14
Ab initio gene identification: prokaryote genome annotation with GeneScan and GLIMMER; Aggarwal G et al.; We compare the annotation of three complete genomes using the ab initio methods of gene identification GeneScan and GLIMMER . The annotation given in GenBank, the standard against which these are compared, has been made using GeneMark . We find a number of novel genes which are predicted by both methods used here, as well as a number of genes that are predicted by GeneMark, but are not identified by either of the nonconsensus methods that we have used . The three organisms studied here are all prokaryotic species with fairly compact genomes . The Fourier measure forms the basis for an efficient non-consensus method for gene prediction, and the algorithm GeneScan exploits this measure . We have bench-marked this program as well as GLIMMER using 3 complete prokaryotic genomes . An effort has also been made to study the limitations of these techniques for complete genome analysis . GeneScan and GLIMMER are of comparable accuracy insofar as gene-identification is concerned, with sensitivities and specificities typically greater than 0.9 . The number of false predictions (both positive and negative) is higher for GeneScan as compared to GLIMMER, but in a significant number of cases, similar results are provided by the two techniques . This suggests that there could be some as-yet unidentified additional genes in these three genomes, and also that some of the putative identifications made hitherto might require re-evaluation . All these cases are discussed in detail.

EMBO J, 2002 Apr 2, 21(7), 1811 - 20
Trm7p catalyses the formation of two 2'-O-methylriboses in yeast tRNA anticodon loop; Pintard L et al.; The genome of Saccharomyces cerevisiae encodes three close homologues of the Escherichia coli 2'-O-rRNA methyltransferase FtsJ/RrmJ, designated Trm7p, Spb1p and Mrm2p . We present evidence that Trm7p methylates the 2'-O-ribose of nucleotides at positions 32 and 34 of the tRNA anticodon loop, both in vivo and in vitro . In a trm7Delta strain, which is viable but grows slowly, translation is impaired, thus indicating that these tRNA modifications could be important for translation efficiency . We discuss the emergence of a family of three 2'-O-RNA methyltransferases in Eukaryota and one in Prokaryota from a common ancestor . We propose that each eukaryotic enzyme is located in a different cell compartment, in which it would methylate a different RNA that can adopt a very similar secondary structure.

J Biochem (Tokyo), 2002 Apr, 131(4), 541 - 6
Acidic phospholipids with unsaturated fatty acids inhibit the binding of origin recognition complex to origin DNA; Lee JR et al.; Origin Recognition Complex (ORC) is a candidate initiator of chromosomal DNA replication in eukaryotes . We recently reported that cardiolipin inhibits the interaction of Origin Recognition Complex ORC with origin DNA, as is the case of DnaA, the initiator of chromosomal DNA replication in prokaryotes . We report here that another acidic phospholipid, phosphatidylglycerol (PG), also inhibits the interaction . Synthetic PG with only unsaturated fatty acids inhibits ORC-binding to origin DNA more strongly than PG with only saturated fatty acids . On the other hand, phosphatidylcholine (neutral phospholipid) does not affect the ORC-origin interaction, regardless of the presence of saturated or unsaturated fatty acids . These results suggest that an acidic moiety and unsaturated fatty acids are important factors for the inhibitory effect of phospholipids on ORC binding to origin DNA, as is the case for DnaA . The inhibitory effect of cardiolipin on ORC binding to origin DNA was more apparent at 30 degrees C than at 4 degrees C . Furthermore, chlorpromazine restored the ORC-origin interaction in the presence of cardiolipin . Since the presence of unsaturated fatty acids, low incubation temperatures, and the addition of chlorpromazine all decrease membrane fluidity, these results suggest that membrane fluidity is important for the inhibitory effect of acidic phospholipids on ORC-binding to origin DNA, as is the case for DnaA.

World J Gastroenterol, 2002 Apr, 8(2), 308 - 11
A study of recombinant protective H.pylori antigens; Jiang Z et al.; AIM: To construct a recombinant vector which can express M (r)26000 outer membrane protein (OMP) from Helicobacter pylori (Hp), and to obtain the vaccine protecting against Hp infection and a diagnostic reagent kit quickly detecting Hp infection . METHODS: The gene encoding the structural M(r)26000 outer membrane protein of Hp was amplified from Hp chromosomal DNA by PCR, and inserted in the prokaryotic expression vector pET32a (+), which was transformed into the Top10 E . coli strain . Recombinant vector was selected, identified and transformed into BL-21(DE3) E . coli strain . The recombinant fusion proteins were expressed . The antigenicity of recombinant protein was studied by ELISA or immunoblotting and immunized Balb/c mice . RESULTS: The gene of M(r)26000 OMP was amplified to be 594 base pairs, 1.1% of the cloned genes was mutated and 1.51% of amino acid residues was changed, but there was homogeneity between them . The recombinant fusion protein encoded objective polypeptides of 198 amino acid residues, corresponding to calculated molecular masses of M (r)26000 . The level of soluble expression products was about 38.96% of the total cell protein . After purification by Ni-NTA agarose resin columniation, the purity of objective protein became about 90% . The ELISA results showed that recombinant fusion protein could be recognized by patient serum infected with Hp and rabbit serum immunized with the recombinant protein . Furthermore,Balb/c mice immunized with the recombinant protein were protected against H.pylori infection . CONCLUSION: M (r)26000 OMP may be a candidate vaccine preventing Hp infection.

J Biol Chem, 2002 Jun 14, 277(24), 21851 - 61 Epub 2002 Mar 29.
A novel family of calmodulin-binding transcription activators in multicellular organisms; Bouche N et al.; Screening of cDNA expression libraries derived from plants exposed to stress, with 35S-labeled recombinant calmodulin as a probe, revealed a new family of proteins containing a transcription activation domain and two types of DNA-binding domains designated the CG-1 domain and the transcription factor immunoglobulin domain, ankyrin repeats, and a varying number of IQ calmodulin-binding motifs . Based on domain organization and amino acid sequence comparisons, similar proteins, with the same domain organization, were identified in the genomes of other multicellular organisms including human, Drosophila, and Caenorhabditis, whereas none were found in the complete genomes of single cell eukaryotes and prokaryotes . This family of proteins was designated calmodulin-binding transcription activators (CAMTAs) . Arabidopsis thaliana contains six CAMTA genes (AtCAMTA1-AtCAMTA6) . The transcription activation domain of AtCAMTA1 was mapped by testing a series of protein fusions with the DNA-binding domain of the bacterial LexA transcription factor and two reporter genes fused to LexA recognition sequences in yeast cells . Two human proteins designated HsCAMTA1 and HsCAMTA2 were also shown to activate transcription in yeast using the same reporter system . Subcellular fractionation of Arabidopsis tissues revealed the presence of CAMTAs predominantly in the nucleus . Calmodulin binding assays identified a region of 25 amino acids capable of binding calmodulin with high affinity (K(d) = 1.2 nm) in the presence of calcium . We suggest that CAMTAs comprise a conserved family of transcription factors in a wide range of multicellular eukaryotes, which possibly respond to calcium signaling by direct binding of calmodulin.

J Chem Ecol, 2002 Feb, 28(2), 317 - 31
Antimicrobial activity of exocrine glandular secretion of Chrysomela larvae; Gross J et al.; The exocrine glandular secretions of leaf beetle larvae of the taxon Chrysomela are well-known defensive devices used against some generalist predators . Salicylaldehyde is the major repellent component of secretions emitted by larvae of Chrysomela vigintipunctata and C . lapponica, which feed on salicin-rich Salicaceae . In this study, we examined whether salicylaldehyde is also active against the entomopathogenic fungus Metarhizium anisopliae . The germination and growth of this fungus was strongly inhibited when salicylaldehyde was applied directly onto the blastospores . The salicylaldehyde concentration of the larval secretions of the tested willow feeding Chrsysomela larvae was much higher than the one necessary to display this antifungal activity . Additionally, salicylaldehyde was shown to reduce germination and growth of M . anisopliae via the gas phase over a distance of 45 mm . Further studies on the antimicrobial activity of the salicylaldehyde-containing secretions of Chrysomela larvae revealed that they act nonspecifically against prokaryotic (Escherichia coli) and eukaryotic cells (Saccharomyces cerevisiae and Trichoplusia ni) . All antimicrobial and cytotoxic effects detected proved to be due to salicylaldehyde . The larval secretions of the birch-feeding allospecies of C . lapponica, that do not contain salicylaldehyde, but mainly carboxylic acids and their esters, showed no detectable effects on bacteria or fungi and no cytotoxic effects against insect cells . The results are discussed with respect to their ecological relevance.

Protein Expr Purif, 2002 Apr, 24(3), 338 - 47
Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells; Choo AB et al.; We have previously described the production of a recombinant melittin-based cytolytic immunotoxin (IT), scFv-mel-FLAG, in bacterial cells . While the IT exhibited specific cytotoxicity for a human lymphoblastoid cell line, HMy2, yields from expression were low . Here, we describe a baculovirus expression system for the overexpression and secretion of scFv-mel-FLAG . A novel snake phospholipase A2 inhibitor signal peptide was used to aid in the secretion of the immunotoxin . Sf21 insect cells infected with the recombinant virus secreted soluble scFv-mel-FLAG into the culture medium from which it was purified directly on an affinity column . The final yield of scFv-mel-FLAG was estimated at 3-5 mg/L, which was an improvement of 30-fold compared to expression in the prokaryotic system . The cell binding characteristics of the recombinant IT were assessed by flow cytometry using the antigen expressing cell line HMy2 . ScFv-mel-FLAG bound specifically to HMy2 cells in direct binding assays and this binding was completely inhibited in the presence of an excess of soluble antigen . Significant cytotoxicity for HMy2 cells, measured by leakage of cytosolic LDH, was also observed for the IT at a concentration of 60 pmol/10(4) cells . Cytotoxicity was concentration dependent and was specific for antigen-positive cells . Thus the baculovirus expression system, under the control of a novel secretion signal, can be used for the production of soluble and functional recombinant cytolytic immunotoxins . To our knowledge, this is the first report of expression of a recombinant immunotoxin in the baculovirus expression vector system .

J Mol Biol, 2002 Mar 29, 317(3), 415 - 29
Solution structure of the N-terminal domain of a potential copper-translocating P-type ATPase from Bacillus subtilis in the apo and Cu(I) loaded states; Banci L et al.; A putative partner of the already characterized CopZ from Bacillus subtilis was found, both proteins being encoded by genes located in the same operon . This new protein is highly homologous to eukaryotic and prokaryotic P-type ATPases such as CopA, Ccc2 and Menkes proteins . The N-terminal region of this protein contains two soluble domains constituted by amino acid residues 1 to 72 and 73 to 147, respectively, which were expressed both separately and together . In both cases only the 73-147 domain is folded and is stable both in the copper(I)-free and in the copper(I)-bound forms . The folded and unfolded state is monitored through the chemical shift dispersion of 15N-HSQC spectra . In the absence of any structural characterization of CopA-type proteins, we determined the structure of the 73-147 domain in the 1-151 construct in the apo state through 1H, 15N and 13C NMR spectroscopies . The structure of the Cu(I)-loaded 73-147 domain has been also determined in the construct 73-151 . About 1300 meaningful NOEs and 90 dihedral angles were used to obtain structures at high resolution both for the Cu(I)-bound and the Cu(I)-free states (backbone RMSD to the mean 0.35(+/-0.06) A and 0.39(+/-0.07) A, respectively) . The structural assessment shows that the structures are accurate . The protein has the typical betaalpha(betabeta)alphabeta folding with a cysteine in the C-terminal part of helix alpha1 and the other cysteine in loop 1 . The structures are similar to other proteins involved in copper homeostasis . Particularly, between BsCopA and BsCopZ, only the charges located around loop 1 are reversed for BsCopA and BsCopZ, thus suggesting that the two proteins could interact one with the other . The variability in conformation displayed by the N-terminal cysteine of the CXXC motif in a number of structures of copper transporting proteins suggests that this may be the cysteine which binds first to the copper(I) carried by the partner protein .

J Biol Chem, 2002 Jun 14, 277(24), 21431 - 9 Epub 2002 Mar 28.
Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics; Daugherty M et al.; The biosynthesis of CoA from pantothenic acid (vitamin B5) is an essential universal pathway in prokaryotes and eukaryotes . The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown . Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC ), phosphopantothenoylcysteine decarboxylase (EC ), phosphopantetheine adenylyltransferase (EC ), and dephospho-CoA kinase (EC ) . Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis . The individual human enzymes were overexpressed in E . coli and purified, and the corresponding activities were experimentally verified . In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes . Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized . This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.

Mol Biol Evol, 2002 Apr, 19(4), 521 - 252
Do introns favor or avoid regions of amino acid conservation?
Endo T, Fedorov A, de Souza SJ, Gilbert W.
Are intron positions correlated with regions of high amino acid conservation? For a set of ancient conserved proteins, with intronless prokaryotic but intron-containing eukaryotic homologs, multiple sequence alignments identified residues invariant throughout evolution . Intron positions between codons show no preferences . However, introns lying after the first base of a codon prefer conserved regions, markedly in glycines . Because glycines are in excess in conserved regions, this behavior could reflect phase-one introns entering glycine residues randomly in the ancestral sequences . Examination of intron positions within codons of evolutionarily invariable amino acids showed that roughly 50% of these introns are bordered by guanines at both 5'- and 3'-ends, 25% have a G only before the intron, and 5% have a G only after the intron, whereas about 20% are bordered by nonguanine bases.

BMC Genomics . 2002;3(1):4 . Epub 2002 Feb 05.
Bootstrap, Bayesian probability and maximum likelihood mapping: exploring new tools for comparative genome analyses; Zhaxybayeva O et al.; BACKGROUND: Horizontal gene transfer (HGT) played an important role in shaping microbial genomes . In addition to genes under sporadic selection, HGT also affects housekeeping genes and those involved in information processing, even ribosomal RNA encoding genes . Here we describe tools that provide an assessment and graphic illustration of the mosaic nature of microbial genomes . RESULTS: We adapted the Maximum Likelihood (ML) mapping to the analyses of all detected quartets of orthologous genes found in four genomes . We have automated the assembly and analyses of these quartets of orthologs given the selection of four genomes . We compared the ML-mapping approach to more rigorous Bayesian probability and Bootstrap mapping techniques . The latter two approaches appear to be more conservative than the ML-mapping approach, but qualitatively all three approaches give equivalent results . All three tools were tested on mitochondrial genomes, which presumably were inherited as a single linkage group . CONCLUSIONS: In some instances of interphylum relationships we find nearly equal numbers of quartets strongly supporting the three possible topologies . In contrast, our analyses of genome quartets containing the cyanobacterium Synechocystis sp . indicate that a large part of the cyanobacterial genome is related to that of low GC Gram positives . Other groups that had been suggested as sister groups to the cyanobacteria contain many fewer genes that group with the Synechocystis orthologs . Interdomain comparisons of genome quartets containing the archaeon Halobacterium sp . revealed that Halobacterium sp . shares more genes with Bacteria that live in the same environment than with Bacteria that are more closely related based on rRNA phylogeny . Many of these genes encode proteins involved in substrate transport and metabolism and in information storage and processing . The performed analyses demonstrate that relationships among prokaryotes cannot be accurately depicted by or inferred from the tree-like evolution of a core of rarely transferred genes; rather prokaryotic genomes are mosaics in which different parts have different evolutionary histories . Probability mapping is a valuable tool to explore the mosaic nature of genomes.

Mol Microbiol, 2002 Mar, 43(5), 1309 - 18
An rRNA mutation identifies the apicoplast as the target for clindamycin in Toxoplasma gondii; Camps M et al.; Toxoplasma gondii is a protozoan sensitive to several inhibitors of prokaryotic translation (e.g . clindamycin, macrolides and tetracyclines) . A priori, two prokaryotic-like organelles, the 'apicoplast' (a non-photosynthetic plastid) and the mitochondrion, are likely targets for these drugs . Without using overt mutagenesis, we selected two independent clones (ClnR-4 and ClnR-21) with strong and stable clindamycin resistance . Several lines with substantial but lower levels of resistance were also isolated with (XR-46) or without (ClnR-23) overt mutagenesis . The ClnR-4 and ClnR-21 mutants uniquely possess a G-->U point mutation at position 1857 of the apicoplast large-subunit rRNA, whereas no mutation was identified in this region for ClnR-23 or XR-46 . Position 1857 corresponds to position 2061 in Escherichia coli where it is predicted to bind clindamycin . The mutation is present in all the apicoplast rDNA copies (an estimated 12 per organelle), indicative of a strong selective advantage in the presence of clindamycin . In the absence of drug, however, such a mutation is unlikely to be neutral, as the G is a critical contributor to the transpeptidation reaction and absolutely conserved in all kingdoms . This may explain why ClnR-4 shows a slight growth defect in vitro . These mutants provide direct genetic evidence that apicoplast translation is the target for clindamycin in Toxoplasma . Further, their sensitivity profiles to other antibiotics specific for the large ribosomal subunit (macrolides and chloramphenicol) and, intriguingly, the small subunit (doxycycline) argue that these drugs also target the apicoplast ribosome.

DNA Seq, 2001 Nov, 12(4), 229 - 38
A 6940 bp DNA fragment from Desulfovibrio gigas contains genes coding for lipoproteins, universal stress response and transcriptional regulator protein homologues; Silva G et al.; The nucleotide sequence of a 6940 bp DNA fragment from Desulfovibrio gigas, containing seven ORFs was determined . ORF-1 encodes a probable lipoprotein having high similarities with lytic transglycosylases . ORF-2 encodes a polypeptide that does not show homologies with proteins deposited in the database, but it contains the consensus pattern of class II aminoacyl-tRNA synthetases . The putative protein encoded by ORF-3 possesses high similarities with universal stress response proteins from the UspA family . Northern blot analysis of ORF-3 shows that it is constitutively and abundantly expressed . ORF-4 encodes a probable helix-turn-helix-containing DNA-binding protein, given the presence of the helix-turn-helix motif, characteristic of this class of proteins . Its N-terminal region has high identity with its counterparts from proteins belonging to the RRF2 family . Northern blot analysis shows that ORF-4 is transcribed as a single mRNA in contrast to its orthologue from Desulfovibrio vulgaris . ORF-5 encodes a putative fusion protein as its N- and C-termini show significant homologies with molybdenum formylmethanofuran dehydrogenase and molybdopterin biosynthesis proteins, respectively . ORF-7 encodes a prokaryotic lipoprotein having homologies with multidrug efflux and DNA damage-inducible proteins, and it is constitutively and abundantly expressed.

Biochemistry, 2002 Apr 2, 41(13), 4193 - 201
Role of dimerization in KH/RNA complexes: the example of Nova KH3; Ramos A et al.; The K homology module, one of the most common RNA-binding motifs, is present in multiple copies in both prokaryotic and eukaryotic regulatory proteins . Increasing evidence suggests that self-aggregation of KH modules has a functional role . We have used a combination of techniques to characterize the behavior in solution of the third KH domain of Nova-1, a paradigmatic KH protein . The possibility of working on the isolated module allowed us to observe specifically the homodimerization and RNA-binding properties of KH domains . We provide conclusive evidence that self-association of Nova-1 KH3 occurs in solution even in the absence of RNA . Homodimerization involves a specific protein/protein interface . We also studied the dynamical behavior of Nova-1 KH3 in isolation and in complex with RNA . These data provide a model for the mechanism of KH/RNA recognition and suggest functional implications of dimerization in KH complexes . We discuss our findings in the context of the whole KH family and suggest a generalized mode of interaction.

Biotechniques, 2002 Mar, 32(3), 522 - 4, 526-7
Cell sorting of formalin-treated pathogenic Mycobacterium paratuberculosis expressing GFP; Harris NB et al.; GFP is widely used as a molecular tool for the study of microbial pathogens . However, the manipulation of these pathogenic microorganisms poses a health threat to the laboratory worker, requiring biosafety level II or III containment . Although the GFPfluorophore is tolerant toformalin, a thorough analysis of this treatment on fluorescent output in prokaryotic systems has not been described . In addition, the analysis of microorganisms expressing GFP often depends on specialized equipment, which may not be housed in biosafety level II or III laboratories . Therefore, we sought to develop a safe and effective method for manipulating the GFP-expressing pathogenic bacterium Mycobacterium avium subsp, paratuberculosis (M . paratuberculosis) utilizing a formalin treatment that would permit the analysis of GFP fluorescence without requiring stringent biosafety containment . We demonstrate that formalin-treated M . paratuberculosis expresses 50% less fluorescence than viable cells, but this reduction is still compatible with spectrofluorometry and cell sorting . Furthermore, plasmid DNA that expresses GFP can be recovered efficiently from nonviable, sorted fluorescent cells . This approach is flexible, provides an additional margin of safety for laboratory personnel, and can be easily applied to other pathogenic microorganisms expressing GFP.

Mikrobiologiia, 2002 Jan-Feb, 71(1), 5 - 22
{Microbial geochemical calcium cycle}; Zavarzin GA; The participation of microorganisms in the geochemical calcium cycle is the most important factor maintaining neutral conditions on the Earth . This cycle has profound influence on the fate of inorganic carbon, and, thereby, on the removal of CO2 from the atmosphere . The major part of calcium deposits was formed in the Precambrian, when prokaryotic biosphere predominated . After that, calcium recycling based on biogenic deposition by skeletal organisms became the main process . Among prokaryotes, only a few representatives, e.g., cyanobacteria, exhibit a special calcium function . The geochemical calcium cycle is made possible by the universal features of bacteria involved in biologically mediated reactions and is determined by the activities of microbial communities . In the prokaryotic system, the calcium cycle begins with the leaching of igneous rock predominantly through the action of the community of organotrophic organisms . The release of carbon dioxide to the soil air by organotrophic aerobes leads to leaching with carbonic acid and soda salinization . Under anoxic conditions, of major importance is the organic acid production by primary anaerobes (fermentative microorganisms) . Calcium carbonate is precipitated by secondary anaerobes (sulfate reducers) and to a smaller degree by methanogens . The role of the cyanobacterial community in carbonate deposition is exposed by stromatolites, which are the most common organo-sedimentary Precambrian structures . Deposition of carbonates in cyanobacterial mats as a consequence of photoassimilation of CO2 does not appear to be a significant process . It is argued that carbonates were deposited at the boundary between the "soda continent", which emerged as a result of subaerial leaching with carbonic acid, and the ocean containing Ca2+ . Such ecotones provided favorable conditions for the development of the benthic cyanobacterial community, which was a precursor of stromatolites.

Curr Microbiol, 2002 Apr, 44(4), 267 - 72
Characterization of the Porphyromonas gingivalis FtsZ containing a novel GTPase activity; Akifusa S et al.; The FtsZ protein is a GTPase that is essential for cell division . We have cloned, sequenced, and expressed the FtsZ (PgFtsZ) gene from the Porphyromonas gingivalis, an oral, anaerobic, rod-shaped bacterium implicated in progressive periodontal disease . The PgFtsZ gene consisted of 1374 bp and coded for an acidic protein with a calculated molecular mass of 50,253 Da . The deduced amino acid sequence exhibited a significant homology with E . coli FtsZ (54% identical residues) . Like other prokaryotic FtsZs, PgFtsZ possessed the clear motifs for GTP binding (GGGTGTG) and hydrolysis (NLDFADV) . When PgFtsZ was overexpressed in E . coli, cell division was inhibited . Recombinant PgFtsZ was purified to homogeneity and characterized . The purified PgFtsZ exhibited GTPase activity even in the absence of Mg2+, and completely retained its activity with EDTA . Furthermore, Na+ and K+ ions inhibited its GTPase activity in a dose-dependent manner . These results suggest that PgFtsZ contains an atypical GTPase activity that has not been previously described.

Microbes Infect, 2002 Mar, 4(3), 309 - 17
The gut microflora and intestinal epithelial cells: a continuing dialogue; Neish AS; The mammalian intestinal epithelium effectively performs its physiological functions in a microbe-rich environment, while the prokaryotic population thrives amidst efficient cellular defenses . Recent delineation of the mechanisms by which bacteria communicate with their eukaryotic hosts and the cellular sites that microbial signals act in may shed light on these complex interactions.

Lipids, 2002 Feb, 37(2), 209 - 16
Biosynthesis of arachidonic acid in the oleaginous microalga Parietochloris incisa (Chlorophyceae): radiolabeling studies; Bigogno C et al.; The fresh-water green alga Parietochloris incisa is the richest plant source of the PUFA arachidonic acid (20:4n-6, AA) . To elucidate the biosynthesis of AA in this alga we labeled cultures of P . incisa with radioactive precursors . Pulse chase labeling with acetate resulted in its incorporation via the de novo biosynthesis pathway of FA . However, labeled acetate was also utilized for the elongation of C16 and C18 PUFA . Labeling with {1-14C}oleic acid has shown that the first steps of the lipid-linked FA desaturations utilize cytoplasmic lipids . PC and diacylglyceryltrimethylhomoserine are the major lipids involved as acyl carriers for the delta12 and delta6 desaturations of oleic acid, leading sequentially to linoleic and gamma-linolenic acids . The latter is released from its lipid carrier and elongated to 20:3n-6, which is reincorporated primarily into PE and PC and finally desaturated to AA . Galactolipids, mostly monogalctosyldiacyl-glycerol (MGDG), serve as substrates for the chloroplastic delta12 desaturase and, apparently, the omega3 desaturation, common to higher plants and many green algae . The predominant sequence desaturates the 18:1/16:0 molecular species of MGDG stepwise to the 18:3n-3/16:3n-3 molecular species similar to the prokaryotic pathway of higher plants and green algae.

Arch Microbiol, 2002 Mar, 177(3), 201 - 8 Epub 2002 Jan 22.
Phototrophic consortia: model systems for symbiotic interrelations between prokaryotes; Overmann J et al.; Most symbiotic prokaryotes known to date have been found in association with eukaryotes, whereas only few (3.5%) bacteria or archaea are known that have established interactions with other prokaryotes . As revealed by direct microscopic investigations, however, multiple morphotypes of highly structured associations of different prokaryotes exist in nature . These so-called consortia appear to represent the most developed type of bacterial interaction . Phototrophic consortia are associations of green sulfur bacteria that surround a central chemotrophic bacterium . In some natural environments, almost all cells of green sulfur bacteria occur in the associated state, i.e . as epibionts of phototrophic consortia . In contrast to earlier speculations, the central bacterium belongs to the beta-Proteobacteria . Within the consortia, the green sulfur bacterial epibionts grow photolithoautotrophically, utilizing exogenous sulfide as photosynthetic electron donor . The entire consortium does not appear to be independent of organic carbon compounds, since it exhibits chemotaxis towards 2-oxoglutarate and, as demonstrated by microautoradiography, can also incorporate this compound . Intact consortia exhibit a scotophobic response in which the bacteriochlorophylls of the epibionts function as light sensors, whereas the chemotrophic central bacterium confers motility upon the association . Hence, a signal exchange must occur between the different bacteria . Based on their 16S rRNA gene sequences, the epibionts represent distinct phylotypes that are often only distantly related to known species of green sulfur bacteria . Since phototrophic consortia have recently become available in enrichment cultures, they can now serve as suitable model systems for the investigation of the molecular mechanisms of cell-cell recognition and signal exchange, and for studies of the coevolution of nonrelated prokaryotes.

FEBS Lett, 2002 Mar 6, 514(1), 70 - 3
Construction of the 'minimal' SRP that interacts with the translating ribosome but not with specific membrane receptors in Escherichia coli; Avdeeva ON et al.; Escherichia coli signal recognition particle (SRP) consists of 4.5S RNA and Ffh protein . In contrast to eukaryotes, it remains unclear whether translation arrest takes place in prokaryotic cells . To study this problem we constructed a fusion of the M domain of Ffh protein with a cleavable affinity tag . This mutant Ffh, in a complex with 4.5S RNA, can bind signal peptide at the translating ribosome but is unable to bind the membrane . This SRP-ribosome complex should accumulate in the cell if translation is arrested . To test this, the complex was purified from the cells by ultracentrifugation and affinity chromatography . The composition of the complex was analyzed and found to consist of ribosomal RNAs and proteins, the Ffh M domain and 4.5S RNA . The accumulation of this complex in the cell in significant amounts indicated that SRP-mediated translation arrest did occur in bacterial cells.

FEBS Lett, 2002 Mar 6, 514(1), 30 - 3
A tripeptide discriminator for stop codon recognition; Nakamura Y et al.; Only recently has it been established that a tripeptide in the bacterial release factors (RFs), RF1 and RF2, is responsible for the stop codon recognition . This functional mimic of the anticodon of tRNA is referred to as a tripeptide 'anticodon' or a tripeptide discriminator . Here we review the experimental background and process leading to this discovery, and strengthen functional evidence for the tripeptide determinant for deciphering stop codons in mRNAs in prokaryotes.

Physiol Plant, 2002 Feb, 114(2), 241 - 250
Differential expression and characterization of three metallothionein-like genes in Cavendish banana (Musa acuminata); Liu P et al.; Metallothioneins (MTs) are cysteine-rich polypeptides that are involved in metal detoxification and homeostasis in both prokaryotes and eukaryotes . In this study, we report the isolation and characterization of three members (MT2A, MT2B and MT3) of the MT-like gene family from ripening banana fruit and their differential expression in various banana organs and during fruit development and ripening . All members of the MT-like gene encode small cysteine-rich polypeptides of 65-79 amino acid residues . MT2A shared a high sequence similarity (54-77%) with several type-2 MTs in plants, while MT3 was highly homologous (51-61%) with type-3 MTs . The three members expressed differentially in various organs but transcripts were generally more abundant in reproductive than vegetative organs . During fruit development, the MT2A transcript was barely detectable in ovary but increased to a high level in young fruit at 20 days after shooting (DAS) and declined gradually thereafter as fruit developed . In contrast, both MT2B and MT3 expressed poorly in young fruits (20-60 DAS) and transcripts were detected only in fruits at later stages of development . As ripening progressed, expression of MT2A decreased but that of MT3 increased . Expression of MT members during ripening appeared to be differentially regulated by ethylene, whose levels were low in FG and TY fruit but surged climacteristically in MG and declined sharply as ripening advanced further . Exogenous application of ethylene at 5 ppm or higher concentrations down-regulated MT2A expression and the inhibitory effect of ethylene could be partially suppressed by the presence of norbornadiene, an inhibitor of ethylene action . Ethylene had no effect on transcript accumulation of MT2B and MT3 . However, MT3 expression was greatly enhanced in response to metals such as CdSO4, CuSO4 and ZnSO4 . These results suggest that increased MT3 expression may be associated with excess metal ions present in ripening fruit tissues . This study also provided evidence, for the first time, that ethylene and metals play a regulatory role in expression of MT-like genes in banana.

Appl Biochem Biotechnol, 2002 Jan, 97(1), 33 - 44
Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E; Lin FP et al.; The bifunctional activities of alpha-amylase and pullulanase are found in the cloned recombinant amylopullulanase . It was encoded in a 2.9-kb DNA fragment that was amplified using polymerase chain reaction from the chromosomal DNA of Thermoanaerobacter ethanolicus 39E . An estimated 109-kDa recombinant protein was obtained from the cloned gene under the prokaryotic expression system . The optimum pH of the recombinant amylopullulanase was 6.0 . The most stable pH for the alpha-amylase and pullulanase activity was 5.5 and 5.0, respectively . The optimum temperature for the alpha-amylase activity was 90 degrees C, while its most stable temperature was 80 degrees C . Regarding pullulanase activity, the optimum temperature and its most stable temperature were found to be 80 and 75 degrees C, respectively . Pullulan was found to be the best substrate for the enzyme . The enzyme was activated and stabilized by the presence of Ca2+, whereas EDTA, N-bromosuccinimide, and alpha-cyclodextrin inhibited its bifunctional activities . A malto-2-4-oligosaccharide was the major product obtained from the enzymatic reaction on soluble starch, amylose, amylopectin, and glycogen . A single maltotriose product was found in the pullulan hydrolysis reaction using this recombinant amylopullulanase . Kinetic analysis of the enzyme indicated that the Km values of alpha-amylase and pullulanase were 1.38 and 3.79 mg/mL, respectively, while the Vmax values were 39 and 98 micromol/(min x mg of protein), respectively.

Cell Biochem Biophys, 2001, 34(3), 349 - 81
Mechanosensitive channels in archaea; Kloda A et al.; The ubiquity of mechanosensitive (MS) channels triggered a search for their functional homologues in Archaea, the third domain of the phylogenetic tree . Two types of MS channels have been identified in the cell membranes of Haloferax volcanii using the patch clamp technique . Recently MS channels were identified and cloned from two archaeal species occupying different environmental habitats . These studies demonstrate that archaeal MS channels share structural and functional homology with bacterial MS channels . The mechanical force transmitted via the lipid bilayer alone activates all to date known prokaryotic MS channels . This implies the existence of a common gating mechanism for bacterial as well as archaeal MS channels according to the bilayer model . Based on recent evidence that the bilayer model also applies to eukaryotic MS channels, mechanosensory transduction probably originated along with the appearance of the first life forms according to simple biophysical principles . In support of this hypothesis the phylogenetic analysis revealed that prokaryotic MS channels of large and small conductance originated from a common ancestral molecule resembling the bacterial MscL channel protein . Furthemore, bacterial and archaeal MS channels share common structural motifs with eukaryotic channels of diverse function indicating the importance of identified structures to the gating mechanism of this family of channels . The comparative approach used throughout this review should contribute towards understanding of the evolution and molecular basis of mechanosensory transduction in general.

J Biol Chem, 2002 May 31, 277(22), 20051 - 8 Epub 2002 Mar 15.
A novel prokaryotic phospholipase A2 . Characterization, gene cloning, and solution structure; Sugiyama M et al.; Until now, phospholipase A(2) (PLA(2); EC 3.1.14) has been found only from eukaryotic sources . In the present study, we found a secreted PLA(2), which is produced by a soil bacterium, Streptomyces violaceoruber A-2688, demonstrating that the enzyme is the first phospholipase A(2) identified in prokaryote . After characterization of the novel PLA(2), a gene encoding the enzyme was cloned, sequenced, and overexpressed using a Streptomyces host-vector system . The amino acid sequence showed that the prokaryotic PLA(2) has only four cysteines and less homology to the eukaryotic ones, which have 12-16 cysteines . The solution structures of the prokaryotic PLA(2), bound and unbound with calcium(II) ion, were determined by using the NMR technique and structure calculation . The overall structure of the S . violaceoruber PLA(2), which is composed of only five alpha-helices, is completely different from those of eukaryotic PLA(2)s, which consist of beta-sheets and alpha-helices . The structure of the calcium-binding domain is obviously distinct from that without the ion; the ligands for the calcium(II) ion are the two carboxylates of Asp(43) (monodentate) and Asp(65) (bidentate), the carbonyl oxygen of Leu(44), and three water molecules . A calcium-binding experiment showed that the calcium dissociation constant ( approximately 5 mm) for the prokaryotic PLA(2) is much larger than those of eukaryotic ones.

J Biol Chem, 2002 May 31, 277(22), 20059 - 69 Epub 2002 Mar 15.
The crystal structure of prokaryotic phospholipase A2; Matoba Y et al.; In this study, the x-ray crystal structures of the calcium-free and calcium-bound forms of phospholipase A(2) (PLA(2)), produced extracellularly by Streptomyces violaceoruber, were determined by using the multiple isomorphous replacement and molecular replacement methods, respectively . The former and latter structures were refined to an R-factor of 18.8% at a 1.4-A resolution and an R-factor of 15.0% at a 1.6-A resolution, respectively . The overall structure of the prokaryotic PLA(2) exhibits a novel folding topology that demonstrates that it is completely distinct from those of eukaryotic PLA(2)s, which have been already determined by x-ray and NMR analyses . Furthermore, the coordination geometry of the calcium(II) ion apparently deviated from that of eukaryotic PLA(2)s . Regardless of the evolutionary divergence, the catalytic mechanism including the calcium(II) ion on secreted PLA(2) seems to be conserved between prokaryotic and eukaryotic cells . Demonstrating that the overall structure determined by x-ray analysis is almost the same as that determined by NMR analysis is useful to discuss the catalytic mechanism at the molecular level of the bacterial PLA(2).

Eur J Biochem, 2002 Mar, 269(6), 1662 - 9
DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene-expression and metabolic regulation of both topoisomerase I and DNA gyrase; Snoep JL et al.; DNA of prokaryotes is in a nonequilibrium structural state, characterized as 'active' DNA supercoiling . Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested {Menzel, R . & Gellert, M . (1983) Cell 34, 105-113} . We here report on a new method for quantifying homeostatic control of the high-energy state of in vivo DNA . The method involves making small perturbation in the expression of topoisomerase I, and measuring the effect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase . In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomerase I expression was measured {part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff, H.V . & Snoep, J.L . (1999) Eur . J . Biochem . 266, 865-877} . Of the two regulatory mechanisms via which homeostasis is conferred, regulation of enzyme activity or regulation of enzyme expression, we quantified the first to be responsible for 72% and the latter for 28% . The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomerase I (7%) . On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quantified the homeostatic control of DNA supercoiling at 0.87 . A 10% manipulation of either topoisomerase I or DNA gyrase activity results in a 1.3% change of DNA supercoiling only . We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wild-type Escherichia coli is almost complete and subtle (i.e . involving at least three regulatory mechanisms).

J Theor Biol, 2001 Nov 21, 213(2), 159 - 70
Analysis of a circular code model; Lacan J et al.; A circular code has been identified in the protein (coding) genes of both eukaryotes and prokaryotes by using a statistical method called trinucleotide frequency (TF) method {Arques & Michel (1996) . J . theor . Biol . 182, 45-58} . Recently, a probabilistic model based on the nucleotide frequencies with a hypothesis of absence of correlation between successive bases on a DNA strand, has been proposed by Koch & Lehmann {(1997) . J . theor . Biol . 189, 171-174} for constructing some particular circular codes . Their interesting method which we call here nucleotide frequency (NF) method, reveals several limits for constructing the circular code observed with protein genes.

Chest, 2002 Mar, 121(3 Suppl), 91S - 97S
Mode of action of RNA/DNA oligonucleotides: progress in the development of gene repair as a therapy for alpha(1)-antitrypsin deficiency; Metz R et al.; We describe a technology developed for the site-specific correction of a single base carried on an episome or chromosome in prokaryotic and eukaryotic cells . Critical to the development of this technology as a therapeutic device for treating genetic disorders, like alpha(1)-antitrypsin deficiency, is the establishment of a standardized assay to study its mode of action and structure-activity relationships (SARs) . To this end, a positive-selection system in Escherichia coli has been developed to assess RNA/DNA oligonucleotide (RDO)-directed repair activity . We demonstrate that RDO-directed repair requires the concerted action of the two following repair proteins: the pairing protein RecA; and the mismatch recognition protein, MutS . SAR studies demonstrate that the RDO molecule is functionally asymmetric . The RNA-containing strand enables strand-pairing and stabilization of the molecule, and the DNA-containing strand confers the information transfer.

Clin Exp Rheumatol, 2002 Jan-Feb, 20(1), 45 - 51
Autoantibody detection in scleroderma patients . Diagnostic and analytical performances of a new coupled particle light scattering immunoassay; Bizzaro N et al.; OBJECTIVE: We evaluated the diagnostic and analytical performance of the Coupled Particle Light Scattering technology applied to the detection of anti-topoisomerase I (anti-Scl70) and anti-CENP-B autoantibodies . METHODS: The Scl70 antigen was obtained by recombinant DNA procedures using a prokaryotic expression system; CENP-B was a Baculovirus-expressed recombinant protein . Anti-centromere and anti-Scl70 antibodies were assayed in serum samples from 288 patients, of whom 123 had systemic sclerosis/scleroderma and 165 constituted the control groups (including patients with other connective tissue diseases, viral infections, Lyme disease, and healthy subjects) . RESULTS: The sensitivity was 98.8% (confidence interval, 96-101%) for anti-Scl70 and 100% (99.6-100%) for anti-CENP-B; the specificity was 99.0% (98-100%) and 100% (99.9-100%) for anti-Scl70 and anti-CENP-B, respectively . The intra-assay coefficient variations (CV) ranged from 3.8 to 9.2% for anti-Scl70, and from 2.8 to 8.0% for anti-CENP-B . Inter-assay CVs were 8.1 to 12.0% for anti-Scl70, and 4.7 to 10.5% for anti-CENP-B . In 3 patients, coexpression of both antibodies was observed . CONCLUSION: The results of this study demonstrate that the light scattering technology is also applicable to the detection of autoantibodies to intracellular antigens for the diagnosis of autoimmune rheumatic diseases.

Plant Physiol, 2002 Mar, 128(3), 885 - 95
Digalactosyldiacylglycerol synthesis in chloroplasts of the Arabidopsis dgd1 mutant; Klaus D et al.; Galactolipid biosynthesis in plants is highly complex . It involves multiple pathways giving rise to different molecular species . To assess the contribution of different routes of galactolipid synthesis and the role of molecular species for growth and photosynthesis, we initiated a genetic approach of analyzing double mutants of the digalactosyldiacylglycerol (DGDG) synthase mutant dgd1 with the acyltransferase mutant, act1, and the two desaturase mutants, fad2 and fad3 . The double mutants showed different degrees of growth retardation: act1,dgd1 was most severely affected and growth of fad2,dgd1 was slightly reduced, whereas fad3,dgd1 plants were very similar to dgd1 . In act1,dgd1, lipid and chlorophyll content were reduced and photosynthetic capacity was affected . Molecular analysis of galactolipid content, fatty acid composition, and positional distribution suggested that the growth deficiency is not caused by changes in galactolipid composition per se . Chloroplasts of dgd1 were capable of synthesizing monogalactosyldiacylglycerol, DGDG, and tri- and tetragalactosyldiacylglycerol . Therefore, the reduced growth of act1,dgd1 and fad2,dgd1 cannot be explained by the absence of DGDG synthase activity from chloroplasts . Molecular analysis of DGDG accumulating in the mutants during phosphate deprivation suggested that similarly to the residual DGDG of dgd1, this additional lipid is synthesized in association with chloroplast membranes through a pathway independent of the mutations, act1, dgd1, fad2, and fad3 . Our data imply that the severe growth defect of act1,dgd1 is caused by a reduced metabolic flux of chloroplast lipid synthesis through the eukaryotic and prokaryotic pathway as well as by the reduction of photosynthetic capacity caused by the destabilization of photosynthetic complexes.

J Biol Chem, 2002 May 24, 277(21), 18257 - 65 Epub 2002 Mar 11.
Error prone translesion synthesis past gamma-hydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells; Kanuri M et al.; 8-Hydroxy-5,6,7,8-tetrahydropyrimido{1,2-a}purin- 10(3H)-one,3-(2'-deoxyriboside) (1,N(2)-gamma-hydroxypropano deoxyguanosine, gamma-HOPdG) is a major DNA adduct that forms as a result of exposure to acrolein, an environmental pollutant and a product of endogenous lipid peroxidation . gamma-HOPdG has been shown previously not to be a miscoding lesion when replicated in Escherichia coli . In contrast to those prokaryotic studies, in vivo replication and mutagenesis assays in COS-7 cells using single stranded DNA containing a specific gamma-HOPdG adduct, revealed that the gamma-HOPdG adduct was significantly mutagenic . Analyses revealed both transversion and transition types of mutations at an overall mutagenic frequency of 7.4 x 10(-2)/translesion synthesis . In vitro gamma-HOPdG strongly blocks DNA synthesis by two major polymerases, pol delta and pol epsilon . Replicative blockage of pol delta by gamma-HOPdG could be diminished by the addition of proliferating cell nuclear antigen, leading to highly mutagenic translesion bypass across this adduct . The differential functioning and processing capacities of the mammalian polymerases may be responsible for the higher mutation frequencies observed in this study when compared with the accurate and efficient nonmutagenic bypass observed in the bacterial system.

J Bacteriol, 2002 Apr, 184(7), 1998 - 2004
Analysis of the Bacillus subtilis spoIIIJ gene and its Paralogue gene, yqjG; Murakami T et al.; The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene . Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans . A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer . On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins . We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation . In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B . subtilis translocase homologues play an important role in maintaining the viability of the cell . This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth . spoIIIJ mutations have already been established to block sporulation at stage III . In contrast, disruption of yqjG did not interfere with sporulation . We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level . Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells . This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

Glycobiology, 2002 Feb, 12(2), 17R - 27R
Complex glycosylation of Skp1 in Dictyostelium: implications for the modification of other eukaryotic cytoplasmic and nuclear proteins; West CM et al.; Recently, complex O-glycosylation of the cytoplasmic/nuclear protein Skp1 has been characterized in the eukaryotic microorganism Dictyostelium . Skp1's glycosylation is mediated by the sequential action of a prolyl hydroxylase and five conventional sugar nucleotide-dependent glycosyltransferase activities that reside in the cytoplasm rather than the secretory compartment . The Skp1-HyPro GlcNAcTransferase, which adds the first sugar, appears to be related to a lineage of enzymes that originated in the prokaryotic cytoplasm and initiates mucin-type O-linked glycosylation in the lumen of the eukaryotic Golgi apparatus . GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of beta1,3-linked Gal and alpha1,2-linked Fuc . The architecture of this enzyme resembles that of certain two-domain prokaryotic glycosyltransferases . The catalytic domains are related to those of a large family of prokaryotic and eukaryotic, cytoplasmic, membrane-bound, inverting glycosyltransferases that modify glycolipids and polysaccharides prior to their translocation across membranes toward the secretory pathway or the cell exterior . The existence of these enzymes in the eukaryotic cytoplasm away from membranes and their ability to modify protein acceptors expose a new set of cytoplasmic and nuclear proteins to potential prolyl hydroxylation and complex O-linked glycosylation.

FEMS Microbiol Lett, 2002 Jan 22, 207(1), 55 - 61
MOSC domains: ancient, predicted sulfur-carrier domains, present in diverse metal-sulfur cluster biosynthesis proteins including Molybdenum cofactor sulfurases; Anantharaman V et al.; Using computational analysis, a novel superfamily of beta-strand-rich domains was identified in the Molybdenum cofactor sulfurase and several other proteins from both prokaryotes and eukaryotes . These MOSC domains contain an absolutely conserved cysteine and occur either as stand-alone forms such as the bacterial YiiM proteins, or fused to other domains such as a NifS-like catalytic domain in Molybdenum cofactor sulfurase . The MOSC domain is predicted to be a sulfur-carrier domain that receives sulfur abstracted by the pyridoxal phosphate-dependent NifS-like enzymes, on its conserved cysteine, and delivers it for the formation of diverse sulfur-metal clusters . The identification of this domain may clarify the mechanism of biogenesis of various metallo-enzymes including Molybdenum cofactor-containing enzymes that are compromised in human type II xanthinuria.

Indian J Biochem Biophys, 2001 Oct, 38(5), 289 - 93
A unique group of self-splicing introns in bacteriophage T4; Khan AU et al.; We describe in this review, the salient splicing features of group I introns of bacteriophage T4 and propose, a hypothetical model to fit in the self-splicing of nrdB intron of T4 phage . Occurrence of non-coding sequences in prokaryotic cells is a rare event while it is common in eukaryotic cells, especially the higher eukaryotes . Therefore, T4 bacteriophage can serve as a good model system to study the evolutionary aspects of splicing of introns . Three genes of T4 phage were found to have stretches of non-coding sequences which belonged to the group IA type introns of self-splicing nature.

Arch Biochem Biophys, 2002 Mar 1, 399(1), 19 - 36
Molecular evolution and structure--function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases; Fink RC et al.; This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species . Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes . Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs . Among eukaryotes, the plant SODs group together . Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%) . Tree topologies are similar and reflect the taxonomic classification of the corresponding species . Intron number and position in the Cu/Zn Sod genes are highly conserved in plants . Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven . In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species . The link between the phylogenetic relationships and SOD functions remains unclear . Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes . Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.

Nature, 2002 Mar 7, 416(6876), 76 - 81
Questioning the evidence for Earth's oldest fossils; Brasier MD et al.; Structures resembling remarkably preserved bacterial and cyanobacterial microfossils from about 3,465-million-year-old Apex cherts of the Warrawoona Group in Western Australia currently provide the oldest morphological evidence for life on Earth and have been taken to support an early beginning for oxygen-producing photosynthesis . Eleven species of filamentous prokaryote, distinguished by shape and geometry, have been put forward as meeting the criteria required of authentic Archaean microfossils, and contrast with other microfossils dismissed as either unreliable or unreproducible . These structures are nearly a billion years older than putative cyanobacterial biomarkers, genomic arguments for cyanobacteria, an oxygenic atmosphere and any comparably diverse suite of microfossils . Here we report new research on the type and re-collected material, involving mapping, optical and electron microscopy, digital image analysis, micro-Raman spectroscopy and other geochemical techniques . We reinterpret the purported microfossil-like structure as secondary artefacts formed from amorphous graphite within multiple generations of metalliferous hydrothermal vein chert and volcanic glass . Although there is no support for primary biological morphology, a Fischer--Tropsch-type synthesis of carbon compounds and carbon isotopic fractionation is inferred for one of the oldest known hydrothermal systems on Earth.

Nature, 2002 Mar 7, 416(6876), 73 - 6
Laser--Raman imagery of Earth's earliest fossils; Schopf JW et al.; Unlike the familiar Phanerozoic history of life, evolution during the earlier and much longer Precambrian segment of geological time centred on prokaryotic microbes . Because such microorganisms are minute, are preserved incompletely in geological materials, and have simple morphologies that can be mimicked by nonbiological mineral microstructures, discriminating between true microbial fossils and microscopic pseudofossil 'lookalikes' can be difficult . Thus, valid identification of fossil microbes, which is essential to understanding the prokaryote-dominated, Precambrian 85% of life's history, can require more than traditional palaeontology that is focused on morphology . By combining optically discernible morphology with analyses of chemical composition, laser--Raman spectroscopic imagery of individual microscopic fossils provides a means by which to address this need . Here we apply this technique to exceptionally ancient fossil microbe-like objects, including the oldest such specimens reported from the geological record, and show that the results obtained substantiate the biological origin of the earliest cellular fossils known.

Curr Biol, 2002 Mar 5, 12(5), 369 - 77
How myxobacteria glide; Wolgemuth C et al.; BACKGROUND: Many microorganisms, including myxobacteria, cyanobacteria, and flexibacteria, move by gliding . Although gliding always describes a slow surface-associated translocation in the direction of the cell's long axis, it can result from two very different propulsion mechanisms: social (S) motility and adventurous (A) motility . The force for S motility is generated by retraction of type 4 pili . A motility may be associated with the extrusion of slime, but evidence has been lacking, and how force might be generated has remained an enigma . Recently, nozzle-like structures were discovered in cyanobacteria from which slime emanated at the same rate at which the bacteria moved . This strongly implicates slime extrusion as a propulsion mechanism for gliding . RESULTS: Here we show that similar but smaller nozzle-like structures are found in Myxococcus xanthus and that they are clustered at both cell poles, where one might expect propulsive organelles . Furthermore, light and electron microscopical observations show that slime is secreted in ribbons from the ends of cells . To test whether the slime propulsion hypothesis is physically reasonable, we construct a mathematical model of the slime nozzle to see if it can generate a force sufficient to propel M . xanthus at the observed velocities . The model assumes that the hydration of slime, a cationic polyelectrolyte, is the force-generating mechanism . CONCLUSIONS: The discovery of nozzle-like organelles in various gliding bacteria suggests their role in prokaryotic gliding . Our calculations and our observations of slime trails demonstrate that slime extrusion from such nozzles can account for most of the observed properties of A motile gliding.

Res Microbiol, 2002 Jan-Feb, 153(1), 7 - 12
Life and death of dried prokaryotes; Billi D et al.; The removal of water through air drying damages membranes, proteins and nucleic acids and is lethal to the majority of organisms . Nevertheless, some vegetative cells of bacteria and cyanobacteria survive extreme desiccation . Understanding the mechanisms of their desiccation tolerance is an important issue in cell biology and holds promise for the metabolic engineering of desiccation-sensitive cells.

OMICS, 2002, 6(1), 115 - 21
A theoretical limit to coding space in chromosomes of bacteria; Jackson JH et al.; A mathematical model of cluster patterns for mapped genes with known phenotypes in Escherichia coli predicted thatfunctional genes may account for a maximum of two-thirds of the total chromosomal space . The corollary prediction was that one-third of the chromosome comprised noncoding space . Open reading frame (ORF) analyses for 15 phylogenetically diverse bacterial genomes and for 30 fully sequenced prokaryotic genomes supported the gene cluster model prediction of a two-thirds tendency for coding space . Our results suggest that only 3-4% of unassigned ORFs in E . coli represent genes with potential phenotype and that ORFs marking novel genes in prokaryotes are far fewer than previously thought.

BMC Bioinformatics . 2002;3(1):5 . Epub 2002 Feb 05.
Re-annotation of genome microbial coding-sequences: finding new genes and inaccurately annotated genes; Bocs S et al.; BACKGROUND: Analysis of any newly sequenced bacterial genome starts with the identification of protein-coding genes . Despite the accumulation of multiple complete genome sequences, which provide useful comparisons with close relatives among other organisms during the annotation process, accurate gene prediction remains quite difficult . A major reason for this situation is that genes are tightly packed in prokaryotes, resulting in frequent overlap . Thus, detection of translation initiation sites and/or selection of the correct coding regions remain difficult unless appropriate biological knowledge (about the structure of a gene) is imbedded in the approach . RESULTS: We have developed a new program that automatically identifies biologically significant candidate genes in a bacterial genome . Twenty-six complete prokaryotic genomes were analyzed using this tool, and the accuracy of gene finding was assessed by comparison with existing annotations . This analysis revealed that, despite the enormous effort of genome program annotators, a small but not negligible number of genes annotated within the framework of sequencing projects are likely to be partially inaccurate or plainly wrong . Moreover, the analysis of several putative new genes shows that, as expected, many short genes have escaped annotation . In most cases, these new genes revealed frameshifts that could be either artifacts or genuine frameshifts . Some entirely unexpected new genes have also been identified . This allowed us to get a more complete picture of prokaryotic genomes . The results of this procedure are progressively integrated into the SWISS-PROT reference databank . CONCLUSIONS: The results described in the present study show that our procedure is very satisfactory in terms of gene finding accuracy . Except in few cases, discrepancies between our results and annotations provided by individual authors can be accounted for by the nature of each annotation process or by specific characteristics of some genomes . This stresses that close cooperation between scientists, regular update and curation of the findings in databases are clearly required to reduce the level of errors in genome annotation (and hence in reducing the unfortunate spreading of errors through centralized data libraries).

Extremophiles, 2002 Feb, 6(1), 1 - 14
Archaeal DNA replication: spotlight on a rapidly moving field; Bohlke K et al.; The replication of DNA is a fundamental step in the cell cycle, which must be coordinated with cell division to ensure that the daughter cells inherit the same genomic material as the parental cell . The recently published complete genome sequences of some archaeal species together with preliminary biochemical studies suggest that the Archaea quite likely duplicate their chromosome by using replication machinery that seems to be a simplified version of the eukaryotic machinery, although their metabolic facets and their cellular morphology are prokaryotic-like . This review is focused on recent progress on the structural and functional analysis of proteins and enzymes involved in the initiation and elongation steps of DNA replication in Archaea . Differences between the genome replication apparatus of the Euryarchaea and the Crenarchaea (the two main phylogenetic divisions of the Archaea domain) are highlighted.

J Biol Chem, 2002 May 17, 277(20), 17464 - 75 Epub 2002 Mar 04.
NEMO trimerizes through its coiled-coil C-terminal domain; Agou F et al.; NEMO/IkappaB kinase (IKK) gamma is the regulatory component of the IKK complex comprising the two protein kinases, IKKalpha and IKKbeta . To investigate the self-assembly properties of NEMO and to understand further the mechanism of activation of the IKK complex, we purified wild-type and mutant NEMO expressed in Escherichia coli . In the absence of its IKK partners, recombinant NEMO (rNEMO) is a metastable functional monomer correctly folded, according to its fluorescence and far-UV CD spectra, which is binding specifically to the IKK complex . A minor fraction of rNEMO was found tightly associated with DnaK (E . coli Hsp70) . We also examined the interaction of NEMO with prokaryotic and eukaryotic Hsp70, and we showed that the Hsp70-NEMO complex forms a supramolecular structure probably corresponding to an assembly intermediate . In vivo cross-linking experiments indicate that native NEMO in association with IKK is in equilibrium between a dimeric and a trimeric form . Similarly to native NEMO, a NEMO mutant deleted from its IKK binding N-terminal domain (residues 242-388) forms a stable trimeric coiled-coil, suggesting that the association of NEMO with IKK or with Hsp70 prevents incorrect interdomain pairing reactions that could lead to aggregation or to an non-native oligomeric state of rNEMO . We propose a model in which the activation of the IKK complex occurs through the trimerization of NEMO upon binding to a not yet identified upstream activator.

Syst Appl Microbiol, 2001 Dec, 24(4), 490 - 9
Stable low molecular weight RNA analyzed by staircase electrophoresis, a molecular signature for both prokaryotic and eukaryotic microorganisms; Velazquez E et al.; Low-molecular weight RNA (LMW RNA) analysis using staircase electrophoresis was performed for several species of eukaryotic and prokaryotic microorganisms . According to our results, the LMW RNA profiles of archaea and bacteria contain three zones: 5S RNA, class 1 tRNA and class 2 tRNA . In fungi an additional band is included in the LMW RNA profiles, which correspond to the 5.8S RNA . In archaea and bacteria we found that the 5S rRNA zone is characteristic for each genus and the tRNA profile is characteristic for each species . In eukaryotes the combined 5.8S and 5S rRNA zones are characteristic for each genus and, as in prokaryotes, tRNA profiles are characteristic for each species . Therefore, stable low molecular weight RNA, separated by staircase electrophoresis, can be considered a molecular signature for both prokaryotic and eukaryotic microorganisms . Analysis of the data obtained and construction of the corresponding dendrograms afforded relationships between genera and species; these were essentially the same as those obtained with 16S rRNA sequencing (in prokaryotes) and 18S rRNA sequencing (in eukaryotes).

Appl Environ Microbiol, 2002 Mar, 68(3), 1265 - 79
Molecular and culture-based analyses of prokaryotic communities from an agricultural soil and the burrows and casts of the earthworm Lumbricus rubellus; Furlong MA et al.; The microbial populations in no-till agricultural soil and casts of the earthworm Lumbricus rubellus were examined by culturing and molecular methods . Clone libraries of the 16S rRNA genes were prepared from DNA isolated directly from the soil and earthworm casts . Although no single phylum dominated the soil library of 95 clones, the largest numbers of clones were from Acidobacteria (14%), Cytophagales (13%), Chloroflexi (8%), and gamma-Proteobacteria (8%) . While the cast clone library of 102 clones was similar to the soil library, the abundances of several taxa were different . Representatives of the Pseudomonas genus as well as the Actinobacteria and Firmicutes increased in number, and one group of unclassified organisms found in the soil library was absent in the cast library . Likewise, soil and cast archaeal 16S rRNA gene libraries were similar, although the abundances of some groups were different . Two hundred and thirty aerobic bacteria were also isolated on general heterotrophic media from casts, burrows, and soil . The cast isolates were both phenotypically and genotypically different from the soil isolates . The cast isolates were more likely to reduce nitrate, grow on acetate and Casamino Acids, and utilize fewer sugars than the soil isolates . On the basis of their ribotypes, the cast isolates were dominated by Aeromonas spp . (28%), which were not found in the soil isolates, and other gamma-Proteobacteria (49%) . In contrast, the soil isolates were mostly Actinobacteria (53%), Firmicutes (16%), and gamma-Proteobacteria (19%) . Isolates obtained from the sides of earthworm burrows were not different from the soil isolates . Diversity indices for the collections of isolates as well as rRNA gene libraries indicated that the species richness and evenness were decreased in the casts from their levels in the soil . These results were consistent with a model where a large portion of the microbial population in soil passes through the gastrointestinal tract of the earthworm unchanged while representatives of some phyla increase in abundance.

Hepatology, 2002 Mar, 35(3), 658 - 64
Antibodies to conformational epitopes of soluble liver antigen define a severe form of autoimmune liver disease; Ma Y et al.; Prevalence and clinical relevance of antibodies to soluble liver antigen (tRNP((Ser)Sec)/SLA) in autoimmune hepatitis (AIH) have been investigated using partially purified or prokaryotically expressed antigen . The aim of this study was to improve the detection of anti-tRNP((Ser)Sec)/SLA by establishing an immunoassay that was able to identify antibodies directed to conformational epitopes and to investigate the clinical implication of this autoantibody in autoimmune liver disease . By using eukaryotically expressed tRNP((Ser)Sec)/SLA as target in a radioligand assay (RLA), 81 patients with autoimmune liver disease (AILD) (33 type 1 AIH, 31 type 2 AIH, and 17 autoimmune sclerosing cholantitis {ASC}), 147 pathologic, and 56 healthy controls were investigated . RLA results were compared with those obtained using a commercial enzyme-linked immunosorbent assay (ELISA) and immunoblot . Reactivity to tRNP((Ser)Sec)/SLA was present in 58% of patients with type 1 and type 2 AIH, 41% with ASC, but in only 3 pathologic controls . RLA was similarly disease-specific but remarkably more sensitive than ELISA and immunoblot . A prospective study showed that anti-tRNP((Ser)Sec)/SLA-positive patients run a severe clinical course, having worse histology, needing longer to achieve remission, relapsing and requiring liver transplantation or dying more frequently than anti-tRNP((Ser)Sec)/SLA negative patients . Anti-tRNP((Ser)Sec)/SLA production was favored by the possession of DR3 and A1-B8-DR3 in AIH type 1 and ASC, and prevented by the possession of A2 in all 3 types of AILD, particularly in type 2 AIH . In conclusion, anticonformational tRNP((Ser)Sec)/SLA reactivity is frequent in type 1 and type 2 AIH and ASC, defining patients with a worse prognosis.

Gene Expr, 2002, 10(1-2), 59 - 78
The 3' end formation in small RNAs; Perumal K et al.; Small RNAs are a major class of RNAs along with transfer RNAs, ribosomal RNAs, and messenger RNAs . They vary in size from less than 100 nucleotides to several thousand nucleotides and have been identified and characterized both in prokaryotes and eukaryotes . Small RNAs participate in a variety of cellular functions including regulating RNA synthesis, RNA processing, guiding modifications in RNA, and in transport of proteins . Small RNAs are generated by a series of posttranscriptional processing steps following transcription . While RNA 5' end structure, 5' cap formation, and RNA processing mechanisms have been fairly well characterized, the 3' end processing is poorly understood . Recent data point to an emerging theme in small RNAs metabolism in which the 3' end processing is mediated by the exosome, a large multienzyme complex . In addition to removal of nucleotides by the exosome, there is simultaneous rebuilding of the 3' end of some small RNA by adenylation and/or uridylation . This review presents a picture of both degradative and rebuilding reactions operative on the 3' end of some small RNA molecules in prokaryotes and eukaryotes.

Comput Chem, 2002 Feb, 26(3), 195 - 206
Using a Euclid distance discriminant method to find protein coding genes in the yeast genome; Zhang CT et al.; The Euclid distance discriminant method is used to find protein coding genes in the yeast genome, based on the single nucleotide frequencies at three codon positions in the ORFs . The method is extremely simple and may be extended to find genes in prokaryotic genomes or eukaryotic genomes with less introns . Six-fold cross-validation tests have demonstrated that the accuracy of the algorithm is better than 93% . Based on this, it is found that the total number of protein coding genes in the yeast genome is less than or equal to 5579 only, about 3.8-7.0% less than 5800-6000, which is currently widely accepted . The base compositions at three codon positions are analyzed in details using a graphic method . The result shows that the preference codons adopted by yeast genes are of the RGW type, where R, G and W indicate the bases of purine, non-G and A/T, whereas the 'codons' in the intergenic sequences are of the form NNN, where N denotes any base . This fact constitutes the basis of the algorithm to distinguish between coding and non-coding ORFs in the yeast genome . The names of putative non-coding ORFs are listed here in detail.

Adv Protein Chem, 2001, 59, 413 - 29
Clp ATPases and their role in protein unfolding and degradation; Hoskins JR et al.; Although much has been learned about the structure and function of Clp chaperones and their role in proteolysis, the mechanism of protein unfolding catalyzed by Clp ATPases and the mechanism of translocation of the unfolded proteins from Clp ATPases to partner proteases remain unsolved puzzles . However, models in which mechanical force is used to destabilize the structure of the substrate in a processive and directional manner are probable . It also seems likely that when ClpA ATPases are associated with proteases, unfolding is coupled to extrusion of the unfolded protein into the proteolytic cavity . In summary, it is anticipated that the large family of Clp ATPases will accomplish their many important cellular functions by similar mechanisms and what has been learned by studying the prokaryotic members reviewed here will shed a great deal of light on all members of the family.

Mol Cell, 2002 Feb, 9(2), 329 - 39
The sigma(70)-like motif: a eukaryotic RNA binding domain unique to a superfamily of proteins required for ribosome biogenesis; Wehner KA et al.; Little is understood about the role of nucleolar RNA binding proteins in ribosome biogenesis, although there is a clear need for them based on the strict folding requirements of the pre-rRNA . We have identified a superfamily of RNA binding proteins whose members are required for different stages of ribosome biogenesis . The Imp4 superfamily is composed of five individual families (Imp4, Rpf1, Rpf2, Brx1, and Ssf) that all possess the sigma(70)-like motif, a eukaryotic RNA binding domain with prokaryotic origins . The Imp4 superfamily members associate with RNAs that are consistent with their distinct roles in ribosome biogenesis and suggest the mechanisms by which they function.

Genome Biol . 2002;3(2):REVIEWS0003 . Epub 2002 Jan 29.
Exploring prokaryotic diversity in the genomic era; Hugenholtz P; Our understanding of prokaryote biology from study of pure cultures and genome sequencing has been limited by a pronounced sampling bias towards four bacterial phyla - Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes - out of 35 bacterial and 18 archaeal phylum-level lineages . This bias is beginning to be rectified by the use of phylogenetically directed isolation strategies and by directly accessing microbial genomes from environmental samples.

Trends Cell Biol, 2002 Mar, 12(3), 146 - 50
Functional diversity of protein C-termini: more than zipcoding?
Chung JJ, Shikano S, Hanyu Y, Li M.
The carboxylated (C)-terminus of proteins, which includes the single terminal alpha-carboxyl group and preceding residues, is uniquely positioned to serve as a recognition signature for a variety of cell-biological processes, including protein targeting, subcellular anchoring and the static and dynamic formation of macromolecular complexes . The terminal sequence motifs can be processed by posttranslational modifications, thereby providing a means to increase sequence diversity and to regulate interactions . Several classes of protein domains have been identified that are either designed for or are capable of interacting with protein C-termini - these include PDZ and TPR domains . The interactions between these protein domains and various terminal epitopes play an important role in specifying cell-biological functions . The combination of diversity and the plasticity of the chemistry of C-termini provides mechanisms for spatial and temporal specificity that are exploited by a variety of biological processes, ranging from specifying prokaryotic protein degradation to nucleating mammalian neuronal signaling complexes . Understanding the diverse functions of protein C-termini might also provide an important indexing criterion for functional proteomics.

Protein Expr Purif, 2002 Mar, 24(2), 234 - 41
Enhancement of poplar glutaredoxin expression by optimization of the cDNA sequence; Rouhier N et al.; Glutaredoxins are low-molecular-weight oxidore ductases that play an important role in redox regulation in eukaryotic and prokaryotic cells . Because of their low abundance, these proteins are poorly characterized in plants . Furthermore, very poor yields have been obtained with the expression systems prepared so far, and in addition, the recombinant products contain a His-tag which can interfere with the biochemical characterization . In order to obtain more information about those important regulatory proteins in plants, a cDNA coding for an extended glutaredoxin has been introduced into the expression plasmid pET-3d and the resulting construction has been used to transform Escherichia coli strain BL21(DE3) in the presence of plasmid helper pSBET or not . Initially poor or ineffective protein expression has been improved by successively cloning a N-terminus truncated form of the protein, introducing silent mutations both at the 5' and at the 3' ends of the nucleotide sequence, and finally altering the 3' end in order to change the C-terminus amino acid sequence of the protein . The first modifications have allowed us to produce the protein in large amounts but essentially in an insoluble form which could be resolubilized and purified . On the other hand, changing the C-terminus sequence resulted in protein preparations of high purity and in a soluble form . The recombinant proteins were biochemically active and the yield varied between 6 and 14 mg of homogeneous protein per liter of culture .

Rapid Commun Mass Spectrom, 2002, 16(5), 453 - 61
Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry of bacteriochlorophylls from Chlorobiaceae: characteristic fragmentations; Airs RL et al.; Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry/mass spectrometry (APCI-LC/MS/MS) has been applied to the study of bacteriochlorophylls c, d, and e of phototrophic prokaryotes . Cultures of Chlorobiaceae containing bacteriochlorophyll c, d or e were examined using a high-resolution high-performance liquid chromatography (HPLC) method and APCI-LC/MS/MS employing post-column addition of formic acid . The results reveal complex distributions of bacteriochlorophyll homologues, with some closely eluting species giving isobaric protonated molecules . On-line LC/MS/MS studies reveal characteristic fragment ions for bacteriochlorophylls c, d, and e . Fragmentations involving loss of the extended alkyl substituents that are unique to bacteriochlorophylls c, d and e and their derivatives have been rationalised by studying the phaeophorbides and the results applied to the direct study of the bacteriochlorophylls .

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 524 - 5 Epub 2002 Feb 21.
Crystallization and preliminary X-ray characterization of the acylphosphatase-like domain from the Escherichia coli hydrogenase maturation factor HypF; Rosano C et al.; Maturation of prokaryotic hydrogenase involves several protein factors, among which is the accessory protein HypF, which hosts the consensus sequence of acylphosphatases and a sequence motif common to proteins catalyzing O-carbamoylations . The specific functions of HypF are largely unknown, although it has been observed that CN(-) and CO ligands at the hydrogenase Ni,Fe active centre originate from carbamoylphosphate . The HypF N-terminal domain (91 residues, acylphosphatase-like domain) has been crystallized in two different crystal forms belonging to the orthorhombic P2(1)2(1)2(1) space group (unit-cell parameters a = 35.5, b = 59.8, c = 87.6 A) and to the rhombohedral space group R32 (unit-cell parameters a = b = 58.1, c = 155.6 A in the hexagonal setting).

J Biol Chem, 2002 May 3, 277(18), 16167 - 71 Epub 2002 Feb 20.
Direct evidence for nitric oxide production by a nitric-oxide synthase-like protein from Bacillus subtilis; Adak S et al.; Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology . Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown . We therefore cloned, expressed, and purified a NOS-like protein from Bacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions . bsNOS was dimeric, bound l-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite from l-Arg when incubated with NADPH and a mammalian NOS reductase domain . Stopped-flow analysis showed that ferrous bsNOS reacted with O(2) to form a transient heme Fe(II)O(2) species in the presence of either Arg or the reaction intermediate N-hydroxy-l-arginine . In the latter case, disappearance of the Fe(II)O(2) species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS . This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO . NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O(2) intermediate during the reaction . Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS . This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS.

Eur J Biochem, 2002 Feb, 269(4), 1078 - 85
Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division; Chaba R et al.; A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra . Sequencing of the clone indicated 100% identity with the published pknA sequence of M . tuberculosis strain H37Rv . PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of approximately 97 kDa . The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin . In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese-dependent, and sodium orthovanadate can inhibit this activity . Phosphoamino-acid analysis indicated that PknA phosphorylates at serine and threonine residues . PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone . A comparison of the nucleotide-derived amino-acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation . Protein--protein interaction studies revealed that PknA is capable of phosphorylating at least a approximately 56-kDa soluble protein from E . coli . Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E . coli cells, supporting its regulatory role in cell division.

Eur J Biochem, 2002 Feb, 269(4), 1067 - 71
Phosphorylation and dephosphorylation of histidine residues in proteins; Klumpp S et al.; Protein phosphorylation is a key mechanism for intracellular signal transduction in both prokaryotic and eukaryotic cells . Vertebrate proteins are prevalently phosphorylated on side chains that contain a hydroxyl group, such as serine, threonine and tyrosine residues . In the past decade, however, an increasing number of examples of histidine phosphorylation has been described . Because acid treatment of phosphoproteins during purification and detection of phosphoamino acid analysis is routine, O-phosphomonoesters have been studied more often, and the existence of acid-labile phosphates has been largely overlooked . The latter class of N-phosphoamidates may well be more widespread than is generally believed, even though the O-phosphates remain the major class in terms of quantity and extent of distribution in proteins . Phosphohistidine currently is estimated to be 10- to 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine {Matthews, H.R . (1995) Pharmac . Ther . 67, 323-350.} . This minireview briefly summarizes the extensive knowledge of the key mechanisms and functions of phosphohistidine in bacteria . It also describes the still limited, yet increasing, data from homologs of the bacterial two-component system . Finally, novel mechanisms of phosphorylation and dephosphorylation of histidine residues not related to the two-component system are described.

Infect Immun, 2002 Mar, 70(3), 1530 - 7
Interaction of an uuter membrane protein of enterotoxigenic Escherichia coli with cell surface heparan sulfate proteoglycans; Fleckenstein JM et al.; We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic Escherichia coli (ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells . The epithelial cell receptor(s) for Tia has not been identified . Here we show that Tia interacts with cell surface heparan sulfate proteoglycans . Recombinant E . coli expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells . Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia) . Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate . Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia . In addition, we also observed heparin binding to both immobilized rTia and recombinant E . coli expressing Tia . Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide . Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins . These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.

Infect Immun, 2002 Mar, 70(3), 1129 - 35
Site-specific proteolysis of the MALP-404 lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentans; Davis KL et al.; The mature MALP-404 surface lipoprotein of Mycoplasma fermentans comprises a membrane-anchored N-terminal lipid-modified region responsible for macrophage activation (P . F . Muhlradt, M . Kiess, H . Meyer, R . Sussmuth, and G . Jung, J . Exp . Med . 185:1951-1958, 1997) and an external hydrophilic region that contains the selective lipoprotein-associated (SLA) motif defining a family of lipoproteins from diverse but selective prokaryotes, including mycoplasmas (M . J . Calcutt, M . F . Kim, A . B . Karpas, P . F . Muhlradt, and K . S . Wise, Infect . Immun . 67:760-771, 1999) . This family generally corresponds to a computationally defined group of orthologs containing the basic membrane protein (BMP) domain . Two discrete lipid-modified forms of the abundant MALP product which vary dramatically in ratio among isolates of M . fermentans occur on the mycoplasma surface: (i) MALP-404, the full-length mature product, and (ii) MALP-2, the Toll-like receptor 2-mediated macrophage-activating lipopeptide containing the N-terminal 14 residues of the mature lipoprotein . The role of posttranslational processing in the biogenesis of MALP-2 from the prototype MALP-404 SLA-containing lipoprotein was investigated . Detergent phase fractionation of cell-bound products and N-terminal sequencing of a newly discovered released fragment (RF) demonstrated that MALP-404 was subject to site-specific proteolysis between residues 14 and 15 of the mature lipoprotein, resulting in the cell-bound MALP-2 and soluble RF products . This previously unknown mechanism of posttranslational processing among mycoplasmas suggests that specific cleavage of some surface proteins may confer efficient "secretion" of extracellular products by these organisms, with concurrent changes in the surface phenotype . This newly identified form of variation may have significant implications for host adaptation by mycoplasmas, as well as other pathogens expressing lipoproteins of the SLA (BMP) family.

Biochem Pharmacol, 2002 Feb 1, 63(3), 399 - 407
Selective inhibition of the activities of both eukaryotic DNA polymerases and DNA topoisomerases by elenic acid; Mizushina Y et al.; (R)-(-)-Elenic acid (R-2,4-dimethyl-22-(p-hydroxyphenyl)-docos-3(E)-enoic acid) (EA) is a DNA topoisomerase II inhibitor found in an Indonesian sponge, Plakinastrella sp . We found and report here that it is a potent inhibitor of calf DNA polymerase alpha (IC(50)=7.7 microM) and rat DNA polymerase beta (IC(50)=12.9 microM) . EA did not bind to DNA directly . EA did not influence the activities of DNA polymerases such as plant DNA polymerases I and II and prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I, or other DNA metabolic enzymes such as human immunodeficiency virus (HIV) reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I . Interestingly, EA was also an inhibitor of DNA topoisomerases I and II, although the enzymatic characteristics including modes of action, amino acid sequences and three-dimensional structures were markedly different from those of DNA polymerases . EA could prevent the growth of NUGC-3 cancer cells, and the LD(50) value was 22.5 microM . The cells were halted at G1 and G2/M phase in the cell cycle . From these results, the action mode of EA is discussed.

Biochem J, 2002 Mar 1, 362(Pt 2), 395 - 9
Inhibitory effects of acidic phospholipids on the binding of origin-recognition complex to origin DNA; Lee JR et al.; Origin-recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, shares certain biochemical characteristics with DnaA, the initiator of chromosomal DNA replication in prokaryotes . These similarities include origin-specific DNA binding, ATP binding and ATPase activity . DnaA interacts with acidic phospholipids, such as cardiolipin, and its activity is regulated by these phospholipids . In this study, we examined whether Saccharomyces cerevisiae ORC also interacts with phospholipids . Among the various phospholipids tested, ORC was found to bind specifically to cardiolipin . This binding was inhibited by excess concentrations of salts but unaffected by ATP, adenosine 5'-{gamma-thio}triphosphate or the origin DNA . Cardiolipin weakly inhibited the ATP-binding activity of ORC, whereas it strongly inhibited ORC binding to origin DNA . Acidic phospholipids other than cardiolipin (phosphatidylglycerol and phosphatidylinositol) weakly inhibited ORC binding to origin DNA . Furthermore, total phospholipids extracted from yeast nuclear membranes inhibited ORC binding to origin DNA . We consider that phospholipids may modulate initiation of DNA replication in eukaryotes in a similar manner to that found in prokaryotes.

J Steroid Biochem Mol Biol, 2001 Dec, 79(1-5), 289 - 97
A comparative approach to structure-function studies of mammalian aromatases; Conley A et al.; To date, structure--function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450 . Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity . We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes . Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay . Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis . All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function . The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively . In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A . Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B' and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites . Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes . Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism . These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom . These studies are currently in progress.

Int J Biochem Cell Biol, 2002 Mar, 34(3), 298 - 307
A graphic representation of protein sequence and predicting the subcellular locations of prokaryotic proteins; Feng ZP et al.; Zp curve, a three-dimensional space curve representation of protein primary sequence based on the hydrophobicity and charged properties of amino acid residues along the primary sequence is suggested . Relying on the Zp parameters extracted from the three components of the Zp curve and the Bayes discriminant algorithm, the subcellular locations of prokaryotic proteins were predicted . Consequently, an accuracy of 81.5% in the cross-validation test has been achieved using 13 parameters extracted from the curve for the database of 997 prokaryotic proteins . The result is slightly better than that of using the neural network method (80.9%) based on the amino acid composition for the same database . By jointing the amino acid composition and the Zp parameters, the overall predictive accuracy 89.6% can be achieved . It is about 3% higher than that of the Bayes discriminant algorithm based merely on the amino acid composition for the same database . The prediction is also performed with a larger dataset derived from the version 39 SWISS-PROT databank and two datasets with different sequence similarity . Even for the dataset of non-sequence similarity, the improvement can be of 4.4% in the cross-validation test . The results indicate that the Zp parameters are effective in representing the information within a protein primary sequence . The method of extracting information from the primary structure may be useful for other areas of protein studies.

Int J Biochem Cell Biol, 2002 Mar, 34(3), 263 - 8
In vitro interaction of eukaryotic elongation factor 2 with synthetic oligoribonucleotide that mimics GTPase domain of rat 28S ribosomal RNA; He WJ et al.; Eukaryotic elongation factor 2 (eEF2) catalyzed the translocation of peptidyl-tRNA from the ribosomal A site to the P site . In this paper, the interaction between eEF2 and GTD RNA, a synthetic oligoribonucleotide that mimicked the GTPase domain of rat 28S ribosomal RNA, was studied in vitro . The purified eEF2 could bind to GTD RNA, forming a stable complex . Transfer RNA competed with GTD RNA in binding to eEF2, whereas poly(A), poly(U) and poly(I, C) did not interfere with the interaction between eEF2 and GTD RNA, demonstrating that the tertiary structure of RNA might be necessary for the recognition of and binding to eEF2 . The complex formation of eEF2 with GTD RNA was inhibited by SRD RNA, a synthetic oligoribonucleotide mimic of Sarcin/Ricin domain RNA of rat 28S RNA . Similarly, GTD RNA inhibited the interaction between eEF2 and SRD RNA . This fact implies that these small oligoribonucleotides probably share similar recognition or binding identity elements in their tertiary structures . In addition, the binding of eEF2 to GTD RNA could be obviously weakened by the ADP-ribosylation of eEF2 with diphtheria toxin . These results indicate that eEF2 behaves differently from prokaryotic EF-G in binding to ribosomal RNA.

J Photochem Photobiol B, 2002 Feb, 66(1), 73 - 80
Involvement of reactive oxygen species in the UV-B damage to the cyanobacterium Anabaena sp; He YY et al.; Reactive oxygen species (ROS) are involved the damage of living organisms under environmental stress including UV radiation . Cyanobacteria, photoautotrophic prokaryotic organisms, also suffer from increasing UV-B due to the depletion of the stratospheric ozone layer . The increased UV-B induces the production of ROS in vivo detected by using the ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) . Ascorbic acid and N-acetyl-L-cysteine (NAC) scavenged ROS effectively, while alpha-tocopherol acetate or pyrrolidine dithiocarbamate (PDTC) did not . The presence of rose bengal and hypocrellin A increased the ROS level by photodynamic action in the visible light . The presence of the herbicide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), increased ROS production slightly, and ROS formation was greatly enhanced by the addition of methyl viologen due to the fact that this redox system diverts electrons from PSI to oxygen and thus forms ROS . UV-B induces ROS generation by photodynamic action and inhibition of the electron transport by damaging the electron receptors or enzymes associated with the electron transport chain during photosynthesis.

Curr Opin Biotechnol, 2002 Feb, 13(1), 20 - 4
Metabolomics and microarrays for improved understanding of phenotypic characteristics controlled by both genomics and environmental constraints; Phelps TJ et al.; Advances in our understanding of functional genomics are best addressed by integrative studies that include measurements of mRNA, proteins, and low molecular weight metabolites over time and varied conditions . Bioinformatics can then be used to relate this data to the genome . Current technology allows for comprehensive and rapid mRNA expression profiling and mass spectrophotometric measurement of low molecular weight intermediates and metabolic products . In prokaryotic organisms, this combination provides a potentially powerful tool for identifying gene function and regulatory networks even in the absence of a combined proteomic approach.

Mol Microbiol, 2002 Jan, 43(1), 239 - 46
Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control; Moll I et al.; It is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions . However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5'-terminal AUG that seems to be the only necessary and constant element . Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery . Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome-IF2-fMet-tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways . In fact, the faithful translation of leaderless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evolutionarily conserved function of the translational apparatus.

J Appl Microbiol, 2002, 92(1), 134 - 9
mlpB, a gene encoding a new lipoprotein in Myxococcus xanthus; Martinez-Canamero M et al.; AIMS: To search for and study the genes involved in the regulation of phosphate in the soil developmental bacterium Myxococcus xanthus . METHODS AND RESULTS: The mlpB gene encoding a 149 residue polypeptide was identified while screening for genes with products related to phosphate metabolism . The amino terminal 19 residues of MlpB encode a typical prokaryotic signal sequence with a putative lipoprotein cleavage site . CONCLUSIONS: In this study, a new myxobacterial putative lipoprotein is reported . The data suggest that MlpB may be involved in the secretion of phosphate-related proteins . SIGNIFICANCE AND IMPACT OF THE STUDY: Soil bacteria have complex regulatory systems for using inorganic phosphate . This nutrient is limiting in the environment, and has a critical importance for growth and in the initiation of differentiation for developmental bacteria . A number of proteins are involved in all these processes, including membrane lipoproteins, which are being increasingly studied in M . xanthus.

J Mol Evol, 2002 Mar, 54(3), 333 - 45
Evolved RNA secondary structure and the rooting of the universal tree of life; Caetano-Anolles G; The origin and diversification of RNA secondary structure were traced using cladistic methods . Structural components were coded as polarized and ordered multi-state characters, following a model of character state transformation outlined by considerations in statistical mechanics . Several classes of functional RNA were analyzed, including ribosomal RNA (rRNA) . Considerable phylogenetic signal was present in their secondary structure . The intrinsically rooted phylogenies reconstructed from evolved RNA structure depicted those derived from nucleic acid sequence at all taxonomical levels, and grouped organisms in concordance with traditional classification, especially in the archaeal and eukaryal domains . Natural selection appears therefore to operate early in the information flow that originates in sequence and ends in an adapted phenotype . When examining the hierarchical classification of the living world, phylogenetic analysis of secondary structure of the small and large rRNA subunits reconstructed a universal tree of life that branched in three monophyletic groups corresponding to Eucarya, Archaea, and Bacteria, and was rooted in the eukaryotic branch . Ribosomal characters involved in the translational cycle could be easily traced and showed that transfer RNA (tRNA) binding domains in the large rRNA subunit evolved concurrently with the rest of the rRNA molecule . Results suggest it is equally parsimonious to consider that ancestral unicellular eukaryotes or prokaryotes gave rise to all extant life forms and provide a rare insight into the early evolution of nucleic acid and protein biosynthesis.

Protein Sci, 2002 Mar, 11(3), 614 - 24
Crystal structure of a cyanobacterial phytochrome response regulator; Im YJ et al.; The two-component signal transduction pathway widespread in prokaryotes, fungi, molds, and some plants involves an elaborate phosphorelay cascade . Rcp1 is the phosphate receiver module in a two-component system controlling the light response of cyanobacteria Synechocystis sp . via cyanobacterial phytochrome Cph1, which recognizes Rcp1 and transfers its phosphoryl group to an aspartate residue in response to light . Here we describe the crystal structure of Rcp1 refined to a crystallographic R-factor of 18.8% at a resolution of 1.9 A . The structure reveals a tightly associated homodimer with monomers comprised of doubly wound five-stranded parallel beta-sheets forming a single-domain protein homologous with the N-terminal activator domain of other response regulators (e.g., chemotaxis protein CheY) . The three-dimensional structure of Rcp1 appears consistent with the conserved activation mechanism of phosphate receiver proteins, although in this case, the C-terminal half of its regulatory domain, which undergoes structural changes upon phosphorylation, contributes to the dimerization interface . The involvement of the residues undergoing phosphorylation-induced conformational changes at the dimeric interface suggests that dimerization of Rcp1 may be regulated by phosphorylation, which could affect the interaction of Rcp1 with downstream target molecules.

J Mol Biol, 2001 Nov 30, 314(3), 335 - 52
Regulated assembly of transcription factors and control of transcription initiation; Beckett D; Proteins that function in regulation of transcription initiation are typically homo or hetero-oligomeric . Results of recent biophysical studies of transcription regulators indicate that the assembly of these proteins is often subject to regulation . This regulation of assembly dictates the frequency of transcription initiation via its influence on the affinity of a transcription regulator for DNA and its affect on target site selection . Factors that modulate transcription factor assembly include binding of small molecules, post-translational modification, DNA binding and interactions with other proteins . Here, the results of recent structural and/or thermodynamic studies of a number of transcription regulators that are subject to regulated assembly are reviewed . The accumulated data indicate that this phenomenon is ubiquitous and that mechanisms utilized in eukaryotes and prokaryotes share common features .

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 2082 - 7 Epub 2002 Feb 12.
Long-term microclimatic stress causes rapid adaptive radiation of kaiABC clock gene family in a cyanobacterium, Nostoc linckia, from "Evolution Canyons" I and II, Israel; Dvornyk V et al.; Cyanobacteria are the only prokaryotes known thus far possessing regulation of physiological functions with approximate daily periodicity, or circadian rhythms, that are controlled by a cluster of three genes, kaiA, kaiB, and kaiC . Here we demonstrate considerably higher genetic polymorphism and extremely rapid evolution of the kaiABC gene family in a filamentous cyanobacterium, Nostoc linckia, permanently exposed to the acute natural environmental stress in the two microsite evolutionary models known as "Evolution Canyons," I (Mount Carmel) and II (Upper Galilee) in Israel . The family consists of five distinct subfamilies (kaiI-kaiV) comprising at least 20 functional genes and pseudogenes . The obtained data suggest that the duplications of kai genes have adaptive significance, and some of them are evolutionarily quite recent (approximately 80,000 years ago) . The observed patterns of within- and between-subfamily polymorphisms indicate that positive diversifying, balancing, and purifying selections are the principal driving forces of the kai gene family's evolution.

Nucleic Acids Res, 2002 Feb 15, 30(4), 866 - 75
Integration sites for genetic elements in prokaryotic tRNA and tmRNA genes: sublocation preference of integrase subfamilies; Williams KP; Most classical integrases of prokaryotic genetic elements specify integration into tRNA or tmRNA genes . Sequences shared between element and host integration sites suggest that crossover can occur at any of three sublocations within a tRNA gene, two with flanking symmetry (anticodon-loop and T-loop tDNA) and the third at the asymmetric 3' end of the gene . Integrase phylogeny matches this classification: integrase subfamilies use exclusively either the symmetric sublocations or the asymmetric sublocation, although tRNA genes of several different aminoacylation identities may be used within any subfamily . These two familial sublocation preferences imply two modes by which new integration site usage evolves . The tmRNA gene has been adopted as an integration site in both modes, and its distinctive structure imposes some constraints on proposed evolutionary mechanisms.

Structure (Camb), 2002 Jan, 10(1), 69 - 79
Crystal structures of E . coli nicotinate mononucleotide adenylyltransferase and its complex with deamido-NAD; Zhang H et al.; Nicotinamide/Nicotinate mononucleotide (NMN/NaMN) adenylyltransferase is an indispensable enzyme in both de novo biosynthesis and salvage of NAD+ and NADP+ . In prokaryotes, it is absolutely required for cell survival, thus representing an attractive target for the development of new broad-spectrum antibacteria inhibitors . The crystal structures of E . coli NaMN adenylyltransferase (NMNAT) and its complex with deamido-NAD (NaAD) revealed that ligand binding causes large conformational changes in several loop regions around the active site . The enzyme specifically recognizes the deamidated pyridine nucleotide through interactions between nicotinate carboxylate with several protein main chain amides and a positive helix dipole . Comparison of E . coli NMNAT with those from archaeal organisms revealed extensive differences in the active site architecture, enzyme-ligand interaction mode, and bound dinucleotide conformations . The bacterial NaMN adenylyltransferase structures described here provide a foundation for structure-based design of specific inhibitors that may have therapeutic potential.

Structure (Camb), 2002 Jan, 10(1), 8 - 9
Polypeptide release factors in prokaryotes and eukaryotes: same function, different structure; Kisselev L; Although the eukaryotic (eRF1) and prokaryotic (RF2) polypeptide release (translation termination) factors are functionally similar, they turn out to be very different in overall shape and architecture and in the location of key functional elements.

Curr Opin Struct Biol, 2002 Feb, 12(1), 98 - 106
Prokaryotic transcription regulators: more than just the helix-turn-helix motif; Huffman JL et al.; Over the past two years, the structures of many prokaryotic transcriptional regulators have been solved, and several of them have revealed the structural mechanism of gene regulation . The crystal structure of BmrR-TPP-DNA reveals a novel mechanism of transcription activation, whereby the drug-bound protein activates the bmr promoter by local DNA unwinding and base pair disruption . Myristoyl-CoA induces FadR by a three-helix pushing mechanism, whereas TetR employs a helical pendulum motion to regulate expression . The structures of AbrB, and DNA complexes of Rob and MuR unveil a novel DNA-binding motif, 'the looped-hinge helix', and new uses of the helix-turn-helix and winged helix motifs in DNA binding.

Structure (Camb), 2002 Feb, 10(2), 153 - 64
Evidence of intradomain and interdomain flexibility in an OmpR/PhoB homolog from Thermotoga maritima; Buckler DR et al.; Two-component systems, the predominant signal transduction strategy used by prokaryotes, involve phosphorelay from a sensor histidine kinase (HK) to an intracellular response regulator protein (RR) that typically acts as a transcription regulator . RRs are modular proteins, usually composed of a conserved regulatory domain, which functions as a phosphorylation-activated switch, and an attached DNA binding effector domain . The crystal structure of a Thermotoga maritima transcription factor, DrrD, has been determined at 1.5 A resolution, providing the first structural information for a full-length member of the OmpR/PhoB subfamily of RRs . A small interdomain interface occurs between alpha 5 of the regulatory domain and an antiparallel sheet of the effector domain . The lack of an extensive interface in the unphosphorylated protein distinguishes DrrD from other structurally characterized multidomain RRs and suggests a different mode of interdomain regulation.

Curr Biol, 2002 Feb 5, 12(3), 199 - 203
Archaeal guide RNAs function in rRNA modification in the eukaryotic nucleus; Speckmann WA et al.; In eukaryotes, many Box C/D small nucleolar RNAs base pair with ribosomal RNA through short complementary guide sequences, thereby marking up to 100 individual nucleotides of ribosomal RNA for 2'-O-methylation . Function of the eukaryotic Box C/D RNAs depends upon interaction with at least six known proteins . Box C/D RNAs are not known to exist in Bacteria but were recently identified in Archaea by biochemical analysis and computational genomic screens and have likely evolved independently in Archaea and Eukarya for more than 2000 million years . We have microinjected Box C/D RNAs from Pyrococcus furiosus, a hyperthermophilic archaeon, into the nuclei of oocytes from the aquatic frog Xenopus laevis . Our results show that Box C/D RNAs derived from this prokaryote are retained in the nucleus, localize to nucleoli, and interact with the X . laevis Box C/D RNA binding proteins fibrillarin, Nop56, and Nop58 . Furthermore, we have demonstrated the ability of archaeal Box C/D RNAs to direct site-specific 2'-O-methylation of ribosomal RNA . Our studies have revealed the remarkable ability of archaeal Box C/D RNAs to assemble into functional RNA-protein complexes in the eukaryotic nucleus.

J Virol, 2002 Mar, 76(5), 2543 - 7
Identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle; Mohan KV et al.; Peroxisomes are unimembrane, respiratory organelles of the cell . Transport of cellular proteins to the peroxisomal matrix requires a type 1 peroxisomal targeting signal (PTS1) which essentially constitutes a tripeptide from the consensus sequence S/T/A/G/C/N-K/R/H-L/I/V/M/A/F/Y . Although PTS-containing proteins have been identified in eukaryotes, prokaryotes, and parasites, viral proteins with such signals have not been identified so far . We report here the first instance of a virus, the rotavirus, which causes infantile diarrhea worldwide, containing a functional C-terminal PTS1 in one of its proteins (VP4) . Analysis of 153 rotavirus VP4-deduced amino acid sequences identified five groups of conserved C-terminal PTS1 tripeptide sequences (SKL, CKL, GKL, CRL, and CRI), of which CRL is represented in approximately 62% of the sequences . Infection of cells by a CRL-containing representative rotavirus (SA11 strain) and confocal immunofluorescence analysis revealed colocalization of VP4 with peroxisomal markers and morphological changes of peroxisomes . Further, transient cellular expression of green fluorescent protein (GFP)-fused VP4CRL resulted in transport of VP4 to peroxisomes, whereas the chimera lacking the PTS1 signal, GFP-VP4DeltaCRL, resulted in diffuse cytoplasmic staining, suggesting a CRL-dependent targeting of the protein . The present study therefore demonstrates hitherto unreported organelle involvement, specifically of the peroxisomes, in rotaviral infections as demonstrated by using the SA11 strain of rotavirus and opens a new line of investigation toward understanding viral pathogenesis and disease mechanisms.

Genesis, 2002 Jan, 32(1), 19 - 26
Codon-improved Cre recombinase (iCre) expression in the mouse; Shimshek DR et al.; By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines . The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals . Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre . Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice . However, Cre induction after administration of tamoxifen yielded only low Cre activity . Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse .

Proteins, 2002 Mar 1, 46(4), 355 - 67
Classification of the caspase-hemoglobinase fold: detection of new families and implications for the origin of the eukaryotic separins; Aravind L et al.; A comprehensive sequence and structural comparative analysis of the caspase-hemoglobinase protein fold resulted in the delineation of the minimal structural core of the protease domain and the identification of numerous, previously undetected members, including a new protease family typified by the HetF protein from the cyanobacterium Nostoc . The first bacterial homologs of legumains and hemoglobinases were also identified . Most proteins containing this fold are known or predicted to be active proteases, but multiple, independent inactivations were noticed in nearly all lineages . Together with the tendency of caspase-related proteases to form intramolecular or intermolecular dimers, this suggests a widespread regulatory role for the inactive forms . A classification of the caspase-hemoglobinase fold was developed to reflect the inferred evolutionary relationships between the constituent protein families . Proteins containing this domain were so far detected almost exclusively in bacteria and eukaryotes . This analysis indicates that caspase-hemoglobinase-fold proteases and their inactivated derivatives are widespread in diverse bacteria, particularly those with a complex development, such as Streptomyces, Anabaena, Mesorhizobium, and Myxococcus . The eukaryotic separin family was shown to be most closely related to the mainly prokaryotic HetF family . The phyletic patterns and evolutionary relationships between these proteins suggest that they probably were acquired by eukaryotes from bacteria during the primary, promitochondrial endosymbiosis . A similar scenario, supported by phylogenetic analysis, seems to apply to metacaspases and paracaspases, with the latter, perhaps, being acquired in an independent horizontal transfer to the eukaryotes . The acquisition of the caspase-hemoglobinase-fold domains by eukaryotes might have been critical in the evolution of important eukaryotic processes, such as mitosis and programmed cell death .

Life Sci, 2001 Dec 28, 70(6), 659 - 67
Localization of metallothionein I-II immunoreactivity in bovine pituitary gland; Zatt P et al.; Metallothioneins belong to a family of shock proteins characterized by an unusual high content of cystein, absence of aromatic amino acids and high metal content (Zinc and Copper) . Metallothioneins are ubiquitously present in a large variety of prokaryotic and eukaryotic species as well as in all mammalian organs and tissues examined thus far . To the best of our knowledge this is the first report describing the presence of metallothioneins in the pituitary gland . Metallothioneins were identified immunohistochemically and chromatographically both in the neuro and adenohypophysis of the bovine pituitary gland . Metallothioneins are highly expressed in the neurohypophyseal glial cells, and in a subpopulation of folliculo-stellate cells located in the pars intermedia of the adenohypophysis . While the specific role of these proteins in the pituitary gland remains to be established, we hypothesize that, besides their protective action against free radicals, hypophyseal metallothioneins might be involved in the regulation of metal ion homeostasis with putative implication in release of hypothalamic peptide hormones in the neurohypophysis and synthesis/release of alpha-MSH by POMC-cells located in the pars intermedia of the adenohypophysis.

J Biol Chem, 2002 Apr 19, 277(16), 14321 - 8 Epub 2002 Feb 06.
A dynamic RecA filament permits DNA polymerase-catalyzed extension of the invading strand in recombination intermediates; Xu L et al.; Recombination-dependent replication is an essential housekeeping function in prokaryotes and eukaryotes, serving, for example, to restart DNA replication after the repair of a double-strand break . Little is known about the interplay between the recombination and replication machinery when recombination intermediates are used as substrates for DNA replication . We show here that recombination intermediates formed between linear duplex and supercoiled plasmid DNAs are substrates for a generalized strand displacement DNA synthesis reaction in which the 3'-OH of the invading strand in the recombination intermediate is used as a primer . DNA synthesis is driven by negative superhelicity and is inhibited if disassembly of the RecA filament is prevented . Thus, assembly and disassembly of RecA filaments in the same direction facilitates filament clearance from the 3'-end of the invading strand, allowing DNA synthesis to occur from recombination intermediates.

Trends Plant Sci, 2002 Feb, 7(2), 72 - 7
Protein import into cyanelles; Steiner JM et al.; "Cyanelles" are peptidoglycan-armored plastids of glaucocystophyte algae with close morphological and biochemical resemblance to endosymbiotic cyanobacteria . Genome sequencing and phylogenetic analysis have placed cyanelles on the earliest branch of phototrophic eukaryotes after the singular primary endosymbiotic event, the closest relatives to cyanobacteria among extant plastids . This model is supported by similar mechanisms for the targeting of nucleus-encoded cyanelle and chloroplast precursor proteins whose genes were transferred to the nucleus after the endosymbiotic event . As in chloroplasts, a prokaryote-type Sec preprotein translocase is shown to operate in cyanelle thylakoid membranes.

Folia Microbiol (Praha), 2001, 46(4), 292 - 6
Use of yeast two-hybrid system for detection of Bacillus subtilis FtsZ protein partners; Prepiak P et al.; Yeast two-hybrid system was modified to allow easy detection of prokaryotic protein-protein interactions . Three plasmids (pGBR1, pGBR2, pGBR3) with the ClaI restriction site shifted in the three possible reading frames in fusion with GAL4 activating domain were constructed . The modified plasmids were used for identification of protein partners of FtsZ from Bacillus subtilis . Among partners of FtsZ the FtsA protein and a globular part of the SpoIIE protein were identified . The protein interactions were quantified by measurements of beta-galactosidase activity in yeast cells using 4-methylumbelliferyl beta-D-galactopyranoside as fluorogenic substrate.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1831 - 5 Epub 2002 Feb 05.
Solution structure and stability of the anti-sigma factor AsiA: implications for novel functions; Urbauer JL et al.; Anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors . The bacteriophage T4 anti-sigma factor AsiA is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of Escherichia coli, sigma(70) . AsiA is an all-helical, symmetric dimer in solution . The solution structure of the AsiA dimer reveals a novel helical fold for the protomer . Furthermore, the AsiA protomer, surprisingly, contains a helix-turn-helix DNA binding motif, predicting a potential new role for AsiA . The AsiA dimer interface includes a substantial hydrophobic component, and results of hydrogen/deuterium exchange studies suggest that the dimer interface is the most stable region of the AsiA dimer . In addition, the residues that form the dimer interface are those that are involved in binding to sigma(70) . The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to sigma(70), where an AsiA protomer is displaced from the dimer via the interaction of sigma(70) with the same residues in AsiA that constitute the dimer interface.

Chembiochem, 2001 Sep 3, 2(9), 612 - 27
A biosynthetic classification of fungal and streptomycete fused-ring aromatic polyketides; Thomas R; Polyketides are one of the largest and most structurally diverse classes of naturally occurring compounds, ranging from simple aromatic metabolites to complex macrocyclic lactones . Fungi and filamentous bacteria, particularly the actinomycetes, are major sources of polycyclic aromatic structures, which include many clinically important antibiotics and other useful metabolites . These fused-ring polyketides are formed by the action of polyketide synthases (PKSs), which catalyse the assembly, folding and cross-linking of poly-beta-ketoacyl intermediates . In view of the taxonomic gulf between the eukaryotic fungi and prokaryotic bacteria, it is not surprising that they are rarely found to produce structurally identical fused-ring metabolites . A review of {(13)C(2)}acetate incorporation data has revealed consistent differences in the reported cyclisation patterns, which require regiospecifically distinct cross-linking of otherwise identical linear polyketide precursors . This observation provides the basis for a structural and biosynthetic classification of microbial fused-ring polyketides, which has a number of useful ramifications.

Trends Microbiol, 2002 Feb, 10(2), 87 - 93
The functions of Ca(2+) in bacteria: a role for EF-hand proteins?
Michiels J, Xi C, Verhaert J, Vanderleyden J.
In bacteria, Ca(2+) is implicated in a wide variety of cellular processes, including the cell cycle and cell division . Dedicated influx and efflux systems tightly control the low cytoplasmic Ca(2+) levels in prokaryotes . Additionally, the growing number of proteins containing various Ca(2+)-binding motifs supports the importance of Ca(2+), which controls various protein functions by affecting protein stability, enzymatic activity or signal transduction . The existence of calmodulin-like proteins (containing EF-hand motifs) in bacteria is a long-standing hypothesis . Analysis of the prokaryotic protein sequences available in the databases has revealed the presence of several calmodulin-like proteins containing two or more authentic EF-hand motifs, suggesting that calmodulin-like proteins could be involved in Ca(2+) regulation in bacteria.

FEMS Immunol Med Microbiol, 2002 Jan 14, 32(2), 97 - 103
Mycoplasma bovis induces apoptosis of bovine lymphocytes; Vanden Bush TJ et al.; We report Mycoplasma bovis induces apoptotic death of bovine lymphocytes . Using flow cytometry analyzed propidium iodide inclusion we observed a loss in viable lymphocytes upon incubation of freshly isolated bovine PBMCs with M . bovis . The use of annexin V staining as well as TUNEL assays corroborated these findings . In addition, these assays indicated that the M . bovis induced lymphocyte death is apoptotic in nature . Subsequent experiments demonstrated that the prokaryotic protein production inhibitor chloramphenicol inhibited lymphocyte death induced by M . bovis, showing that M . bovis protein production is necessary for the induction of lymphocyte death, and that the death is not dependent upon the addition of apoptotic inducers as shown with other mycoplasmas . We also show that M . bovis is different from other bovine mycoplasmas (both pathogenic and non-pathogenic) with regards to this characteristic.

J Biochem (Tokyo), 2002 Feb, 131(2), 267 - 75
Real-time kinetic analyses of the interaction of ricin toxin A-chain with ribosomes prove a conformational change involved in complex formation; Honjo E et al.; Ricin toxin A-chain (RTA), a ribosome-inactivating protein from seeds of the castor bean plant (Ricinus communis), inactivates eukaryotic ribosomes by hydrolyzing the N-glycosidic bond of a single adenosine residue in a highly conserved loop of 28S rRNA, but does not act on prokaryotic ribosomes . We investigated the interaction of rat liver 80S ribosomes with RTA using an optical biosensor based on surface plasmon resonance (BIAcore instrument), which allows real-time recording of the interaction . RTA was coupled to the dextran gel matrix on the sensor chip surface through a single thiol group that is not involved in the enzymatic action . The interaction of rat ribosomes with RTA, which was greatly affected by the Mg(2+) concentration and ionic strength, was usually measured at 5 mM Mg(2+), 50 mM KCl, and pH 7.5 . The modes of interaction of intact and RTA-depurinated rat liver ribosomes with the immobilized RTA were virtually the same, while no considerable interaction was observed for Escherichia coli ribosomes . The interaction was not influenced by the presence of 5 mM adenine, which is higher than the reported dissociation constant (1 mM) for the adenine-RTA complex . These results demonstrate that binding of the target adenine with the active site of RTA does not contribute much to the total interaction of ribosomes and RTA . Global analyses of association and dissociation data with several binding models, taking account of mass transport, allowed us to conclude that the data were unable to fit a simple 1:1 binding model, but were best described by a model including a conformational change involved in high affinity complex formation.

World J Gastroenterol, 2001 Oct, 7(5), 642 - 6
Prokaryotical expression of structural and non-structural proteins of hepatitis G virus; Xia NS et al.; AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents . METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli . Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins . RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting . One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting . No obvious expression was found in the other six clones . All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form . Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively . Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera . Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification . CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins . A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

In Silico Biol, 2002, 2(1), 1 - 4
EXProt--a database for EXPerimentally verified Protein functions; Ursing BM et al.; EXProt (database for EXPerimentally verified Protein functions) is a new non-redundant database containing protein sequences for which the function has been experimentally verified . It is a selection of 3976 entries from the Prokaryotes section of the EMBL Nucleotide Sequence Database, Release 66, and 375 entries from the Pseudomonas Community Annotation Project (PseudoCAP) . The entries in EXProt all have a unique ID number and provide information about the organism, protein sequence, functional annotation, link to entry in original database, and if known, gene name and link to references in PubMed/Medline . The EXProt web page provides further details of the database and a link to a BLAST search (blastp & blastx) of the database . The EXProt entries are indexed in SRS and can be searched by means of keywords . Authors can be reached by email (exprot(cmbi.kun.nl).

Aviakosm Ekolog Med, 2000, 34(5), 65 - 71
{Development of approaches to studying the structural and functional organization of protein molecules aboard the International space station}; Kalinin IuT et al.; To solve the problems of biological safety of the cosmonaut on long-duration space mission and prediction of changes in macroorganism as a whole and constituting protein molecules under these conditions, it is important to study the influence of spaceflight factors (SFF) on microorganisms-carriers of modeled structures, protein molecules and pro- and eukaryotic genes in particular . Within the framework of scientific cooperation NAUKA-NASA, the authors proposed a model system of prokaryotic producers of pro- and eukaryotic proteins--staphylococcus alpha-toxin (SAT)--as a key protein in pathogenesis of staphylococcal infections, and human leukocytic interferon (HuIFN-alpha) as one of the homeostasis regulating proteins with well-studied structural and functional properties . Recombinant strains of E . coli with either a single or duplicated HuIFN-alpha 2b gene or other genes of the HuIFN-alpha family: HuIFN-alpha 8a, HuIFN-alpha 10a and HuIFN-alpha 14a were selected as producers of SAT and HuIFN-alpha . This biotechnologic system allows imitation and assessment of the SFF mutagenic effect both at the levels of genome of strain-carrier and gene-insertion and expressed CAT and HuIFN-alpha molecules including transcription, translation, assembly and post-translatory modifications of the target-protein . The developed methodology allows determination of highly mutable and conservative regions in the primary structure of a hypothetical protein associated with its functional activity, prediction of specific amino acid substitutes in these regions, and comparison of test calculations with a pool of natural mutations in the family of proteins under study . The structural/functional analysis of proteins and HuIFN-alpha genes made it possible to isolate and systematize functionally significant areas in the structure of hypothetical protein HuUFN-alpha, on the basis of which the most probable amino acid substitutions were prognosticated . This will present a possibility to identify expectable mutation events in HuIFN-alpha proteins, which so far have not been found in natural genes of the human interferon . Comparison of results of SFF modeling and space experiments aboard the International Space Station with monitoring of HuIFN-alpha mutant forms will help estimation of the extent of influence of the spaceflight factors on evolution of protein molecules.

Gene, 2002 Jan 9, 282(1-2), 19 - 31
SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus alpha-amylase; Schlatter S et al.; The Bacillus stearothermophilus alpha-amylase (amyS) is a heat-stable monomeric exoenzyme which catalyses random hydrolysis of 1,4-alpha-glucosidic linkages in polyglucosans . The Bacillus alpha-amylase was engineered for use as an intracellular (AmyS(Delta S)) as well as a secreted reporter protein (SAMY; secreted alpha-amylase) in mammalian cells . The 5' end of amyS containing the prokaryotic secretion signal was either deleted (amyS(Delta S)) or replaced by a murine immunoglobulin secretion signal . SAMY was cloned under control of the cytomegalovirus promoter (P(CMV)) in a mammalian expression vector or the promoter of the human elongation factor 1 alpha (P(EF1 alpha)) in a lentiviral expression context . A variety of mammalian and human cell lines growing as monolayers, in suspension or as three-dimensional spheroids were transfected/transduced with SAMY- or amyS(Delta S)-encoding expression/lentiviral vectors and alpha-amylase activity was measured in cell lysates and culture supernatants . These experiments showed that SAMY and AmyS(Delta S) were either secreted or remained intracellular as highly sensitive reporter enzymes . SAMY expression and detection was fully compatible with established SEAP (human secreted alkaline phosphatase) and u-PA(LMW) (low molecular weight urokinase-type plasminogen activator) reporter systems and could be used to quantify expression of up to three independent genes in one culture supernatant.

Biochemistry, 2002 Feb 5, 41(5), 1602 - 8
A vibrational spectroscopic investigation of interactions of agonists with GluR0, a prokaryotic glutamate receptor; Cheng Q et al.; We have used Fourier transform infrared spectroscopy to provide a detailed picture of the interactions between the carboxylate groups of the ligands, glutamate, serine, and glutamine, with the ligand-binding domain of a prokaryotic ionotropic glutamate receptor (GluR0) . The vibrational spectra indicate that the noncovalent interactions between the 1C(alpha)-carboxylate moiety of the ligand and the protein are stronger for glutamate than for serine and glutamine . These results correlate well with the higher affinity of glutamate for GluR0-S1S2 relative to the affinities of serine and glutamine . In addition, all three ligands induce similar changes in the vibrational spectra and intrinsic fluorescence of the protein, which indicates that all three ligands induce the same structural changes in the protein . These results are consistent with the recent crystal structures of the glutamate and serine bound forms of GluR0-S1S2 and in addition provide insights into the structure of the glutamine bound form of the protein.

J Cell Biochem, 2002, 84(3), 484 - 96
Multiple collagen I gene regulatory elements have sites of stress-induced DNA duplex destabilization and nuclear scaffold/matrix association potential; Mielke C et al.; The availability of the complete nucleotide sequences of numerous prokaryotic and eukaryotic organisms should stimulate the development and application of computer-based approaches for studying genome organization and function . Earlier work has shown that distinct regulatory DNA elements can be identified by computational analysis as sites of stress-induced DNA duplex destabilization (SIDD) . Here we report the results of computational and experimental analyses of previously identified regulatory elements in the murine alpha1(I) collagen (Col1a1) gene domain . We found that several distal 5' DNase I-hypersensitive sites (HSs) which function in the chromatin loop organization of the Col1a1 gene are characterized by strongly destabilized SIDD profiles . Elements in the proximal 5' promoter and first intron which differentially regulate Col1a1 promoter activity in different collagen-producing cell types also contain SIDD sites . All 5' elements associated with destabilized sites are shown to have nuclear matrix binding activity in an in vitro binding assay . Other putative regulatory elements in the transcribed and 3'-flanking regions of the Col1a1 gene, including both of its polyadenylation sites, are also associated with SIDD peaks . The human COL1A1 gene has periodic SIDD peaks within the transcribed region, suggesting that abundantly expressed genes may require SIDDs acting as topological sinks during transcription . The 5' ends of the murine Col1a1 and the homologous human gene revealed similar SIDD profiles, but limited DNA sequence similarity, indicating that some DNA functions are evolutionarily conserved by preserving higher order DNA structural properties rather than nucleotide sequence . Our results show that destabilized SIDD profiles are a common feature of eukaryotic regulatory DNA elements with such diverse functions as chromatin organization, cell-specific transcriptional enhancement, and initiation and termination of transcription . They demonstrate the usefulness of computational analyses that predict SIDD properties in reliably identifying DNA elements involved in the structural organization of the eukaryotic genome and the regulation of its expression .

Nucleic Acids Res, 2001 Dec 15, 29(24), 5058 - 66
Flavones inhibit the hexameric replicative helicase RepA; Xu H et al.; Helicases couple the hydrolysis of nucleoside triphosphates (NTPs) to the unwinding of double-stranded nucleic acids and are essential in DNA metabolism . Thus far, no inhibitors are known for helicases except heliquinomycin isolated from Streptomyces sp . As the three-dimensional structure of the hexameric replicative DNA helicase RepA encoded by the broad host-range plasmid RSF1010 is known, this protein served as a model helicase to search for inhibitory compounds . The commercially available flavone derivatives luteolin, morin, myricetin and dimyricetin (an oxidation product of myricetin) inhibited the ATPase and double-stranded DNA unwinding activities of RepA . Dimyricetin was the most effective inhibitor for both activities . Single-stranded DNA-dependent RepA ATPase activity is inhibited non-competitively by all four compounds . This finding contrasts the inhibition of phosphoinositide 3-kinase by flavones that fit into the ATP binding pocket of this enzyme . Myricetin also inhibited the growth of a Gram-positive and a Gram-negative bacterial species . As we found other hexameric and non-hexameric prokaryotic helicases to be differentially sensitive to myricetin, flavones may provide substructures for the design of molecules helpful for unraveling the mechanism of helicase action and of novel pharmacologically useful molecules.

Int J Parasitol, 2002 Feb, 32(2), 187 - 97
Molecular cloning and analysis of the Cryptosporidium parvum aminopeptidase N gene; Padda RS et al.; Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro . Additionally, proteases have been implicated in the processing of parasite adhesion molecules found on the surface of sporozoites and merozoites . In this study, we cloned and expressed the C . parvum aminopeptidase N gene by screening a large insert, P1 artificial chromosome library with a probe identified from a Cryptosporidium genome survey-sequencing project . Analysis of the predicted protein encoded by the 2.3 kb gene demonstrated a high degree of homology with prokaryotic and eukaryotic aminopeptidases . The 783 amino acid sequence predicted a M(r) of approximately 89,000 . The active site sequence was found to be highly conserved when compared with other Apicomplexan aminopeptidases . Motifs commonly found in aminopeptidases of this class and a unique single Arg-Gly-Asp (RGD) tripeptide motif predictive of cell adhesion were identified . The aminopeptidase N mRNA was expressed in infective sporozoites and during the infection of human HCT-8 enterocytes as revealed by reverse transcription PCR.

Zhonghua Kou Qiang Yi Xue Za Zhi, 2001 Jan, 36(1), 45 - 7
{Expression of rat amelogenin gene in Escherichia coli}; Sui W et al.; OBJECTIVE: To detect the expression of amelogenin (Am) gene in Escherichia coli (E . coli) . METHODS: The cDNA fragmain of Am gene was obtained with EcoRI and Xhol I from the plasmid PUC18/Am . The fragment was inserted into prokaryotic gene fusion vector PGEX-4T-2 and an expression plasmid PGEX-4T-2/Am was constructed . PGEX-4T-2/Am was induced by IPTG for 4 h . RESULTS: 15% SDS-PAGE revealed a new forein protein band near 27,000 . CONCLUSION: The constructed plasmid expresses Am gene in E . coli.

J Mol Biol, 2002 Jan 25, 315(4), 687 - 97
The structure of Escherichia coli cytosine deaminase; Ireton GC et al.; Cytosine deaminase (CD) catalyzes the deamination of cytosine, producing uracil . This enzyme is present in prokaryotes and fungi (but not multicellular eukaryotes) and is an important member of the pyrimidine salvage pathway in those organisms . The same enzyme also catalyzes the conversion of 5-fluorocytosine to 5-fluorouracil; this activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor . The enzyme is of widespread interest both for antimicrobial drug design and for gene therapy applications against tumors . The structure of Escherichia coli CD has been determined in the presence and absence of a bound mechanism-based inhibitor . The enzyme forms an (alphabeta)(8) barrel structure with structural similarity to adenosine deaminase, a relationship that is undetectable at the sequence level, and no similarity to bacterial cytidine deaminase . The enzyme is packed into a hexameric assembly stabilized by a unique domain-swapping interaction between enzyme subunits . The active site is located in the mouth of the enzyme barrel and contains a bound iron ion that coordinates a hydroxyl nucleophile . Substrate binding involves a significant conformational change that sequesters the reaction complex from solvent .

Mol Genet Genomics, 2001 Dec, 266(4), 527 - 36 Epub 2001 Oct 10.
The Aspergillus niger cypA gene encodes a cyclophilin that mediates sensitivity to the immunosuppressant cyclosporin A; Derkx PM et al.; Here we report the cloning and characterization of a gene, cypA, from Aspergillus niger that encodes a peptidyl prolyl cis-trans isomerase (PPIase) belonging to the cyclophilin family . Sequencing of both genomic and cDNA clones revealed two ORFs in cypA, one encoding a 19-kDa protein of 174 amino acid residues and the other a 24-kDa protein of 219 amino acid residues, with overall identities of 27-77% to the homologous cyclophilins from prokaryotic and eukaryotic organisms . Expression of the 19-kDa CYPA-(His)(6) in E . coli shows that the purified protein has PPIase activity which is inhibited by cyclosporin A . Northern analysis shows two specific cypA transcripts, the smaller transcript encodes the cytosolic 19-kDa CYPA protein, the larger transcript encodes the putative mitochondrial 24 kDa CYPA protein . The transcript for the cytosolic CYPA is expressed at a higher basal level than that for the mitochondrial protein . The presence of tunicamycin, DTT or cyclosporin A in the medium does not affect the expression level of cypA . Its expression is however slightly induced by heat shock . Growing A . niger mycelium in the presence of cyclosporin A leads to an increase in hyphal branching prior to growth arrest . Overexpression of cypA under the control of its own promoter in A . niger results in increased sensitivity to cyclosporin A, suggesting that cypA encodes the cellular target for cyclosporin A in A . niger.

Nucleic Acids Res . 2002 Feb 1;30(3):E14.
Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro; Larson ED et al.; The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes . Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult . We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick . The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide . Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick . Mismatch correction in either strand of a prototype G.T mismatch was achieved by placing a nick 10-40 bp away from the targeted base . This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein . All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.

Nucleic Acids Res, 2002 Feb 1, 30(3), 636 - 42
Action of prokaryotic enhancer over a distance does not require continued presence of promoter-bound sigma54 subunit; Bondarenko V et al.; The mechanism by which an enhancer activates transcription over large distances has been investigated . Activation of the glnAp2 promoter by the NtrC-dependent enhancer in Escherichia coli was analyzed using a purified system supporting multiple-round transcription in vitro . Our results suggest that the enhancer-promoter interaction and the initiation complex must be formed de novo during every round of transcription . No protein remained bound to the promoter after RNA polymerase escaped into elongation . Furthermore, the rate of initiation during the first and subsequent rounds of transcription were very similar, suggesting that there was no functional 'memory' facilitating multiple rounds of transcription . These studies exclude the hypothesis that enhancer action during multiple-round transcription involves the memory of the initial activation event.

Int J Biochem Cell Biol, 2002 Feb, 34(2), 116 - 29
Ribonuclease III: new sense from nuisance; Conrad C et al.; RNases play an important role in the processing of precursor RNAs, creating the mature, functional RNAs . The ribonuclease III family currently is one of the most interesting families of endoribonucleases . Surprisingly, RNase III is involved in the maturation of almost every class of prokaryotic and eukaryotic RNA . We present an overview of the various substrates and their processing . RNase III contains one of the most prominent protein domains used in RNA-protein recognition, the double-stranded RNA binding domain (dsRBD) . Progress in the understanding of this domain is summarized . Furthermore, RNase III only recently emerged as a key player in the new exciting biological field of RNA silencing, or RNA interference . The eukaryotic RNase III homologues which are likely involved in this process are compared with the other members of the RNase III family.

In Silico Biol, 2002, 2(1), 19 - 33
Transmembrane topology prediction methods: a re-assessment and improvement by a consensus method using a dataset of experimentally-characterized transmembrane topologies; Ikeda M et al.; We selected 10 transmembrane (TM) prediction methods (KKD, TMpred, TopPred II, DAS, TMAP, MEMSAT 2, SOSUI, PRED-TMR2, TMHMM 2.0 and HMMTOP 2.0) and re-assessed its prediction performance using a reliable dataset with 122 entries of experimentally-characterized TM topologies . Then, we improved prediction performance by a consensus prediction method . Prediction performance during re-assessment and consensus prediction were based on four attributes: (i) the number of transmembrane segments (TMSs), (ii) the number of TMSs plus TMS-position, (iii) N-tail location and (iv) TM topology . We noted that hidden Markov model-based methods dominate over other methods by individual prediction performance for all four attributes . In addition, all top-performing methods generally were model-based . Among prokaryotic sequences, HMMTOP 2.0 solely topped among other methods with prediction accuracies ranging from 64% to 86% across all attributes . However, among eukaryotic sequences, prediction performance for all the attributes was relatively poor compared with prokaryotic ones . On the other hand, our results showed that our proposed consensus prediction method significantly improved prediction performance by, at least, an additional nine percentage points particularly among prokaryotic sequences for the number of TMS (84%), number of TMS and position (80%), and TM topology attributes (74%) . Although our consensus prediction method improved also the prediction performance among eukaryotic sequences, the obtained accuracies for all attributes were relatively lower than that obtained by prokaryotic counterparts particularly for TM topology.

J Am Acad Dermatol, 2002 Feb, 46(2), 228 - 41
Tacrolimus and pimecrolimus: from clever prokaryotes to inhibiting calcineurin and treating atopic dermatitis; Nghiem P et al.; Tacrolimus ointment, a topical inhibitor of the phosphatase calcineurin, has recently been approved in the United States for use in the treatment of atopic dermatitis . It is the first topical immune suppressant that is not one of the hydrocortisone derivatives, important allies in dermatology for nearly 50 years . Although tacrolimus is less able to penetrate thick skin than glucocorticoids, it does not cause dermal atrophy, an important advantage over the hydrocortisone class . Pimecrolimus (ASM 981), a newer calcineurin inhibitor closely related to tacrolimus, is also being developed for atopic dermatitis therapy . Pimecrolimus has an altered skin penetration profile but the same mechanism of action as tacrolimus . In this review we chronicle the discovery of the calcineurin inhibitors, their presumed evolutionary role as a bacterial "smart bomb" against fungi, molecular and cellular mechanisms of action in the immune system, systemic and topical side effects, efficacy in atopic dermatitis, and future applications within the specialty of dermatology . Particular attention is given to the issues of systemic absorption of tacrolimus, the conditions in which absorption can become a concern, efficacy relative to glucocorticoids, and the choice of 0.03% or 0.1% tacrolimus ointment for use in adults and children.

Genome Biol . 2002;3(1):RESEARCH0005 . Epub 2001 Dec 14.
Assessment of the relationship between signal intensities and transcript concentration for Affymetrix GeneChip arrays; Chudin E et al.; BACKGROUND: Affymetrix microarrays have become increasingly popular in gene-expression studies; however, limitations of the technology have not been well established for commercially available arrays . The hybridization signal has been shown to be proportional to actual transcript concentration for specialized arrays containing hundreds of distinct probe pairs per gene . Additionally, the technology has been described as capable of distinguishing concentration levels within a factor of 2, and of detecting transcript frequencies as low as 1 in 2,000,000 . Using commercially available arrays, we assessed these representations directly through a series of 'spike-in' hybridizations involving four prokaryotic transcripts in the absence and presence of fixed eukaryotic background . The contribution of probe-target interactions to the mismatch signal was quantified under various analyte concentrations . RESULTS: A linear relationship between transcript abundance and signal was consistently observed between 1 pM and 10 pM transcripts . The signal ceased to be linear above the 10 pM level and commenced saturating around the 100 pM level . The 0.1 pM transcripts were virtually undetectable in the presence of eukaryotic background . Our measurements show that preponderance of the signal for mismatch probes derives from interactions with the target transcripts . CONCLUSIONS: Landmark studies outlining an observed linear relationship between signal and transcript concentration were carried out under highly specialized conditions and may not extend to commercially available arrays under routine operating conditions . Additionally, alternative metrics that are not based on the difference in the signal of members of a probe pair may further improve the quantitative utility of the Affymetrix GeneChip array.

J Med Chem, 2002 Jan 31, 45(3), 663 - 9
Correlation of the antibacterial activities of cationic peptide antibiotics and cationic steroid antibiotics; Ding B et al.; The antibacterial activities of cationic steroid antibiotics and cationic peptide antibiotics have been compared . Depolarization of bacterial membranes, activation of bacterial stress-related gene promoters, and changes in bacterial morphologies caused by these antibiotics suggest that cationic steroid and peptide antibiotics share mechanistic aspects . Modified cationic steroid antibiotics display improved selectivity for prokaryotic cells over eukaryotic cells presumably due to increased charge recognition.

Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1420 - 5 Epub 2002 Jan 22.
The origin of the eukaryotic cell: a genomic investigation; Hartman H et al.; We have collected a set of 347 proteins that are found in eukaryotic cells but have no significant homology to proteins in Archaea and Bacteria . We call these proteins eukaryotic signature proteins (ESPs) . The dominant hypothesis for the formation of the eukaryotic cell is that it is a fusion of an archaeon with a bacterium . If this hypothesis is accepted then the three cellular domains, Eukarya, Archaea, and Bacteria, would collapse into two cellular domains . We have used the existence of this set of ESPs to test this hypothesis . The evidence of the ESPs implicates a third cell (chronocyte) in the formation of the eukaryotic cell . The chronocyte had a cytoskeleton that enabled it to engulf prokaryotic cells and a complex internal membrane system where lipids and proteins were synthesized . It also had a complex internal signaling system involving calcium ions, calmodulin, inositol phosphates, ubiquitin, cyclin, and GTP-binding proteins . The nucleus was formed when a number of archaea and bacteria were engulfed by a chronocyte . This formation of the nucleus would restore the three cellular domains as the Chronocyte was not a cell that belonged to the Archaea or to the Bacteria.

Genetics, 2002 Jan, 160(1), 271 - 7
Maternal effect for DNA mismatch repair in the mouse; Gurtu VE et al.; DNA mismatch repair (DMR) functions to maintain genome stability . Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences . Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of approximately 10% was observed . To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F(1) progeny from Pms2(-/-) female mice mated with wild-type males . Our analysis indicates that MSI in the female germ line was approximately 9% . MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient . We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions . The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote . The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.

Biochemistry, 2002 Jan 29, 41(4), 1123 - 8
Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA; Gross A et al.; This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface . The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface . Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision . Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface . To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate . The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface . Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates . The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer.

Planta, 2001 Dec, 214(2), 250 - 6
Sucrose-phosphate phosphatase from Anabaena sp . strain PCC 7120: isolation of the protein and gene revealed significant structural differences from the higher-plant enzyme; Cumino A et al.; The present study describes the first isolation and characterization of a prokaryotic protein and gene for sucrose-phosphate phosphatase (SPP), the enzyme that catalyzes the terminal step in sucrose synthesis . For gene isolation, a 2,015-bp DNA fragment containing an open reading frame with about 31% amino acid identity to Synechocystis SPS was amplified from Anabaena sp . PCC 7120 DNA . Surprisingly, expression of the putative gene in Escherichia coli demonstrated that it encoded an SPP protein . The expressed protein cross-reacted with antibodies against the native form of Anabaena SPP and its biochemical properties were identical to those of the enzyme purified from the cyanobacterial cells . Comparisons of the Anabaena SPP with the higher-plant enzyme revealed important differences in the C-terminal region, molecular mass, subunit composition and immunoreactivity . Nevertheless, two conserved motifs, including four invariant aspartate residues similar to those found in members of the phosphohydrolase superfamily, were identified in the Anabaena SPP deduced amino acid sequence.

J Biol Chem, 2002 Mar 22, 277(12), 10739 - 45 Epub 2002 Jan 17.
In the absence of the first membrane-spanning segment of subunit 4(b), the yeast ATP synthase is functional but does not dimerize or oligomerize; Soubannier V et al.; The N-terminal portion of the mitochondrial b-subunit is anchored in the inner mitochondrial membrane by two hydrophobic segments . We investigated the role of the first membrane-spanning segment, which is absent in prokaryotic and chloroplastic enzymes . In the absence of the first membrane-spanning segment of the yeast subunit (subunit 4), a strong decrease in the amount of subunit g was found . The mutant ATP synthase did not dimerize or oligomerize, and mutant cells displayed anomalous mitochondrial morphologies with onion-like structures . This phenotype is similar to that of the null mutant in the ATP20 gene that encodes subunit g, a component involved in the dimerization/oligomerization of ATP synthase . Our data indicate that the first membrane-spanning segment of the mitochondrial b-subunit is not essential for the function of the enzyme since its removal did not directly alter the oxidative phosphorylation . It is proposed that the unique membrane-spanning segment of subunit g and the first membrane-spanning segment of subunit 4 interact, as shown by cross-linking experiments . We hypothesize that in eukaryotic cells the b-subunit has evolved to accommodate the interaction with the g-subunit, an associated ATP synthase component only present in the mitochondrial enzyme.

Zhonghua Yi Xue Za Zhi, 2001 Feb 10, 81(3), 154 - 7
{Molecular cloning of the tumor associated antigen recognized by monoclonal antibody 3H11}; Chen D et al.; OBJECTIVE: Monoclonal antibody (McAb) 3H11 can specifically bind to different cancer cells from different tissues . Meanwhile, McAb 3H11 labeled with radioactive isotopes has been successfully applied to detect primary cancer and metastatic cancer in clinical imaging . Molecular cloning of the antigen recognized by McAb 3H11 will be significant for understanding tumor development . METHODS: Based on the fragment of the McAb 3H11 antigen cloned by Immunoscreening MGC803 cDNA expression library, the whole length of the cDNA molecule was gotten by RACE and nested PCR . RESULTS: Sequence data of the cDNA molecule indicates that there was no homologous gene in GenBank . Northern blot experiments showed that mRNA of McAb 3H11 antigen extensively distributes in different cancer cells and tissues but not in corresponding normal tissues . Moreover in producing antibodies of the antigen expressed prokaryotically, it was found that the immunogenicity of the antigen was quite low in mammalian . CONCLUSION: The antigen recognized by McAb 3H11 acts as a regulator in embryo cells and regains expression in tumor cells; and it may be associated with low differentiation and high proliferation.

Plasmid, 2002 Jan, 47(1), 26 - 35
Mobile elements as a combination of functional modules; Toussaint A et al.; Prokaryotic mobile elements have traditionally been classified as bacteriophages, plasmids, and transposons . We propose here a global classification of these and other bacterial and archaeal mobile elements based on their modular structure . This would allow for setting up interconnected databases where mobile elements could be stored as combinations of functional modules . Such a database would be very helpful . It would, for instance, allow for analyzing the phylogeny of individual blocks within an element, to understand how modules get associated and properly express the functions they carry in various bacterial hosts . Modules of practical importance, as for instance those that encode toxins or other virulence factors, could be identified and compared, and probes devised to test bacterial populations for the presence of such modules .

Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 553 - 6
{High level expression and purification of Cysticercosis cellulose annexin32 in Escherichia coli}; Guo YJ et al.; In previous work, the cDNA encoding Cysticercus cellulose annexin32 has been cloned . With PCR method, two different restriction Sites were added to each end of the cDNA respectively . Then, the cDNA was inserted into prokaryotic expression vector pJLA-503 . After inducing, most foreign protein was expressed in soluble form, which was up to 35% of the total protein of the bacteria . Subsequently, the recombinant Annexin32 was purified with (NH4)2SO4 stepwise precipitation, DEAE-Sepharose FF and Sephacryl S-200 HR chromatography . The final pure protein can been shown as a single band in SDS-PAGE, and the biological activity was verified by Western blot and anticoagulation activity assay.

Funct Integr Genomics, 2000 May, 1(1), 35 - 43
Transactivation of a target gene through feedforward loop activation in plants; Schwechheimer C et al.; The expression of many genes encoding transcriptional activators in prokaryotes and eukaryotes is upregulated through positive feedback activation . During positive feedback activation, a transcriptional activator binds to its own promoter and thus increases its own expression as well as the expression of its target genes . In the simplest case, increased levels of the transcriptional activator can be directly correlated with increased expression of its target genes . In this study, we present a gene expression system, designated feedforward loop (FFL) system, which makes use of this kind of positive feedback regulation for the expression of plant transgenes . We show in transient and stable transformation experiments that such a system is functional and that it can be used to obtain high level gene expression in plants . We also provide evidence that the transgene is ubiquitously expressed when using the FFL system . Finally, we discuss the possibilities of using FFL gene expression systems for applications in plant molecular genetics and biotechnology.

Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1152 - 7 Epub 2002 Jan 15.
Eukaryotic-type elongator tRNAMet of Trypanosoma brucei becomes formylated after import into mitochondria; Tan TH et al.; The mitochondrion of Trypanosoma brucei lacks tRNA genes . Its translation system therefore depends on the import of cytosolic, nucleus-encoded tRNAs . Thus, most trypanosomal tRNAs function in both the cytosol and the mitochondrion, and all are of the eukaryotic type . This is also the case for the elongator tRNA(Met), whereas the only other trypanosomal tRNA(Met), the eukaryotic initiator, is found exclusively in the cytosol . Unlike their cytosolic counterparts, organellar initiator tRNAs(Met) carry a formylated methionine . This raises the question of how initiation of translation works in trypanosomal mitochondria, where only elongator tRNA(Met) is found . Using in organello charging and formylation assays, we show that unexpectedly a fraction of elongator tRNA(Met) becomes formylated after import into mitochondria . Furthermore, in vitro experiments with mitochondrial extracts demonstrate that only the trypanosomal elongator and not the initiator tRNA(Met) is recognized by the formylation activity . Finally, RNA interference assays identify the gene encoding the trypanosomal formylase activity . Whereas the predicted protein is homologous to prokaryotic and mitochondrial methionyl-tRNA(Met) formyltransferases, it has about twice the mass of any of these proteins.

J Biol Chem, 2002 Mar 29, 277(13), 11208 - 16 Epub 2002 Jan 15.
Cupiennin 1, a new family of highly basic antimicrobial peptides in the venom of the spider Cupiennius salei (Ctenidae); Kuhn-Nentwig L et al.; A new family of antimicrobial peptides was isolated from the venom of Cupiennius salei . The peptides were purified to homogeneity, and the sequence of cupiennin 1a was determined by Edman degradation: GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH(2) . The amino acid sequences of cupiennin 1b, c, and d were obtained by a combination of sequence analysis and mass spectrometric measurements of comparative tryptic peptide mapping . All peptides consist of 35 amino acid residues and are characterized by a more hydrophobic N-terminal chain region and a C terminus composed preferentially of polar and charged residues . The total charge of all cupiennins calculated under physiological conditions is +8, and their C terminus, formed by a glutamic acid residue, is amidated . Conformational studies of the peptides revealed a high helix forming potential . Antimicrobial assays on bacteria with cupiennin 1a, 1d, and synthesized cupiennins 1a* and 1d* showed minimal inhibitory concentrations for bacteria in the submicromolar range . Their lytic effect on human red blood cells was lower by a factor of 8 to 14 than the highly hemolytic melittin . Cupiennin 1a, 1b, 1d, 1a*, and 1d* showed pronounced insecticidal activity . The immediate biological effects and the structural properties of the isolated cupiennins indicate a membrane-destroying mode of action on prokaryotic as well as eukaryotic cells.

Genome Inform Ser Workshop Genome Inform, 2001, 12, 34 - 43
Characterizing the relationship between protein-fusion and gene co-expression; Gunther CS et al.; A pair of distinct proteins in one organism may most closely match different parts of the same protein in another organism . A comparison of all proteins from the genome of Saccharomyces cerevisiae and all proteins from 24 prokaryotic genomes yields 1010 pairs of yeast proteins whose homologs are parts of one protein from a prokaryotic genome . Marcotte et al . (Science 285:751-3) showed that proteins related in this manner are more likely to interact than proteins chosen at random . In this paper, we investigated whether genes coding for such proteins are also likely to be concurrently transcribed . We identified 1010 fused pairs of proteins encoded in the yeast genome and analyzed expression of the corresponding genes at the transcriptional level . We found that the transcriptional profiles of fused gene pairs are significantly closer than those of randomly selected pairs . This finding is reproducible and established by multiple distance metrics . Moreover, such pairs frequently share additional biologically relevant properties . Thus, while protein fusion patterns are not predictive of co-expression, they are an important element in explaining co-expression . This justifies the use of curated protein fusion events to help characterize gene co-expression clusters.

J Bacteriol, 2002 Feb, 184(3), 859 - 60
Integrative genetic element that reverses the usual target gene orientation; Zhao S et al.; A genetic element integrating site specifically into a prokaryotic gene usually carries a copy of the 3' portion of that gene that restores the active gene even as the original is disrupted . A cryptic element in Mesorhizobium loti instead carries a copy of the 5' end of the tRNA gene into which it integrated . This has implications for the evolution of new integrase-site combinations.

Genome Biol . 2001;2(12):REVIEWS3013 . Epub 2001 Nov 23.
Family-B G-protein-coupled receptors; Harmar AJ; SUMMARY: All G-protein-coupled receptors (GPCRs) share a common molecular architecture (with seven putative transmembrane segments) and a common signaling mechanism, in that they interact with G proteins (heterotrimeric GTPases) to regulate the synthesis of intracellular second messengers such as cyclic AMP, inositol phosphates, diacylglycerol and calcium ions . Historically, GPCRs have been classified into six families, which were thought to be unrelated; three of these are found in vertebrates . Recent work has identified several new GCPR families and suggested the possibility of a common evolutionary origin for all of them . Family B (the secretin-receptor family or 'family 2') of the GPCRs is a small but structurally and functionally diverse group of proteins that includes receptors for polypeptide hormones, molecules thought to mediate intercellular interactions at the plasma membrane and a group of Drosophila proteins that regulate stress responses and longevity . Family-B GPCRs have been found in all animal species investigated, including mammals, Caenorhabditis elegans and Drosophila melanogaster, but not in plants, fungi or prokaryotes . In this article, I describe the structures and functions of family-B GPCRs and propose a simplified nomenclature for these proteins.

Plant Physiol, 2002 Jan, 128(1), 150 - 9
Elemental sulfur and thiol accumulation in tomato and defense against a fungal vascular pathogen; Williams JS et al.; The occurrence of fungicidal, elemental S is well documented in certain specialized prokaryotes, but has rarely been detected in eukaryotes . Elemental S was first identified in this laboratory as a novel phytoalexin in the xylem of resistant genotypes of Theobroma cacao, after infection by the vascular, fungal pathogen Verticillium dahliae . In the current work, this phenomenon is demonstrated in a resistant line of tomato, Lycopersicon esculentum, in response to V . dahliae . A novel gas chromatography-mass spectroscopy method using isotope dilution analysis with 34S internal standard was developed to identify unambiguously and quantify 32S in samples of excised xylem . Accumulation of S in vascular tissue was more rapid and much greater in the disease-resistant than in the disease-susceptible line . Levels of S detected in the resistant variety (approximately 10 microg g-1 fresh weight excised xylem) were fungitoxic to V . dahliae (spore germination was inhibited >90% at approximately 3 microg mL-1) . Scanning electron microscopy-energy dispersive x-ray microanalysis confirmed accumulation of S in vascular but not in pith cells and in greater amounts and frequency in the Verticillium spp.-resistant genotype . More intensive localizations of S were occasionally detected in xylem parenchyma cells, vessel walls, vascular gels, and tyloses, structures in potential contact with and linked with defense to V . dahliae . Transient increases in concentrations of sulfate, glutathione, and Cys of vascular tissues from resistant but not susceptible lines after infection may indicate a perturbation of S metabolism induced by elemental S formation; this is discussed in terms of possible S biogenesis.

J Biol Chem, 2002 Mar 22, 277(12), 9997 - 10002 Epub 2002 Jan 11.
Mitochondrial ferredoxin is required for heme A synthesis in Saccharomyces cerevisiae; Barros MH et al.; Heme A is a prosthetic group of all eukaryotic and some prokaryotic cytochrome oxidases . This heme differs from heme B (protoheme) at two carbon positions of the porphyrin ring . The synthesis of heme A begins with farnesylation of the vinyl group at carbon C-2 of heme B . The heme O product of this reaction is then converted to heme A by a further oxidation of a methyl to a formyl group on C-8 . In a previous study (Barros, M . H., Carlson, C . G., Glerum, D . M., and Tzagoloff, A . (2001) FEBS Lett . 492, 133-138) we proposed that the formyl group is formed by an initial hydroxylation of the C-8 methyl by a three-component monooxygenase consisting of Cox15p, ferredoxin, and ferredoxin reductase . In the present study three lines of evidence confirm a requirement of ferredoxin in heme A synthesis . 1) Temperature-conditional yah1 mutants grown under restrictive conditions display a decrease in heme A relative to heme B . 2) The incorporation of radioactive delta-aminolevulinic acid into heme A is reduced in yah1 ts but not in the wild type after the shift to the restrictive temperature; and 3) the overexpression of Cox15p in cytochrome oxidase mutants that accumulate heme O leads to an increased mitochondrial concentration of heme A . The increase in heme A is greater in mutants that overexpress Cox15p and ferredoxin . These results are consistent with a requirement of ferredoxin and indirectly of ferredoxin reductase in hydroxylation of heme O.

Am J Physiol Endocrinol Metab, 2002 Feb, 282(2), E239 - 46
Fatty acid transport: the roads taken; Schaffer JE; Efficient uptake and channeling of long-chain fatty acids (LCFAs) are critical cellular functions . Although spontaneous flip-flop of nonionized LCFAs from one leaflet of a bilayer to the other is rapid, evidence is emerging that proteins are important mediators and/or regulators of trafficking of LCFAs into and within cells . Genetic screens have led to the identification of proteins that are required for fatty acid import and utilization in prokaryotic organisms . In addition, functional screens have elucidated proteins that facilitate fatty acid import into mammalian cells . Although the mechanisms by which these proteins mediate LCFA import are not well understood, studies in both prokaryotic and eukaryotic organisms provide compelling evidence that uptake of LCFAs across cellular membranes is coupled to esterification by acyl-CoA synthetases . This review will summarize results of studies of non-protein-mediated and protein-mediated LCFA transport and discuss how these different mechanisms may contribute to cellular metabolism.

FEMS Microbiol Lett, 2002 Jan 2, 206(1), 1 - 8
Protein kinases and protein phosphatases in prokaryotes: a genomic perspective; Kennelly PJ; For many years, the regulation of protein structure and function by phosphorylation and dephosphorylation was considered a relatively recent invention that arose independently in each phylogenetic domain . Over time, however, incidents of apparent domain trespass involving the presence of 'eukaryotic' protein kinases or protein phosphatases in prokaryotic organisms were reported with increasing frequency . Today, genomics has provided the means to examine the phylogenetic distribution of 'eukaryotic' protein kinases and protein phosphatases in a comprehensive and systematic manner . The results of these genome searches challenge previous conceptions concerning the origins and evolution of this versatile regulatory mechanism.

Plant Mol Biol, 2001 Dec, 47(6), 795 - 804
An OGG1 orthologue encoding a functional 8-oxoguanine DNA glycosylase/lyase in Arabidopsis thaliana; Garcia-Ortiz MV et al.; Repair of the ubiquitous mutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG) is initiated in eukaryotes by DNA glycosylases/lyases, such as yeast Ogg1, that do not share significant sequence identity with their prokaryotic counterparts, typified by Escherichia coli MutM (Fpg) protein . The unexpected presence of a functional mutM orthologue in the model plant Arabidopsis thaliana has brought into question the existence of functional OGG1 orthologues in plants . We report here the cDNA cloning, expression and functional characterization of AtOGG1, an Arabidopsis thaliana gene widely expressed in different plant tissues which encodes a 40.3 kDa protein with significant sequence identity to yeast and human Ogg1 proteins . Purified AtOgg1 enzyme specifically cleaves duplex DNA containing an 8-OxoG:C mispair, and the repair reaction proceeds through an imine intermediate

Eur J Biochem, 2002 Jan, 269(1), 175 - 83
Inhibition of nuclear pre-mRNA splicing by antibiotics in vitro; Hertweck M et al.; A number of antibiotics have been reported to disturb the decoding process in prokaryotic translation and to inhibit the function of various natural ribozymes . We investigated the effect of several antibiotics on in vitro splicing of a eukaryotic nuclear pre-mRNA (beta-globin) . Of the eight antibiotics studied, erythromycin, Cl-tetracycline and streptomycin were identified as splicing inhibitors in nuclear HeLa cell extract . The K(i) values were 160, 180 and 230 microm, respectively . Cl-tetracycline-mediated and streptomycin-mediated splicing inhibition were in the molar inhibition range for hammerhead and human hepatitis delta virus ribozyme self-cleavage (tetracycline), of group-I intron self-splicing (streptomycin) and inhibition of RNase P cleavage by some aminoglycosides . Cl-tetracycline and the aminocyclitol glycoside streptomycin were found to have an indirect effect on splicing by unspecific binding to the pre-mRNA, suggesting that the inhibition is the result of disturbance of the correct folding of the pre-mRNA into the splicing-compatible tertiary structure by the charged groups of these antibiotics . The macrolide, erythromycin, the strongest inhibitor, had only a slight effect on formation of the presplicing complexes A and B, but almost completely inhibited formation of the splicing-active C complex by binding to nuclear extract component(s) . This results in direct inhibition of the second step of pre-mRNA splicing . To our knowledge, this is the first report on specific inhibition of nuclear splicing by an antibiotic . The functional groups involved in the interaction of erythromycin with snRNAs and/or splicing factors require further investigation.

J Biol Chem, 2002 Mar 29, 277(13), 11450 - 5 Epub 2002 Jan 07.
Stoichiometry and structural effect of the cyclic nucleotide binding to cyclic AMP receptor protein; Won HS et al.; Cyclic AMP receptor protein (CRP) is a homodimeric protein, which is activated by cAMP binding to function as a transcriptional regulator of many genes in prokaryotes . Until now, the actual number of cAMP molecules that can be bound by CRP in solution has been ambiguous . In this work, we performed a nuclear magnetic resonance study on CRP to investigate the stoichiometry of cyclic nucleotide binding to CRP . A series of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the protein in the absence and in the presence of cAMP or cGMP were analyzed . The addition of cAMP to CRP induced a biphasic spectral change up to 4 equivalents, whereas the cGMP addition made a monophasic change up to 2 equivalents . Altogether, the results not only established for the first time that CRP possesses two cyclic AMP-binding sites in each monomer, even in a solution without DNA, but also suggest that the syn-cAMP binding sites of the CRP dimer can be formed by an allosteric conformational change of the protein upon the binding of two anti-cAMPs at the N-terminal domain . In addition, a residue-specific inspection of the spectral changes provides some new structural information about the cAMP-induced allosteric activation of CRP.

Biochemistry, 2002 Jan 15, 41(2), 521 - 9
GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers; Scheffers DJ et al.; The essential prokaryotic cell division protein FtsZ is a tubulin homologue that forms a ring at the division site . FtsZ forms polymers in a GTP-dependent manner . Recent biochemical evidence has shown that FtsZ forms multimeric structures in vitro and in vivo and functions as a self-activating GTPase . Structural analysis of FtsZ points to an important role for the highly conserved tubulin-like loop 7 (T7-loop) in the self-activation of GTP hydrolysis . The T7-loop was postulated to form the active site together with the nucleotide-binding site on an adjacent FtsZ monomer . To characterize the role of the T7-loop of Escherichia coli FtsZ, we have mutagenized residues M206, N207, D209, D212, and R214 . All the mutant proteins, except the R214 mutant, are severely affected in polymerization and GTP hydrolysis . Charged residues D209 and D212 cannot be substituted with a glutamate residue . All mutants interact with wild-type FtsZ in vitro, indicating that the T7-loop mutations do not abolish FtsZ self-association . Strikingly, in mixtures of wild-type and mutant proteins, most mutants are capable of inhibiting wild-type GTP hydrolysis . We conclude that the T7-loop is part of the active site for GTP hydrolysis, formed by the association of two FtsZ monomers.

RNA, 2001 Dec, 7(12), 1683 - 92
A mechanism for stop codon recognition by the ribosome: a bioinformatic approach; Ivanov V et al.; Protein synthesis in ribosomes requires two kinds of tRNAs: initiation and elongation . The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes) . The latter participates in the synthesis proper, recognizing the sense codons . Synthesis is also assisted by special proteins: initiation, elongation, and termination factors . The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon . No termination tRNA capable of recognizing stop codons by their anticodons is known . The termination factors are thought to do this . In the large ribosomal RNA, we found two sites that, like tRNAs, contain the anticodon hairpin but with triplets complementary to stop codons . One site is hairpin 69 from domain IV; the other site is hairpin 89, domain V . By analogy, we call them termination tRNAs: Ter-tRNA1 and Ter-tRNA2, respectively, even though they transport no amino acids, and suggest that they directly pair to stop codons . The termination factors only aid in this recognition, making it specific and reliable . A strong argument in favor of our hypothesis comes from vertebrate mitochondria . They are known to acquire two new stop codons, AGA and AGG . In the standard code, these are two out of six arginine codons . We revealed that the corresponding anticodons, UCU and CCU, have evolved in Ter-tRNA1 of these mitochondria.

Biochim Biophys Acta, 2001 Nov 1, 1506(3), 147 - 62
Comparative genomics and bioenergetics; Castresana J; Bacterial and archaeal complete genome sequences have been obtained from a wide range of evolutionary lines, which allows some general conclusions about the phylogenetic distribution and evolution of bioenergetic pathways to be drawn . In particular, I searched in the complete genomes for key enzymes involved in aerobic and anaerobic respiratory pathways and in photosynthesis, and mapped them into an rRNA tree of sequenced species . The phylogenetic distribution of these enzymes is very irregular, and clearly shows the diverse strategies of energy conservation used by prokaryotes . In addition, a thorough phylogenetic analysis of other bioenergetic protein families of wide distribution reveals a complex evolutionary history for the respective genes . A parsimonious explanation for these complex phylogenetic patterns and for the irregular distribution of metabolic pathways is that the last common ancestor of Bacteria and Archaea contained several members of every gene family as a consequence of previous gene or genome duplications, while different patterns of gene loss occurred during the evolution of every gene family . This would imply that the last universal ancestor was a bioenergetically sophisticated organism . Finally, important steps that occurred during the evolution of energetic machineries, such as the early evolution of aerobic respiration and the acquisition of eukaryotic mitochondria from a proteobacterium ancestor, are supported by the analysis of the complete genome sequences.

Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 579 - 92
Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification; Rehm BH; The development of efficient DNA sequencing methods has led to the achievement of the DNA sequence of entire genomes from (to date) 55 prokaryotes, 5 eukaryotic organisms and 10 eukaryotic chromosomes . Thus, an enormous amount of DNA sequence data is available and even more will be forthcoming in the near future . Analysis of this overwhelming amount of data requires bioinformatic tools in order to identify genes that encode functional proteins or RNA . This is an important task, considering that even in the well-studied Escherichia coli more than 30% of the identified open reading frames are hypothetical genes . Future challenges of genome sequence analysis will include the understanding of gene regulation and metabolic pathway reconstruction including DNA chip technology, which holds tremendous potential for biomedicine and the biotechnological production of valuable compounds . The overwhelming volume of information often confuses scientists . This review intends to provide a guide to choosing the most efficient way to analyze a new sequence or to collect information on a gene or protein of interest by applying current publicly available databases and Web services . Recently developed tools that allow functional assignment of genes, mainly based on sequence similarity of the deduced amino acid sequence, using the currently available and increasing biological databases will be discussed.

Virus Genes, 2001 Dec, 23(3), 347 - 59
Comparison of the eIF-2alpha homologous proteins of seven ranaviruses (Iridoviridae); Essbauer S et al.; The alpha-subunit of the eukaryotic initiation factor 2 (eIF-2alpha) is a key component of the translation machinery of the cell . In response to cellular stress such as viral infections, eIF-2alpha is phosphorylated by double-stranded RNA-dependent protein kinase (PKR) leading to the inhibition of cellular protein synthesis . The importance of eIF-2alpha as a regulatory mechanism for protein synthesis is illustrated by the wide variety of strategies employed by viruses to down-regulate PKR . Thus, Vaccinia virus encodes K3L protein, which resembles eIF-2alpha and acts as a pseudo-substrate inhibitor of PKR . Nucleotide sequencing of the genome of epizootic haematopoietic necrosis virus (EHNV), a member of the genus ranavirus of Iridoviridae, has revealed an eIF-2alpha equivalent gene . We have cloned and sequenced eIF-2alpha genes of several iridoviruses of fishes and frogs . The eIF-2alpha open reading frames and deduced proteins of the iridoviruses investigated exhibit a high degree of homology of both nucleotide and amino acid sequences . At the N-terminus, the iridoviral eIF-2alpha shows significant homology to the N-termini of cellular initiation factor 2-alpha of various species, to full-length poxviral eIF-2alpha proteins, and to the S1 domain of ribosomal proteins . Comparison of amino acid sequences of corresponding iridoviral proteins with eIF-2alpha homologous proteins of poxviruses and eukaryotes has revealed a high conservation of motifs . A phylogenetic analysis of eukaryotic eIF-2alpha and poxvirus and iridovirus eIF-2alpha sequences has demonstrated the relationship of these iridoviruses . In order to investigate the role of the eIF-2alpha equivalent, respective genes have been expressed in prokaryotic and eukaryotic (insect, fish and chicken cell) systems . The iridoviral eIF-2alpha protein has a molecular weight of 31 kDa and is cytoplasmic . The cellular and viral protein synthesis of iridoviruses is probably regulated by a mechanism similar to that of Vaccinia virus . Frog-virus 3, the type species of the genus ranavirus of Iridoviridae, has a unique translational efficiency and, moreover, down-regulates the cellular protein synthesis of infected cells.

Virus Genes, 2001 Dec, 23(3), 331 - 7
Identification and phylogeny of a non-specific endonuclease gene of white spot syndrome virus of shrimp; Witteveldt J et al.; White spot syndrome virus (WSSV) is a taxonomically unclassified virus which causes a disease in shrimps worldwide . A 936 bp long open reading frame (ORF) was found on a 7.2 kb HindIII fragment of the DNA genome of WSSV located adjacent to the ribonucleotide reductase small subunit gene . This putative ORF showed homology to prokaryotic and eukaryotic endonucleases, which contain a non-specific endonuclease motif . Alignment with viral and eukaryotic endonuclease ORFs revealed that most catalytically and structurally important amino acid residues were present in the putative WSSV non-specific endonuclease gene . An unrooted parsimonous phylogenetic tree of non-specific endonucleases indicated that the WSSV ORF was located in a well bootstrap supported clade containing only arthopods, including one of WSSV's natural hosts, Penaeus japonicus . A similar conjunction was found for the only other viral homologue, present in Fowlpox virus, which was also found in a well bootstrap-supported clade with its natural host, Gallus gallus . This clustering of virus and host suggests that both WSSV and Fowlpox virus may have acquired their nuclease genes from their respective natural hosts . Because the motif for non-specific nucleases is found in only two viruses, this gene cannot be used to clarify the taxonomic position of WSSV . However, the presence of this type of nuclease rarely found in viruses adds a novel feature to WSSV.

Science, 2002 Jan 4, 295(5552), 117 - 9
Intracellular iron minerals in a dissimilatory iron-reducing bacterium; Glasauer S et al.; Among prokaryotes, there are few examples of controlled mineral formation; the formation of crystalline iron oxides and sulfides {magnetite (Fe3O4) or greigite (Fe3S4)} by magnetotactic bacteria is an exception . Shewanella putrefaciens CN32, a Gram-negative, facultative anaerobic bacterium that is capable of dissimilatory iron reduction, produced microscopic intracellular grains of iron oxide minerals during growth on two-line ferrihydrite in a hydrogen-argon atmosphere . The minerals, formed at iron concentrations found in the soil and sedimentary environments where these bacteria are active, could represent an unexplored pathway for the cycling of iron by bacteria.

Zhonghua Zhong Liu Za Zhi, 1999 Mar, 21(2), 93 - 5
{Preparation of monoclonal antibodies to human vascular endothelial growth factor(VEGF121) and identification of its expression on gastric carcinoma cell line MGC803}; Tian X et al.; OBJECTIVE: To further elucidate the source of VEGF in solid tumors . METHODS: Traditional hybridoma technology was used to prepare VEGF monoclonal antibodies (McAb); it's activity was measured by 3H-TdR incorporation on human umbilical vein endothelial cells (HUVEC) . VEGF gene was amplified by PCR with cDNA as template from MGC803 cell lines . The PCR products were cloned into fusion protein prokaryotic expression vector PGEX2T which expressed in E . coli XL-1 blue . Western blot analysis was used to identify expression of VEGF . RESULTS: The McAbs could specifically bind to VEGF in ELISA . One of the McAbs(5C5) could neutralize the mitogenic activity of VEGF121 on HUVEC in a dose-dependent manner . The VEGF gene fragment obtained from RT-PCR cloned to PGEX2T vector expressed a protein product which reacted specifically with McAb 5C5 . CONCLUSION: Gastric carcinoma is shown to express VEGF . It may be a major source of VEGF in human solid tumor . Monoclonal antibody against VEGF may be of value in the treatment of cancer.

Chin Med J (Engl), 2000 Aug, 113(8), 714 - 9
Bacterially produced human B7-1 protein encompassing its complete extracellular domain maintains its costimulatory activity in vitro; Shen W et al.; OBJECTIVE: To investigate which of the two immunoglobulin (Ig)-like domains, immunoglobulin variable region homologous domain IgV (hB7-1 IgV), or immunoglobulin constant region homologous domain IgC (hB7-1 IgC) on human B7-1 molecule contain the receptor binding sites, and to evaluate if the B7-1 molecule expressed in bacteria has biological activity . METHODS: PCR was used to amplify three fragments of hB7-1 IgV, hB7-1 IgC and complete extracellular region of human B7-1 containing both the IgV and IgC domains (hB7-1 IgV + IgC) . Three recombinants, pQE9-hB7-1 IgV, pQE9-hB7-1 IgC and pQE9-Hb7-1 (IgV + IgC) were generated by cloning the PCR products into a prokaryote expression plasmid (pQE-9) and were introduced into the host stain M15 . The relevant target hexahistidine-tagged proteins were identified by SDS-PAGE and Western blotting . With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7-1 proteins and {3H}-TdR incorporation . RESULTS: Three recombinant proteins of human B7-1, hB7-1 IgV, hB7-1 IgC and hB7-1 (IgV + IgC) were produced and detected in both soluble and inclusive body forms from engineered bacterial cells . With the presence of anti-CD3 antibody, T lymphocytes proliferated when co-stimulated by bacterially produced hB7-1 (IgV + IgC), but not by either hB7-1 IgV or hB7-1 IgC . CONCLUSIONS: Functional glycoprotein human B7-1 could be produced in bacterial cells . Both extracellular immunoglobulin-like domains are necessary for B7-1 to react with its counter receptors.

Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 1070 - 5 Epub 2002 Jan 02.
Directed molecular evolution of ADP-glucose pyrophosphorylase; Salamone PR et al.; ADP-glucose pyrophosphorylase catalyzes a rate-limiting reaction in prokaryotic glycogen and plant starch biosynthesis . Despite sharing similar molecular size and catalytic and allosteric regulatory properties, the prokaryotic and higher plant enzymes differ in higher-order protein structure . The bacterial enzyme is encoded by a single gene whose product of ca . 50,000 Da assembles into a homotetrameric structure . Although the higher plant enzyme has a similar molecular size, it is made up of a pair of large subunits and a pair of small subunits, encoded by different genes . To identify the basis for the evolution of AGPase function and quaternary structure, a potato small subunit homotetrameric mutant, TG-15, was subjected to iterations of DNA shuffling and screened for enzyme variants with up-regulated catalytic and/or regulatory properties . A glycogen selection/screening regimen of buoyant density gradient centrifugation and iodine vapor colony staining on glucose-containing media was used to increase the stringency of selection . This approach led to the isolation of a population of AGPase small subunit homotetramer enzymes with enhanced affinity toward ATP and increased sensitivity to activator and/or greater resistance to inhibition than TG-15 . Several enzymes displayed a shift in effector preference from 3-phosphoglycerate to fructose-6 phosphate or fructose-1,6-bis-phosphate, effectors used by specific bacterial AGPases . Our results suggest that evolution of AGPase, with regard to quaternary structure, allosteric effector selectivity, and effector sensitivity, can occur through the introduction of a few point mutations alone with low-level recombination hastening the process.

J Neural Transm Suppl, 2001, (61), 35 - 45
Expression of the multidrug resistance P glycoprotein (Pgp) and multidrug resistance associated protein (MRP1) in Down syndrome brains; Engidawork E et al.; Transport by ATP-dependent efflux pumps such as P glycoprotein (Pgp) and multidrug resistance associated protein (MRP), encoded by multidrug resistant (MDR) associated genes, is an increasingly recognized mechanism by which cells maintain substrate homeostasis and evade drug therapy . Pgp and MRP are members of the so-called ATP binding cassette (ABC) transporters superfamily, which are associated with many biological processes in both prokaryotes and eukaryotes, as well as clinical problems . The observation of upregulated sequences that are homologous to the Mycobacterium smegmatis phage resistance (mpr) gene and putative ABC transporters subunits in fetal Down syndrome (DS) using the gene hunting technique, subtractive hybridization formed the Rationale for this study . The expression of Pgp and MRP1 is therefore investigated in different brain regions of controls and adult DS patients with western blot technique . No apparent changes were observed between controls and DS in levels of Pgp in all brain regions examined . By contrast, MRP1 detection using the rat monoclonal antibody (MRPr1) produced a significant elevation in DS temporal cortex (P < 0.01) and parietal cortex (P < 0.05) . Although MRP1 detected with the mouse monoclonal antibody (MRPm6) tended to increase in most of the regions of DS brain, it failed to reach significance level . Age or postmortem interval did not correlate with protein levels in both controls as well as DS . Taken together, the current data provide evidence for the presence of MDR related pumps in different regions of the human brain . In addition, overexpression of MRP1 in DS brain may have some relevance to the disorder either by deranging substrate homeostasis or limiting drug access.

Int Microbiol, 2001 Jun, 4(2), 67 - 71
Evolution of the genomic systems of prokaryotes and its momentous consequences; Sonea S et al.; The earliest self-reproducing cell on Earth, our common ancestor, was probably as small as present-day bacteria . It gave rise to a very large and durable clone whose descendants must have been the only living occupants of the oceans for about one thousand million years . They reached astronomical numbers of separate, disjunct cells, and synthesized many new genes . Their small volume could not accommodate ever larger genomes and useful new genes replaced resident, less successful sequences, thus increasing diversity and the number of strains with highly specialized, distinct, bioenergetic potentialities . Also, selective pressure favored strains able to participate successfully in division of labor and in the sharing of diverse abilities in mixed communities, counterbalancing the limited capacities of individual genomes . Lateral gene transfer mechanisms appeared and were progressively improved, furthering the development of diversity . The prokaryotes' constructive evolution resulted in the formation of a worldwide web of genetic information, and a global bacterial superbiosystem (superorganism) . By contrast, eukaryotic evolution of organisms has been typically Darwinian . Diversification of eukaryotic organisms was, however, considerably enriched and accelerated by symbioses with prokaryotes . The more broadly diversified bioenergetic potential of prokaryotes considerably increased the diversity of eukaryotes . Without their participation, our biosphere would have remained much less diverse and less dynamic . Environmental homeostasis has been maintained all along by guided bacterial evolution.

Int Microbiol, 2001 Jun, 4(2), 59 - 66
Roger Yate Stanier, 1916-1982: a transcendent journey; Glazer AN; The Tenth International Symposium on Phototrophic Prokaryotes (Barcelona, 26-31 August 2000) was the latest in a series of conferences initiated by Roger Stanier in 1971 to create ties within the community of scientists working with cyanobacteria or green and purple bacteria . Consonant with Stanier's own work, the subjects of these conferences range broadly from systematics and ecology through genetics, biochemistry and physiology . The effort to define comprehensively the place of bacteria in the living world was the leitmotif in Stanier's work, the subject of one of his earliest papers (in 1941), and revisited for the final time in his autobiographical memoir of 1980 . Salvador Luria noted that Stanier "...always pursued broad naturalistic interests along with chemical ones, deliberately emphasizing morphology and ecology side by side with biochemistry." Chronologically, Stanier's work addressed taxonomic and nutritional aspects of the cytophagas, enzyme induction and patterns of regulation of enzyme synthesis, aromatic degradative pathways, characterization of what would subsequently be called 70S bacterial ribosomes, the regulation of bacteriochlorophyll synthesis by nonsulfur purple bacteria, protection by carotenoids against photooxidative damage, the path of carbon in heterotrophy, the molecular basis of streptomycin dependence, the life cycle of Caulobacter, the taxonomy of pseudomonads, and, for the last 12 years of his life, wide-ranging studies of the cyanobacteria.

Biol Chem, 2001 Nov, 382(11), 1521 - 39
An overview of endosymbiotic models for the origins of eukaryotes, their ATP-producing organelles (mitochondria and hydrogenosomes), and their heterotrophic lifestyle; Martin W et al.; The evolutionary processes underlying the differentness of prokaryotic and eukaryotic cells and the origin of the latter's organelles are still poorly understood . For about 100 years, the principle of endosymbiosis has figured into thoughts as to how these processes might have occurred . A number of models that have been discussed in the literature and that are designed to explain this difference are summarized . The evolutionary histories of the enzymes of anaerobic energy metabolism (oxygen-independent ATP synthesis) in the three basic types of heterotrophic eukaryotes those that lack organelles of ATP synthesis, those that possess mitochondria and those that possess hydrogenosomes--play an important role in this issue . Traditional endosymbiotic models generally do not address the origin of the heterotrophic lifestyle and anaerobic energy metabolism in eukaryotes . Rather they take it as a given, a direct inheritance from the host that acquired mitochondria . Traditional models are contrasted to an alternative endosymbiotic model (the hydrogen hypothesis), which addresses the origin of heterotrophy and the origin of compartmentalized energy metabolism in eukaryotes.

J Mol Microbiol Biotechnol, 2002 Jan, 4(1), 93 - 9
Sequence analysis and structure prediction of 23S rRNA:m1G methyltransferases reveals a conserved core augmented with a putative Zn-binding domain in the N-terminus and family-specific elaborations in the C-terminus; Bujnicki JM et al.; N1-methylation of G748 within 23S ribosomal RNA results in resistance to the macrolide tylosin in Streptomyces . In contrast, the Escherichia coli mutant lacking N1-methylation of G745 exhibits increased resistance to viomycin, in addition to severe defects of growth characteristics . Both methylated guanines are located in hairpin 35, in domain II of prokaryotic 23S rRNA . G748 and G745 are modified by related S-adenosylmethionine-dependent methyltransferases (MTases), TlrB and RrmA respectively . Earlier sequence comparisons allowed identification of the AdoMet-binding site, however the catalytic site and the target-recognition region of these enzymes could not be delineated unambiguously . In this work, we carried out sequence-to-structure threading of the rRNA:m1G MTase family against the database of known structures to Identify those "missing regions" . Our analysis confirms the earlier prediction of the AdoMet-binding site, but suggests a different location of the putative catalytic center than was previously postulated . We predict that RrmA and TlrB possess two regions that may be responsible for specific interactions with their target nucleic acid sequences: a putative Zn-finger domain in the N-terminus and the variable domain close to the C-terminus, which indicates that 23S rRNA MTases exhibit the primary structural organization distinct from other nucleic acid MTases, despite sharing the common catalytic domain.

Mikrobiologiia, 2001 Sep-Oct, 70(5), 629 - 35
{Characteristics of in vivo isolation of free fatty acids by prokaryotic and eukaryotic algae under elevated and reduced temperature}; Sushchik NN et al.; The paper describes the composition of extracellular free fatty acids (FFAs) and intracellular fatty acids (FAs) in the enrichment cultures of the prokaryotic alga Spirulina platensis and the eukaryotic alga Chlorella vulgaris grown at optimal, supraoptimal, and suboptimal growth temperatures . With increasing growth temperature, the degree of unsaturation of the intracellular FAs of both algae decreased, while that of the extracellular FFAs of S . platensis increased . The composition of the extracellular FFAs of C . vulgaris practically did not depend on the growth temperature.

Appl Microbiol Biotechnol, 2001 Nov, 57(4), 451 - 9
Biotechnological production of pyruvic acid; Li Y et al.; Pyruvic acid is an important organic acid widely used in the chemical and drug, as well as agrochemical, industries . Compared with the chemical method, biotechnological production of pyruvic acid is an alternative approach because of the low cost . An overview of biotechnological production of pyruvate, including direct fermentative production employing eukaryotic and prokaryotic microorganisms, production by a resting cell method and an enzymatic method as well as the recovery of pyruvate, is discussed . A multi-vitamin auxotrophic yeast strain, Torulopsis glabrata . has been used in the commercial production of pyruvate; emphasis is therefore placed on the mechanism and characteristics of pyruvate production by this strain.

Antonie Van Leeuwenhoek, 2001 Oct, 80(1), 1 - 10
Development of a PCR test for the detection of Curtobacterium flaccumfaciens pv . flaccumfaciens; Guimaraes PM et al.; A chromosomal DNA library of the bacterial pathogen of bean, Curtobacterium flaccumfaciens pv.flaccumfaciens NCPPB 559 was constructed in the plasmid pGEM-7Zf(+) . Several clones were identified that hybridised to all Curtobacterium flaccumfaciens pathovars including: C . f betae, C . f flaccumfaciens, C . f oortii, C . f . poinsettiae and, in addition, to some strains of Clavibacter michiganensis subsp . insidiosus and Clavibacter michiganensis subsp . One of these clones (pPMP-26), after subsequent digestion with restriction endonucleases EcoRI/SacI, yielded a fragment of approximately 0.2 Kb (pPMP-26D) that hybridised specifically to C . f flaccumfaciens and not to any of the other plant pathogenic members of the order Actinomycetales or any of the other prokaryotic bean pathogens tested . This fragment was subcloned and sequenced, analysis of the resultant 198 bp sequence showed that no significant homology existed with any other sequence currently deposited in public databases . Further analysis of these data facilitated the design of PCR primers which were subsequently tested against a wide range of plant pathogenic actinomycetes and other prokaryotic bean pathogens . Results show that these primers are highly specific for all strains of C . f flaccumfaciens with no cross-reaction to strains from any other bacterial taxa tested.

Trends Microbiol, 2002 Jan, 10(1), 31 - 8
The molecular ecology of microbial eukaryotes unveils a hidden world; Moreira D et al.; In spite of the great success of small-subunit ribosomal RNA (SSU rRNA)-based studies for the analysis of environmental prokaryotic diversity, this molecular approach has seldom been applied to microbial eukaryotes . Recent molecular surveys of the smallest eukaryotic planktonic fractions at different oceanic surface regions and in deep-sea Antarctic samples revealed an astonishing protist diversity . Many of the phylotypes found in the photic region affiliate with photosynthetic groups that are known to contain picoeukaryotic representatives in the range 1-2 microm . Surprisingly, a vast diversity of presumably heterotrophic or mixotrophic lineages is also found . Among these, several novel lineages of heterokonts, and a large diversity of alveolates clustering in two major groups (Groups I and II), are present at all depths in the water column . Many of these new phylotypes appear biogeographically ubiquitous . These initial studies suggest that a wide diversity of small eukaryotes remains to be discovered not only in the ocean but also in other environments . For both ecology and evolutionary studies, it is predicted that environmental molecular identification of eukaryotes will have a profound impact in the immediate future.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 163 - 4 Epub 2001 Dec 21.
Crystallization and preliminary X-ray diffraction analysis of monkey dimeric dihydrodiol dehydrogenase; El-Kabbani O et al.; Dihydrodiol dehydrogenase catalyzes the NADP(+)-linked oxidation of trans-dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues . The dimeric form of mammalian dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins . Monkey kidney dimeric dihydrodiol dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method . The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6(1)22 or P6(5)22 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, alpha = beta = 90, gamma = 120 degrees.

Sci STKE . 2001 Feb 27;2001(71):PE1.
A progress report on translational control in eukaryotes; Kozak M; An extraordinary amount of regulation goes into making sure that protein expression is controlled correctly . Several layers of regulation function to achieve the proper levels and proper timing of protein expression . Much is known about the protein machinery involved in translation, but we are lagging behind in understanding the mechanisms of control in eukaryotes . Kozak reviews the new second edition of Translational Control of Gene Expression, which attempts to catalog the mechanisms used by prokaryotes and eukaryotes, and the viruses that infect them and subvert their translational machinery.

Sci STKE . 2000 Dec 12;2000(62):PE1.
Regulation of enzymatic activity and gene expression by membrane fluidity; Los DA et al.; Changes in the cellular environment can lead to alterations in the fluidity of the membranes of prokaryotes and eukaryotes . Changes in temperature and osmotic conditions are two of the best-studied stresses that can affect membrane fluidity . Los and Murata discuss the types of sensors that detect these changes in membrane fluidity and the types of signals that are generated.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15078 - 83
The double par locus of virulence factor pB171: DNA segregation is correlated with oscillation of ParA; Ebersbach G et al.; Prokaryotic plasmids and chromosomes encode partitioning (par) loci that segregate DNA to daughter cells before cell division . Recent database analyses showed that almost all known par loci encode an ATPase and a DNA-binding protein, and one or more cis-acting regions where the proteins act . All par-encoded ATPases belong to one of two protein superfamilies, Walker-type and actin-like ATPases . This property was recently used to divide par loci into Types I and II loci . We show here that the Escherichia coli virulence factor pB171 encodes a double par locus that consists of one Type I and one Type II locus . Separately, each locus stabilized a test-plasmid efficiently . Together, the two loci mediated even more efficient plasmid stabilization . The par loci have a unique genetic organization in that they share a common central region at which the two different DNA-binding proteins probably act . Interestingly, a fusion protein consisting of the Walker-type ParA ATPase and Gfp was functional and oscillated in nucleoid regions on a time scale of minutes . ParA-green fluorescent protein (Gfp) oscillation depended on both ParB and parC but was independent of minCDE . Point mutations in the Walker A box motif simultaneously abolished plasmid stabilization and ParA-Gfp oscillation . These observations raise the possibility that ParA oscillation is prerequisite for active plasmid segregation.

Nucleic Acids Res, 2002 Jan 1, 30(1), 349 - 50
Sentra, a database of signal transduction proteins; Maltsev N et al.; Sentra is a database of signal transduction proteins with the emphasis on microbial signal transduction . The database was updated to include classes of signal transduction systems modulated by either phosphorylation or methylation reactions such as PAS proteins and serine/threonine kinases, as well as the classical two-component histidine kinases and methyl-accepting chemotaxis proteins . Currently, Sentra contains signal transduction proteins from 43 completely sequenced prokaryotic genomes as well as sequences from SWISS-PROT and TrEMBL . Signal transduction proteins are annotated with information describing conserved domains, paralogous and orthologous sequences, and conserved chromosomal gene clusters . The newly developed user interface supports flexible search capabilities and extensive visualization of the data.

Nucleic Acids Res, 2002 Jan 1, 30(1), 142 - 4
Comparative Genometrics (CG): a database dedicated to biometric comparisons of whole genomes; Roten CA et al.; The ever increasing rate at which whole genome sequences are becoming accessible to the scientific community has created an urgent need for tools enabling comparison of chromosomes of different species . We have applied biometric methods to available chromosome sequences and posted the results on our Comparative Genometrics (CG) web site . By genometrics, a term coined by Elston and Wilson {GENET: Epidemiol . (1990), 7, 17-19}, we understand a biometric analysis of chromosomes . During the initial phase, our web site displays, for all completely sequenced prokaryotic genomes, three genometric analyses: the DNA walk {Lobry (1999) Microbiology Today, 26, 164-165} and two complementary representations, i.e . the cumulative GC- and TA-skew analyses, capable of identifying, at the level of whole genomes, features inherent to chromosome organization and functioning . It appears that the latter features are taxon-specific . Although primarily focused on prokaryotic chromosomes, the CG web site contains genometric information on paradigm plasmids, phages, viruses and eukaryotic organelles . Relevant data and methods can be readily used by the scientific community for further analyses as well as for tutorial purposes . Our data posted at the CG web site are freely available on the World Wide Web at http://www.unil.ch/comparativegenometrics.

J Virol, 2002 Jan, 76(2), 499 - 506
Localization of the single-stranded RNA-binding domains of bluetongue virus nonstructural protein NS2; Fillmore GC et al.; The S2 gene of bluetongue virus, serotype 17, has been cloned, and the nonstructural protein NS2 has been expressed . Synthetic peptides matching regions within the amino acid sequence of NS2 were used to map three single-stranded RNA (ssRNA)-binding regions within the protein . A prokaryotic expression system was then used to generate a series of deletion mutants with the ssRNA-binding domains of NS2 removed, singly and in different combinations . These truncated proteins were expressed on a large scale and purified to near homogeneity . The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays . As a result, the three ssRNA-binding domains of BTV nonstructural protein NS2 have been conclusively localized, and removal of these three domains completely abrogates the ability of NS2 to bind to ssRNA.

J Biol Chem, 2002 Mar 8, 277(10), 8524 - 30 Epub 2001 Dec 19.
Structure of human NMN adenylyltransferase . A key nuclear enzyme for NAD homeostasis; Garavaglia S et al.; Nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase alpha/beta-phosphodiesterases superfamily, catalyzes a universal step (NMN + ATP = NAD + PP(i)) in NAD biosynthesis . Localized within the nucleus, the activity of the human enzyme is greatly altered in tumor cells, rendering it a promising target for cancer chemotherapy . By using a combination of single isomorphous replacement and density modification techniques, the human NMNAT structure was solved by x-ray crystallography to a 2.5-A resolution, revealing a hexamer that is composed of alpha/beta-topology subunits . The active site topology of the enzyme, analyzed through homology modeling and structural comparison with other NMNATs, yielded convincing evidence for a substrate-induced conformational change . We also observed remarkable structural conservation in the ATP-recognition motifs GXXXPX(T/H)XXH and SXTXXR, which we take to be the universal signature for NMNATs . Structural comparison of human and prokaryotic NMNATs may also lead to the rational design of highly selective antimicrobial drugs.

Bioinformatics, 2001 Dec, 17(12), 1123 - 30
A probabilistic method for identifying start codons in bacterial genomes; Suzek BE et al.; As the pace of genome sequencing has accelerated, the need for highly accurate gene prediction systems has grown . Computational systems for identifying genes in prokaryotic genomes have sensitivities of 98-99% or higher (Delcher et al., Nucleic Acids Res., 27, 4636-4641, 1999) . These accuracy figures are calculated by comparing the locations of verified stop codons to the predictions . Determining the accuracy of start codon prediction is more problematic, however, due to the relatively small number of start sites that have been confirmed by independent, non-computational methods . Nonetheless, the accuracy of gene finders at predicting the exact gene boundaries at both the 5' and 3' ends of genes is of critical importance for microbial genome annotation, especially in light of the important signaling information that is sometimes found on the 5' end of a protein coding region . In this paper we propose a probabilistic method to improve the accuracy of gene identification systems at finding precise translation start sites . The new system, RBSfinder, is tested on a validated set of genes from Escherichia coli, for which it improves the accuracy of start site locations predicted by computational gene finding systems from the range 67-77% to 90% correct.

FEMS Microbiol Lett, 2001 Dec 18, 205(2), 329 - 35
Epsilon-proteobacterial diversity from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge; Corre E et al.; The prokaryotic phylogenetic diversity was determined for a sample associated with an in situ growth chamber deployed for 5 days on a Mid-Atlantic Ridge hydrothermal vent (23 degrees 22'N, 44 degrees 57'W) . The DNA was extracted from the sample and the 16S rDNA amplified by PCR . No Archaea were detected in the sample . Eighty-seven clones containing bacterial 16S rDNA inserts were selected . Based on restriction fragment length polymorphism analysis, 47 clones were unique, however, based on comparative sequence analysis some of these were very similar, and thus only 22 clones were selected for full sequence and phylogenetic analysis . The phylotypes were dominated by epsilon-Proteobacteria (66%) . The remainder formed a novel lineage within the Proteobacteria (33%) . One clone formed a distinct deeply branching lineage, and was a distant relative of the Aquificales . This report further expands the growing evidence that epsilon-Proteobacteria are important members in biogeochemical cycling at deep-sea hydrothermal ecosystems, participating as epibionts and free living bacteria.

Biochim Biophys Acta, 2001 Dec 12, 1541(1-2), 120 - 34
Function of a chloroplast SRP in thylakoid protein export; Eichacker LA et al.; Protein export systems derived from prokaryotes are used to transport proteins into or across the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoid membrane . Signal recognition particle (SRP) and its receptor are essential components used exclusively for cotranslational export of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and export of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes . An organellar SRP in chloroplasts (cpSRP) participates in cotranslational targeting of chloroplast synthesized integral thylakoid proteins . Remarkably, cpSRP is also used to posttranslationally localize a subset of nuclear encoded thylakoid proteins . Recent work has begun to reveal the basis for cpSRP's unique ability to function in co- and posttranslational protein localization, yet much is left to question . This review will attempt to highlight these advances and will also focus on the role of other soluble and membrane components that are part of this novel organellar SRP targeting pathway.

Biochim Biophys Acta, 2001 Dec 12, 1541(1-2), 80 - 90
Post-translational protein translocation into thylakoids by the Sec and DeltapH-dependent pathways; Mori H et al.; Two distinct protein translocation pathways that employ hydrophobic signal peptides function in the plant thylakoid membrane . These two systems are precursor specific and distinguished by their energy and component requirements . Recent studies have shown that one pathway is homologous to the bacterial general export system called Sec . The other one, called the DeltapH-dependent pathway, was originally considered to be unique to plant thylakoids . However, it is now known that homologous transport systems are widely present in prokaryotes and even present in archaea . Here we review these protein transport pathways and discuss their capabilities and mechanisms of operation.

Biochim Biophys Acta, 2001 Dec 12, 1541(1-2), 64 - 79
Toc, Tic, and chloroplast protein import; Jarvis P et al.; The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes . Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides . Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way . They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex . The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane . Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively . The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process . The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro . Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.

J Biol Chem, 2002 Feb 15, 277(7), 4959 - 65 Epub 2001 Dec 03.
The domain organization and properties of individual domains of DNA topoisomerase V, a type 1B topoisomerase with DNA repair activities; Belova GI et al.; Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase . It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities . The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C . The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain . Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized . Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations . Topo34 has strong DNA-binding ability but shows no topoisomerase activity . Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair . Thus, Topo V has at least two active sites capable of processing AP DNA . The significance of multiple HhH motifs for the Topo V processivity is discussed.

Curr Biol, 2001 Dec 11, 11(24), R1022 - 4
Molecular evolution: actin's long lost relative found; Egelman EH; The bacterial protein MreB has been identified as a prokaryotic homolog of the eukaryotic cytoskeletal protein actin . While we still know little about MreB's function, the structural similarities and differences between MreB and actin provide more insight into the remarkable properties of actin.

Environ Mol Mutagen, 2001, 38(2-3), 105 - 10
Why do cells have multiple error-prone DNA polymerases?
Friedberg EC.
Recent years have witnessed the emergence of a plethora of so-called novel DNA polymerases in both eukaryotic and prokaryotic cells . Many of these DNA polymerases are characterized by poor replicational fidelity and low processivity, and are devoid of 3' --> 5' exonuclease activity . This article describes the discovery of these error-prone polymerases and what is known about their biological function .

J Cell Biochem, 2001, 81(S36), 117 - 128
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 .

Bioessays, 2001 Dec, 23(12), 1148 - 58
Bacterial ion channels and their eukaryotic homologues; Koprowski P et al.; Due to the relative ease of obtaining their crystal structures, bacterial ion channels provide a unique opportunity to analyse structure and function of their eukaryotic homologues . This review describes prokaryotic channels whose structures have been determined . These channels are KcsA, a bacterial homologue of eukaryotic potassium channels, MscL, a bacterial mechanosensitive ion channel and ClC0, a prokaryotic homologue of the eukaryotic ClC family of anion-selective channels . General features of their structure and function are described with a special emphasis on the advantages that these channels offer for understanding the properties of their eukaryotic homologues . We present amino-acid sequences of eukaryotic proteins related in their primary sequences to bacterial mechanosensitive channels . The usefulness of bacterial mechanosensitive channels for the studies on general principles of mechanosensation is discussed .

Biochem J, 2002 Jan 1, 361(Pt 1), 41 - 7
The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins; Oberer M et al.; NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2 . ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter P(parDE) and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE . Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and (3)J(Halpha-NH) scalar coupling constants showed that the N-terminal domain of ParD consists of a short beta-ribbon followed by three alpha-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors . (15)N longitudinal (T(1)) and transverse (T(2)) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain . An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein . This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins.

Science, 2001 Dec 14, 294(5550), 2372 - 5
A prokaryotic voltage-gated sodium channel; Ren D et al.; The pore-forming subunits of canonical voltage-gated sodium and calcium channels are encoded by four repeated domains of six-transmembrane (6TM) segments . We expressed and characterized a bacterial ion channel (NaChBac) from Bacillus halodurans that is encoded by one 6TM segment . The sequence, especially in the pore region, is similar to that of voltage-gated calcium channels . The expressed channel was activated by voltage and was blocked by calcium channel blockers . However, the channel was selective for sodium . The identification of NaChBac as a functionally expressed bacterial voltage-sensitive ion-selective channel provides insight into both voltage-dependent activation and divalent cation selectivity.

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