Dev Cell, 2001 Dec, 1(6), 733 - 42 Morphological coupling in development: lessons from prokaryotes; Rudner DZ et al.; At certain junctures in development, gene transcription is coupled to the completion of landmark morphological events . We refer to this dependence on morphogenesis for gene expression as "morphological coupling." Three examples of morphological coupling in prokaryotes are reviewed in which the activation of a transcription factor is tied to the assembly of a critically important structure in development. Gene, 2001 Dec 12, 280(1-2), 97 - 105 New Mos1 mariner transposons suitable for the recovery of gene fusions in vivo and in vitro; Goyard S et al.; The Drosophila Mos1 element can be mobilized in species ranging from prokaryotes to protozoans and vertebrates, and the purified transposase can be used for in vitro transposition assays . In this report we developed a 'mini-Mos1' element and describe a number of useful derivatives suitable for transposon mutagenesis in vivo or in vitro . Several of these allow the creation and/or selection of tripartite protein fusions to a green fluorescent protein-phleomycin resistance (GFP-PHLEO) reporter/selectable marker . Such X-GFP-PHLEO-X fusions have the advantage of retaining 5' and 3' regulatory information and N- and C-terminal protein targeting domains . A Mos1 derivative suitable for use in transposon-insertion mediated linker insertion (TIMLI) mutagenesis is described, and transposons bearing selectable markers suitable for use in the protozoan parasite Leishmania were made and tested . A novel 'negative selection' approach was developed which permits in vitro assays of transposons lacking bacterial selectable markers . Application of this assay to several Mos1 elements developed for use in insects suggests that the large mariner pM{cn} element used previously in vivo is poorly active in vitro, while the Mos1-Act-EGFP transposon is highly active. BMC Evol Biol . 2001 Oct 20;1(1):8. Genome trees constructed using five different approaches suggest new major bacterial clades; Wolf YI et al.; BACKGROUND: The availability of multiple complete genome sequences from diverse taxa prompts the development of new phylogenetic approaches, which attempt to incorporate information derived from comparative analysis of complete gene sets or large subsets thereof . Such attempts are particularly relevant because of the major role of horizontal gene transfer and lineage-specific gene loss, at least in the evolution of prokaryotes . RESULTS: Five largely independent approaches were employed to construct trees for completely sequenced bacterial and archaeal genomes: i) presence-absence of genomes in clusters of orthologous genes; ii) conservation of local gene order (gene pairs) among prokaryotic genomes; iii) parameters of identity distribution for probable orthologs; iv) analysis of concatenated alignments of ribosomal proteins; v) comparison of trees constructed for multiple protein families . All constructed trees support the separation of the two primary prokaryotic domains, bacteria and archaea, as well as some terminal bifurcations within the bacterial and archaeal domains . Beyond these obvious groupings, the trees made with different methods appeared to differ substantially in terms of the relative contributions of phylogenetic relationships and similarities in gene repertoires caused by similar life styles and horizontal gene transfer to the tree topology . The trees based on presence-absence of genomes in orthologous clusters and the trees based on conserved gene pairs appear to be strongly affected by gene loss and horizontal gene transfer . The trees based on identity distributions for orthologs and particularly the tree made of concatenated ribosomal protein sequences seemed to carry a stronger phylogenetic signal . The latter tree supported three potential high-level bacterial clades,: i) Chlamydia-Spirochetes, ii) Thermotogales-Aquificales (bacterial hyperthermophiles), and ii) Actinomycetes-Deinococcales-Cyanobacteria . The latter group also appeared to join the low-GC Gram-positive bacteria at a deeper tree node . These new groupings of bacteria were supported by the analysis of alternative topologies in the concatenated ribosomal protein tree using the Kishino-Hasegawa test and by a census of the topologies of 132 individual groups of orthologous proteins . Additionally, the results of this analysis put into question the sister-group relationship between the two major archaeal groups, Euryarchaeota and Crenarchaeota, and suggest instead that Euryarchaeota might be a paraphyletic group with respect to Crenarchaeota . CONCLUSIONS: We conclude that, the extensive horizontal gene flow and lineage-specific gene loss notwithstanding, extension of phylogenetic analysis to the genome scale has the potential of uncovering deep evolutionary relationships between prokaryotic lineages. Eur J Biochem, 2001 Dec, 268(23), 6256 - 62 Use of site-specific recombination as a probe of nucleoprotein complex formation in chromatin; Schwikardi M et al.; DNA transactions in eukaryotes require that proteins gain access to target sequences packaged in chromatin . Further, interactions between distinct nucleoprotein complexes are often required to generate higher-order structures . Here, we employed two prokaryotic site-specific recombination systems to investigate how chromatin packaging affects the assembly of nucleoprotein structures of different complexities at more than 30 genomic loci . The dynamic nature of chromatin permitted protein-DNA and DNA-DNA interactions for sites of at least 34 bp in length . However, the assembly of higher-order nucleoprotein structures on targets spanning 114 bp was impaired . This impediment was maintained over at least 72 h and was not affected by the transcriptional status of chromatin nor by inhibitors of histone deacetylases and topoisomerases . Our findings suggest that nucleosomal linker-sized DNA segments become accessible within hours for protein binding due to the dynamic nature of chromatin . Longer segments, however, appear refractory for complete occupancy by sequence-specific DNA-binding proteins . The results thus also provide an explanation why simple recombination systems such as Cre and Flp are proficient in eukaryotic chromatin. Biochemistry, 2001 Dec 11, 40(49), 14976 - 84 Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain; Sun W et al.; The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways . A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition . RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding . However, it is not known whether the catalytic domain requires the dsRBD for activity . This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III{DeltadsRBD}) can accurately cleave small processing substrates in vitro . Optimal activity of RNase III{DeltadsRBD} is observed at low salt concentrations (<60 mM Na(+)), either in the presence of Mg(2+) (>25 mM) or Mn(2+) ( approximately 5 mM) . At 60 mM Na(+) and 5 mM Mn(2+) the catalytic efficiency of RNase III{DeltadsRBD} is similar to that of RNase III at physiological salt concentrations and Mg(2+) . In the presence of Mg(2+) RNase III{DeltadsRBD} is less efficient than the wild-type enzyme, due to a higher K(m) . Similar to RNase III, RNase III{DeltadsRBD} is inhibited by high concentrations of Mn(2+), which is due to metal ion occupancy of an inhibitory site on the enzyme . RNase III{DeltadsRBD} retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25) . Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved . Low (micromolar) concentrations of ethidium bromide block RNase III{DeltadsRBD} cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition . Finally, RNase III{DeltadsRBD} is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III . These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection. Acta Biochim Pol, 2001, 48(2), 495 - 510 Mg2+ ions do not induce expansion of the melted DNA region in the open complex formed by Escherichia coli RNA polymerase at a cognate synthetic Pa promoter . A quantitative KMnO4 footprinting study; Loziinski T et al.; Footprinting studies of prokaryotic open transcription complexes (RPO), based on oxidation of pyrimidine residues by KMnO4 and/or OsO4 at a single oxidant dose, have suggested that the extent of DNA melting in the transcription bubble region increases in the presence of Mg . In this work, quantitative KMnO4 footprinting in function of the oxidant dose of RPO, using Escherichia coli RNA polymerase (E(sigma)70) at a fully functional synthetic promoter Pa having -35 and -10 consensus hexamers, has been used to determine individual rate constants of oxidation of T residues in this region at 37degrees C in the absence of Mg2+ and in the presence of 10 mM MgCl2, and to evaluate therefrom the effect of Mg2+ on the extent of DNA melting . Population distributions of end-labeled DNA fragments corresponding to oxidized Ts were quantified and analyzed according to the single-hit kinetic model . Pseudo-first order reactivity rate constants, ki, thus obtained demonstrated that Mg2+ ions bound to RPO merely enhanced the reactivity of all 11 oxidizable thymines between the +3 and -11 promoter sites by a position-dependent factor: 3-4 for those located close to the transcription start point +1 in either DNA strand, and about 1.6 for those located more distantly therefrom . On the basis of these observations, we conclude that Mg2+ ions bound to RPO at Pa do not influence the length of the melted DNA region and propose that the higher reactivity of thymines results mainly from lower local repulsive electrostatic barriers to MnO4 diffusion around carboxylate binding sites in the catalytic center of RPO and promoter DNA phosphates. Acta Biochim Pol, 2001, 48(2), 367 - 81 Reduction of bacterial genome size and expansion resulting from obligate intracellular lifestyle and adaptation to soil habitat; Stepkowski T et al.; Prokaryotic organisms are exposed in the course of evolution to various impacts, resulting often in drastic changes of their genome size . Depending on circumstances, the same lineage may diverge into species having substantially reduced genomes, or such whose genomes have undergone considerable enlargement . Genome reduction is a consequence of obligate intracellular lifestyle rendering numerous genes expendable . Another consequence of intracellular lifestyle is reduction of effective population size and limited possibility of gene acquirement via lateral transfer . This causes a state of relaxed selection resulting in accumulation of mildly deleterious mutations that can not be corrected by recombination with the wild type copy . Thus, gene loss is usually irreversible . Additionally, constant environment of the eukaryotic cell renders that some bacterial genes involved in DNA repair are expandable . The loss of these genes is a probable cause of mutational bias resulting in a high A+T content . While causes of genome reduction are rather indisputable, those resulting in genome expansion seem to be less obvious . Presumably, the genome enlargement is an indirect consequence of adaptation to changing environmental conditions and requires the acquisition and integration of numerous genes . It seems that the need for a great number of capabilities is common among soil bacteria irrespective of their phylogenetic relationship . However, this would not be possible if soil bacteria lacked indigenous abilities to exchange and accumulate genetic information . The latter are considerably facilitated when housekeeping genes are physically separated from adaptive loci which are useful only in certain circumstances. Protoplasma, 2001, 218(1-2), 95 - 103 Production of a recombinant human basic fibroblast growth factor with a collagen binding domain; Andrades JA et al.; Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair . Basic FGF is found in abundance in tissues such as brain, kidney, and cartilage . This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli . A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of a purification tag, a protease-sensitive linker and collagen binding domain, and a cDNA sequence encoding the active fragment of hbFGF . The expressed hbFGF-F1 and hbFGF-F2 (it contains the collagen binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured by a glutathione redox system and protracted dialysis under various experimental conditions . The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a nickel-nitrilotriacetic acid metal chelate column . The biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of human vein endothelial cells, monitored by {3H}thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control . Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF . The high-affinity binding was demonstrated by the binding of {3H}collagen to the rhbFGF-F2 protein immobilized on a Ni-nitrilotriacetic acid column . The rhbFGF-F2 fusion protein bound to collagen-coated surfaces with high affinity . Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for their high-yield production, purification, and renaturation from microorganisms . Furthermore, we demonstrate that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type I collagen . These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins for specific biomedical applications. Genome Res, 2001 Dec, 11(12), 2101 - 14 Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila; Boudet N et al.; The DEAD box RNA helicase (RH) proteins are homologs involved in diverse cellular functions in all of the organisms from prokaryotes to eukaryotes . Nevertheless, there is a lack of conservation in the splicing pattern in the 53 Arabidopsis thaliana (AtRHs), the 32 Caenorhabditis elegans (CeRHs) and the 29 Drosophila melanogaster (DmRHs) genes . Of the 153 different observed intron positions, 4 are conserved between AtRHs, CeRHs, and DmRHs, and one position is also found in RHs from yeast and human . Of the 27 different AtRH structures with introns, 20 have at least one predicted ancient intron in the regions coding for the catalytic domain . In all of the organisms examined, we found at least one gene with most of its intron predicted to be ancient . In A . thaliana, the large diversity in RH structures suggests that duplications of the ancestral RH were followed by a high number of intron deletions and additions . The very high bias toward phase 0 introns is in favor of intron addition, preferentially in phase 0 . Results from this comparative study of the same gene family in a plant and in two animals are discussed in terms of the general mechanisms of gene family evolution. Curr Opin Microbiol, 2001 Dec, 4(6), 639 - 46 Organelle fission in eukaryotes; Osteryoung KW; The cellular machineries that power chloroplast and mitochondrial division in eukaryotes carry out the topologically challenging job of constricting and severing these double-membraned organelles . Consistent with their endosymbiotic origins, mitochondria in protists and chloroplasts in photosynthetic eukaryotes have evolved organelle-targeted forms of FtsZ, the prokaryotic ancestor of tubulin, as key components of their fission complexes . In fungi, animals and plants, mitochondria no longer utilize FtsZ for division, but several mitochondrial division proteins that localize to the outer membrane and intermembrane space, including two related to the filament-forming dynamins, have been identified in yeast and animals . Although the reactions that mediate organelle division are not yet understood, recent progress in uncovering the constituents of the organelle division machineries promises rapid advancement in our understanding of the biochemical mechanisms underlying the distinct but related processes of chloroplast and mitochondrial division in eukaryotes. Curr Opin Microbiol, 2001 Dec, 4(6), 634 - 8 Bacterial ancestry of actin and tubulin; van den Ent F et al.; The structural and functional resemblance between the bacterial cell-division protein FtsZ and eukaryotic tubulin was the first indication that the eukaryotic cytoskeleton may have a prokaryotic origin . The bacterial ancestry is made even more obvious by the findings that the bacterial cell-shape-determining proteins Mreb and Mbl form large spirals inside non-spherical cells, and that MreB polymerises in vitro into protofilaments very similar to actin . Recent advances in research on two proteins involved in prokaryotic cytokinesis and cell shape determination that have similar properties to the key components of the eukaryotic cytoskeleton are discussed. FEMS Microbiol Lett, 2001 Nov 13, 204(2), 223 - 31 Phylogenetic and structural analyses of the oxa1 family of protein translocases; Yen MR et al.; Mitochondrial Oxa1p homologs have been shown to function in protein export and membrane insertion in bacteria, mitochondria and chloroplasts, but their mode of action, organismal distribution and evolutionary origins are poorly understood . All sequenced homologs of Oxa1p were retrieved from the databases and multiply aligned . All organisms with a fully sequenced genome possess at least one Oxa1p homolog showing that the family is truly ubiquitous . Most prokaryotes possess just one Oxa1p homolog, but several Gram-positive bacteria and one archaeon possess two, and eukaryotes may have as many as six . Although these proteins vary in length over a 5-fold range, they exhibit a common hydrophobic core region of about 200 residues . Multiple sequence alignments reveal conserved residues and provide the basis for structural and phylogenetic analyses that serve to characterize the Oxa1 family. J Biol Chem, 2002 Feb 8, 277(6), 3857 - 62 Epub 2001 Nov 29. Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts; Uchiumi T et al.; Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes . We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities . The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11 . The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain . The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G . The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity . The presence of either E . coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation. Free Radic Biol Med, 2001 Dec 1, 31(11), 1405 - 16 NF kappa B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells; Zhou LZ et al.; The ability to induce cellular defense mechanisms in response to environmental challenges is a fundamental property of eukaryotic and prokaryotic cells . We have previously shown that oxidative challenges lead to an increase in antioxidant enzymes, particularly glutathione peroxidase (GPx) and catalase (CAT), in mouse skeletal muscle . The focus of the current studies is the transcriptional regulatory mechanisms responsible for these increases . Sequence analysis of the mouse GPx and CAT genes revealed putative binding motifs for NF kappa B and AP-1, transcriptional regulators that are activated in response to oxidative stress in various tissues . To test whether NF kappa B or AP-1 might be mediating the induction of GPx and CAT in muscle cells subjected to oxidative stress, we first characterized their activation by pro-oxidants . Electrophoretic mobility shift assays showed that oxidative stress led to increases in the DNA binding of NF kappa B in differentiated muscle cells . The NF kappa B complexes included a p50/p65 heterodimer, a p50 homodimer, and a p50/RelB heterodimer . AP-1 was also activated, but with slower kinetics than that of NF kappa B . The major component of the AP-1 complexes was a heterodimer composed of c-jun/fos . To test for redox regulation of NF kappa B- or AP-1-dependent transcriptional activation, muscle cells expressing either kappa B/luciferase or TRE/luciferase reporter constructs were subjected to oxidative stress . Pro-oxidant treatment resulted in increased luciferase activity in cells expressing either construct . To test whether NF kappa B mediates oxidant-induced increases of GPx and CAT expression, we transfected cells with either a transdominant inhibitor (I kappa B alpha) or a dominant-negative inhibitor (Delta SP) of NF kappa B . Both inhibitors blocked the induction of antioxidant gene expression by more than 50% . In summary, our results suggest that NF kappa B and AP-1 are important mediators of redox-responsive gene expression in skeletal muscle, and that at least NF kappa B is actively involved in the upregulation of the GPx and CAT in response to oxidative stress. Curr Genet, 2001 Oct, 40(3), 157 - 71 Yeast--a panacea for the structure-function analysis of membrane proteins? Bill RM. In recent years, the scientific community has begun to realise that the structure-function analysis of membrane proteins has lagged considerably behind that of their soluble counterparts . A boom in the field of membrane protein biology has resulted in the tailoring of techniques for the cloning, expression, purification and characterisation of these somewhat intractable proteins and most notably in the optimisation of several alternative host systems for this purpose . This Review Article summarises the use of yeast as a host . Compared with other hosts, it is clear that yeast combines the advantages of eukaryotes with the ease of handling of prokaryotes . Moreover, this organism provides membrane protein biologists with a panacea for structure-function analyses, not least because the tools of yeast genetics are at their disposal. EMBO J, 2001 Dec 3, 20(23), 6583 - 90 V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis; Moser J et al.; Processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls . tRNA-dependent catalysis generally is associated with protein biosynthesis . An exception is the reduction of glutamyl-tRNA to glutamate-1-semialdehyde by the enzyme glutamyl-tRNA reductase . This reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes . The crystal structure of glutamyl-tRNA reductase from the archaeon Methanopyrus kandleri in complex with the substrate-like inhibitor glutamycin at 1.9 A resolution reveals an extended yet planar V-shaped dimer . The well defined interactions of the inhibitor with the active site support a thioester-mediated reduction process . Modeling the glutamyl-tRNA onto each monomer reveals an extensive protein-tRNA interface . We furthermore propose a model whereby the large void of glutamyl-tRNA reductase is occupied by glutamate-1-semialdehyde-1,2-mutase, the subsequent enzyme of this pathway, allowing for the efficient synthesis of 5-aminolevulinic acid, the common precursor of all tetrapyrroles. J Biomed Sci, 1994 Mar, 1(2), 119 - 124 Serological Responses of Patients with Nasopharyngeal Carcinoma to an N-Terminal Epstein-Barr Virus DNA Polymerase Protein Expressed in Prokaryotic Cells; Liu MY et al.; A 1.7-kb cDNA clone, pGEM-cDP, was isolated from a cDNA library of IUdR-induced p3HR1 cells . It contains the upstream nucleotide sequence of the Epstein-Barr virus (EBV) DNA polymerase gene from 156,859 to 155,088, and was subcloned into expression vector pET3cp* by the polymerase chain reaction, giving the plasmid pDP1 . Using a T7 RNA polymerase expression system, a 77-kD polypeptide was produced from pDP1 in Escherichia coli and specific hyperimmune serum was generated in mice . The truncated EBV DNA polymerase was shown to possess the authentic antigenicity by an indirect immunofluorescence assay and by immunoblotting using EBV-containing cells as antigens . Serum from nasopharyngeal carcinoma (NPC) patients and healthy donors was examined for antibodies against the 77-kD polypeptide by Western blot analyses and ELISAs . About 70% NPC patients were positive, while less than 15% of healthy persons showed weak reactivities in ELISAs . Eur J Biochem, 2001 Nov, 268(22), 5667 - 75 Fusogenic potential of prokaryotic membrane lipids . Implication in vaccine development; Ahmad N et al.; Development of protective immunity against many pathogens, particularly viruses, requires fine orchestration of both humoral- and cell mediated-immunity . The immunization of animals with soluble antigens usually leads to the induction of humoral immune responses . In contrast, the activation of a cell-mediated immune response against exogenous antigens has always been a challenge, requiring special strategies to expose them to the proteasome, a multifunctional protease complex in the cytosol of the target cells . The degradation of the protein by the cytosolic proteolytic system forms a cardinal step for the induction of cytotoxic T lymphocytes (CTLs) . In the present study, we report that a potent primary CTL response against a soluble protein, ovalbumin, can be induced in mice by encapsulating it in the liposomes comprised of Escherichia coli membrane lipids . These lipids were shown to induce strong membrane-membrane fusion as evident from resonance energy transfer and content mixing assays . Furthermore, the fusion of these liposomes with living cells (J774 A1) was demonstrated to result in effective transfer of a fluorescent lipid probe to the plasma membrane of the cells . Moreover, ricin A, a protein synthesis inhibitor that does not cross plasma membrane, was demonstrated to gain access to the cytosol when it was encapsulated in these liposomes . Finally, the liposomes were demonstrated to behave like efficient vehicles for the in vivo delivery of the antigens to the target cells resulting in the elicitation of antigen reactive CD8+ T cell responses. Protein Expr Purif, 2001 Dec, 23(3), 369 - 73 The expression of soluble, full-length, recombinant human TSH receptor in a prokaryotic system; Busuttil BE et al.; For the first time soluble, full-length, recombinant, human thyroid-stimulating hormone (TSH) receptor (TSHR) has been expressed in a prokaryotic system . The full-length TSHR cDNA, obtained from normal human thyroid, was cloned into a pQE-9 vector, sequenced, and confirmed to be identical to the published sequence, to be full length, and to be in frame . Expression of the receptor was as a fusion protein with a hexahistidine tail at the amino terminal, in an Escherichia coli expression system . Approximately 2.5 mg of protein per liter of bacterial culture was recovered from the cell homogenate, after a single passage through a nickel-nitrilotriacetic acid resin column . An estimated 60% increase in purity of a band of expected size, 87 kDa, was observed upon gel electrophoresis and staining with Coomassie blue, after the single purification step . Immunoreactivity of the 87-kDa protein with Graves' sera was confirmed by Western blotting . J Mol Endocrinol, 2001 Dec, 27(3), 321 - 8 Expression of the human androgen receptor in eukaryotic cells using a recombinant adenovirus vector yields high levels of the soluble, functional receptor protein; Zoppi S et al.; The androgen receptor (AR) and closely related members of the steroid receptor family have proven difficult to obtain in native form in large quantities . In the case of the human AR (hAR), high-level expression in prokaryotic or non-mammalian cells leads to the synthesis of a high proportion of non-binding, insoluble, or degraded forms of the receptor protein . To circumvent these difficulties, we have constructed a recombinant adenovirus that directs the expression of hAR under the control of a potent, constitutive promoter . Infection of eukaryotic cells with this recombinant virus leads to the synthesis of large quantities of the intact AR . In contrast to expression methods designed to direct the full-length AR in bacteria, yeast, and insect cells, AR expressed in mammalian cells using this adenoviral vector accumulates at high levels, retains many properties of the native AR, and is not rapidly proteolyzed . This method will prove useful for large-scale preparations of hAR for use in functional and structural studies. Cell, 2001 Nov 16, 107(4), 437 - 49 Control of intrinsic transcription termination by N and NusA: the basic mechanisms; Gusarov I et al.; Intrinsic transcription termination plays a crucial role in regulating gene expression in prokaryotes . After a short pause, the termination signal appears in RNA as a hairpin that destabilizes the elongation complex (EC) . We demonstrate that negative and positive termination factors control the efficiency of termination primarily through a direct modulation of hairpin folding and, to a much lesser extent, by changing pausing at the point of termination . The mechanism controlling hairpin formation at the termination point relies on weak protein interactions with single-stranded RNA, which corresponds to the upstream portion of the hairpin . Escherichia coli NusA protein destabilizes these interactions and thus promotes hairpin folding and termination . Stabilization of these contacts by phage lambda N protein leads to antitermination. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1998 Oct, 20(5), 371 - 6 {Human interleukin-13 cDNA cloning and expression}; Sun Z et al.; OBJECTIVE: To construct an efficient expression system for HIL-13 in prokaryotic cells . METHODS: After Amplified by RT-PCR, The cDNA fragment encoding HIL-13 was inserted into pGEX-2T plasmid and expressed under induced condition . RESULTS: HIL-13 cDNA was obtained . Recombinant plasmid pGEX-2T HIL-13 was constructed and sequenced, the inserted fragment of the recombinant plasmid was confirmed to be HIL-13 cDNA . HIL-13 was expressed as a fusion protein with glutathione-s-transferase (GST-HIL-13, MW = 39,000) . CONCLUSIONS: (1) HIL-13 cDNA has been obtained from T lymphocytes . (2) The rate of expression of HIL-13 in prokaryotic cells is about 37%. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1998 Oct, 20(5), 361 - 6 {Synthesis of hirudin variant 1 (HV1) gene and primary study of expression in yeast}; Zan Y et al.; OBJECTIVE: Hirudin is an extremely efficient and specific thrombin inhibitor . It is clinically used to prevent the formation of thrombus . In this research the hirudin gene was put into yeast system for expression to evaluate the feasibility of artificially synthesized gene expressed in eukaryotic system and study the factor affecting expression level . METHODS: According to the amino acid sequence of hirudin variant 1 (HV1), the genetic code saccharomyces cerevisiae was used to design and synthesize the HV1 gene . Amplified by PCR, it was inserted into cloning vector pBS-SK(+) and sequenced . Ligation with the signal peptide gene of yeast alpha factor the correct HV1 gene was inserted into yeast expression vector pYC-DE . The recombinant plasmid was transformed into the cell of S . cerevisiae BJ1990 to carry out the primary expression experiment . RESULTS: In cultured supernatant of screened positive clone the hirudin activity was detected to be 30 ATU/ml . The expression level was higher than HV2 in yeast and HV1 in prokaryotic system . The N terminal amino acid sequence completely matches with natural hirudin . CONCLUSIONS: It was proved by this study that the synthesized hirudin gene had been expressed in yeast successfully . This result showed that it was a better way to carry out the expression in yeast using synthesized HV1 gene and a stronger promoter. J Bacteriol, 2001 Dec, 183(24), 7190 - 7 Assembly of an FtsZ mutant deficient in GTPase activity has implications for FtsZ assembly and the role of the Z ring in cell division; Mukherjee A et al.; FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells . Both FtsZ and tubulin have a GTPase activity associated with polymerization . Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop . In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site . In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ . FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers . The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap . These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo . Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division. J Bacteriol, 2001 Dec, 183(24), 7076 - 86 Characterization of the cydAB-encoded cytochrome bd oxidase from Mycobacterium smegmatis; Kana BD et al.; The cydAB genes from Mycobacterium smegmatis have been cloned and characterized . The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes . The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter . At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M . smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase . Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm . The d-heme could be restored by transformation of the M . smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis . Inactivation of cydA had no effect on the ability of M . smegmatis to exit from stationary phase at 37 or 42 degrees C . The growth rate of the cydA mutant was tested under oxystatic conditions . Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation) . These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M . smegmatis cultured at 1% air saturation . Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat . In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M . smegmatis. Biochem Biophys Res Commun, 2001 Nov 30, 289(2), 485 - 90 Matrix Gla protein accumulates at the border of regions of calcification and normal tissue in the media of the arterial vessel wall; Spronk HM et al.; Vitamin K-dependent matrix Gla protein (MGP) has been suggested to play a role in the inhibition of soft-tissue calcification . Here we report the expression of recombinant prokaryotic MGP as part of a fusion protein and the preparation of two antibodies that specifically recognize MGP . Monoclonal antibodies were raised against synthetic peptides homologous to the sequences 3-15 and 63-75 of human MGP . Both antibodies recognize recombinant and synthetic human MGP . Immunohistochemical analysis showed that MGP was associated with the extracellular matrix of noncalcified bone and with chondrocytes in cartilage . In the healthy human arterial vessel wall, MGP antigen was demonstrated in association with smooth muscle cells and elastic laminae of the tunica media and with the extracellular matrix of the adventitia . Colocalization with the elastic laminae was lost at sites of medial calcification; in both human and rat arteries, high amounts of MGP were found in the extracellular matrix at borders of intimal and medial calcification . Our data demonstrate the close association between MGP and calcification . It is suggested that undercarboxylated MGP is biologically inactive and that poor vascular vitamin K status may form a risk factor for vascular calcification. Curr Opin Mol Ther, 1999 Apr, 1(2), 244 - 51 Non-viral amplification systems for gene transfer: vectors based on alphaviruses; Smerdou C et al.; Non-viral self-replicating vectors based on defective viral genomes have been developed for a number of different alphaviruses including Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE) . These vectors can be used for gene delivery as naked RNA or DNA . Recombinant alphavirus RNA can be synthesized in vitro from plasmids containing the alphavirus replicon under the control of a prokaryotic promoter such as SP6 or T7 . These self-replicating RNAs have been able to induce protective immune responses in vivo, probably due to the high level of expression of the recombinant antigen in the transfected cells . However, alphavirus vectors based on the direct delivery DNA are probably a better choice due to their higher stability and lower production cost . In these vectors, the alphavirus replicon is placed under the control of a RNA polymerase II promoter . These vectors are more efficient than conventional plasmids in inducing both humoral and cellular immune responses in small animals, allowing the use of significant smaller amounts of DNA for immunization . In addition, due to the transient nature of the alphavirus replicons, possible problems associated with DNA integration into host chromosomes are eliminated. Protein Sci, 2001 Dec, 10(12), 2426 - 38 Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy; Li W et al.; Translation initiation factor 1A (aIF-1A) from the archaeon Methanococcus jannaschii was expressed in Escherichia coli, purified, and characterized in terms of its structure and dynamics using multidimensional NMR methods . The protein was found to be a member of the OB-fold family of RNA-associated proteins, containing a barrel of five beta-strands, a feature that is shared with the homologous eukaryotic translation initiation factor 1A (eIF-1A), as well as the prokaryotic translation initiation factor IF1 . External to the beta barrel, aIF-1A contains an alpha-helix at its C-terminal and a flexible loop at its N-terminal, features that are qualitatively similar to those found in eIF-1A, but not present in prokaryotic IF1 . The structural model of aIF-1A, when used in combination with primary sequence information for aIF-1A in divergent species, permitted the most-conserved residues on the protein surface to be identified, including the most likely candidates for direct interaction with the 16S ribosomal RNA and other components of the translational apparatus . Several of the conserved surface residues appear to be unique to the archaea . Nitrogen-15 relaxation and amide exchange rate data were used to characterize the internal motions within aIF-1A, providing evidence that the protein surfaces that are most likely to participate in intermolecular interactions are relatively flexible . A model is proposed, suggesting some specific interactions that may occur between aIF-1A and the small subunit of the archaeal ribosome. Nucleic Acids Res . 2001 Nov 15;29(22):E112. Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches; Rosenow C et al.; High density oligonucleotide arrays have been used extensively for expression studies of eukaryotic organisms . We have designed a prokaryotic high density oligonucleotide array using the complete Escherichia coli genome sequence to monitor expression levels of all genes and intergenic regions in the genome . Because previously described methods for preparing labeled target nucleic acids are not useful for prokaryotic cell analysis using such arrays, a mRNA enrichment and direct labeling protocol was developed together with a cDNA synthesis protocol . The reproducibility of each labeling method was determined using high density oligonucleotide probe arrays as a read-out methodology and the expression results from direct labeling were compared to the expression results from the cDNA synthesis . About 50% of all annotated E.coli open reading frames are observed to be transcribed, as measured by both protocols, when the cells were grown in rich LB medium . Each labeling method individually showed a high degree of concordance in replica experiments (95 and 99%, respectively), but when each sample preparation method was compared to the other, approximately 32% of the genes observed to be expressed were discordant . However, both labeling methods can detect the same relative gene expression changes when RNA from IPTG-induced cells was labeled and compared to RNA from uninduced E.coli cells. Nucleic Acids Res, 2001 Nov 15, 29(22), 4724 - 35 RNAMotif, an RNA secondary structure definition and search algorithm; Macke TJ et al.; RNA molecules fold into characteristic secondary and tertiary structures that account for their diverse functional activities . Many of these RNA structures are assembled from a collection of RNA structural motifs . These basic building blocks are used repeatedly, and in various combinations, to form different RNA types and define their unique structural and functional properties . Identification of recurring RNA structural motifs will therefore enhance our understanding of RNA structure and help associate elements of RNA structure with functional and regulatory elements . Our goal was to develop a computer program that can describe an RNA structural element of any complexity and then search any nucleotide sequence database, including the complete prokaryotic and eukaryotic genomes, for these structural elements . Here we describe in detail a new computational motif search algorithm, RNAMotif, and demonstrate its utility with some motif search examples . RNAMotif differs from other motif search tools in two important aspects: first, the structure definition language is more flexible and can specify any type of base-base interaction; second, RNAMotif provides a user controlled scoring section that can be used to add capabilities that patterns alone cannot provide. Gene, 2001 Oct 31, 278(1-2), 253 - 64 Prokaryotic structural maintenance of chromosomes (SMC) proteins: distribution, phylogeny, and comparison with MukBs and additional prokaryotic and eukaryotic coiled-coil proteins; Soppa J; Structural maintenance of chromosomes (SMC) proteins are known to be essential for chromosome segregation in some prokaryotes and in eukaryotes . A systematic search for the distribution of SMC proteins in prokaryotes with fully or partially sequenced genomes showed that they form a larger family than previously anticipated and raised the number of known prokaryotic homologs to 54 . Secondary structure predictions revealed that the length of the globular N-terminal and C-terminal domains is extremely well conserved in contrast to the hinge domain and coiled-coil domains which are considerably shorter in several bacterial species . SMC proteins are present in all gram-positive bacteria and in nearly all archaea while they were found in less than half of the gram-negative bacteria . Phylogenetic analyses indicate that the SMC tree roughly resembles the 16S rRNA tree, but that cyanobacteria and Aquifex aeolicus obtained smc genes by lateral transfer from archaea . Fourteen out of 22 smc genes located in fully sequenced genomes seem to be co-transcribed with a second gene out of six different gene families, indicating that the deduced gene products might be involved in similar functions . The SMC proteins were compared with other prokaryotic proteins with long coiled-coil domains . The lengths of different protein domains and signature sequences allowed to differentiate SMCs, MukBs, which were found to be confined to gamma proteobacteria, and two subfamilies of COG 0419 including the SbcC nuclease from E . coli . A phylogenetic analysis was performed including the prokaryotic coiled-coil proteins as well as SMCs and Rad18 proteins from selected eukaryotes. J Biol Chem, 2002 Jan 18, 277(3), 1762 - 9 Epub 2001 Nov 08. The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain; Francin M et al.; Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain . Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties . A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme . The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA . A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not . As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo . Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles. Carbohydr Res, 2001 Nov 21, 336(3), 237 - 42 NMR-based identification of cell wall anionic polymers of Spirilliplanes yamanashiensis VKM Ac-1993(T); Shashkov AS et al.; The cell wall of Spirilliplanes yamanashiensis VKM Ac-1993(T) contains four anionic polymers, viz., three teichoic acids and a sugar-1-phosphate polymer . The following are the structures of the teichoic acids: poly{-6-beta-D-glucopyranosyl-(1-->2)-glycerol phosphate} (PI), 1,3-poly(glycerol phosphate) bearing N-acetyl-alpha-D-glucosamine residues at O-2 (70%) (PII), and poly{-6-N-acetyl-alpha-D-glucosaminyl-(1-->2)-glycerol phosphate} (PIII) . The repeating unit of the fourth polymer (PIV) has the structure of -6-alpha-D-GlcpNAc-(1-->6)-alpha-D-GlcpNAc-1-P- with a 3-O-methyl-alpha-D-mannopyranosyl residues at position 3 of some 6-phosphorylated N-acetylglucosamine residues (50%) . Polymers PI, PIII and PIV have not hitherto been found in prokaryotic cell walls. Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 460 - 2 {Cloning, expression and purification of the chicken growth and differentiation factor-8}; Yang W et al.; Growth and Differentiation Factor-8(GDF-8) is a new member of TGF-beta super-family . It has been shown that GDF-8 is specifically expressed in skeleton muscle in mouse and its function is to inhibit the growth of muscle cell, so it is named as Myostatin . Here, we amplified 3'half-length GDF-8 cDNA from chicken skeleton muscle by RT-PCR, and cloned it into the prokaryotic expression vector pTrcHisB, which was then transformed into E . coli Top10 cells . The recombinant 6 x His-GDF-8 fusion protein expressed in the Top10 cells was purified by Ni(+)-Affinity Chromatography for future study. Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 392 - 5 {Cloning of secondary lymphoid-tissue chemokine (SLC) and its expression in prokaryotic system}; Wang DN et al.; The total RNA from lymphoid tissue in Chinese was extracted, and the gene encoding the mature peptide of secondary lymphoid-tissue chemokine (SLC) was cloned by RT-PCR . Nucleotide sequence analysis showed that there is only one nucleotide different from that reported, but it doesn't alter the amino acid encoded . The SLC cDNA was inserted into an expression vector pET-28a(+) under T7 promoter and constructed recombinant plasmid pET28a-SLC . pET28a-SLC was transformed to E . coli BL21(DE3) and the expression strain was gotten . After inducing with IPTG for 3-5 hours the bacterium were sonicated . After centrifuging the supernatant was analysed by SDS-PAGE . An obvious expression band about 18 kD can be seen . The expressed product was purified by Ni2+ affinity chromatography column, and the purity is up to 90 percent. J Biomed Sci, 2001 Nov-Dec, 8(6), 439 - 45 Prospects of chimeric RNA-DNA oligonucleotides in gene therapy; Wu XS et al.; A strategy called targeted gene repair was developed to facilitate the process of gene therapy using a chimeric RNA-DNA oligonucleotide . Experiments demonstrated the feasibility of using the chimeric oligonucleotide to introduce point conversion in genes in vitro and in vivo . However, barriers exist in the low and/or inconstant frequency of gene repair . To overcome this difficulty, three main aspects should be considered . One is designing a more effective structure of the oligonucleotide . Trials have included lengthening the homologous region, displacing the mismatch on the chimeric strand and inventing a novel thioate-modified single-stranded DNA, which was demonstrated to be more active than the primary chimera in cell-free extracts . The second aspect is optimizing the delivery system . Producing synthetic carriers for efficient and specific transfection is demanding, especially for treatment in vivo where targeting is difficult . The third and most important aspect lies in the elucidation of the mechanism of the strategy . Investigation of the mechanism of strand exchange between the oligonucleotide molecule and double-stranded DNA in prokaryotes may greatly help to understand the mechanism of gene repair in eukaryotes . The development of this strategy holds great potential for the treatment of genetic defects and other purposes . Cell, 2001 Nov 2, 107(3), 373 - 86 Structure of the 80S ribosome from Saccharomyces cerevisiae--tRNA-ribosome and subunit-subunit interactions; Spahn CM et al.; A cryo-EM reconstruction of the translating yeast 80S ribosome was analyzed . Computationally separated rRNA and protein densities were used for docking of appropriately modified rRNA models and homology models of yeast ribosomal proteins . The core of the ribosome shows a remarkable degree of conservation . However, some significant differences in functionally important regions and dramatic changes in the periphery due to expansion segments and additional ribosomal proteins are evident . As in the bacterial ribosome, bridges between the subunits are mainly formed by RNA contacts . Four new bridges are present at the periphery . The position of the P site tRNA coincides precisely with its prokaryotic counterpart, with mainly rRNA contributing to its molecular environment . This analysis presents an exhaustive inventory of an eukaryotic ribosome at the molecular level. Microbiology, 2001 Nov, 147(Pt 11), 3171 - 82 Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: evidence for rapid evolution of genetic heterogeneity; Ting CS et al.; Prochlorococcus is a major photosynthetic prokaryote in nutrient-limited, open ocean environments and an important participant in the global carbon cycle . This phototroph is distinct from other members of the cyanobacterial lineage to which it belongs because it utilizes a chlorophyll a2/b(2) light-harvesting complex as its major antenna, instead of phycobilisomes . Recently, genes encoding the phycobiliprotein phycoerythrin were identified in several Prochlorococcus isolates, thus making it the only extant photosynthetic prokaryote to possess a chlorophyll a/b antenna as well as phycobiliprotein genes . In order to understand the evolution of phycobiliproteins in this genus, the authors have sequenced the phycoerythrin genes of two isolates that are the most deeply branching in the Prochlorococcus lineage and share the highest degree of 16S rDNA sequence similarity to phycobilisome-containing marine SYNECHOCOCCUS: Sequence analyses suggest that within the Prochlorococcus lineage, the selective forces shaping the evolution of the phycoerythrin gene set have not been uniform . Although strains that are most closely related to marine Synechococcus possess genes (cpeB, cpeA) encoding both subunits of phycoerythrin, a more recently evolved strain is shown to lack cpeA and to possess a degenerate form of cpeB . Differences in phycoerythrin gene sequences between Prochlorococcus and Synechococcus appear to be consistent with a model of elevated mutation rates rather than relaxed selection . This suggests that although phycoerythrin is not a major constituent of the light-harvesting apparatus in Prochlorococcus, as it is in Synechococcus, the cpeB and cpeA genes are still under selection, albeit a different type of selection than in Synechococcus . The evolution of the Prochlorococcus light-harvesting antenna complex provides an important system for understanding the origins and scope of phylogenetic diversity in ocean ecosystems. Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13577 - 82 Epub 2001 Nov 06. Bacillus subtilis arsenate reductase is structurally and functionally similar to low molecular weight protein tyrosine phosphatases; Bennett MS et al.; Arsenate is an abundant oxyanion that, because of its ability to mimic the phosphate group, is toxic to cells . Arsenate reductase (EC; encoded by the arsC gene in bacteria) participates to achieve arsenate resistance in both prokaryotes and yeast by reducing arsenate to arsenite; the arsenite is then exported by a specific transporter . The crystal structure of Bacillus subtilis arsenate reductase in the reduced form with a bound sulfate ion in its active site is solved at 1.6-A resolution . Significant structural similarity is seen between arsenate reductase and bovine low molecular weight protein tyrosine phosphatase, despite very low sequence identity . The similarity is especially high between their active sites . It is further confirmed that this structural homology is relevant functionally by showing the phosphatase activity of the arsenate reductase in vitro . Thus, we can understand the arsenate reduction in the light of low molecular weight protein tyrosine phosphatase mechanism and also explain the catalytic roles of essential residues such as Cys-10, Cys-82, Cys-89, Arg-16, and Asp-105 . A "triple cysteine redox relay" is proposed for the arsenate reduction mechanism. J Biol Chem, 2002 Jan 18, 277(3), 1649 - 52 Epub 2001 Nov 06. Identification of the ubiquinone-binding domain in the disulfide catalyst disulfide bond protein B; Xie T et al.; Disulfide bond (Dsb) formation is catalyzed in the periplasm of prokaryotes by the Dsb proteins . DsbB, a key enzyme in this process, generates disulfides de novo by using the oxidizing power of quinones . To explore the mechanism of this newly described enzymatic activity, we decided to study the ubiquinone-protein interaction and identify the ubiquinone-binding domain in DsbB by cross-linking to photoactivatable quinone analogues . When purified Escherichia coli DsbB was incubated with an azidoubiquinone derivative, 3-azido-2-methyl-5-{(3)H}methoxy-6-decyl-1,4-benzoquinone ({(3)H}azido-Q), and illuminated with long wavelength UV light, the decrease in enzymatic activity correlated with the amount of 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone (azido-Q) incorporated into the protein . One azido-Q-linked peptide with a retention time of 33.5 min was obtained by high performance liquid chromatography of the V8 digest of {(3)H}azido-Q-labeled DsbB . This peptide has a partial NH(2)-terminal amino acid sequence of NH(2)-HTMLQLY corresponding to residues 91-97 . This sequence occurs in the second periplasmic domain of the inner membrane protein DsbB in a loop connecting transmembrane helices 3 and 4 . We propose that the quinone-binding site is within or very near to this sequence. J Mol Biol, 2001 Nov 2, 313(4), 673 - 81 Protein family and fold occurrence in genomes: power-law behaviour and evolutionary model; Qian J et al.; Global surveys of genomes measure the usage of essential molecular parts, defined here as protein families, superfamilies or folds, in different organisms . Based on surveys of the first 20 completely sequenced genomes, we observe that the occurrence of these parts follows a power-law distribution . That is, the number of distinct parts (F) with a given genomic occurrence (V) decays as F=aV(-b), with a few parts occurring many times and most occurring infrequently . For a given organism, the distributions of families, superfamilies and folds are nearly identical, and this is reflected in the size of the decay exponent b . Moreover, the exponent varies between different organisms, with those of smaller genomes displaying a steeper decay (i.e . larger b) . Clearly, the power law indicates a preference to duplicate genes that encode for molecular parts which are already common . Here, we present a minimal, but biologically meaningful model that accurately describes the observed power law . Although the model performs equally well for all three protein classes, we focus on the occurrence of folds in preference to families and superfamilies . This is because folds are comparatively insensitive to the effects of point mutations that can cause a family member to diverge beyond detectable similarity . In the model, genomes evolve through two basic operations: (i) duplication of existing genes; (ii) net flow of new genes . The flow term is closely related to the exponent b and can accommodate considerable gene loss; however, we demonstrate that the observed data is reproduced best with a net inflow, i.e . with more gene gain than loss . Moreover, we show that prokaryotes have much higher rates of gene acquisition than eukaryotes, probably reflecting lateral transfer . A further natural outcome from our model is an estimation of the fold composition of the initial genome, which potentially relates to the common ancestor for modern organisms . Supplementary material pertaining to this work is available from J Membr Biol, 2001 Oct 1, 183(3), 175 - 82 Signal-sequence trap in mammalian and yeast cells: a comparison; Galliciotti G et al.; Membrane associated and secreted proteins are translated as precursors containing a signal peptide that allows protein-insertion into the membrane of the endoplasmic reticulum and is co-translationally removed in the lumen . The ability of the signal peptide to direct a polypeptide into the secretory pathway is exploited in methods developed to select cDNAs encoding such proteins . Different strategies are known in which cDNA libraries can be screened for signal peptides by the ability of the latter to rescue the translocation of signal sequence-less proteins . In one method, a cDNA library is tested for interleukin 2 receptor alpha chain translocation to the membrane in COS cells, in another one for invertase secretion from yeast . In this work, we compared the two systems by testing six mouse signal peptides in COS and yeast cells . All of them were functional in the mammalian system, whereas only three of them in yeast . Two other sequences needed the 5' cDNA sequence flanking the ATG codon to be removed in order to enable protein translocation . Although the structure of signal sequences and the functioning of the secretory machinery are well conserved from prokaryotes to eukaryotes, it seems evident that not all signal peptides can be interchanged between different proteins and organisms . In particular, signal peptides that are functional in the mammalian system do not necessarily lead to protein translocation in yeast. Protist, 2001 Sep, 152(3), 193 - 201 Diplonemid glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prokaryote-to-eukaryote lateral gene transfer; Qian Q et al.; Lateral gene transfer refers to the movement of genetic information from one genome to another, and the integration of that foreign DNA into its new genetic environment . There are currently only a few well-supported cases of prokaryote-to-eukaryote transfer known that do not involve mitochondria or plastids, but it is not clear whether this reflects a lack of such transfer events, or poor sampling of diverse eukaryotes . One gene where this process is apparently active is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), where lateral transfer has been implicated in the origin of euglenoid and kinetoplastid genes . We have characterised GAPDH genes from diplonemids, heterotrophic flagellates that are closely related to kinetoplastids and euglenoids . Two distinct classes of diplonemid GAPDH genes were found in diplonemids, however, neither class is closely related to any other euglenozoan GAPDH . One diplonemid GAPDH is related to the cytosolic gapC of eukaryotes, although not to either euglenoids or kinetoplastids, and the second is related to cyanobacterial and proteobacterial gap3 . The bacterial gap3 gene in diplonemids provides one of the most well-supported examples of lateral gene transfer from a bacterium to a eukaryote characterised to date, and may indicate that diplonemids have acquired a novel biochemical capacity through lateral transfer. J Biotechnol, 2002 Jan 31, 93(1), 59 - 71 Selective surface adhesion of the toxic microalga Alexandrium minutum induced by contact with substituted polystyrene derivatives; La Barre S et al.; On the basis of observations that biospecific random copolymers (RACS) could induce phenotypic changes on contact with selected eukaryotic or prokaryotic cell lines, polystyrene derivatives of known compositions and obtained by random substitutions of sodium sulfonate and of sulfamides of aspartic acid dimethyl ester, phenylalanine and leucine, were placed in contact with swimming dinophytes of the PSP toxin producing species Alexandrium minutum and of the non-toxic species Heterocapsa triquetra . A . minutum cells exhibited higher adhesion for the random copolymer made up of polystyrene (29%), polystyrene aspartic acid dimethyl ester sulfamide (47%) and polystyrene sodium sulfonate (24%), than for samples of this series with different compositions . In contrast to this, A . minutum adhesion remained very low throughout the phenylalanine and leucine copolymer series . These results indicate that the cell-substrate adhesion phenomenon is dependent upon the final composition of the copolymer, i.e . that it is composition-specific . Taxonomic specificity was then demonstrated by presenting the PSAspOMe copolymer series with cells of the non toxic species H . triquetra (Peridinialia) related to A . minutum (Gonyaulacacea), and by observing no specific association, i.e . no signal above background levels at any composition . Specific ligand-cell adhesion is evidenced for the first time between biospecific RACS and phytoplankton, which may inspire a new generation of structures to be used in aquaculture as protective nets over shellfish clusters, or as selective filtering devices to assist in shellfish depuration from toxic microalgae. Biochim Biophys Acta, 2001 Oct 31, 1521(1-3), 30 - 8 Possible interaction sites of mRNA, tRNA, translation factors and the nascent peptide in 5S, 5.8S and 28S rRNA in in vivo assembled eukaryotic ribosomal complexes; Sloma MS et al.; We have investigated possible interaction sites for mRNA, tRNA, translation factors and the nascent peptide on 5S, 5.8S and 28S rRNA in in vivo assembled translational active mouse ribosomes by comparing the chemical footprinting patterns derived from native polysomes, salt-washed polysomes (mainly lacking translational factors) and salt-washed runoff ribosomes (lacking mRNA, tRNA and translational factors) . Several ligand-induced footprints were observed in 28S rRNA while no reactivity changes were seen in 5S and 5.8S rRNA . Footprints derived from mRNA, tRNA and/or the nascent peptide chain were observed in domain I of 28S rRNA (hairpin 23), in domain II (helix 37/38 and helices 42 and 43 and in the eukaryotic expansion segment 15), in domain IV (helices 67 and 74) and in domain V (helices 94 and 96 and in the peptidyl transferase ring) . Some of the protected sites were homologous to sites previously suggested to be involved in mRNA, tRNA and/or peptide binding in in vitro assembled prokaryotic complexes . Additional footprints were located in regions that have not previously been found involved in ligand binding . Part of these sites could derive from the nascent peptide in the exit channel of the ribosome. EMBO J, 2001 Nov 1, 20(21), 6060 - 70 Mechanism for the switch of phi29 DNA early to late transcription by regulatory protein p4 and histone-like protein p6; Camacho A et al.; Bacteriophage phi29 gene expression takes place from four major promoters, three of them (A2b, A2c and A3) clustered within 219 bp at a central region of the genome . Transcription regulation of these promoters involves both a highly specific DNA-binding protein (p4) and a low specificity DNA-binding protein (p6) functionally related to prokaryotic histone-like proteins . Protein p6 forms extended oligomeric arrays along the phage DNA . In contrast, protein p4 binds specifically upstream of late promoter A3 and early promoter A2c . We have analysed the concomitant binding of p6 and p4 and found that the proteins cooperate with each other in the binding to the central region of the genome, resulting in a ternary p4-p6-DNA complex that affects local DNA topology . Through this complex, protein p6 exerts a direct role in the repression of promoter A2c, impeding unwinding of the DNA strands needed for open complex formation . In contrast, protein p6 functions by reinforcing the positioning of protein p4 in the repression of promoter A2b and activation of promoter A3, thereby facilitating p4-mediated transcription regulation. J Biomol Screen, 2001 Aug, 6(4), 233 - 43 Identification of inhibitors of bacterial transcription/translation machinery utilizing a miniaturized 1536-well format screen; Kariv I et al.; This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format . The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement . This multicomponent system permits identification of inhibitors at different steps in this pathway . Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems . Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives . The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time . The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively . This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff . Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format. Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13443 - 8 Epub 2001 Oct 30. The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes; Zak E et al.; During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes . Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system . In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane . We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp . PCC 6803 . Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems . Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data . Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation . Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells. Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13177 - 82 Epub 2001 Oct 30. Intron distribution difference for 276 ancient and 131 modern genes suggests the existence of ancient introns; Fedorov A et al.; o introns delineate elements of protein tertiary structure? This issue is crucial to the debate about the role and origin of introns . We present an analysis of the full set of proteins with known three-dimensional structures that have homologs with intron positions recorded in GenBank . A computer program was generated that maps on a reference sequence the positions of all introns in homologous genes . We have applied this program to a set of 665 nonredundant protein sequences with defined three-dimensional structures in the Protein Data Bank (PDB), which yielded 8,217 introns in 407 proteins . For the subset of proteins corresponding to ancient conserved regions (ACR), we find that there is a correlation of phase-zero introns with the boundary regions of modules and no correlation for the phase-one and phase-two positions . However, for a subset of proteins without prokaryotic counterparts (131 non-ACR proteins), a set of presumably modern proteins (or proteins that have diverged extremely far from any ancestral form), we do not find any correlation of phase-zero intron positions with three-dimensional structure . Furthermore, we find an anticorrelation of phase-one intron positions with module boundaries: they actually have a preference for the interior of modules . This finding is explicable as a preference for phase-one introns to lie in glycines, between G/G sequences, the preference for glycines being anticorrelated with the three-dimensional modules . We interpret this anticorrelation as a sign that a number of phase-one introns, and hence many modern introns, have been inserted into G/G "protosplice" sequences. Annu Rev Cell Dev Biol, 2001, 17, 215 - 53 Molecular bases of circadian rhythms; Harmer SL et al.; Circadian rhythms are found in most eukaryotes and some prokaryotes . The mechanism by which organisms maintain these roughly 24-h rhythms in the absence of environmental stimuli has long been a mystery and has recently been the subject of intense research . In the past few years, we have seen explosive progress in the understanding of the molecular basis of circadian rhythms in model systems ranging from cyanobacteria to mammals . This review attempts to outline these primarily genetic and biochemical findings and encompasses work done in cyanobacteria, Neurospora, higher plants, Drosophila, and rodents . Although actual clock components do not seem to be conserved between kingdoms, central clock mechanisms are conserved . Somewhat paradoxically, clock components that are conserved between species can be used in diverse ways . The different uses of common components may reflect the important role that the circadian clock plays in adaptation of species to particular environmental niches. Res Microbiol, 2001 Oct, 152(8), 717 - 23 Disruption of the hup gene encoding a histone-like protein HS1 and detection of HS12 of Streptomyces lividans; Yokoyama E et al.; When the latter half of the hup gene encoding a histone-like protein HS1 of Streptomyces lividans TK24 was replaced by the kanamycin resistance gene, the hup mutant EY1 grew slowly in liquid medium and this delay was overcome by introduction of the complete hup . EY1 sporulated normally on solid medium, with no serious defects as observed in hupAB mutants of Escherichia coli . Therefore, HS1 probably has a role in growth in the presence of liquid medium and this organism may possess another histone-like protein with functions overlapping those of HS1 . We cloned the hup2 gene encoding another histone-like protein HS12, which has two motifs of prokaryotic histone-like protein and eukaryotic histone H1 . The amount of HS12 increased in EY1, determined by western blotting analysis using an anti-His-tagged HS12 polyclonal antibody . We are entertaining the notion that the increased amount of HS12 partially suppressed the defects caused by the hup mutation. Chromosoma, 2001 Sep, 110(5), 352 - 9 Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences; Skovorodkin IN et al.; The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences . However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants . In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3' overhang . Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3' telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres . Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication . To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified alpha1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae . Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells . Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months . In contrast, alpha1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection . Hence, DNA replication of the alpha1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres. Mol Med, 2001 Jul, 7(7), 442 - 53 Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas; Pavelic K et al.; BACKGROUND: Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes . Loss of FHIT function may be important in the development and/or progression of various types of cancer . MATERIALS AND METHODS: We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC) . Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, {3H}-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections . RESULTS: The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation . Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues . The observed changes increased with progression of these lesions . We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci . We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC) . The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months . FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant . Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05) . CONCLUSIONS: Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation . Aberrant FHIT gene could be a prognostic marker in lung cancer. Mol Genet Genomics, 2001 Oct, 266(2), 216 - 22 Transposition of IS10 from the host Escherichia coli genome to a plasmid may lead to cloning artefacts; Kovarik A et al.; During recloning of Nicotiana tabacum L . repetitive sequence R8.3 in Escherichia coli, a modified clone that differed from the original by the insertion of an IS10 sequence was unintentionally produced . The insert was flanked by a 9-bp direct repeat derived from the R8.3 sequence, the 9-bp duplication of acceptor DNA in the site of insertion being a characteristic of IS10 transposition events . A database search using the FASTA program showed IS10 and other prokaryotic IS elements inserted into numerous eukaryotic clones . Unexpectedly, the IS10, which is not a natural component of the E . coli genome, appeared to be by far the most frequent contaminant of DNA databases among several IS sequences tested . In the GenEMBL database, the IS10 query sequence yielded positive scores with more than 500 eukaryotic clones . Insertions of shortened IS10 sequences having only one intact terminal inverted repeat were commonly found . Most full-length IS10 insertions (32 out of 40 analyzed) were flanked by 9-bp direct repeats having the consensus 5'-NPuCNN-NGPyN-3' with a strong preference for 5'-TGCTNA-GNN-3' . One insertion was flanked by an inverted repeat of more than 400 bp in length . PCR amplification and Southern analysis revealed the presence of IS10 sequences in E . coli strains commonly used for DNA cloning, including some reported to be Tn10-free . No IS10-specific PCR product was obtained with N . tabacum or human DNA . Our data suggest that transposition of IS10 elements may accompany cloning steps, particularly into large BAC vectors . This might lead to the relatively frequent contamination of DNA databases by this bacterial sequence . It is estimated that one in approximately every thousand eukaryotic clone in the databases is contaminated by IS-derived sequences . We recommend checking submitted sequences for the presence of IS10 and other IS elements . In addition, DNA databases should be corrected by removing contaminating IS sequences. Cell Tissue Res, 2001 Oct, 306(1), 157 - 65 Cellular location of (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol, a potent antimicrobial metabolite produced by the Caribbean sponge Haliclona vansoesti; Richelle-Maurer E et al.; The Caribbean sponge Haliclona vansoesti has been found to contain large amounts of a new sphingosine derivative, (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol (compound 1) . To determine the localization of this compound within the organism, cell distribution and quantitative determination of the aminodiol content of cell fractions obtained by differential centrifugation have been performed . Results show that choanocytes and archaeocytes are the major sponge cell types and that H . vansoesti harbour small photosynthetic symbionts (cyanobacteria) and few heterotrophic bacteria . Reverse-phase HPLC analyses of the cell fractions reveal that the aminodiol 1 is not associated with the prokaryotic endobionts but with the sponge cells, in particular the archaeocytes . This is clearly established by the positive significant correlation existing between the numbers of archaeocytes and the amounts of aminodiol 1 . The mean aminodiol concentration is estimated to be 2 microg/10(5) archaeocytes . The aminodiol 1 is also found in substantial amounts in primary cell cultures, so that cell culture can be envisaged as an option for its production . Sponge cell suspensions display potent antibacterial and antiyeast activities, in correlation with their aminodiol content, indicating that this compound is at least in part responsible for these activities in the sponge . The release of the aminodiol I into the external medium suggests that this substance may be involved in the defence mechanisms of the sponge. J Cell Sci, 2001 Oct, 114(Pt 19), 3557 - 64 Noradrenaline and alpha-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells; Lacoste A et al.; Expression of heat shock proteins (hsp) is a homeostatic mechanism induced in both prokaryotic and eukaryotic cells in response to metabolic and environmental insults . A growing body of evidence suggests that in mammals, the hsp response is integrated with physiological responses through neuroendocrine signaling . In the present study, we have examined the effect of noradrenaline (NA) on the hsp70 response in mollusc immune cells . Oyster and abalone hemocytes transfected with a gene construct containing a gastropod hsp70 gene promoter linked to the luciferase reporter-gene were exposed to physiological concentrations of NA, or to various alpha- and beta-adrenoceptor agonists and antagonists . Results show that NA and alpha-adrenergic stimulations induced the expression of luciferase in transfected mollusc immunocytes . Furthermore, exposure of hemocytes to NA or to the alpha-adrenoceptor agonist phenylephrine (PE) resulted in the expression of the inducible isoform of the hsp70 protein . Pertussis toxin (PTX), the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor calphostin C, the Ca(2+)-dependent PKC inhibitor Go 6976 and the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 blocked the PE-mediated induction of the hsp70 gene promoter . These results suggest that alpha-adrenergic signaling induces the transcriptionnal upregulation of hsp70 in mollusc hemocytes through a PTX-sensitive G-protein, PLC, Ca(2+)-dependent PKC and PI 3-kinase . Thus, a functional link exists between neuroendocrine signaling and the hsp70 response in mollusc immune cells. Curr Opin Genet Dev, 2001 Dec, 11(6), 660 - 6 Gene expression and molecular evolution; Akashi H; The combination of complete genome sequence information and estimates of mRNA abundances have begun to reveal causes of both silent and protein sequence evolution . Translational selection appears to explain patterns of synonymous codon usage in many prokaryotes as well as a number of eukaryotic model organisms (with the notable exception of vertebrates) . Relationships between gene length and codon usage bias, however, remain unexplained . Intriguing correlations between expression patterns and protein divergence suggest some general mechanisms underlying protein evolution. FEBS Lett, 2001 Oct 19, 507(1), 11 - 5 Calf thymus Hsc70 and Hsc40 can substitute for DnaK and DnaJ function in protein renaturation but not in bacteriophage DNA replication; Ziemienowicz A et al.; Calf thymus (ct) Hsc70 has been shown previously to reactivate heat-inactivated prokaryotic and eukaryotic enzymes, while DnaK was able to reactivate solely prokaryotic enzymes . Here, we report on isolation from calf thymus of a DnaJ homolog, ctHsc40, and on testing of its cooperative function in three different assays: (i) reactivation of heat-inactivated DNA polymerases, (ii) stimulation of the ATPase activity of ctHsc70 chaperone, and (iii) replication of bacteriophage lambda DNA . Surprisingly, ctHsc70/ctHsc40 chaperones were found to reactivate the denatured prokaryotic and eukaryotic enzymes but not to promote bacteriophage lambda DNA replication, suggesting species specificity in DNA replication. Int J Radiat Biol, 2001 Oct, 77(10), 1007 - 21 Proliferating cell nuclear antigen (PCNA): ringmaster of the genome; Paunesku T et al.; Proliferating cell nuclear antigen (PCNA) protein is one of the central molecules responsible for decisions of life and death of the cell . The PCNA gene is induced by p53, while PCNA protein interacts with p53-controlled proteins Gadd45, MyD118, CR6 and, most importantly, p21, in the process of deciding cell fate . If PCNA protein is present in abundance in the cell in the absence of p53, DNA replication occurs . On the other hand, if PCNA protein levels are high in the cell in the presence of p53, DNA repair takes place . If PCNA is rendered non-functional or is absent or present in low quantities in the cell, apoptosis occurs . The evolution from prokaryotes to eukaryotes involved a change of function of PCNA from a 'simple' sliding clamp protein of the DNA polymerase complex to an executive molecule controlling critical cellular decision pathways . The evolution of multicellular organisms led to the development of multicellular processes such as differentiation, senescence and apoptosis . PCNA, already an essential molecule in the life of single cellular organisms, then became a protein critical for the survival of multicellular organisms. Proteomics, 2001 Apr, 1(4), 516 - 21 Identification of differentially regulated proteins in metronidozole resistant Helicobacter pylori by proteome techniques; McAtee CP et al.; Resistance to metronidazole (MTZ) is common among Helicobacter pylori strains in many societies, and results from loss of function mutations in genes for one or more cellular nitroreductases . When functional, these enzymes convert MTZ from a harmless prodrug to mutagenic and bacteriocidal products (probably hydroxylamine-type compounds), and in the process may generate active reactive oxygen metabolites . Here we examine the protein profiles of a derivative of strain 26695 that is resistant to moderate levels of MTZ because of mutation in rdxA (HP0954), the gene for the most important of these nitroreductases . The strain was grown with and without 18 micrograms/mL of MTZ to assess whether sublethal exposure triggers an adaptive response . Bacterial lysates were subjected to two-dimensional (2-D) electrophoresis and protein bands were identified by mass spectrometry and sequence analysis . Several proteins were decreased at least two-fold during growth with MTZ, yet the levels of various isoforms of alkylhydroperoxide reductase (AHP) (encoded by ahpC HP1563) were increased . AHP is an essential enzyme, and had been linked to resistance to oxygen toxicity in various prokaryotic and eukaryotic systems; we propose that the ability of an rdxA mutant strain to increase AHP abundance during exposure to MTZ is critically important in the realization of the resistance phenotype . More generally, these results highlight the potential of proteome analysis to tracing out how pathogenic bacteria cope with the challenges imposed on them by therapy or host responses to infection. Proteomics, 2001 Mar, 1(3), 409 - 23 New perspectives in the Escherichia coli proteome investigation; Tonella L et al.; Escherichia coli is a model organism for biochemical and biological studies as it is one of the best characterised prokaryote . Two-dimensional polyacrylamide gel electrophoresis, computer image analysis and different protein identification techniques gave rise, in 1995, to the Escherichia coli SWISS-2D PAGE database . In the E . coli 3.5-10 SWISS-2D PAGE map, 40% of the E . coli proteome was displayed . The present study demonstrated that the use of narrow range pH gradients is able to potentially display up to a few copies of protein per E . coli cell . Moreover, the six new E . coli SWISS-2D PAGE maps (pH 4-5, 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11) presented here displayed altogether more than 70% of the entire E . coli proteome. Proteomics, 2001 Feb, 1(2), 248 - 61 Prokaryotic glycosylation; Schaffer C et al.; With the advances of molecular biology and with improved analytical techniques a significant change of perception has taken place regarding prokaryotic glycoproteins . Glycosylation of proteins from prokaryotes is no longer considered a specific feature of certain organisms but has been demonstrated for many archaea and bacteria . Besides the occurrence of glycosylated enzymes, antigens and other cell envelope components, surface layer (S-layer) glycoproteins represent the best-studied examples of glycosylated prokaryotic proteins . They are widely distributed among archaeal wild-type strains, but among bacteria they have been mainly observed with Gram-positive organisms . There is, in general, an enormous increase of reports on the presence of glycosylated proteins among prokaryotes . For their isolation and characterization a great number of methods are available, aiming at the identification of the covalent linkage between the carbohydrate and the polypeptide portion . So far, several differences in structure and biosynthesis have been observed in comparison to eukaryotic glycoproteins . In this review we introduce a protocol which has been successfully applied to the investigation of the complex structures, linkage units, and polypeptide consensus sequences of glycosylated bacterial S-layer proteins. Proteomics, 2001 Feb, 1(2), 194 - 9 Use of antibodies for detection of phosphorylated proteins separated by two-dimensional gel electrophoresis; Kaufmann H et al.; Protein phosphorylation and dephosphorylation are key regulatory mechanisms in prokaryotic and eukaryotic cells . Considering the role of phosphorylation in many human diseases, it appears a major challenge to refine on the methods to analyze the phospho-proteome . Here we review the use of monoclonal antibodies directed against specific phosphorylated amino acid residues to visualize phosphoproteins separated by two-dimensional gel electrophoresis . Strategies are described how this method can successfully be applied to create phospho-proteome maps of mammalian cells. Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1643 - 5 Epub 2001 Oct 25. Crystallization and preliminary X-ray analysis of bacterial cytosine deaminase; Ireton GC et al.; Cytosine deaminase (CD) is found in prokaryotes and fungi (but not higher eukaryotes) and catalyzes the deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively . The former activity is an important function within the pyrimidine-salvage pathway, while the latter activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor . Recombinant bacterial CD from Escherichia coli has been subcloned, overexpressed, purified and crystallized for structural analysis . The crystals belong to space group R32, with unit-cell parameters a = b = 109.1, c = 240 A and diffract to at least 1.5 A resolution at a synchrotron X-ray source . There is one enzyme subunit per asymmetric unit and the Matthews coefficient V(M) is 2.8 A(3) Da(-1), corresponding to a solvent content of 56% . Selenomethionine-containing protein has been prepared and crystallized for MAD phasing. Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1634 - 8 Epub 2001 Oct 25. Crystallization of redox-insensitive Oct1 POU domain with different DNA-response elements; Remenyi A et al.; The POU domain of the human Oct1 transcription factor has been crystallized with two different POU-dimer-binding DNA elements . Protein-DNA cocrystals suitable for structural analysis could be obtained only with a redox-insensitive version of the POU domain . The recombinant protein expression in a prokaryotic host was adjusted for fast purification . Optimized crystals were obtained by systematically varying the length of the oligonucleotide and by modifying cryofreezing procedures . These steps are generally applicable to the preparation of protein-DNA complexes for structural studies. Mol Cell Biochem, 2001 Jun, 222(1-2), 173 - 82 Effects of glutathione on chromium-induced DNA crosslinking and DNA polymerase arrest; O'Brien T et al.; Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH) . These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication . Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNA-Cr-DNA crosslinks (Cr-DDC) and guanine-specific arrests of both prokaryotic and mammalian DNA polymerases . GSH not only acts as a reductant of Cr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Cr-induced DNA damage and polymerase arrests . Co-incubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA . GSH co-treatment with Cr (III) also led to a decrease in the degree of Cr-induced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding . DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but were markedly reduced when GSH was added to the reaction mixture . Pre-formed polymerase-arresting lesions (Cr-DDC) were not removed by subsequent addition of GSH . Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests . The inhibitory effect of GSH on Cr-induced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis . Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene by Taq polymerase . This was almost completely prevented by co-treatment with GSH and Cr (III) . These results indicate that Cr-induced DNA interstrand crosslinks, and not DNA-Cr-GSH crosslinks, are the principal lesions responsible for blocking DNA replication . Moreover, the formation of DNA-Cr-GSH crosslinks may actually preclude the formation of the polymerase arresting lesions. Dis Aquat Organ, 2001 Sep 12, 46(2), 147 - 52 Detection of 'Candidatus Xenohaliotis californiensis' (Rickettsiales-like prokaryote) inclusions in tissue squashes of abalone (Haliotis spp.) gastrointestinal epithelium using a nucleic acid fluorochrome; Moor JD et al.; Rickettsiales-like prokaryotes appear to be etiologic agents of a number of newly described diseases of fish and shellfish . 'Candidatus Xenohaliotis californiensis' is a Rickettsiales-like prokaryote responsible for withering syndrome, a fatal disease of wild and farmed Eastern Pacific abalone, Haliotis spp . The bacterium proliferates in gastrointestinal epithelial cells, forming large intracytoplasmic inclusions . We describe a method of rapidly detecting and assessing the intensity of 'Candidatus Xenohaliotis californiensis' infections in abalone gastrointestinal tissue using the nucleic acid-specific fluorochrome Hoechst 33258 . In excised tissue pieces dried onto slides, rehydrated in the Hoechst stain and viewed with ultraviolet light, the large bacterial inclusions were strongly fluorescent and could be easily distinguished from smaller host cell nuclei . This provided a rapid, inexpensive alternative to paraffin section microscopy or molecular techniques, allowing detection of the pathogen within minutes of tissue excision . Comparison of the fluorochrome method with conventional histological analysis for the ability to detect inclusions in 109 samples was 90% accurate, with discrepancies due to false negative diagnosis of low-level infections . An alternative nucleic acid-specific fluorochrome, propidium iodide, showed a staining pattern identical to that of Hoechst 33258 . These methods should prove useful for the rapid detection of inclusion-forming Rickettsiales-like prokaryotes in tissues from many host species. J Mol Evol, 2001 Dec, 53(6), 622 - 33 Independent evolution of heavy metal-associated domains in copper chaperones and copper-transporting atpases; Jordan IK et al.; Copper chaperones are small cytoplasmic proteins that bind intracellular copper (Cu) and deliver it to Cu-dependent enzymes such as cytochrome oxidase, superoxide dismutase, and amine oxidase . Copper chaperones are similar in sequence and structure to the Cu-binding heavy metal-associated (HMA) domains of Cu-transporting ATPases (Cu-ATPases), and the genes for copper chaperones and Cu-ATPases are often located in the same operon . Phylogenetic analysis shows that Cu chaperones and HMA domains of Cu-ATPases represent ancient and distinct lineages that have evolved largely independently since their initial separation . Copper chaperone-Cu-ATPase operons appear to have evolved independently in different prokaryotic lineages, probably due to a strong selective pressure for coexpression of these genes. J Mol Evol, 2001 Dec, 53(6), 615 - 21 The differential killing of genes by inversions in prokaryotic genomes; Mackiewicz P et al.; We have elaborated a method which has allowed us to estimate the direction of translocation of orthologs which have changed, during the phylogeny, their positions on chromosome in respect to the leading or lagging role of DNA strands . We have shown that the relative number of translocations which have switched positions of genes from the leading to the lagging DNA strand is lower than the number of translocations which have transferred genes from the lagging strand to the leading strand of prokaryotic genomes . This paradox could be explained by assuming that the stronger mutation pressure and selection after inversion preferentially eliminate genes transferred from the leading to the lagging DNA strand. Nature, 2001 Oct 25, 413(6858), 809 - 13 Ion conduction pore is conserved among potassium channels; Lu Z et al.; Potassium channels, a group of specialized membrane proteins, enable K+ ions to flow selectively across cell membranes . Transmembrane K+ currents underlie electrical signalling in neurons and other excitable cells . The atomic structure of a bacterial K+ channel pore has been solved by means of X-ray crystallography . To the extent that the prokaryotic pore is representative of other K+ channels, this landmark achievement has profound implications for our general understanding of K+ channels . But serious doubts have been raised concerning whether the prokaryotic K+ channel pore does actually represent those of eukaryotes . Here we have addressed this fundamental issue by substituting the prokaryotic pore into eukaryotic voltage-gated and inward-rectifier K+ channels . The resulting chimaeras retain the respective functional hallmarks of the eukaryotic channels, which indicates that the ion conduction pore is indeed conserved among K+ channels. Biochem Biophys Res Commun, 2001 Nov 2, 288(3), 509 - 14 Detection and characterization of glutathione S-transferase activity in rice EF-1betabeta'gamma and EF-1gamma expressed in Escherichia coli; Kobayashi S et al.; Plant elongation factor EF-1 consists of four subunits (EF-1alphabetabeta'gamma) . EF-1alpha . GTP catalyses the binding of aminoacyl-tRNA to the ribosome . EF-1beta and EF-1beta' catalyze the GDP/GTP exchange on EF-1alpha . GDP . However, the function of EF-1gamma, a subunit detected in eukaryotes, but not in prokaryotes remained unknown . This report demonstrates that rice EF-1betabeta'gamma and recombinant EF-1gamma possess glutathione S-transferase (GST) activity . The EF-1betabeta'gamma- or EF-1gamma-dependent GST activity is about one-fiftieth of the rice GST activity . The Km values of EF-1betabeta'gamma, EF-1gamma, and rice GST for glutathione and 1-chloro-2,4-dinitrobenzene are of about the same order . Although recombinant EF-1gamma is heat labile, active EF-1gamma was obtained by purifying it in the presence of 20% glycerol . J Mol Evol, 2001 Oct-Nov, 53(4-5), 377 - 86 Accumulation of species-specific amino acid replacements that cause loss of particular protein functions in Buchnera, an endocellular bacterial symbiont; Shigenobu S et al.; Endosymbiotic bacteria live in animal cells and are transmitted vertically at the time of the host's reproduction . In view of their small and asexual populations with infrequent chances of recombination, these endocellular bacteria are expected to accumulate mildly deleterious mutations . Previous studies showed that the DNA sequences of these bacteria evolved faster than those of free-living bacteria . In this study, we compared all the ORFs of Buchnera, an endocellular bacterial symbiont of aphids, with those of 34 other prokaryotic organisms and estimated the effect of the accelerated evolution of Buchnera on the functions of its proteins . It was revealed that Buchnera proteins contain many mutations at the sites where sequences are conserved in their orthologues in many other organisms . In addition, amino acid replacements at the conserved sites are mostly changes to physicochemically different amino acids . These results suggest that functions and conformations of Buchnera proteins have been seriously impaired or strongly modified . Indeed, extensive loss of functional motifs was observed in some Buchnera proteins . In many Buchnera proteins mutations were not detected evenly throughout each molecule but tended to accumulate in some functional units, possibly leading to loss of specific functions . As Buchnera has an unusual and limited gene repertory, it is conceivable that the manner of interactions among its proteins has been changed, and thus, functional constraints over their amino acid residues have also been changed during evolution . This may account for the loss of some functional units only in the Buchnera proteins . We obtained evidence that amino acid replacements in Buchnera were not always deleterious, but neutral or, in some cases, even positively selected. J Mol Evol, 2001 Oct-Nov, 53(4-5), 340 - 50 Defining the core of nontransferable prokaryotic genes: the euryarchaeal core; Nesbo CL et al.; If lateral gene transfer (LGT) has affected all genes over the course of prokaryotic evolution, reconstruction of organismal phylogeny is compromised . However, if a core of genes is immune to transfer, then the evolutionary history of that core might be our most reliable guide to the evolution of organisms . Such a core should be preferentially included in the subset of genes shared by all organisms, but where universally conserved genes have been analyzed, there is too little phylogenetic signal to allow determination of whether or not they indeed have the same history (Hansmann and Martin 2000; Teichmann and Mitchison 1999) . Here we look at a more restricted set, 521 homologous genes (COGs) simultaneously present in four sequenced euryarchaeal genomes . Although there is overall little robust phylogenetic signal in this data set, there is, among well-supported trees, strong representation of all three possible four-taxon topologies . "Informational" genes seem no less subject to LGT than are "operational genes," within the euryarchaeotes . We conclude that (i) even in this collection of conserved genes there has been extensive LGT (orthologous gene replacement) and (ii) the notion that there is a core of nontransferable genes (the "core hypothesis") has not been proven and may be unprovable. J Biol Chem, 2002 Jan 4, 277(1), 50 - 8 Epub 2001 Oct 23. Escherichia coli gamma-glutamylcysteine synthetase . Two active site metal ions affect substrate and inhibitor binding; Kelly BS et al.; Gamma-glutamylcysteine synthetase (gamma-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes . Although all eukaryotic gamma-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial gamma-GCS is resistant to BSO . We have confirmed the latter finding with Escherichia coli gamma-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms . We also find that substitution of Mn2+ for Mg2+ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation . Similarly, the specificity of E . coli gamma-GCS for its amino acid substrates is broadened in the presence of Mn2+, and the rate of reaction for some very poor substrates is improved . These results suggest that divalent metal ions have a role in amino acid binding to E . coli gamma-GCS . Electron paramagnetic resonance (EPR) studies carried out with Mn2+ show that E . coli gamma-GCS binds two divalent metal ions; Kd values for Mn2+ are 1.1 microm and 82 microm, respectively . Binding of l-glutamate or l-BSO to the two Mn2+/gamma-GCS species produces additional upfield and downfield X-band EPR hyperfine lines at 45 G intervals, a result indicating that the two Mn2+ are spin-coupled and thus apparently separated by 5 A or less in the active site . Additional EPR studies in which Cu2+ replaced Mg2+ or Mn2+ suggest that Cu2+ is bound by one N and three O ligands in the gamma-GCS active site . The results are discussed in the context of the catalytic mechanism of gamma-GCS and its relationship to the more fully characterized glutamine synthetase reaction. J Bacteriol, 2001 Nov, 183(22), 6699 - 706 Integron integrases possess a unique additional domain necessary for activity; Messier N et al.; Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase . Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III) . An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif . We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases . The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases) . Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215 . We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA . W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding . Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion). Biochemistry, 2001 Oct 30, 40(43), 12950 - 8 Ligand-induced changes in the Streptomyces lividans TipAL protein imply an alternative mechanism of transcriptional activation for MerR-like proteins; Chiu ML et al.; TipAL is a Streptomyces transcriptional activator assigned to the MerR/SoxR family based both on homology within its putative DNA recognition domain and the fact that its operator binding sites lie within a region of its promoter normally occupied by RNA polymerase . The tipA gene is also independently translated as the C-terminal ligand-binding domain of TipAL (TipAS; residues 111-254) . Both TipAS and TipAL share broad recognition specificity for cyclic thiopeptide antibiotics . The molecular mechanism by which TipAL catalyzes prokaryotic transcriptional activation at the tipA promoter (ptipA) in response to thiostrepton was studied using a combination of analytical ultracentrifugation (AU), circular dichroism (CD), optical waveguide lightmode spectroscopy (OWLS; a sensitive in situ binding assay), and mutational analyses . AU showed that TipAL, but not TipAS, was a dimer in solution in the presence or absence of thiostrepton . This indicated that activation of TipAL by thiostrepton was not mediated by changes in multimerization and mapped the dimerization domain to its N-terminal 110 amino acids, presumably within amino acids predicted to form a coil-coil domain (residues 77-109) . CD spectra showed that TipAL had more alpha-helical content than TipAS, probably because of the presence of the additional N-terminal region . The helicity of TipAL and TipAS both increased slightly after binding thiostrepton demonstrating conformation changes upon thiostrepton binding . OWLS experiments determined the overall binding constants via measurements of association and dissociation rates for both TipA proteins and RNA polymerase with ptipA . Thiostrepton slightly enhanced the rate of specific association of TipAL with ptipA, but drastically lowered the rate of dissociation from the binding site . TipAL-thiostrepton increased the affinity of RNA polymerase for ptipA more than 10-fold . In conjunction with genetic experiments, we propose that, while there are some similarities, the mechanism by which TipAL activates transcription is distinctly different from the established MerR/SoxR paradigm. Biochemistry, 2001 Oct 30, 40(43), 12754 - 60 The assembly of the PsaD subunit into the membranal photosystem I complex occurs via an exchange mechanism; Minai L et al.; PsaD is a peripheral stromal-facing subunit of photosystem I (PSI), a multisubunit complex of the thylakoid membranes . PsaD plays a major role in both the function and assembly of PSI . Past studies with radiolabeled PsaD indicated that PsaD is able to assemble in vitro specifically into the PSI complex . To unravel the mechanism by which this assembly takes place, the following steps were taken . (i) Mature PsaD of spinach and PsaD of the prokaryotic caynobacterium Mastigocladus laminosus, both bearing a six-histidine tag at their C-termini, were overexpressed in Escherichia coli and purified to homogeneity . (ii) The purified recombinant protein was introduced into the isolated PSI complex . (iii) Following incubation, the PsaD that assembled into PSI was separated from the nonassembled PsaD by