Prokaryotes

Plant Physiol, 2003 Jun, 132(2), 748 - 56 Epub 2003 Apr 17.
Expression of antisense acyl carrier protein-4 reduces lipid content in Arabidopsis leaf tissue; Branen JK et al.; Arabidopsis plants were transformed with acyl carrier protein (ACP)-4 in antisense conformation driven by the cauliflower mosaic virus 35S promoter . It was hypothesized that reduction of ACP4 in leaf tissue would result in a reduction in lipid biosynthesis and, in addition, affect fatty acid composition and leaf physiology . Several transgenic lines have been generated with reduced ACP4 protein in leaf tissue . Dramatic reductions in ACP4 resulted in a reduction of leaf lipid content (22%-60%) based on fresh leaf weight and a bleached appearance and reduced photosynthetic efficiency . In addition, a decrease in 16:3 as a percentage of the total fatty acid composition was noted . There were no changes in leaf lipid class distribution; however, there was a decrease in the relative amount of 16:3 in monogalactosyldiacylglycerol . These results suggest that ACP4 plays a major role in the biosynthesis of fatty acids for chloroplast membrane development . Alterations in the ACP isoform profile of Arabidopsis leaf also appear to alter the flow of fatty acids between the prokaryotic and eukaryotic pathways for assembly of galactolipids . However, it has not yet been determined if the changes in fatty acid composition are due to changes in the profile of ACP isoforms, or if they are actually a reaction to a reduction in fatty acid precursors.

Biochem Biophys Res Commun, 2003 Jun 27, 306(2), 530 - 6
Characterization of quercetin binding site on DNA gyrase; Plaper A et al.; Gyrases are DNA topology modifying enzymes present only in prokaryotes which makes them an attractive target for antibacterial drugs . Quercetin, one of the most abundant natural flavonoids, inhibits supercoiling activity of bacterial gyrase and induces DNA cleavage . It has been generally assumed that the mechanism of flavonoid inhibition is based on interaction with DNA . We show that quercetin binds to the 24 kDa fragment of gyrase B of Escherichia coli with a K(D) value of 15 microM and inhibits ATPase activity of gyrase B . Its binding site overlaps with ATP binding pocket and could be competitively replaced by either ATP or novobiocin . The structural model of quercetin-gyrase complex was prepared, based on the close similarity with ATP and quercetin binding sites of the src family tyrosine kinase Hck . We propose that quercetin inhibits gyrases through two different mechanisms based either on interaction with DNA or with ATP binding site of gyrase.

BMC Genomics . 2003 Jun 12;4(1):23.
The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy; Postier BL et al.; BACKGROUND: DNA microarrays are a powerful tool with important applications such as global gene expression profiling . Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g . over-expression of protein) . We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project . RESULTS: Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp . PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis . The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs . Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes . The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface . We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile . The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs . The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress . CONCLUSIONS: The technique detailed here minimizes the cost and effort to replicate a PCR-generated DNA gene fragment library and facilitates several downstream processes (e.g . directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling.

J Pept Sci, 2003 May, 9(5), 300 - 11
Antitumour activity and specificity as a function of substitutions in the lipophilic sector of helical lactoferrin-derived peptide; Yang N et al.; A peptide L5 (PAWRKAFRWAWRMLKKAA), derived from the N-terminal alpha-helical region of bovine lactoferrin (LFB 14-31), that is highly active against several tumour cell lines was reported earlier . In this study, a number of L5 analogues were designed in order to investigate how subsequent replacements of the aromatic amino acids in L5 with three amino acids representing different structural parameters influenced antitumour activity and tumour cell specificity relative to normal human cells . The Trp residues were substituted by Lys, Ile or Ala, while the Phe residue was substituted with Ala . The resulting peptides were investigated for their activity against prokaryotic cells, four tumour cell lines, human lung fibroblasts and human erythrocytes . Most of the peptides were highly active against both E . coli and S . aureus . The peptides were more active against the tumour cell lines than against normal eukaryotic cells but the activity against normal fibroblasts varied more among the peptides than did their antitumour activities . The results revealed that aromatic residues located opposite the cationic sector in L5 were more critical for antitumour activity than were aromatic residues located adjacent to the cationic sector . The biological responses for the peptides against tumour cell lines, fibroblasts, S . aureus (but not E . coli), were highly correlated with the amino acid descriptors used in our QSAR model . The result obtained from the QSAR study identified specific structural features that were important for lytic activity and membrane specificity . Certain structural properties in positions 3, 9 and 11 were shown to be important for antitumour activity, while additional structural properties in position 7 were found to be important with respect to tumour cell specificity . This information may offer a possibility for de novo design of an antitumour peptide with an improved therapeutic index.

J Biochem (Tokyo), 2003 May, 133(5), 671 - 7
Enzymological characterization of EpoA, a laccase-like phenol oxidase produced by Streptomyces griseus; Endo K et al.; Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water . While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known . Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus . EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA) . Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3) . Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates . Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM . Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity . The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively . The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min . EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.

Genome Biol . 2003;4(6):R38 . Epub 2003 May 28.
A phylogenetic study of cytochrome b561 proteins; Verelst W et al.; BACKGROUND: As an antioxidant and cofactor to numerous metabolic enzymes, ascorbate has an essential role in plants and animals . Cytochromes b561 constitute a class of intrinsic membrane proteins involved in ascorbate regeneration . Despite their importance in ascorbate metabolism, no evolutionary analysis has been presented so far on this newly described protein family . RESULTS: Cytochromes b561 have been identified in a large number of phylogenetically distant species, but are absent in fungi and prokaryotes . Most species contain three or four cytochrome b561 paralogous proteins, and the encoding genes usually have four or five exons . At the protein level, sequence similarities are rather low between cytochromes b561 within a single species (34-45% identity), and among phylogenetically distant species (around 30% identity) . However, particular structural features characterizing this protein family are well conserved in members from all species investigated . These features comprise six transmembrane helices, four strictly conserved histidine residues, probably coordinating the two heme molecules, and putative ascorbate and monodehydro-ascorbate (MDHA) substrate-binding sites . Analysis of plant cytochromes b561 shows a separation between those from monocotyledonous and dicotyledonous species in a phylogenetic tree . CONCLUSIONS: All cytochromes b561 have probably evolved from a common ancestral protein before the separation of plants and animals . Their phyletic distribution mirrors the use of ascorbate as primary antioxidant, indicating their role in ascorbate homeostasis and antioxidative defense . In plants, the differentiation into four cytochrome b561 isoforms probably occurred before the separation between monocots and dicots.

Microb Ecol . 2003 Jun 13; {Epub ahead of print}
Structure and Seasonal Dynamics of Hyporheic Zone Microbial Communities in Free-Stone Rivers of the Western United States; Feris KP et al.; The hyporheic zone of a river is characterized by being nonphotic, exhibiting chemical/redox gradients, and having a heterotrophic food web based on the consumption of organic carbon entrained from surface waters . Hyporheic microbial communities constitute the base of food webs in these environments and are important for maintaining a functioning lotic ecosystem . While microbial communities of rivers dominated by fine-grained sediments are relatively well studied, little is known about the structure and seasonal dynamics of microbial communities inhabiting the predominantly gravel and cobble hyporheic zones of rivers of the western United States . Here, we present the first molecular analysis of hyporheic microbial communities of three different stream types (based on mean base discharge, substratum type, and drainage area), in Montana . Utilizing 16S rDNA phylogeny, DGGE pattern analysis, and qPCR, we have analyzed the prokaryotic communities living on the 1.7 to 2.36 mm grain-size fraction of hyporheic sediments from three separate riffles in each stream . DGGE analysis showed clear seasonal community patterns, indicated similar community composition between different riffles within a stream (95.6-96.6% similarity), and allowed differentiation between communities in different streams . Each river supported a unique complement of species; however, several phylogenetic groups were conserved between all three streams including Pseudomonads and members of the genera Aquabacterium, Rhodoferax, Hyphomicrobium, and Pirellula . Each group showed pronounced seasonal trends in abundance, with peaks during the Fall . The Hyphomicrobium group was numerically dominant throughout the year in all three streams . This work provides a framework for investigating the effects of various environmental factors and anthropogenic effects on microbial communities inhabiting the hyporheic zone.

Res Microbiol, 2003 May, 154(4), 289 - 94
Evolutionary insights from studies on viruses of hyperthermophilic archaea; Prangishvili D; The morphological diversity of viruses which parasitize hyperthermophilic archaea thriving at temperatures > or = 80 degrees C appears to exceed that of viruses of prokaryotes living at lower temperatures . Based on assumptions of the existence of viruses in the prebiotic phase of evolution and hot origins of cellular life, we suggest that this remarkable diversity could have its source in ancestral diversity of viral morphotypes in hot environments . Attempts are made to trace evolutionary relationships of viruses of hyperthermophilic archaea with other viruses.

Comput Biol Chem, 2003 Feb, 27(1), 49 - 58
Automated annotation of microbial proteomes in SWISS-PROT; Gattiker A et al.; Large-scale sequencing of prokaryotic genomes demands the automation of certain annotation tasks currently manually performed in the production of the SWISS-PROT protein knowledgebase . The HAMAP project, or 'High-quality Automated and Manual Annotation of microbial Proteomes', aims to integrate manual and automatic annotation methods in order to enhance the speed of the curation process while preserving the quality of the database annotation . Automatic annotation is only applied to entries that belong to manually defined orthologous families and to entries with no identifiable similarities (ORFans) . Many checks are enforced in order to prevent the propagation of wrong annotation and to spot problematic cases, which are channelled to manual curation . The results of this annotation are integrated in SWISS-PROT, and a website is provided at http://www.expasy.org/sprot/hamap/.

Infect Genet Evol, 2002 Jul, 1(4), 297 - 301
Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase; Kedzierski L et al.; Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes . Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis . The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment . Furthermore, it has been shown that ASL is not subject to positive natural selection . We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P . falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium . The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution . Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships . We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed . Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa.

Plant J, 2003 Jun, 34(6), 856 - 67
The Arabidopsis thaliana CUTA gene encodes an evolutionarily conserved copper binding chloroplast protein; Burkhead JL et al.; The Arabidopsis thaliana CUTA gene encodes a 182-amino-acid-long putative precursor of a chloroplast protein with high sequence similarity to evolutionarily conserved prokaryotic proteins implicated in copper tolerance . Northern analysis indicates that AtCUTA mRNA is expressed in all major tissue types . Analysis of cDNA clones and RT-PCR with total mRNA revealed alternative splicing of AtCUTA by retention of an intron . The intron-containing mRNA encodes a truncated 156-amino-acid protein as a result of stop codons in the included intron . The sequence of AtCutAp encoded by the fully spliced transcript suggests that the precursor consists of three domains: an N-terminal chloroplast transit sequence of 70 residues, followed by a domain with prokaryotic signal-sequence-like characteristics and finally the most conserved C-terminal domain . The N-terminal chloroplast transit sequence was functional to route a passenger protein into isolated pea chloroplasts with possible sorting to the envelope . Chloroplast localization was confirmed by Western blot analysis of isolated and fractionated chloroplasts . Recombinant AtCutA protein was expressed in Escherichia coli without the N-terminal 70-amino-acid chloroplast transit sequence . This recombinant AtCutAp was routed to the bacterial periplasm of E . coli . Purified recombinant AtCutAp is tetrameric and selectively binds Cu(II) ions with an affinity comparable to that reported for mammalian prion proteins.

Structure (Camb), 2003 Jun, 11(6), 677 - 90
Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-complementing protein; Mathews II et al.; Like thymidylate synthase (TS) in eukaryotes, the thymidylate synthase-complementing proteins (TSCPs) are mandatory for cell survival of many prokaryotes in the absence of external sources of thymidylate . Details of the mechanism of this novel family of enzymes are unknown . Here, we report the structural and functional analysis of a TSCP from Thermotoga maritima and its complexes with substrate, analogs, and cofactor . The structures presented here provide a basis for rationalizing the TSCP catalysis and reveal the possibility of the design of an inhibitor . We have identified a new helix-loop-strand FAD binding motif characteristic of the enzymes in the TSCP family . The presence of a hydrophobic core with residues conserved among the TSCP family suggests a common overall fold.

Parasite Immunol, 2003 Feb, 25(2), 69 - 77
Characterization of a recombinant immunomodulatory protein from the salivary glands of Dermacentor andersoni; Alarcon-Chaidez FJ et al.; The gene encoding a 36-kDa (p36) immunomodulatory protein present in saliva of Dermacentor andersoni was cloned in prokaryotic and eukaryotic expression vectors . A polymerase chain reaction (PCR)-generated cDNA lacking signal peptide was cloned into the Escherichia coli expression vector pET28 and a similar sequence was cloned into pIB/V5-His-TOPO expression vector for stable transfection of insect cells, High 5 trade mark . The 26-kDa molecular mass of p36 expressed by bacteria is in agreement with that predicted from the translated full-length cDNA sequence . Eukaryotic-cell-expressed p36 consisted of multiple forms with molecular masses between 34 and 36 kDa . These multiple forms were attributed to differences in post-translational modifications . N-linked mannose was detected on insect-cell-expressed and tick-derived p36 . Multiple bands remained after endoglycosidase removal of N-linked sugars, indicating the presence of other modifications . Both bacterial- and insect-cell-expressed p36 reacted on immunoblots with polyclonal antibodies raised against tick-derived p36 . Insect-cell-expressed p36 suppressed T-lymphocyte-mitogen-driven in vitro proliferation of splenocytes from tick-naive mice in a dose-dependent manner . Bacterial-cell-expressed p36 lacked immunomodulatory activity.

Biotechnol Prog, 2003 May-Jun, 19(3), 727 - 33
New system for positive selection of recombinant plasmids and dual expression in yeast and bacteria based on the restriction ribonuclease RegB; Saida F et al.; By coupling the toxic restriction endoribonuclease RegB, from the bacteriophage T4, to the prokaryotic T7 and the eukaryotic GAL1 promoters, we constructed a two-function plasmid called pTOXR-1 . This plasmid is a zero-background cloning vector . It allows an efficient positive selection of recombinant plasmids without the need to completely digest, dephosphorylate, or purify the vector prior to the ligation step . The pTOXR-1 positive selection system requires no special Escherichia coli strains, no special culture media, and no addition of inducer to the selective plates . In addition, since this vector carries all signals required for both prokaryotic and eukaryotic expression, it allows the overproduction of heterologous proteins, fused to a polyhistidine tag, in the bacterium E . coli and in the yeast Saccharomyces cerevisiae from a single plasmid . Hence, this vector may be a useful time-saving tool for one-step cloning and versatile protein expression in bacteria and yeast.

Appl Environ Microbiol, 2003 Jun, 69(6), 3663 - 7
Linkage of high rates of sulfate reduction in Yellowstone hot springs to unique sequence types in the dissimilatory sulfate respiration pathway; Fishbain S et al.; Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment . Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89 degrees C . Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.

Appl Environ Microbiol, 2003 Jun, 69(6), 3181 - 91
Analysis of the sulfate-reducing bacterial and methanogenic archaeal populations in contrasting Antarctic sediments; Purdy KJ et al.; The distribution and activity of communities of sulfate-reducing bacteria (SRB) and methanogenic archaea in two contrasting Antarctic sediments were investigated . Methanogenesis dominated in freshwater Lake Heywood, while sulfate reduction dominated in marine Shallow Bay . Slurry experiments indicated that 90% of the methanogenesis in Lake Heywood was acetoclastic . This finding was supported by the limited diversity of clones detected in a Lake Heywood archaeal clone library, in which most clones were closely related to the obligate acetate-utilizing Methanosaeta concilii . The Shallow Bay archaeal clone library contained clones related to the C(1)-utilizing Methanolobus and Methanococcoides and the H(2)-utilizing Methanogenium: Oligonucleotide probing of RNA extracted directly from sediment indicated that archaea represented 34% of the total prokaryotic signal in Lake Heywood and that Methanosaeta was a major component (13.2%) of this signal . Archaea represented only 0.2% of the total prokaryotic signal in RNA extracted from Shallow Bay sediments . In the Shallow Bay bacterial clone library, 10.3% of the clones were SRB-like, related to Desulfotalea/Desulforhopalus, Desulfofaba, Desulfosarcina, and Desulfobacter as well as to the sulfur and metal oxidizers comprising the Desulfuromonas cluster . Oligonucleotide probes for specific SRB clusters indicated that SRB represented 14.7% of the total prokaryotic signal, with Desulfotalea/Desulforhopalus being the dominant SRB group (10.7% of the total prokaryotic signal) in the Shallow Bay sediments; these results support previous results obtained for Arctic sediments . Methanosaeta and Desulfotalea/Desulforhopalus appear to be important in Lake Heywood and Shallow Bay, respectively, and may be globally important in permanently low-temperature sediments.

Mutat Res, 2003 Jun, 543(3), 235 - 49
The evolution of cell death programs as prerequisites of multicellularity; Huettenbrenner S et al.; One of the hallmarks of multicellularity is that the individual cellular fate is sacrificed for the benefit of a higher order of life-the organism . The accidental death of cells in a multicellular organism results in swelling and membrane-rupture and inevitably spills cell contents into the surrounding tissue with deleterious effects for the organism . To avoid this form of necrotic death the cells of metazoans have developed complex self-destruction mechanisms, collectively called programmed cell death, which see to an orderly removal of superfluous cells . Since evolution never invents new genes but plays variations on old themes by DNA mutations, it is not surprising, that some of the genes involved in metazoan death pathways apparently have evolved from homologues in unicellular organisms, where they originally had different functions . Interestingly some unicellular protozoans have developed a primitive form of non-necrotic cell death themselves, which could mean that the idea of an altruistic death for the benefit of genetically identical cells predated the invention of multicellularity . The cell death pathways of protozoans, however, show no homology to those in metazoans, where several death pathways seem to have evolved in parallel . Mitochondria stands at the beginning of several death pathways and also determines, whether a cell has sufficient energy to complete a death program . However, the endosymbiotic bacterial ancestors of mitochondria are unlikely to have contributed to the recent mitochondrial death machinery and therefore, these components may derive from mutated eukaryotic precursors and might have invaded the respective mitochondrial compartments . Although there is no direct evidence, it seems that the prokaryotic-eukaryotic symbiosis created the space necessary for sophisticated death mechanisms on command, which in their distinct forms are major factors for the evolution of multicellular organisms.

Mol Microbiol, 2003 Jun, 48(5), 1389 - 400
RelE toxins from bacteria and Archaea cleave mRNAs on translating ribosomes, which are rescued by tmRNA; Christensen SK et al.; RelE of Escherichia coli is a global inhibitor of translation that is activated by nutritional stress . Activation of RelE depends on Lon-mediated degradation of RelB, the antagonist that neutralizes RelE . In vitro, RelE cleaves synthetic mRNAs positioned at the ribosomal A-site . We show here that in vivo overexpression of RelE confers cleavage of mRNA and tmRNA in their coding regions . RelE-mediated cleavage depended on translation of the RNAs and occurred at both sense and stop codons . RelE cleavage of mRNA and tmRNA was also induced by amino acid starvation . An ssrA deletion strain was hypersensitive to RelE, whereas overproduction of tmRNA counteracted RelE toxicity . After neutralization of RelE by RelB, rapid recovery of translation required tmRNA, indicating that tmRNA alleviated RelE toxicity by rescuing ribosomes stalled on damaged mRNAs . RelE proteins from Gram-positive Bacteria and Archaea cleaved tmRNA with a pattern similar to that of E . coli RelE, suggesting that the function and target of RelE may be conserved across the prokaryotic domains.

Mol Microbiol, 2003 Jun, 48(5), 1157 - 69
Translational repression mechanisms in prokaryotes; Schlax PJ et al.; Translational repression results from a complex choreography of macromolecular interactions interfering with the formation of translational initiation complexes . The relationship between the rate and extent of formation of these interactions to form repressed mRNA complexes determines the extent of repression . A novel analysis of repression mechanisms is presented here and it indicates that the reversibility of repressed complex formation influences the steady state balance of the distribution of translationally active and inactive complexes and therefore has an impact on the efficiency of repression . Reviewed here is evidence for three distinct translational repression mechanisms, regulating expression of the transcription factor sigma32, threonine tRNA synthetase and ribosomal proteins on the alpha operon in Escherichia coli . Efficient regulation of expression in these systems makes use of specific mRNA structures in quite different ways.

J Am Chem Soc, 2003 May 21, 125(20), 6078 - 84
Iron-sulfur cluster biosynthesis . Characterization of frataxin as an iron donor for assembly of {2Fe-2S} clusters in ISU-type proteins; Yoon T et al.; ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of {2Fe-2S} centers prior to delivery to an apo target protein . The intermediate {2Fe-2S} ISU-bound cluster is formed by delivery of iron and sulfur to the apo ISU, with the latter delivered through an IscS-mediated reaction . The identity of the iron donor has thus far not been established . In this paper we demonstrate human frataxin to bind from six to seven iron ions . Iron binding to frataxin has been quantitated by iron-dependent fluorescence measurements {K(D)(Fe(3+)) approximately 11.7 microM; (K(D)(Fe(2+)) approximately 55.0 microM} and isothermal titration calorimetry (ITC) {K(D)(Fe(3+)) approximately 10.2 microM} . Enthalpies and entropies for ferric ion binding were determined from calorimetric measurements . Both fluorescence (K(D) 0.45 microM) and ITC measurements (K(D) 0.15 microM) demonstrate holo frataxin to form a complex with ISU with sub-micromolar binding affinities . Significantly, apo frataxin does not bind to ISU, suggesting an important role for iron in cross-linking the two proteins and/or stabilizing the structure of frataxin that is recognized by ISU . Holo frataxin is also shown to mediate the transfer of iron from holo frataxin to nucleation sites for {2Fe-2S} cluster formation on ISU . We have demonstrated elsewhere {J . Am . Chem . Soc . 2002, 124, 8774-8775} that this iron-bound form of ISU is viable for assembly of holo ISU, either by subsequent addition of sulfide or by NifS-mediated sulfur delivery . Provision of holo frataxin and inorganic sulfide is sufficient for cluster assembly in up to 70% yield . With NifS as a sulfur donor, yields in excess of 70% of holo ISU were obtained . Both UV-vis and CD spectroscopic characteristics were found to be consistent with those of previously characterized ISU proteins . The time course for cluster assembly was monitored from the 456 nm absorbance of holo ISU formed during the {2Fe-2S} cluster assembly reaction . A kinetic rate constant k(obs) approximately 0.075 min(-)(1) was determined with 100 microM ISU, 2.4 mM Na(2)S, and 40 microM holo frataxin in 50 mM Tris-HCl (pH 7.5) with 4.3 mM DTT . Similar rates were obtained for NifS-mediated sulfur delivery, consistent with iron release from frataxin as a rate-limiting step in the cluster assembly reaction.

Sci STKE . 2003 Jun 03;2003(185):pe22.
Suboperonic regulatory signals; Adhya S; In prokaryotes, the genome is necessarily small in size, thus creating challenges for gene regulation . Adhya discusses how polycistronic operons can be regulated at the suboperonic level to allow genes to be independently regulated within an operon . This permits the cells to respond to different environmental conditions and allows the genes within operons to encode proteins involved in divergent cellular processes and still be regulated according to the cell's needs . Suboperonic control leads to discoordinate gene expression and can occur through transcriptional regulatory events or translational regulatory events mediated by proteins or cis- or trans-acting RNAs.

J Biol Chem, 2003 Aug 15, 278(33), 30497 - 505 Epub 2003 Jun 03.
Analysis of the open region and of DNA-protein contacts of archaeal RNA polymerase transcription complexes during transition from initiation to elongation; Spitalny P et al.; The archaeal transcriptional machinery is polymerase II (pol II)-like but does not require ATP or TFIIH for open complex formation . We have used enzymatic and chemical probes to follow the movement of Pyrococcus RNA polymerase (RNAP) along the glutamate dehydrogenase gene during transcription initiation and transition to elongation . RNAP was stalled between registers +5 and +20 using C-minus cassettes . The upstream edge of RNAP was in close contact with the archaeal transcription factors TATA box-binding protein/transcription factor B in complexes stalled at position +5 . Movement of the downstream edge of the RNAP was not detected by exonuclease III footprinting until register +8 . A first structural transition characterized by movement of the upstream edge of RNAP was observed at registers +6/+7 . A major transition was observed at registers +10/+11 . In complexes stalled at these positions also the downstream edge of RNA polymerase started translocation, and reclosure of the initially open complex occurred indicating promoter clearance . Between registers +11 and +20 both RNAP and transcription bubble moved synchronously with RNA synthesis . The distance of the catalytic center to the front edge of the exo III footprint was approximately 12 nucleotides in all registers . The size of the RNA-DNA hybrid in an early archaeal elongation complex was estimated between 9 and 12 nucleotides . For complexes stalled between positions +10 and +20 the size of the transcription bubble was around 17 nucleotides . This study shows characteristic mechanistic properties of the archaeal system and also similarities to prokaryotic RNAP and pol II.

BMC Bioinformatics . 2003 Jun 03;4(1):21.
EasyGene--a prokaryotic gene finder that ranks ORFs by statistical significance; Larsen TS et al.; BACKGROUND: Contrary to other areas of sequence analysis, a measure of statistical significance of a putative gene has not been devised to help in discriminating real genes from the masses of random Open Reading Frames (ORFs) in prokaryotic genomes . Therefore, many genomes have too many short ORFs annotated as genes . RESULTS: In this paper, we present a new automated gene-finding method, EasyGene, which estimates the statistical significance of a predicted gene . The gene finder is based on a hidden Markov model (HMM) that is automatically estimated for a new genome . Using extensions of similarities in Swiss-Prot, a high quality training set of genes is automatically extracted from the genome and used to estimate the HMM . Putative genes are then scored with the HMM, and based on score and length of an ORF, the statistical significance is calculated . The measure of statistical significance for an ORF is the expected number of ORFs in one megabase of random sequence at the same significance level or better, where the random sequence has the same statistics as the genome in the sense of a third order Markov chain . CONCLUSIONS: The result is a flexible gene finder whose overall performance matches or exceeds other methods . The entire pipeline of computer processing from the raw input of a genome or set of contigs to a list of putative genes with significance is automated, making it easy to apply EasyGene to newly sequenced organisms . EasyGene with pre-trained models can be accessed at http://www.cbs.dtu.dk/services/EasyGene.

Int J Parasitol, 2003 May, 33(5-6), 621 - 40
Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression; Dalton JP et al.; The control of helminth diseases of people and livestock continues to rely on the widespread use of anti-helminthic drugs . However, concerns with the appearance of drug resistant parasites and the presence of pesticide residues in food and the environment, has given further incentive to the goal of discovering molecular vaccines against these pathogens . The exponential rate at which gene and protein sequence information is accruing for many helminth parasites requires new methods for the assimilation and analysis of the data and for the identification of molecules capable of inducing immunological protection . Some promising vaccine candidates have been discovered, in particular cathepsin L proteases from Fasciola hepatica, aminopeptidases from Haemonchus contortus, and aspartic proteases from schistosomes and hookworms, all of which are secreted into the host tissues or into the parasite intestine where they play important roles in host-parasite interactions . Since secreted proteins, in general, are exposed to the immune system of the host they represent obvious candidates at which vaccines could be targeted . Therefore, in this article, we consider the potential values and uses of algorithms for characterising cDNAs amongst the collated helminth genomic information that encode secreted proteins, and methods for their selective isolation and cloning . We also review the variety of prokaryotic and eukaryotic cell expression systems that have been employed for the production and downstream purification of recombinant proteins in functionally active form, and provide an overview of the parameters that must be considered if these recombinant proteins are to be commercialised as vaccine therapeutics in humans and/or animals.

Arch Biochem Biophys, 2003 Jun 15, 414(2), 141 - 9
Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase; Spreitzer RJ; Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon . Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site . However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies . The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood . Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions . Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco . Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity . Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity . It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes.

Trends Microbiol, 2003 May, 11(5), 192 - 5
Is the bacterial ferrous iron transporter FeoB a living fossil?
Hantke K.
The cytoplasmic membrane protein FeoB of Escherichia coli, Helicobacter pylori, Legionella pneumophila and Synechocystis sp . strain PCC 6803 is necessary for Fe(2+) uptake . The C-terminal part of FeoB is predicted to contain 8-12 membrane-spanning helices . The N-terminal domain shows much similarity to eukaryotic and prokaryotic G proteins and, indeed, GTPase activity is necessary for Fe(2+) transport . Four of the five characteristic conserved G protein motifs have been identified in FeoB proteins . Whether FeoB is involved directly, via its Me(2+) binding site, or indirectly in Fe(2+) transport, remains to be investigated.

Chaos, 2001 Mar, 11(1), 142 - 159
Design principles for elementary gene circuits: Elements, methods, and examples; Savageau MA; The control of gene expression involves complex circuits that exhibit enormous variation in design . For years the most convenient explanation for these variations was historical accident . According to this view, evolution is a haphazard process in which many different designs are generated by chance; there are many ways to accomplish the same thing, and so no further meaning can be attached to such different but equivalent designs . In recent years a more satisfying explanation based on design principles has been found for at least certain aspects of gene circuitry . By design principle we mean a rule that characterizes some biological feature exhibited by a class of systems such that discovery of the rule allows one not only to understand known instances but also to predict new instances within the class . The central importance of gene regulation in modern molecular biology provides strong motivation to search for more of these underlying design principles . The search is in its infancy and there are undoubtedly many design principles that remain to be discovered . The focus of this three-part review will be the class of elementary gene circuits in bacteria . The first part reviews several elements of design that enter into the characterization of elementary gene circuits in prokaryotic organisms . Each of these elements exhibits a variety of realizations whose meaning is generally unclear . The second part reviews mathematical methods used to represent, analyze, and compare alternative designs . Emphasis is placed on particular methods that have been used successfully to identify design principles for elementary gene circuits . The third part reviews four design principles that make specific predictions regarding (1) two alternative modes of gene control, (2) three patterns of coupling gene expression in elementary circuits, (3) two types of switches in inducible gene circuits, and (4) the realizability of alternative gene circuits and their response to phased environmental cues . In each case, the predictions are supported by experimental evidence . These results are important for understanding the function, design, and evolution of elementary gene circuits . (c) 2001 American Institute of Physics.

Mol Biol Evol, 2003 Jul, 20(7), 1098 - 112 Epub 2003 May 30.
The molecular evolution of catalatic hydroperoxidases: evidence for multiple lateral transfer of genes between prokaryota and from bacteria into eukaryota; Klotz MG et al.; The past decade has produced an increasing number of reports on horizontal gene transfer between prokaryotic organisms . Only recently, with the flood of available whole genome sequence data and a renewed intensity of the debate about the universal tree of life, a very few reports on lateral gene transfer (LGT) from prokaryotes into the Eukaryota have been published . We have investigated and report here on the molecular evolution of the gene families that encode catalatic hydroperoxidases . We have found that this process included not only frequent horizontal gene transfer among prokaryotes but also several lateral gene transfer events between bacteria and fungi and between bacteria and the protistan ancestor of the alga/plant lineage.

Mol Biol Evol, 2003 Oct, 20(10), 1598 - 602 Epub 2003 May 30.
Horizontal gene transfer accelerates genome innovation and evolution; Jain R et al.; Horizontal gene transfer (HGT) spreads genetic diversity by moving genes across species boundaries . By rapidly introducing newly evolved genes into existing genomes, HGT circumvents the slow step of ab initio gene creation and accelerates genome innovation . However, HGT can only affect organisms that readily exchange genes (exchange communities) . In order to define exchange communities and understand the internal and external environmental factors that regulate HGT, we analyzed approximately 20,000 genes contained in eight free-living prokaryotic genomes . These analyses indicate that HGT occurs among organisms that share similar factors . The most significant are genome size, genome G/C composition, carbon utilization, and oxygen tolerance.

J Biol Chem, 2003 Aug 29, 278(35), 33056 - 9 Epub 2003 May 30.
The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad; Bieganowski P et al.; Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide . NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate . All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia . In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia . Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H . C., and Brenner, C . (2003) J . Biol . Chem . 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1 . Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity . Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues . Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.

J Bacteriol, 2003 Jun, 185(12), 3527 - 37
The Stigmatella aurantiaca homolog of Myxococcus xanthus high-mobility-group A-type transcription factor CarD: insights into the functional modules of CarD and their distribution in bacteria; Cayuela ML et al.; Transcriptional factor CarD is the only reported prokaryotic analog of eukaryotic high-mobility-group A (HMGA) proteins, in that it has contiguous acidic and AT hook DNA-binding segments and multifunctional roles in Myxococcus xanthus carotenogenesis and fruiting body formation . HMGA proteins are small, randomly structured, nonhistone, nuclear architectural factors that remodel DNA and chromatin structure . Here we report on a second AT hook protein, CarD(Sa), that is very similar to CarD and that occurs in the bacterium Stigmatella aurantiaca . CarD(Sa) has a C-terminal HMGA-like domain with three AT hooks and a highly acidic adjacent region with one predicted casein kinase II (CKII) phosphorylation site, compared to the four AT hooks and five CKII sites in CarD . Both proteins have a nearly identical 180-residue N-terminal segment that is absent in HMGA proteins . In vitro, CarD(Sa) exhibits the specific minor-groove binding to appropriately spaced AT-rich DNA that is characteristic of CarD or HMGA proteins, and it is also phosphorylated by CKII . In vivo, CarD(Sa) or a variant without the single CKII phosphorylation site can replace CarD in M . xanthus carotenogenesis and fruiting body formation . These two cellular processes absolutely require that the highly conserved N-terminal domain be present . Thus, three AT hooks are sufficient, the N-terminal domain is essential, and phosphorylation in the acidic region by a CKII-type kinase can be dispensed with for CarD function in M . xanthus carotenogenesis and fruiting body development . Whereas a number of hypothetical proteins homologous to the N-terminal region occur in a diverse array of bacterial species, eukaryotic HMGA-type domains appear to be confined primarily to myxobacteria.

Biochem J, 2003 Aug 15, 374(Pt 1), 247 - 54
Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens; Moll RG; The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail . A . ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes . In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions . Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions . The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP-RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions . The SRP RNA of A . ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius . A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic supernatants, indicating a transiently formed complex during archaeal SRP targeting . The FtsY protein precipitated in cytosolic supernatants with anti-FtsY antisera as a homomeric protein lacking accessory protein components . However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.

FASEB J, 2003 Jun, 17(9), 961 - 83
The dsRNA binding protein family: critical roles, diverse cellular functions; Saunders LR et al.; The dsRNA binding proteins (DRBPs) comprise a growing family of eukaryotic, prokaryotic, and viral-encoded products that share a common evolutionarily conserved motif specifically facilitating interaction with dsRNA . Proteins harboring dsRNA binding domains (DRBDs) have been reported to interact with as little as 11 bp of dsRNA, an event that is independent of nucleotide sequence arrangement . More than 20 DRBPs have been identified and reportedly function in a diverse range of critically important roles in the cell . Examples include the dsRNA-dependent protein kinase PKR that functions in dsRNA signaling and host defense against virus infection and DICER, which is implicated in RNA interference (RNAi) -mediated gene silencing . Other DRBPs such as Staufen, adenosine deaminase acting on RNA (ADAR), and spermatid perinuclear RNA binding protein (SPNR) are known to play essential roles in development, translation, RNA editing, and stability . In many cases, homozygous and even heterozygous disruption of DRBPs in animal models results in embryonic lethality . These results implicate the recognition of dsRNA as an evolutionarily conserved mechanism important in the regulation of gene expression and in host defense and underscore the diversity of essential biological tasks performed by dsRNA-related processes in the cell.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 625 - 30
Cytochromes P450: novel drug targets in the war against multidrug-resistant Mycobacterium tuberculosis; Munro AW et al.; Novel drug strategies are desperately needed to combat the global threat posed by multidrug-resistant strains of Mycobacterium tuberculosis (Mtb) . The genome sequence of Mtb has revealed an unprecedented number of cytochrome P450 enzymes in a prokaryote, suggesting fundamental physiological roles for many of these enzymes . Several azole drugs (known inhibitors of cytochromes P450) have been shown to have potent anti-mycobacterial activity, and the most effective azoles have extremely tight binding constants for one of the Mtb P450s (CYP121) . The structure of CYP121 has been determined at atomic resolution, revealing novel features of P450 structure, including mixed haem conformations and putative proton-relay pathways from protein surface to haem iron . The structure provides both a platform for investigation of structure/mechanism of cytochrome P450, and for design of inhibitor molecules as novel anti-tubercular agents.

J Gen Virol, 2003 Jun, 84(Pt 6), 1629 - 39
Evidence that avian reovirus sigmaA protein is an inhibitor of the double-stranded RNA-dependent protein kinase; Gonzalez-Lopez C et al.; The results of a previous study demonstrated that avian reovirus is highly resistant to the antiviral effects of interferon and suggested that the double-stranded RNA (dsRNA)-binding sigmaA protein might play an important role in that resistance . To gather more evidence on the interferon-inhibitory activity of sigmaA protein, its gene was cloned into the prokaryotic maltose-binding protein (MBP) gene fusion vector pMalC and into the recombinant vaccinia virus WRS2 . The two recombinant sigmaA proteins displayed a dsRNA-binding affinity similar to that of sigmaA protein synthesized in avian reovirus-infected cells . Interestingly, MBP-sigmaA, but not MBP, was able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates by blocking the activation of endogenous dsRNA-dependent enzymes . In addition, transient expression of sigmaA protein in HeLa cells rescued gene expression of a vaccinia virus mutant lacking the E3L gene, and insertion of the sigmaA-encoding gene into vaccinia virus conferred protection for the virus against interferon in chicken cells . Further studies demonstrated that expression of recombinant sigmaA in mammalian cells interfered with dsRNA-dependent protein kinase (PKR) function . From these results we conclude that sigmaA is capable of reversing the interferon-induced antiviral state by down-regulating PKR activity in a manner similar to other virus-encoded dsRNA-binding proteins.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 7099 - 104 Epub 2003 May 27.
The BglF sensor recruits the BglG transcription regulator to the membrane and releases it on stimulation; Lopian L et al.; The Escherichia coli BglF protein is a sugar-sensor that controls the activity of the transcriptional antiterminator BglG by reversibly phosphorylating it, depending on beta-glucoside availability . BglF is a membrane-bound protein, whereas BglG is a soluble protein, and they are both present in the cell in minute amounts . How do BglF and BglG find each other to initiate signal transduction efficiently? Using bacterial two-hybrid systems and the Far-Western technique, we demonstrated unequivocally that BglG binds to BglF and to its active site-containing domain in vivo and in vitro . Measurements by surface plasmon resonance corroborated that the affinity between these proteins is high enough to enable their stable binding . To visualize the subcellular localization of BglG, we used fluorescence microscopy . In cells lacking BglF, the BglG-GFP fusion protein was evenly distributed throughout the cytoplasm . In contrast, in cells producing BglF, BglG-GFP was localized to the membrane . On addition of beta-glucoside, BglG-GFP was released from the membrane, becoming evenly distributed throughout the cell . Using mutant proteins and genetic backgrounds that impede phosphorylation of the Bgl proteins, we demonstrated that BglG-BglF binding and recruitment of BglG to the membrane sensor requires phosphorylation but does not depend on the individual phosphorylation sites of the Bgl proteins . We suggest a mechanism for rapid response to environmental changes by preassembly of signaling complexes, which contain transcription regulators recruited by their cognate sensors-kinases, under nonstimulating conditions, and release of the regulators to the cytoplasm on stimulation . This mechanism might be applicable to signaling cascades in prokaryotes and eukaryotes.

J Biol Chem, 2003 Aug 29, 278(35), 33049 - 55 Epub 2003 May 27.
Eukaryotic NAD+ synthetase Qns1 contains an essential, obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase; Bieganowski P et al.; NAD+ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADP-ribosyl)ation . NAD+ synthetase catalyzes the final step in NAD+ synthesis in the well characterized de novo, salvage, and import pathways . It has been long known that eukaryotic NAD+ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD+ synthetases are ammonia-dependent . Earlier, we discovered that glutamine-dependent NAD+ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases . Here we show yeast glutamine-dependent NAD+ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD+ synthetase active-site residues for function in vivo . Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD+ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity . Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo . These data indicate that the nitrilase-related domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 A from one active site to another within Qns1 monomers.

Mol Cell, 2003 May, 11(5), 1349 - 60
Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis; Kato M et al.; DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase . The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA . We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site . NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide . The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase . These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase . The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.

Protein Expr Purif, 2003 Jun, 29(2), 230 - 4
Expression, purification, and characterization of arginine kinase from the sea cucumber Stichopus japonicus; Guo SY et al.; The arginine kinase gene of sea cucumber Stichopus japonicus was cloned and inserted into the prokaryotic expression plasmid pET-21b . The protein was expressed in a soluble and functional form in Escherichia coli and purified by Blue Sepharose CL-6B, DEAE-32, and Sephadex G-100 chromotography with a final yield of 83 mgL(-1) of LB medium . The specific activity, electrophoretic mobility, and isoelectric focusing were all identical with those of arginine kinase that was purified from sea cucumber muscle . The fluorescence emission spectrum of arginine kinase had a maximum fluorescence at a wavelength of 330 nm upon excitation at 295 nm . These results are the first report of this purified protein.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Jun 25, 790(1-2), 285 - 316
Synthesis and chromatographic purification of recombinant human pituitary hormones; Ribela MT et al.; Recombinant DNA-derived proteins and, in particular, human pituitary hormones, are increasingly used for research, diagnostic and therapeutic purposes . This trend has demanded new synthetic approaches and improved purification techniques . The type and sequence of the purification steps have to be selected in accordance with the cloning and protein expression strategy, the host organism and cellular localization of the protein of interest, with a view to producing the desired product at a required purity, biological activity and acceptable cost . This review article describes and analyzes the main synthetic and purification strategies that have been used for the production of recombinant human growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle-stimulating hormone, giving special consideration to the few published downstream processes utilized by the biotechnology industry . Practically all types of prokaryotic and eukaryotic organisms utilized for this purpose are also reviewed.

Bioessays, 2003 Jun, 25(6), 569 - 76
The architecture of polarized cell growth: the unique status of elongating plant cells; Baluska F et al.; Polarity is an inherent feature of almost all prokaryotic and eukaryotic cells . In most eukaryotic cells, growth polarity is due to the assembly of actin-based growing domains at particular locations on the cell periphery . A contrasting scenario is that growth polarity results from the establishment of non-growing domains, which are actively maintained at opposite end-poles of the cell . This latter mode of growth is common in rod-shaped bacteria and, surprisingly, also in the majority of plant cells, which elongate along the apical-basal axes of plant organs . The available data indicate that the non-growing end-pole domains of plant cells are sites of intense endocytosis and recycling . These actin-enriched end-poles serve also as signaling platforms, allowing bidirectional exchange of diverse signals along the supracellular domains of longitudinal cell files . It is proposed that these actively remodeled end-poles of elongating plant cells remotely resemble neuronal synapses .

J Mol Microbiol Biotechnol, 2003, 5(3), 190 - 8
Radiation and functional specialization of the family-3 glycoside hydrolases; Cournoyer B et al.; A phylogenetic analysis of the glycoside hydrolases of family 3 (GH3s) was conducted in order to infer particular trends in its evolution: functional specialization, gene transfer events, gene duplications and paralogous evolution, and gene deletions . The phylogenetic analysis of GH3s revealed six clusters, i.e., A, B, C, D, E, and F that could fit the definition of 3 sub-families, i.e., AB, AB' and AB" . While the sub-families AB' and AB" contain a single cluster, F and E, respectively, the AB sub-family is sub-divided into four clusters . Global analysis of the GH3 phylogenetic tree suggests a primary burst of amplification of the GH3s that might have led to these sub-families . Specializations, gene transfers, and gene duplications among each of these sub-families and phylogenetic clusters might then have occurred and have been inferred . The fine comparison of the enzyme properties and phylogenetic relationships of GH3s allowed to detect common functional groups that belong to the same cluster (D, E or F), or sub-cluster (A1, A2 or B2) . The prokaryotic and eukaryotic beta-xylosidases and beta-glucosidases belong to the AB and AB' sub-families, and the N-acetylglucosaminidases are in sub-family AB" (in cluster E) . In some instances (B1, B2, C1, C2, and C3), the lack of data and/or the high heterogeneity of the hydrolytic properties did not allow to infer a particular link between an enzyme functional group and a phylogenetic cluster, suggesting the emergence of some highly specialized GH3s .

J Mol Microbiol Biotechnol, 2003, 5(3), 172 - 89
Phylogenomic analysis of the Giardia intestinalis transcarboxylase reveals multiple instances of domain fusion and fission in the evolution of biotin-dependent enzymes; Jordan IK et al.; Sequencing of the gene encoding a pyruvate carboxylase-like protein from the amitochondrial eukaryote Giardia intestinalis revealed a 1,338 aa protein composed of acetyl-CoA carboxyltransferase (ACCT), pyruvate carboxyltransferase (PycB), and biotin carboxyl carrier protein (BCCP) domains, linked in a single polypeptide chain . This particular domain combination has been previously seen only in the methylmalonyl-CoA:pyruvate transcarboxylase from Propionibacterium freudenreichii, where each of these domains is encoded by an individual gene and forms a separate subunit . To get an insight into the evolutionary origin and biochemical function of the G . intestinalis enzyme, we compared its domain composition to those of other biotin-dependent enzymes and performed a phylogenetic analysis of each of its domains . The results obtained indicate that: (1) evolution of the BCCP domain included several domain fusion events, leading to the ACCT-BCCP and PycB-BCCP domain combinations; (2) fusions of the PycB and BCCP domains in pyruvate carboxylases and oxaloacetate decarboxylases occurred on several independent occasions in different prokaryotic lineages, probably due to selective pressure towards co-expression of these genes, and (3) because newly sequenced biotin-dependent enzymes are often misannotated in sequence databases, their annotation as either carboxylases, decarboxylases, or transcarboxylases has to rely on detailed analysis of their domain composition, operon organization of the corresponding genes, gene content in the particular genome, and phylogenetic analysis .

J Biol Chem, 2003 Aug 1, 278(31), 28787 - 92 Epub 2003 May 23.
Involvement of a mate chaperone (TorD) in the maturation pathway of molybdoenzyme TorA; Ilbert M et al.; As many prokaryotic molybdoenzymes, the trimethylamine oxide reductase (TorA) of Escherichia coli requires the insertion of a bis(molybdopterin guanine dinucleotide)molybdenum cofactor in its catalytic site to be active and translocated to the periplasm . We show in vitro that the purified apo form of TorA was activated weakly when an appropriate bis(molybdopterin guanine dinucleotide)molybdenum source was provided, whereas addition of the TorD chaperone increased apoTorA activation up to 4-fold, allowing maturation of most of the apoprotein . We demonstrate that TorD alone is sufficient for the efficient activation of apoTorA by performing a minimal in vitro assay containing only the components for the cofactor synthesis, apoTorA and TorD . Interestingly, incubation of apoTorA with TorD before cofactor addition led to a significant increase of apoTorA activation, suggesting that TorD acts on apoTorA before cofactor insertion . This result is consistent with the fact that TorD binds to apoTorA and probably modifies its conformation in the absence of cofactor . Therefore, we propose that TorD is involved in the first step of TorA maturation to make it competent to receive the cofactor.

Trends Biochem Sci, 2003 May, 28(5), 224 - 6
HEPN: a common domain in bacterial drug resistance and human neurodegenerative proteins; Grynberg M et al.; A novel domain - HEPN (higher eukarytoes and prokaryotes nucleotide-binding domain) - found in several bacterial species is also present in the human protein, sacsin, a chaperonin implicated in an early-onset neurodegenerative disease . The distant structural similarity suggests that this domain might be involved in nucleotide binding.

J Biol Chem, 2003 Aug 8, 278(32), 30294 - 301 Epub 2003 May 22.
Membrane topology of a metabotropic glutamate receptor; Bhave G et al.; The metabotropic glutamate receptors (mGluRs) have been predicted to have a classical seven transmembrane domain structure similar to that seen for members of the G-protein-coupled receptor (GPCR) superfamily . However, the mGluRs (and other members of the family C GPCRs) show no sequence homology to the rhodopsin-like GPCRs, for which this seven transmembrane domain structure has been experimentally confirmed . Furthermore, several transmembrane domain prediction algorithms suggest that the mGluRs have a topology that is distinct from these receptors . In the present study, we set out to test whether mGluR5 has seven true transmembrane domains . Using a variety of approaches in both prokaryotic and eukaryotic systems, our data provide strong support for the proposed seven transmembrane domain model of mGluR5 . We propose that this membrane topology can be extended to all members of the family C GPCRs.

In Silico Biol, 2003, 3(1-2), 101 - 15 Epub 2003 Mar 30.
Parametric stability evaluation in computer experiments on the mathematical model of Drosophila control gene subnetwork; Tchuraev RN et al.; Using the method of generalized threshold models, the problem is formulated and solved to evaluate the parametric stability of the model of a gene subnetwork controlling the early ontogenesis of the fruit fly Drosophila melanogaster . Computer experiments have been performed to test the parametric stability of the model . Quantitative evaluations have been obtained for parametric stability of the Drosophila gene subnetwork in nuclei along the embryo's anterior-posterior axis . The results of computer experiments have been compared with the previous research data on "sensitivity" of functioning regimes to random changes of the parameters in the models of prokaryotic and eukaryotic systems, namely the system controlling the lambda-phage development and the subsystem controlling the flower morphogenesis of Arabidopsis thaliana . The obtained results confirm high parametric stability of gene networks that control the development of organisms.

In Silico Biol, 2003, 3(1-2), 17 - 31 Epub 2003 Mar 16.
Methods of horizontal gene transfer determination using phylogenetic data; Lyubetsky VA et al.; A new approach for comparative analysis of multiple trees reconstructed for representative protein families is proposed . This approach is based on the hypothesis of gene duplication, gene loss and horizontal gene transfer and makes use of stochastic methods and optimization . We present a species tree of 40 prokaryotic organisms obtained by our algorithm on the basis of 132 clusters of orthologous groups of proteins (COGs) from the GenBank of the National Center for Biotechnology Information (USA) . We also present a computer technology intended to determine horizontally transferred genes . Some application results of the technology, based on comparative analysis of protein and species trees, are given.

J Biochem (Tokyo), 2003 Apr, 133(4), 541 - 52
Molecular action mode of Hippospongic acid A, an inhibitor of gastrulation of starfish embryos; Mizushina Y et al.; Hippospongic acid A (HA-A) is a novel natural triterpene metabolite that exhibits inhibitory activity against the gastrulation of starfish embryos isolated from a marine sponge, Hippospongia sp . We succeeded in chemically synthesizing the natural enantiomer and the racemate HA-A . In this study, we examined its action mode in vitro . HA-A was a rare compound that could selectively but uniformly inhibit the activities of all the vertebrate DNA polymerases tested such as alpha, beta, delta, epsilon, eta, kappa, and lambda, in the IC(50) range of 5.9-17.6 microM, and interestingly also those of human DNA topoisomerases I and II (IC(50) = 15-25 microM) . HA-A exhibited no inhibitory effect on DNA polymerases from insects, plants and prokaryotes, or on many other DNA metabolic enzymes . HA-A was an inhibitor specific to DNA polymerases and DNA topoisomerases from vertebrates, but not selective as to a subclass species among the enzymes . Since DNA polymerase beta is the smallest, we used it to analyze the biochemical relationship with HA-A . Biochemical, BIAcore and computer modeling analyses demonstrated that HA-A bound selectively to the N-terminal 8 kDa DNA template-binding domain of DNA polymerase beta, and HA-A inhibited the ssDNA binding activity . HA-A could prevent the growth of NUGC-3 cancer cells at both the G1 and G2/M phases, and induce apoptosis in the cells . The LD(50) value was 9.5 microM, i.e . in the same range as for the enzyme inhibition . Therefore, we concluded that one molecular basis of the gastrulation of starfish embryos is a process that requires DNA polymerases and DNA topoisomerases, and subsequently the gastrulation was inhibited by HA-A . We also discussed the in vivo role of HA-A.

Zhonghua Yu Fang Yi Xue Za Zhi, 2003 Jan, 37(1), 29 - 32
{Cloning, sequencing and expressing of microneme protein 1 partial gene in toxoplasma gondii ZS2 isolate}; Yang HL et al.; OBJECTIVE: To construct a recombinant prokaryotic expression vector (plasmid) containing microneme protein 1 (MIC1) partial gene in toxoplasma gondii (T . gondii) ZS2 isolate . The gene was expressed in varied Escherichia coli (E . coli) after sequencing . METHODS: The gene fragment coding MIC 1 from the genomic DNA of T . gondii ZS2 isolate was amplified by polymerase chain reaction (PCR) . The gene was inserted to a prokaryotic expression vector pWR450-1 by digesting with restriction enzymes and linking reaction . The positive clone was screened on LB plates containing ampicillin and identified by restrictive enzyme digestion, PCR amplification and sequence analysis . The recombinant plasmid was transferred into E . coli TG1, JM109 (DE3) and DH5 alpha, and was expressed under the induction of IPTG . The expression products were identified by SDS-PAGE . The MIC1 gene structure was analyzed and compared in homology with the gene sequence of RH isolate using computer software . RESULTS: The recombinant plasmid pWR450-1/MIC1, after cloning from acquired 471 bp MIC1 gene fragment and amplified from the genome gene ZS2, was complete homologous to the sequence of RH isolate, reflecting its highly conservative . The gene could be expressed as fusion protein with 70,000 in varied E . coli . CONCLUSION: Recombinant plasmid pWR450-MIC1 was successfully constructed and could be expressed in different strains of E . coli, laying a foundation for research on its structure and function.

Genetika, 2003 Apr, 39(4), 453 - 73
{Variation and evolution of meiosis}; Bogdanov IuF; Meiosis arose in the evolution of primitive unicellular organisms as a part of sexual process . One type of meiosis, the so-called classical type, predominates in all kingdoms of eukaryotes . Meiosis is controlled by hundreds of genes, both shared with mitosis and specifically meiotic ones . In a wide range of taxa, which in some cases include kingdoms, meiotic genes and features obey Vavilov's law of homologous variation series . Synaptonemal complexes (SCs) temporarily binding homologous chromosomes at prophase I, ensure precise and equal crossing over and interference . SC proteins have 60-80% homology within the class of mammals but differ from the corresponding proteins in fungi and plants . Thus, nonhomologous SC proteins perform similar functions in different taxa . Some recombination enzymes in fungi and insects have common epitopes . The molecular mechanism of recombination is inherited by eukaryotes from prokaryotes and operates in special compartments: SC recombination nodules . Chiasmata, i.e., physical crossovers of nonsister chromatids, are preserved in bivalents until metaphase I due to local cohesion of sister chromatids in the remaining SC fragments . Owing to chiasmata, homologous chromosomes participate in meiosis I in pairs rather than individually, which, along with unipolarity of kinetochores (only in meiosis 1), ensures segregation of homologous chromosomes . The appearance of SC and chiasmata played a key role in the evolution of unicellular organisms since it promoted the development of a progressive type of meiosis . Some lower eukaryotes retain primitive meiosis types . These primitive modes of meiosis also occur in the sex of some insects that is heterozygous for sex chromosomes . I suggest an explanation for these cases . Mutations at meiotic genes impair meiosis; however, due to the preservation of archaic meiotic genes in the genotype, bypass metabolic pathways arise, which provide partial rescue of the traits damaged by mutations . Individual blocks of genetic program of meiotic regulation have probably evolved independently.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1999 Dec, 13(4), 358 - 61
{Efficient expression of human papillomavirus type 16 (HPV16) capsid proteins L1 and L2 in E . coli}; Wang W et al.; OBJECTIVE: To study the expression of HPV16 capsid proteins L1 and L2 in E . coli . METHODS: The plasmids pET-30a-L1 and pET-30a-L2 were constructed from the pET-30a vector, and transducted into the BL21 . The fusion proteins 6 x His-L1 and 6 x His-L2 were expressed when induced by adding of IPTG . SDS-PAGE and Western blot analyses were used to detect the expressed proteins . RESULTS: Fusion proteins 6 x His-L1 and 6 x His-L2 were expressed efficiently in E . coli, the Mr were about 60,000 and 97,000 respectively, 6 x His-L1 degraded more than 6 x His-L2 . CONCLUSION: L1 and L2 proteins were expressed at a high level in prokaryotic expression system, and 12 protein were more stable than L1 protein.

Cell Biol Int, 2003, 27(5), 429 - 38
Cytoskeletons in prokaryotes; Mayer F; Not only eukaryotes, but also prokaryotes possess a cytoskeleton . Tubulin-related bacterial protein FtsZ, and actin-related bacterial proteins MreB/Mbl have recently been described as constituents of bacterial cytoskeletons . Genes coding for MreB/Mbl could only be found in elongated bacteria, not in coccoid forms . It was speculated that constituents of today's eukaryotic cytoskeleton (tubulin, actin) may have evolved from prokaryotic precursor proteins closely related to today's bacterial proteins FtsZ and MreB/Mbl.Prior to the description of proteins MreB/Mbl, evidence had been obtained for the existence of a shape-preserving cytoskeleton ubiquitously present in all bacteria . In the meantime, structural studies allow to speculate on a possible role of bacterial elongation factor Tu (EF-Tu) as a structural element in such a "cytoskeletal web" . EF-Tu was long known to form fibrillar structures in vitro; now experimental data accumulate, pointing towards formation of intracellular protofilaments containing EF-Tu, and networks thereof as well . In addition, results of these structural studies suggest a so far unknown mode of complex formation of EF-Tu with active ribosomes: ribosomes/polysomes were seen to be attached to intracellular protofilaments . Implications for the understanding of EF-Tu-ribosome interaction, and a role of such a kind of putative protofilaments as a general site of attachment for cellular functional macromolecules are discussed . The notion is discussed that an EF-Tu-containing cytoskeletal web might have been the "primary" or "basic" kind of prokaryotic cytoskeleton, already in existence prior to the "invention" of precursors of today's MreB.

FEMS Microbiol Lett, 2003 May 16, 222(1), 59 - 68
Origin and evolution of transmembrane Chl-binding proteins: hydrophobic cluster analysis suggests a common one-helix ancestor for prokaryotic (Pcb) and eukaryotic (LHC) antenna protein superfamilies; Garczarek L et al.; All chlorophyll (Chl)-binding proteins constituting the photosynthetic apparatus of both prokaryotes and eukaryotes possess hydrophobic domains, corresponding to membrane-spanning alpha-helices (MSHs) . Hydrophobic cluster analysis of representative members of the different Chl protein superfamilies revealed that all Chl proteins except the five-helix reaction center II proteins and the small subunits of photosystem I possess related domains . As a major conclusion, we found that the eukaryotic antennae likely share a common precursor with the prokaryotic Chl a/b antennae from Chl-b-containing oxyphotobacteria . From these data, we propose a global scheme for the evolution of these proteins from a one-MSH ancestor.

RNA, 2003 Jun, 9(6), 644 - 7
Metabolite-binding RNA domains are present in the genes of eukaryotes; Sudarsan N et al.; Genetic control by metabolite-binding mRNAs is widespread in prokaryotes . These riboswitches are typically located in noncoding regions of mRNA, where they selectively bind their target compound and subsequently modulate gene expression . We have identified mRNA elements in fungi and in plants that match the consensus sequence and structure of thiamine pyrophosphate-binding domains of prokaryotes . In Arabidopsis, the consensus motif resides in the 3'-UTR of a thiamine biosynthetic gene, and the isolated RNA domain binds the corresponding coenzyme in vitro . These results suggest that metabolite-binding mRNAs are possibly involved in eukaryotic gene regulation and that some riboswitches might be representatives of an ancient form of genetic control.

Environ Microbiol, 2003 Jun, 5(6), 492 - 502
Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 degrees N); Nercessian O et al.; To evaluate possible compositional changes in archaeal communities at a deep-sea hydrothermal vent field scale, we examined five different samples obtained after deploying in situ collectors for different times on three spatially separated venting sulphide structures on the East Pacific Rise (13 degrees N) . Direct cell counts and whole-cell hybridizations with fluorescently labelled 16S rRNA-based oligonucleotide probes revealed that the relative abundance of archaeal populations represented from 14 to 33% of the prokaryotic community . 16S rDNA sequence analysis of the archaeal clone libraries indicated that a large percentage of clones were closely related to known archaeal isolates recovered from similar habitats . Among the 24 different phylotypes identified, Thermococcales-related sequences were dominant in all the libraries that also included representative genera of orders Methanopyrales, Methanococcales, Archaeoglobales and Desulfurococcales . The presence of most of these phylogenetic groups was confirmed in enrichment cultures performed at temperatures from 60 to 90 degrees C . Additional sequences with no known cultivated relatives grouped with the Marine group I Crenarchaeota, Korarchaeota and Deep-sea Hydrothermal Vent Euryarchaeota (DHVE) within which a novel lineage was identified . Furthermore, the archaeal community composition was distinct from vent to vent within the same vent field and varied within short time scales . This study provides new insights into microbial diversity and distribution at deep-sea hydrothermal vents.

Eur J Biochem, 2003 Jun, 270(11), 2505 - 12
NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea; Tossavainen H et al.; The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy . Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-hand motifs arranged in a compact globular structure . A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-hands, close together . The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.

Eur J Biochem, 2003 Jun, 270(11), 2404 - 11
Sulfation of hydroxychlorobiphenyls . Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases; Sugahara T et al.; As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish . By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs . These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing . Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences . Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST . These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family . Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE . Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls . Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined . A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C . Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.

DNA Seq, 2003 Feb, 14(1), 75 - 7
Cloning and prokaryotic expression of a cDNA encoding a putative mitochondrial malate dehydrogenase in Oryza sativa; Lin CF et al.; Malate dehydrogenase (MDH) has been characterized as a key player in oxaloacetate (OAA) biosynthesis mechanism in citrate acid cycle that generates reducing powers for further assimilation in the whole cell . Here we present the cloning, characterization and prokaryotic expression of a putative Mdh (OsmMDH) in Oryza sativa . Sequence alignment shows that there is a high homology between the deduced amino acid sequence of OsmMDH and MDH portein in Eucalyptus gunnii (80%), as well as between the deduced amino acid sequence of OsmMDH and other MDHs . Moreover, pI and the mitochondrial location of OsmMDH are predicted . The tissue-specific expression pattern of OsmMDH reveals that it is abundant in young panicle and immature seed, while its expression level is mush lower in leaf and root . Its expression in E . coli BL21 as a fusion gene is studied further.

Genetics, 2003 May, 164(1), 81 - 94
The Mus81/Mms4 endonuclease acts independently of double-Holliday junction resolution to promote a distinct subset of crossovers during meiosis in budding yeast; de los Santos T et al.; Current models for meiotic recombination require that crossovers derive from the resolution of a double-Holliday junction (dHJ) intermediate . In prokaryotes, enzymes responsible for HJ resolution are well characterized but the identification of a eukaryotic nuclear HJ resolvase has been elusive . Indirect evidence suggests that MUS81 from humans and fission yeast encodes a HJ resolvase . We provide three lines of evidence that Mus81/Mms4 is not the major meiotic HJ resolvase in S . cerevisiae: (1) MUS81/MMS4 is required to form only a distinct subset of crossovers; (2) rather than accumulating, dHJ intermediates are reduced in an mms4 mutant; and (3) expression of a bacterial HJ resolvase has no suppressive effect on mus81 meiotic phenotypes . Our analysis also reveals the existence of two distinct classes of crossovers in budding yeast . Class I is dependent upon MSH4/MSH5 and exhibits crossover interference, while class II is dependent upon MUS81/MMS4 and exhibits no interference . mms4 specifically reduces crossing over on small chromosomes, which are known to undergo less interference . The correlation between recombination rate and degree of interference to chromosome size may therefore be achieved by modulating the balance between class I/class II crossovers.

Plasmid, 2003 May, 49(3), 269 - 80
The replicon theory 40 years: an EMBO workshop held in Villefranche sur Mer, France, January 18-23, 2003; Nordstrom K; It is now 40 years since Jacob, Brenner, and Cuzin presented their Replicon Theory at a Cold Spring Harbor Symposium . The theory was based on their fundamental studies of the sexual system of Escherichia coli which led to the realisation that only specific sequences are able to replicate . They introduced the concept of a replicon consisting of a replicator (a DNA sequence) and a structural gene for an initiator protein . They also proposed a model for how replication of the bacterial chromosome might fit into the bacterial cell cycle . To commemorate the anniversary, an EMBO Workshop was organised in Villefranche on the Riviera of France . During the Workshop, the state of the art of cell-cycle studies of prokaryotic and eukaryotic organisms was presented and discussed in the presence of two of the fathers of the Replicon Theory, Jacob and Cuzin.

Proteomics, 2003 May, 3(5), 764 - 76
Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns; Birch RM et al.; The reliable identification and analysis of the low abundance proteins expressed by a cell remains a key challenge in the study of cellular proteomes . The analysis of low abundance proteins is a particular problem when using two-dimensional gel electrophoresis (2-DE) to resolve the cellular proteins since the technology is unable to display the wide dynamic range of protein levels typically synthesized by cells . We have investigated the use of reactive dye compounds for the enrichment of low abundance cellular proteins prior to analysis by 2-DE . The capacity of reactive dye compounds to bind specific protein species was used as the basis for a general chromatographic tool for protein enrichment . Six reactive dye compounds were investigated in detail for the analysis of Escherichia coli proteins . Whole bacterial cell lysates were passed down columns prepared with the reactive dye compounds . The bound proteins were eluted with 1.5 M NaCl and analyzed by 2-DE . Distinctive protein profiles were observed for the bound proteins recovered from the different reactive dye compounds . Selected proteins enriched by these methods were identified by peptide mass mapping . The enrichment procedure developed using reactive dye compounds were used to investigate acid-induced changes in the proteome of E . coli grown at either pH 7.0 or pH 5.8 . Increased levels of expression were observed for a number of proteins (for example, GdhA, PanC, ProC, TkrA, EF-TS and YodA) were observed for E . coli grown at pH 5.8 . Five identified proteins (AroG, FabI, GlyA, PurA and EF-Tu) showed reduced levels of synthesis for bacteria grown at pH 5.8 compared to pH 7.0 . In the case of PanC and FabI the altered expression profiles were only reliably demonstrated using the enrichment protocols . One theme emerging from these data was that the expression of proteins concerned with one-carbon metabolism was perturbed at pH 5.8, which may point to a previously unrecognized affect of low pH stress on the physiology of E . coli cells . We conclude that the prefractionation of cell lysates on reactive dye columns will serve as a valuable generic tool for the analysis of low abundance proteins expressed by both prokaryotic and eukaryotic cells.

Nippon Ishinkin Gakkai Zasshi, 2003, 44(2), 107 - 14
{Evaluation of targets for antifungal drugs using a system to regulate gene expression}; Nakayama H et al.; A system to regulate gene expression is a convenient tool to explore gene function(s) not only in prokaryote but also in eukaryote . Such manipulation tools are scarce in the medical mycology field due to its complexity and diploidity . Although systems to regulate gene expression have been constructed, most of them are restricted in their application to particular culture conditions due to the nature of the promoter used . This motivated us to establish a new regulatable expression system that can function regardless of culture conditions, including in a host . In this review, a new system using tetracycline or its derivative as a molecular switch is introduced, which can function in several culture conditions, and in a host . We also show that the system can be applied to the selection of antifungal drug targets, which is the first step in a target-based strategy for drug discovery.

Mol Plant Microbe Interact, 2003 Apr, 16(4), 271 - 80
The sypA, sypS, and sypC synthetase genes encode twenty-two modules involved in the nonribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv . syringae B301D; Scholz-Schroeder BK et al.; Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail . Syringopeptin, produced by Pseudomonas syringae pv . syringae, functions as a virulence determinant in the plant-pathogen interaction . A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size . Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively . Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation . Within the aminoacyl adenylation domain is a region responsible for substrate specificity . Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups . This clustering reflects the substrate amino acids predicted to be recognized by each of the respective modules based on placement of the syringopeptin NRPS (nonribosomal peptide synthetase) system in the linear (type A) group . Finally, SypC contains two C-terminal thioesterase domains predicted to catalyze the release of syringopeptin from the synthetase and peptide cyclization to form the lactone ring . The syringopeptin synthetases, which carry 22 NRPS modules, represent the largest linear NRPS system described for a prokaryote.

Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6771 - 6 Epub 2003 May 12.
Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion; Carabeo RA et al.; Cholesterol, a lipid not normally found in prokaryotes, was identified in purified Chlamydia trachomatis elementary bodies and in the chlamydial parasitophorous vacuole (inclusion) membrane of infected HeLa cells . Chlamydiae obtained eukaryotic host cell cholesterol both from de novo synthesis or low-density lipoprotein . Acquisition of either de novo-synthesized cholesterol or low-density lipoprotein-derived cholesterol was microtubule-dependent and brefeldin A-sensitive, indicating a requirement for the Golgi apparatus . Transport also required chlamydial protein synthesis, indicative of a pathogen-directed process . The cholesterol trafficking pathway appears to coincide with a previously characterized delivery of sphingomyelin to the inclusion in that similar pharmacological treatments inhibited transport of both sphingomyelin and cholesterol . These results support the hypothesis that sphingomyelin and cholesterol may be cotransported via a Golgi-dependent pathway and that the chlamydial inclusion receives cholesterol preferentially from a brefeldin A-sensitive pathway of cholesterol trafficking from the Golgi apparatus to the plasma membrane.

Genome Res, 2003 Jun, 13(6A), 1155 - 7 Epub 2003 May 12.
Phylogenetically older introns strongly correlate with module boundaries in ancient proteins; Fedorov A et al.; The hypothesis that some (but not all) introns were used to construct ancient genes by exon shuffling of modules at the earliest stages of evolution is supported by the finding of an excess of phase-zero intron positions in the boundary regions of such modules in 276 ancient proteins (defined as common to eukaryotes and prokaryotes) . Here we show further that as phase-zero intron positions are shared by distant taxa, and thus are truly phylogenetically ancient, their excess in the boundaries becomes greater, rising to an 80% excess if shared by four out of the five taxa: vertebrates, invertebrates, fungi, plants, and protists.

Mol Phylogenet Evol, 2003 Jun, 27(3), 504 - 9
Evolution of the prokaryotic protein translocation complex: a comparison of archaeal and bacterial versions of SecDF; Eichler J; Protein translocation across the prokaryotic plasma membrane occurs at the translocon, an evolutionarily conserved membrane-embedded proteinaceous complex . Together with the core components SecYE, prokaryotic translocons also contain auxilliary proteins, such as SecDF . Alignment of bacterial and archaeal SecDF protein sequences reveals the presence of a similar number of homologous regions within each protein . Moreover, the conserved sequence domains in the archaeal proteins are located in similar positions as their bacterial counterparts . When these domains are, however, compared along Bacteria-Archaea lines, a much lower degree of similarity is detected . In Bacteria, SecDF are thought to modulate the membrane association of SecA, the ATPase that provides the driving force for bacterial protein secretion . As no archaeal version of SecA has been detected, the sequence differences reported here may reflect functional differences between bacterial and archaeal SecDF proteins, and by extension, between the bacterial and archaeal protein translocation processes . Moreover, the apparent absence of SecDF in several completed archaeal genomes suggests that differences may exist in the process of protein translocation within the archaeal domain itself.

J Mol Biol, 2003 May 23, 329(1), 59 - 68
Interplay between DnaA and SeqA proteins during regulation of bacteriophage lambda pR promoter activity; Slominska M et al.; DnaA and SeqA proteins are main regulators (positive and negative, respectively) of the chromosome replication in Escherichia coli . Nevertheless, both these replication regulators were found recently to be also transcription factors . Interestingly, both DnaA and SeqA control activity of the bacteriophage lambdap(R) promoter by binding downstream of the transcription start site, which is unusual among prokaryotic systems . Here we asked what are functional relationships between these two transcription regulators at one promoter region . Both in vivo and in vitro studies revealed that DnaA and SeqA can activate the p(R) promoter independently and separately rather than in co-operation, however, increased concentrations of one of these proteins negatively influenced the transcription stimulation mediated by the second regulator . This may suggest a competition between DnaA and SeqA for binding to the p(R) regulatory region . The physiological significance of this DnaA and SeqA-mediated regulation of p(R) is demonstrated by studies on lambda plasmid DNA replication in vivo.

J Mol Biol, 2003 May 23, 329(1), 35 - 43
A mechanism for ParB-dependent waves of ParA, a protein related to DNA segregation during cell division in prokaryotes; Hunding A et al.; Prokaryotic plasmids encode partitioning (par) loci involved in segregation of DNA to daughter cells at cell division . A functional fusion protein consisting of Walker-type ParA ATPase and green fluorescent protein (Gfp) oscillates back and forth within nucleoid regions with a wave period of about 20 minutes . A model is discussed which is based on cooperative non-specific binding of ParA to the nucleoid, and local ParB initiated generation of ParA oligomer degradation products, which act autocatalytically on the degradation reaction . The model yields self-initiated spontaneous pattern formation, based on Turing's mechanism, and these patterns are destroyed by the degradation products, only to initiate a new pattern at the opposite nucleoid region . A recurrent wave thus emerges . This may be a particular example of a more general class of pattern forming mechanisms, based on protein oligomerization upon a template (membranes, DNA a.o.) with resulting enhanced NTPase function in the oligomer state, which may bring the oligomer into an unstable internal state . An effector initializes destabilization of the oligomer to yield degradation products, which act as seeds for further degradation in an autocatalytic process . We discuss this mechanism in relation to recent models for MinDE oscillations in E.coli and to microtubule degradation in mitosis . The study points to an ancestral role for the presented pattern types in generating bipolarity in prokaryotes and eukaryotes.

Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6843 - 8 Epub 2003 May 09.
Mitochondrial succinic-semialdehyde dehydrogenase of the gamma-aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants; Bouche N et al.; The gamma-aminobutyrate (GABA) shunt is a metabolic pathway that bypasses two steps of the tricarboxylic acid cycle, and it is present in both prokaryotes and eukaryotes . In plants the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase and the mitochondrial enzymes GABA transaminase and succinic-semialdehyde dehydrogenase (SSADH) . The activity of the GABA shunt in plants is rapidly enhanced in response to various biotic and abiotic stresses . However the physiological role of this pathway remains obscure . To elucidate its role in plants, we analyzed Arabidopsis T-DNA knockout mutants of SSADH, the ultimate enzyme of the pathway . Four alleles of the ssadh mutation were isolated, and these exhibited a similar phenotype . When exposed to white light (100 micromol of photons per m2 per s), they appear dwarfed with necrotic lesions . Detailed spectrum analysis revealed that UV-B has the most adverse effect on the mutant phenotype, whereas photosynthetic active range light has a very little effect . The ssadh mutants are also sensitive to heat, as they develop necrosis when submitted to such stress . Moreover, both UV and heat cause a rapid increase in the levels of hydrogen peroxide in the ssadh mutants, which is associated with enhanced cell death . Surprisingly, our study also shows that trichomes are hypersensitive to stresses in ssadh mutants . Our work establishes a role for the GABA shunt in preventing the accumulation of reactive oxygen intermediates and cell death, which appears to be essential for plant defense against environmental stress.

Science, 2003 Jun 20, 300(5627), 1922 - 6 Epub 2003 May 08.
Crystal structure of the potassium channel KirBac1.1 in the closed state; Kuo A et al.; The KirBac1.1 channel belongs to the inward-rectifier family of potassium channels . Here we report the structure of the entire prokaryotic Kir channel assembly, in the closed state, refined to a resolution of 3.65 angstroms . We identify the main activation gate and structural elements involved in gating . On the basis of structural evidence presented here, we suggest that gating involves coupling between the intracellular and membrane domains . This further suggests that initiation of gating by membrane or intracellular signals represents different entry points to a common mechanistic pathway.

Science, 2003 May 9, 300(5621), 939 - 44
The ecology of arsenic; Oremland RS et al.; Arsenic is a metalloid whose name conjures up images of murder . Nonetheless, certain prokaryotes use arsenic oxyanions for energy generation, either by oxidizing arsenite or by respiring arsenate . These microbes are phylogenetically diverse and occur in a wide range of habitats . Arsenic cycling may take place in the absence of oxygen and can contribute to organic matter oxidation . In aquifers, these microbial reactions may mobilize arsenic from the solid to the aqueous phase, resulting in contaminated drinking water . Here we review what is known about arsenic-metabolizing bacteria and their potential impact on speciation and mobilization of arsenic in nature.

Folia Parasitol (Praha), 2003 Mar, 50(1), 3 - 18
Spermidine metabolism in parasitic protozoa--a comparison to the situation in prokaryotes, viruses, plants and fungi; Kaiser AE et al.; Targeting polyamines of parasitic protozoa in chemotherapy has attracted attention because polyamines might reveal novel drug targets for antiparasite therapies (Muller et al . 2001) . The biological function of the triamine spermidine in parasitic protozoa has not been studied in great detail although the results obtained mainly imply three different functions, i.e., cell proliferation, cell differentiation, and biosynthesis of macromolecules . Sequence information from the malaria genome project databases and inhibitor studies provide evidence that the current status of spermidine research has to be extended since enzymes of spermidine metabolism are present in the parasite (Kaiser et al . 2001) . Isolation and characterisation of these enzymes, i.e., deoxyhypusine synthase (EC 1.1.1.249) (DHS) and homospermidine synthase (EC 2.5.1.44) (HSS) might lead to valuable new targets in drug therapy . Currently research on spermidine metabolism is based on the deposition of the deoxyhypusine synthase nucleic acid sequence in GenBank while the activity of homospermidine synthase was deduced from inhibitor studies . Spermidine biosynthesis is catalyzed by spermidine synthase (EC 2.5.1.16) which transfers an aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine . Spermidine is also an important precursor in the biosynthesis of the unusual amino acid hypusine (Wolff et al . 1995) and the uncommon triamine homospermidine in eukaryotes, in particular in pyrrolizidine alkaloid-producing plants (Ober and Hartmann 2000) . Hypusine is formed by a two-step enzymatic mechanism starting with the transfer of an aminobutyl moiety from spermidine to the epsilon-amino group of one of the lysine residues in the precursor protein of eukaryotic initiation factor eIF5A by DHS (Lee and Park 2000) . The second step of hypusinylation is completed by deoxyhypusine hydroxylase (EC 1.14.9929) (Abbruzzese et al . 1985) . Homospermidine formation in eukaryotes parallels deoxyhypusine formation in the way that in an NAD(+)-dependent reaction an aminobutyl moiety is transferred from spermidine . In the case of homospermidine synthase, however the acceptor is putrescine . Thus the triamine homospermidine consists of two symmetric aminobutyl moieties while there is one aminobutyl and one aminopropyl moiety present in spermidine . Here, we review the metabolism of the triamine spermidine with particular focus on the biosynthesis of hypusine and homospermidine in parasitic protozoa, i.e., Plasmodium, Trypanosoma and Leishmania, compared to that in prokaryotes i.e., Escherichia coli, a phytopathogenic virus and pyrrolizidine alkaloid-producing plants (Asteraceae) and fungi.

Biol Proced Online, 1998 May 14, 1, 70 - 80
Expression of a prokaryotic P-type ATPase in E . coli Plasma Membranes and Purification by Ni2+-affinity chromatography; Geisler M; In order to characterize the P-type ATPase from Synechocystis 6803 {Geisler (1993) et al . J . Mol . Biol . 234, 1284} and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E . coli strain . The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E . coli plasma membranes representing about 20-25% of the membrane protein . The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described . ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC(50)= 119 &mgr;M) and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

Appl Environ Microbiol, 2003 May, 69(5), 2463 - 83
Biodiversity, community structural shifts, and biogeography of prokaryotes within Antarctic continental shelf sediment; Bowman JP et al.; 16S ribosomal DNA (rDNA) clone library analysis was conducted to assess prokaryotic diversity and community structural changes within a surficial sediment core obtained from an Antarctic continental shelf area (depth, 761 m) within the Mertz Glacier Polynya (MGP) region . Libraries were created from three separate horizons of the core (0- to 0.4-cm, 1.5- to 2.5-cm, and 20- to 21-cm depth positions) . The results indicated that at the oxic sediment surface (depth, 0 to 0.4 cm) the microbial community appeared to be dominated by a small subset of potentially r-strategist (fast-growing, opportunistic) species, resulting in a lower-than-expected species richness of 442 operational taxonomic units (OTUs) . At a depth of 1.5 to 2.5 cm, the species richness (1,128 OTUs) was much higher, with the community dominated by numerous gamma and delta proteobacterial phylotypes . At a depth of 20 to 21 cm, a clear decline in species richness (541 OTUs) occurred, accompanied by a larger number of more phylogenetically divergent phylotypes and a decline in the predominance of Proteobacteria . Based on rRNA and clonal abundance as well as sequence comparisons, syntrophic cycling of oxidized and reduced sulfur compounds appeared to be the dominant process in surficial MGP sediment, as phylotype groups putatively linked to these processes made up a large proportion of clones throughout the core . Between 18 and 65% of 16S rDNA phylotypes detected in a wide range of coastal and open ocean sediments possessed high levels of sequence similarity (>95%) with the MGP sediment phylotypes, indicating that many sediment prokaryote phylotype groups defined in this study are ubiquitous in marine sediment.

J Microbiol Methods, 2003 Jul, 54(1), 1 - 11
Low-shear modeled microgravity: a global environmental regulatory signal affecting bacterial gene expression, physiology, and pathogenesis; Nickerson CA et al.; Bacteria inhabit an impressive variety of ecological niches and must adapt constantly to changing environmental conditions . While numerous environmental signals have been examined for their effect on bacteria, the effects of mechanical forces such as shear stress and gravity have only been investigated to a limited extent . However, several important studies have demonstrated a key role for the environmental signals of low shear and/or microgravity in the regulation of bacterial gene expression, physiology, and pathogenesis {Chem . Rec . 1 (2001) 333; Appl . Microbiol . Biotechnol . 54 (2000) 33; Appl . Environ . Microbiol . 63 (1997) 4090; J . Ind . Microbiol . 18 (1997) 22; Curr . Microbiol . 34(4) (1997) 199; Appl . Microbiol . Biotechnol . 56(3-4) (2001) 384; Infect Immun . 68(6) (2000) 3147; Cell 109(7) (2002) 913; Appl . Environ . Microbiol . 68(11) (2002) 5408; Proc . Natl . Acad . Sci . U . S . A . 99(21) (2002) 13807} . The response of bacteria to these environmental signals, which are similar to those encountered during prokaryotic life cycles, may provide insight into bacterial adaptations to physiologically relevant conditions . This review focuses on the current and potential future research trends aimed at understanding the effect of the mechanical forces of low shear and microgravity analogues on different bacterial parameters . In addition, this review also discusses the use of microgravity technology to generate physiologically relevant human tissue models for research in bacterial pathogenesis.

Curr Opin Microbiol, 2003 Apr, 6(2), 157 - 62
Ribosome-tethered molecular chaperones: the first line of defense against protein misfolding?
Craig EA, Eisenman HC, Hundley HA.
Folding of many cellular proteins is facilitated by molecular chaperones . Analysis of both prokaryotic and lower eukaryotic model systems has revealed the presence of ribosome-associated molecular chaperones, thought to be the first line of defense against protein aggregation as translating polypeptides emerge from the ribosome . However, structurally unrelated chaperones have evolved to carry out these functions in different microbes . In the yeast Saccharomyces cerevisiae, an unusual complex of Hsp70 and J-type chaperones associates with ribosome-bound nascent chains, whereas in Escherichia coli the ribosome-associated peptidyl-prolyl-cis-trans isomerase, trigger factor, plays a predominant role.

Curr Opin Microbiol, 2003 Apr, 6(2), 101 - 8
Polarisation of prokaryotic chromosomes; Lobry JR et al.; In many prokaryotes, asymmetrical mutational or selective pressures have caused compositional skews between complementary strands of replication arms, especially sensitive in the distribution of guanine and cytosine . In Escherichia coli, most of the guanine/cytosine skew is caused by mutation rates differing on leading and lagging strands, but contribution of skewed functionally important guanine-rich motifs (Chi and Rag sites), which control chromosome repair or positioning, is noticeable . Interference between replication and gene expression plays a minor role . The situation may be different in other bacteria . Studies of chromosome processing and bacterial taxonomy might profit from consideration of chromosome polarisation.

Cell, 2003 May 2, 113(3), 369 - 81
Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis; Meyer AS et al.; Chaperonins use ATPase cycling to promote conformational changes leading to protein folding . The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction . The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid . Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear . We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis . Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC . Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding . Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.

Int Microbiol, 2003 Mar, 6(1), 11 - 6 Epub 2003 Feb 12.
Genetic basis of microbial carotenogenesis; Sieiro C et al.; The synthesis of carotenoids begins with the formation of a phytoene from geranylgeranyl pyrophosphate, a well conserved step in all carotenogenic organisms and catalyzed by a phytoene synthase, an enzyme encoded by the crtB ( spy) genes . The next step is the dehydrogenation of the phytoene, which is carried out by phytoene dehydrogenase . In organisms with oxygenic photosynthesis, this enzyme, which accomplishes two dehydrogenations, is encoded by the crtP genes . In organisms that lack oxygenic photosynthesis, dehydrogenation is carried out by an enzyme completely unrelated to the former one, which carries out four dehydrogenations and is encoded by the crtI genes . In organisms with oxygenic photosynthesis, dehydrogenation of the phytoene is accomplished by a zeta-carotene dehydrogenase encoded by the crtQ ( zds) genes . In many carotenogenic organisms, the process is completed with the cyclization of lycopene . In organisms exhibiting oxygenic photosynthesis, this step is performed by a lycopene cyclase encoded by the crtL genes . In contrast, anoxygenic photosynthetic and non-photosynthetic organisms use a different lycopene cyclase, encoded by the crtY ( lyc) genes . A third and unrelated type of lycopene beta-cyclase has been described in certain bacteria and archaea . Fungi differ from the rest of non-photosynthetic organisms in that they have a bifunctional enzyme that displays both phytoene synthase and lycopene cyclase activity . Carotenoids can be modified by oxygen-containing functional groups, thus originating xanthophylls . Only two enzymes are necessary for the conversion of beta-carotene into astaxanthin, using several ketocarotenoids as intermediates, in both prokaryotes and eukaryotes . These enzymes are a beta-carotene hydroxylase ( crtZ genes) and a beta-carotene ketolase, encoded by the crtW (bacteria) or bkt (algae) genes.

J Nutr, 2003 May, 133(5 Suppl 1), 1510S - 6S
Novel roles for iron regulatory proteins in the adaptive response to iron deficiency; Eisenstein RS et al.; Iron regulatory proteins (IRP) modulate the use of mRNA-encoding proteins that are involved in the transport, storage and use of iron . Several new potential mRNA targets for IRP were recently identified: divalent metal transporter-1 (DMT-1) and ferroportin, which are critical regulators of iron absorption in the gut and of iron cycling between various tissues of the body . Although this may extend the reach of IRP to other processes that are important for maintaining body iron homeostasis, the extent to which IRP modulate other physiological processes that are altered in response to changes in iron availability is not clear . However, in the past several years, targets for IRP and IRP-like proteins were identified in eukaryotes and prokaryotes in the tricarboxylic acid (TCA) cycle and electron-transport chain . In mammals, this includes the mRNA that encodes the TCA-cycle enzyme mitochondrial aconitase (m-acon) . Recent work established that m-acon expression is translationally regulated by iron in a manner that is strongly correlated with IRP RNA-binding activity . Interestingly, these studies also demonstrate that IRP regulate their mRNA targets in a hierarchical manner . The changes in m-acon synthesis and abundance in liver during iron deficiency fail to affect TCA-cycle capacity but are associated with a significant upregulation of mitochondrial export of radiolabeled citrate . We conclude that IRP are required for the regulation of physiological pathways that include but are not limited to iron metabolism, and as such, IRP are critical factors in the adaptive response to iron deficiency.

J Mol Biol, 2003 May 16, 328(5), 995 - 1010
Towards understanding human mitochondrial leucine aminoacylation identity; Sohm B et al.; Specific recognition of tRNAs by aminoacyl-tRNA synthetases is governed by sets of aminoacylation identity elements, well defined for numerous prokaryotic systems and eukaryotic cytosolic systems . Only restricted information is available for aminoacylation of human mitochondrial tRNAs, despite their particularities linked to the non-classical structures of the tRNAs and their involvement in a growing number of human neurodegenerative disorders linked to mutations in the corresponding tRNA genes . A major difficulty to be overcome is the preparation of active in vitro transcripts enabling a rational mutagenic analysis, as is currently performed for classical tRNAs . Here, structural and aminoacylation properties of in vitro transcribed tRNA(Leu(UUR)) are presented . Solution probing using a combination of enzymatic and chemical tools revealed only partial folding into an L-shaped structure, with an acceptor branch but with a floppy anticodon branch . Optimization of aminoacylation conditions allowed charging of up to 75% of molecules, showing that, despite its partially relaxed structure, in vitro transcribed tRNA(Leu(UUR)) is able to adapt to the synthetase . In addition, mutational analysis demonstrates that the discriminator base as well as residue A14 are important leucine identity elements . Thus, human mitochondrial leucylation is dependent on rules similar to those that apply in Escherichia coli . The impact of a subset of pathology-related mutations on aminoacylation and on tRNA structure, has been explored . These variants do not show significant structural rearrangements and either do not affect aminoacylation (mutations T3250C, T3271C, C3303T) or lead to marked effects . Interestingly, two variants with a mutation at the same position (A3243G and A3243T) lead to markedly different losses in aminoacylation efficiencies (tenfold and 300-fold, respectively).

Genome Res, 2003 May, 13(5), 773 - 80
Genome-wide in silico identification of transcriptional regulators controlling the cell cycle in human cells; Elkon R et al.; Dissection of regulatory networks that control gene transcription is one of the greatest challenges of functional genomics . Using human genomic sequences, models for binding sites of known transcription factors, and gene expression data, we demonstrate that the reverse engineering approach, which infers regulatory mechanisms from gene expression patterns, can reveal transcriptional networks in human cells . To date, such methodologies were successfully demonstrated only in prokaryotes and low eukaryotes . We developed computational methods for identifying putative binding sites of transcription factors and for evaluating the statistical significance of their prevalence in a given set of promoters . Focusing on transcriptional mechanisms that control cell cycle progression, our computational analyses revealed eight transcription factors whose binding sites are significantly overrepresented in promoters of genes whose expression is cell-cycle-dependent . The enrichment of some of these factors is specific to certain phases of the cell cycle . In addition, several pairs of these transcription factors show a significant co-occurrence rate in cell-cycle-regulated promoters . Each such pair indicates functional cooperation between its members in regulating the transcriptional program associated with cell cycle progression . The methods presented here are general and can be applied to the analysis of transcriptional networks controlling any biological process.

EMBO J, 2003 May 1, 22(9), 2297 - 306
The integral membrane protein p16.7 organizes in vivo phi29 DNA replication through interaction with both the terminal protein and ssDNA; Serna-Rico A et al.; Remarkably little is known about the in vivo organization of membrane-associated prokaryotic DNA replication or the proteins involved . We have studied this fundamental process using the Bacillus subtilis phage phi29 as a model system . Previously, we demonstrated that the phi29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated phi29 DNA replication . Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the phi29 terminal protein . In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C-terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein . Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA-binding mode . These and previous results indicate that p16.7 encompasses four distinct modules . An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo phi29 DNA replication is presented.

EMBO J, 2003 May 1, 22(9), 2117 - 26
Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC; Xu Y et al.; Using model strains in which we ectopically express the cyanobacterial clock protein KaiC in cells from which the clock genes kaiA, kaiB and/or kaiC are deleted, we found that some features of circadian clocks in eukaryotic organisms are conserved in the clocks of prokaryotic cyanobacteria, but others are not . One unexpected difference is that the circadian autoregulatory feedback loop in cyanobacteria does not require specific clock gene promoters as it does in eukaryotes, because a heterologous promoter can functionally replace the kaiBC promoter . On the other hand, a similarity between eukaryotic clock proteins and the cyanobacterial KaiC protein is that KaiC is phosphorylated in vivo . The other essential clock proteins KaiA and KaiB modulate the status of KaiC phosphorylation; KaiA inhibits KaiC dephosphorylation and KaiB antagonizes this action of KaiA . Based upon an analysis of clock mutants, we conclude that the circadian period in cyanobacteria is determined by the phosphorylation status of KaiC and also by the degradation rate of KaiC . These observations are integrated into a model proposing rhythmic changes in chromosomal status.

Curr Opin Struct Biol, 2003 Apr, 13(2), 244 - 8
Actin's prokaryotic homologs; Egelman EH; Actin is one of the most abundant and conserved eukaryotic proteins . Remarkably, two prokaryotic homologs of actin, MreB and ParM, have only recently been identified . MreB and ParM polymerize into filaments and play important roles in the control of bacterial cell shape and plasmid segregation, respectively . Whereas the eukaryotic actins display a remarkable degree of conservation (e.g . no amino acid changes in muscle actin from chickens to humans), the two bacterial proteins have as much sequence similarity to each other ( approximately 11% sequence identity) as they do to actin . It is possible that the interesting properties of eukaryotic F-actin may account for the unusual degree of conservation among the actins, whereas the bacterial proteins have had fewer constraints over the course of evolution.

J Theor Biol, 2003 May 21, 222(2), 139 - 49
Statistics of trinucleotides in coding sequences and evolution; Takeuchi F et al.; The aim of this paper is to give measurements indicative of evolutional stages of the species . Two types of statistics of trinucleotides in coding regions are analysed for 27 species . The first one is the codon space, the nucleotide ratio for each of the three codon positions . We apply principal component analysis on this space and extract two principal components faithfully describing the original distribution of the codon space . The first principal component corresponds to the GC content . The second principal component classifies the species into three evolutional groups, Archaea, Bacteria and Eukaryota . The second statistics is the real and theoretical frequency of amino acids . The real frequency of an amino acid in a coding sequence is its frequency in the translated protein . The theoretical frequency is the expected frequency calculated from the ratio of nucleotides . We introduce the discrepancy between these two frequencies as an index of non-randomness of nucleotides in the sequence . This index of non-randomness divides the species into two groups: eukaryotes having smaller non-randomness (i.e . being more random) and prokaryotes having higher non-randomness.

Mol Biol (Mosk), 2003 Mar-Apr, 37(2), 300 - 14
{Prokaryotic DNA methyltransferases: the structure and the mechanism of interaction with DNA}; Gromova ES et al.; The review considers current views on the function of DNA methyltransferases (MTases) that belong to prokaryotic type II restriction-modification systems . A commonly accepted classification of MTases is described along with their primary and tertiary structures and molecular mechanisms of their specific interaction with DNA (including methylation) . MTase inhibitors are also considered . Special emphasis is placed on the flipping of the target heterocyclic base out of the double helix and on the methods employed in its analysis . Base flipping is a fundamentally new type of DNA conformational changes and is also of importance in the case of other DNA-operating enzymes . MTases show unique sequence homology, and are similar in structure of functional centers and in the mechanism of methylation . These data contribute to the understanding of the general biological significance of methylation, since prokaryotic and eukaryotic MTases are structurally and functionally similar.

Int Rev Cytol, 2003, 224, 57 - 110
The apicoplast: a plastid in Plasmodium falciparum and other Apicomplexan parasites; Foth BJ et al.; Apicomplexan parasites cause severe diseases such as malaria, toxoplasmosis, and coccidiosis (caused by Plasmodium spp., Toxoplasma, and Eimeria, respectively) . These parasites contain a relict plastid-termed "apicoplast"--that originated from the engulfment of an organism of the red algal lineage . The apicoplast is indispensable but its exact role in parasites is unknown . The apicoplast has its own genome and expresses a small number of genes, but the vast majority of the apicoplast proteome is encoded in the nuclear genome . The products of these nuclear genes are posttranslationally targeted to the organelle via the secretory pathway courtesy of a bipartite N-terminal leader sequence . Apicoplasts are nonphotosynthetic but retain other typical plastid functions such as fatty acid, isoprenoid and heme synthesis, and products of these pathways might be exported from the apicoplast for use by the parasite . Apicoplast pathways are essentially prokaryotic and therefore excellent drug targets . Some antibiotics inhibiting these molecular processes are already in chemotherapeutic use, whereas many new drugs will hopefully spring from our growing understanding of this intriguing organelle.

Prikl Biokhim Mikrobiol, 2003 Mar-Apr, 39(2), 133 - 59
{Multiple functions of corrinoids in prokaryote biology}; Ryzhkova EP; Data on more than 30 metabolic processes and biochemical reactions, involving corrinoids, which have been described in prokaryotes thus far, are reviewed . These pathways (central or specific, catabolic or anabolic) are inherent in bacteria and archebacteria of diverse phylogenetic lineages, comprising several physiological groups . Particular emphasis is placed on the role of corrinoid-dependent transmethylation in acetogenesis and methanogenesis and on the contribution of adenosylcobalamin in DNA metabolism.

J Plant Res, 2003 Jun, 116(3), 241 - 52 Epub 2003 Apr 29.
Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species; Schmulling T et al.; Cytokinin oxidases/dehydrogenases (CKX) catalyze the irreversible degradation of the cytokinins isopentenyladenine, zeatin, and their ribosides in a single enzymatic step by oxidative side chain cleavage . To date the sequences of 17 fully annotated CKX genes are known, including two prokaryotic genes . The CKX gene families of Arabidopsis thaliana and rice comprise seven and at least ten members, respectively . The main features of CKX genes and proteins are summarized in this review . Individual proteins differ in their catalytic properties, their subcellular localization and their expression domains . The evolutionary development of cytokinin-catabolizing gene families and the individual properties of their members indicate an important role for the fine-tuned control of catabolism to assure proper regulation of cytokinin functions . The use of CKX genes as a tool in studies of cytokinin biology and biotechnological applications is discussed.

BMC Genomics . 2003 Apr 29;4(1):16.
A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast; Noskov VN et al.; BACKGROUND: Transformation-associated recombination (TAR) cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes . The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (approximately 60 bp) 5' and 3' gene targeting sequences (hooks) . RESULT: TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating sequence (ARS) that can function as the origin of replication in yeast, which prevents wide application of the method . In this paper, we describe a novel TAR cloning system that allows isolation of genomic regions lacking yeast ARS-like sequences . ARS is inserted into the TAR vector along with URA3 as a counter-selectable marker . The hooks are placed between the TATA box and the transcription initiation site of URA3 . Insertion of any sequence between hooks results in inactivation of URA3 expression . That inactivation confers resistance to 5-fluoroorotic acid, allowing selection of TAR cloning events against background vector recircularization events . CONCLUSION: The new system greatly expands the area of application of TAR cloning by allowing isolation of any chromosomal region from eukaryotic and prokaryotic genomes regardless of the presence of autonomously replicating sequences.

Mol Cell, 2003 Apr, 11(4), 848 - 50
Distressing bacteria: structure of a prokaryotic detox program; de la Cueva-Mendez G; MazF and MazE are components of a chromosomal toxin-antitoxin system of Escherichia coli . In this issue of Molecular Cell, Kamada et al . describe the crystal structure of a MazE/MazF heterohexamer and propose that the mechanism of toxin-antidote recognition is common to other homologous chromosomal and plasmid-borne systems.

World J Gastroenterol, 2003 May, 9(5), 1119 - 22
Recombinant Helicobacter pylori catalase; Bai Y et al.; AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori (H . pylori) and assay the activity of H . pylori catalase . METHODS: The catalase DNA was amplified from H . pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E.coli strain which expressed catalase recombinant protein . The activity of H . pylori catalase was assayed by the Beers and Sizers . RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research . The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 degrees and the activity of H . pylori catalase was high in the BL21 (DE3) E.coli strain . CONCLUSION: A clone expressing high activity H . pylori catalase is obtained, laying a good foundation for further studies.

J Proteome Res, 2003 Mar-Apr, 2(2), 183 - 90
Prediction of enzyme family classes; Chou KC et al.; Classes of newly found enzyme sequences are usually determined either by biochemical analysis of eukaryotic and prokaryotic genomes or by microarray chips . These experimental methods are both time-consuming and costly . With the explosion of protein sequences entering into databanks, it is highly desirable to explore the feasibility of selectively classifying newly found enzyme sequences into their respective enzyme classes by means of an automated method . This is indeed important because knowing which family or subfamily an enzyme belongs to may help deduce its catalytic mechanism and specificity, giving clues to the relevant biological function . In this study, a bioinformatical analysis was conducted for 2640 oxidoreductases classified into 16 subclasses according to the different types of substrates they act on during the catalytic process . Although it is an extremely complicated problem and might involve the knowledge of 3-dimensional structure as well as many other physical chemistry factors, some quite promising results have been obtained indicating that the family or subfamily of an enzyme is predictable to a considerable degree by means of sequence-based approach alone if a good training dataset can be established.

Invest Ophthalmol Vis Sci, 2003 May, 44(5), 1953 - 61
Perfusion with the olfactomedin domain of myocilin does not affect outflow facility; Goldwich A et al.; PURPOSE: Mutations in the MYOC gene coding for myocilin are associated with elevated intraocular pressure (IOP), and recombinant myocilin, produced in a prokaryotic expression system, has been reported to affect aqueous outflow facility . This study was conducted to test whether perfusion with a fragment of recombinant myocilin (containing the full-length olfactomedin domain), produced in a eukaryotic expression system, affects facility . METHODS: 293 EBNA cells were transfected by a vector containing the BM40 signal peptide, a human cDNA coding for myocilin, and a polyhistidine tag (HisTag) sequence . Recombinant protein was isolated by Ni-chelate chromatography, and characterized, and perfused into cultured anterior segments of human and porcine eyes . RESULTS: Recombinant myocilin was secreted as a approximately 55-kDa intact protein and two fragments arising from cleavage of the recombinant protein at amino acid 215 . The C-terminal fragment, containing the entire olfactomedin domain, was successfully isolated . When perfused into human and porcine eyes, this C-terminal fragment did not appreciably affect outflow facility . CONCLUSIONS: Although the olfactomedin domain appears to be important for the function of myocilin, perfusion with a recombinant myocilin fragment containing this domain does not change outflow facility . It is possible that both the olfactomedin and N-terminal domains (including the leucine zipper) must be present for myocilin to have full function . Alternatively, posttranslational modifications of myocilin may have a major impact on protein function.

Trends Biochem Sci, 2003 Apr, 28(4), 170 - 3
TRASH: a novel metal-binding domain predicted to be involved in heavy-metal sensing, trafficking and resistance; Ettema TJ et al.; We describe a previously undetected domain - TRASH - containing a well-conserved cysteine motif that we anticipate to be involved in metal coordination . TRASH is encoded by multiple prokaryotic genomes and is present in transcriptional regulators, cation-transporting ATPases and hydrogenases, and is also present as a stand-alone module . The observed domain associations and conserved genome context of TRASH-encoding genes in prokaryotic genomes suggest that TRASH constitutes a novel component in metal trafficking and heavy-metal resistance . The role of the multiple copies of TRASH that are present in vertebrate proteins remains to be elucidated.

Plant J, 2003 May, 34(3), 377 - 82
The Shine-Dalgarno-like sequence is a negative regulatory element for translation of tobacco chloroplast rps2 mRNA: an additional mechanism for translational control in chloroplasts; Plader W et al.; Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation . The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon) . Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined . Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation . Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation . Scanning mutagenesis assays confirmed the above result . Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation . In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.

Biopolymers, 2003, 71(1), 28 - 48
Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S; Lee DL et al.; The cyclic beta-sheet structure possessed by the 10-residue antibiotic peptide gramicidin S was taken as the structural framework for the de novo design of biologically active peptides with membrane-active properties . We have shown from previous studies that gramicidin S is a broad-spectrum antibiotic effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, but is toxic to human red blood cells . We tested the effect of ring size on antimicrobial activity and hemolytic activity on peptides varying from 4 to 16 residues . Interestingly, we were able to dissociate hemolytic activity and antimicrobial activity by increasing the ring size of the peptide to 14 residues (peptide GS14) . Furthermore, we increased specificity for microbial membranes while decreasing toxicity to red blood cells by substituting enantiomers (D-amino acids for L-amino acids and vice versa) into the GS14 sequence . The enantiomeric substitutions all disrupted beta-sheet structure in benign medium and decreased peptide amphipathicity . The least amphipathic peptide, produced by substituting a D-Lys at position 4 of GS14 (peptide GS14K4), also had the highest therapeutic index, i.e., highest degree of specificity for microbial cells over human cells . Solution structures of GS14 analogs solved by NMR spectroscopy showed that the D-amino acid side chain was located on the nonpolar face of GS14K4 . Another analog, a beta-sheet peptide with reduced amphipathicity (peptide GS14 K3L4), also had a lysine (lysine 3) on the nonpolar face as determined by the NMR structure . Both GS14K4 and GS14 K3L4 had reduced amphipathicity relative to GS14 and much higher therapeutic indices . Finally, the alteration of the nonpolar face hydrophobicity of GS14K4 analogs provided a range of activities and specificities, where the peptides with the intermediate hydrophobicities among the series had the highest therapeutic indices . The optimal peptide hydrophobicities varied depending on the microorganism being tested, with higher hydrophobicity requirements against Gram-positive bacteria and yeast compared with Gram-negative microorganisms . The net result of these studies suggests that it is possible to rationally design a cyclic membrane-active antimicrobial peptide with high specificity towards prokaryotic (bacterial and fungal) membranes and minimal toxicity to eukaryotic (e.g., mammalian) membranes .

Arch Microbiol, 2003 Jun, 179(6), 409 - 15 Epub 2003 Apr 24.
A novel type of lycopene epsilon-cyclase in the marine cyanobacterium Prochlorococcus marinus MED4; Stickforth P et al.; Chlorophyll- b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of alpha- and beta-carotenoids with green algae and higher plants . The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, epsilon- and beta-lycopene cyclase . All cyanobacteria studied so far possess a single beta-cyclase . Here, two different Prochlorococcus sp . MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases . Whereas one is both functionally and in sequence highly similar to the beta-cyclase of Synechococcus sp . strain PCC 7942 and other cyanobacteria, the other showed several intriguing features . It acts as a bifunctional enzyme catalyzing the formation of epsilon- as well as of beta-ionone end groups . Expression of this cyclase in E . coli resulted in the simultaneous accumulation of alpha- beta-, delta-, and epsilon-carotene . Such an activity is in contrast to all lycopene epsilon-cyclases known so far, including those of the higher plants . Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either beta- or epsilon-end groups . These two genes are suggested to be designated as crtL-b and crtL-e . The results indicate that both enzymes might have originated from duplication of a single gene . Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.

Gene Expr Patterns, 2003 May, 3(2), 147 - 52
Darmin is a novel secreted protein expressed during endoderm development in Xenopus; Pera EM et al.; Endoderm development is an area of intense interest in developmental biology, but progress has been hampered by the lack of specific markers for differentiated endodermal cells . In an unbiased secretion cloning screen of Xenopus gastrula embryos we isolated a novel gene, designated Darmin . Darmin encodes a secreted protein of 56 kDa containing a peptidase M20 domain characteristic of the glutamate carboxypeptidase group of zinc metalloproteases . We also identified homologous Darmin genes in other eukaryotes and in prokaryotes suggesting that Darmin is the founding member of a family of evolutionarily conserved proteins . Xenopus Darmin showed zygotic expression in the early endoderm and later became restricted to the midgut . By secretion cloning of Xenopus cleavage-stage embryos we isolated another novel protein, designated Darmin-related (Darmin-r) due to its sequence similarity with Darmin . Darmin-r was maternally expressed and showed at later stages expression in the lens and pronephric glomus . The endoderm-specific expression of Darmin makes this gene a useful marker for the study of endoderm development.

Anal Biochem, 2003 May 15, 316(2), 223 - 31
Detection and prevention of protein aggregation before, during, and after purification; Bondos SE et al.; The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers . A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks . We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete . This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting . Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility . The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification . Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications . This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins . From the results presented, this technique can be applied to a variety of proteins.

J Exp Bot, 2003 May, 54(386), 1351 - 60
Molecular and biochemical characterization of cytosolic phosphoglucomutase in wheat endosperm (Triticum aestivum L . cv . Axona); Davies EJ et al.; Evidence from a number of plant tissues suggests that phosphoglucomutase (PGM) is present in both the cytosol and the plastid . The cytosolic and plastidic isoforms of PGM have been partially purified from wheat endosperm (Triticum aestivum L . cv . Axona) . Both isoforms required glucose 1,6-bisphosphate for their activity with K(a) values of 4.5 micro M and 3.8 micro M for cytosolic and plastidic isoforms, respectively, and followed normal Michaelis-Menten kinetics with glucose 1-phosphate as the substrate with K(m) values of 0.1 mM and 0.12 mM for the cytosolic and plastidic isoforms, respectively . A cDNA clone was isolated from wheat endosperm that encodes the cytosolic isoform of PGM . The deduced amino acid sequence shows significant homology to PGMs from eukaryotic and prokaryotic sources . PGM activity was measured in whole cell extracts and in amyloplasts isolated during the development of wheat endosperm . Results indicate an approximate 80% reduction in measurable activity of plastidial and cytosolic PGM between 8 d and 30 d post-anthesis . Northern analysis showed a reduction in cytosolic PGM mRNA accumulation during the same period of development . The implications of the changes in PGM activity during the synthesis of starch in developing endosperm are discussed.

Trends Microbiol, 2003 Apr, 11(4), 166 - 70
Were Gram-positive rods the first bacteria?
Koch AL.
At some point in the evolution of life, the domain Bacteria arose from prokaryotic progenitors . The cell that gave rise to the first bacterium has been given the name (among several other names) "last universal ancestor (LUA)" . This cell had an extensive, well-developed suite of biochemical strategies that increased its ability to grow . The first bacterium is thought to have acquired a covering, called a sacculus or exoskeleton, that made it stress-resistant . This protected it from rupturing as a result of turgor pressure stress arising from the success of its metabolic abilities . So what were the properties of this cell's wall? Was it Gram-positive or Gram-negative? And was it a coccus or a rod?

J Mol Biol, 2003 May 2, 328(3), 555 - 66
Crystal structure of a full-length LysR-type transcriptional regulator, CbnR: unusual combination of two subunit forms and molecular bases for causing and changing DNA bend; Muraoka S et al.; The LysR-type transcriptional regulator (LTTR) proteins are one of the most common transcriptional regulators in prokaryotes . Here we report the crystal structure of CbnR, which is one of the LTTRs derived from Ralstonia eutropha NH9 . This is the first crystal structure of a full-length LTTR . CbnR was found to form a homo-tetramer, which seems to be a biologically active form . Surprisingly, the tetramer can be regarded as a dimer of dimers, whereby each dimer is composed of two subunits in different conformations . In the CbnR tetramer, the DNA-binding domains are located at the V-shaped bottom of the main body of the tetramer, and seem to be suitable to interact with a long stretch of the promoter DNA, which is approximately 60bp . Interaction between the four DNA-binding domains and the two binding sites on the target DNA is likely to bend the target DNA along the V-shaped bottom of the CbnR tetramer . The relaxation of the bent DNA, which occurs upon inducer binding to CbnR, seems to be associated with a quaternary structure change of the tetramer.

Biochem Biophys Res Commun, 2003 Apr 25, 304(1), 78 - 85
The inhibitory action of pyrrolidine alkaloid, 1,4-dideoxy-1,4-imino-D-ribitol, on eukaryotic DNA polymerases; Mizushina Y et al.; The pyrrolidine alkaloids mimicking the structures of pentose with nitrogen in the ring are known to be inhibitors of glycosidases . We report here that a compound belonging to this category is an inhibitor of eukaryotic DNA polymerases . Among the eight naturally occurring pyrrolidine alkaloids we tested, only one compound, 1,4-dideoxy-1,4-imino-D-ribitol (DRB), which was purified from the mulberry tree (Morus alba), strongly inhibited the activities of eukaryotic DNA polymerases with IC50 values of 21-35 microM, and had almost no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, and bovine deoxyribonuclease I . Kinetic studies showed that inhibition of both DNA polymerases alpha and beta by DRB was competitive with respect to dNTP substrate . Whereas DNA polymerase alpha inhibition was noncompetitive with the template-primer, the inhibition of DNA polymerase beta was found to be competitive with the template-primer . The K(i) values of DNA polymerases alpha and beta for the template-primer were smaller than those for dNTP substrate . Therefore, the affinity of DRB was suggested to be higher at the template-primer binding site than at the dNTP substrate-binding site, although DRB is an analogue of deoxyribose consisting of dNTP . Computational analyses of the eight pyrrolidine alkaloids revealed a remarkable difference in the distribution of positive and negative electrostatic charges on the surface of molecules . The relationship between the structure of DRB and the inhibition of eukaryotic DNA polymerases is discussed.

Anal Chem, 2003 Apr 1, 75(7), 1741 - 7
Selective extraction and characterization of a histidine-phosphorylated peptide using immobilized copper(II) ion affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Napper S et al.; Phosphorylation is the predominant posttranslational modification involved in regulating enzymatic activity and mediating signal transduction in prokaryotic and eukaryotic cells . Selective enrichment of phosphorylated peptides prior to mass spectrometric analysis facilitates identification of phosphorylated proteins, determination of specific phosphorylated residues, and characterization of the conditions under which phosphorylation occurs . Such protocols have been established for peptides containing residues that form phosphoesters, such as serine and threonine, using immobilized metal-ion affinity chromatography . Despite the importance of histidine phosphorylation in two-component signal transduction pathways, similar protocols for peptides containing phosphorylated histidine (P-His) residues have proven elusive, due to the instability of these modifications and the propensity of unphosphorylated histidines to interact with immobilized metals ions . We describe a method for the selective extraction of a P-His-containing peptide using immobilized copper(II) ions and disposable metal-chelating pipet tips (ZipTipMC, Millipore) . The method is contingent upon pH-dependent interactions between the phosphate group and immobilized copper(II) ions . Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with postsource decay confirms the identity and phosphorylation state of the extracted peptide . Peptides containing unphosphorylated histidine residues or other phosphorylated amino acids are not retained, demonstrating the specificity of the method for P-His-containing peptides.

J Allergy Clin Immunol, 2003 Apr, 111(4), 889 - 96
Biological activity of IgE specific for cross-reactive carbohydrate determinants; Foetisch K et al.; BACKGROUND: The clinical relevance of IgE specific for cross-reactive carbohydrate determinants (CCDs) has been a matter of controversy . Until now, no convincing experiments have been performed to test the biologic significance of individual multivalent allergens that carry multiple carbohydrate epitopes . OBJECTIVE: We sought to contribute to the understanding of the role of CCD-specific IgE antibodies and to study whether CCD-specific IgE antibodies are able to activate basophils using different multivalent glycoproteins as antigens . METHODS: Purified natural tomato beta-fructofuranosidase (nLyc e 2) and rLyc e 2.02 expressed in Escherichia coli were compared by means of histamine release tests . In addition, native and deglycosylated horseradish peroxidase and a neoglycoprotein consisting of a defined glycopeptide (Manalpha1-6{Xylbeta1-2}Manbeta1-4GlcNAcbeta1-4{Fucalpha1-3}GlcNAc) coupled to BSA were used in histamine release assays using stripped basophils from normal donors resensitized with IgE from CCD-reactive patients with food allergy to tomato . RESULTS: Ten CCD-positive and 2 CCD-negative sera from patients with tomato allergy underwent histamine release testing by using the glycoproteins and nonglycosylated controls as antigens, respectively . All sera induced histamine release with tomato extract (up to 100%), confirming the allergic status of the donors . Four of the CCD-positive sera induced releases ranging from 12% to 82% with all of the glycoproteins but not with the nonglycosylated or monovalent controls . All other sera showed no response or only very weak response to the glycoproteins . CONCLUSION: Approximately one third of the CCD-positive sera from patients with tomato allergy have biologically relevant CCD-specific IgE antibodies . Therefore the general claim that CCD-specific IgE is clinically irrelevant has to be reconsidered critically . Hence IgE specific for CCDs should be taken into consideration in the diagnosis and therapy of certain allergies . In the subgroup of patients sensitized to CCDs, the use of natural allergens should be preferred over the use of recombinant allergens expressed in prokaryotic organisms.

FEMS Yeast Res, 2002 May, 2(2), 123 - 35
Preliminary characterisation of DML1, an essential Saccharomyces cerevisiae gene related to misato of Drosophila melanogaster; Gurvitz A et al.; A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein . Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ . Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1Delta heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad . DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were {rho(0)} lacking mtDNA . In addition, meiotic transmission of centromeric plasmids also appeared to be impaired . Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part . Inheritance of mtDNA could also be restored using misato cDNA . Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology . We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively . Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement.

J Biol Chem, 2003 Jun 20, 278(25), 22466 - 74 Epub 2003 Apr 07.
Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule; Kysela B et al.; The DNA ligase IV.XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells . We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate . The effect is specific for ds ligation, and LX/DNA binding is not influenced by the substrate length . Ku stimulates LX ligation at concentrations resulting in 1-2 Ku molecules bound per substrate, whereas multiply Ku-bound DNA molecules inhibit ds ligation . The combined footprint of DNA with Ku and LX bound is the sum of each individual footprint suggesting that the two complexes are located in tandem at the DNA end . Inhibition of Ku translocation by the presence of cis-platinum adducts on the DNA substrate severely inhibits ligation by LX . Fluorescence resonance energy transfer analysis using fluorophore-labeled Ku and DNA molecules showed that, as expected, Ku makes close contact with the DNA end and that addition of LX can disrupt this close contact . Finally, we show that recruitment of LX by Ku is impaired in an adenylation-defective mutant providing further evidence that LX interacts directly with the DNA end, possibly via the 5'-phosphate as shown for prokaryotic ligases . Taken together, our results suggest that, when LX binds to a Ku-bound DNA molecule, it causes inward translocation of Ku and that freedom to move inward on the DNA is essential to Ku stimulation of LX activity.

Biol Bull, 2003 Apr, 204(2), 205 - 9
From genes to genomes: beyond biodiversity in Spain's Rio Tinto; Amaral Zettler LA et al.; Spain's Rio Tinto, or Red River, an example of an extremely acidic (pH 1.7-2.5) environment with a high metal content, teems with prokaryotic and eukaryotic microbial life . Our recent studies based on small-subunit rRNA genes reveal an unexpectedly high eukaryotic phylogenetic diversity in the river when compared to the relatively low prokaryotic diversity . Protists can therefore thrive in and dominate extremely acidic, heavy-metal-laden environments . Further, because we have discovered protistan acidophiles closely related to neutrophiles, we can hypothesize that the transition from neutral to acidic environments occurs rapidly over geological time scales . How have these organisms adapted to such environments? We are currently exploring the alterations in physiological mechanisms that might allow for growth of eukaryotic microbes at acid extremes . To this end, we are isolating phylogenetically diverse protists in order to characterize and compare ion-transporting ATPases from cultured acidophiles with those from neutrophilic counterparts . We predict that special properties of these ion transporters allow protists to survive in the Rio Tinto.

Biol Bull, 2003 Apr, 204(2), 109 - 13
Ultraviolet-light-absorbing tunic cells in didemnid ascidians hosting a symbiotic photo-oxygenic prokaryote, Prochloron; Maruyama T et al.; Coral reef invertebrates that host phototrophic symbionts are thought to protect themselves and their symbionts with mycosporine-like amino acids (MAAs)-UV-absorbing substances that act as sunscreens (Dunlap, W . C., and J . M . Shick, 1998 . J . Phycol . 34: 418-430) . However, the histological distribution of MAAs in the host tissues has not yet been visualized . We have localized the UV-absorbing substances in the tissues of two colonial didemnid ascidians-Lissoclinum patella and Diplosoma sp.-that contain the symbiotic photo-oxygenic prokaryote Prochloron sp . Cross-sections of unfixed tissue from these ascidians were examined by UV-light microscopy at 320 or 330 nm, wavelengths at which UV light is absorbed by MAAs . Within the tunic, the gelatinous integument of the colony, UV light was exclusively absorbed by a particular type of cell, the tunic bladder cell . Tunic bladder cells with strong UV absorption were denser in the upper tunic, which lies over a colony's zooids, than in the basal tunic underlying the zooid . In the upper tunic, those cells with strong UV absorption were most dense near the surface . The tunic bladder cell is highly vacuolated, and the vacuole contains strong acid, which destabilizes MAAs . Furthermore, the UV-absorbing portion of tunic bladder cells seemed to be cup-shaped, indicating that the MAAs are not localized in the vacuole, but in the cytoplasm . These results strongly suggest that didemnid ascidians accumulate MAAs in tunic bladder cells as a protection against UV radiation.

Front Biosci, 2003 May 01, 8, d652 - 60
Maltose transport through the inner membrane of E . coli; Fetsch EE et al.; The maltose transport complex of E.coli is one of the most well-characterized members of the ATP-Binding Cassette (ABC) protein superfamily . ABC proteins represent the largest superfamily of transmembrane proteins in prokaryotes and eukaryotes, performing diverse functions from ion transport by the cystic fibrosis transmembrane regulator to multiple drug efflux by the P-glycoprotein transporter and sugar transport by the maltose transporter . Characterization of the mechanism of transport for ABC transporters is currently being investigated both biochemically and structurally, however some uncertainty remains as to how the individual subunits of these multisubunit transporters interact . This review discusses the current knowledge of the mechanism of maltose transport, as it relates to the ABC superfamily of transporters as a whole.

Neurosurgery, 2003 May, 52(5), 1154 - 65; discussion 1165-7
Engineering of the extracellular matrix: working toward neural stem cell programming and neurorestoration--concept and progress report; Liu CY et al.; In the concept of neurorestoration, cellular and structural elements that have been lost are replaced, and their function is restored . Central to this therapeutic strategy is the transplantation of neural progenitor cells such as clonogenically expanded stem cells . Stem cells make decisions regarding fate and patterning in response to external environmental signals . The therapeutic effectiveness of neural stem cells may be facilitated by the ability to manipulate these signals in a temporal and spatially appropriate fashion . Artificial deoxyribonucleic acid and artificial protein technology combines elements of protein engineering, molecular biology, and recombinant deoxyribonucleic acid technology to produce proteins with functional domains derived from naturally occurring proteins and represents a potentially powerful tool to modulate stem cell behavior . To this end, we have developed three artificial extracellular matrix proteins that incorporate the active domain of hJagged1 and hDelta1 into an elastin backbone . hJagged1 and hDelta1 are members of the DSL family of ligands to the Notch receptor, a signaling system that is very important in development and is the strongest known signal to instruct neural progenitor cells to choose glial fates over neuronal fates . The successful cloning of the artificial genes was confirmed by test digestions with appropriate restriction enzymes as well as direct deoxyribonucleic acid sequencing . In addition, we have demonstrated that all three artificial extracellular matrix proteins express at a high level in a prokaryotic host . This report describes the concept and progress in an entirely novel and previously unreported approach to modulate neural stem cell behavior . Its future application could include in vitro processing of stem cells before transplantation, supporting and programming the cells after transplantation, as well as the development of bioactive biomaterials.

Di Yi Jun Yi Da Xue Xue Bao, 2003 Apr, 23(4), 293 - 5, 309
Construction of the E.coli clone expressing adhesin BabA of Helicobacter pylori and evaluation of the adherence activity of BabA; Bai Y et al.; OBJECTIVE: To construct a recombinant E.coli strain that highly expresses blood group Ag-binding adhesin (BabA) of Helicobacter pylori (Hp) and to assess the adherence activity of Hp BabA . METHODS: The gene fragment encoding BabA was amplified from Hp chromosomal DNA by PCR technique and inserted into prokaryotic expression vector pET-22b (+), which was then transformed into BL21 (DE3) E.coli strain for the expression of BabA recombinant protein . The adherence activity of Hp BabA obtained was assayed by counting under light microscope . RESULTS: DNA sequence analysis showed that the sequence of babA2 DNA was in agreement with that published in GenBank . The BabA recombinant protein amounted to 34.8% of the total protein of the bacterium after IPTG induction for 3 h at 37 degrees Celsius, and BabA-mediated adherence was confirmed in vitro . CONCLUSION: A clone expressing biologically active Hp BabA has been obtained, which may facilitate further study of the function of the adhesin.

FEMS Microbiol Rev, 2003 Apr, 27(1), 17 - 34
Phages of the marine cyanobacterial picophytoplankton; Mann NH; Cyanobacteria of the genera Synechococcus and Prochlorococcus dominate the prokaryotic component of the picophytoplankton in the oceans . It is still less than 10 years since the discovery of phages that infect marine Synechococcus and the beginning of the characterisation of these phages and assessment of their ecological impact . Estimations of the contribution of phages to Synechococcus mortality are highly variable, but there is clear evidence that phages exert a significant selection pressure on Synechococcus community structure . In turn, there are strong selection pressures on the phage community, in terms of both abundance and composition . This review focuses on the factors affecting the diversity of cyanophages in the marine environment, cyanophage interactions with their hosts, and the selective pressures in the marine environment that affect cyanophage evolutionary biology.

BMC Genomics . 2003 Apr 02;4(1):12.
Analysis of two large functionally uncharacterized regions in the Methanopyrus kandleri AV19 genome; Jensen LJ et al.; BACKGROUND: For most sequenced prokaryotic genomes, about a third of the protein coding genes annotated are "orphan proteins", that is, they lack homology to known proteins . These hypothetical genes are typically short and randomly scattered throughout the genome . This trend is seen for most of the bacterial and archaeal genomes published to date . RESULTS: In contrast we have found that a large fraction of the genes coding for such orphan proteins in the Methanopyrus kandleri AV19 genome occur within two large regions . These genes have no known homologs except from other M . kandleri genes . However, analysis of their lengths, codon usage, and Ribosomal Binding Site (RBS) sequences shows that they are most likely true protein coding genes and not random open reading frames . CONCLUSIONS: Although these regions can be considered as candidates for massive lateral gene transfer, our bioinformatics analysis suggests that this is not the case . We predict many of the organism specific proteins to be transmembrane and belong to protein families that are non-randomly distributed between the regions . Consistent with this, we suggest that the two regions are most likely unrelated, and that they may be integrated plasmids.

Biochem J, 2003 Jul 15, 373(Pt 2), 635 - 40
Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases; Guranowski A et al.; Dinucleoside 5',5"'- P (1), P ( n )-polyphosphates, and particularly the diadenosine compounds, have been implicated in extracellular purinergic signalling and in various intracellular processes, including DNA metabolism, tumour suppression and stress responses . If permitted to accumulate, they may also be toxic . One approach to understanding their function is through the various specific degradative enzymes that regulate their levels . Eight adenosine-5'- O -phosphorylated polyols (derivatives of glycerol, erythritol and pentaerythritol) and 11 adenosine-5'- O -phosphorothioylated polyols (derivatives of glycerol, erythritol, pentaerythritol, butanediol and pentanediol) have been tested as inhibitors of specific diadenosine tetraphosphate (Ap(4)A) hydrolases . Of these two groups of novel nucleotides, the adenosine-5'- O -phosphorothioylated polyols were generally stronger inhibitors than their adenosine-5'- O -phosphorylated counterparts . 1,4-Di(adenosine-5'- O -phosphorothio) erythritol appeared to be the strongest inhibitor of ( asymmetrical ) Ap(4)A hydrolases (EC 3.6.1.17) from both lupin and human, with K (i) values of 0.15 microM and 1.5 microM respectively . Of eight adenosine-5'- O -phosphorylated polyols, 1,4-di(adenosine-5'- O -phospho) erythritol was the only compound that inhibited the lupin enzyme . Two derivatives of pentaerythritol, di(adenosine-5'- O -phosphorothio)-di(phosphorothio) pentaerythritol and tri(adenosine-5'- O -phosphorothio)-phosphorothio-pentaerythritol, proved to be the strongest inhibitors of the prokaryotic ( symmetrical ) Ap(4)A hydrolase (EC 3.6.1.41) so far reported . The estimated K (i) values were 0.04 microM and 0.08 microM respectively . All of these inhibitors were competitive with respect to Ap(4)A . These new selectively acting Ap(4)A analogues should prove to be valuable tools for further studies of Ap(4)A function and of the enzymes involved in its metabolism.

J Am Chem Soc, 2003 Apr 23, 125(16), 4686 - 7
Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne {3 + 2} cycloaddition; Speers AE et al.; Toward the goal of assigning function to the tens of thousands of protein products encoded by eukaryotic and prokaryotic genomes, the field of proteomics requires new technologies that can functionally characterize proteins within the dynamic environment of the cell, where these biomolecules are subject to myriad posttranslational modifications and the actions of endogenous activators and inhibitors . Here, we report an advanced strategy for activity-based protein profiling (ABPP) that addresses this important need . We show that several enzymes can be labeled in an activity-based manner both in vitro and in vivo by an azido-sulfonate ester probe and that these labeling events can be detected in whole proteomes by copper-catalyzed ligation with a rhodamine-alkyne reagent . This click chemistry-based strategy for ABPP represents a unique and versatile method for functional proteome analysis.

Int Rev Cytol, 2003, 225, 273 - 323
Biogenesis and cellular dynamics of aminoglycerophospholipids; Birner R et al.; Aminoglycerophospholipids phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho) comprise about 80% of total cellular phospholipids in most cell types . While the major function of PtdCho in eukaryotes and PtdEtn in prokaryotes is that of bulk membrane lipids, PtdSer is a minor component and appears to play a more specialized role in the plasma membrane of eukaryotes, e.g., in cell recognition processes . All three aminoglycerophospholipid classes are essential in mammals, whereas prokaryotes and lower eukaryotes such as yeast appear to be more flexible regarding their aminoglycerophospholipid requirement . Since different subcellular compartments of eukaryotes, namely the endoplasmic reticulum and mitochondria, contribute to the biosynthetic sequence of aminoglycerophospholipid formation, intracellular transport, sorting, and specific function of these lipids in different organelles are of special interest.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4399 - 404 Epub 2003 Apr 07.
A complex microbiota from snowball Earth times: microfossils from the Neoproterozoic Kingston Peak Formation, Death Valley, USA; Corsetti FA et al.; A thin carbonate unit associated with a Sturtian-age ( approximately 750-700 million years ago) glaciogenic diamictite of the Neoproterozoic Kingston Peak Formation, eastern California, contains microfossil evidence of a once-thriving prokaryotic and eukaryotic microbial community (preserved in chert and carbonate) . Stratiform stromatolites, oncoids, and rare columnar stromatolites also occur . The microbial fossils, which include putative autotrophic and heterotrophic eukaryotes, are similar to those found in chert in the underlying preglacial units . They indicate that microbial life adapted to shallow-water carbonate environments did not suffer the significant extinction postulated for this phase of low-latitude glaciation and that trophic complexity survived through snowball Earth times.

Proteins, 2003 May 15, 51(3), 453 - 69
Atomic resolution structure of prokaryotic phospholipase A2: analysis of internal motion and implication for a catalytic mechanism; Matoba Y et al.; We have found a secreted phospholipase A(2) (PLA(2), EC 3.1.1.4) from Streptomyces violaceoruber A-2688, which is the first PLA(2) identified in prokaryote, and determined its tertiary structure by NMR and X-ray analyses . In this study, we collected the X-ray diffraction data of the bacterial PLA(2) at room temperature (297 K) using conventional MoK(alpha) radiation and refined the structure at a 1.05 A resolution . The atomic resolution analysis led us to introduce disordered conformations and hydrogen atoms into a full anisotropic model . The molecular motion, which is expressed as the sum of rigid-body motion and internal motion of protein, is roughly estimated as the thermal motion when the X-ray diffraction data are collected at room temperature . In this study, we applied a TLS (rigid-body motion in terms of translation, libration, and screw motions) model to analyze the rigid-body motion of the bacterial PLA(2) and calculated the internal motion by subtracting the estimate of the rigid-body motion from the observed anisotropic temperature factor . We also subjected the TLS model to estimate the internal motion of the bovine pancreatic PLA(2) using the anisotropic temperature factor deposited in the Protein Data Bank . Both results indicate that the localization of regions exhibiting larger internal motion in the bacterial PLA(2) is almost the same as that in the bovine pancreatic PLA(2), suggesting that although the tertiary structure of the bacterial PLA(2) is strikingly different from that of the bovine pancreatic PLA(2), the internal motion, which is associated with the calcium(II) ion-binding, phospholipid-binding, and allosteric interfacial activation, is commonly observed in both PLA(2)s .

Genome Res, 2003 May, 13(5), 875 - 82 Epub 2003 Apr 14.
Genomic gene clustering analysis of pathways in eukaryotes; Lee JM et al.; Genomic clustering of genes in a pathway is commonly found in prokaryotes due to transcriptional operons, but these are not present in most eukaryotes . Yet, there might be clustering to a lesser extent of pathway members in eukaryotic genomes, that assist coregulation of a set of functionally cooperating genes . We analyzed five sequenced eukaryotic genomes for clustering of genes assigned to the same pathway in the KEGG database . Between 98% and 30% of the analyzed pathways in a genome were found to exhibit significantly higher clustering levels than expected by chance . In descending order by the level of clustering, the genomes studied were Saccharomyces cerevisiae, Homo sapiens, Caenorhabditis elegans, Arabidopsis thaliana, and Drosophila melanogaster . Surprisingly, there is not much agreement between genomes in terms of which pathways are most clustered . Only seven of 69 pathways found in all species were significantly clustered in all five of them . This species-specific pattern of pathway clustering may reflect adaptations or evolutionary events unique to a particular lineage . We note that although operons are common in C . elegans, only 58% of the pathways showed significant clustering, which is less than in human . Virtually all pathways in S . cerevisiae showed significant clustering.

Arch Ophthalmol, 2003 Apr, 121(4), 478 - 82
Serological association between Chlamydia pneumoniae infection and age-related macular degeneration; Kalayoglu MV et al.; BACKGROUND: Age-related macular degeneration (ARMD) is a leading cause of blindness in the United States, but the mechanisms that initiate and promote the disease remain ill defined . There are several risk factors that ARMD shares with atherosclerosis, and these diseases may have similar pathogenic mechanisms that involve inflammation . Chlamydia pneumoniae, a prokaryotic pathogen that causes chronic inflammation is now emerging as a risk factor in the development of cardiovascular diseases . It is therefore plausible that this microorganism also contributes to the pathogenesis of ARMD . METHODS: To examine if C pneumoniae infection is associated with ARMD, serum samples from 25 consecutive patients with ARMD and from 18 without the disease were collected and assayed for the presence of the antibodies to C pneumoniae elementary bodies, Chlamydia trachomatis heat shock protein 60 (cHsp60), C trachomatis heat shock protein 10 (cHsp10), Escherichia coli GroEL, and E coli GroES . RESULTS: A serological association was found between ARMD and anti-C pneumoniae antibodies (P =.047) but not between ARMD and the anti-C trachomatis or anti-E coli heat shock protein antibodies . The association remained statistically significant after adjusting for age and smoking, both established risk factors for ARMD . CONCLUSIONS: These data indicate that C pneumoniae infection may be associated with ARMD . Further studies on larger cohorts of individuals are necessary to determine if this pathogen plays a role in the pathogenesis of ARMD.

FEMS Microbiol Lett, 2003 Apr 11, 221(1), 39 - 47
Characterisation of IS901 integration sites in the Mycobacterium avium genome; Inglis NF et al.; Data are presented on the identification and characterisation of 17 chromosomal integration loci of the insertion element IS901 in the Mycobacterium avium (cervine strain JD88/118) genome . Thirteen of these integration loci have been mapped to their corresponding positions on the M . avium strain 104 (an IS901(-) strain) genome (The Institute for Genome Research (TIGR) unfinished genome-sequencing project) . Sequence data for both upstream and downstream sequence flanking regions were obtained for 12 insertion loci, while upstream sequence was obtained for five others . A consensus IS901 insertion target sequence compiled from all 17 integration sites was in broad agreement with earlier reports that were based on only two such loci . Analysis of IS901 integration site flanking sequences revealed that, like IS900 in M . avium subspecies paratuberculosis, IS901 inserts preferentially between a putative ribosome-binding sequence (RBS) and the translational start codon of an open reading frame (ORF) . In BLAST X and BLAST P searches of the GenBank database, these ORFs were shown to share significant homologies with a number of other prokaryotic genes.

FEMS Microbiol Lett, 2003 Apr 11, 221(1), 17 - 23
Immunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids; Motta MC et al.; Research about the kinetoplast of trypanosomatids has yielded valuable information about the organization of extranuclear structure . However, the ultrastructural localization of nucleic acids within these protozoa remains uncertain . We have applied cytochemical and immunocytochemical approaches to precisely identify DNA and RNA in lower endosymbiont-bearing trypanosomatids . Using the Terminal deoxynucleotidyl Transferase (TdT) immunogold technique, we showed that nuclear DNA is seen associated with the nuclear envelope during the trypanosomatid cell cycle . By combining the TdT technique with the acetylation method, which improves the contrast between structures containing fibrils and granules, we have demonstrated that the nucleolus of endosymbiont-bearing trypanosomatids is composed of two constituents: a granular component and a DNA-positive fibrillar zone . Moreover, we revealed that DNA of endosymbiotic bacteria consisted of electron-dense filaments which are usually in close contact with the prokaryote envelope . Using a Lowicryl post-embedding immunogold labeling procedure with anti-RNA antibodies, we showed the presence of RNA not only over the cytoplasm, the interchromatin spaces and the nucleolus, but also over the kinetoplast and virus-like particles present in Crithidia desouzai.

FEMS Microbiol Lett, 2003 Apr 11, 221(1), 1 - 6
Mechanisms of replication and telomere resolution of the linear plasmid prophage N15; Ravin NV; The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends . Upon infection of an Escherichia coli cell, the phage DNA circularizes via cohensive ends . A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres) . Purified protelomerase alone processes circular and linear plasmid DNA containing the target site telRL to produce linear double-stranded DNA with covalently closed ends in vitro . N15 protelomerase is necessary for replication of the linear prophage through its action as a telomere-resolving enzyme . Replication of circular N15-based miniplasmids requires the only gene repA that encodes multidomain protein homologous to replication proteins of bacterial plasmids replicated by theta-mechanism, particularly, phage P4 alpha-replication protein . Replication of the N15 prophage is initiated at an internal ori site located within repA . Bidirectional replication results in formation of the circular head-to-head, tail-to-tail dimer molecule . Then the N15 protelomerase cuts both duplicated telomeres generating two linear plasmid molecules with covalently closed ends . The N15 prophage replication thus appears to follow the mechanism distinct from that employed by poxviruses and could serve as a model for other prokaryotic replicons with hairpin ends, and particularly, for linear plasmids and chromosomes of Borrelia burgdorferi.

Eur J Biochem, 2003 Apr, 270(8), 1699 - 709
Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes; Storm P et al.; In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes . Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature') . Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity . The lateral organization of five unsaturated A . laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs . Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses . Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids . The anionic precursor phosphatidic acid (PA) was without effects . Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers . The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes . It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.

Nat Struct Biol, 2003 May, 10(5), 379 - 85
Two crystal structures demonstrate large conformational changes in the eukaryotic ribosomal translocase; Jorgensen R et al.; Two crystal structures of yeast translation elongation factor 2 (eEF2) were determined: the apo form at 2.9 A resolution and eEF2 in the presence of the translocation inhibitor sordarin at 2.1 A resolution . The overall conformation of apo eEF2 is similar to that of its prokaryotic homolog elongation factor G (EF-G) in complex with GDP . Upon sordarin binding, the three tRNA-mimicking C-terminal domains undergo substantial conformational changes, while the three N-terminal domains containing the nucleotide-binding site form an almost rigid unit . The conformation of eEF2 in complex with sordarin is entirely different from known conformations observed in crystal structures of EF-G or from cryo-EM studies of EF-G-70S complexes . The domain rearrangements induced by sordarin binding and the highly ordered drug-binding site observed in the eEF2-sordarin structure provide a high-resolution structural basis for the mechanism of sordarin inhibition . The two structures also emphasize the dynamic nature of the ribosomal translocase.

Bioinformatics, 2003 Apr 12, 19(6), 681 - 5
Genome-wide analysis of Bkm sequences (GATA repeats): predominant association with sex chromosomes and potential role in higher order chromatin organization and function; Subramanian S et al.; MOTIVATION: Bkm (Banded krait minor) satellite DNA sequences (GATA repeats) have been shown to be associated with the sex determining chromosomes of various eukaryotes and have been implicated in the evolution and differentiation of sex chromosomes in snakes . The objective of the study is to analyze the GATA repeats of human genome specifically, the Y-chromosome, and other model organisms to understand the possible function and potential role in higher order chromatin organization . RESULTS: Our extensive analysis of GATA repeats in the prokaryotic and eukaryotic genomes, which have been completely sequenced so far, has revealed that GATA repeats are absent in prokaryotes and have been gradually accumulated in higher organisms during the course of evolution . In human, the Y-chromosome has the highest GATA repeat density, which predominantly exists in the Yq centromeric region . Generally, occurrence of repeats in the genomes decreases steadily as the length of the repeat increases . In contrast, we report, that the occurrence of GATA repeats increases as the length of the repeat increases from six tandem repeats onwards and peaks at (GATA)(10-12) . This has not been observed with any other simple repeat . Distribution of (GATA)(10-12) along the chromosome and their close proximity to Matrix Associated Regions (GATA-MAR) suggests that it may be demarking chromatin domains for a coordinated expression of genes residing in these domains.

Biosystems, 2003 May, 69(2-3), 187 - 209
A computational model of symbiotic composition in evolutionary transitions; Watson RA et al.; Several of the major transitions in evolutionary history, such as the symbiogenic origin of eukaryotes from prokaryotes, share the feature that existing entities became the components of composite entities at a higher-level of organization . This composition of pre-adapted extant entities into a new whole is a fundamentally different source of variation from the gradual accumulation of small random variations, and it has some interesting consequences for issues of evolvability . Intuitively, the pre-adaptation of sets of features in reproductively independent specialists suggests a form of 'divide and conquer' decomposition of the adaptive domain . Moreover, the compositions resulting from one level may become the components for compositions at the next level, thus scaling-up the variation mechanism . In this paper, we explore and develop these concepts using a simple abstract model of symbiotic composition to examine its impact on evolvability . To exemplify the adaptive capacity of the composition model, we employ a scale-invariant fitness landscape exhibiting significant ruggedness at all scales . Whilst innovation by mutation and by conventional evolutionary algorithms becomes increasingly more difficult as evolution continues in this landscape, innovation by composition is not impeded as it discovers and assembles component entities through successive hierarchical levels.

Biosystems, 2003 May, 69(2-3), 163 - 85
Prokaryote and eukaryote evolvability; Poole AM et al.; The concept of evolvability covers a broad spectrum of, often contradictory, ideas . At one end of the spectrum it is equivalent to the statement that evolution is possible, at the other end are untestable post hoc explanations, such as the suggestion that current evolutionary theory cannot explain the evolution of evolvability . We examine similarities and differences in eukaryote and prokaryote evolvability, and look for explanations that are compatible with a wide range of observations . Differences in genome organisation between eukaryotes and prokaryotes meets this criterion . The single origin of replication in prokaryote chromosomes (versus multiple origins in eukaryotes) accounts for many differences because the time to replicate a prokaryote genome limits its size (and the accumulation of junk DNA) . Both prokaryotes and eukaryotes appear to switch from genetic stability to genetic change in response to stress . We examine a range of stress responses, and discuss how these impact on evolvability, particularly in unicellular organisms versus complex multicellular ones . Evolvability is also limited by environmental interactions (including competition) and we describe a model that places limits on potential evolvability . Examples are given of its application to predator competition and limits to lateral gene transfer . We suggest that unicellular organisms evolve largely through a process of metabolic change, resulting in biochemical diversity . Multicellular organisms evolve largely through morphological changes, not through extensive changes to cellular biochemistry.

Dev Cell, 2003 Apr, 4(4), 459 - 65
Function, diversity, and evolution of signal transduction in prokaryotes; Harshey RM et al.; Major areas covered at the Bacterial Locomotion and Signal Transduction (BLAST) meeting included the clustering of chemoreceptors and its significance to signal amplification, organelle biogenesis, motility, developmental responses mediated by "chemotaxis" operons, and advances in two-component signaling mechanisms.

J Mol Med, 2003 Mar, 81(3), 205 - 13 Epub 2003 Mar 18.
Efficient dose-dependent and time-dependent protein transduction of pancreatic carcinoma cells in vitro and in vivo using purified VP22-EGFP fusion protein; Boenicke L et al.; We constructed a prokaryotic vector expressing a truncated VP22-EGFP gene and purified this fusion protein from Escherichia coli cultures using nickel resin . Application of purified VP22-EGFP protein to human pancreatic carcinoma cells showed a highly efficient time-dependent and dose-dependent uptake and resulted in green fluorescence predominantly located in the nuclei of treated cells . Purified VP22-EGFP efficiently translocated into deeper layers of pancreatic tumor cell spheroids . Homogeneous uptake into the whole tumor was observed after peritumoral injection in human pancreatic tumors in SCID mice . We conclude that the direct application of purified VP22 fusion proteins offers a new, peptide-mediated and potentially systemic therapy for pancreatic cancer . This opens the possibility of achieving specific antitumor effects induced by fused apoptosis-enhancing proteins.

Nucleic Acids Res, 2003 Apr 15, 31(8), 2234 - 41
Revised Escherichia coli selenocysteine insertion requirements determined by in vivo screening of combinatorial libraries of SECIS variants; Sandman KE et al.; To investigate the stringency of the Escherichia coli selenocysteine insertion sequence (SECIS) requirements, libraries of SECIS variants were screened via a novel method in which suppression of the selenocysteine (Sec) opal codon was coupled to bacteriophage plaque formation . The SECIS variant libraries were designed with a mostly paired lower stem, so that randomization could be focused on the upper stem and loop regions . We identified 19 functional non-native SECIS sequences that violated the expected pairing requirements for the SECIS upper stem . All of the SECIS variants were shown to permit Sec insertion in phage (by chemical modification of the Sec residue) and fused to lacZalpha (by beta-galactosidase assay) . The diminished pairing of the upper stem appears to be mitigated by the overall stem stability; a given upper stem variant has significantly higher readthrough in the context of a paired, rather than unpaired, lower stem . These results suggest an unexpected downstream sequence flexibility in prokaryotic selenoprotein expression.

Nucleic Acids Res, 2003 Apr 15, 31(8), 2187 - 95
GenDB--an open source genome annotation system for prokaryote genomes; Meyer F et al.; The flood of sequence data resulting from the large number of current genome projects has increased the need for a flexible, open source genome annotation system, which so far has not existed . To account for the individual needs of different projects, such a system should be modular and easily extensible . We present a genome annotation system for prokaryote genomes, which is well tested and readily adaptable to different tasks . The modular system was developed using an object-oriented approach, and it relies on a relational database backend . Using a well defined application programmers interface (API), the system can be linked easily to other systems . GenDB supports manual as well as automatic annotation strategies . The software currently is in use in more than a dozen microbial genome annotation projects . In addition to its use as a production genome annotation system, it can be employed as a flexible framework for the large-scale evaluation of different annotation strategies . The system is open source.

Planta, 2003 Apr, 216(6), 969 - 75 Epub 2003 Jan 28.
Ultrasensitive behavior in the synthesis of storage polysaccharides in cyanobacteria; Gomez-Casati DF et al.; The glycogen synthetic pathway operates ultrasensitively as a function of the ADPglucose pyrophosphorylase (ADPGlcPPase) allosteric effectors, 3-phosphoglycerate and Pi, in permeabilized cells of the cyanobacterium Anabaena PCC 7120 . In vitro data previously showed that the ultrasensitive behavior of ADPGlcPPase depends upon cross-talk between the two allosteric effectors, the enzyme's response being additionally modulated by molecular crowding {D.F . Gomez Casatiet al . (2000) Biochem J 350:139-147} . In the present work we show, experimentally and with a mathematical model, that alpha-1,4-glucan synthesis is also ultrasensitive in cells due to the propagation of the switch-like behavior of ADPGlcPPase to the synthetic pathway . Amplifications of up to 20-fold in storage-polysaccharide synthesis can be achieved with a modest 6.7-fold increase in 3-phosphoglycerate in the presence of 5 mM Pi in contrast to the 30-fold necessary in its absence . This is the first time that this phenomenon has been reported to occur in the glycogen synthetic pathway of a photosynthetic prokaryote . The implications of the results for plant cell physiology during light-dark transitions are discussed.

Planta, 2003 Apr, 216(6), 951 - 60 Epub 2002 Dec 18.
Cyanobacterial alkaline/neutral invertases . Origin of sucrose hydrolysis in the plant cytosol?
Vargas W, Cumino A, Salerno GL.
The aim of this work was to investigate the occurrence of invertase (Inv) in cyanobacteria . We describe the first isolation and characterization of prokaryotic alkaline/neutral Inv (A/N-Inv) genes . Two genes (invA and invB) were identified in Anabaena sp . PCC 7120, which share about 50-56% identity with plant A/N-Inv and encode proteins of about 53-55 kDa . The identification of these proteins was confirmed by biochemical and immunological studies with recombinant proteins and with the enzymes isolated from Anabaena cells . Expression analysis supported the important role of A/N-Inv in nitrogen-fixing growth conditions . Nevertheless, A/N-Inv activities were shown in all filamentous and unicellular cyanobacteria investigated, regardless of their capacity to fix dinitrogen . Searches in complete sequenced genomes showed that A/N-Inv homologues are restricted to cyanobacterial species and plants . In particular, filamentous nitrogen-fixing strains display two A/N-Inv genes and unicellular strains have only one . Phylogenetic analysis leads us to suggest that modern plant A/N-Inv might have originated from an orthologous ancestral gene after the endosymbiotic origin of chloroplasts.

Russ J Immunol, 2002 Jul, 7(2), 135 - 42
Polyprenols as possible factors that determine an instructive role of the innate immunity in the acquired immune response; Pronin AV et al.; Polyprenols are an integral part of all living cells including prokaryotic and eukaryotic ones . These compounds take part in biosynthesis of glycoproteins . We have found that phosphates of polyprenols may act as effective antiviral agents with a wide spectrum of activity . One of such antiviral agents received from Pinus sativum polyprenols was named phosprenyl . Earlier we showed that phosprenyl expressed direct antiviral effect, while having mild immunomodulatory activity . In the present study we further evaluated influence of phosprenyl on the immune system . The drug was found to inhibit an early phase of IL-1 and Con A interaction in spleen cells as well as lypoxigenase activity and expression of IL-2 receptors . At the same time, phosprenyl induced NK cell activity and early TNF-alpha production . Basing on all these data we proposed that polyprenols could be considered as a "label" which grants a possibility to the innate immune system to recognize infection at the early stages and govern the acquired immunity.

Russ J Immunol, 2000 Oct, 5(3), 259 - 266
Immunodetection of Murine Lymphotoxins in Eukaryotic Cells; Boitchenko VE et al.; Lymphotoxins alpha and beta (LTalpha and LTbeta) are members of tumor necrosis factor superfamily . LT heterotrimers exist on the surface of lymphocytes and signal through LTbeta receptor while soluble LTalpha homotrimer can signal through TNF receptors p55 and p75 . LT-, as well as TNF-mediated signaling are important for the organogenesis and maintenance of microarchitecture of secondary lymphoid organs in mice and has been implicated in the mechanism of certain inflammatory syndromes in humans . In this study we describe the generation of eukaryotic expression plasmids encoding murine LTalpha and LTbeta genes and a prokaryotic expression construct for murine LTalpha . Using recombinant proteins expressed by these vectors as tools for antisera selection, we produced and characterized several polyclonal antibodies capable of detecting LT proteins in eukaryotic cells.

Nature, 2003 Apr 10, 422(6932), 633 - 7
Noise in eukaryotic gene expression; Blake WJ et al.; Transcription in eukaryotic cells has been described as quantal, with pulses of messenger RNA produced in a probabilistic manner . This description reflects the inherently stochastic nature of gene expression, known to be a major factor in the heterogeneous response of individual cells within a clonal population to an inducing stimulus . Here we show in Saccharomyces cerevisiae that stochasticity (noise) arising from transcription contributes significantly to the level of heterogeneity within a eukaryotic clonal population, in contrast to observations in prokaryotes, and that such noise can be modulated at the translational level . We use a stochastic model of transcription initiation specific to eukaryotes to show that pulsatile mRNA production, through reinitiation, is crucial for the dependence of noise on transcriptional efficiency, highlighting a key difference between eukaryotic and prokaryotic sources of noise . Furthermore, we explore the propagation of noise in a gene cascade network and demonstrate experimentally that increased noise in the transcription of a regulatory protein leads to increased cell-cell variability in the target gene output, resulting in prolonged bistable expression states . This result has implications for the role of noise in phenotypic variation and cellular differentiation.

Virus Res, 2003 Apr, 92(2), 165 - 70
Prokaryotic and eukaryotic translational machineries respond differently to the frameshifting RNA signal from plant or animal virus; Sung D et al.; Many mutational and structural analyses of the RNA signals propose a hypothesis that programmed frameshifting occurs by a specific interaction between ribosome and frameshifting signals comprised of a shifty site and a downstream RNA structure, in which the exact nature of the interaction has not yet been proven . To address this question, we analyzed the frameshifting sequence elements from animal or plant virus in yeast and Escherichia coli . Frameshifting efficiencies varied in yeast, but not in E . coli, depending on the specific conformation of mouse mammary tumor virus (MMTV) RNA pseudoknot . Similar changes in frameshifting efficiencies were observed in yeast, but not in E . coli, for the mutations in frameshifting sequence elements from cereal yellow dwarf virus serotype RPV (CYDV-RPV) . The differential response of MMTV or CYDV-RPV frameshifting signal to prokaryotic and eukaryotic translational machineries implies that ribosome pausing alone is insufficient to mediate frameshifting, and additional events including specific interaction between ribosome and RNA structural element are required for efficient frameshifting . These results supports the hypothesis that frameshifting occurs by a specific interaction between ribosome and frameshifting signal.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 143 - 51
Structure and mechanism of soluble glucose dehydrogenase and other PQQ-dependent enzymes; Oubrie A; This paper discusses recent X-ray structures of several pyrroloquinoline quinone (PQQ)-dependent proteins in relation to their proposed modes of action . In addition, a detailed analysis of redox-related structural changes in the soluble PQQ-dependent glucose dehydrogenase is presented . A sequence comparison of that enzyme with a number of homologues shows that PQQ-dependent enzymes are much more widespread than has been assumed so far . In particular, the presence of a PQQ-dependent enzyme in at least one archaeon opens up the possibility that PQQ has been involved in prokaryotic metabolism since the early days of the evolution of bacterial life on earth.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 61 - 5
Human mitochondrial branched chain aminotransferase: structural basis for substrate specificity and role of redox active cysteines; Conway ME et al.; Crystal structures of the fold type IV pyridoxal phosphate (PLP)-dependent human mitochondrial branched chain aminotransferase (hBCATm) reaction intermediates have provided a structural explanation for the kinetically determined substrate specificity of hBCATm . The isoleucine side chain in the ketimine intermediate occupies a hydrophobic binding pocket that can be defined by three surfaces . Modeling of amino acids on the ketimine structure shows that the side chains of nonsubstrate amino acids such as the aromatic amino acids, alanine, or aspartate either are unable to interact through van der Waals' interactions or have steric clashes . The structural and biochemical basis for the sensitivity of the mammalian BCAT to reducing agents has also been elucidated . Two cysteine residues in hBCATm, Cys315 and Cys318 (CXXC), are part of a redox-controlled mechanism that can regulate hBCATm activity . The residues surrounding Cys315 and Cys318 show considerable sequence conservation in the prokaryotic and eukaryotic BCAT sequences, however, the CXXC motif is found only in the mammalian proteins . The results suggest that the BCAT enzymes may join the list of enzymes that can be regulated by redox status.

World J Gastroenterol, 2003 Apr, 9(4), 714 - 6
Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma; Hu HY et al.; AIM: To construct and express ODC recombinant gene for further exploring its potential use in early diagnosis of colorectal carcinoma . METHODS: Total RNA was extracted from colon cancer tissues and amplified by reverse-transcription PCR with two primers, which span the whole coding region of ODC . The synthesized ODC cDNA was cloned into vector pQE-30 at restriction sites BamH I and Sal I which constituted recombinant expression plasmid pQE30-ODC . The sequence of inserted fragment was confirmed by DNA sequencing, the fusion protein including 6His-tag was facilitated for purification by Ni-NTA chromatographic column . RESULTS: ODC expression vector was constructed and confirmed with restriction enzyme digestion and subsequent DNA sequencing . The DNA sequence matching on NCBI Blast showed 99 % affinity . The vector was transformed into E . coli M15 and expressed . The expressed ODC protein was verified with Western blotting . CONCLUSION: The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in colorectal carcinoma.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4463 - 8 Epub 2003 Apr 04.
Amino acids determining enzyme-substrate specificity in prokaryotic and eukaryotic protein kinases; Li L et al.; The binding between a PK and its target is highly specific, despite the fact that many different PKs exhibit significant sequence and structure homology . There must be, then, specificity-determining residues (SDRs) that enable different PKs to recognize their unique substrate . Here we use and further develop a computational procedure to discover putative SDRs (PSDRs) in protein families, whereby a family of homologous proteins is split into orthologous proteins, which are assumed to have the same specificity, and paralogous proteins, which have different specificities . We reason that PSDRs must be similar among orthologs, whereas they must necessarily be different among paralogs . Our statistical procedure and evolutionary model identifies such residues by discriminating a functional signal from a phylogenetic one . As case studies we investigate the prokaryotic two-component system and the eukaryotic AGC (i.e., cAMP-dependent PK, cGMP-dependent PK, and PKC) PKs . Without using experimental data, we predict PSDRs in prokaryotic and eukaryotic PKs, and suggest precise mutations that may convert the specificity of one PK to another . We compare our predictions with current experimental results and obtain considerable agreement with them . Our analysis unifies much of existing data on PK specificity . Finally, we find PSDRs that are outside the active site . Based on our results, as well as structural and biochemical characterizations of eukaryotic PKs, we propose the testable hypothesis of "specificity via differential activation" as a way for the cell to control kinase specificity.

Cell Mol Life Sci, 2003 Feb, 60(2), 229 - 40
Evolutionary and functional implications of the complex glycosylation of Skp1, a cytoplasmic/nuclear glycoprotein associated with polyubiquitination; West CM; Protein degradation is regulatory for the cell cycle, signal transduction and gene transcription . A critical step is the selective marking of the target protein, resulting in polyubiquitination by one of a large number of E3-ubiquitin ligases . Both target marking and E3-ubiquitin ligase activity are associated with common as well as unusual posttranslational modifications . For example, hydroxylation of Pro-residues and modification of Asn-residues by high-mannose sugar chains can target the modified proteins for rapid polyubiquitination in the mammalian cytoplasm . Both prolyl hydroxylation and glycosylation also occur on Skp1, a subunit of the SCF class of E3-ubiquitin ligases, from Dictyostelium . In this case, a pentasaccharide containing Gal, Fuc and N-acetyl-D-glucosmine (GlcNAc) is attached to the HyPro-residue . The sugars are added sequentially by enzymes that reside in the cytoplasm rather than the secretory pathway . Two of the glycosyltransferases appear to be positioned in ancient evolutionary lineages that bridge prokaryotes and eukaryotes . The first, which attaches GlcNAc to HyPro, is related to enzymes that form alpha-GalNAc- and alpha-GlcNAc-Ser/Thr linkages in the Golgi . GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of beta1,3-linked Gal and alpha1,2-linked Fuc, using an architecture resembling that of two-domain prokaryotic glycosyltransferases involved in glycosaminoglycan synthesis . Mutational and pharmacological perturbation of glycosylation alters the subcellular localization of Skpl and growth properties ofcells . Prolyl hydroxylation and complex O-glycosylation provide the cell with new options for epigenetic regulation of protein turnover in its cytoplasmic and nuclear compartments.

Life Sci Space Res, 1976, 14, 351 - 4
Performance of fungi in low temperature and hypersaline environments; Siegel SM et al.; During the past ten years we have observed a broad array of stress capabilities in common fungi including ability to grow in aqueous ammonia and other alkaline solutions, in acids, in the presence of heavy metals, and in various salt media at low temperature . This report is concerned primarily with (a) the performance of Aspergillaceae in a variety of saturated salts, (b) distinctive roles for K+ and Rb+ ions, and (c) the lowest temperatures at which growth in nutrient brines has been observed, namely 267 degrees K in as little as 14 days . We also describe a novel solid medium based upon gelatin, glycerol and water in which fungal cultures growing at 248 degrees K can be directly examined under oil-immersion magnification . The performance capabilities of the fungi show that tolerance or adaptability to harsh and extreme physical-chemical environments cannot be considered a unique feature of prokaryotic life forms . Salt flats, brine pools and other natural hypersaline environments have long been recognized as real ecological niches harboring a range of biota from pseudomonad bacteria and green algae to specialized crustaceans . A notable omission in this ecological record is the fungi, although the group is known to include marine forms.

Mol Microbiol, 2003 Apr, 48(2), 287 - 94
The Lrp family of transcriptional regulators; Brinkman AB et al.; Genome analysis has revealed that members of the Lrp family of transcriptional regulators are widely distributed among prokaryotes, both bacteria and archaea . The archetype Leucine-responsive Regulatory Protein from Escherichia coli is a global regulator involved in modulating a variety of metabolic functions, including the catabolism and anabolism of amino acids as well as pili synthesis . Most Lrp homologues, however, appear to act as specific regulators of amino acid metabolism-related genes . Like most prokaryotic transcriptional regulators, Lrp-like regulators consist of a DNA-binding domain and a ligand-binding domain . The crystal structure of the Pyrococcus furiosus LrpA revealed an N-terminal domain with a common helix-turn-helix fold, and a C-terminal domain with a typical alphabeta-sandwich fold . The latter regulatory domain constitutes a novel ligand-binding site and has been designated RAM . Database analysis reveals that the RAM domain is present in many prokaryotic genomes, potentially encoding (1) Lrp-homologues, when fused to a DNA-binding domain (2) enzymes, when fused as a potential regulatory domain to a catalytic domain, and (3) stand-alone RAM modules with unknown function . The architecture of Lrp regulators with two distinct domains that harbour the regulatory (effector-binding) site and the active (DNA-binding) site, and their separation by a flexible hinge region, suggests a general allosteric switch of Lrp-like regulators.

Cell Microbiol, 2003 Apr, 5(4), 225 - 31
Redox proteins in mammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes; Punj V et al.; Mammalian cell mitochondria are believed to have prokaryotic ancestry . Mitochondria are not only the powerhouse of energy generation within the eukaryotic cell but they also play a major role in inducing apoptotic cell death through release of redox proteins such as cytochrome c and the apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity . Recent evidence indicates that some present day prokaryotes release redox proteins that induce apoptosis in mammalian cells through stabilization of the tumour suppressor protein p53 . p53 interacts with mitochondria either directly or through activation of the genes for pro-apoptotic proteins such as Bax or NOXA or genes that encode redox enzymes responsible for the production of reactive oxygen species (ROS) . The analogy between the ancient ancestors of present day bacteria, the mitochondria, and the present day bacteria with regard to their ability to release redox proteins for triggering mammalian cell death is an interesting example of functional conservation during the hundreds of millions of years of evolution . It is possible that the ancestors of the present day prokaryotes released redox proteins to kill the ancestors of the eukaryotes . During evolution of the mitochondria from prokaryotes as obligate endosymbionts, the mitochondria maintained the same functions to programme their own host cell death.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Apr, 35(4), 317 - 24
Prediction of prokaryotic promoters based on prediction of transcriptional units; Lin JC et al.; Identification of promoters is very important in understanding gene regulating relationships in an organism, and computational identification of promoters has been a long standing problem in computational biology . A new method was presented to predict promoter regions in prokaryotic organism . The method predicted transcription unit (TU) first and the TU was divided into singlet that contains only one single gene in a TU, and operon that contains more than one gene . Based on these predicted TUs, promoter was predicted for each TU using hidden Markov model including explicit state duration density . Both predicted TUs and promoters were satisfying.

Plant Cell, 2003 Apr, 15(4), 1020 - 33
Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth; Bonaventure G et al.; Acyl-acyl carrier protein thioesterases determine the amount and type of fatty acids that are exported from the plastids . To better understand the role of the FATB class of acyl-acyl carrier protein thioesterases, we identified an Arabidopsis mutant with a T-DNA insertion in the FATB gene . Palmitate (16:0) content of glycerolipids of the mutant was reduced by 42% in leaves, by 56% in flowers, by 48% in roots, and by 56% in seeds . In addition, stearate (18:0) was reduced by 50% in leaves and by 30% in seeds . The growth rate was reduced in the mutant, resulting in 50% less fresh weight at 4 weeks compared with wild-type plants . Furthermore, mutant plants produced seeds with low viability and altered morphology . Analysis of individual glycerolipids revealed that the fatty acid composition of prokaryotic plastid lipids was largely unaltered, whereas the impact on eukaryotic lipids varied but was particularly severe for phosphatidylcholine, with a >4-fold reduction of 16:0 and a 10-fold reduction of 18:0 levels . The total wax load of fatb-ko plants was reduced by 20% in leaves and by 50% in stems, implicating FATB in the supply of saturated fatty acids for wax biosynthesis . Analysis of C(18) sphingoid bases derived from 16:0 indicated that, despite a 50% reduction in exported 16:0, the mutant cells maintained wild-type levels of sphingoid bases, presumably at the expense of other cell components . The growth retardation caused by the fatb mutation was enhanced in a fatb-ko act1 double mutant in which saturated fatty acid content was reduced further . Together, these results demonstrate the in vivo role of FATB as a major determinant of saturated fatty acid synthesis and the essential role of saturates for the biosynthesis and/or regulation of cellular components critical for plant growth and seed development.

Genome Res, 2003 Apr, 13(4), 693 - 702
Informatics for unveiling hidden genome signatures; Abe T et al.; With the increasing amount of available genome sequences, novel tools are needed for comprehensive analysis of species-specific sequence characteristics for a wide variety of genomes . We used an unsupervised neural network algorithm, a self-organizing map (SOM), to analyze di-, tri-, and tetranucleotide frequencies in a wide variety of prokaryotic and eukaryotic genomes . The SOM, which can cluster complex data efficiently, was shown to be an excellent tool for analyzing global characteristics of genome sequences and for revealing key combinations of oligonucleotides representing individual genomes . From analysis of 1- and 10-kb genomic sequences derived from 65 bacteria (a total of 170 Mb) and from 6 eukaryotes (460 Mb), clear species-specific separations of major portions of the sequences were obtained with the di-, tri-, and tetranucleotide SOMs . The unsupervised algorithm could recognize, in most 10-kb sequences, the species-specific characteristics (key combinations of oligonucleotide frequencies) that are signature features of each genome . We were able to classify DNA sequences within one and between many species into subgroups that corresponded generally to biological categories . Because the classification power is very high, the SOM is an efficient and fundamental bioinformatic strategy for extracting a wide range of genomic information from a vast amount of sequences.

Genome Res, 2003 Apr, 13(4), 617 - 23
Nonrandom tripeptide sequence distributions at protein carboxyl termini; Gatto GJ Jr et al.; The availability of complete genome sequences enables the statistical analysis of sequence features without significant database-imposed bias . The carboxyl termini of proteins often contain regions associated with protein targeting and enhanced translational termination . We analyzed the frequency of occurrence of C-terminal tripeptides in representative archaeal, bacterial, and eukaryotic genomes . The sequence distribution in prokaryotic genomes nearly matches that generated by the randomization of the observed tripeptide set . In contrast, eukaryotic genomes contain large numbers of overrepresented sequences . Some of these correspond to highly repeated sequences from either duplicated endogenous genes or transposon open reading frames . Gratifyingly, others represent previously known targeting signals or sequences associated with an increase in translational termination efficiency . However, a number of overrepresented tripeptides have not been previously noted and may represent novel functional sequences . For example, the sequence XSS may enhance translational termination efficiency in plants, whereas FWC may be a targeting or processing signal for certain amino acid permeases in yeast.

J Bacteriol, 2003 Apr, 185(8), 2538 - 47
Homologue of macrophage-activating lipoprotein in Mycoplasma gallisepticum is not essential for growth and pathogenicity in tracheal organ cultures; Markham PF et al.; While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential . The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed . Its gene was cloned, and the DNA sequence was determined . Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M . pneumoniae and M . genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M . agalactiae and M . fermentans . The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens . Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M . fermentans and lipoprotein P48 of M . agalactiae . The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis . Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end . A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M . gallisepticum cells . A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene . The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins . There was no difference between the p47(-) mutant and wild-type M . gallisepticum in pathogenicity in chicken tracheal organ cultures . Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.

Curr Opin Drug Discov Devel, 2003 Mar, 6(2), 204 - 17
Recent advances in the development of immunostimulatory oligonucleotides; Uhlmann E et al.; Some immune cells recognize distinct molecular structures present in pathogens through specific pattern recognition receptors that are able to distinguish prokaryotic DNA from vertebrate DNA . The detection of invading microbial DNA is based on the recognition of unmethylated deoxycytidyl-deoxyguanosin dinucleotide (CpG) motifs . Synthetic oligonucleotides (ODNs) containing these CpG motifs are able to activate both innate and acquired immune responses through a signaling pathway involving Toll-like receptor 9 (TLR9) . Depending on the sequence, length, as well as number and positions of CpG motifs in an ODN, distinct immunostimulatory profiles can be observed . These immunostimulatory profiles can be further modified and fine-tuned by appropriate chemical modifications, leading to preclinical and clinical development of CpG ODNs in cancer, allergy, asthma and infectious diseases.

J Biol Chem, 2003 Jun 13, 278(24), 22014 - 22 Epub 2003 Mar 31.
Multiple splice variants encode a novel adenylyl cyclase of possible plastid origin expressed in the sexual stage of the malaria parasite Plasmodium falciparum; Muhia DK et al.; We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfACalpha . The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle) . The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants . One intron has three alternative 3'-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons . Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant . Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl cyclase . These results identify a novel mechanism in P . falciparum for the generation of multiple isoforms of a key, membrane-bound signaling molecule from a single genomic copy . Comparisons of the catalytic domains of PfACalpha and a second putative P . falciparum adenylyl cyclase (PfACbeta) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae) . In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases.

Biophys J, 2003 Apr, 84(4), 2709 - 14
The effects of intense submicrosecond electrical pulses on cells; Deng J et al.; A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range . The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide . Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy . For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability . For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses . Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses . These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses . The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.

Biophys J, 2003 Apr, 84(4), 2467 - 73
Increased bending rigidity of single DNA molecules by H-NS, a temperature and osmolarity sensor; Amit R et al.; Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing . It plays a key role in cell response to changes in temperature and osmolarity . Force-extension measurements of single, twist-relaxed lambda-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates . Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15-20 bp . These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity . These effects are not observed above 32 degrees C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing . Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell.

Arch Biochem Biophys, 2003 Apr 15, 412(2), 259 - 66
The membrane-interactive tail of cytochrome b(5) can function as a stop-transfer sequence in concert with a signal sequence to give inversion of protein topology in the endoplasmic reticulum; Kaderbhai MA et al.; Sequence analyses of the C-terminal membrane intercalative region of the rat cytochrome b(5) indicated that this domain has, in addition to a signal sequence, a combined element of the classic stop-transfer sequence typically found in a variety of transmembrane proteins . Such bitopic protein arrangements arise by tandem but topogenically displaced activities of cleavable/noncleavable signal and stop-transfer sequences . A fusion precursor comprising an N-terminally linked prokaryotic signal sequence and the full-length of mammalian cytochrome b(5), including its C-terminal membrane insertion sequence, was engineered to investigate the outcome of this combination of signals on the targeting and topology of the cytochrome b(5) in the endoplasmic reticulum membrane . Precytochrome b(5) was cotranslationally translocated across the endoplasmic reticulum membrane . The signal-processed cytochrome b(5) was integrally anchored in the membrane with the globular domain facing the lumen . Thus, the topology of the signal sequence-directed cytochrome b(5) in the microsomal vesicle was reversed with respect to that of the native form . Posttranslational incubation of the precytochrome b(5) with microsomes resulted in a "loose" incorporation of the unprocessed form onto the surface of the vesicle . Our findings suggest that the membrane-insertion sequence of cytochrome b(5) has a functional stop-transfer sequence . We discuss the implications of these findings with respect to selective targeting of cytochrome b(5) to the endoplasmic reticulum membrane in the view that signal and stop-transfer sequences are often interchangeable or combined for topogenic functions.

Mol Cell, 2003 Mar, 11(3), 807 - 15
Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice; Cerritelli SM et al.; Although ribonucleases H (RNases H) have long been implicated in DNA metabolism, they are not required for viability in prokaryotes or unicellular eukaryotes . We generated Rnaseh1(-/-) mice to investigate the role of RNase H1 in mammals and observed developmental arrest at E8.5 in null embryos . A fraction of the mainly nuclear RNase H1 was targeted to mitochondria, and its absence in embryos resulted in a significant decrease in mitochondrial DNA content, leading to apoptotic cell death . This report links RNase H1 to generation of mitochondrial DNA, providing direct support for the strand-coupled mechanism of mitochondrial DNA replication . These findings also have important implications for therapy of mitochondrial dysfunctions and drug development for the structurally related RNase H of HIV.

Mol Cell, 2003 Mar, 11(3), 659 - 69
Lack of a robust unfoldase activity confers a unique level of substrate specificity to the universal AAA protease FtsH; Herman C et al.; FtsH, a member of the AAA family of proteins, is the only membrane ATP-dependent protease universally conserved in prokaryotes, and the only essential ATP-dependent protease in Escherichia coli . We investigated the mechanism of degradation by FtsH . Other well-studied ATP-dependent proteases use ATP to unfold their substrates . In contrast, both in vitro and in vivo studies indicate that degradation by FtsH occurs efficiently only when the substrate is a protein of low intrinsic thermodynamic stability . Because FtsH lacks robust unfoldase activity, it is able to use the protein folding state of substrates as a criterion for degradation . This feature may be key to its role in the cell and account for its ubiquitous distribution among prokaryotic organisms.

Mol Cell, 2003 Mar, 11(3), 571 - 5
Kleisins: a superfamily of bacterial and eukaryotic SMC protein partners; Schleiffer A et al.; We describe a superfamily of eukaryotic and prokaryotic proteins (kleisins) that includes ScpA, Scc1, Rec8, and Barren . Scc1 interacts with SMC proteins through N- and C-terminal domains to form a ring-like structure . Since these are the only domains conserved among kleisins, we suggest that ring formation with SMC proteins may define this family.

Biochemistry, 2003 Apr 8, 42(13), 3666 - 73
Extending the structure of an ABC transporter to atomic resolution: modeling and simulation studies of MsbA; Campbell JD et al.; Molecular modeling and simulation approaches have been use to generate a complete model of the prokaryotic ABC transporter MsbA from Escherichia coli, starting from the low-resolution structure-based Calpha trace (PDB code 1JSQ) . MsbA is of some biomedical interest as it is homologous to mammalian transporters such as P-glycoprotein and TAP . The quality of the MsbA model is assessed using a combination of molecular dynamics simulations and static structural analysis . These results suggest that the approach adopted for MsbA may be of general utility for generating all atom models from low-resolution crystal structures of membrane proteins . Molecular dynamics simulations of the MsbA model inserted in a fully solvated octane slab (a membrane mimetic environment) reveal that while the monomer is relatively stable, the dimer is unstable and undergoes significant conformational drift on a nanosecond time scale . This suggests that the MsbA crystal dimer may not correspond to the MsbA dimer in vivo . An alternative model of the dimer is discussed in the context of available experimental data.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Sep, 16(3), 215 - 8
{Expression of the 3a and 3d fusion protein of hepatitis A virus in prokaryotic cell and antigenicity analysis}; Cheng H et al.; OBJECTIVE: To fusionaly express the HAV 3a (located in 1403-1456 aa) and 3d located in 1719-1764 aa cDNA gene fragments in prokaryotic system; to investigate the antigenicity and application of recombinant protein . METHODS: By using PCR technique, 3a and 3d gene fragments were cloned . Choosing pET-30a as the expressive vector, the recombinant plasmid Pet-3ad was constructed and pET-3ad was expressed in Escherichia coli after inducing by IPTG . By affinity chromatography, purified recombinant protein was obtained . By using Western blot analysis and indirect ELISA to detect its antigenic activity . RESULTS: Recombinant plasmid pET-3ad was proved to construct successfully by enzyme-digestion and sequence measurement . Recombinant protein P3ad(18,000) was obtained in BL21(DE3) and purified after Ni+ affinity chromatography . Western blot analysis and indirect ELISA showed that P3ad had specific antigenicity . CONCLUSIONS: Recombinant plasmid pET-3ad was proved to construct successfully by enzyme-digestion (Nco I/Hind III) and sequence measurement . Recombinant protein P3ad(18,000) was obtained in BL21(DE3) and purified after Ni+ affinity chromatography . Specific immunoblotting appeared at 18,000 by western blot analysis, which showed the recombinant protein P3ad had specific antigenicity, indirect ELISA further proved its antigenicity.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Dec, 16(4), 315 - 8
{Translational control of human interferon alpha 1 gene expression in E.coli}; Wang J et al.; OBJECTIVE: To increase prokaryotic expression level of IFN-alpha 1C gene through the quantitative theory of translational control and the translational enhancer sequence . METHODS: Stepwise polymerase chain reaction (PCR) was used to alter the 5 terminal cDNA sequence of IFN-alpha 1C in three different grades of base mutation . In this way, the free energy (Delta G) of the secondary structure in translational initiation region (TIR) was decreased gradually . An expression plasmid (pBVE) was constructed to contain the translational enhancer cDNA sequence by modifying pBV220 upstream of the SD region . RESULTS: The expression levels of three kinds of IFN-alpha 1C modified gene were all increased . Furthermore, it presented an increasing trend with decreasing in delta G varying from -50,241.6 to -22,190.0 J/mol . The highest expression was 2.43 x 10(8) U/L, covering twelve times more than its original cDNA . IFN-alpha 1C gene and its modified cDNA was inserted into pBVE as reporting genes . E.Coli cells harbouring pBVE/IFN-alpha 1Cs cDNA produced two to five times more IFN than cells harbouring pBV220/IFN-alpha 1Cs . CONCLUSIONS: pBVE containing translational enhancer is a high level prokaryotic expression vector . The theory of quantitative translational control can effectively be used to enhance the IFN-alpha 1C gene expression level in E.coli.

Eur J Oral Sci, 2002 Oct, 110(5), 358 - 65
Porcine kallikrein-4 activation, glycosylation, activity, and expression in prokaryotic and eukaryotic hosts; Ryu O et al.; Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel . We isolated native KLK4 from developing pig enamel and expressed four recombinant forms . Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems . Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified . The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4 . These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction . Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations . Deglycosylation was also associated with a loss of proteolytic activity . We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry . Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.

J Mol Evol, 2003 Apr, 56(4), 446 - 57
Evolutionary affiliations within the superfamily of ketosynthases reflect complex pathway associations; Moffitt MC et al.; Type I polyketide synthases are known to produce a wide range of medically and industrially important polyketides . The ketosynthase (KS) domain is required for the condensation of an extender unit onto the growing polyketide chain during polyketide biosynthesis . KSs represent a superfamily of complex biosynthetic pathway-associated enzymes found in prokaryotes, fungi, and plants . Although themselves functionally conserved, KSs are involved in the production of a structurally diverse range of metabolites . Degenerate oligonucleotide primers, designed for the amplification of KS domains, amplified KS domains from a range of organisms including cyanobacterial and dinoflagellates . KS domains detected in dinoflagellate cultures appear to have been amplified from the less than 3- micro m filtrate of the nonaxenic culture . Phylogenetic analysis of sequences obtained during this study enabled the specific identification of KS domains of hybrid or mixed polyketide synthase/peptide synthetase complexes, required for the condensation of an extender unit onto an amino acid starter unit . The primer sets described in this study were also used for the detection of novel KS domains directly from environmental samples . The ability to predict function based on primary molecular structure will be critical for future discovery and rational engineering of polyketides.

Physiol Rev, 2003 Apr, 83(2), 433 - 73
Actin binding proteins: regulation of cytoskeletal microfilaments; dos Remedios CG et al.; The actin cytoskeleton is a complex structure that performs a wide range of cellular functions . In 2001, significant advances were made to our understanding of the structure and function of actin monomers . Many of these are likely to help us understand and distinguish between the structural models of actin microfilaments . In particular, 1) the structure of actin was resolved from crystals in the absence of cocrystallized actin binding proteins (ABPs), 2) the prokaryotic ancestral gene of actin was crystallized and its function as a bacterial cytoskeleton was revealed, and 3) the structure of the Arp2/3 complex was described for the first time . In this review we selected several ABPs (ADF/cofilin, profilin, gelsolin, thymosin beta4, DNase I, CapZ, tropomodulin, and Arp2/3) that regulate actin-driven assembly, i.e., movement that is independent of motor proteins . They were chosen because 1) they represent a family of related proteins, 2) they are widely distributed in nature, 3) an atomic structure (or at least a plausible model) is available for each of them, and 4) each is expressed in significant quantities in cells . These ABPs perform the following cellular functions: 1) they maintain the population of unassembled but assembly-ready actin monomers (profilin), 2) they regulate the state of polymerization of filaments (ADF/cofilin, profilin), 3) they bind to and block the growing ends of actin filaments (gelsolin), 4) they nucleate actin assembly (gelsolin, Arp2/3, cofilin), 5) they sever actin filaments (gelsolin, ADF/cofilin), 6) they bind to the sides of actin filaments (gelsolin, Arp2/3), and 7) they cross-link actin filaments (Arp2/3) . Some of these ABPs are essential, whereas others may form regulatory ternary complexes . Some play crucial roles in human disorders, and for all of them, there are good reasons why investigations into their structures and functions should continue.

Physiol Rev, 2003 Apr, 83(2), 417 - 32
Interaction of mycoplasmas with host cells; Rottem S; The mycoplasmas form a large group of prokaryotic microorganisms with over 190 species distinguished from ordinary bacteria by their small size, minute genome, and total lack of a cell wall . Owing to their limited biosynthetic capabilities, most mycoplasmas are parasites exhibiting strict host and tissue specificities . The aim of this review is to collate present knowledge on the strategies employed by mycoplasmas while interacting with their host eukaryotic cells . Prominant among these strategies is the adherence of mycoplasma to host cells, identifying the mycoplasmal adhesins as well as the mammalian membrane receptors; the invasion of mycoplasmas into host cells including studies on the role of mycoplasmal surface molecules and signaling mechanisms in the invasion; the fusion of mycoplasmas with host cells, a novel process that raises intriguing questions of how microinjection of mycoplasma components into eukaryotic cells subvert and damage the host cells . The observations of diverse interactions of mycoplasmas with cells of the immune system and their immunomodulatory effects and the discovery of genetic systems that enable mycoplasmas to rapidly change their surface antigenic composition have been important developments in mycoplasma research over the past decade, showing that mycoplasmas possess an impressive capability of maintaining a dynamic surface architecture that is antigenically and functionally versatile, contributing to the capability of the mycoplasmas to adapt to a large range of habitats and cause diseases that are often chronic in nature.

J Mol Biol, 2003 Apr 11, 327(5), 901 - 9
Classification of 29 families of secondary transport proteins into a single structural class using hydropathy profile analysis; Lolkema JS et al.; A classification scheme for membrane proteins is proposed that clusters families of proteins into structural classes based on hydropathy profile analysis . The averaged hydropathy profiles of protein families are taken as fingerprints of the 3D structure of the proteins and, therefore, are able to detect more distant evolutionary relationships than amino acid sequences . A procedure was developed in which hydropathy profile analysis is used initially as a filter in a BLAST search of the NCBI protein database . The strength of the procedure is demonstrated by the classification of 29 families of secondary transporters into a single structural class, termed ST{3} . An exhaustive search of the database revealed that the 29 families contain 568 unique sequences . The proteins are predominantly from prokaryotic origin and most of the characterized transporters in ST{3} transport organic and inorganic anions and a smaller number are Na(+)/H(+) antiporters . All modes of energy coupling (symport, antiport, uniport) are found in structural class ST{3} . The relevance of the classification for structure/function prediction of uncharacterised transporters in the class is discussed.

Environ Microbiol, 2003 Apr, 5(4), 256 - 66
Sedimentary microbial community dynamics in a regulated stream: East Fork of the Little Miami River, Ohio; Sutton SD et al.; A field study was conducted in the Lower East Fork of the Little Miami River, a regulated stream in Clermont county, Ohio, to determine how changes in streamflow, water temperature and photo-period affect sediment microbial community structure . Surface sediment cores were collected from sampling stations spanning 60 river kilometers three to four times per year between October 1996 and October 1999 . During the final year of the field study, water temperature, water depth, conductivity, total suspended solids, dissolved organic carbon, instantaneous streamflow velocity, sediment grain size and sediment organic matter were determined . Total microbial biomass was measured using the phospholipid phosphate technique (PLP) and ranged between 2 and 134 nmol PLP * g(-1) dry weight sediment with a mean of 25 nmol PLP * g(-1) . Microbial community structure was determined using the phospholipid fatty acid analysis and indicated seasonal shifts in sedimentary microbial community composition . January to June sedimentary microbial biomass was predominately prokaryotic (60% +/- 2), whereas microeukaryotes dominated samples collected during the late summer (55% +/- 2.4) and fall (60% +/- 2) . These changes were correlated with stream discharge and water temperature . Microbial community structure varied spatially about a reservoir with prokaryotic biomass dominant at upstream stations and eukaryotic biomass dominant at downstream stations . These findings reveal that sedimentary microbial communities in streams are dynamic responding to the seasonal variation of environmental factors.

Appl Microbiol Biotechnol, 2003 Mar, 61(1), 1 - 9 Epub 2003 Jan 14.
Termite symbiotic systems: efficient bio-recycling of lignocellulose; Ohkuma M; Termites thrive in great abundance in terrestrial ecosystems and play important roles in biorecycling of lignocellulose . Together with their microbial symbionts, they efficiently decompose lignocellulose . In so-called lower termites, a dual decomposing system, consisting of the termite's own cellulases and those of its gut protists, was elucidated at the molecular level . Higher termites degrade cellulose apparently using only their own enzymes, because of the absence of symbiotic protists . Termite gut prokaryotes efficiently support lignocellulose degradation . However, culture-independent molecular studies have revealed that the majority of these gut symbionts have not yet been cultivated, and that the gut symbiotic community shows a highly structured spatial organization . In situ localization of individual populations and their functional interactions are important to understand the nature of symbioses in the gut . In contrast to cellulose, lignin degradation does not appear to be important in the gut of wood-feeding termites . Soil-feeding termites decompose humic substances in soil at least partly, but little is known about the decomposition . Fungus-growing termites are successful in the almost complete decomposition of lignocellulose in a sophisticated cooperation with basidiomycete fungi cultivated in their nest . A detailed understanding of efficient biorecycling systems, such as that for lignocellulose, and the symbioses that provide this efficiency will benefit applied microbiology and biotechnology.

Arch Microbiol, 2003 May, 179(5), 377 - 80 Epub 2003 Mar 26.
Rhodospirillum rubrum has a family I pyrophosphatase: purification, cloning, and sequencing; Romero I et al.; The cytoplasmic pyrophosphatase of the photosynthetic bacterium Rhodospirillum rubrum was purified to electrophoretic homogeneity . The enzyme is a homohexamer of 20-kDa monomers . The gene was cloned and sequenced . Alignment of the deduced 179-amino-acid protein with known bacterial pyrophosphatases revealed conservation of all residues in the active site . Attempts to obtain an insertion mutant of the cytoplasmic pyrophosphatase gene did not yield any cell completely devoid of cytoplasmic pyrophosphatase activity . The mutants obtained showed 50% of the enzymatic activity and grew in twice the generation time of wild-type cells . This suggests that the membrane-bound pyrophosphatase of Rsp . rubrum is not sufficient for a normal growth rate, whereas the cytoplasmic enzyme is essential for growth . The characteristics of the gene and the encoded protein fit those of prokaryotic family I pyrophosphatases.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 197 - 200
'Candidatus pasteuria usgae' sp . nov., an obligate endoparasite of the phytoparasitic nematode Belonolaimus longicaudatus; Giblin-Davis RM et al.; Taxonomically relevant characteristics of a fastidiously Gram-positive, obligately endoparasitic prokaryote (strain S-1) that uses the phytoparasitic sting nematode Belonolaimus longicaudatus as its host are reviewed . 16S rDNA sequence similarity (> or = 93%) confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans and corroborates morphological, morphometric and host range evidence suggesting a novel taxon . The 16S rDNA sequence of strain S-1 has greatest similarity (96%) to the 16S rDNA sequences of both Pasteuria penetrans from root-knot nematodes (Meloidogyne species) and the recently reported strain of Pasteuria isolated from the soybean cyst nematode Heterodera glycines . Because the obligately endoparasitic nature of prokaryotes in the genus Pasteuria prevents isolation of definitive type strains, strain S-1 is proposed as 'Candidatus Pasteuria usgae' sp . nov.

Hum Mutat, 2003 Apr, 21(4), 370 - 8
Phenylketonuria: genotype-phenotype correlations based on expression analysis of structural and functional mutations in PAH; Pey AL et al.; When analyzed in the context of the phenylalanine hydroxylase (PAH) three-dimensional structure, only a minority of the PKU mutations described world-wide affect catalytic residues . Consistent with these observations, recent data point to defective folding and subsequent aggregation/degradation as a predominant disease mechanism for several mutations . In this work, we use a combined approach of expression in eukaryotic cells at different temperatures and a prokaryotic system with co-expression of chaperonins to elucidate and confirm structural consequences for 18 PKU mutations . Three mutations are located in the amino terminal regulatory domain and 15 in the catalytic domain . Four mutations were found to abolish the specific activity in all conditions . Two are catalytic mutations (Y277D and E280K) and two are severe structural defects (IVS10-11G>A and L311P) . All the remaining mutations (D59Y, I65T, E76G, P122Q, R158Q, G218V, R243Q, P244L, R252W, R261Q, A309V, R408Q, R408W, and Y414C) are folding defects causing reduced stability and accelerated degradation, although some of them probably affect residues involved in regulation . In these cases, we have demonstrated that the amount of mutant PAH protein and residual activity could be modulated by in vitro experimental conditions, and therefore the observed in vivo metabolic variation may be explained by interindividual variation in the quality control systems . The results derived provide an experimental framework to define the mutation severity relating genotype to phenotype . They also explain the observed inconsistencies for some mutations in patients with similar genotype and different phenotypes .

Nucleic Acids Res, 2003 Apr 1, 31(7), 1944 - 54
What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence; Mannhaupt G et al.; The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively . Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process . The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes . The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus . Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism . While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.

Mol Biol Evol, 2003 Apr, 20(4), 471 - 83 Epub 2003 Mar 05.
G+C3 structuring along the genome: a common feature in prokaryotes; Daubin V et al.; The heterogeneity of gene nucleotide content in prokaryotic genomes is commonly interpreted as the result of three main phenomena: (1) genes undergo different selection pressures both during and after translation (affecting codon and amino acid choice); (2) genes undergo different mutational pressure whether they are on the leading or lagging strand; and (3) genes may have different phylogenetic origins as a result of lateral transfers . However, this view neglects the necessity of organizing genetic information on a chromosome that needs to be replicated and folded, which may add constraints to single gene evolution . As a consequence, genes are potentially subjected to different mutation and selection pressures, depending on their position in the genome . In this paper, we analyze the structuring of different codon usage measures along completely sequenced bacterial genomes . We show that most of them are highly structured, suggesting that genes have different base content, depending on their location on the chromosome . A peculiar pattern of genome structure, with a tendency toward an A+T-enrichment near the replication terminus, is found in most bacterial phyla and may reflect common chromosome constraints . Several species may have lost this pattern, probably because of genome rearrangements or integration of foreign DNA . We show that in several species, this enrichment is associated with an increase of evolutionary rate and we discuss the evolutionary implications of these results . We argue that structural constraints acting on the circular chromosome are not negligible and that this natural structuring of bacterial genomes may be a cause of overestimation in lateral gene transfer predictions using codon composition indices.

J Biol Chem, 2003 Jun 20, 278(25), 22250 - 6 Epub 2003 Mar 24.
The mechanism of regulation of bacteriophage lambda pR promoter activity by Escherichia coli DnaA protein; Glinkowska M et al.; Apart from its function as an initiator of DNA replication, the Escherichia coli DnaA protein is also a specific transcription factor . It activates and represses a number of promoters . However, mechanisms of transcription stimulation by DnaA remained unknown . Bacteriophage lambda pR promoter is one of the promoters activated by DnaA . It was reported previously that DnaA binds downstream of the pR promoter and perhaps interacts with the RNA polymerase beta subunit . Here we demonstrate that DnaA positively regulates transcription from pR by stimulation of two steps in transcription initiation: RNA polymerase binding to the promoter region and promoter escape . For this transcription activation, two weak DnaA boxes located downstream of pR are necessary and sufficient . Such a mechanism of transcription activation and location of the activator-binding sites relative to the transcription start point are unusual in prokaryotes . Changes in the distance between the transcription start point and the first DnaA box by 5 and 10 bp and alterations in the orientation of these boxes did not abolish the stimulation of transcription by DnaA, but the efficiency of the promoter activation was different for various mutations . It seems plausible that formation of higher order nucleoprotein structures, involving DNA looping, is necessary for effective stimulation of the pR promoter . At high concentrations, DnaA is a repressor of pR rather than an activator . This repression was found to be because of inhibition of RNA polymerase binding to the promoter region.

J Biol Chem, 2003 Jun 6, 278(23), 21267 - 75 Epub 2003 Mar 24.
Binding discrimination of MutS to a set of lesions and compound lesions (base damage and mismatch) reveals its potential role as a cisplatin-damaged DNA sensing protein; Fourrier L et al.; The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin . It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions . This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis . Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions . The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate . The rate of association, kon, for binding to the 1,2-d(GpG) adduct was 3.1 x 104 m-1 s-1 and the specificity of binding was essentially dependent on koff . DNA duplexes containing a single 1,2-d(ApG), 1,3-d(GpCpG) adduct, and an interstrand cross-link of cisplatin were not preferentially recognized . Among 12 DNA substrates, each containing a different cisplatin compound lesion derived from replicative misincorporation of one base opposite either of the 1,2-intrastrand adducts, 10 were specifically recognized including those that are more likely formed in vivo based on cisplatin mutation spectra . Moreover, among these lesions, two compound lesions formed when an adenine was misincorporated opposite a 1,2-d(GpG) adduct were not substrates for the MutY-dependent mismatch repair pathway . The ability of MutS to sense differentially various platinated DNA substrates suggests that cisplatin compound lesions formed during misincorporation of a base opposite either adducted base of both 1,2-intrastrand cross-links are more plausible critical lesions for MMR-mediated cisplatin cytotoxicity.

Free Radic Biol Med, 2003 Apr 1, 34(7), 862 - 72
Recombinant peroxiredoxin 5 protects against excitotoxic brain lesions in newborn mice; Plaisant F et al.; The pathophysiology of brain lesions associated with cerebral palsy is multifactorial and likely involves excess release of glutamate and excess production of free radicals, among other factors . Theoretically, antioxidants could limit the severity of these brain lesions . Peroxiredoxins are a family of peroxidases widely distributed in eukaryotes and prokaryotes . Peroxiredoxin 5 (PRDX5) is a recently discovered mammalian member of this family of antioxidant enzymes that is able to reduce hydrogen peroxide and alkyl hydroperoxides . The present study was designed to examine the neuroprotective effects of recombinant PRDX5 against neonatal excitotoxic challenge in both in vivo and in vitro experiments . For in vivo experiments, mice (postnatal day 5) were injected intraneopallially with ibotenate acting on NMDA and metabotropic receptors, or S-bromowillardiine acting on AMPA-kainate receptors to produce excitotoxic stress and brain lesions . Systemically administered recombinant PRDX5 provided protection against ibotenate-induced excitotoxic stress . Brain lesions of animals given ibotenate and PRDX5 were up to 63% smaller than that given ibotenate alone . However, PRDX5 provided no prevention from lesions induced with S-bromowillardiine . A mutated recombinant PRDX5 that is devoid of peroxidase activity was also tested and showed no protection against lesions induced by either ibotenate or S-bromowillardiine . Two classical antioxidants, N-acetylcysteine and catalase-PEG, provided the same neuroprotective effect as PRDX5 . For in vitro experiments, neocortical neurons were exposed to 300 microM NMDA alone, NMDA plus recombinant PRDX5, or NMDA, recombinant PRDX5 and dithiothreitol, a classical electron donor for peroxiredoxins . Recombinant PRDX5 plus dithiothreitol displayed a synergistic neuroprotective effect on NMDA-induced neuronal death . These findings indicate that reactive oxygen species production participates in the formation of NMDA receptor-mediated brain lesions in newborn mice and that antioxidant compounds, such as PRDX5, provide some neuroprotection in these models.

Biochemistry, 2003 Apr 1, 42(12), 3409 - 18
A group II intron inserted into a bacterial heat-shock operon shows autocatalytic activity and unusual thermostability; Adamidi C et al.; Group II intron RNAs fold into catalytically active structures that catalyze their own self-splicing and subsequent transposition into DNA . Because of their remarkable enzymatic properties, it has been of interest to find new group II introns with novel properties . Here we report the cloning, sequencing, and mechanistic characterization of a new group II intron from the bacterium Azotobacter vinelandii (the AV intron) . Although it bears the characteristics of the group IIB1 class, the AV intron is unusually G-C rich, and it has unusual insertion sequences and a minimal dependence on the EBS2-IBS2 tertiary interaction . The AV intron is the first bacterial intron that has been found to reside in a housekeeping gene which, in this case, encodes a heat-shock protein (hsp60) . Consistent with a potential role in heat-shock regulation, kinetic analysis reveals that AV intron self-splicing is activated only at elevated temperatures . This suggests a novel pathway for the regulation of heat shock in prokaryotes and provides a first example of a thermally tolerant group II intron RNA.

DNA Seq, 2002 Dec, 13(6), 363 - 7
Isolation and sequence analysis of the rat dihydrolipoamide succinyltransferase gene; Nakano K et al.; The dihydrolipoamide succinyltransferase (DLST) gene of the alpha-ketoglutarate dehydrogenase complex (alpha-KGDC) was isolated from a rat genomic DNA library and sequenced . This gene was composed of 15 exons and 14 introns like the human DLST gene . Sequence analysis of the promoter-regulatory region of the rat DLST gene-(Dlst) showed the possible presence of a CAAT box-sequence and of the sequences for an AP-2 site and three Sp1 sites, but no TATA box-sequence was evidenced . The nucleotide sequences of introns 1 and 4 of the rat Dlst were significantly homologous to those of introns 1 and 4 of the human DLST gene . The sequence analysis of the rat Dlst suggested that the exon coding for the E3- and/or E1-binding domain may have been lost from the gene during evolution in eukaryotic DLST, possibly after mitochondrial symbiosis because prokaryotic DLST possesses the E3- and/or E1-binding domain.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3713 - 8 Epub 2003 Mar 21.
Structural elements of metal selectivity in metal sensor proteins; Pennella MA et al.; Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR are homologous zinccobalt-responsive and nickelcobalt-responsive transcriptional repressors in vivo, respectively, and members of the ArsRSmtB superfamily of prokaryotic metal sensor proteins . We show here that Zn(II) is the most potent negative allosteric regulator of czr operatorpromoter binding in vitro with the trend Zn(II)>Co(II)Ni(II), whereas the opposite holds for the binding of NmtR to the nmt operatorpromoter, Ni(II)>Co(II)>Zn(II) . Characterization of the metal coordination complexes of CzrA and NmtR by UVvisible and x-ray absorption spectroscopies reveals that metals that form four-coordinate tetrahedral complexes with CzrA {Zn(II) and Co(II)} are potent regulators of DNA binding, whereas metals that form five- or six-coordinate complexes with NmtR {Ni(II) and Co(II)} are the strongest allosteric regulators in this system . Strikingly, the Zn(II) coordination complexes of CzrA and NmtR cannot be distinguished from one another by x-ray absorption spectroscopy, with the best fit a His-3-carboxylate complex in both cases . Inspection of the primary structures of CzrA and NmtR, coupled with previous functional data, suggests that three conserved His and one Asp from the C-terminal alpha5 helix donate ligands to create a four-coordinate complex in both CzrA and NmtR, with NmtR uniquely capable of expanding its coordination number in the Ni(II) and Co(II) complexes by recruiting additional His ligands from a C-terminal extension of the alpha5 helix.

J Biol Chem, 2003 May 30, 278(22), 20259 - 67 Epub 2003 Mar 21.
The cohesin SMC3 is a target the for beta-catenin/TCF4 transactivation pathway; Ghiselli G et al.; The structural maintenance of chromosome protein SMC3 is a component of the cohesin complex that mediates sister chromatid cohesion and segregation in prokaryotes and eukaryotes . It is also present extracellularly in the form of a chondroitin sulfate proteoglycan known as bamacan . We have found previously that SMC3 expression is elevated in a large fraction of human colon carcinomas . The additional finding that the protein is significantly increased in the intestinal polyps of ApcMin/+ mice has led us to hypothesize that SMC3 expression is linked to activation of the APC/beta-catenin/TCF4 pathway . The immunohistochemical analysis of colon adenocarcinomas from clinical specimens revealed that beta-catenin and SMC3 antigens co-localize with maximal stain intensity within the transformed areas . Cloning and sequencing of 1578 bp of the human SMC3 promoter unveiled the presence of seven putative consensus sequences for beta-catenin/TCF4 binding, two of which are conserved in the mouse Smc3 promoter . Transient transfection experiments in HCT116 and SW480 human colon carcinoma cells using deletion and mutated promoter constructs in luciferase reporter vectors confirmed that the putative sites, the first located at -48 bp and the second located at -701 bp, are susceptible to beta-catenin/TCF4 transactivation . Co-transfection with a beta-catenin expression vector enhanced the promoter activity whereas E-cadherin had the opposite effect . Binding of beta-catenin/TCF4 complexes from SW480 nuclear extracts to these sequences was confirmed by electrophoretic shift and supershift mobility assays . Altogether these results are consistent with the idea that the beta-catenin/TCF4 transactivation pathway contributes to SMC3 overexpression in intestinal tumorigenesis.

Annu Rev Biochem, 2003, 72, 367 - 94 Epub 2003 Mar 19.
Temporal and spatial regulation in prokaryotic cell cycle progression and development; Ryan KR et al.; Bacteria exhibit a high degree of intracellular organization, both in the timing of essential processes and in the placement of the chromosome, the division site, and individual structural and regulatory proteins . We examine the temporal and spatial regulation of the Caulobacter cell cycle, bacterial chromosome segregation and cytokinesis, and Bacillus subtilis sporulation . Mechanisms that control timing of cell cycle and developmental events include transcriptional cascades, regulated phosphorylation and proteolysis of signal transduction proteins, transient genetic asymmetry, and intercellular communication . Surprisingly, many signal transduction proteins are dynamically localized to specific subcellular addresses during the cell division cycle and sporulation, and proper localization is essential for their function . The Min proteins that govern division site selection in Escherichia coli may be the first example of a system that generates positional information de novo.

Bioinformatics, 2003 Mar 22, 19(5), 593 - 9
The Z curve database: a graphic representation of genome sequences; Zhang CT et al.; MOTIVATION: Genome projects for many prokaryotic and eukaryotic species have been completed and more new genome projects are being underway currently . The availability of a large number of genomic sequences for researchers creates a need to find graphic tools to study genomes in a perceivable form . The Z curve is one of such tools available for visualizing genomes . The Z curve is a unique three-dimensional curve representation for a given DNA sequence in the sense that each can be uniquely reconstructed given the other . The Z curve database for more than 1000 genomes have been established here . RESULTS: The database contains the Z curves for archaea, bacteria, eukaryota, organelles, phages, plasmids, viroids and viruses, whose genomic sequences are currently available . All the 3-dimensional Z curves and their three component curves are stored in the database . The applications of the Z curve database on comparative genomics, gene prediction, computation of G+C content with a windowless technique, prediction of replication origins and terminations of bacterial and archaeal genomes and study of local deviations from the Chargaff Parity Rule 2 etc . are presented in detail . The Z curve database reported here is a treasure trove in which biologists could find useful biological knowledge.

Bioinformatics, 2003 Mar 22, 19(5), 549 - 52
Triplet repeats in human genome: distribution and their association with genes and other genomic regions; Subramanian S et al.; MOTIVATION: Simple sequence repeats (SSRs) or microsatellite repeats are found abundantly in many prokaryotic and eukaryotic genomes . Among SSRs, triplet repeats are of special significance because some of them have been linked to various genetic disorders . The objective of the study is to analyze the triplet repeats of complete human genome and to identify the genes that contain the triplet repeats in their coding region . The analysis will help us to identify the candidate genes that have potential for repeat expansion . RESULTS: We have analyzed triplet repeats in the complete human genome from the publicly available sequences . Our analysis revealed that AGC and CCG repeat were predominantly present in the coding regions of the genome while UTRs and the upstream sequences contained CCG repeats in relative abundance . Analysis of density of triplet repeats (bp/Mb) revealed that AAT and AAC were the abundant repeats whereas ACT and ACG were the rare repeats found in human genome . We could identify about 2135 known or predicted genes that were associated with at least one of the triplet repeat types . A large proportion of putative transcripts that were identified by gene finding programs were found to be associated with triplet repeats . These transcripts will be the candidate genes for analysis of triplet repeat expansion and a possible association with disease phenotypes . Identification of 171 genes which contain a minimum of ten repeat units will be of particular interest in future in correlating their association with any disease phenotype due to the expansion potential of repeats present in them . The list of genes and other details of analysis are given in the online supplementary data .

Protein Expr Purif, 2003 Mar, 28(1), 111 - 9
Pyroglutamyl-peptidase I: cloning, sequencing, and characterisation of the recombinant human enzyme; Dando PM et al.; Pyroglutamyl-peptidase I (EC 3.4.19.3) is well known from bacteria and archaea, but has not previously been cloned or sequenced from any vertebrate . We describe the cloning and sequencing of the human (AJ278828) and mouse (AJ278829) forms of pyroglutamyl-peptidase I . The deduced amino acid sequences each consist of 209 residues and show approximately 30% identity with bacterial forms of the enzyme . They show clear homology to the enzyme from prokaryotes and place the mammalian forms of the enzyme in peptidase family C15 of the MEROPS database . The catalytic residues Glu81, Cys144, and His166 in the enzyme from Bacillus amyloliquefaciens are all conserved in the human sequence . A simple cartoon model of the human protein was constructed on the basis of the published crystal structures of pyroglutamyl-peptidase I forms from Thermococcus litoralis and B . amyloliquefaciens . The human enzyme was expressed by use of a baculovirus vector in Spodoptera frugiperda cells . The recombinant protein was enzymatically active and had properties similar to those described for the naturally occurring mammalian enzyme . Gel-filtration chromatography of the active enzyme gave a molecular mass of about 24kDa, showing that the enzyme is active as the monomer . This contrasted with indications that the prokaryotic enzymes may be tetrameric . Recombinant human pyroglutamyl-peptidase I was active on pGlu-aminomethylcoumarin in the range pH 6-9, with maximal activity being seen at pH 7.0-8.5; it showed an absolute requirement for a thiol-reducing agent . In crude preparations, the enzyme was completely stable for 90 min at 50 degrees C . The enzyme was inhibited by transition metal ions including Ni(2+), Zn(2+), and Cu(2+), and by sulfhydryl-blocking agents . Reversible inhibition was seen with 2-pyrrolidone (K(i)=50 microM), and surprisingly, with N-ethylmaleimide (K(i)=30 microM).

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Mar 25, 786(1-2), 7 - 15
Purification of human alpha-L-fucosidase precursor expressed in Escherichia coli as a glutathione S-transferase fusion protein; de Carlos A et al.; Alpha-L-fucosidase (FUC) is a glycosidase involved in the degradation of fucose-containing glycoconjugates . A cDNA representing the complete sequence of human FUC was inserted into the prokaryotic expression vector pGEX-2T . High levels of the glutathione S-transferase (GST) fusion protein were detected in Escherichia coli cells after induction with isopropyl thio-beta-D-galactopyranoside . The GST-FUC protein was mostly found as inclusion bodies and attempts to optimise its expression as a soluble form were unsuccessful . Nevertheless, the recombinant protein was purified by affinity chromatography on glutathione-sepharose and its fucosidase activity was characterised . After thrombin cleavage of the GST tag, the FUC precursor protein was purified by electro-elution.

J Biol Chem, 2003 May 23, 278(21), 19558 - 64 Epub 2003 Mar 19.
Transcription arrest at a lesion in the transcribed DNA strand in vitro is not affected by a nearby lesion in the opposite strand; Kalogeraki VS et al.; Cis-syn cyclobutane pyrimidine dimers (CPDs) are the most frequently formed lesions in UV-irradiated DNA . CPDs are repaired by the nucleotide excision repair pathway . Additionally, they are subject to transcription-coupled DNA repair . In the general model for transcription-coupled DNA repair, an RNA polymerase arrested at a lesion on the transcribed DNA strand facilitates repair by recruiting the repair machinery to the site of the lesion . Consistent with this model, transcription experiments in vitro have shown that CPDs in the transcribed DNA strand interfere with the translocation of prokaryotic and eukaryotic RNA polymerases . Here, we study the behavior of RNA polymerase when transcribing a template that contains two closely spaced lesions, one on each DNA strand . Similar DNA templates containing no CPD, or a single CPD on either the transcribed or the nontranscribed strand were used as controls . Using an in vitro transcription system with purified T7 RNA polymerase (T7 RNAP) or rat liver RNAP II, we characterized transcript length and efficiency of transcription in vitro . We also tested the sensitivity of the arrested RNAP II-DNA-RNA ternary complex, at a CPD in the transcribed strand, to transcription factor TFIIS . The presence of a nearby CPD in the nontranscribed strand did not affect the behavior of either RNA polymerase nor did it affect the reverse translocation ability of the RNAP II-arrested complex . Our results additionally indicate that the sequence context of a CPD affects the efficiency of T7 RNAP arrest more significantly than that of RNAP II.

Biophys Chem, 2003, 100(1-3), 577 - 91
Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: implications of crowding; Grunewald K et al.; Electron cryotomography has unique potential for three-dimensional visualization of macromolecular complexes at work in their natural environment . This approach is based on reconstructing three-dimensional volumes from tilt series of electron micrographs of cells preserved in their native states by vitrification . Resolutions of 5-8 nm have already been achieved and the prospects for further improvement are good . Since many intracellular activities are conducted by complexes in the megadalton range with dimensions of 20-50 nm, current resolutions should suffice to identify many of them in tomograms . However, residual noise and the dense packing of cellular constituents hamper interpretation . Recently, tomographic data have been collected on vitrified eukaryotic cells (Medalia et al., Science (2002) in press) . Their cytoplasm was found to be markedly less crowded than in the prokaryotes previously studied, in accord with differences in crowding between prokaryotic and eukaryotic cells documented by other (indirect) biophysical methods . The implications of this observation are twofold . First, complexes should be more easily identifiable in tomograms of eukaryotic cytoplasm . This applies both to recognizing known complexes and characterizing novel complexes . An example of the latter-a 5-fold symmetric particle is-given . Second, electron cryotomography offers an incisive probe to examine crowding in different cellular compartments.

J Proteome Res, 2002 Jul-Aug, 1(4), 345 - 50
Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine; Jiang H et al.; Quantitative comparison of protein expression levels in 2D gels is complicated by the variables associated with protein separation and mass spectrometric responses . Metabolic labeling allows cells from different experiments to be mixed prior to analysis . This approach has been reported for prokaryotic cells . Here, we demonstrate that metabolic labeling can also be successfully applied to the eukaryote Saccharormyces cerevisiae . Yeast leucine auxotrophs grown on synthetic complete media containing natural abundance Leu or D10-Leu were mixed prior to 2D gel separation and MALDI analysis of the digested proteins . D10-Leu labeling provided an effective internal calibrant for peptide MS analysis, and the number of Leu residues yielded an additional parameter for peptide identification at low mass resolution (1000) . Metabolic incorporation of D10-Leu into yeast proteins was found to be quantitative since the intensities of the peptide peaks corresponded to those expected on the basis of the percent label in the media . Thus, D10-Leu labeling should provide reliable data for comparing proteomes both quantitatively and qualitatively from wild-type and nonessential-gene-null-mutant strains of S . cerevisiae . Given the central role played by yeast in our understanding of eukaryotic gene and protein expression, it is anticipated that the quantitative expressional proteomic method outlined here will have widespread applications.

J Biol Chem, 2003 Mar 14, 278(11), 9678 - 82
The herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) promotes strand invasion; Nimonkar AV et al.; ICP8, the herpes simplex virus type-1 single-strand DNA-binding protein, was recently shown to promote strand exchange in conjunction with the viral replicative helicase (Nimonkar, A . V., and Boehmer, P . E . (2002) J . Biol . Chem . 277, 15182-15189) . Here we show that ICP8 also catalyzes strand invasion in an ATP-independent manner . Thus, ICP8 promotes the assimilation of a single-stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop . Invasion of a homologous duplex by single-stranded DNA requires homology at either 3' or 5' end of the invading strand . The reaction is dependent on the free energy of supercoiling and alters the topology of the acceptor plasmid . Hence, strand invasion products formed by ICP8 are resistant to the action of restriction endonucleases that cleave outside of the area of pairing . The ability to catalyze strand invasion is a novel activity of ICP8 and the first demonstration of a eukaryotic viral single-strand DNA-binding protein to promote this reaction . In this regard ICP8 is functionally similar to the prototypical prokaryotic recombinase RecA and its eukaryotic homologs . This strand invasion activity of ICP8 coupled with DNA synthesis may explain the high prevalence of branched DNA structures during viral replication.

J Mass Spectrom, 2003 Mar, 38(3), 257 - 64
Detection of tyrosine phosphorylated peptides via skimmer collision-induced dissociation/ion trap mass spectrometry; Zolodz MD et al.; Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes . Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate . Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine . Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways . Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations . This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer . Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216 . This method is gentle enough that the protonated molecule of the intact peptide is still observed . In-trap CID was employed for the verification of the phosphotyrosine immonium ion . Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry .

FEMS Microbiol Lett, 2003 Mar 14, 220(1), 81 - 8
Truffle thio-flavours reversibly inhibit truffle tyrosinase; Zarivi O et al.; Tyrosinase is an enzyme having two copper atoms at the reactive site occurring in prokaryotic and eukaryotic organisms . In animals tyrosinase is responsible for pigmentation, in plants for protection of injured tissues or, as in fungi, to harden cell walls . Some of us have previously shown that tyrosinase is involved in truffle development and differentiation . Here we present the purification, the molecular properties and the reversible inhibition of Tuber melanosporum tyrosinase by dimethyl-sulfide and bis{methylthio}methane, the main flavour compounds of black and whitish truffles . The MW(r) is 39000 . L-3,4-dihydroxyphenylalanine and L-tyrosine stain corresponding bands as expected for a true tyrosinase . Phenylthiourea, diethyldithiocarbamate and mimosine inhibit L-tyrosine and L-3,4-dihydroxyphenylalanine oxidation.

Trends Parasitol, 2003 Mar, 19(3), 103 - 5
The mosquito genome: perspectives and possibilities; Land KM; Anopheles gambiae is the mosquito vector responsible for transmitting Plasmodium falciparum, a malaria parasite of humans . With the emergence of genome projects for a variety of prokaryotic and eukaryotic microorganisms, there has been a long-standing interest in sequencing the genomes of the malaria parasite and its insect vector . This tour de force effort has now been completed and reported . The alignment of putative orthologs in An . gambiae with those of Drosophila melanogaster highlights several similarities and differences . These findings could have implications in: (1) identifying new targets for insecticide development; (2) strengthening our understanding of the developmental biology of mosquitoes; and (3) possibly controlling pathogen transmission . A brief overview of these interesting findings and the implications for further studies will be discussed here.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 4328 - 33 Epub 2003 Mar 17.
ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery; Gao H et al.; Chloroplast division in plant cells is orchestrated by a complex macromolecular machine with components positioned on both the inner and outer envelope surfaces . The only plastid division proteins identified to date are of endosymbiotic origin and are localized inside the organelle . Employing positional cloning methods in Arabidopsis in conjunction with a novel strategy for pinpointing the mutant locus, we have identified a gene encoding a new chloroplast division protein, ARC5 . Mutants of ARC5 exhibit defects in chloroplast constriction, have enlarged, dumbbell-shaped chloroplasts, and are rescued by a wild-type copy of ARC5 . The ARC5 gene product shares similarity with the dynamin family of GTPases, which mediate endocytosis, mitochondrial division, and other organellar fission and fusion events in eukaryotes . Phylogenetic analysis showed that ARC5 is related to a group of dynamin-like proteins unique to plants . A GFP-ARC5 fusion protein localizes to a ring at the chloroplast division site . Chloroplast import and protease protection assays indicate that the ARC5 ring is positioned on the outer surface of the chloroplast . Thus, ARC5 is the first cytosolic component of the chloroplast division complex to be identified . ARC5 has no obvious counterparts in prokaryotes, suggesting that it evolved from a dynamin-related protein present in the eukaryotic ancestor of plants . These results indicate that the chloroplast division apparatus is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes.

J Theor Biol, 2003 Apr 7, 221(3), 401 - 10
Genome size and operon content; Cherry JL; Prokaryotic genes are often organized into operons, clusters of genes that are transcribed together . Because all genes in an operon must be transcribed in the same direction, this organization will be reflected in a tendency for nearby genes to have the same orientation . This tendency can be used to estimate the degree to which the genes in a genome are clustered into operons . Application of the technique to Escherichia coli yields results that are similar to estimates based on detailed examination of the genome and empirical knowledge about particular operons . Results for Saccharomyces cerevisiae are consistent with the near absence of polycistronic transcripts in eukaryotes . The method is easily applied to other genomes that have been sequenced and annotated . Analysis of 26 bacterial and archaeal genomes indicates that the degree of clustering varies widely among prokaryotes . Comparison of these genomes shows that those containing more genes tend to have less clustering of genes into operons . This observation may have implications concerning the evolution of operons .

Zhejiang Da Xue Xue Bao Yi Xue Ban, 2003 Feb, 32(1), 13 - 6
{Cloning, expression and identification of flaB gene from a clinical isolate of Helicobacter pylori}; Liang SH et al.; OBJECTIVE: To clone Helicobacter pylori flagellin B gene (flaB) to construct prokaryotic expression system of the gene and to identify immunity of the fusion protein . METHODS: The flaB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR . The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning . The expression vector pET32a with inserted flaB gene was constructed . FlaB fusion protein was expressed in E.coli strain BL21DE3 inducted by IPTG at different dosages . Western blot was applied to determine immunoreactivity and immunogenicity of the fusion protein and antibody against whole cell of H.pylori and rabbit antiserum immunized with the fusion protein, respectively . RESULTS: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned flaB gene was from 96.31% approximate, equals 97.73%, while the homology of its putative amino acid sequence was as high as 99.41% approximate, equals 100% . The expression output of FlaB fusion protein in pET32a-flaB-BL21DE3 system was approximately 40% of the total bacterial proteins . FlaB fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce specific antibody with high titer after the animal was immunized with the protein . CONCLUSION: A prokaryotic expression system of H . pylori flaB gene with high efficiency has been established successfully . The expressed FlaB fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine and detect kit.

Zhejiang Da Xue Xue Bao Yi Xue Ban, 2003 Feb, 32(1), 4 - 8
{Construction of prokaryotic expression system of ureB gene from a clinical isolate of Helicobacter pylori and identification of immunogenicity of the fusion protein}; Chen Z et al.; OBJECTIVE: To clone Helicobacter pylori ureB gene, to construct prokaryotic expression system of the gene and to identify immunogenicity of the fusion protein . METHODS: The ureB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR . The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning . The expression vector pET32a with inserted ureB gene was constructed . ureB fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages . Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein . RESULTS: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned ureB gene was from 96.88% approximate, equals 97.82%, while the homology of its putative amino acid sequence was as high as 99.65% approximate, equals 99.82% . The expression output of UreB protein in pET32a-ureB-BL21DE3 system was approximately 40%of the total bacterial proteins . UreB protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce high titer antibody after the animal was immunized with the protein . CONCLUSION: An expression system with high efficiency of H.pylori ureB gene has been established successfully . The expressed UreB protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.

Nat Neurosci, 2003 Apr, 6(4), 353 - 61
Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel; Sesti F et al.; MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells . Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization . The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier) . Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels . Homologous channels in mammals and plants are expected to function similarly.

Nat Struct Biol, 2003 Apr, 10(4), 280 - 4
Potassium channel gating observed with site-directed mass tagging; Kelly BL et al.; Potassium channels allow the selective flow of K+ ions across otherwise impermeable membranes . During a process called gating, these channels undergo a conformational change that proceeds from a closed to an open state . The closed state of KcsA, a prokaryotic potassium channel, has been structurally well characterized with equilibrium structural techniques . However, attempts to obtain a structural description of the gating transition of the channel have been hampered because the open state is only transiently occupied and, therefore, not readily accessible to such techniques . Here we describe a non-equilibrium technique that we call site-directed mass tagging and use this technique to probe the conformational change that KcsA undergoes during gating . The results indicate that KcsA is a dynamically modular molecule; the extracellular half of the membrane-spanning region is held rigid during gating, while the intracellular half undergoes a significant conformational change.

Crit Rev Microbiol, 2003, 29(1), 37 - 78
Increasing diversity within Chlamydiae; Corsaro D et al.; In recent years, 16S ribosomal DNA analyses has allowed the recognition of new chlamydia organisms, requiring the creation of new species, genera, and families within this unique, deep lineage of prokaryotes . The trachoma and psittaci groups chlamydiae are now recognized as separate genera, Chlamydia and Chlamydophila, respectively, and biovars of each group have been elevated to the species rank . Simkania and Parachlamydia have been associated with human respiratory infections, while Waddlia seems to be implicated in abortion in bovins . DNA amplification studies targeting the 16S rDNA have revealed a richer diversity within chlamydiae, identifying new lineages from both environmental and clinical samples . Further studies will be of interest to both examine the ecology and evaluate the clinical importance of these novel chlamydiae . Herein, we provide a summary of literature and our data about the novel chlamydial lineages.

Curr Issues Mol Biol, 2003 Jan, 5(1), 17 - 25
Genomics and bacterial metabolism; Downs DM; The field of bacterial metabolism and physiology is arguably the oldest in microbiology . Much of our understanding of biological processes and molecular paradigms has its roots In early studies of prokaryotic physiology . After a period of declining interest in metabolic studies (prompted by the insurgence of molecular techniques), genomic technologies are revitalizing the study of bacterial metabolism and physiology . These new technologies bring a means to approach metabolic questions with a global perspective . When used in combination with classical and molecular techniques, emerging global technologies will make it feasible to understand the complex integration of metabolic processes that result in an efficient physiology . At the same time, without increased computational capabilities, the massive amounts of data generated by these technologies threaten to overwhelm, rather than facilitate, this work . For genomic technologies to reach their potential for increasing our understanding of bacterial metabolism, microbiologists must become more collaborative and multidisciplinary than at any time in our history.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 4197 - 202 Epub 2003 Mar 12.
A gain-of-function mutation in ftsA bypasses the requirement for the essential cell division gene zipA in Escherichia coli; Geissler B et al.; ZipA and FtsA are recruited independently to the FtsZ cytokinetic ring (Z ring) and are essential for cell division of Escherichia coli . The molecular role of FtsA in cell division is unknown; however, ZipA is thought to stabilize the Z ring, anchor it to the membrane, and recruit downstream cell division proteins . Here we demonstrate that the requirement for ZipA can be bypassed completely by a single alteration in a conserved residue of FtsA (FtsA*) . Cells with ftsA* in single copy in place of WT ftsA or with ftsA* alone on a multicopy plasmid divide mostly normally, whether they are zipA+ or zipA- . Experiments with ftsQAZ and ftsQA*Z on multicopy plasmids indicate that ftsQAZzipA+ and ftsQA*ZzipA- cells divide fairly normally, whereas ftsQAZzipA- cells divide poorly and ftsQA*ZzipA+ cells display a phenotype that suggests their septa are unusually stable . In support of the idea that ftsA* stabilizes Z rings, single-copy ftsA* confers resistance to excess MinC, which destabilizes Z rings . The inhibitory effect of excess ZipA on division is also suppressed by ftsA* . These results suggest that the molecular mechanism of the FtsA* bypass is to stabilize FtsZ assembly via a parallel pathway and that FtsA* can replace the multiple functions of ZipA . This is an example of a complete functional replacement of an essential prokaryotic cell division protein by another and may explain why most bacteria can divide without an obvious ZipA homolog.

Microbiology, 2003 Mar, 149(Pt 3), 589 - 600
Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica; Augstein A et al.; Adaptation of cells to acetic acid requires a hitherto unknown number of proteins . Studies on the GPR1 gene and its encoded protein in the ascomycetous fungus Yarrowia lipolytica have revealed an involvement of this protein in the molecular processes of adaptation to acetic acid . Gpr1p belongs to a novel family of conserved proteins in prokaryotic and eukaryotic organisms that is characterized by the two motifs (A/G)NPAPLGL and SYG(X)FW (GPR1_FUN34_YaaH protein family) . Analysis of four trans-dominant mutations and N-terminal deletion analysis of Gpr1p identified the amino acid sequence FGGTLN important for function of this protein in Y . lipolytica . Deletion of GPR1 slowed down adaptation to acetic acid, but had no effect on growth in the presence of acetic acid . Expression of GPR1 is induced by acetic acid and moderately repressed by glucose . It was shown by subcellular fractionation that Gpr1p is an integral membrane protein, which is also suggested by the presence of five to six putative transmembrane spanning regions . Fluorescence microscopy confirmed a localization to the plasma membrane . A model is presented describing a hypothetical function of Gpr1p during adaptation to acetic acid.

Biochem J, 2003 Jun 1, 372(Pt 2), 279 - 90
N-acetylglutamate and its changing role through evolution; Caldovic L et al.; N -Acetylglutamate (NAG) fulfils distinct biological roles in lower and higher organisms . In prokaryotes, lower eukaryotes and plants it is the first intermediate in the biosynthesis of arginine, whereas in ureotelic (excreting nitrogen mostly in the form of urea) vertebrates, it is an essential allosteric cofactor for carbamyl phosphate synthetase I (CPSI), the first enzyme of the urea cycle . The pathway that leads from glutamate to arginine in lower organisms employs eight steps, starting with the acetylation of glutamate to form NAG . In these species, NAG can be produced by two enzymic reactions: one catalysed by NAG synthase (NAGS) and the other by ornithine acetyltransferase (OAT) . In ureotelic species, NAG is produced exclusively by NAGS . In lower organisms, NAGS is feedback-inhibited by L-arginine, whereas mammalian NAGS activity is significantly enhanced by this amino acid . The NAGS genes of bacteria, fungi and mammals are more diverse than other arginine-biosynthesis and urea-cycle genes . The evolutionary relationship between the distinctly different roles of NAG and its metabolism in lower and higher organisms remains to be determined . In humans, inherited NAGS deficiency is an autosomal recessive disorder causing hyperammonaemia and a phenotype similar to CPSI deficiency . Several mutations have been recently identified in the NAGS genes of families affected with this disorder.

Microb Ecol, 2003 Mar, 45(3), 203 - 17 Epub 2003 Mar 14.
Life under nutrient limitation in oligotrophic marine environments: an eco/physiological perspective of Sphingopyxis alaskensis (formerly Sphingomonas alaskensis); Cavicchioli R et al.; The oceans of the world are nutrient-limited environments that support a dynamic diversity of microbial life . Heterotrophic prokaryotes proliferate in oligotrophic regions and affect nutrient transformation and remineralization thereby impacting directly on the all marine biota . An important challenge in studying the microbial ecology of oligotrophic environments has been the isolation of ecologically important species . This goal has been recognized not only for its relevance in defining the dynamics of community composition, but for enabling physiological studies of competitive species and inferring their impact on the microbial food web . This review describes the successful isolation attempts of the ultramicrobacterium, Sphingopyxis alaskensis (formerly described as Sphingomonas alaskensis) using extinction dilution culturing methods . It then provides a comprehensive perspective of the unique physiological and genetic properties that have been identified that distinguish it from typical copiotrophic species . These properties are described through studies of the growth phase and growth rate control of macromolecular synthesis, stress resistance and global gene expression (proteomics) . We also discuss the importance of integrating ecological and physiological approaches for studying microorganisms in marine environments.

Biofizika, 2003 Jan-Feb, 48(1), 27 - 34
{Analysis of exciton parameters in DNA . Exciton waves in DNA as one of the reasons of mutagenesis}; Vasil'kov SL; Formulae were obtained for the quantitative analysis of the following parameters of excitons in DNA: 1) the lifetime of electronic excitation; 2) the numbers of exciton runs along the DNA sequence; 3) the energy loss by an exciton for one run; 4) the maximum length of the DNA sequence capable of deactivating an exciton for one run . The maximum and minimum ranges for the constant of electronic excitation migration was determined to meet the requirement of inductive-resonance energy transfer for the case of strong interaction . The constant of exciton energy migration was shown to depend on the activation energy, which is equal to the energy of absorbed quantum . An analytical formula was derived to determine the number of quanta the DNA molecule is able to absorb, depending on its length, without nonlinear effects (without overlapping of spatial areas of electronic excitation) . By this formula, DNA sequences consisting of only identical AT and GC nucleotide pairs and aggregate AT + GC (in the ratio 1:1) DNA sequences ranging from 1 up to 10(10) base pairs were analyzed . The results of the analysis suggest that the overlapping of spatial areas of electronic excitation induced by a single ultraviolet quantum occurs in short DNA sequences characteristic of prokaryotes . To achieve the same effects on long DNA sequences specific for eukaryotes, DNA must synchronously absorb a great number of ultraviolet quanta . Based on the above results, the following conclusions were made: 1) disturbances in the normal activity of DNA and RNA polymerases may be due to electromagnetic field, which is caused by the oscillatory relaxation of vibronic levels of nucleotides . The energy enters the vibronic levels of nucleotides from an exciton running along the DNA sequence; 2) the increase in the noncoding DNA sequences in eukaryotes due to evolution is a way of DNA protection from undesirable mutations; 3) prokaryotes must possess a greater potentiality and a higher rate of mutagenesis in comparison with eukaryotes, which is proved by their greater diversity in nature.

Biochemistry, 2003 Mar 18, 42(10), 2952 - 9
Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation; Mehan RS et al.; The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes . The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex . Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers . However, the locations and extents of the contact regions on the receptor surface remain ambiguous . The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex . The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains . When the bulky, anionic probe 5-fluorescein-maleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations . By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain . The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region . These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase . Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation . A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity . Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex . By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.

Rev Med Virol, 2003 Mar-Apr, 13(2), 111 - 21
Cloning of herpesviral genomes as bacterial artificial chromosomes; Adler H et al.; Herpesviruses, which are important pathogens for both animals and humans, have large and complex genomes with a coding capacity for up to 225 open reading frames (ORFs) . Due to the large genome size and the slow replication kinetics in vitro of some herpesviruses, mutagenesis of viral genes in the context of the viral genome by conventional recombination methods in cell culture has been difficult . Given that mutagenesis of viral genes is the basic strategy to investigate function, many of the herpesvirus ORFs could not be defined functionally . Recently, a completely new approach for the construction of herpesvirus mutants has been developed, based on cloning of the virus genome as a bacterial artificial chromosome (BAC) in E . coli . This technique allows the maintenance of viral genomes as a plasmid in E . coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells . Any genetic modification of the viral genome in E . coli using prokaryotic recombination proteins is possible, thereby allowing the generation of mutant viruses and facilitating the analysis of herpesvirus genomes cloned as infectious BACs . In this review, we describe the principle of cloning a viral genome as a BAC using murine gammaherpesvirus 68 (MHV-68), a mouse model for gammaherpesvirus infections, as an example .

Nucleic Acids Res, 2003 Mar 15, 31(6), 1725 - 34
RPA is an initiation factor for human chromosomal DNA replication; Szuts D et al.; The initiation of chromosomal DNA replication in human cell nuclei is not well understood because of its complexity . To allow investigation of this process on a molecular level, we have recently established a cell-free system that initiates chromosomal DNA replication in an origin-specific manner under cell cycle control in isolated human cell nuclei . We have now used fractionation and reconstitution experiments to functionally identify cellular factors present in a human cell extract that trigger initiation of chromosomal DNA replication in this system . Initial fractionation of a cytosolic extract indicates the presence of at least two independent and non-redundant initiation factors . We have purified one of these factors to homogeneity and identified it as the single-stranded DNA binding protein RPA . The prokaryotic single-stranded DNA binding protein SSB cannot substitute for RPA in the initiation of human chromosomal DNA replication . Antibodies specific for human RPA inhibit the initiation step of human chromosomal DNA replication in vitro . RPA is recruited to DNA replication foci and becomes phosphorylated concomitant with the initiation step in vitro . These data establish a direct functional role for RPA as an essential factor for the initiation of human chromosomal DNA replication.

Glycobiology, 2003 Feb, 13(2), 51 - 71 Epub 2002 Nov 01.
Characterization by NMR and molecular modeling of the binding of polyisoprenols and polyisoprenyl recognition sequence peptides: 3D structure of the complexes reveals sites of specific interactions; Zhou GP et al.; The objective of these studies was to test the hypothesis that proteins that contain potential polyisoprenyl recognition sequences (PIRSs) in their transmembrane-spanning domain can bind to the polyisoprenyl (PI) glycosyl carrier lipids undecaprenyl phosphate (C55-P) and dolichyl phosphate (C95-P) . A number of prokaryotic and eukaryotic glycosyltransferases that utilize PI coenzymes contain a conserved PIRS postulated to be the active PI binding domain . To study this problem, we first determined the 3D structure of a PIRS peptide, NeuE, by homonuclear 2D 1H-nuclear magnetic resonance (NMR) spectroscopy . Experimentally generated distance constraints derived from nuclear Overhauser enhancement and torsion angle constraints derived from coupling constants were used for restrained molecular dynamics and energy minimization calculations . Molecular models of the NeuE peptide were built based on calculations of energy minimization using the DGII program NMRchitect . 3D models of dolichol (C95) and C95-P were built based on our 2D 1H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the AMBER (assisted modeling with energy refinements) force field . Our energy minimization studies were carried out on a conformational model of dolichol that was originally derived from small-angle X-ray scattering and molecular mechanics methods . These results revealed that the PIs are conformationally nearly identical tripartite molecules, with their three domains arranged in a coiled, helical structure . Analyses of the intermolecular cross-peaks in the 2D NOESY spectra of PIRS peptides in the presence of PIs confirmed a highly specific interaction and identified key contact amino acids in the NeuE peptide that constituted a binding motif for interacting with the PIs . These studies also showed that subtle conformational changes occurred within both the PIs and the NeuE peptide after binding . 3D structures of the resulting molecular complexes revealed that each PI could bind more than one PIRS peptide . These studies thus represent the first evidence for a direct physical interaction between specific contact amino acids in the PIRS peptides and the PIs and supports the hypothesis of a bifunctional role for the PIs . The central idea is that these superlipids may serve as a structural scaffold to organize and stabilize in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in biosynthetic and translocation processes.

Planta, 2003 Mar, 216(5), 874 - 80 Epub 2002 Nov 26.
Isolation and characterization of a myo-inositol 1-phosphate synthase cDNA from developing sesame (Sesamum indicum L.) seeds: functional and differential expression, and salt-induced transcription during germination; Chun JA et al.; A cDNA (SeMIPS1) encoding myo-inositol 1-phosphate synthase (EC 5.5.1.4) (MIPS) has been characterized from sesame (Sesamum indicum L . cv . Dan-Baek) seeds and its functional expression analyzed . The SeMIPS1 protein was highly homologous with those from other plant species (88-94%), while a much lower degree of sequence homology (53-62%) was found with other organisms such as humans, mouse, algae, yeast, Drosophila, bacteria and other prokaryotes . A yeast-based complementation assay in yeast mutants containing a disrupted INO1gene for yeast MIPS confirmed that the SeMIPS1 gene encodes a functional MIPS . Phylogenetic analysis suggested that the SeMIPS1 gene diverged as a different subfamily or family member . Southern hybridization revealed several copies of the SeMIPS1 gene present in the sesame genome and northern blotting indicated that expression of the SeMIPS1gene may be organ specific . Salt stress during sesame seed germination had an adverse influence on transcription of SeMIPS1and greatly reduced transcript levels as the duration of exposure to a saline environment increased and NaCl concentration increased . Germination initiation of sesame seeds was severely delayed as NaCl level increased . These results suggest that expression of SeMIPS1 is down-regulated by salt stress during sesame seed germination.

Microbiology, 2003 Feb, 149(Pt 2), 295 - 304
Prokaryotic motility structures; Bardy SL et al.; Prokaryotes use a wide variety of structures to facilitate motility . The majority of research to date has focused on swimming motility and the molecular architecture of the bacterial flagellum . While intriguing questions remain, especially concerning the specialized export system involved in flagellum assembly, for the most part the structural components and their location within the flagellum and function are now known . The same cannot be said of the other apparati including archaeal flagella, type IV pili, the junctional pore, ratchet structure and the contractile cytoskeleton used by a variety of organisms for motility . In these cases, many of the structural components have yet to be identified and the mechanism of action that results in motility is often still poorly understood . Research on the bacterial flagellum has greatly aided our understanding of not only motility but also protein secretion and genetic regulation systems . Continued study and understanding of all prokaryotic motility structures will provide a wealth of knowledge that is sure to extend beyond the bounds of prokaryotic movement.

Mol Microbiol, 2003 Mar, 47(6), 1485 - 94
Mechanisms of iron regulation in mycobacteria: role in physiology and virulence; Rodriguez GM et al.; The role of iron in mycobacteria as in other bacteria goes beyond the need for this essential cofactor . Limitation of this metal triggers an extensive response aimed at increasing iron acquisition while coping with iron deficiency . In contrast, iron-rich environments prompt these prokaryotes to induce synthesis of iron storage molecules and to increase mechanisms of protection against iron-mediated oxidative damage . The response to changes in iron availability is strictly regulated in order to maintain sufficient but not excessive and potentially toxic levels of iron in the cell . This response is also linked to other important processes such as protection against oxidative stress and virulence . In bacteria, iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage . In mycobacteria, this role is fulfilled by the iron-dependent regulator IdeR . IdeR is an essential protein in Mycobacterium tuberculosis, the causative agent of human tuberculosis . It functions as a repressor of iron acquisition genes, but is also an activator of iron storage genes and a positive regulator of oxidative stress responses.

Appl Environ Microbiol, 2003 Mar, 69(3), 1797 - 9
Strong synergy between a eukaryotic antimicrobial peptide and bacteriocins from lactic acid bacteria; Luders T et al.; The antimicrobial effect obtained upon combining the prokaryotic antimicrobial peptides (AMPs; more commonly referred to as bacteriocins) pediocin PA-1, sakacin P, and curvacin A (all produced by lactic acid bacteria {LAB}) with the eukaryotic AMP pleurocidin (from fish) has been investigated . The three LAB AMPs alone were active against gram-positive Listeria ivanovii bacteria at nanomolar concentrations, whereas they were inactive against gram-negative Escherichia coli bacteria . Pleurocidin alone was active against both of these types of bacteria at micromolar concentrations . Little if any synergy between the LAB AMPs and pleurocidin against the gram-positive L . ivanovii strain was obtained . In contrast, the LAB AMPs and pleurocidin acted highly synergistically against the gram-negative E . coli strain . Nanomolar concentrations of LAB AMPs increased the growth inhibitory potency of pleurocidin by about fourfold . When micromolar concentrations of LAB AMPs were combined with 2 micro g of pleurocidin/ml, 100% growth inhibition was attained, whereas pleurocidin alone at a concentration of 2 micro g/ml gave no growth inhibition . Most noteworthy, when high concentrations (128 micro g/ml) of pleurocidin in the absence of LAB AMPs were used over a long period of incubation (1 week), some growth of E . coli was observed, whereas 16 micro g of pleurocidin/ml completely abolished growth in the presence of 64 to 128 ng of LAB AMPs/ml over the same period of time . The results clearly demonstrate that combining eukaryotic and prokaryotic AMPs can greatly increase the specific activity and broaden the target-cell range of these peptides.

Appl Environ Microbiol, 2003 Mar, 69(3), 1748 - 58
In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes; Behrens S et al.; Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes . In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp . strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya) . For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry . The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models . For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M . sedula were grouped into class V and VI (46% of the whole data set) . For E . coli, 45% of all probes of the data set belong to class III and IV . A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules . In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3' half of helix 47 (positions 1320 to 1345) were generally inaccessible . Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S . cerevisiae.

Genome Biol . 2003;4(2):P1 . Epub 2003 Jan 10.
Positional clustering of differentially expressed genes on human chromosomes 20, 21 and 22; Megy K et al.; BACKGROUND: Clusters of genes co-expressed are known in prokaryotes (operons) and were recently described in several eukaryote organisms, including Human . According to some studies, these clusters consist of housekeeping genes, whereas other studies suggest that these clustered genes exhibit similar tissue specificity . Here we further explore the relationship between co-expression and chromosomal co-localization in the human genome by analyzing the expression status of the genes along the best-annotated chromosomes 20, 21 and 22 . METHODS: Gene expression levels were estimated according to their publicly available ESTs and gene differential expressions were assessed using a previously described and validated statistical test . Gene sequences for chromosomes 20, 21 and 22 were taken from the Ensembl annotation . RESULTS: We identified clusters of genes specifically expressed in similar tissues along chromosomes 20, 21 and 22 . These co-expression clusters occurred more frequently than expected by chance and may thus be biologically significant . CONCLUSION: The co-expression of co-localized genes might be due to higher chromatin structures influencing the gene availability for transcription in a given tissue or cell type.

Cell Mol Biol (Noisy-le-grand), 2002 Nov, 48(7), 777 - 81
Assessment of the stannous fluoride and phytic acid effect in the yeast Saccharomyces cerevislae; Lima-Filho GL et al.; Stannous fluoride (SnF2) is a powerful reducing agent in 99mTc-labelled radiopharmaceuticals for nuclear medicine procedures . SnF2 may enhance reactive oxidative species (ROS) in prokaryotic cells . Phytic acid (PA) is a wide-ranging regulator of many important cellular functions such as intracellular regulations of surface receptions channels and it is known to have antioxidant and chelating properties . In order to analyze whether membrane transporters of the facilitator or the ABC type (SNQ1 and SNQ2) have an influence on Sn2+ toxicity in yeast we used the respective mutants and compared their responses to the wild type (WT) . Since ABC transporters are YAP1p transcription activator inducible, we included a yap1 mutant in our Sn2+ toxicity assay . Finally, we tested the PA influence on Sn2+ toxicity in these strains . Yeast cells in stationary growth phase were exposed to different concentrations of SnF2 (ranging from 2 to 6 mg/ml) and PA (0.1 M) for one hour . The snq1 mutant exhibited the highest sensitivity to SnF2 while the snq2 and snq3/yap1 mutants had an equally intermediate sensitivity . The presence of PA was not able to produce a significant protection against the cytotoxicity of SnF2 . This is probably due to its reduced chelating power in complex liquid media Our results with yeast support the genotoxic effects described for SnF2 in bacteria andindicate that the biological effect of this reducing agent could be related to the generation of reactive oxygen species.

Oncogene, 2003 Mar 6, 22(9), 1340 - 8
Elimination of the vertebrate Escherichia coli Ras-like protein homologue leads to cell cycle arrest at G1 phase and apoptosis; Gohda J et al.; Homologues of the Escherichia coli (E . coli) Ras-like protein (ERA), a GTP-binding protein with RNA binding activity, have recently been found in various species, including human, mouse, and Antirrhinum majus . Depletion of prokaryotic ERA blocks cell division without affecting chromosome segregation . However, the physiological function of eukaryotic ERA is largely unknown . We have performed a genetic analysis of chicken ERA (GdERA) in DT40 cells . Depletion of GdERA diminished the growth rate of the cells, accompanied by an accumulation of apoptotic cells . The analysis of cell cycle indicates that the elimination of GdERA caused arrest at G1 phase, but not at M phase, which highlights the distinct role of vertebrate ERA in the cell cycle progression compared to prokaryotic ERA . Furthermore, human ERA (HsERA) rescued the phenotype of GdERA-deficient cells, whereas a mutant of HsERA deprived of RNA-binding activity did not . These data suggest that vertebrate ERA regulates the G1 phase progression via an as yet unknown molecular mechanism, which involves RNA recognition by ERA.

J Bacteriol, 2003 Mar, 185(6), 1831 - 40
Analysis of genome plasticity in pathogenic and commensal Escherichia coli isolates by use of DNA arrays; Dobrindt U et al.; Genomes of prokaryotes differ significantly in size and DNA composition . Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants . Pathogenic and nonpathogenic E . coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information . To analyze the genetic diversity of pathogenic and commensal E . coli isolates, a whole-genome approach was applied . Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E . coli K-12 strain MG1655 was investigated in 26 E . coli isolates, including various extraintestinal and intestinal pathogenic E . coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains . Additionally, the presence of virulence-associated genes of E . coli was determined using a DNA "pathoarray" developed in our laboratory . The frequency and distributional pattern of genomic variations vary widely in different E . coli strains . Up to 10% of the E . coli K-12-specific ORFs were not detectable in the genomes of the different strains . DNA sequences described for extraintestinal or intestinal pathogenic E . coli are more frequently detectable in isolates of the same origin than in other pathotypes . Several genes coding for virulence or fitness factors are also present in commensal E . coli isolates . Based on these results, the conserved E . coli core genome is estimated to consist of at least 3,100 translatable ORFs . The absence of K-12-specific ORFs was detectable in all chromosomal regions . These data demonstrate the great genome heterogeneity and genetic diversity among E . coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.

J Bacteriol, 2003 Mar, 185(6), 1803 - 7
The Escherichia coli mazEF suicide module mediates thymineless death; Sat B et al.; In 1954, Cohen and Barner discovered that a thymine auxotrophic (thyA) mutant of Escherichia coli undergoes cell death in response to thymine starvation . This phenomenon, called thymineless death (TLD), has also been found in many other organisms, including prokaryotes and eukaryotes . Though TLD has been studied intensively, its molecular mechanism has not yet been explained . Previously we reported on the E . coli mazEF system, a regulatable chromosomal suicide module that can be triggered by various stress conditions . MazF is a stable toxin, and MazE is an unstable antitoxin . Here, we show that cell death that is mediated by the mazEF module can also be activated by thymine starvation . We found that TLD depends on E . coli mazEF and that under thymine starvation, the activity of the mazEF promoter P(2) is significantly reduced . Our results, which describe thymine starvation as a trigger for a built-in death program, have implications for programmed cell death in both prokaryotes and eukaryotes.

Pharmacol Rev, 2003 Mar, 55(1), 27 - 55
Mechanisms of antimicrobial peptide action and resistance; Yeaman MR et al.; Antimicrobial peptides have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum, ranging from prokaryotes to humans . Yet, recurrent structural and functional themes in mechanisms of action and resistance are observed among peptides of widely diverse source and composition . Biochemical distinctions among the peptides themselves, target versus host cells, and the microenvironments in which these counterparts convene, likely provide for varying degrees of selective toxicity among diverse antimicrobial peptide types . Moreover, many antimicrobial peptides employ sophisticated and dynamic mechanisms of action to effect rapid and potent activities consistent with their likely roles in antimicrobial host defense . In balance, successful microbial pathogens have evolved multifaceted and effective countermeasures to avoid exposure to and subvert mechanisms of antimicrobial peptides . A clearer recognition of these opposing themes will significantly advance our understanding of how antimicrobial peptides function in defense against infection . Furthermore, this understanding may provide new models and strategies for developing novel antimicrobial agents, that may also augment immunity, restore potency or amplify the mechanisms of conventional antibiotics, and minimize antimicrobial resistance mechanisms among pathogens . From these perspectives, the intention of this review is to illustrate the contemporary structural and functional themes among mechanisms of antimicrobial peptide action and resistance.

J Struct Biol, 2003 Feb, 141(2), 97 - 102
Increasing the diffraction limit and internal order of a membrane protein crystal by dehydration; Kuo A et al.; It is notoriously difficult to produce crystals of membrane proteins that diffract to sufficient resolution for structural studies by X-ray crystallography . Crystals of a prokaryotic CLC chloride channel that were initially unacceptable for structural analysis improved in both quality and diffraction limit by a process of dehydration . The loss of water decreased the dimensions of the unit cell axes by up to 25 A, improved the diffraction limit from 8.0 to 4.0 A, and decreased the mosaicity to values of approximately 1 degrees . Dehydration of integral membrane protein crystals should be one of the procedures included in the initial screening for appropriate crystals and as a method of improving the diffraction limits of existing crystals.

Trends Genet, 2003 Mar, 19(3), 162 - 7
Lateralization defects and ciliary dyskinesia: lessons from algae; El Zein L et al.; Flagella and cilia are two very similar organelles that "beat" to move cells and to propel fluid over tissues . They are highly conserved, being found in organisms ranging from prokaryotes to plant and animal eukaryotes . In humans, cilia are present in almost every organ, and several human conditions involve dysfunctional cilia; for example, lateralization defects, where the positions of organs are reversed, and primary ciliary dyskinesia, a rare condition where patients suffer from recurrent respiratory infections . In this article, we will discuss how information gained from studies on algae has aided research into these human diseases . These studies found a variety of functions that was previously unsuspected, renewing interest in cilia.

Trends Genet, 2003 Mar, 19(3), 155 - 61
Bacterial translational control at atomic resolution; Romby P et al.; Translational regulation allows rapid adaptation of protein synthesis to environmental conditions . In prokaryotes, the synthesis of many RNA-binding proteins is regulated by a translational feedback mechanism involving a competition between their natural substrate and their binding site on mRNA, which are often thought to resemble each other . This article describes the case of threonyl-tRNA synthetase, which represses the translation of its own mRNA . Recent data provide the first opportunity to describe at the atomic level both the extent and the limit of mimicry between the way this enzyme recognizes tRNA(Thr) and its regulatory site in mRNA . The data also give some clues about how the binding of the synthetase to its mRNA inhibits translation.

Free Radic Biol Med, 2003 Mar 1, 34(5), 509 - 20
Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell; Sarti P et al.; The aim of this work is to review the information available on the molecular mechanisms by which the NO radical reversibly downregulates the function of cytochrome c oxidase (CcOX) . The mechanisms of the reactions with NO elucidated over the past few years are described and discussed in the context of the inhibitory effects on the enzyme activity . Two alternative reaction pathways are presented whereby NO reacts with the catalytic intermediates of CcOX populated during turnover . The central idea is that at "cellular" concentrations of NO (</= microM), the redox state of the respiratory chain results in the formation of either the nitrosyl- or the nitrite-derivative of CcOX, with potentially different metabolic implications for the cell . In particular, the role played by CcOX in protecting the cell from excess NO, potentially toxic for mitochondria, is also reviewed highlighting the mechanistic differences between eukaryotes and some prokaryotes.

J Mol Biol, 2003 Mar 14, 327(1), 61 - 73
Crystal structure and evolution of a prokaryotic glucoamylase; Aleshin AE et al.; The first crystal structures of a two-domain, prokaryotic glucoamylase were determined to high resolution from the clostridial species Thermoanaerobacterium thermosaccharolyticum with and without acarbose . The N-terminal domain has 18 antiparallel strands arranged in beta-sheets of a super-beta-sandwich . The C-terminal domain is an (alpha/alpha)(6) barrel, lacking the peripheral subdomain of eukaryotic glucoamylases . Interdomain contacts are common to all prokaryotic Family GH15 proteins . Domains similar to those of prokaryotic glucoamylases in maltose phosphorylases (Family GH65) and glycoaminoglycan lyases (Family PL8) suggest evolution from a common ancestor . Eukaryotic glucoamylases may have evolved from prokaryotic glucoamylases by the substitution of the N-terminal domain with the peripheral subdomain and by the addition of a starch-binding domain.

Am J Kidney Dis, 2003 Mar, 41(3 Suppl 1), S116 - 22
Carnitine and hemodialysis; Bellinghieri G et al.; Carnitine, gamma-trimethyl-beta-hydroxybutyrobetaine, is a small molecule widely present in all cells from prokaryotic to eukaryotic . It is an important element in the beta-oxidation of fatty acids . A lack of carnitine in hemodialysis patients is caused by insufficient carnitine synthesis and particularly by the loss through dialytic membranes, leading in some patients to carnitine depletion with a relative increase of esterified forms . The authors found a decrease in plasma-triglyceride and increase of high-density lipoprotein cholesterol (HDL-Chol) in dialysis patients during carnitine treatment . Many studies have shown that L-carnitine supplementation leads to improvements in several complications seen in uremic patients, including cardiac complications, impaired exercise and functional capacities, muscle symptoms, increased symptomatic intradialytic hypotension, and erythropoietin-resistant anemia, normalizing the reduced carnitine palmitoyl transferase activity in red cells . In addition, carnitine supplementation may improve protein metabolism and insulin resistance . Recently, carnitine supplementation has been approved by the US Food and Drug Administration not only for the treatment, but also for the prevention of carnitine depletion in dialysis patients . Regular carnitine supplementation in hemodialysis patients can improve their lipid metabolism, protein nutrition, antioxidant status, and anemia requiring large doses of erythropoietin, It also may reduce the incidence of intradialytic muscle cramps, hypotension, asthenia, muscle weakness, and cardiomyopathy.

J Mol Evol, 2003 Mar, 56(3), 362 - 70
Effects of GC content and mutational pressure on the lengths of exons and coding sequences; Xia X et al.; It has been hypothesized that the length of an exon tends to increase with the GC content because stop codons are AT-rich and should occur less frequently in GC-rich exons . This prediction assumes that mutation pressure plays a significant role in the occurrence and distribution of stop codons . However, the prediction is applicable not to all exons, but only to the last coding exon of a gene and to single-exon CDS sequences . We classified exons in multiexon genes in eight eukaryotic species into three groups-the first exon, the internal, and the last exon-and computed the Spearman correlation between the exon length and the percentage GC (%GC) for each of the three groups . In only five of the species studied is the correlation for the last coding exon greater than that for the first or internal exons . For the single-exon CDS sequences, the correlation between CDS length and %GC is mostly negative . Thus, eukaryotic genomes do not support the predicted relationship between exon length and %GC . In prokaryotic genomes, CDS length and %GC are positively correlated in each of the 68 completely sequenced prokaryotic genomes in GenBank with genomic GC contents varying from 25 to 68%, except for the wall-less Mycoplasma genitalium and the syphilis pathogen Treponema pallidum . Moreover, the average CDS length and the genomic GC content are also positively correlated . After correcting for genome size, the partial correlation between the average CDS length and the genomic GC content is 0.3217 ( p < 0.025).

EMBO Rep, 2003 Feb, 4(2), 172 - 7
Growth-rate dependent RNA polyadenylation in Escherichia coli; Jasiecki J et al.; RNA polyadenylation occurs not only in eukaryotes but also in bacteria . In prokaryotes, polyadenylated RNA molecules are usually degraded more efficiently than non-modified transcripts . Here we demonstrate that two transcripts, which were shown previously to be substrates for poly(A) polymerase I (PAP I), Escherichia coli lpp messenger RNA and bacteriophage lambda oop RNA, are polyadenylated more efficiently in slowly growing bacteria than in rapidly growing bacteria . Intracellular levels of PAP I varied in inverse proportion to bacterial growth rate . Moreover, transcription from a promoter for the pcnB gene (encoding PAP I) was shown to be more efficient under conditions of low bacterial growth rates . We conclude that efficiency of RNA polyadenylation in E . coli is higher in slowly growing bacteria because of more efficient expression of the pcnB gene . This may allow regulation of the stability of certain transcripts (those subjected to PAP I-dependent polyadenylation) in response to various growth conditions.

EMBO Rep, 2003 Feb, 4(2), 154 - 8
Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin; Matsunaga F et al.; Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria . In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo . We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments . Mapping of replication initiation points further showed that discontinuous DNA replication in P . abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile . Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea . Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

Vestn Ross Akad Med Nauk, 2003, (1), 15 - 20
{Genome-wide non-sequencing strategies for bacterial genome comparison: the necessity and an analysis of the variable bacterial world}; Sverdlov ED; A tremendous success in bacterial genome sequencing has been achieved during the recent years; it resulted in making available, for analysis, multiple sequences of different bacterial genomes, including such pathogens as causative agents of syphilis, typhus, and tuberculosis as well as such organisms like archaebacterias living under extreme conditions . A comparative analysis of bacterial genomes leads to conclusions, which have a general biological value, and, in particular, to the conclusions about mechanisms and evolution rate as well as about the variability of genomes and interrelation between organisms and their habitat . On the other hand, the analysis reveals specific features of separate bacterial species responsible for their pathogenicity and ability to avoid the destruction of the host immune system as well as for adaptation to exist within a certain ecological niche . However, the variability of bacterial genomes is so high that methods, which enable to evaluate the variability without full genome sequencing, are needed to depict adequately the evolution and ecological characteristics of the prokaryotic world and to develop new effective therapeutics and diagnostic tools . The survey covers two approaches to such comparative analysis, i.e . DNA arrays and subtractive hybridization . The advantages and disadvantages of each approach are discussed and the necessity in a new approach combining the positive features of the two mentioned approaches is substantiated.

FEBS Lett, 2003 Feb 27, 537(1-3), 101 - 5
Inhibition of the plant cytokinin transduction pathway by bacterial histidine kinase inhibitors in Catharanthus roseus cell cultures; Papon N et al.; We describe the isolation of two Catharanthus roseus cDNAs encoding proteins putatively involved in the final steps of a 'histidine-to-aspartate' phosphorelay in cytokinin (CK) signaling . The expression of one of these genes, CrRR1, was specifically up-regulated by CKs in C . roseus cell suspensions . We used this system as a biological model to test the activity of bacterial histidine kinase inhibitors . Our data demonstrate that these inhibitors are active on the CK transduction pathway and represent powerful chemical tools to study hormone signal transduction in plants . Moreover, these data suggest a strong conservation of functional features between prokaryotic and plant signaling pathways utilizing histidine kinases.

Rev Physiol Biochem Pharmacol, 2003, 146, 55 - 94 Epub 2002 Dec 17.
Signal recognition particle-dependent protein targeting, universal to all kingdoms of life; Koch HG et al.; The signal recognition particle (SRP) and its membrane-bound receptor represent a ubiquitous protein-targeting device utilized by organisms as different as bacteria and humans, archaea and plants . The unifying concept of SRP-dependent protein targeting is that SRP binds to signal sequences of newly synthesized proteins as they emerge from the ribosome . In eukaryotes this interaction arrests or retards translation elongation until SRP targets the ribosome-nascent chain complexes via the SRP receptor to the translocation channel . Such channels are present in the endoplasmic reticulum of eukaryotic cells, the thylakoids of chloroplasts, or the plasma membrane of prokaryotes . The minimal functional unit of SRP consists of a signal sequence-recognizing protein and a small RNA . The as yet most complex version is the mammalian SRP whose RNA, together with six proteinaceous subunits, undergo an intricate assembly process . The preferential substrates of SRP possess especially hydrophobic signal sequences . Interactions between SRP and its receptor, the ribosome, the signal sequence, and the target membrane are regulated by GTP hydrolysis . SRP-dependent protein targeting in bacteria and chloroplasts slightly deviate from the canonical mechanism found in eukaryotes . Pro- and eukaryotic cells harbour regulatory mechanisms to prevent a malfunction of the SRP pathway.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2495 - 500 Epub 2003 Feb 25.
Origin and evolution of circadian clock genes in prokaryotes; Dvornyk V et al.; Regulation of physiological functions with approximate daily periodicity, or circadian rhythms, is a characteristic feature of eukaryotes . Until recently, cyanobacteria were the only prokaryotes reported to possess circadian rhythmicity . It is controlled by a cluster of three genes: kaiA, kaiB, and kaiC . Using sequence data of approximately 70 complete prokaryotic genomes from the various public depositories, we show here that the kai genes and their homologs have quite a different evolutionary history and occur in Archaea and Proteobacteria as well . Among the three genes, kaiC is evolutionarily the oldest, and kaiA is the youngest and likely evolved only in cyanobacteria . Our data suggest that the prokaryotic circadian pacemakers have evolved in parallel with the geological history of the earth, and that natural selection, multiple lateral transfers, and gene duplications and losses have been the major factors shaping their evolution.

Chem Biol Interact, 2003 Feb 1, 143-144, 621 - 31
The aldo-keto reductase superfamily homepage; Hyndman D et al.; The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates . A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit Investigators have been diligent in submitting sequences of functional proteins to the Web site . With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14) . The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest . Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases . Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase) . Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site . Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity . Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers . To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed . For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated . The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

Mol Microbiol, 2003 Mar, 47(5), 1475 - 83
Cell shape, division and development: the 2002 American Society for Microbiology (ASM) conference on prokaryotic development; Figge RM et al.; In the last decade, the use of cytological techniques, together with the analysis of complete genomes, has dramatically advanced our understanding of bacterial development . Work on several well-developed model systems such as Bacillus subtilis, Caulobacter crescentus, Myxococcus xanthus and Streptomyces spp., has provided us with an in-depth understanding of processes such as sporulation, multicellular behaviour and the bacterial cell cycle . At the same time, these studies have revolutionized our view of the bacterial cell and shown it to be a highly complex entity with spatial and temporal organization . The recent American Society for Microbiology (ASM) conference on prokaryotic development demonstrated that several laboratories have now started to connect data obtained through functional genomic analysis with subcellular organization, thereby generating three-dimensional regulatory networks . This meeting report highlights new findings in the field, such as regulation of protein localization during sporulation and the cell cycle, control of cell-cell interaction and the initiation of cell division.

Eur J Biochem, 2003 Mar, 270(5), 799 - 813
The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily; Hvorup RN et al.; The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily (TC #2.A.66) consists of four previously recognized families: (a) the ubiquitous multi-drug and toxin extrusion (MATE) family; (b) the prokaryotic polysaccharide transporter (PST) family; (c) the eukaryotic oligosaccharidyl-lipid flippase (OLF) family and (d) the bacterial mouse virulence factor family (MVF) . Of these four families, only members of the MATE family have been shown to function mechanistically as secondary carriers, and no member of the MVF family has been shown to function as a transporter . Establishment of a common origin for the MATE, PST, OLF and MVF families suggests a common mechanism of action as secondary carriers catalyzing substrate/cation antiport . Most protein members of these four families exhibit 12 putative transmembrane alpha-helical segments (TMSs), and several have been shown to have arisen by an internal gene duplication event; topological variation is observed for some members of the superfamily . The PST family is more closely related to the MATE, OLF and MVF families than any of these latter three families are related to each other . This fact leads to the suggestion that primordial proteins most closely related to the PST family were the evolutionary precursors of all members of the MOP superfamily . Here, phylogenetic trees and average hydropathy, similarity and amphipathicity plots for members of the four families are derived and provide detailed evolutionary and structural information about these proteins . We show that each family exhibits unique characteristics . For example, the MATE and PST families are characterized by numerous paralogues within a single organism (58 paralogues of the MATE family are present in Arabidopsis thaliana), while the OLF family consists exclusively of orthologues, and the MVF family consists primarily of orthologues . Only in the PST family has extensive lateral transfer of the encoding genes occurred, and in this family as well as the MVF family, topological variation is a characteristic feature . The results serve to define a large superfamily of transporters that we predict function to export substrates using a monovalent cation antiport mechanism.

Pac Symp Biocomput . 2003;:29-40.
Genome-wide analysis of bacterial promoter regions; Eskin E et al.; Identifying prokaryotic promoter sequences is notoriously difficult and for most sequenced bacterial genomes the promoter sequences are still unknown . Since experimental analysis trails behind sequencing, genome-wide computational promoter discovery is often the only realistic way to discover these sequences in newly sequenced bacterial genomes . However, genome-wide samples for promoter discovery may be very large and corrupted complicating promoter discovery . We discuss three aspects of genome-wide promoter discovery: sample generation, signal finding algorithms, and scoring signals . We applied our new MITRA algorithm to analyze samples of divergent and convergent genes in 20 bacterial genomes and found strong putative dyad signals in 17 out of the 20 genomes . Moreover, in 12 out of 20 genomes the found signals are identical or similar to the known regulatory patterns (Pribnow-Gilbert boxes and CRP binding sites) . Since many of putative signals correspond to previously known elements of bacterial transcriptional regulation, the remaining discovered signals are good candidates for unknown regulatory elements.

Plant Mol Biol, 2003 Feb, 51(3), 385 - 99
Characterization of Arabidopsis plastid sigma-like transcription factors SIG1, SIG2 and SIG3; Privat I et al.; The plastid genome is transcribed by nucleus-encoded (NEP) and plastid-encoded (PEP) RNA polymerases . PEP is a prokaryotic-type enzyme whose activity is regulated by sigma-like transcription initiation factors that are nucleus-encoded . cDNAs coding for six different potential a-like factors have been cloned and sequenced recently . However, functional analyses of these factors are still limited . We have used an anti-sense approach in order to study the function of SIG1, SIG2 and SIG3 . Only SIG2 anti-sense plants show a visible phenotype characterized by chlorophyll deficiency . Surprisingly, this phenotype is different from the phenotype of SIG2 knockout plants in that the chlorophyll deficiency is limited to cotyledons . In later developmental stages, the SIG2 anti-sense plants can overcome SIG2 mRNA under-expression by adjusting SIG2 protein levels to that of wild-type plants, suggesting that SIG2 expression is also regulated at the post-transcriptional level . The efficient recovery of the wild-type phenotype could also be supported by partial take-over of SIG2 function by one of the six other sigma factors . A good candidate for such substitution of SIG2 function represents SIG3 . SIG3 is constitutively expressed during plant development and its specificity in promoter discrimination is less pronounced than that of SIG1 and SIG2 . Finally, SIG3 protein is enhanced in SIG2 anti-sense plants when compared to wild-type plants . SIG2 is present as a soluble factor while SIG3 is partly attached to the plastid membranes . We suggest that membrane localization is necessary for efficient SIG3 function . Therefore, SIG3 cannot substitute for SIG2 function in early chloroplast biogenesis, when plastid membranes are not yet made up.

J Biol Chem, 2003 May 2, 278(18), 15771 - 7 Epub 2003 Feb 21.
RNA polyadenylation and degradation in cyanobacteria are similar to the chloroplast but different from Escherichia coli; Rott R et al.; The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II . The poly(A) tails are homogenous, containing only adenosines in most of the growth conditions . In the chloroplast, however, the same enzyme, PNPase, polyadenylates and degrades the RNA molecule; there is no equivalent for the E . coli poly(A) polymerase enzyme . Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E . coli or the chloroplast . We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E . coli . No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich tails . These heterogeneous tails were found in the amino acid coding region, the 5' and 3' untranslated regions of mRNAs, as well as in rRNA and the single intron located at the tRNA(fmet) . Furthermore, unlike E . coli, the inactivation of PNPase or RNase II genes caused lethality . Together, our results show that the RNA polyadenylation and degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E . coli.

Ai Zheng, 2003 Feb, 22(2), 136 - 9
{Expression of a new cloned nitroreductase gene NOR1 and purification of expressed product}; Nie XM et al.; BACKGROUND & OBJECTIVE: NOR(1)is a good candidate of tumor suppressor/susceptibility gene associated with nasopharyngeal carcinoma . This study was designed to construct the prokaryotic expression vector and to investigate the expression of nitroreductase gene NOR(1)in Escherichia coli and to purify expressed product . METHODS: Total RNA was subtracted from normal nasopharyngeal carcinoma tissue . The full length of NOR(1)gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHIand XhoI restriction endonucleases . The plasmid pGEX-4T-2 was also digested with BamHI and XhoI,then the NOR(1)gene was inserted into vector pGEX-4T-2 . The recombinant expression vector pGEX-4T-2/NOR(1)was identified by sequencing and digested with restriction enzymes . E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express GST fusion protein . The result was confirmed by Western blot analysis and the purified targeted protein was obtained by affinity chromatography . RESULTS: The 1.25kb NOR(1)gene was successfully isolated . After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) . It existed not only in supernatant but also in precipitation of broken bacteria . The result was confirmed by Western blot analysis,and the purified targeted protein was obtained by affinity chromatography . CONCLUSION: The successes in construction of expression vector of NOR(1), expression and purification of GST/NOR(1)fusion protein make it possible to prepare for the polyantibodies for NOR(1).

Biochemistry, 2003 Mar 4, 42(8), 2422 - 30
Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity; Kravchuk AV et al.; Eukaryotic phosphatidylinositol-specific phospholipase Cs (PI-PLCs) utilize calcium as a cofactor during catalysis, whereas prokaryotic PI-PLCs use a spatially conserved guanidinium group from Arg69 . In this study, we aimed to construct a metal-dependent mutant of a bacterial PI-PLC and characterize the catalytic role of the metal ion, seeking an enhanced understanding of the functional differences between these two positively charged moieties . The following results indicate that a bona fide catalytic metal binding site was created by the single arginine-to-aspartate mutation at position 69: (1) The R69D mutant was activated by Ca(2+), and the activation was specific for R69D, not for other mutants at this position . (2) Titration of R69D with Ca(2+), monitored by (15)N-(1)H HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca(2+) to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme . (3) Upon Ca(2+) activation, the thio effect of the S(P)-isomer of the phosphorothioate analogue (k(O)/k(Sp) = 4.4 x 10(5)) approached a value similar to that of the WT enzyme, suggesting a structural and functional similarity between the two positively charged moieties (Arg69 and Asp69-Ca(2+)) . The R(P)-thio effect (k(O)/k(Rp) = 9.4) is smaller than that of the WT enzyme by a factor of 5 . Consequently, R69D-Ca(2+) displays higher stereoselectivity (k(Rp)/k(Sp) = 47,000) than WT (k(Rp)/k(Sp) = 7600) . (4) Results from additional mutagenesis analyses suggest that the Ca(2+) binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117 . Our studies provide new insight into the mechanism of metal-dependent and metal-independent PI-PLCs.

Biochemistry, 2003 Mar 4, 42(8), 2275 - 81
A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants; Knowlton JR et al.; Transcription factor NusG is present in all prokaryotes, and orthologous proteins have also been identified in yeast and humans . NusG contains a 27-residue KOW motif, found in ribosomal protein L24 where it interacts with rRNA . NusG in Escherichia coli (EcNusG) is an essential protein and functions as a regulator of Rho-dependent transcription termination, phage lambda N and rRNA transcription antitermination, and phage HK022 Nun termination . Relative to EcNusG, Aquifex aeolicus NusG (AaNusG) and several other bacterial NusG proteins contain a variable insertion sequence of approximately 70 residues in the central region of the molecule . Recently, crystal structures of AaNusG in space groups P2(1) and I222 have been reported; the authors conclude that there are no conserved dimers among the contacting molecules in the crystals {Steiner, T., Kaiser, J . T., Marinkovic, S., Huber, R., and Wahl, M . C . (2002) EMBO J . 21, 4641-4653} . We have independently determined the structures of AaNusG also in two crystal forms, P2(1) and C222(1), and surprisingly found that AaNusG molecules form domain-swapped dimers in both crystals . Additionally, polymerization is also observed in the P2(1) crystal . A unique "ball-and-socket" junction dominates the intermolecular interactions within both oligomers . We believe that this interaction is a clue to the function of the molecule and propose a spring-loaded state in the functional cycle of NusG . The importance of the ball-and-socket junction for the function of NusG is supported by the functional analysis of site-directed mutants.

Sheng Li Xue Bao, 2003 Feb 25, 55(1), 110 - 3
{Preparation and indentification of polyclonal antiserum against angiotensinogen}; Meng QJ et al.; For studying the expression and distribution of angiotensinogen (AGT), the C-teminus of rat AGT gene was expressed in E.coli . Rabbits were immunized with expressed AGT protein and sera from different rabbits were raised . ELISA showed a high titre (1:25600) of the antiserum . With the antiserum, Western blotting recognized not only the prokaryotic expressed AGT, but also the endogenous AGT protein in liver tissue of both rats and humans . Using this antiserum, immunohistochemistry showed the expression of AGT protein in islet cells of human pancreas as well as in epithelium of human bile duct . These results suggest that the prokaryotic expressed AGT protein is an effective immunogen for the preparation of anti-AGT antiserum . Our present work provides an important tool for study of the pathophysiological role of AGT as well as local renin-angiotensin system.

Trends Microbiol, 2003 Feb, 11(2), 61 - 6
Genome-specific higher-order background models to improve motif detection; Marchal K et al.; Motif detection based on Gibbs sampling is a common procedure used to retrieve regulatory motifs in silico . Using a species-specific background model was previously shown to increase the robustness of the algorithm . Here, we demonstrate that selecting a non-species-adapted background model can have an adverse effect on the results of motif detection . The large differences in the average nucleotide composition of prokaryotic sequences exacerbate the problem of exchanging background models . Therefore, we have developed complex background models for all prokaryotic species with available genome sequences.

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