Prokaryotes

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 a sucrose gradient . Differential Western blot analysis was used to determine whether the native and the recombinant PsaD were present as free or assembled proteins of the PSI complex . Antibodies that can recognize only the recombinant PsaD (anti-his) or both the native and recombinant PsaD (anti-PsaD) were used . The findings indicated that an exchange mechanism enables the assembly of a newly introduced PsaD into PSI . The latter replaces the PsaD subunit that is present in situ within the complex . In vivo studies that followed the assembly of PsaD in Chlamydomonas reinhardtii cells supported this in vitro-characterized exchange mechanism . In C . reinhardtii, in the absence of synthesis and assembly of new PSI complexes, newly synthesized PsaD assembled into pre-existing PSI complexes.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4129 - 33
The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system; Chang C et al.; The simple gas ethylene affects numerous physiological processes in the growth and development of higher plants . With the use of molecular genetic approaches, we are beginning to learn how plants perceive ethylene and how this signal is transduced . Components of ethylene signal transduction are defined by ethylene response mutants in Arabidopsis thaliana . The genes corresponding to two of these mutants, etr1 and etr1, have been cloned . The ETR1 gene encodes a homolog of two-component regulators that are known almost exclusively in prokaryotes . The two-component regulators in prokaryotes are involved in the perception and transduction of a wide range of environmental signals leading to adaptive responses . The CTR1 gene encodes a homolog of the Raf family of serine/threonine protein kinases . Raf is part of a mitogen-activated protein kinase cascade known to regulate cell growth and development in mammals, worms, and flies . The ethylene response pathway may, therefore, exemplify a conserved protein kinase cascade regulated by a two-component system . The dominance of all known mutant alleles of ETR1 may be due to either constitutive activation of the ETR1 protein or dominant interference of wild-type activity . The discovery of Arabidopsis genes encoding proteins related to ETR1 suggests that the failure to recover recessive etr1 mutant alleles may be due to the presence of redundant genes.

Proc Natl Acad Sci U S A, 1990 Jan, 87(1), 71 - 4
Do thylakoids really contain phosphatidylcholine?
Dorne AJ, Joyard J, Douce R.
Isolated intact spinach chloroplasts were incubated with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) under mild experimental conditions in which only the phosphatidylcholine localized in the cytosolic leaflet of the outer envelope membrane can be hydrolyzed . Thylakoids, which were protected from phospholipase C degradation, were subsequently prepared from the phospholipase C-treated chloroplasts and found to be devoid of phosphatidylcholine . Previously reported occurrences of phosphatidylcholine in thylakoid preparations probably reflect contamination of the thylakoids by envelope membranes . In the present work, contamination of thylakoids by envelope membranes was determined by measuring the 1,2-diacylglycerol 3-beta-galactosyltransferase {monogalactosyldiacylglycerol (MGDG) synthase; UDPgalactose: 1,2-diacylglycerol 3-beta-D-galactosyltransferase, EC 2.4.1.46} in the different chloroplast subfractions . We conclude that phosphatidylcholine is not present in highly purified thylakoids . Phosphatidylcholine is also absent from prokaryotic cyanobacterial membranes, and our results are in agreement with the endosymbiotic origin of higher plant chloroplasts.

Biophys J, 2001 Nov, 81(5), 2886 - 96
Protonation studies and multivariate curve resolution on oligodeoxynucleotides carrying the mutagenic base 2-aminopurine; Gargallo R et al.; 2-Aminopurine (P) is a mutagen causing A.T to G.C transitions in prokaryotic systems . To study the base-pairing schemes between P and cytosine (C) or thymine (T), two self-complementary dodecamers containing P paired with either C or T were synthesized, and their protonation equilibria were studied by acid-base titrations and melting experiments . The mismatches were incorporated into the self-complementary sequence d(CGCPCCGGXGCG), where X was C or T . Spectroscopic data obtained from molecular absorption, circular dichroism (CD), and molecular fluorescence spectroscopy were analyzed by a factor-analysis-based method, multivariate curve resolution based on the alternating least squares optimization procedure (MCR-ALS) . This procedure allows determination of the number of acid-base species or conformations present in an acid-base or melting experiment and the resolution of the concentration profiles and pure spectra for each of them . Acid-base experiments have shown that at pH 7, 150 mM ionic strength, and 37 degrees C, both C and P are deprotonated . At pH near 4, the majority of species shows C protonated and P deprotonated . Finally, at pH values near 3, the majority of species shows both protonated C and P . These results are in agreement with NMR studies showing a wobble geometry for the P x C base pair and a Watson-Crick geometry for the P x T base pair at neutral pH . Melting experiments were carried out to confirm the proposed acid-base distribution profile . For the sequence including the P x T mismatch, only one transition was observed at neutral pH . However, for the sequence including the P x C mismatch, two transitions were detected by CD but only one by molecular absorption . This behavior agrees with that observed by other authors for oligonucleotides of similar sequence and suggests the following sequence of conformational changes during melting: duplex --> hairpin --> random coil.

Eur J Biochem, 2001 Oct, 268(20), 5386 - 96
Analysis of a putative voltage-gated prokaryotic potassium channel; Ungar D et al.; Most of the completely sequenced prokaryotic genomes contain genes of potassium channel homologues, but there is still not much known about the role of these proteins in prokaryotes . Here we describe the large-scale overproduction and purification of a prokaryotic voltage-gated potassium channel homologue, Kch, from Escherichia coli . After successful overproduction of the protein, a specific increase in the potassium permeability of the cells was found . Kch could be purified in large amounts using classical purification methods to prevent aggregation of the protein . The physiological state of the protein was revealed to be a homotetramer and the protein was shown to be localized to the cytoplasmic membrane of the cells . In the course of the localization studies, we found a specific increase in the density of the cytoplasmic membrane on Kch production . This was linked to the observed increase in the protein to lipid ratio in the membranes . Another observed change in the membrane composition was an increase in the cardiolipin to phosphatidylglycerol ratio, which may indicate a specific cardiolipin requirement of Kch . On the basis of some of our results, we discuss a function for Kch in the maintenance of the membrane potential in E . coli.

FEBS Lett, 2001 Oct 12, 506(3), 257 - 61
A vesicle transport system inside chloroplasts; Westphal S et al.; Intracellular transport via membrane vesicle traffic is a well known feature of eukaryotic cells . Yet, no vesicle transport system has been described for prokaryotes or organelles of prokaryotic origin, such as chloroplasts and mitochondria . Here we show that chloroplasts possess a vesicle transport system with features similar to vesicle traffic in homotypic membrane fusion . Vesicle formation and fusion is affected by specific inhibitors, e.g . nucleotide analogues, protein phosphatase inhibitors and Ca2+ antagonists . This vesicle transfer is an ongoing process in mature chloroplasts indicating that it represents an important new pathway in the formation and maintenance of the thylakoid membranes.

J Mol Biol, 2001 Oct 12, 313(1), 215 - 28
Anatomy of Escherichia coli ribosome binding sites; Shultzaberger RK et al.; During translational initiation in prokaryotes, the 3' end of the 16S rRNA binds to a region just upstream of the initiation codon . The relationship between this Shine-Dalgarno (SD) region and the binding of ribosomes to translation start-points has been well studied, but a unified mathematical connection between the SD, the initiation codon and the spacing between them has been lacking . Using information theory, we constructed a model that treats these three components uniformly by assigning to the SD and the initiation region (IR) conservations in bits of information, and by assigning to the spacing an uncertainty, also in bits . To build the model, we first aligned the SD region by maximizing the information content there . The ease of this process confirmed the existence of the SD pattern within a set of 4122 reviewed and revised Escherichia coli gene starts . This large data set allowed us to show graphically, by sequence logos, that the spacing between the SD and the initiation region affects both the SD site conservation and its pattern . We used the aligned SD, the spacing, and the initiation region to model ribosome binding and to identify gene starts that do not conform to the ribosome binding site model . A total of 569 experimentally proven starts are more conserved (have higher information content) than the full set of revised starts, which probably reflects an experimental bias against the detection of gene products that have inefficient ribosome binding sites . Models were refined cyclically by removing non-conforming weak sites . After this procedure, models derived from either the original or the revised gene start annotation were similar . Therefore, this information theory-based technique provides a method for easily constructing biologically sensible ribosome binding site models . Such models should be useful for refining gene-start predictions of any sequenced bacterial genome .

J Biol Chem, 2002 Jan 11, 277(2), 1388 - 97 Epub 2001 Oct 12.
Solution structure of the fibronectin type III domain from Bacillus circulans WL-12 chitinase A1; Jee JG et al.; Growing evidence suggests that horizontal gene transfer plays an integral role in the evolution of bacterial genomes . One of the debated examples of horizontal gene transfer from animal to prokaryote is the fibronectin type III domain (FnIIID) . Certain extracellular proteins of soil bacteria contain an unusual cluster of FnIIIDs, which show sequence similarity to those of animals and are likely to have been acquired horizontally from animals . Here we report the solution structure of the FnIIID of chitinase A1 from Bacillus circulans WL-12 . To the best of our knowledge, this is the first tertiary structure to be reported for an FnIIID from a bacterial protein . The structure of the domain shows significant similarity to FnIIIDs from animal proteins . Sequence comparisons with FnIIIDs from other soil bacteria proteins show that the core-forming residues are highly conserved and, thus, are under strong evolutionary pressure . Striking similarities in the tertiary structures of bacterial FnIIIDs and their mammalian counterparts may support the hypothesis that the evolution of the FnIIID in bacterial carbohydrases occurred horizontally . The total lack of surface-exposed aromatic residues also suggests that the role of this FnIIID is different from those of other bacterial beta-sandwich domains, which function as carbohydrate-binding modules.

Plant Physiol, 2001 Oct, 127(2), 529 - 42
Cellulose in cyanobacteria . Origin of vascular plant cellulose synthase?
Nobles DR, Romanovicz DK, Brown RM Jr.
Although cellulose biosynthesis among the cyanobacteria has been suggested previously, we present the first conclusive evidence, to our knowledge, of the presence of cellulose in these organisms . Based on the results of x-ray diffraction, electron microscopy of microfibrils, and cellobiohydrolase I-gold labeling, we report the occurrence of cellulose biosynthesis in nine species representing three of the five sections of cyanobacteria . Sequence analysis of the genomes of four cyanobacteria revealed the presence of multiple amino acid sequences bearing the DDD35QXXRW motif conserved in all cellulose synthases . Pairwise alignments demonstrated that CesAs from plants were more similar to putative cellulose synthases from Anabaena sp . Pasteur Culture Collection 7120 and Nostoc punctiforme American Type Culture Collection 29133 than any other cellulose synthases in the database . Multiple alignments of putative cellulose synthases from Anabaena sp . Pasteur Culture Collection 7120 and N . punctiforme American Type Culture Collection 29133 with the cellulose synthases of other prokaryotes, Arabidopsis, Gossypium hirsutum, Populus alba x Populus tremula, corn (Zea mays), and Dictyostelium discoideum showed that cyanobacteria share an insertion between conserved regions U1 and U2 found previously only in eukaryotic sequences . Furthermore, phylogenetic analysis indicates that the cyanobacterial cellulose synthases share a common branch with CesAs of vascular plants in a manner similar to the relationship observed with cyanobacterial and chloroplast 16s rRNAs, implying endosymbiotic transfer of CesA from cyanobacteria to plants and an ancient origin for cellulose synthase in eukaryotes.

Plant Cell, 2001 Oct, 13(10), 2373 - 84
A role for initiation codon context in chloroplast translation; Esposito D et al.; To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the -1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii . In prokaryotes, the -1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA . We found that in vivo, none of the chloroplast mutations affected mRNA stability . However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon . These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo . Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo . Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.

J Biol Chem, 2001 Dec 14, 276(50), 46917 - 24 Epub 2001 Oct 10.
Substrate water exchange in photosystem II depends on the peripheral proteins; Hillier W et al.; The (18)O exchange rates for the substrate water bound in the S(3) state were determined in different photosystem II sample types using time-resolved mass spectrometry . The samples included thylakoid membranes, salt-washed Triton X-100-prepared membrane fragments, and purified core complexes from spinach and cyanobacteria . For each sample type, two kinetically distinct isotopic exchange rates could be resolved, indicating that the biphasic exchange behavior for the substrate water is inherent to the O(2)-evolving catalytic site in the S(3) state . However, the fast phase of exchange became somewhat slower (by a factor of approximately 2) in NaCl-washed membrane fragments and core complexes from spinach in which the 16- and 23-kDa extrinsic proteins have been removed, compared with the corresponding rate for the intact samples . For CaCl(2)-washed membrane fragments in which the 33-kDa manganese stabilizing protein (MSP) has also been removed, the fast phase of exchange slowed down even further (by a factor of approximately 3) . Interestingly, the slow phase of exchange was little affected in the samples from spinach . For core complexes prepared from Synechocystis PCC 6803 and Synechococcus elongatus, the fast and slow exchange rates were variously affected . Nevertheless, within the experimental error, nearly the same exchange rates were measured for thylakoid samples made from wild type and an MSP-lacking mutant of Synechocystis PCC 6803 . This result could indicate that the MSP has a slightly different function in eukaryotic organisms compared with prokaryotic organisms . In all samples, however, the differences in the exchange rates are relatively small . Such small differences are unlikely to arise from major changes in the metal-ligand structure at the catalytic site . Rather, the observed differences may reflect subtle long range effects in which the exchange reaction coordinates become slightly altered . We discuss the results in terms of solvent penetration into photosystem II and the regional dielectric around the catalytic site.

Mutat Res, 2001 Nov 1, 487(1-2), 67 - 71
SUVi and BACH1: a new subfamily of mammalian helicases?
Menichini P, Linial M.
The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in prokaryotes and eukaryotes . Members of this family are genes responsible for cancer predisposition disorders like Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome . Here, we show the sequence homologies between two recently identified mammalian helicases, namely SUVi and BACH1 . These two genes also share strong homologies with other members of the RecQ family . On the basis of published data and sequence analysis we suggest that SUVi/BACH1 may represent a novel subfamily of mammalian helicases, functioning in the processing of lesions induced by different genotoxic agents.

Biochem Biophys Res Commun, 2001 Oct 19, 288(1), 69 - 74
Structural and functional characterization of human NAD kinase; Lerner F et al.; NADP is essential for biosynthetic pathways, energy, and signal transduction . Its synthesis is catalyzed by NAD kinase . Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms . We identified a human NAD kinase cDNA and the corresponding gene using available database information . A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli . The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa . The gene is expressed in most human tissues, but not in skeletal muscle . Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa) . The catalytically active homotetramer is highly selective for its substrates, NAD and ATP . It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route .

J Protein Chem, 2001 May, 20(4), 311 - 5
Expression and purification of recombinant hemoglobin I from Lucina pectinata; Rosado-Ruiz T et al.; Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric sulfide complex needed to transport H2S to the bacterial endosymbiont . To further study HbI, expression studies of this protein were performed in Escherichia coli . This is the first time that the recombinant HbI was produced using a recombinant DNA expression system . Hemoglobin I cDNA was amplified and cloned into the TOPO-PBAD expression vector, which contains a fusion tag of six histidine residues (6XHis tag) . Plasmid clone sequence analysis was carried out in order to ensure that the insert was in the correct reading frame for proper protein expression in E . coli . The expression of recombinant HbI was optimal when induced for 5 hr with 0.002% of L-arabinose as detected by Western blot analysis . The proto-porphyrin group was inserted into the recombinant HbI . Purification of the heme-bound recombinant protein was performed under native conditions by affinity chromatography using Ni-NTA and Probond resins . The sodium dithionite-reduced recombinant protein presented a shift from the Soret band at 413-435 nm, indicating the presence of the heme group in the adequate amino acid environment of HbI . These results indicate that recombinant HbI from Lucina pectinata can be successfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding.

Mol Genet Metab, 2001 Sep-Oct, 74(1-2), 147 - 59
Queuosine modification of tRNA: a case for convergent evolution; Morris RC et al.; Queuosine is a hypermodified nucleoside found in position 34, the anticodon wobble position, of four tRNA species . This modification is distributed with near uniformity across all life forms found on this planet . Yet the molecular mechanisms involved with accomplishing this ubiquitous posttranscriptional modification of tRNA are dramatically different between prokaryotic and eukaryotic organisms, which suggests that these were formed by convergent evolution of a fundamental life process essential to nearly all life forms . This minireview describes the differences between these modification systems and points to a new direction for developing research on the molecular function queuosine-modified tRNA in diverse species .

J Immunol, 2001 Oct 15, 167(8), 4616 - 26
Intracisternally localized bacterial DNA containing CpG motifs induces meningitis; Deng GM et al.; Unmethylated CpG motifs are frequently found in bacterial DNA, and have recently been shown to exert immunostimulatory effects on leukocytes . Since bacterial infections in the CNS will lead to local release of prokaryotic DNA, we wanted to investigate whether such an event might trigger meningitis . To that end, we have intracisternally injected mice and rats with bacterial DNA and oligonucleotides containing CpG motifs . Histopathological signs of meningitis were evident within 12 h and lasted for at least 14 days, and were characterized by an influx of monocytic, Mac-3(+) cells and by a lack of T lymphocytes . To study the mechanisms whereby unmethylated CpG DNA gives rise to meningitis, we deleted the monocyte/macrophage population leading to abrogation of brain inflammation . Also, interaction with NF-kappaB using antisense technology led to down-regulation of proinflammatory cytokine production and frequency of meningitis . Furthermore, specific interactions with vascular selectin expression and inhibition of NO synthase led to a significant amelioration of meningitis, altogether indicating that this condition is dependent on macrophages and their products . In contrast, neutrophils, NK cells, T/B lymphocytes, IL-12, and complement system were not instrumental in meningitis triggered by bacterial DNA containing CpG motifs . This study proves that bacterial DNA containing unmethylated CpG motifs induces meningitis, and indicates that this condition is mediated in vivo by activated macrophages.

FEBS Lett, 2001 Sep 28, 506(1), 6 - 10
The polymerization mechanism of the bacterial cell division protein FtsZ; Scheffers D et al.; Bacteria and archaea usually divide symmetrically by formation of a septum in the middle of the cell . A key event in cell division is the assembly of the FtsZ ring . FtsZ is the prokaryotic homolog of tubulin and forms polymers in the presence of guanine nucleotides . Here, we specifically address the polymerization of FtsZ and the role of nucleotide hydrolysis in polymer formation and stabilization . Recent structural and biochemical results are discussed and a model for FtsZ polymerization, similar to that for tubulin, is presented.

Structure (Camb), 2001 Oct, 9(10), 941 - 53
Structure of E . coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase reveals similarity to the purine nucleoside phosphorylases; Lee JE et al.; BACKGROUND: 5'-methylthioadenosine/S-adenosyl-homocysteine (MTA/AdoHcy) nucleosidase catalyzes the irreversible cleavage of 5'-methylthioadenosine and S-adenosylhomocysteine to adenine and the corresponding thioribose, 5'-methylthioribose and S-ribosylhomocysteine, respectively . While this enzyme is crucial for the metabolism of AdoHcy and MTA nucleosides in many prokaryotic and lower eukaryotic organisms, it is absent in mammalian cells . This metabolic difference represents an exploitable target for rational drug design . RESULTS: The crystal structure of E . coli MTA/AdoHcy nucleosidase was determined at 1.90 A resolution with the multiwavelength anomalous diffraction (MAD) technique . Each monomer of the MTA/AdoHcy nucleosidase dimer consists of a mixed alpha/beta domain with a nine-stranded mixed beta sheet, flanked by six alpha helices and a small 3(10) helix . Intersubunit contacts between the two monomers present in the asymmetric unit are mediated primarily by helix-helix and helix-loop hydrophobic interactions . The unexpected presence of an adenine molecule in the active site of the enzyme has allowed the identification of both substrate binding and potential catalytic amino acid residues . CONCLUSIONS: Although the sequence of E . coli MTA/AdoHcy nucleosidase has almost no identity with any known enzyme, its tertiary structure is similar to both the mammalian (trimeric) and prokaryotic (hexameric) purine nucleoside phosphorylases . The structure provides evidence that this protein is functional as a dimer and that the dual specificity for MTA and AdoHcy results from the truncation of a helix . The structure of MTA/AdoHcy nucleosidase is the first structure of a prokaryotic nucleoside N-ribohydrolase specific for 6-aminopurines.

Trends Biochem Sci, 2001 Oct, 26(10), 582 - 4
CHASE: an extracellular sensing domain common to transmembrane receptors from prokaryotes, lower eukaryotes and plants; Mougel C et al.; A novel extracellular ligand-binding domain, termed CHASE, is described in sensory adenylyl and diguanylate cyclases, and histidine kinases, in several bacterial species, Dictyostelium and plants . The CHASE domain is predicted to sense stimuli that are specific for the developmental program of an organism.

Pharmacol Toxicol, 2001 Sep, 89(3), 113 - 22
Gene induction by Phenobarbital: an update on an old question that receives key novel answers; Corcos L et al.; Phenobarbital has long been used as a sedative and antiepileptic drug . The drug is the representative of a myriad of lipophilic molecules able to evoke a pleiotropic response in the liver and also in prokaryotes and flies . A great deal of novel information has been obtained in recent years regarding the mechanism of cytochrome P450 (CYP) gene induction by phenobarbital . Most importantly, a nuclear orphan receptor, the constitutive androstane receptor has been identified as a primary determinant of the transcriptional activation of CYP genes in response to phenobarbital-like inducers in mammals . Another nuclear receptor, the pregnane X receptor can also mediate some of the phenobarbital response, but the functional overlap of the two inductive pathways is only partial . The response of mammalian CYP2B genes to phenobarbital was abolished in the liver of mice carrying a null allele of the constitutive androstane receptor gene, whereas that of CYP3A genes was lost in pregnane X receptor knock-out mice.

Peptides, 2001 Oct, 22(10), 1555 - 77
A peptide profile of the Bacillus subtilis genome; Zuber P; Bacillus subtilis is known to produce an abundance of small polypeptides . Several of these have antimicrobial activity and others are pheromones or extracellular factors that affect internal signal transduction systems . The completion of the B . subtilis genomic nucleotide sequence has revealed 345 small polypeptide open-reading frames (of 85 codons or less), 81% of which are of unknown function . A significant number of these reside in prophage genomes or phage-like elements where they can be organized into large operons . It is likely that many more exist in the genome of B . subtilis but are "hidden" entirely or partially within other reading frames, or possess non-conventional translation start signals and have escaped detection . The discovery of so many small polypeptide orfs (SPORFs) and the likelihood of many more pose a challenging problem for those undertaking the complete functional analysis of genes that constitute prokaryotic genomes . A survey of known and potential peptide-encoding reading frames is presented herein as an attempt to classify those that are found in the B . subtilis genome according to function inferred from homology searches and to conservation among products of other microbial genomes.

Plant Mol Biol, 2001 Oct, 47(3), 353 - 66
Analysis of barley chloroplast psbD light-responsive promoter elements in transplastomic tobacco; Thum KE et al.; The plastid gene psbD encodes D2, a photosystem II reaction center chlorophyll-binding protein . psbD is transcribed from a conserved chloroplast promoter that is activated by blue, white, or UV-A light . In this study, various forms of the barley (Hordeum vulgare L.) chloroplast psbD-LRP were fused to the uidA reporter gene and introduced into the tobacco (Nicotiana tabacum L.) plastid genome through homologous recombination . Primer extension analysis of transcripts from the psbD-LRP-uidA construct showed that the barley psbD-LRP was activated in tobacco by blue or white light . Transcription from this construct was also regulated by circadian cycling indicating that the barley psbD-LRP could respond to light modulated regulatory pathways in tobacco . Mutation of the psbD-LRP prokaryotic -10 promoter element reduced transcription to very low levels in all light regimes . In contrast, mutation of a prokaryotic -35 promoter element had no effect on transcription from the psbD-LRP . Deletion or mutation of an upstream activating element, the AAG-box (-36 to -64), also reduced transcription from the construct to very low levels . In contrast, deletion of the upstream PGT-box (-71 to -100) did not alter promoter activation by blue light, or responsiveness to circadian cycling . These in vivo studies confirm the importance of the psbD-LRP -10 promoter element and AAG-box in light regulation and demonstrate that these elements are sufficient to mediate circadian cycling of the barley psbD promoter.

Methods Enzymol, 2001, 342, 143 - 58
Escherichia coli ribonuclease III: affinity purification of hexahistidine-tagged enzyme and assays for substrate binding and cleavage; Amarasinghe AK et al.; It is now evident that members of the RNase III family of nucleases have central roles in prokaryotic and eukaryotic RNA maturation and decay pathways . Ongoing research is uncovering new roles for RNase III homologs . For example, the phenomena of RNA interference (RNAi) and posttranscriptional gene silencing (PTGS) involve dsRNA processing, carried out by an RNase III homolog . We anticipate an increased focus on the mechanism, regulation, and biological roles of RNase III orthologs . Although the differences in the physicochemical properties of RNase III orthologs, and distinct substrate reactivity epitopes and ionic requirements for optimal activity, may mean that the protocols describe here are not strictly transferrable, the affinity purification methodology, and substrate preparation and use should be generally applicable.

Nat Biotechnol, 2001 Oct, 19(10), 974 - 7
A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli; Cottingham IR et al.; The increasing use of peptides as pharmaceutical agents, especially in the antiviral and anti-infective therapeutic areas, requires cost-effective production on a large scale . Many peptides need carboxy amidation for full activity or prolonged bioavailability . However, this modification is not possible in prokaryotes and must be done using recombinant enzymes or by expression in transgenic milk . Methods employing recombinant enzymes are appropriate for small-scale production, whereas transgenic milk expression is suitable for making complex disulfide-containing peptides required in large quantity . Here we describe a method for making amidated peptides using a modified self-cleaving vacuolar membrane ATPase (VMA) intein expression system . This system is suitable for making amidated peptides at a laboratory scale using readily available constructs and reagents . Further improvements are possible, such as reducing the size of the intein to improve the peptide yields (the VMA intein comprises 454 amino acids) and, if necessary, secreting the fusion protein to ensure correct N-terminal processing to the peptide . With such developments, this method could form the basis of a large-scale cost-effective system for the bulk production of amidated peptides without the use of recombinant enzymes or the need to cleave fusion proteins.

J Struct Biol, 2001 Aug, 135(2), 205 - 18
Analysis of the interaction between the eukaryotic chaperonin CCT and its substrates actin and tubulin; Llorca O et al.; Two mechanisms have thus far been characterized for the assistance by chaperonins of the folding of other proteins . The first and best described is that of the prokaryotic chaperonin GroEL, which interacts with a large spectrum of proteins . GroEL uses a nonspecific mechanism by which any conformation of practically any unfolded polypeptide interacts with it through exposed, hydrophobic residues . ATP binding liberates the substrate in the GroEL cavity where it is given a chance to fold . A second mechanism has been described for the eukaryotic chaperonin CCT, which interacts mainly with the cytoskeletal proteins actin and tubulin . Cryoelectron microscopy and biochemical studies have revealed that both of these proteins interact with CCT in quasi-native, defined conformations . Here we have performed a detailed study of the docking of the actin and tubulin molecules extracted from their corresponding CCT:substrate complexes obtained from cryoelectron microscopy and image processing to localize certain regions in actin and tubulin that are involved in the interaction with CCT . These regions of actin and tubulin, which are not present in their prokaryotic counterparts FtsA and FtsZ, are involved in the polymerization of the two cytoskeletal proteins . These findings suggest coevolution of CCT with actin and tubulin in order to counteract the folding problems associated with the generation in these two cytoskeletal protein families of new domains involved in their polymerization .

J Struct Biol, 2001 Aug, 135(2), 170 - 5
Review: nucleotide-dependent conformational changes of the chaperonin containing TCP-1; Melki R; Current biochemical and structural studies on the conformational changes induced by the nature of nucleotide bound to the chaperonin containing testis complex polypeptide 1 (CCT) are examined to see how consistent the data are . This exercise suggests that the biochemical and structural data are in good agreement . CCT clearly appears as a folding nano-machine fueled by ATP . A careful comparison of the biochemical and structural data, however, highlights a number of points that remain to be carefully documented in order to better understand the nature of the conformational changes in CCT that yield folded target proteins . Special effort should be made to clearly answer the points listed at the end of this review in order to obtain the dynamic sequence of events yielding folded proteins in the eukaryotic cytoplasm similar to what has been obtained for prokaryotes .

Clin Immunol, 2001 Oct, 101(1), 100 - 5
IgG autoantibodies in patients with anti-epiligrin cicatricial pemphigoid recognize the G domain of the laminin 5 alpha-subunit; Lazarova Z et al.; Anti-epiligrin cicatricial pemphigoid (AECP) is a mucosal-predominant, subepithelial blistering disease characterized by IgG anti-basement membrane autoantibodies to laminin 5 (alpha3beta3gamma2) . This and prior studies found that autoantibodies from most patients recognize the alpha-subunit of this laminin isoform . Accordingly, sera from 10 representative patients were tested against prokaryotic recombinants of this polypeptide in epitope mapping studies . cDNAs spanning the full length of the alpha-subunit were generated by PCR, directionally cloned into the pGEX-4T-3 vector, and expressed as glutathione-S-transferase fusion proteins of appropriate size and immunoreactivity . Sera from 9 of 10 AECP patients immunoblotted fusion proteins corresponding to subdomains G2, G3, G4, and G5 at the carboxyl terminus of the laminin 5 alpha-subunit . Serum from 1 patient (and that from normal volunteers) showed no reactivity to any fusion proteins; no sera bound recombinant glutathione-S-transferase alone . Immunoadsorption of patient sera with fusion proteins corresponding to the G domain substantially reduced basement membrane autoantibody titers . IgG from patients with this form of cicatricial pemphigoid recognize the portion of laminin 5 thought to play a key role in promoting keratinocyte adhesion to epidermal basement membrane .

Nucleic Acids Res, 2001 Oct 1, 29(19), 4079 - 88
Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting; Shigemoto K et al.; Translational recoding of mRNA through a -1 ribosomal slippage mechanism has been observed in RNA viruses and retrotransposons of both eukaryotes and prokaryotes . Whilst this provides a potentially powerful mechanism of gene regulation, the utilization of -1 translational frameshifting in regulating mammalian gene expression has remained obscure . Here we report a mammalian gene, Edr, which provides the first example of -1 translational recoding in a eukaryotic cellular gene . In addition to bearing functional frameshift elements that mediate expression of distinct polypeptides, Edr bears both CCHC zinc-finger and putative aspartyl protease catalytic site retroviral-like motifs, indicative of a relic retroviral-like origin for Edr . These features, coupled with conservation of Edr as a single copy gene in mouse and man and striking spatio-temporal regulation of expression during embryogenesis, suggest that Edr plays a functionally important role in mammalian development.

Nucleic Acids Res, 2001 Oct 1, 29(19), 3928 - 38
A computational approach to identify genes for functional RNAs in genomic sequences; Carter RJ et al.; Currently there is no successful computational approach for identification of genes encoding novel functional RNAs (fRNAs) in genomic sequences . We have developed a machine learning approach using neural networks and support vector machines to extract common features among known RNAs for prediction of new RNA genes in the unannotated regions of prokaryotic and archaeal genomes . The Escherichia coli genome was used for development, but we have applied this method to several other bacterial and archaeal genomes . Networks based on nucleotide composition were 80-90% accurate in jackknife testing experiments for bacteria and 90-99% for hyperthermophilic archaea . We also achieved a significant improvement in accuracy by combining these predictions with those obtained using a second set of parameters consisting of known RNA sequence motifs and the calculated free energy of folding . Several known fRNAs not included in the training datasets were identified as well as several hundred predicted novel RNAs . These studies indicate that there are many unidentified RNAs in simple genomes that can be predicted computationally as a precursor to experimental study . Public access to our RNA gene predictions and an interface for user predictions is available via the web.

Genome Biol . 2001;2(9):RESEARCH 0033 . Epub 2001 Aug 30.
Two C or not two C: recurrent disruption of Zn-ribbons, gene duplication, lineage-specific gene loss, and horizontal gene transfer in evolution of bacterial ribosomal proteins; Makarova KS et al.; BACKGROUND: Ribosomal proteins are encoded in all genomes of cellular life forms and are, generally, well conserved during evolution . In prokaryotes, the genes for most ribosomal proteins are clustered in several highly conserved operons, which ensures efficient co-regulation of their expression . Duplications of ribosomal-protein genes are infrequent, and given their coordinated expression and functioning, it is generally assumed that ribosomal-protein genes are unlikely to undergo horizontal transfer . However, with the accumulation of numerous complete genome sequences of prokaryotes, several paralogous pairs of ribosomal protein genes have been identified . Here we analyze all such cases and attempt to reconstruct the evolutionary history of these ribosomal proteins . RESULTS: Complete bacterial genomes were searched for duplications of ribosomal proteins . Ribosomal proteins L36, L33, L31, S14 are each duplicated in several bacterial genomes and ribosomal proteins L11, L28, L7/L12, S1, S15, S18 are so far duplicated in only one genome each . Sequence analysis of the four ribosomal proteins, for which paralogs were detected in several genomes, two of the ribosomal proteins duplicated in one genome (L28 and S18), and the ribosomal protein L32 showed that each of them comes in two distinct versions . One form contains a predicted metal-binding Zn-ribbon that consists of four conserved cysteines (in some cases replaced by histidines), whereas, in the second form, these metal-chelating residues are completely or partially replaced . Typically, genomes containing paralogous genes for these ribosomal proteins encode both versions, designated C+ and C-, respectively . Analysis of phylogenetic trees for these seven ribosomal proteins, combined with comparison of genomic contexts for the respective genes, indicates that in most, if not all cases, their evolution involved a duplication of the ancestral C+ form early in bacterial evolution, with subsequent alternative loss of the C+ and C- forms in different lineages . Additionally, evidence was obtained for a role of horizontal gene transfer in the evolution of these ribosomal proteins, with multiple cases of gene displacement 'in situ', that is, without a change of the gene order in the recipient genome . CONCLUSIONS: A more complex picture of evolution of bacterial ribosomal proteins than previously suspected is emerging from these results, with major contributions of lineage-specific gene loss and horizontal gene transfer . The recurrent theme of emergence and disruption of Zn-ribbons in bacterial ribosomal proteins awaits a functional interpretation.

Biochem Soc Symp, 2001, (68), 45 - 68
Contribution of molecular chaperones to protein folding in the cytoplasm of prokaryotic and eukaryotic cells; Naylor DJ et al.; While it is clear that many unfolded proteins can attain their native state spontaneously in vitro, the efficiency of such folding is usually limited to conditions far removed from those encountered within cells . Two properties of the cellular environment are expected to enhance strongly the propensity of incompletely folded polypeptides to misfold and aggregate: the crowding effect caused by the high concentration of macromolecules, and the close proximity of nascent polypeptide chains emerging from polyribosomes . However, in the living cell, non-productive protein folding is in many, if not most, cases prevented by the action of a highly conserved set of proteins termed molecular chaperones . In the cytoplasm, the Hsp70 (heat-shock protein of 70 kDa) and chaperonin families of molecular chaperones appear to be the major contributors to efficient protein folding during both normal conditions and adverse conditions such as heat stress . Hsp70 chaperones recognize and shield short, hydrophobic peptide segments in the context of non-native polypeptides and probably promote folding by decreasing the concentration of aggregation-prone intermediates . In contrast, the chaperonins interact with and globally enclose collapsed folding intermediates in a central cavity where efficient folding can proceed in a protected environment . For a number of proteins, folding requires the co-ordinated action of both of these molecular chaperones.

Protein Expr Purif, 2001 Oct, 23(1), 128 - 33
High-level expression and one-step purification of cyclic amidohydrolase family enzymes; Kim GJ et al.; The cyclic amidohydrolase family enzymes, including hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in prokaryotic and eukaryotic cells . With the increasing demand for the elucidation of enzyme structures and functions, along with industrial applications, the research on the family enzymes has recently been proliferating, but the related enzymes had been purified conventionally by multistep purification procedures . Here, we reported the expression in Escherichia coli cells of maltose-binding protein-fused family enzymes and their one-step purification . The expression levels of the fusion proteins account for 20-35% of the total protein in E . coli, allowing approximately 2-3 mg of the purified proteins by affinity chromatography to be obtained per 0.3 L of bacterial culture . As more promising results, their nascent biochemical properties, after the cleavage of the fusion proteins with Factor Xa, in terms of oligomeric structure, optimal pH, specific activity, and kinetic property, were also conserved as those from the native enzymes . The availability of the family enzymes to fusion strategy shows potential as a convenient procedure to recombinant protein purification and accelerates the structure-function study of the related family enzymes .

Biotechniques . 2001 Sep;31(3):636, 638, 640, passim.
Bioinformatics: use in bacterial vaccine discovery; Zagursky RJ et al.; Bioinformatics has now become a common laboratory name for groups studying genomic sequences . It is composed of many different, yet interrelated scientific fields such as genomics, proteomics, and transcriptional profiling . The availability of complete genomic sequences, especially prokaryotic organisms, allows one to rapidly identify, analyze, and clone genes of interest . For bacterial vaccine discovery, one can "mine" the genomic sequence for potential surface targets using various algorithms, characterize these gene targets, and produce primers for cloning, all before one enters the wet laboratory . This review will focus on various genomic mining tools/algorithms available for predicting open reading frames and their associated annotation (if known), physical and functional characterization, and cellular localization . Finally, examples are given of how all of this is being used for the identification of potential bacterial vaccine candidates.

Biofactors, 2001, 14(1-4), 53 - 9
Functional analysis of prokaryotic SELB proteins; Thanbichler M et al.; Since the discovery of selenocysteine as the 21st amino acid considerable progress has been made in elucidating the system responsible for its insertion into proteins . Elongation factor SELB, whose amino-terminal part shows homology to EF-Tu, was found to be the key component mediating delivery of selenocysteyl-tRNA(Sec) to the ribosomal A site . It exhibits a distinct tertiary structure comprising binding sites for guanosine nucleotides, the cognate tRNA, an mRNA secondary structure (SECIS element) and presumably ribosomal components . The kinetics of interaction of SELB with its ligands have been studied in detail . GDP was found to bind with about 20-fold lower affinity than GTP and to be in rapid exchange, which obviates the need for a guanosine nucleotide exchange factor . The affinity of SELB for the SECIS element is in the range of 1 nM and further increases upon binding of selenocysteyl-tRNA(Sec) to the protein . This supports the model that SELB forms a tight quaternary complex on the SECIS element which is loosened after insertion of the tRNA into the ribosomal A site and the concomitant hydrolysis of GTP.

Biofactors, 2001, 14(1-4), 17 - 24
Selenocysteine incorporation directed from the 3'UTR: characterization of eukaryotic EFsec and mechanistic implications; Berry MJ et al.; The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms . After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes . A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid . The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA . Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.

Protein Sci, 2001 Oct, 10(10), 1970 - 9
Comparing function and structure between entire proteomes; Liu J et al.; More than 30 organisms have been sequenced entirely . Here, we applied a variety of simple bioinformatics tools to analyze 29 proteomes for representatives from all three kingdoms: eukaryotes, prokaryotes, and archaebacteria . We confirmed that eukaryotes have relatively more long proteins than prokaryotes and archaes, and that the overall amino acid composition is similar among the three . We predicted that approximately 15%-30% of all proteins contained transmembrane helices . We could not find a correlation between the content of membrane proteins and the complexity of the organism . In particular, we did not find significantly higher percentages of helical membrane proteins in eukaryotes than in prokaryotes or archae . However, we found more proteins with seven transmembrane helices in eukaryotes and more with six and 12 transmembrane helices in prokaryotes . We found twice as many coiled-coil proteins in eukaryotes (10%) as in prokaryotes and archaes (4%-5%), and we predicted approximately 15%-25% of all proteins to be secreted by most eukaryotes and prokaryotes . Every tenth protein had no known homolog in current databases, and 30%-40% of the proteins fell into structural families with >100 members . A classification by cellular function verified that eukaryotes have a higher proportion of proteins for communication with the environment . Finally, we found at least one homolog of experimentally known structure for approximately 20%-45% of all proteins; the regions with structural homology covered 20%-30% of all residues . These numbers may or may not suggest that there are 1200-2600 folds in the universe of protein structures . All predictions are available at http://cubic.bioc.columbia.edu/genomes.

J Bacteriol, 2001 Oct, 183(20), 6028 - 35
Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes; Klein M et al.; A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria . In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined . In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny . For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences . The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions . A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes . Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica . The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits . This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor . Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.

J Bacteriol, 2001 Oct, 183(20), 5826 - 33
Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans; Margelis S et al.; Glutamine synthetase (GS), EC 6.3.1.2, is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine . We have isolated the Aspergillus nidulans glnA gene encoding GS and have shown that glnA encodes a highly expressed but not highly regulated mRNA . Inactivation of glnA results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid . Even when supplemented with high levels of glutamine, strains lacking a functional glnA gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy . Heterologous expression of the prokaryotic Anabaena glnA gene from the A . nidulans alcA promoter allowed full complementation of the A . nidulans glnADelta mutation . However, the A . nidulans fluG gene, which encodes a protein with similarity to prokaryotic GS, did not replace A . nidulans glnA function when similarly expressed . Our studies with the glnADelta mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression . Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.

Toxicol In Vitro, 2001 Aug-Oct, 15(4-5), 469 - 75
Whole cell biosensors--electrochemical and optical approaches to ecotoxicity testing; Bentley A et al.; Two different approaches to the interrogation of prokaryotic and eukaryotic cells have been explored, with the objective of developing biosensor based rapid ecotoxicity test protocols for use in a wide range of applications . Prokaryotic cells and some eukaryotic cell types lend themselves to interrogation by mediated amperometry, a technique that allows metabolic activity of the cell to be monitored by accessing cellular redox events with a chemical mediator . Reduction of the mediator by the cell is followed by re-oxidation at an electrode surface poised at a fixed potential . The resulting current flow is proportional to the metabolic status of the cell . A commercial ecotoxicity test, CellSense, employing such whole cell biosensors has now been developed . An alternative approach to interrogation of vertebrate cells has been the use of luminescent reporter genes to determine changes in the metabolic status of the cells following environmental challenge . Several clones have been established of epithelial cells from the bluegill sunfish (BF-2), transfected with the pCIneoLuc plasmid, that express luciferase constitutively . Protocols using these transformed cells are being developed to provide an alternative to the standard neutral red retention test.

Structure (Camb), 2001 Sep, 9(9), 859 - 67
The conformation of bound GMPPNP suggests a mechanism for gating the active site of the SRP GTPase; Padmanabhan S et al.; BACKGROUND: The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that mediates cotranslational targeting of secreted and membrane proteins to the membrane . Targeting is regulated by GTP binding and hydrolysis events that require direct interaction between structurally homologous "NG" GTPase domains of the SRP signal recognition subunit and its membrane-associated receptor, SR alpha . Structures of both the apo and GDP bound NG domains of the prokaryotic SRP54 homolog, Ffh, and the prokaryotic receptor homolog, FtsY, have been determined . The structural basis for the GTP-dependent interaction between the two proteins, however, remains unknown . RESULTS: We report here two structures of the NG GTPase of Ffh from Thermus aquaticus bound to the nonhydrolyzable GTP analog GMPPNP . Both structures reveal an unexpected binding mode in which the beta-phosphate is kinked away from the binding site and magnesium is not bound . Binding of the GTP analog in the canonical conformation found in other GTPase structures is precluded by constriction of the phosphate binding P loop . The structural difference between the Ffh complex and other GTPases suggests a specific conformational change that must accompany movement of the nucleotide from an "inactive" to an "active" binding mode . CONCLUSIONS: Conserved side chains of the GTPase sequence motifs unique to the SRP subfamily may function to gate formation of the active GTP bound conformation . Exposed hydrophobic residues provide an interaction surface that may allow regulation of the GTP binding conformation, and thus activation of the GTPase, during the association of SRP with its receptor.

J Mol Biol, 2001 Sep 21, 312(3), 425 - 38
Transgenic plastids in basic research and plant biotechnology; Bock R; Facile methods of genetic transformation are of outstanding importance for both basic and applied research . For many years, transgenic technologies for plants were restricted to manipulations of the nuclear genome . More recently, a second genome of the plant cell has become amenable to genetic engineering: the prokaryotically organized circular genome of the chloroplast . The possibility to directly manipulate chloroplast genome-encoded information has paved the way to detailed in vivo studies of virtually all aspects of plastid gene expression . Moreover, plastid transformation technologies have been intensely used in functional genomics by performing gene knockouts and site-directed mutageneses of plastid genes . These studies have contributed greatly to our understanding of the physiology and biochemistry of biogenergetic processes inside the plastid compartment . Plastid transformation technologies have also stirred considerable excitement among plant biotechnologists, since transgene expression from the plastid genome offers a number of most attractive advantages, including high-level foreign protein expression and transgene containment due to lack of pollen transmission . This review describes the generation of plants with transgenic plastids, summarizes our current understanding of the transformation process and highlights selected applications of transplastomic technologies in basic and applied research .

Curr Med Chem, 2001 Dec, 8(14), 1713 - 26
The role of genomics in antibacterial target discovery; Buysse JM; Complete DNA sequence information has now been obtained for several prokaryotic genomes, defining the entire genetic complement of these organisms . The collection of genomic data has provided new insights into the molecular architecture of bacterial cells, revealing the basic genetic and metabolic structures that support viability of the organisms . Genomic information has also revealed new avenues for inhibition of bacterial growth and viability, expanding the number of possible drug targets for antibiotic discovery . This review examines how genomic sciences and experimental tools are applied to antibacterial target discovery, the necessary first step in the development of new antibiotic classes . Significant advances have been realized in the development of functional genomic, comparative genomic, and proteomic methods for the analysis of completed genomes . The combination of these methods can be used to systematically parse the genome and identify targets worthy of inhibitor screens . Two basic categories of targets emerge from this exercise, comprising in vitro essential targets required for bacterial viability on synthetic media and in vivo essential targets required to establish and maintain infection within a host organism . Current use of genomic information is focused primarily on a definition of all in vitro essential targets that satisfy criteria of selectivity, spectrum, and novelty . As the genomes of additional bacterial pathogens are solved, it will be possible to select in vivo essential targets common to groups of select pathogens (e.g., bacterial agents of community acquired pneumonia) or even pathogen-specific targets . Consideration of host-pathogen interactions, defined at the level of gene expression for each organism, might provide novel therapeutic options in the future.

Rev Neurosci, 2001, 12(3), 217 - 87
The deprivation syndrome is the driving force of phylogeny, ontogeny and oncogeny; Heininger K; Energy is the motor of life . Energy ensures the organism's survival and competitive advantage for reproductive success . For almost 3 billion years, unicellular organisms were the only life form on earth . Competition for limited energy resources and raw materials exerted an incessant selective pressure on organisms . In the adverse environment and due to their 'feast and famine' life style, hardiness to a variety of stressors, particularly to nutrient deprivation, was the selection principle . Both resistance and mutagenic adaptation to stressors were established as survival strategies by means of context-specific processes creating stability or variability of DNA sequence . The conservation of transduction pathways and functional homology of effector molecules clearly bear witness that the principles of life established during prokaryotic and eukaryotic unicellular evolution, although later diversified, have been unshakably cast to persist during metazoan phylogenesis . A wealth of evidence suggests that unicellular organisms evolved the phenomena of differentiation and apoptosis, sexual reproduction, and even aging, as responses to environmental challenges . These evolutionary accomplishments were elaborated from the dichotomous resistance/mutagenesis response and sophisticated the capacity of cells to tune their genetic information to changing environmental conditions . Notably, the social deprivation responses, differentiation and apoptosis, evolved as intercellularly coordinated events: a multitude of differentiation processes were elaborated from sporulation, the prototypic stress resistance response, while apoptosis, contrary to current concepts, is no altruistic cell suicide but was programmed as a mutagenic survival response; this response, however, is socially thwarted leading into mutagenic error catastrophe . In the hybrid differentiation-apoptosis process, cytocide and cannibalism of apoptotic cells thus serve the purpose of fueling the survival of the selfish genes in the differentiating cells . However, successful mutagenesis, although repressed, persisted in the asocial stress response of carcinogenesis as a regression to primitive unicellular behavior following failure of intercellular communication . While somatic mutagenesis was largely prevented, Metazoa elaborated germ cell mutagenesis as an evolutionary vehicle . Genetic competence, a primitive, stress-induced mating behavior, evolved into sexual reproduction which harnessed mutagenesis by subjecting highly mutable germ cells to a rigid viability selection . These processes were programmatically fixed as life- and cell-cycle events but retained their deprivation response phenotypes . Thus, the differentiation-apoptosis tandem evolved as the 'clay' to mold the specialized structures and functions of a multicellular organism while sexual reproduction elaborated the principle of quality-checked mutagenesis to create the immense diversity of Metazoa following the Cambrian explosion . Throughout these events, reactive oxygen and nitrogen species, which are regulated by energy homeostasis, shape the genetic information in a regulated but random, uncoded process providing the fitness-related feedback of phenotype to genotype . The interplay of genes and environment establishes a dynamic stimulus-response feedback cycle which, in animate nature, may be the organizing principle to contrive the reciprocal duality of energy and matter.

Biol Pharm Bull, 2001 Sep, 24(9), 982 - 7
Galactosyldiacylglycerol, a mammalian DNA polymerase alpha-specific inhibitor from a sea alga, Petalonia bingbamiae; Mizushina Y et al.; The glycolipid galactosyldiacylglycerol (GDG), containing C16:0 and C18:1 fatty acids, was isolated from the sea alga Petalonia bingbamiae as a potent inhibitor of the activities of mammalian DNA polymerase alpha (pol . alpha) . GDG, however, had no effect on pol . alpha from a fish or a higher plant . The inhibition of pol . alpha by GDG was dose-dependent with an IC50 value of 54 microM . The compound did not influence the activities of other replicative DNA polymerases such as mammalian pol . delta, or repair-related enzymes such as mammalian pol . beta . GDG also did not influence the activities of prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, DNA polymerases from the higher plant, cauliflower, or DNA metabolic enzymes such as calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type 1 reverse transcriptase and deoxyribonuclease 1 . Kinetic analysis of the compound showed that pol . alpha was non-competitively inhibited with respect to both the DNA template and the nucleotide substrate . In this study, we demonstrated the structure-function relationship in the selective inhibition of pol . alpha by the glycolipid group.

Nucleic Acids Res, 2001 Sep 15, 29(18), 3835 - 40
Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells; Tada S et al.; DNA helicase B is a major DNA helicase in mouse FM3A cells . A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B . In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein . By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members . A database search revealed similarity between DNA helicase B and the alpha subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast . The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.

Bioorg Med Chem, 2001 Oct, 9(10), 2601 - 8
Aminoglycoside binding to human and bacterial A-Site rRNA decoding region constructs; Ryu DH et al.; The 16S bacterial ribosomal A-site decoding rRNA region is thought to be the pharmacological target for the aminoglycoside antibiotics . The clinical utility of aminoglycosides could possibly depend on the preferential binding of these drugs to the prokaryotic A-site versus the corresponding A-site from eukaryotes . However, quantitative aminoglycoside binding experiments reported here on prokaryotic and eukaryotic A-site RNA constructs show that there is little in the way of differential binding affinities of aminoglycosides for the two targets . The largest difference in affinity is 4-fold in the case of neomycin, with the prokaryotic A-site construct exhibiting the higher binding affinity . Mutational studies revealed that decoding region constructs retaining elements of non-Watson-Crick (WC) base pairing, specifically bound aminoglycosides with affinities in the muM range . These studies are consistent with the idea that aminoglycoside antibiotics can specifically bind to RNA molecules as long as the latter have non-A form structural elements allowing access of aminoglycosides to the narrow major groove.

Bioorg Med Chem, 2001 Oct, 9(10), 2565 - 70
Inducible regulation of the S . cerevisiae cell cycle mediated by an RNA aptamer-ligand complex; Grate D et al.; Previous studies have shown that the introduction of a ligand-binding RNA (aptamer) into the 5'-UTR of an mRNA can confer regulated expression of both prokaryotic and eukaryotic reporter genes . The current report shows that aptamer insertion into the 5'-UTR of a cyclin transcript in S . cerevisiae renders cell-cycle control dependent upon the presence or absence of the target ligand . A malachite green binding motif, defined by an asymmetric internal loop flanked by short RNA helices, was inserted immediately upstream of the CLB2 start codon . Progression through the cell cycle is dramatically slowed and elongated bud morphology develops when tetramethylrosamine (a fluorescent malachite green analogue) is added to the aptamer-containing strain . Quantification of CLB2 expression at the RNA and protein levels by RT-PCR and Western blot analysis, respectively, demonstrates that the aptamer ligand regulates transcript translatability rather than stability . One-dimensional NMR spectroscopy shows that the malachite green binding aptamer undergoes a dramatic ligand-dependent change in structure with many nucleotides folding to adopt a well-defined conformation . These results are consistent with a model in which translational initiation is blocked by ligand-induced conformational changes in the 5'-UTR.

FEMS Microbiol Lett, 2001 Sep 11, 203(1), 1 - 9
Enzymology and molecular biology of prokaryotic sulfite oxidation; Kappler U et al.; Despite its toxicity, sulfite plays a key role in oxidative sulfur metabolism and there are even some microorganisms which can use it as sole electron source . Sulfite is the main intermediate in the oxidation of sulfur compounds to sulfate, the major product of most dissimilatory sulfur-oxidizing prokaryotes . Two pathways of sulfite oxidation are known: (1) direct oxidation to sulfate catalyzed by a sulfite:acceptor oxidoreductase, which is thought to be a molybdenum-containing enzyme; (2) indirect oxidation under the involvement of the enzymes adenylylsulfate (APS) reductase and ATP sulfurylase and/or adenylylsulfate:phosphate adenylyltransferase with APS as an intermediate . The latter pathway allows substrate phosphorylation and occurs in the bacterial cytoplasm . Direct oxidation appears to have a wider distribution; however, a redundancy of pathways has been described for diverse photo- or chemotrophic, sulfite-oxidizing prokaryotes . In many pro- and also eukaryotes sulfite is formed as a degradative product from molecules containing sulfur as a heteroatom . In these organisms detoxification of sulfite is generally achieved by direct oxidation to sulfate.

Glycobiology, 2001 Sep, 11(9), 107R - 118R
Biosynthesis of the arabinogalactan-peptidoglycan complex of Mycobacterium tuberculosis; Crick DC et al.; The compositional complexity of the mycobacterial cell envelope differentiates Mycobacterium species from most other prokaryotes . Historically, research in this area has focused on the elucidation of the structure of the mycobacterial cell envelope with the result that the structures of the mycolic acid-arabinogalactan-peptidoglycan complex from M . tuberculosis are fairly well understood . However, the current impetus for studying M . tuberculosis and other pathogenic mycobacteria is the need to identify targets for the development of new drugs . Therefore, emphasis has been shifting to the study of cell envelope biosynthesis and the identification of enzymes that are essential to the viability of M . tuberculosis . The publication of the complete M . tuberculosis genome in 1998 has greatly aided these studies . To date, thirteen enzymes involved in the synthesis of the arabinogalactan-peptidoglycan complex of M . tuberculosis have been identified and at least partially characterized . Eleven of these enzymes were reported subsequent to the publication of the M . tuberculosis genome, a clear indication of the rapid evolution of knowledge stimulated by the sequencing of the genome . In this article we review the current understanding of M . tuberculosis arabinogalactan-peptidoglycan structure and biosynthesis.

Mol Microbiol, 2001 Sep, 41(5), 1037 - 51
Deficiency of essential GTP-binding protein ObgE in Escherichia coli inhibits chromosome partition; Kobayashi G et al.; GTP-binding proteins are involved in cell proliferation, development, signal transduction, protein elongation, etc . and construct the GTPase superfamily, whose structures and sequence motifs (G-1 to G-5) are highly conserved from prokaryote to eukaryote . Obg of Bacillus subtilis and Obg homologues of other bacteria belong to the GTPase superfamily and have been suggested as being essential for cell growth, development and monitoring of intracellular levels of GTP . We identified the Obg homologue in Escherichia coli, a protein previously known as YhbZ, which we have renamed ObgE . Double cross-over experiments showed that the obgE gene is essential for growth in E . coli . From characterization of the obgE temperature-sensitive mutant, we found that DNA replication was not inhibited, that the nucleoids did not partition and instead remained in the middle of cell, and that the cells elongated . Overproduction of ObgE also resulted in aberrant chromosome segregation . These data suggested that ObgE is involved directly or indirectly in E . coli chromosome partitioning . Characterization studies showed that ObgE is abundant in normal cells, partially associated with the membrane and does not associate with ribosomes such as in Obg of B . subtilis . We purified ObgE protein from a cell extract of E . coli, and the purified ObgE had GTPase activity and DNA-binding ability.

Prog Nucleic Acid Res Mol Biol, 2001, 68, 207 - 21
DNA substrates containing defined oxidative base lesions and their application to study substrate specificities of base excision repair enzymes; Ide H; Reactive oxygen species generate structurally diverse base lesions in DNA . These lesions are primarily removed by base excision repair (BER) enzymes in prokaryotic and eukaryotic cells . Biochemical properties of BER enzymes such as substrate specificity, enzymatic parameters, and action mechanisms can be best studied by employing defined oligonucleotide and DNA substrates . Currently available methods are listed to prepare defined DNA substrates containing oxidative base damage and analogs . BER enzymes for oxidative base damage are classified into two subgroups that recognize pyrimidine lesions (Endo III homologs) and purine lesions (Fpg homologs), though E . coli Fpg exhibits weak repair activity for certain pyrimidine damage . Recently, several interesting findings have been reported in relation to the substrate specificity of BER enzymes . Saccharomyces cerevisiae Endo III homologs (NTG1 and NTG2) have been shown to recognize formamidopyrimidine (Fapy) lesions that are derived from purine . Endo III and Endo VIII have a very weak activity to dihydrothymine in comparison with thymine glycol . Excision of 7,8-dihydro-8-oxoguanine by Fpg and human OGG1 is paired-base-dependent, whereas that of Fapy is essentially paired-base-independent . The repair efficiency of BER enzymes is affected by surrounding sequence contexts . In general, the sequence context effect appears to be more pronounced for Fpg homologs than Endo III homologs.

Proc Natl Acad Sci U S A, 2001 Sep 11, 98(19), 10960 - 5
Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana; Awai K et al.; In Arabidopsis, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3) . The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope . The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways . Type A atMGD1 gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme . Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide . atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network . Transient expression of green fluorescent protein fusions in Arabidopsis leaves and in vitro import experiments show that type B precursors are targeted to plastids, owing to a different mechanism . Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion . Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (atMGD2) and roots (atMGD3), where they conceivably influence the eukaryotic structure prominence in MGDG . In addition, their expression of type B enzymes is enhanced under phosphate deprivation.

J Biol Chem, 2001 Nov 16, 276(46), 42869 - 80 Epub 2001 Sep 11.
A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase; Abadjieva A et al.; Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis . The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents . The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly . ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway . The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active . Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed . This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R . W., Jacobson, J . A., Weiss, R . L., and Davis, R . H . (1986) J . Biol . Chem . 261, 5848-5852) . In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase . Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase . Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway . Hypotheses regarding the biological significance of this interaction are discussed.

Infect Immun, 2001 Oct, 69(10), 6193 - 200
The opdB locus encodes the trypsin-like peptidase activity of Treponema denticola; Fenno JC et al.; High levels of Treponema denticola in subgingival dental plaque are associated with severe periodontal disease . T . denticola, along with Porphyromonas gingivalis and Bacteroides forsythus, are the only cultivatable oral microorganisms that produce significant amounts of "trypsin-like" peptidase activity . The ability of subgingival plaque to hydrolyze N-alpha-benzoyl-DL-arginine-2-naphthylamide (BANA) is associated with high levels of one or more of these organisms . The purpose of this study was to identify the gene encoding trypsin-like activity in T . denticola and thus facilitate molecular-level studies of its potential role in disease . Using published peptide sequences of a T . denticola surface-associated oligopeptidase with BANA-hydrolyzing activity, we identified the gene, designated opdB, in an apparently noncoding region of the T . denticola genome unannotated contigs (11/2000; The opdB gene begins with a TTG start codon and encodes a 685-residue peptide with high homology to the oligopeptidase B family in prokaryotes and eukaryotes . An isogenic T . denticola opdB mutant was constructed by allelic replacement mutagenesis using an ermF/AM gene cassette . The mutant lacked BANA-hydrolyzing activity and had a slightly slower growth rate than the parent strain . This mutant will be used in future studies of interactions of T . denticola with host cells and tissue.

Environ Microbiol, 2001 Jul, 3(7), 450 - 9
16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection; Schmid M et al.; Recently, anaerobic ammonium-oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep-branching lineage within the Planctomycetales . This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candidatus 'Brocadia anammoxidans' and Candidatus 'Kuenenia stuttgartiensis' . In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB . The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b) . This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FISH) and thus present in the mature 16S rRNA . In contrast with the genera Pirellula, Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length . Phylogenetic analysis of the obtained 23S rRNA-genes confirmed the deep branching of the AAOB within the Planctomycetales and allowed the design of additional specific FISH probes . Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes . Quantitative FISH experiments with cells of Candidatus 'Brocadia anammoxidans' that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration . Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.

J Biol Chem, 2001 Nov 23, 276(47), 43958 - 69 Epub 2001 Sep 10.
The large subunit of the mammalian mitochondrial ribosome . Analysis of the complement of ribosomal proteins present; Koc EC et al.; Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry . Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled . The human mitochondrial 39 S subunit has 48 distinct proteins . Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36 . Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes . No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases . The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes . Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes . All but two of the proteins has a clear homolog in D . melanogaster while all can be found in the genome of C . elegans . Ten of the 20 mitochondrial specific 39 S proteins have homologs in S . cerevisiae . Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Mar-Apr, (2), 76 - 80
{Expression of hepatitis A virus recombinant proteins in Escherichia coli}; Ternovoi VA et al.; On the basis of coding regions on the fragments of genes P1 and P2 of hepatitis A virus (HAV) recombinant proteins of this virus have been synthesized in the prokaryotic expressing system of E . coli, isolated and studied with the use of sera obtained from hepatitis A patients . The capacity of HAV recombinant proteins for binding with the sera of patients with hepatitis A in the acute stage has been shown with the use of immunoblotting and the indirect solid-phase enzyme immunoassay . The results obtained in this investigation are discussed in the light of the possible use of recombinant proteins for the detection of HAV markers.

J Mol Microbiol Biotechnol, 2001 Oct, 3(4), 601 - 10
Biochemical and physiological studies of the small heat shock protein Lo18 from the lactic acid bacterium Oenococcus oeni; Delmas F et al.; The small heat shock protein (smHSP) family has been extensively studied in eukaryotic cells . SmHSP assemble into large multimeric structures and possess chaperone activity that can prevent protein aggregation in vitro . Few studies on prokaryotic smHSP are actually available and no smHSP from lactic acid bacteria has been characterized at a biochemical level to date . Here we report on the Lo18 membrane-associated smHSP from the lactic acid bacterium Oenococcus oeni . Using size exclusion chromatography, nondenaturing pore-exclusion PAGE and in vitro and in vivo cross-linking experiments, the multimeric structure of Lol8 from O . oeni or expressed in Escherichia coli was investigated . In vitro, Lo18 is able to form a trimer and a higher oligomer which could be a dodecamer . Experiments strongly suggest that the same structures exist in vivo . First, Lo18 prevented thermal aggregation of citrate synthase and lactate dehydrogenase even at 60degreesC . These findings showed that the prokaryotic smHSP Lo18 can function as a molecular chaperone in vitro . Second, Lo18 did not protect lactate dehydrogenase from thermal inactivation and did not assist in enzymatic activity restoration after thermal aggregation, suggesting that other components may be needed for the refolding of the enzyme in an active conformation . Third, we showed that membrane association of Lo18 depends on the temperature upshift . Moreover, expression of this smHSP was induced by administration of a membrane fluidiser, the benzyl alcohol, suggesting that Lo18 expression could be regulated by the level of membrane fluidity.

Nature, 2001 Sep 6, 413(6851), 39 - 44
Prokaryotic origin of the actin cytoskeleton; van den Ent F et al.; It was thought until recently that bacteria lack the actin or tubulin filament networks that organize eukaryotic cytoplasm . However, we show here that the bacterial MreB protein assembles into filaments with a subunit repeat similar to that of F-actin-the physiological polymer of eukaryotic actin . By elucidating the MreB crystal structure we demonstrate that MreB and actin are very similar in three dimensions . Moreover, the crystals contain protofilaments, allowing visualization of actin-like strands at atomic resolution . The structure of the MreB protofilament is in remarkably good agreement with the model for F-actin, showing that the proteins assemble in identical orientations . The actin-like properties of MreB explain the finding that MreB forms large fibrous spirals under the cell membrane of rod-shaped cells, where they are involved in cell-shape determination . Thus, prokaryotes are now known to possess homologues both of tubulin, namely FtsZ, and of actin.

Annu Rev Microbiol, 2001, 55, 709 - 42
Horizontal gene transfer in prokaryotes: quantification and classification; Koonin EV et al.; Comparative analysis of bacterial, archaeal, and eukaryotic genomes indicates that a significant fraction of the genes in the prokaryotic genomes have been subject to horizontal transfer . In some cases, the amount and source of horizontal gene transfer can be linked to an organism's lifestyle . For example, bacterial hyperthermophiles seem to have exchanged genes with archaea to a greater extent than other bacteria, whereas transfer of certain classes of eukaryotic genes is most common in parasitic and symbiotic bacteria . Horizontal transfer events can be classified into distinct categories of acquisition of new genes, acquisition of paralogs of existing genes, and xenologous gene displacement whereby a gene is displaced by a horizontally transferred ortholog from another lineage (xenolog) . Each of these types of horizontal gene transfer is common among prokaryotes, but their relative contributions differ in different lineages . The fixation and long-term persistence of horizontally transferred genes suggests that they confer a selective advantage on the recipient organism . In most cases, the nature of this advantage remains unclear, but detailed examination of several cases of acquisition of eukaryotic genes by bacteria seems to reveal the evolutionary forces involved . Examples include isoleucyl-tRNA synthetases whose acquisition from eukaryotes by several bacteria is linked to antibiotic resistance, ATP/ADP translocases acquired by intracellular parasitic bacteria, Chlamydia and Rickettsia, apparently from plants, and proteases that may be implicated in chlamydial pathogenesis.

Annu Rev Microbiol, 2001, 55, 333 - 56
Novel thiols of prokaryotes; Fahey RC; Glutathione metabolism is associated with oxygenic cyanobacteria and the oxygen-utilizing purple bacteria, but is absent in many other prokaryotes . This review focuses on novel thiols found in those bacteria lacking glutathione . Included are glutathione amide and its perthiol, produced by phototrophic purple sulfur bacteria and apparently involved in their sulfide metabolism . Among archaebacteria, coenzyme M (2-mercaptoethanesulfonic acid) and coenzyme B (7-mercaptoheptanoylthreonine phosphate) play central roles in the anaerobic production of CH4 and associated energy conversion by methanogens, whereas the major thiol in the aerobic phototrophic halobacteria is gamma-glutamylcysteine . The highly aerobic actinomycetes produce mycothiol, a conjugate of N-acetylcysteine with a pseudodisaccharide of glucosamine and myo-inositol, AcCys-GlcNalpha(1 --> 1)Ins, which appears to play an antioxidant role similar to glutathione . Ergothioneine, also produced by actinomycetes, remains a mystery despite many years of study . Available data on the biosynthesis and metabolism of these and other novel thiols is summarized and key areas for additional study are identified.

Annu Rev Microbiol, 2001, 55, 105 - 37
Big bacteria; Schulz HN et al.; A small number of prokaryotic species have a unique physiology or ecology related to their development of unusually large size . The biomass of bacteria varies over more than 10 orders of magnitude, from the 0.2 microm wide nanobacteria to the largest cells of the colorless sulfur bacteria, Thiomargarita namibiensis, with a diameter of 750 microm . All bacteria, including those that swim around in the environment, obtain their food molecules by molecular diffusion . Only the fastest and largest swimmers known, Thiovulum majus, are able to significantly increase their food supply by motility and by actively creating an advective flow through the entire population . Diffusion limitation generally restricts the maximal size of prokaryotic cells and provides a selective advantage for microm-sized cells at the normally low substrate concentrations in the environment . The largest heterotrophic bacteria, the 80 x 600 microm large Epulopiscium sp . from the gut of tropical fish, are presumably living in a very nutrient-rich medium . Many large bacteria contain numerous inclusions in the cells that reduce the volume of active cytoplasm . The most striking examples of competitive advantage from large cell size are found among the colorless sulfur bacteria that oxidize hydrogen sulfide to sulfate with oxygen or nitrate . The several-cm-long filamentous species can penetrate up through the ca 500-microm-thick diffusive boundary layer and may thereby reach into water containing their electron acceptor, oxygen or nitrate . By their ability to store vast quantities of both nitrate and elemental sulfur in the cells, these bacteria have become independent of the coexistence of their substrates . In fact, a close relative, T . namibiensis, can probably respire in the sulfidic mud for several months before again filling up their large vacuoles with nitrate.

J Bacteriol, 2001 Oct, 183(19), 5668 - 74
Localization of c-di-GMP-binding protein with the linear terminal complexes of Acetobacter xylinum; Kimura S et al.; Specific labeling of a single row of cellulose-synthesizing complexes (terminal complexes, TC subunits, TCs, or TC arrays) in Acetobacter xylinum by antibodies raised against a 93-kDa protein (the cyclic dignanylic acid-binding protein) has been demonstrated by using the sodium dodecyl sulfate (SDS)-freeze-fracture labeling (FRL) technique . The antibodies to the 93-kDa protein specifically recognized the TC subunits on the protoplasmic fracture (PF) face of the outer membrane in A . xylinum; however, nonlabeled TCs were also observed . Two types of TC subunits (particles or pits) are observed on the PF face of the outer membrane: (i) immunogold-labeled TCs showing a line of depressions (pits) with an indistinct particle array and (ii) nonlabeled TC subunits with a distinct single row of particle arrays . The evidence indicates that the labeling patterns differ with respect to the presence or absence of certain TC subunits remaining attached to the replica after SDS treatment . This suggests the presence of at least two TC components, one in the outer membrane and the other in the cytoplasmic membrane . If the TC component in the outer membrane is preferentially fractured and remains attached to the ectoplasmic fracture face (or outer leaflet) of the outer membrane, subsequent replica formation reveals a pit or depression with positive antibody labeling on the PF face of the outer membrane . If the TC component in the outer membrane remains with the PF face (or inner leaflet) of the outer membrane, the innermost TC component is removed during SDS treatment and labeling does not occur . SDS-FRL of TCs in A . xylinum has enabled us to provide the first topological molecular analysis of component proteins in a cellulose-synthesizing TC structure in a prokaryotic organism.

Symbiosis, 1985, 1, 101 - 24
Symbiosis as a mechanism of evolution: status of cell symbiosis theory; Margulis L et al.; Several theories for the origin of eukaryotic (nucleated) cells from prokaryotic (bacterial) ancestors have been published: the progenote, the direct filiation and the serial endosymbiotic theory (SET) . Compelling evidence for two aspects of the SET is now available suggesting that both mitochondria and plastids originated by symbioses with a third type of microbe, probably a Thermoplasma-like archaebacterium ancestral to the nucleocytoplasm . We conclude that not enough information is available to negate or substantiate another SET hypothesis: that the undulipodia (cilia, eukaryotic flagella) evolved from spirochetes . Recognizing the power of symbiosis to recombine in single individual semes from widely differing partners, we develop the idea that symbiosis has been important in the origin of species and higher taxa . The abrupt origin of novel life forms through the formation of stable symbioses is consistent with certain patterns of evolution (e.g punctuated equilibria) described by some paleontologists.

J Paleontol, 1999 Sep, 73(5), 744 - 64
Acritarchs and microfossils from the Mesoproterozoic Bangemall Group, northwestern Australia; Buick R et al.; Three microfossil assemblages occur in the Mesoproterozoic Bangemall Group (1625-1000 Ma) of northwestern Australia, each occupying a different environmental and taphonomic setting . In peritidal environments, benthic prokaryotic filaments and spheroids of matting habit and small size were permineralized by early diagenetic silicification of stromatolitic carbonates . In shallow subtidal environments, benthic filaments of large size and nonmatting habit and planktonic sphaeromorph acritarchs with thin walls and moderate dimensions were compressed in mildly kerogenous shale . In deeper subtidal environments, planktonic megasphaeromorph acritarchs with thick walls were initially entombed in concretionary nodules in highly kerogenous shale and then permineralized by silica during later diagenesis . Taxonomic diversity and numerical abundance evidently decrease offshore . The three assemblages have typical Mesoproterozoic aspects: peritidal benthic habitats were dominated by Siphonophycus-Sphaerophycus-Eosynechococcus-Myxococcoides-Palaeopleurocapsa, shallow subtidal settings were occupied by Siphonophycus-Leiosphaeridia-Pterosphermopsimorpha-Satka, and offshore plankton consisted solely of very large chuarid acritarchs . Because of its taphonomic restriction to mid-intertidal stromatolites, the peritidal assemblage can be equated in microenvironment with a similar assemblage in the Neoproterozoic Draken Conglomerate, suggesting that ecological stasis at the community level can last for intervals up to 900 million years . In the deeper subtidal assemblage, the common chuarid has an unusual mode of preservation, in three dimensions in early diagenetic concretions, revealing that it possesses a thick multilamellate wall . Because of this distinctive ultrastructure, the new genus Crassicorium is erected for these fossils, which are among the oldest indubitable eukaryotes . Very large (34-55 micrometers in diameter) filaments from shallow subtidal habitats are assigned to the emended species Siphonophycus punctatum.

Org Geochem, 1999 Dec, 30(12), 1585 - 7
All-cis hentriaconta-9,15,22-triene in microbial mats formed by the phototrophic prokaryote Chloroflexus; van der Meer MT et al.; All-cis hentriaconta-9,15,22-triene (I) has been isolated from Chloroflexus mats, Yellowstone National Park (USA), and identified by GC-(HR)MS analysis of I and its hydrogenated and DMDS-derivatized products and by 1H and 13C NMR spectroscopy.

Precambrian Res, 1995 Nov, 75(1-2), 65 - 90
Microfossils from the Neoarchean Campbell Group, Griqualand West Sequence of the Transvaal Supergroup, and their paleoenvironmental and evolutionary implications; Altermann W et al.; The oldest filament- and colonial coccoid-containing microbial fossil assemblage now known is described here from drill core samples of stromatolitic cherty limestones of the Neoarchean, approximately 2600-Ma-old Campbell Group (Ghaap Plateau Dolomite, Lime Acres Member) obtained at Lime Acres, northern Cape Province, South Africa . The assemblage is biologically diverse, including entophysalidacean (Eoentophysalis sp.), probable chroococcacean (unnamed colonial coccoids), and oscillatoriacean cyanobacteria (Eomycetopsis cf . filiformis, and Siphonophycus transvaalensis), as well as filamentous fossil bacteria (Archaeotrichion sp.); filamentous possible microfossils (unnamed hematitic filaments) also occur . The Campbell Group microorganisms contributed to the formation of stratiform and domical to columnar stromatolitic reefs in shallow subtidal to intertidal environments of the Transvaal intracratonic sea . Although only moderately to poorly preserved, they provide new evidence regarding the paleoenvironmental setting of the Campbell Group sediments, extend the known time-range of entophysalidacean cyanobacteria by more than 400 million years, substantiate the antiquity and role in stromatolite formation of Archean oscillatoriacean cyanobacteria, and document the exceedingly slow (hypobradytelic) evolutionary rate characteristic of this early evolving prokaryotic lineage.

New Sci, 1994 Apr 9, 142(1920), 21 - 5
Hot bacteria & other ancestors; Day S; NASA: The intestinal parasite Giardia is examined as an evolutionary link in the development of cells with nuclei . Genetic studies of Giardia suggest that eukaryotes may have developed separately from prokaryotes . Advances in cell biology and the study of DNA indicate that Giardia and other diplomonads should be classified as eukaryotes . Evidence is presented for the inclusion of eocytes as a main group in the kingdoms of life . Controversies surrounding the eocyte theory are examined .

J Gravit Physiol, 1998 Jul, 5(1), P137 - 8
Influence of modified gravity on growth and structure of Vaucheria sessilis (Xanthopyceae); Gavrilova OV et al.; The constant presence of gravity force in the evolution of life on Earth caused the adaptation for its pressure and dependence of growth and morphogenesis of high plants on gravity, and appearance of gravitaxises of unicellular organisms . The modern investigations demonstrated the ability even of prokaryotic organisms for sensitivity of gravity modifications Erdmann et al., 1997) . Object of current investigation was siphonaceous alga Vaucheria sessilis with branching thallome without septs . Vaucheria does not demonstrate gravitropic reaction, and does not possess statolite-like structure . It is important to note that graviresponse of such kind of objects was under investigation for the first time . The definite structure of cytoskeleton permits the functions of cytoskeleton elements to be analyzed in course of graviresponse . Siphonaceous algae are new and promising objects in gravitational cell biology . This investigation was intended to compare the graviresponse of Vaucheria detected on different experimental models of modified gravity.

Proc Natl Acad Sci U S A, 1986 Apr, 83, 2138 - 42
Predatory prokaryotes: predation and primary consumption evolved in bacteria; Guerrero R et al.; Two kinds of predatory bacteria have been observed and characterized by light and electron microscopy in samples from freshwater sulfurous lakes in northeastern Spain . The first bacterium, named Vampirococcus, is Gram-negative and ovoidal (0.6 micrometer wide) . An anaerobic epibiont, it adheres to the surface of phototrophic bacteria (Chromatium spp.) by specific attachment structures and, as it grows and divides by fission, destroys its prey . An important in situ predatory role can be inferred for Vampirococcus from direct counts in natural samples . The second bacterium, named Daptobacter, is a Gram-negative, facultatively anaerobic straight rod (0.5 x 1.5 micrometers) with a single polar flagellum, which collides, penetrates, and grows inside the cytoplasm of its prey (several genera of Chromatiaceae) . Considering also the well-known case of Bdellovibrio, a Gram-negative, aerobic curved rod that penetrates and divides in the periplasmic space of many chemotrophic Gram-negative bacteria, there are three types of predatory prokaryotes presently known (epibiotic, cytoplasmic, and periplasmic) . Thus, we conclude that antagonistic relationships such as primary consumption, predation, and scavenging had already evolved in microbial ecosystems prior to the appearance of eukaryotes . Furthermore, because they represent methods by which prokaryotes can penetrate other prokaryotes in the absence of phagocytosis, these associations can be considered preadaptation for the origin of intracellular organelles.

Precambrian Res, 1984, 24, 335 - 49
Proterozoic stromatolitic microbiotas of the 1400-1500 Ma-old Gaoyuzhuang formation near Jixian, northern China; Schopf JW et al.; Two communities of diverse, well-preserved, fossil prokaryotic microorganisms have been discovered in petrographic thin sections of carbonaceous black chert from the ca . 1400-1500 Ma-old Gaoyuzhuang Formation at the stratotype section of the "Sinian Suberathem" near Jixian, northern China . One of these communities is preserved in bedded, essentially flat-laminated, stromatolitic chert; the other occurs in silicified conical stromatolites of the forms Conophyton cylindricum and C . garganicum . The Gaoyuzhuang cherts comprise one of the new microfossiliferous deposits now known from the Precambrian of China; the permineralized microbiotas they contain are among the first such stromatolitic assemblages to be discovered in the Sinian stratotype section.

Speculations Sci Technol, 1984, 7(2), 77 - 81
The origin of the eukaryotic cell; Hartman H; The endosymbiotic hypothesis for the origin of the eukaryotic cell has been applied to the origin of the mitochondria and chloroplasts . However as has been pointed out by Mereschowsky in 1905, it should also be applied to the nucleus as well . If the nucleus, mitochondria and chloroplasts are endosymbionts, then it is likely that the organism that did the engulfing was not a DNA-based organism . In fact, it is useful to postulate that this organism was a primitive RNA-based organism . This hypothesis would explain the preponderance of RNA viruses found in eukaryotic cells . The centriole and basal body do not have a double membrane or DNA . Like all MTOCs (microtubule organising centres), they have a structural or morphic RNA implicated in their formation . This would argue for their origin in the early RNA-based organism rather than in an endosymbiotic event involving bacteria . Finally, the eukaryotic cell uses RNA in ways quite unlike bacteria, thus pointing to a greater emphasis of RNA in both control and structure in the cell . The origin of the eukaryotic cell may tell us why it rather than its prokaryotic relative evolved into the metazoans who are reading this paper.

Phycologia, 1984, 23(2), 203 - 8
Prochloron--a status report; Lewin RA; Prochloron is a genus of prokaryotic algae with photosynthetic pigments like those of chlorophytes . Prochlorophytes are almost invariably found associated as symbionts with marine protochordates (didemnid ascidians), and so far none has been successfully grown in sustained culture away from in host . Based on materials collected from nature, information of various sorts (biochemical, physiological, cytological and fine-structural) has been obtained, indicating many resemblances (and probably close phylogenetic affinities) between prochlorophytes and cyanophytes . Nevertheless they are distinguished by certain unique combinations of characters . Some of the data support the symbiogenesis theory for the origin of green-plant chloroplasts . Other possibilities are briefly discussed.

Geology, 1998 Jun, 26(6), 555 - 8
A billion years of environmental stability and the emergence of eukaryotes: new data from northern Australia; Brasier MD et al.; Carbon isotopes through 6km of fully cored drill holes in 1.7 to 1.5 Ga carbonates of the Mount Isa and McArthur basins, Australia (which host the earliest known eukaryote biomarkers) provide the most comprehensive and best-dated delta 13C stratigraphy yet obtained from such ancient rocks . Both basins reveal remarkably stable temporal delta 13C trends (mean of -0.6% +/- 2% PDB {Peedee belemnite}) and confirm the impression of delta 13C stasis between 2.0 and 1.0 Ga, which, together with other evidence, suggest a prolonged period of stability in crustal dynamics, redox state of surface environments, and planetary climate . This delta 13C stasis is consistent with great stability in the carbon cycle controlled, we suggest, by P limitation of primary productivity . Recent evidence shows that P depletion is a major factor in obligate associations between photosymbionts and host cells . We argue that a billion years of stability in the carbon and nutrient cycles may have been the driving force that propelled prokaryotes toward photosymbiosis and the emergence of the autotrophic eukaryote cell.

J Theor Biol, 1967 Mar, 14(3), 255 - 74
On the origin of mitosing cells
Sagan L.
A theory of the origin of eukaryotic cells ("higher" cells which divide by classical mitosis) is presented . By hypothesis, three fundamental organelles: the mitochondria, the photosynthetic plastids and the (9+2) basal bodies of flagella were themselves once free-living (prokaryotic) cells . The evolution of photosynthesis under the anaerobic conditions of the early atmosphere to form anaerobic bacteria, photosynthetic bacteria and eventually blue-green algae (and protoplastids) is described . The subsequent evolution of aerobic metabolism in prokaryotes to form aerobic bacteria (protoflagella and protomitochondria) presumably occurred during the transition to the oxidizing atmosphere . Classical mitosis evolved in protozoan-type cells millions of years after the evolution of photosynthesis . A plausible scheme for the origin of classical mitosis in primitive amoeboflagellates is presented . During the course of the evolution of mitosis, photosynthetic plastids (themselves derived from prokaryotes) were symbiotically acquired by some of these protozoans to form the eukaryotic algae and the green plants . The cytological, biochemical and paleontological evidence for this theory is presented, along with suggestions for further possible experimental verification . The implications of this scheme for the systematics of the lower organisms is discussed.

J NIH Res, 1993 Mar, 5(3), 65 - 72
On the origin of mitosing cells . 1967
Sagan L.
A theory of the origin of eukaryotic cells ("higher" cells which divide by classical mitosis) is presented . By hypothesis, three fundamental organelles: the mitochondria, the photosynthetic plastids and the (9+2) {9(2)+2} basal bodies {kinetosomes} of flagella {undulipodia} were themselves once free-living (prokaryotic) cells . The evolution of photosynthesis under the anaerobic {anoxic} conditions of the early atmosphere to form anaerobic bacteria, photosynthetic bacteria and eventually blue-green algae (and protoplastids) is described . The subsequent evolution of aerobic metabolism in prokayotes to form aerobic bacteria (protoflagella {undulipodia} and protomitochondria) presumably occurred during the transition to the oxidizing atmosphere . Classical mitosis evolved in protozoan-type cells millions of years after the evolution of photosynthesis . A plausible scheme for the origin of classical mitosis in primitive amoeboflagellates {amoebomastigotes} is presented . During the course of the evolution of mitosis, photosynthetic plastids (themselves derived from prokaryotes) were symbolically acquired by some of these protozoans to form the {"eukaryotic" deleted} algae and the green plants . The cytological, biochemical and paleontological evidence for this theory is presented, along with suggestions for further possible experimental verification . The implications of this scheme for the systematics of the lower {smaller} organisms is discussed.

Adv Space Res, 1998, 21(8-9), 1263 - 8
Cellular responses to gravity: extracellular, intracellular and in-between; Todd P et al.; Our understanding of gravitational effects (inertial effects in the vicinity of 1 x g) on cells has matured to a stage at which it is possible to define, on the basis of experimental evidence, extracellular effects on small cells and intracellular effects on eukaryotic gravisensing cells . Yet undetermined is the nature of response, if any, of those classes of cells that are not governed solely by extracellular physical events (as are prokaryotes) and are devoid of obvious mechanical devices for sensing inertial forces (such as those possessed by certain plant cells and sensory cells of animals) . This "in-between" class of cells needs to be understood on the basis of the combination of intracellular and extracellular gravity-dependent processes that govern experimentally-measurable variables that are relevant to the cell's responses to modified inertial forces . The forces that certain cell types generate or respond to are therefore compared to those imposed by approximately 1 x g in the context of cytoskeletal action and symmetry-breaking pathways.

J Geophys Res, 1997 Oct 25, 102(E10), 23,675 - 86
The limits of life on Earth and searching for life on Mars; Nealson KH; Considerations of basic properties of bacteria such as size, structure, and metabolic versatility allow one to understand how these remarkable life-forms are so adaptable to environments previously thought to be uninhabitable . It is now appreciated that bacteria on Earth can utilize almost any redox couple that yields energy, taking advantage of this energy, while transforming the elements during metabolism . The ability to grow at the expense of inorganic redox couples allows the microbes to occupy niches not available to the more metabolically constrained eukaryotes . Furthermore, the simplicity of the bacterial structure allows them considerably more resistance to environmental variables (pH, salinity, temperature) that are toxic or lethal to more complex organisms . This information can be used to explain the predominance of prokaryotes in extreme environments on Earth, and to speculate as to simple types of metabolism and biogeochemical cycles that may exist on this planet, Mars, and perhaps other non-Earth environments.

Annu Rev Plant Physiol Plant Mol Biol, 1997, 48, 277 - 96
The ethylene response pathway in Arabidopsis; Kieber JJ; The simple gas ethylene influences a diverse array of plant growth and developmental processes including germination, senescence, cell elongation, and fruit ripening . This review focuses on recent molecular genetic studies, principally in Arabidopsis, in which components of the ethylene response pathway have been identified . The isolation and characterization of two of these genes has revealed that ethylene sensing involves a protein kinase cascade . One of these genes encodes a protein with similarity to the ubiquitous Raf family of Ser/Thr protein kinases . A second gene shows similarity to the prokaryotic two-component histidine kinases and most likely encodes an ethylene receptor . Additional elements involved in ethylene signaling have only been identified genetically . The characterization of these genes and mutants will be discussed.

Mar Geol, 1985, 68, 217 - 31
Biomass and community structure of the abyssal microbiota determined from the ester-linked phospholipids recovered from Venezuela Basin and Puerto Rico Trench sediments; Baird BH et al.; Extractible phospholipid fatty acids of abyssal sediment cores from three stations in the Venezuela Basin, transects between them, and a station in the Puerto Rico Trench were analyzed to determine microbial biomass and community composition . Results were compared to abyssal sediments from an area of high-energy boundary currents in the North Atlantic, and estuarine sediments from Apalachee Bay, Florida . Venezuela Basin and Puerto Rico Trench sediments were characterize by low microbial biomass, measured as phospholipid palmitic acid . Venezuela Basin sediments of three different sedimentary regimes showed a remarkably similar microbial community structure, as characterized by fatty acid profiles . Prokaryotic organisms dominated the microbial community, and fatty acids believed to be signatures of anaerobic organisms were present in greater proportions in Venezuela Basin and Puerto Rico Trench sediments than in either the North Atlantic abyssal sediments or shallow-water estuarine sediments.

Annu Rev Earth Planet Sci, 1997, 25, 403 - 34
Sediment bacteria: who's there, what are they doing, and what's new?
Nealson KH.
The prokaryotes (bacteria) comprise the bulk of the biomass and chemical activity in sediments . They are well suited to their role as sediment chemists, as they are the right size and have the required metabolic versatility to oxidize the organic carbon in a variety of different ways . The characteristic vertical nutrient (electron donor and electron acceptor) profiles seen in sediments are produced as a result of microbial activities, with each nutrient a product or reactant of one or more metabolic groups . Thus, understanding the mechanisms by which the chemical environment of a sediment is generated and stabilized requires a knowledge of resident populations, something that has been very difficult to obtain, given the techniques available to microbiologists . however, the new approaches of molecular biology, which have added insights into the phylogenetic relationships of the prokaryotes, have also provided tools whereby sedimentary populations can be examined without the need for culturing the organisms . These techniques, in concert with new methods of microscopy, isolation of new metabolic groups, and the study of new ecosystems, suggest that there is much that will be learned about the microbiology of sedimentary environments in the coming years.

Oncol Rep, 1997 Jul-Aug, 4(4), 691 - 5
Cancer risk in space due to radiation assessed by determining cell lethality and mutation frequencies of prokaryotes and a plasmid during the Second International Microgravity Laboratory (IML-2) Space Shuttle experiment; Harada K et al.; We participated in a space experiment conducted during the 2nd International Microgravity Laboratory Mission (IML-2) project . The aim of our study was to investigate the effects of space radiation, i.e., high-LET (linear energy transfer) cosmic radiation, on living organisms in the 'Realtime Radiation Monitoring Device (RRMD)' . The biological samples, dried E . coli DNA repair-deficient mutant cells and shuttle vector plasmid pZl89 DNA, were prepared and placed in a biospecimen box sandwiched between 'Harzlas' plastic radiation detectors . This box was then loaded into the RRMD sensor unit in the Space Shuttle 'Columbia' and an identical box was left in the NASA John F . Kennedy Space Center (KSC) as a control . 'Columbia' (flight No . STS-65) was launched from KSC in Florida, USA on July 8, 1994 . The mission duration was 14.75 days and after 'Columbia' returned to earth, we studied (i) the lethal and mutagenic effects of high-LET cosmic radiation on E . coli mutants and (ii) the relationship between high-LET cosmic radiation and the mutation frequency of pZ189 DNA . There were virtually no differences between the cell viabilities of the space and control samples of Escherichia coli KMBL3835 (wild-type), KY383 (lexA-), KY385 (recA-) and KY386 (uvrA-), nor between the mutation frequency ratios of the space and control E . coli mutant samples . Furthermore, the survival and mutation frequency of the supF gene of pZ189 DNA space samples did not differ from those of the control samples . We concluded there was no cancer risk during this Space Shuttle flight.

Science, 1993 Apr 30, 260, 640 - 6
Microfossils of the Early Archean Apex chert: new evidence of the antiquity of life; Schopf JW; Eleven taxa (including eight heretofore undescribed species) of cellularly preserved filamentous microbes, among the oldest fossils known, have been discovered in a bedded chert unit of the Early Archean Apex Basalt of northwestern Western Australia . This prokaryotic assemblage establishes that trichomic cyanobacterium-like microorganisms were extant and morphologically diverse at least as early as approximately 3465 million years ago and suggests that oxygen-producing photoautotrophy may have already evolved by this early stage in biotic history.

J Paleontol, 1995 Jan, 69(1 Pt 2), 1 - 37
Paleobiology of the Mesoproterozoic Billyakh Group, Anabar Uplift, northern Siberia; Sergeev VN et al.; Silicified peritidal carbonates of the Mesoproterozoic Kotuikan and Yusmastakh Formations, Anabar Uplift, northeastern Siberia, contain exceptionally well-preserved microfossils . The assemblage is dominated by ellipsoidal akinetes of nostocalean cyanobacteria (Archaeoellipsoides) and problematic spheroidal unicells (Myxococcoides); both are allochthonous and presumably planktonic . The assemblage also includes distinctive mat-forming scytonematacean and entophysalidacean cyanobacteria, diverse short trichomes interpreted as cyanobacterial hormogonia or germinated akinetes, rare longer trichomes, and several types of colonial unicells . Although many taxa in the Kotuikan-Yusmastakh assemblage are long-ranging prokaryotes, the overall character of the assemblage is distinctly Mesoproterozoic, with its major features shared by broadly coeval floras from Canada, China, India, and elsewhere in Siberia . Microfossils also occur in middle to inner shelf shales of the Ust'-Il'ya and lower Kotuikan Formations . Leiosphaerid acritarchs (up to several hundred microns in diameter) characterize this facies . As in other Mesoproterozoic acritarch assemblages, acanthomorphic and other complex forms that typify Neoproterozoic assemblages are absent . The combination in Billyakh assemblages of exceptional preservation and low eukaryotic diversity supports the hypothesis that nucleated organisms diversified markedly near the Mesoproterozoic-Neoproterozoic boundary . The assemblages also demonstrate the antiquity of cyanobacteria capable of cell differentiation and suggest the importance of both changing peritidal substrates and evolving eukaryotes in determining stratigraphic patterns of Proterozoic prokaryotes . The permineralized assemblage contains 33 species belonging to 17 genera . Ten new species or new combinations are proposed: Archaeoellipsoides costatus n . sp., A . elongatus n . comb., A . dolichos n . comb., A . minor n . nom., A . crassus n . comb., A . major n . comb., A . bactroformis n . sp., Veteronostocale medium n . sp., Filiconstrictosus cephalon n . sp., and Partitiofilum yakschinii n . sp.

Symbiosis, 1995, 18, 181 - 210
The microbial community of Ophrydium versatile colonies: endosymbionts, residents, and tenants; Duval B et al.; Ophrydium versatile is a sessile peritrichous ciliate (Kingdom Protoctista, class Oligohymenophora, order Peritrichida, suborder Sessilina) that forms green, gelatinous colonies . Chlorophyll a and b impart a green color to Ophrydium masses due to 400-500 Chlorella-like endosymbionts in each peritrich . Ophrydium colonies, collected from two bog wetlands (Hawley and Leverett, Massachusetts) were analyzed for their gel inhabitants . Other protists include ciliates, mastigotes, euglenids, chlorophytes, and heliozoa . Routine constituents include from 50-100,000 Nitzschia per ml of gel and at least four other diatom genera (Navicula, Pinnularia, Gyrosigma, Cymbella) that may participate in synthesis of the gel matrix . Among the prokaryotes are filamentous and coccoid cyanobacteria, large rod-shaped bacteria, at least three types of spirochetes and one unidentified Saprospira-like organism . Endosymbiotic methanogenic bacteria, observed using fluorescence microscopy, were present in unidentified hypotrichous ciliates . Animals found inside the gel include rotifers, nematodes, and occasional copepods . The latter were observed in the water reservoir of larger Ophrydium masses . From 30-46% of incident visible radiation could be attenuated by Ophrydium green jelly masses in laboratory observations . Protargol staining was used to visualize the elongate macronuclei and small micronucleus of O . versatile zooids and symbiotic algal nuclei . Electron microscopic analysis of the wall of the Chlorella-like symbiont suggests that although the Ophrydium zooids from British Columbia harbor Chlorella vulgaris, those from Hawley Bog contain Graesiella sp . The growth habit in the photic zone and loose level of individuation of macroscopic Ophrydium masses are interpretable as extant analogs of certain Ediacaran biota: colonial protists in the Vendian fossil record.

Neues Jahrb Geol Palaontol Abh, 1995 Feb, 195(1-3), 289 - 302
Taphonomic and evolutionary changes across the Mesoproterozoic-Neoproterozoic transition; Knoll AH et al.; The principal biological distinction between Mesoproterozoic and Neoproterozoic is the abundance and diversity of eukaryotic fossils in the Neoproterozoic rocks, but the two eras also differ in the composition of preserved cyanobacterial assemblages . Evolving eukaryotes provide a partial explanation for observed differences in prokaryotic fossils, but the taphonomic and environmental influences of shifting carbonate depositional pattern are also important.

Adv Space Res, 1995 Mar, 15(3), 237 - 42
Preservation of cell structures in permafrost: a model for exobiology; Soina VS et al.; The present report is the first contribution toward a comprehensive fine-structural study of microbial cells from permafrost . Prokaryotes with a variety of cell wall types demonstrate high stability of cell structure after long-term cryopreservation in frozen soils and sediments of the Arctic . The surface capsular layers that were a salient feature of the cells both in situ and on nutrient media may be an adaptation to low temperature . To the extent that permafrost regions on Earth approximate Martian conditions, preservation of cell structure there can serve as the basis for predictions about preservation in Martian permafrost sediments.

Microb Ecol, 1990, 19, 111 - 8
Water relations and photosynthesis in the cryptoendolithic microbial habitat of hot and cold deserts; Palmer RJ Jr et al.; Two cryptoendolithic microbial communities, lichens in the Ross Desert of Antarctica and cyanobacteria in the Negev Desert, inhabit porous sandstone rocks of similar physical structure . Both rock types adsorb water vapor by physical mechanisms unrelated to biological processes . Yet the two microbial communities respond differently to water stress: cryptoendolithic lichens begin to photosynthesize at a matric water potential of -46.4 megaPascals (MPa) {70% relative humidity (RH) at 8 degrees C}, resembling thallose desert lichens . Cryptoendolithic cyanobacteria, like other prokaryotes, photosynthesize only at very high matric water potentials {> -6.9 MPa, 90% RH at 20 degrees C}.

Geol Mag, 1989, 126(5), 567 - 85
Microfossils from silicified stromatolitic carbonates of the Upper Proterozoic Limestone-Dolomite 'Series', central East Greenland; Green JW et al.; Silicified flake conglomerates and in situ stratiform stromatolites of the Upper Proterozoic (c . 700-800 Ma) Limestone-Dolomite 'Series', central East Greenland, contain well preserved microfossils . Five stratigraphic horizons within the 1200 m succession contain microbial mat assemblages, providing a broad palaeontological representation of late Proterozoic peritidal mat communities . Comparison of assemblages demonstrates that the taxonomy and diversity of mat builder, dweller, and allochthonous populations all vary considerably within and among horizons . The primary mat builder in most assemblages is Siphonophycus inornatum, a sheath-forming prokaryote of probable but not unequivocally established cyanobacterial affinities . An unusual low diversity unit in Bed 17 is dominated by a different builder, Tenuofilum septatum, while a thin cryptalgal horizon in Bed 18 is built almost exclusively by Siphonophycus kestron . Although variable taphonomic histories contribute to observed assemblage variation, most differences within and among horizons appear to reflect the differential success or failure of individual microbial populations in colonizing different tidal flat microenvironments . Twenty-two taxa are recognized, of which two are described as new: Myxococcoides stragulescens n.sp . and Scissilisphaera gradata n . sp.

Adv Space Res, 1996, 17(6-7), 3 - 10
Theories and models on the biology of cells in space; Todd P et al.; A wide variety of observations on cells in space, admittedly made under constraining and unnatural conditions in many cases, have led to experimental results that were surprising or unexpected . Reproducibility, freedom from artifacts, and plausibility must be considered in all cases, even when results are not surprising . The papers in the symposium on "Theories and Models on the Biology of Cells in Space" are dedicated to the subject of the plausibility of cellular responses to gravity--inertial accelerations between 0 and 9.8 m/s2 and higher . The mechanical phenomena inside the cell, the gravitactic locomotion of single eukaryotic and prokaryotic cells, and the effects of inertial unloading on cellular physiology are addressed in theoretical and experimental studies.

Photosynth Res, 1992, 33, 75 - 89
The oldest records of photosynthesis; Awramik SM; There is diverse, yet controversial fossil evidence for the existence of photosynthesis 3500 million years ago . Among the most persuasive evidence is the stromatolites described from low grade metasedimentary rocks in Western Australia and South Africa . Based on the understanding of the paleobiology of stromatolites and using pertinent fossil and Recent analogs, these Early Archean stromatolites suggest that phototrophs evolved by 3500 million years ago . The evidence allows further interpretation that cyanobacteria were involved . Besides stromatolites, microbial and chemical fossils are also known from the same rock units . Some microfossils morphologically resemble cyanobacteria and thus complement the adduced cyanobacterial involvement in stromatolite construction . If cyanobacteria had evolved by 3500 million years ago, this would indicate that nearly all prokaryotic phyla had already evolved and that prokaryotes diversified rapidly on the early Earth.

Adv Space Res, 1992, 12(4), 169 - 80
The seeding of life by comets; Greenberg JM et al.; The evidence that living organisms were already extant on the earth almost 4 Gyr ago and that early bombardment by comets and asteroids created a hostile environment up to about this time has revived the question of how it was possible for prebiotic chemical evolution to have provided the necessary ingredients for life to have developed in the short intervening time . The actual bracketed available temporal space is no more than 0.5 Gyr and probably much less . Was this sufficient time for an earth-based source of the first simple organic precursor molecules to have led to the level of the prokaryotic cell? If not, then the difficulty would be resolved if the ancient earth was impregnated by organic molecular seed from outer space . Curiously, it seems that the most likely source of such seeds was the same a one of the sources of the hostile enviroment, namely the comets which bombarded the earth . With the knowledge of comets gained by the space missions it has become clear that a very large fraction of the chemical composition of comet nuclei consists of quite complex organic molecules . Furthermore it has been demonstrated that comets consist of very fluffy aggregates of interstellar dust whose chemistry derives from photoprocessing of simple ice mixtures in space . Thus, the ultimate source of organics in comets comes from the chemical evolution of interstellar dust . An important and critical justification for assuming that interstellar dust is the ultimate source of prebiotic molecular insertion on the earth is the proof that comets are extremely fluffy aggregates, which have the possibility of breaking up into finely divided fragments when the comet impacts the earth's atmosphere . In the following we will summarize the properties of interstellar dust and the chemical and morphological structure of comets indicated by the most recent interpretations of comet observations . It will be shown that the suitable condition for comets having provided abundant prebiotic molecules as well as the water in which they could have further evolved are consistent with theories of the early earth environment.

Adv Space Res, 1992, 12(4), 143 - 56
The initiation of biological processes on Earth: summary of empirical evidence; Schidlowski M; With the currently available geological record at band, the existence of life on this planet as from at least 3.8 Gyr ago seems so firmly established as to be virtually unassailable . Specifically, various disparate lines of evidence have merged to indicate (1) that the surface of the Archaean Earth had hosted prolific microbial ecosystems as is testified by a quasi-continuous record of microbialites ("stromatolites") and associated microfossils of prokaryotic affinity over 3.5, if not 3.8 Gyr of geological history, and (2) that the sedimentary carbon record has preserved the isotopic signature of autotrophic (notably photosynthetic) carbon fixation over the same time span . With the observed enrichment of isotopically light carbon in sedimentary organic matter largely consonant with the bias in favor of 12C during photosynthesis, the mainstream of the carbon isotope record can be best explained as geochemical manifestation of the isotope discriminating properties of the ribulose-1,5-bisphosphate (RuBP) carboxylase reaction of the Calvin cycle suggesting an extreme degree of evolutionary conservatism in the biochemistry of autotrophic carbon fixation . As a consequence, partial biological control of the geochemical carbon cycle was established already during Early Archaean times and fully operative by the time of formation of the Earth's earliest sediments.

Symbiosis, 1991, 11, 1 - 17
Cristispira from oyster styles: complex morphology of large symbiotic spirochetes; Margulis L et al.; Crystalline styles (digestive organs) of bivalve mollusks provide the habitat for highly motile bacteria . Styles from freshly-collected oysters, Crassostrea virginica, were studied by electron microscopy; Cristispira spirochetes were abundant in these organs . Detailed study reveals these spirochetes to be among the most complex prokaryotic cells known . More than 600 periplasmic flagella and an adhering outer lipoprotein membrane (e.g., a 270 degrees sillon) form the ultrastructural basis for the "crista," first described by light microscopy . Unique rosette structures corresponding to the "chambers" or "ovoid inclusions" of light microscopy were detected at the periphery of all protoplasmic cylinders . Polar organelles and linearly aligned flagellar insertions are conspicuous . In size and complexity, Cristispira more resembles Pillotina, Diplocalyx, Clevelandina and Hollandina (large spirochetes symbiotic in termites) than it does Treponema . Cristispira pectinis (Gross, 1910), the type species; Spirillum ostrea (Noguchi, 1921); and another, less frequent bacterial symbiont are the predominant inhabitants of the dense style matrix . The ultrastructure of the spirillum and an electron micrograph of the third bacterium are shown.

Plant Physiol, 1991, 96, 262 - 8
Purification and characterization of ornithine transcarbamylase from pea (Pisum sativum L.); Slocum RD et al.; Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonacetyl)-L-ornithine-Sepharose 6B affinity chromatography . The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37 degrees C, pH 8.5 . Pea OTC represents approximately 0.05% of the total soluble protein in the leaf . The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography . The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis . These results suggest that the pea OTC is a trimer of identical subunits . The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher . The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.

Plant Physiol, 1992, 100, 1464 - 70
Molecular cloning and evidence for osmoregulation of the delta 1-pyrroline-5-carboxylate reductase (proC) gene in pea (Pisum sativum L.); Williamson CL et al.; Several cDNA clones encoding delta 1-pyrroline-5-carboxylate reductase (P5CR, L-proline:NAD{P}+ 5-oxidoreductase, EC 1.5.1.2), which catalyzes the terminal step in proline biosynthesis, were isolated from a pea leaf library screened with a 32P-labeled Aval fragment of a soybean nodule P5CR cDNA (A.J . Delauney, D.P.S . Verma {1990} Mol Gen Genet 221: 299-305) . DNA sequence analysis of one full-length 1.3-kb clone (pPPS3) indicated that the pea P5CR gene contains a single major open reading frame encoding a polypeptide of 28,242 Da . Genomic analysis suggested that two to three copies of the P5CR gene are present per haploid genome in pea . The primary structure of pea P5CR is 85% identical with that of soybean and exhibits significant homology to human, yeast, and Escherichia coli P5CR . The sequence of one of four highly conserved domains found in all prokaryotic and eukaryotic P5CRs is similar to the consensus sequence for the NAD(P)H-binding site of other enzymes . The pea P5CR cDNA hybridized to two transcripts, 1.3 and 1.1 kb in size, in polyadenylated RNA purified from leaf tissues of mature, light-grown plants (4 weeks old) . Only the 1.3-kb transcript was detected in younger (1 week old) greened seedlings or in etiolated seedlings . In greened seedlings, steady-state levels of this 1.3-kb mRNA increased approximately 5-fold in root tissues within 6 h after plants were irrigated with 0.4 M NaCl, suggesting that expression of the P5CR gene is osmoregulated.

FEMS Microbiol Lett, 1992 Jun 15, 93(3), 209 - 12
The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath); Jahnke LL; Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents . As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed . Methyl sterol content also increased as the growth temperature was lowered . The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively) . The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.

Adv Space Res, 1984, 4(12), 305 - 14
The influence of gravity on structure and function of animals; Ross MD; Gravity is the only environmental parameter that has remained constant during the period of evolution of living matter on Earth . Thus, it must have been a major force in shaping living things . The influence of gravitational loading in evolution of the vertebrate skeleton is well recognized, and scale effects have been studied . This paper, however, considers in addition four pivotal events in early evolution that would seem to have been significant for the later success and diversification of animal life . These are evolution of the cytoskeleton, cell motility (flagellae and cilia), gravity detecting devices (accelerometers), and biomineralization . All are functionally calcium dependent in eukaryotes and all occurred or were foreshadowed in prokaryotes . A major question is why calcium was selected as an ion of great importance to the structure and function of living matter; another is whether gravity played a role in its selection.

Adv Space Res, 1989, 9(11), 111 - 7
Thin film bioreactors in space; Hughes-Fulford M et al.; Studies from the Skylab, SL-3 and D-1 missions have demonstrated that biological organisms grown in microgravity have changes in basic cellular functions such as DNA, mRNA and protein synthesis, cytoskeleton synthesis, glucose utilization and cellular differentiation . Since microgravity could affect prokaryotic and eukaryotic cells at a subcellular and molecular level, space offers us an opportunity to learn more about basic biological systems with one important variable removed . The thin film bioreactor will facilitate the handling of fluids in microgravity, under constant temperature and will allow multiple samples of cells to be grown with variable conditions . Studies on cell cultures grown in microgravity would enable us to identify and quantify changes in basic biological function in microgravity which are needed to develop new applications of orbital research and future biotechnology.

Adv Space Res, 1986, 6(11), 153 - 65
Early and late mammalian responses to heavy charged particles; Ainsworth EJ; This overview summarizes murine results on acute lethality responses, inactivation of marrow CFU-S and intestinal microcolonies, testes weight loss, life span shortening, and posterior lens opacification in mice irradiated with heavy charged particles . RBE-LET relationships for these mammalian responses are compared with results from in vitro studies . The trend is that the maximum RBE for in vivo responses tends to be lower and occurs at a lower LET than for inactivation of V79 and T-1 cells in culture . Based on inactivation cross sections, the response of CFU-S in vivo conforms to expectations from earlier studies with prokaryotic systems and mammalian cells in culture . Effects of heavy ions are compared with fission spectrum neutrons, and the results are consistent with the interpretation that RBEs are lower than for fission neutrons at about the same LET, probably due to differences in track structure . Issues discussed focus on challenges associated with assessments of early and late effects of charged particles based on dose, RBE and LET, and with the concordance or discordance of results obtained with in vivo and in vitro model systems . Models for radiation damage/repair and misrepair should consider effects observed with in vivo as well as in vitro model systems.

J Mol Evol, 1994 Dec, 39(6), 546 - 54
How long did it take for life to begin and evolve to cyanobacteria?
Lazcano A, Miller SL.
There is convincing paleontological evidence showing that stromatolite-building phototactic prokaryotes were already in existence 3.5 x 10(9) years ago . Late accretion impacts may have killed off life on our planet as late as 3.8 x 10(9) years ago . This leaves only 300 million years to go from the prebiotic soup to the RNA world and to cyanobacteria . However, 300 million years should be more than sufficient time . All known prebiotic reactions take place in geologically rapid time scales, and very slow prebiotic reactions are not feasible because the intermediate compounds would have been destroyed due to the passage of the entire ocean through deep-sea vents every 10(7) years or in even less time . Therefore, it is likely that self-replicating systems capable of undergoing Darwinian evolution emerged in a period shorter than the destruction rates of its components (<5 million years) . The time for evolution from the first DNA/protein organisms to cyanobacteria is usually thought to be very long . However, the similarities of many enzymatic reactions, together with the analysis of the available sequence data, suggest that a significant number of the components involved in basic biological processes are the result of ancient gene duplication events . Assuming that the rate of gene duplication of ancient prokaryotes was comparable to today's present values, the development of a filamentous cyanobacterial-like genome would require approximately 7 x 10(6) years--or perhaps much less . Thus, in spite of the many uncertainties involved in the estimates of time for life to arise and evolve to cyanobacteria, we see no compelling reason to assume that this process, from the beginning of the primitive soup to cyanobacteria, took more than 10 million years.

Arch Microbiol, 1984 Oct, 139(2-3), 124 - 9
Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes; Crawford NA et al.; Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts--fructose-1,6-bis-phosphatase (FBPase), sedoheptulose-1,7-bisphosphatase (SBPase), and phosphoribulokinase (PRK)--were partially purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins . Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase . Like their chloroplast counterparts, N . muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f . SBPase was also partially activated by thioredoxin m . PRK, which was present in two regulatory forms in N . muscorum, was activated similarly by thioredoxins f and m . Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts . The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin system, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin . Instead, C . vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5'-AMP . The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system . In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.

Ann N Y Acad Sci, 1987, 503, 515 - 27
Tubulinlike protein from Spirochaeta bajacaliforniensis; Bermudes D et al.; Tubulin proteins are the fundamental subunits of all polymeric microtubule-based eukaryotic structures . Long, hollow structures each composed of 13 protofilaments as revealed by electron microscopy, microtubules (240 angstroms in diameter) are nearly ubiquitous in eukaryotes . These proteins have been the subject of intense biochemical and biophyiscal interest since the early 1970s and are of evolutionary interest as well . If tubulin-based structures (i.e., neurotubules, mitotic spindle tubules, centrioles, kinetosomes, axonemes, etc.) evolved from spirochetes by way of motility symbioses, tubulin homologies with spirochete proteins should be detectable . Tubulin proteins are widely thought to be limited to eukaryotes . Yet both azotobacters and spirochetes have shown immunological cross-reactivity with antitubulin antibodies . In neither of these studies was tubulin isolated nor any specific antigen identified as responsible for the immunoreactivity . Furthermore, although far less uniform in structure than eukaryotic microtubules, various cytoplasmic fibers and tubules (as seen by electron microscopy) have been reported in several types of prokaryotes (e.g., Spirochaeta; large termite spirochetes; treponemes; cyanobacteria; and Azotobacter . This work forms a part of our long-range study of the possible prokaryotic origin of tubulin and microtubules . Spirochetes are helically shaped gram-negative motile prokaryotes . They differ from all other bacterial in that the position of their flagella is periplasmic: their flagella lie between the inner and outer membranes of the gram-negative cell wall . Some of the largest spirochetes have longitudinally aligned 240 angstrom microtubules . Unfortunately, in spite of many attempts, all of the larger spirochetes (family Pillotaceae) with well-defined cytoplasmic tubules and antitubulin immunoreactivity are not cultivable . However, a newly described spirochete species (Spirochaeta bajacaliforniensis) possessing cytoplasmic fibers displays antitubulin immunoreactivity in whole-cell preparations . Since preliminary observations suggested that Spirochaeta bajacaliforniensis proteins may be related to eukaryotic tubulins, their characterization was undertaken . Brain tubulin can be purified by utilizing its ability to polymerize at warm temperatures and to depolymerize in the cold . After several cycles of sedimentation and redissolution the microtubule fraction is comprised of 75% tubulin and 20% high molecular mass microtubule-associated proteins (MAPs) . In this paper we report that components of cell lysates, prepared from a spirochete that contains cytoplasmic fibers (Spirochaeta bajacaliforniensis), also exhibit the property of temperature-dependent cyclical sedimentation . Additionally we report the identification and characterization of the polypeptide responsible for cross-reactivity with antitubulin antiserum.

Ann N Y Acad Sci, 1987, 503, 515 - 27
Tubulinlike protein from Spirochaeta bajacaliforniensis; Bermudes D et al.; Tubulin proteins are the fundamental subunits of all polymeric microtubule-based eukaryotic structures . Long, hollow structures each composed of 13 protofilaments as revealed by electron microscopy, microtubules (240 angstroms in diameter) are nearly ubiquitous in eukaryotes . These proteins have been the subject of intense biochemical and biophysical interest since the early 1970s and are of evolutionary interest as well . If tubulin-based structures (i.e., neurotubules, mitotic spindle tubules, centrioles, kinetosomes, axonemes, etc.) evolved from spirochetes by way of motility symbioses, tubulin homologies with spirochete proteins should be detectable . Tubulin proteins are widely thought to be limited to eukaryotes . Yet both azotobacters and spirochetes have shown immunological cross-reactivity with anitubulin antibodies . In neither of these studies was tubulin isolated nor any specific antigen identified as responsible for the immunoreactivity . Furthermore, although far less uniform in structure than eukaryotic microtubules, various cytoplasmic fibers and tubules (as seen by electron microscopy) have been reported in several types of prokaryotes (e.g., Spirochaeta; large termite spirochetes; treponemes; cyanobacteria; and Azotobacter . This work forms a part of our long-range study of the possible prokaryotic origin of tubulin and microtubules . Spirochetes are helically shaped gram-negative motile prokaryotes . They differ from all other bacteria in that the position of their flagella is periplasmic: their flagella lie between the inner and outer membranes of the gram-negative cell wall . Some of the largest spirochetes have longitudinally aligned 240 angstroms microtubules . Unfortunately, in spite of many attempts, all of the larger spirochetes (family Pillotaceae) with well-defined cytoplasmic tubules and antitubulin immunoreactivity are not cultivable . However, a newly described spirochete species (Spirochaeta bajacaliforniensis) possessing cytoplasmic fibers displays antitubulin immunoreactivity in whole-cell preparations . Since preliminary observations suggested that Spirochaeta bajacaliforniensis proteins may be related to eukaryotic tubulins, their characterization was undertaken . Brain tubulin can be purified by utilizing its ability to polymerize at warm temperatures and to depolymerize in the cold . After several cycles of sedimentation and redissolution the microtubule fraction is composed of 75% tubulin and 20% high molecular mass microtubule-associated proteins (MAPs) . In this paper we report that components of cell lysates, prepared from a spirochete that contains cytoplasmic fibers (Spirochaeta bajacaliforniensis), also exhibit the property of temperature-dependent cyclical sedimentation . Additionally we report the identification and characterization of the polypeptide responsible for cross-reactivity with antitubulin antiserum.

Orig Life Evol Biosph, 1993 Feb, 23(1), 65 - 75
Cryoprotective properties of water in the Earth cryolithosphere and its role in exobiology; Gilichinsky DA et al.; In permanently frozen rocks, water occurs in all the three phases and plays a dual role from the biological point of view . About 93-98% of it is in the solid state . This, alongside with negative temperatures, contributes to cell cryoconservation . The remaining 2-7% is in the unfrozen state and represents thin films enveloping organic-mineral particles . These films play the role of cryoprotectors against cell damage by ice crystals during geologically significant time . Electron microscope examinations of prokaryotes revealed the well preserved outer cell structures, specifically strong envelopes and capsules . The cells are resistant to water phase transitions through 0 degrees C, i.e . to the freezing-thawing stress . The exobiological implication of this phenomenon is determined by the fact that the Earth permafrost at first approximation can he considered as a model of e.g . the Mars one . The latter protects the cells against radiation and simultaneously serves as a cryoconservant . However, most important is the possible presence of unfrozen (= liquid) water as prerequisite for the development of microbial life forms.

Proteins, 2001 Oct 1, 45(1), 40 - 6
Aromatic di-alanine repeats (AdAR) are structural motifs characteristic of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) family; Bruckert F et al.; The aromatic di-alanine repeat is a novel 12-amino acid-long motif constituting alternate small and large hydrophobic residues that mediate the close packing of alpha-helices . A hidden Markov model profile was constructed from the motifs initially described in Soluble N-ethyl maleimide-sensitive factor attachment proteins (SNAP), a family of soluble proteins involved in intracellular membrane fusion . Scanning different sets of protein sequences showed unambiguously that this profile defines a structural motif independent of the tetratrico peptide repeat, another widespread alpha-helical motif . In addition to SNAP, aromatic di-alanine repeats are found in selective LIM homeodomain binding proteins (SLB) and in proteins from the Pyrococcus and Archaeoglobus prokaryotes .

Bioelectromagnetics, 2001 Sep, 22(6), 440 - 8
Intracellular effect of ultrashort electrical pulses; Schoenbach KH et al.; A simple electrical model for biological cells predicts an increasing probability for electric field interactions with cell substructures of prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range . The validity of this hypothesis was verified experimentally by applying electrical pulses with electric field intensities of up to 5.3 MV/m to human eosinophils in vitro . When 3-5 pulses of 60 ns duration were applied to human eosinophils, intracellular granules were modified without permanent disruption of the plasma membrane . In spite of the extreme electrical power levels applied to the cells thermal effects could be neglected because of the ultrashort pulse duration . The intracellular effect extends conventional electroporation to cellular substructures and opens the potential for new applications in apoptosis induction, gene delivery to the nucleus, or altered cell functions, depending on the electrical pulse conditions .

Genome Biol . 2001;2(1):REVIEWS0001 . Epub 2001 Jan 15.
The nitrilase superfamily: classification, structure and function; Pace HC et al.; The nitrilase superfamily consists of thiol enzymes involved in natural product biosynthesis and post-translational modification in plants, animals, fungi and certain prokaryotes . On the basis of sequence similarity and the presence of additional domains, the superfamily can be classified into 13 branches, nine of which have known or deduced specificity for specific nitrile- or amide-hydrolysis or amide-condensation reactions . Genetic and biochemical analysis of the family members and their associated domains assists in predicting the localization, specificity and cell biology of hundreds of uncharacterized protein sequences.

J Virol, 2001 Oct, 75(19), 8968 - 76
Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression; Qing K et al.; Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo . We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K . Y . Qing, X.-S . Wang, D . M . Kube, S . Ponnazhagan, A . Bajpai, and A . Srivastava, Proc . Natl . Acad . Sci . USA 94:10879-10884, 1997) . We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K . Y . Qing, B . Khuntrirat, C . Mah, D . M . Kube, X.-S . Wang, S . Ponnazhagan, S . Z . Zhou, V . J . Dwarki, M . C . Yoder, and A . Srivastava, J . Virol . 72:1593-1599, 1998) . We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C . Mah, K . Y . Qing, B . Khuntrirat, S . Ponnazhagan, X.-S . Wang, D . M . Kube, M . C . Yoder, and A . Srivastava, J . Virol . 72:9835-9841, 1998) . Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52) . FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA . The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe . Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90% . Serine- or threonine-phosphorylated FKBP52 caused approximately 40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis . Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines . These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.

J Biol Chem, 2001 Nov 9, 276(45), 41566 - 75 Epub 2001 Aug 31.
Domain architecture of a high mobility group A-type bacterial transcriptional factor; Padmanabhan S et al.; Myxococcus xanthus transcriptional factor CarD participates in carotenogenesis and fruiting body formation . It is the only reported prokaryotic protein having adjacent "AT-hook" DNA-binding and acidic regions characteristic of eukaryotic high mobility group A (HMGA) proteins . The latter are small, unstructured, nonhistone nuclear proteins that function as architectural factors to remodel DNA and chromatin structure and modulate various DNA binding activities . We find CarD to be predominantly dimeric with two stable domains: (a) an N-terminal domain of defined secondary and tertiary structure which is absent in eukaryotic HMGA proteins; (b) a C-terminal domain formed by the acidic and AT-hook segments and lacking defined structure . CarD, like HMGA proteins, binds specifically to the minor-groove of AT-rich DNA present in two appropriately spaced tracts . As in HMGA proteins, casein kinase II can phosphorylate the CarD acidic region, and this dramatically decreases the DNA binding affinity of CarD . The acidic region, in addition to modulating DNA binding, confers structural stability to CarD . We discuss how the structural and functional plasticity arising from domain organization in CarD could be linked to its role as a general transcriptional factor in M . xanthus.

J Biol Chem, 2001 Nov 2, 276(44), 40926 - 32 Epub 2001 Aug 30.
The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily; Verhees CH et al.; Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars . All but one of the genes that encode the glycolytic enzymes of P . furiosus have previously been identified, either by homology searching of its genome or by reversed genetics . We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P . furiosus and biochemically characterized . The P . furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively . An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h . The N-terminal sequence was used for the identification of the pgiA gene in the P . furiosus genome . The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P . furiosus during growth on maltose and pyruvate . The pgiA gene was functionally expressed in Escherichia coli BL21(DE3) . The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts . In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes . As for the P . furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway . Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.

Circ Res, 2001 Aug 31, 89(5), 461 - 7
Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection; He H et al.; The objective of this study was to identify the mitochondrial proteins that undergo changes in phosphorylation during global ischemia and reperfusion in the isolated rabbit heart . We also assessed whether the cardioprotective intervention of ischemic preconditioning affected mitochondrial protein phosphorylation . We established a reconstituted system using isolated mitochondria and cytosol from control or ischemic hearts . We found that phosphorylation of a 46-kDa protein on a serine residue was increased in ischemia and that phosphorylation was reduced in control or preconditioned hearts . Using 2D gel electrophoresis and mass spectrometry, we have identified the 46-kDa protein as mitochondrial translational elongation factor Tu (EF-Tu(mt)) . These data reveal that ischemia and preconditioning modulate the phosphorylation of EF-Tu(mt) and suggest that the mitochondrial protein synthesis machinery may be regulated by phosphorylation . Phosphorylation of mitochondrial EF-Tu has not been previously described; however, in prokaryotes, EF-Tu phosphorylation inhibits protein translation . We hypothesized that phosphorylation of mitochondrial EF-Tu would inhibit mitochondrial protein translation and attempted to reproduce the effect with inhibition of mitochondrial protein synthesis by chloramphenicol . We found that chloramphenicol pretreatment significantly reduced infarct size, suggesting that mitochondrial protein synthesis is one determinant of myocardial injury during ischemia and reperfusion.

Plant J, 2001 Aug, 27(3), 179 - 89
Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis; Pesaresi P et al.; The prpl11-1 mutant of Arabidopsis thaliana was identified among a collection of T-DNA tagged lines on the basis of a decrease in the effective quantum yield of photosystem II . The mutation responsible was localized to Prpl11, a single-copy nuclear gene that encodes PRPL11, a component of the large subunit of the plastid ribosome . The amino acid sequence of Arabidopsis PRPL11 is very similar to those of L11 proteins from spinach and prokaryotes . In the prpl11-1 mutant, photosensitivity and chlorophyll fluorescence parameters are significantly altered owing to changes in the levels of thylakoid protein complexes and stromal proteins . The abundance of most plastome transcripts examined, such as those of genes coding for the photosystem II core complex and RbcL, is not decreased . Plastid ribosomal RNA accumulates in wild-type amounts, and the assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11 affects neither the abundance of plastid ribosomes nor their assembly into polysomes . However, in vivo translation assays demonstrate that the rate of translation of the large subunit of Rubisco (RbcL) is significantly reduced in prpl11-1 plastids . Our data suggest a major role for PRPL11 in plastid ribosome activity per se, consistent with its location near the GTPase-binding centre of the chloroplast 50S ribosomal subunit . Additional effects of the mutation, including the pale green colour of the leaves and a drastic reduction in growth rate under greenhouse conditions, are compatible with reduced levels of protein synthesis in plastids.

Mol Microbiol, 2001 Aug, 41(4), 873 - 83
Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease; Cromie GA et al.; DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available . However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences . We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid . We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks . Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11 . This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell . Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.

Biol Chem, 2001 Jul, 382(7), 1071 - 5
Singlet molecular oxygen triggers the soxRS regulon of Escherichia coli; Agnez-Lima LF et al.; The electronically excited molecular oxygen (singlet oxygen, 1O2) can be detrimental to cells in several ways, although recent reports indicate that it may play a role as an intercellular signal in eukaryotes . Here we present evidence that 1O2, generated by thermodissociation of disodium 3,3'-(1,4-naphthylidene) diproprionate endoperoxide, activates transcription of genes of the soxRS regulon, and that this induction is paralleled by induction of a soxS'::lacZ operon fusion . The inductions were dependent on a functional soxR gene . These data imply that protective responses, such as induction of the soxRS regulon, may be triggered by diverse environmental oxidative stresses, and that 1O2 may also function as a signal molecule in prokaryotes.

Clin Diagn Lab Immunol, 2001 Sep, 8(5), 922 - 5
New coupled-particle light-scattering assay for detection of Ro/SSA (52 and 60 kilodaltons) and La/SSB autoantibodies in connective tissue diseases; Bizzaro N et al.; The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa {Ro60} and the 52-kDa {Ro52} antibodies) and anti-La/SSB autoantibodies was evaluated . The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system . Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied . Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively . The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively . Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera . Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies . No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sjogren's syndrome . The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.

Acta Crystallogr D Biol Crystallogr, 2001 Sep, 57(Pt 9), 1209 - 18 Epub 2001 Aug 23.
The structure and domain organization of Escherichia coli isocitrate lyase; Britton KL et al.; Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania . Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1 A resolution . E . coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes . Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme . Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzyme's catalytic cycle.

Planta, 2001 Mar, 212(4), 573 - 82
Unusual tolerance to high temperatures in a new herbicide-resistant D1 mutant from Glycine max (L.) Merr . cell cultures deficient in fatty acid desaturation; Alfonso M et al.; The unusual tolerance to heat stress of STR7, an atrazine-resistant mutant isolated from photosynthetic cell-suspension cultures of soybean (Glycine max L . Merr . cv . Corsoy) and characterized previously {M . Alfonso et al . (1996) Plant Physiol 112:1499-1508} has been studied . The STR7 mutant maintained normal growth and fluorescence parameters at higher temperatures than the wild type (WT) . The temperature for 50% inactivation of the oxygen-evolving activity of STR7 thylakoids was 13 degrees C higher than in the WT . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with specific antibodies revealed that the integrity of photosystem II in the STR7 mutant was maintained at higher temperatures than in the WT . This unusual intrinsic tolerance to high temperatures contrasted with the higher sensitivity to heat stress reported as a feature linked to the triazine-resistance trait . The chloroplast membrane of STR7 accumulated an unusually high content of saturated C16:0 and reduced levels of C16:1 and C18:3 unsaturated fatty acids compared with the WT . Among all the lipid classes, chloroplastic lipids synthesized via the prokaryotic pathway (mono-galactosyl-diacyl-glycerol, phosphatidylglycerol and di-galactosyl-diacyl-glycerol), which represented more than 75% of the total lipid classes, showed the most substantial differences in C16:0 and C18:3 levels . In addition, changes in the physicochemical properties of the thylakoid membrane and chloroplast ultrastructure were also detected.

Curr Genet, 2001 Jul, 39(5-6), 377 - 83
A new MFS-transporter gene next to the gibberellin biosynthesis gene cluster of Gibberella fujikuroi is not involved in gibberellin secretion; Voss T et al.; The genes of the gibberellin (GA) biosynthesis pathway in Gibberella fujikuroi are organized in a gene cluster consisting of at least seven genes . Here we report the cloning and characterization of smt, a gene encoding a membrane transporter of the major facilitator super-family 1, which is located next to the GA gene cluster . Since pathway-specific transporters occur frequently in prokaryotic and fungal antibiotic and toxin clusters, smt was thought to be involved in GA secretion . The gene is expressed in mycelium grown under GA-production conditions, but not when the GA biosynthesis is repressed by high amounts of ammonium . To investigate the function of SMT, gene replacement experiments were performed . The smt-mutants did not show any reduction in the GA yield; and gibberellic acid or its precursors did not influence the gene expression . However, sugar alcohols, such as myo-inositol, sorbitol and mannitol, induced the expression of smt . The results demonstrate that the smt gene does not play an essential role in biosynthesis and secretion of GAs in G . fujikuroi, despite the location adjacent to the GA gene cluster.

Prog Nucleic Acid Res Mol Biol, 2001, 67, 65 - 91
The msDNAs of bacteria; Lampson B et al.; msDNAs are small, structurally unique satellite DNAs found in a number of Gram-negative bacteria . Composed of hundreds of copies of single-stranded DNA--hence the name multicopy single-stranded DNA--msDNA is actually a complex of DNA, RNA, and probably protein . These peculiar molecules are synthesized by a reverse transcription mechanism catalyzed by a reverse transcriptase (RT) that is evolutionarily related to the polymerase found in the HIV virus . The genes, including the RT gene, responsible for the synthesis of msDNA are encoded in a retron, a genetic element that is carried on the bacterial chromosome . The retron is, in fact, the first such retroelement to be discovered in prokaryotic cells . This report is a comprehensive review of the many interesting questions raised by this unique DNA and the fascinating answers it has revealed . We have learned a great deal about the structure of msDNA: how it is synthesized, the structure and functions of the RT protein required to make it, its effects on the host cell, the retron element that encodes it, its possible origins and evolution, and even its potential usefulness as a practical genetic tool . Despite the impressive gains in our understanding of the msDNAs, however, the simple, fundamental question of its natural function remains an enduring mystery . Thus, we have much more to learn about the msDNAs of bacteria.

Cell Stress Chaperones, 2001 Jan, 6(1), 71 - 7
The hsp60B gene of Drosophila melanogaster is essential for the spermatid individualization process; Timakov B et al.; The 60-kDa heat shock protein family (Hsp60) is found in prokaryotes, mitochondria, and chloroplasts . The Hsp60 proteins promote proper protein folding by preventing aggregation . In Drosophila melanogaster, the hsp60 gene is essential for a variety of developmental processes, beginning at early embryogenesis . In this study we show that an additional member of the Drosophila hsp60 gene family, hsp60B, is essential in male fertility . In males homozygous for a mutation of the hsp60B gene, developmental processes appeared normal throughout most of spermatogenesis, including spermatocyte growth, meiosis, and spermatid elongation . At these stages, mitochondria also displayed a differentiation process similar to wild-types . However, we found that the mutation disrupted a late stage of spermatogenesis, the spermatid individualization process . In this process, the individualization complex is assembled at spermatid nuclear heads, traverses along spermatid tails, and generates membranes for each of the spermatids in a cyst . Our analysis further shows that the individualization complex in sterile males displayed abnormal morphology as it was traveling along the spermatid tails . The Drosophila Hsp60 proteins are believed to be exclusively localized in the mitochondria . Our observation that the hsp60B mutation displayed no apparent defect in mitochondrial differentiation during spermatogenesis suggests that the Hsp60B protein may operate in a nonmitochondrial location.

Mol Biol (Mosk), 2001 Jul-Aug, 35(4), 718 - 26
{Why termination tRNAs have not been found? Because they are hidden in the large ribosomal RNA}; Ivanov VI et al.; It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation . The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes) . The latter participates in the synthesis proper, recognizing the sense codons . The synthesis is assisted by special proteins: initiation, elongation, and termination factors . The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon . No termination tRNA capable of recognizing stop codons by its anticodon is known . The termination factors are thought to do this . We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons . By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons . The termination factors only condition such a recognition, making it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA) . A strong argument in favor of our hypothesis came from vertebrate mitochondria . They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons) . We revealed that the corresponding anticodons appear in Ter-tRNA1.

Mol Biol (Mosk), 2001 Jul-Aug, 35(4), 647 - 54
{Structure and functions of the prokaryotic elongation factor G}; Gudkov AT; Structural and functional data on elongation factor G (EF-G) are reviewed with regard to nucleotide exchange, GTP hydrolysis, mechanism of action of fusidic acid, and functional roles of the EF-G structural domains in translocation . Biochemical data are correlated with structural dynamics of the EF-G molecule on interaction with various ligands . Data on EF-Tu are also considered, as EF-G and EF-Tu share certain structural and functional features.

Bioinformatics, 2001 Aug, 17(8), 721 - 8
Support vector machine approach for protein subcellular localization prediction; Hua S et al.; MOTIVATION: Subcellular localization is a key functional characteristic of proteins . A fully automatic and reliable prediction system for protein subcellular localization is needed, especially for the analysis of large-scale genome sequences . RESULTS: In this paper, Support Vector Machine has been introduced to predict the subcellular localization of proteins from their amino acid compositions . The total prediction accuracies reach 91.4% for three subcellular locations in prokaryotic organisms and 79.4% for four locations in eukaryotic organisms . Predictions by our approach are robust to errors in the protein N-terminal sequences . This new approach provides superior prediction performance compared with existing algorithms based on amino acid composition and can be a complementary method to other existing methods based on sorting signals . AVAILABILITY: A web server implementing the prediction method is available at SUPPLEMENTARY INFORMATION: Supplementary material is available at http://www.bioinfo.tsinghua.edu.cn/SubLoc/.

FEMS Microbiol Rev, 2001 Aug, 25(4), 405 - 24
The tripartite ATP-independent periplasmic (TRAP) transporters of bacteria and archaea; Kelly DJ et al.; Until recently, extracytoplasmic solute receptor (ESR)-dependent uptake systems were invariably found to possess a conserved ATP-binding protein (the ATP-binding cassette protein or ABC protein), which couples ATP hydrolysis to the translocation of the solute across the cytoplasmic membrane . While it is clear that this class of ABC transporter is ubiquitous in prokaryotes, it is now firmly established that other, unrelated types of membrane transport systems exist which also have ESR components . These systems have been designated tripartite ATP-independent periplasmic (TRAP) transporters, and they form a distinct class of ESR-dependent secondary transporters where the driving force for solute accumulation is an electrochemical ion gradient and not ATP hydrolysis . Currently, the most well characterised TRAP transporter at the functional and molecular level is the high-affinity C4-dicarboxylate transport (Dct) system from Rhodobacter capsulatus . This consists of three proteins; an ESR (DctP) and small (DctQ) and large (DctM) integral membrane proteins . The characteristics of this system are discussed in detail . Homologues of the R . capsulatus DctPQM proteins are present in a diverse range of prokaryotes, both bacteria and archaea, but not in eukaryotes . The deduced structures and possible functions of these homologous systems are described . In addition to the DctP family, other types of ESRs can be associated with TRAP transporters . A conserved family of immunogenic extracytoplasmic proteins is shown to be invariably associated with TRAP systems that contain a large DctQM fusion protein . All of the currently known archaeal systems are of this type . It is concluded that TRAP transporters are a widespread and ancient type of solute uptake system that transport a potentially diverse range of solutes and most likely evolved by the addition of auxiliary proteins to a single secondary transporter.

Extremophiles, 2001 Aug, 5(4), 221 - 8
Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt; Radax C et al.; Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains . DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt . Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters . Similarity values of three clusters to known 16S rRNA genes were less than 90%-95%, suggesting the presence of uncultured novel taxa; two clusters were 98% and 99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to Halobacterium salinarum, respectively . Some rock salt samples, including drilling cores, yielded no amplifiable DNA and no cells or only a few culturable cells . This result suggested a variable distribution of haloarchaea within different strata, probably consistent with the known geologic heterogeneity of Alpine salt deposits . We recently reported identical culturable Halococcus salifodinae strains in Permo-Triassic salt sediments from England, Germany, and Austria; together with the data presented here, those results suggest one plausible scenario to be an ancient continuous hypersaline ocean (Zechstein sea) populated by haloarchaea, whose descendants are found today in the salt sediments . The novelty of the sequences also suggested avoidance of haloarchaeal contaminants during our isolation of strains, preparation of DNA, and PCR reactions.

Mol Genet Genomics, 2001 Jul, 265(5), 771 - 7
Drosophila RNase H1 is essential for development but not for proliferation; Filippov V et al.; Ribonucleases H (RNases H) recognize and specifically degrade RNA that is bound to complementary DNA and are thought to be involved in DNA replication and transcriptional regulation . Though it was previously shown that bacterial RNases H participate in DNA synthesis, none of the known mutations in RNase H genes in either prokaryotes or lower eukaryotes is lethal . Here, we report the characterization of the first loss-of-function mutation in an RNase H1 gene in a metazoan organism, Drosophila melanogaster . Genetic studies of this mutant showed that this gene is essential for metamorphosis in Drosophila . However, disruption of the RNase H1 gene does not affect proliferation, but probably alters the regulation of gene expression . The lethal phenotype of this mutant also demonstrates that RNase H1 activity in Drosophila cannot be provided by other cellular RNase H activities . Analysis of the developmental and spatial expression profiles of a reporter gene placed under the control of the RNase H1 promoter revealed increased expression in several larval tissues . In salivary glands this increase was shown to be inducible by treatment with ecdysone.

Biochimie, 2001 Jul, 83(7), 591 - 9
Glycobiology of surface layer proteins; Schaffer C et al.; Over the last two decades, a significant change of perception has taken place regarding prokaryotic glycoproteins . For many years, protein glycosylation was assumed to be limited to eukaryotes; but now, a wealth of information on structure, function, biosynthesis and molecular biology of prokaryotic glycoproteins has accumulated, with surface layer (S-layer) glycoproteins being one of the best studied examples . With the designation of Archaea as a second prokaryotic domain of life, the occurrence of glycosylated S-layer proteins had been considered a taxonomic criterion for differentiation between Bacteria and Archaea . Extensive structural investigations, however, have demonstrated that S-layer glycoproteins are present in both domains . Among Gram-positive bacteria, S-layer glycoproteins have been identified only in bacilli . In Gram-negative organisms, their presence is still not fully investigated; presently, there is no indication for their existence in this class of bacteria . Extensive biochemical studies of the S-layer glycoprotein from Halobacterium halobium have, at least in part, unravelled the glycosylation pathway in Archaea; molecular biological analyses of these pathways have not been performed, so far . Significant observations concern the occurrence of unusual linkage regions both in archaeal and bacterial S-layer glycoproteins . Regarding S-layer glycoproteins of bacteria, first genetic data have shed some light into the molecular organization of the glycosylation machinery in this domain . In addition to basic S-layer glycoprotein research, the biotechnological application potential of these molecules has been explored . With the development of straightforward molecular biological methods, fascinating possibilities for the expression of prokaryotic glycoproteins will become available . S-layer glycoprotein research has opened up opportunities for the production of recombinant glycosylation enzymes and tailor-made S-layer glycoproteins in large quantities, which are commercially not yet available . These bacterial systems may provide economic technologies for the production of biotechnologically and medically important glycan structures in the future.

FEMS Microbiol Lett, 2001 Aug 21, 202(2), 209 - 13
Polymerase chain reaction assay for the detection of Bacillus cereus group cells; Hansen BM et al.; Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases . Although most cases of diseases caused by the B . cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B . cereus group in food and in the environment . Using 16S rDNA as target, a PCR assay for the detection of B . cereus group cells has been developed . Primers specific for the 16S rDNA of the B . cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control . The PCR procedure was optimized with respect to annealing temperature . When DNA from the B . cereus group bacteria was present, the PCR assay yielded a B . cereus specific fragment, while when non-B . cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products . The PCR analyses with DNA from a number of non-B . cereus confirmed the specificity of the PCR assay.

Growth Factors, 2001, 18(4), 261 - 75
Engineering, expression, and renaturation of a collagen-targeted human bFGF fusion protein; 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 (i) a purification tag, (ii) a protease-sensitive linker/collagen-binding domain, and (iii) cDNA sequence encoding the active fragment of hbFGF . The expressed hbFGF-F1 and hbFGF-F2 (it contains a collagen-binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured using 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 Ni-NTA metal chelate column . The biological activity of the recombinant growth factors was demonstrated by their ability to stimulate proliferation of human vein endothelial cells (HVEC), 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 . Binding of the renatured hbFGF-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column . The high affinity binding was also demonstrated by the binding of {3H}-collagen to the rhbFGF-F2 protein immobilized on a Ni-NTA column . The rhbFGF-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration . 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 its 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 . The clinical effect of rhbFGF-F2 on wound healing is also studied in streptozotocin-induced diabetic rats and evaluated by histological examination comparing with rhbFGF-F1 and commercial bFGF effects . The highly beneficial effects of rhbFGF-F2 on wound healing is suggested to be due to its extremely potent angiogenesis and granulation tissue formation activities, leading to a rapid reepithelialization of the wound . Topical application of rhbFGF-F2 mixed with type I collagen is a more effective method in accelerating closure of full-thickness excisional skin-wound in diabetic rats when compared with the fusion protein alone or commercial hbFGF at the same doses . These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins as well as to develop new strategies for specific biomedical applications.

J Mol Biol, 2001 Aug 24, 311(4), 751 - 9
Crystal structure of human peroxiredoxin 5, a novel type of mammalian peroxiredoxin at 1.5 A resolution; Declercq JP et al.; The peroxiredoxins define an emerging family of peroxidases able to reduce hydrogen peroxide and alkyl hydroperoxides with the use of reducing equivalents derived from thiol-containing donor molecules such as thioredoxin, glutathione, trypanothione and AhpF . Peroxiredoxins have been identified in prokaryotes as well as in eukaryotes . Peroxiredoxin 5 (PRDX5) is a novel type of mammalian thioredoxin peroxidase widely expressed in tissues and located cellularly to mitochondria, peroxisomes and cytosol . Functionally, PRDX5 has been implicated in antioxidant protective mechanisms as well as in signal transduction in cells . We report here the 1.5 A resolution crystal structure of human PRDX5 in its reduced form . The crystal structure reveals that PRDX5 presents a thioredoxin-like domain . Interestingly, the crystal structure shows also that PRDX5 does not form a dimer like other mammalian members of the peroxiredoxin family . In the reduced form of PRDX5, Cys47 and Cys151 are distant of 13.8 A although these two cysteine residues are thought to be involved in peroxide reductase activity by forming an intramolecular disulfide intermediate in the oxidized enzyme . These data suggest that the enzyme would necessitate a conformational change to form a disulfide bond between catalytic Cys47 and Cys151 upon oxidation according to proposed peroxide reduction mechanisms . Moreover, the presence of a benzoate ion, a hydroxyl radical scavenger, was noted close to the active-site pocket . The possible role of benzoate in the antioxidant activity of PRDX5 is discussed .

Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 325 - 8
{Structural and functional study of prokaryotic enhancer-like element VV1 from vaccinia virus}; Cao R et al.; Enhancer VV1 (about 283 bp) is selected as the target to analyze its structure and function systemically . Stepwise deletion experiment is used to identify the functional domain of VV1 element . The results suggest that the 20 bp at 5' terminal and 20 bp at 3' terminal are important to the activity of VV1, for without either of them its activity decreased greatly . Furthermore, the 30-50 bp at 5' terminal is essential to its activity, without which will lead to complete loss of its activity . By random mutagenesis assay it is found that base mutation can regulate the activity of enhancer VV1 positively or negatively . The more the activities of mutants descend, the more mutations take place . For singlebase mutation, the activities change relatively little, and most of the mutations always occur in the 50 bp at the 5' terminal.

Curr Biol, 2001 Jun 5, 11(11), R443 - 5
Cell cycle: connecting DNA replication to sporulation in Bacillus; Michael WM; Checkpoints have been a staple of eukaryotic cell cycle research for the past decade, but little is known about checkpoints in prokaryotes . New work on sporulation in Bacillus fills that gap by showing that such control systems function to coordinate aspects of the bacterial cell cycle.

Protein Sci, 2001 Sep, 10(9), 1905 - 10
N-terminal extension changes the folding mechanism of the FK506-binding protein; Korepanova A et al.; Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein . Here, we investigate the effect of short (three amino acid) N-terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506-binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension . Isothermal equilibrium denaturation experiments demonstrated that the N-terminal extension had a slight destabilizing effect . NMR investigations showed that the N-terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single alpha-helix of FKBP . These structural perturbations probably account for the differential stability . In contrast to the relatively minor equilibrium effects, the N-terminal extension generated a kinetic-folding intermediate that is not observed in the shorter construct . Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects . These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N-terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain.

Biochim Biophys Acta, 2001 Aug 30, 1520(2), 115 - 23
Characterization of a nicotinamide nucleotide transhydrogenase gene from the green alga Acetabularia acetabulum and comparison of its structure with those of the corresponding genes in mouse and Caenorhabditis elegans; Arkblad EL et al.; Proton-pumping nicotinamide nucleotide transhydrogenase (Nnt) is a membrane-bound enzyme that catalyzes the reversible reduction of NADP(+) by NADH . This reaction is linked to proton translocation across the membrane . Depending on metabolic conditions, the enzyme may be involved in NADPH generation, e.g., for detoxification of peroxides and/or free radicals and protection from ischemic damage . Nnt exists in most prokaryotes and in animal mitochondria . It is composed of 2-3 subunits in bacteria and of a single polypeptide in mitochondria . An open question is whether Nnt exists in any photosynthetic eukaryotes and if so, to which class it belongs . In the present study it is demonstrated that, by cloning and sequencing cDNA and genomic copies of its NNT gene, an ancient alga, Acetabularia acetabulum (Chlorophyta, Dasycladales), contains a nuclear-encoded Nnt . In contrast to photosynthetic bacteria, this algal Nnt is composed of a single polypeptide of the class found in animal mitochondria . Excluding a poly(A) tail, NNT cDNA from A . acetabulum is 3688 bp long, consists of eight exons and spans 17 kb . The NNT gene from mouse was also characterized . Subsequently, the gene organization of the A . acetabulum NNT was compared to those of the homologous mouse (100 kb and 21 exons) and Caenorhabditis elegans (5.1 kb and 18 exons) genes.

FASEB J, 2001 Sep, 15(11), 2054 - 6 Epub 2001 Jul 24.
Genome resource utilization during prokaryotic development; Vohradsky J et al.; The distributions of synthesis rates of expressed proteins in a liquid batch culture of the prokaryote S . coelicolor during 3 days' growth have been analyzed by using a law governing the relation between the synthesis rates and the corresponding ranks in a list of rates (the so-called simplified canonical law, scl), which we have found previously to characterize the distribution of prokaryotic protein expression . The scl remains valid throughout development and the two parameters of the distribution, q and r, evolve in a highly characteristic and revealing way . q is a measure of the degree to which available genomic resources are used, in the sense of exploiting their potential diversity . The passage from one developmental phase to another is marked by a sharp peak in q, as these resources are fully mobilized to deal with a crisis (i.e., exhaustion of the habitual food supply) . This is followed by an even more pronounced trough, as the organism briefly focuses its resources on synthesizing just those proteins most essential for survival, especially those hitherto unavailable and needed for metabolizing the new nutrient source . The parameter r indicates redundancy among the most abundantly expressed proteins: higher r corresponds to more diversity; i.e., less duplication of function, hence less robustness . This parameter is relatively steady throughout the development of the culture, except for a pronounced peak during the developmental phase transition . This corresponds to the "emergency mode" characterized by extremely low q, during which a minimum repertoire of proteins is expressed.

Parasitol Res, 2001 Aug, 87(8), 626 - 30
Growth kinetic study of Tetratrichomonas didelphidis isolated from opossum Lutreolina crassicaudata and interaction with a prokaryotic cell; Tasca T et al.; Tetratrichomonas didelphidis is a flagellate protozoan found in the intestine, cecum and colon of opossums, Didelphis marsupialis . This work reports the occurrence of T . didelphidis in another opossum species, Lutreolina crassicaudata . The strain was cultivated in monoxenic culture with Escherichia coli in Diamond (TYM) medium without maltose and with starch solution (trypticase-yeast extract-starch), pH 7.5 at 28 degrees C . The growth kinetic study of T . didelphidis showed a longer time of growth and a higher number of trophozoites when inoculated with E . coli than in axenic cultures, in aerobiosis as well as under anaerobic conditions . Scanning electron microscopy showed that the bacteria adhered throughout the protozoan body and probably evoked endocytic channels, strongly suggesting the existence of endocytosis of rods by T . didelphidis . Our preliminary results suggest that the in vitro culture of T . didelphidis depends on E . coli as a growth-promoting partner, and requires monoxenic cultivation.

J Virol, 2001 Sep, 75(18), 8615 - 23
Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis; Ranjith-Kumar CT et al.; Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3' end of viral RNAs . However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp . In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells . A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity . Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3' terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations . Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.

FEMS Microbiol Lett, 2001 Aug 7, 202(1), 73 - 7
Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species; d'Avila-Levy CM et al.; Crithidia oncopelti, Crithidia deanei and Crithidia desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm . Gelatin-SDS-PAGE analysis was used to characterize cell-associated and extracellular proteinases in these organisms . Our survey indicates that the proteolytic profiles of C . deanei and C . desouzai are identical; that C . oncopelti displays a distinct zymogram; and that species naturally lacking endosymbionts have a more complex extracellular proteolytic activity, which illustrates the heterogeneity of this genus . This is the first report on the presence of cysteine proteinases in the culture supernatant of monoxenic trypanosomatids, and by the use of wild and aposymbiotic strains from C . deanei we also demonstrated that the prokaryote endosymbiont somehow alters quantitatively the expression of extracellular proteinases in this trypanosomatid.

Drug Resist Updat, 1999 Oct, 2(5), 319 - 325
Cell wall targets in methicillin-resistant staphylococci; Labischinski H et al.; Multiresistant staphylococci pose an alarmingly growing problem, especially in serious hospital infections . The recent emergence of strains with reduced susceptibility against vancomycin, the last remaining drug effective against methicillin (multi) resistant Staphylococcus aureus, highlights the urgent need for new antimicrobial agents and new therapeutic regimen . Previously, new drugs were discovered exclusively in bacterial whole cell growth assays . Today's more rational approach depends on the identification of suitable target genes and proteins . These should be bacteria-specific and essential for growth either in vitro or in vivo . Targets within cell wall synthesis and remodeling pathways might be particularly attractive because the bacterial cell wall is a unique structure occurring only in prokaryots; many of the antibiotics in use today have confirmed its 'drugability' . However, several potential targets within this field have not yet been exploited successfully for anti-staphylococcal therapy and some were discovered only recently . After a short summary of known potential targets a set of genes involved in the pentaglycine interpeptide bridge formation of the staphylococcal cell wall will be introduced as interesting targets to combat multiresistant staphylococcal infections .

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2000 Jun, 14(2), 131 - 3
{Preparation of polyclonal antibody to human prion protein using the expressed GST-PrP fusion protein as antigen}; Zhao X et al.; OBJECTIVE: Preparing specific antibody to prion protein . METHODS: Using prokaryotic expressed GST-PrP fusion protein as antigen, rabbits were immunized subcutaneously . RESULTS: ELISA assay revealed that the titer of the prepared antiserum against expressed PrP protein was as high as 1:128 000 . Western blot test showed that the antiserum was able to react with the in vitro expressed intact and different lengths of C-terminus truncated PrP proteins, as well as the native PrP proteins from brain homogenization of human and mouse . CONCLUSIONS: The prokaryotic expressed GST-PrP fusion protein can efficiently elicit in immunized animals the PrP-specific antibody.

Am J Physiol Cell Physiol, 2001 Sep, 281(3), C733 - 9
Escherichia coli as an expression system for K(+) transport systems from plants; Uozumi N; The value of the Escherichia coli expression system has long been established because of its effectiveness in characterizing the structure and function of exogenously expressed proteins . When eukaryotic membrane proteins are functionally expressed in E . coli, this organism can serve as an alternative to eukaryotic host cells . A few examples have been reported of functional expression of animal and plant membrane proteins in E . coli . This mini-review describes the following findings: 1) homologous K(+) transporters exist in prokaryotic cells and in eukaryotic cells; 2) plant K(+) transporters can functionally complement mutant K(+) transporter genes in E . coli; and 3) membrane structures of plant K(+) transporters can be elucidated in an E . coli system . These experimental findings suggest the possibility of utilizing the E . coli bacterium as an expression system for other eukaryotic membrane transport proteins.

Eur J Biochem, 2001 Aug, 268(16), 4562 - 9
Rapid, ATP-dependent degradation of a truncated D1 protein in the chloroplast; Preiss S et al.; The D1 protein constitutes one of the reaction center subunits of photosystem II and turns over rapidly due to photooxidative damage . Here, we studied the degradation of a truncated D1 protein . A plasmid with a precise deletion in the reading frame of the psbA gene encoding D1 was introduced into the chloroplast of Chlamydomonas reinhardtii . A homoplasmic mutant containing the desired gene was able to synthesize the truncated form of the polypeptide, but could not accumulate significant levels of it . As a consequence, other central photosystem II subunits did not assemble within the thylakoid membrane . In vivo pulse-chase experiments showed that the abnormal D1 protein is rapidly degraded in the light . Degradation was delayed in the light in the presence of an uncoupler, or when cells were incubated in the dark . Pulse-chase experiments performed in vitro indicate that an ATP and metal-dependent protease is responsible for the breakdown process . The paper describes the first in vivo and in vitro functional test for ATP-dependent degradation of a defect polypeptide in chloroplasts . The possible involvement of proteases similar to those removing abnormal proteins in prokaryotic organisms is discussed on the basis of proteases recently identified in chloroplasts.

Biochemistry, 2001 Aug 21, 40(33), 9821 - 7
Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues; Sellers VM et al.; The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1) . A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis . The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin . The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max) . The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity . The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate . D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron . On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle . The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction . It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263 . This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.

Environ Toxicol, 2001, 16(4), 321 - 9
Detection of DNA damage in two cell lines from rainbow trout, RTG-2 and RTL-W1, using the comet assay; Nehls S et al.; Screening methods to indicate the genotoxic potential of individual chemicals or environmental mixtures rely mainly on short-term bacterial tests . Differences in the genotoxic response of prokaryotic and eukaryotic cells necessitate the development of nonbacterial screening assays . A promising approach for this purpose could be the comet (single-cell gel electrophoresis) assay performed with fish cells in vitro . In the present study, we evaluated the comet assay with two different fish cell lines from rainbow trout (Oncorhyhnchus mykiss), the fibroblast-like RTG-2 cell line established from gonad tissue, and the epitheloid RTL-W1 cell line established from liver tissue . The cells were exposed in vitro during 2 hr to the genotoxins, 4-nitroquinoline-1-oxide (NQO), and benzo(a)pyrene (BaP), as well as to environmental samples . The LOEC values for NQO were similar in both cell lines, whereas for BaP, the RTL-W1 cells were found to be more sensitive than the RTG-2 cells . The slopes of the concentration-response curves of the two test compounds differed between the two cell lines, with RTG-2 cells showing a steeper slope for NQO, and RTL-W1 cells showing a steeper slope for BaP . When exposed to environmental samples from a remediation site, the RTL-W1 cell line, but not the RTG-2 cell line, indicated a genotoxic potential of the samples . The differences in the genotoxic response pattern of the two cell lines could be only partly explained in relation to metabolic enzymes, cytochrome P4501A, glutathione-S-transferase, and xenobiotic reductase . The findings of this study demonstrate that the comet assay with fish cell lines is suitable as in vitro screening assay in environmental genotoxicity testing, but the choice of test cell line may be critical.

Drug Resist Updat, 2000 Jun, 3(3), 155 - 160
Sulfonamide resistance: mechanisms and trends; Skold O; Sulfonamides were the first drugs acting selectively on bacteria which could be used systemically . Today they are infrequently used, in part due to widespread resistance . The target of sulfonamides, and the basis for their selectivity, is the enzyme dihydropteroate synthase (DHPS) in the folic acid pathway . Mammalian cells are not dependent on endogenous synthesis of folic acid and generally lack DHPS . Instead, they have a folate uptake system which most prokaryotes lack . Laboratory mutants in the dhps (folP) gene can be easily isolated and show a trade off between sulfonamide resistance and DHPS enzyme performance . Clinical resistant mutants, however, have additional compensatory mutations in DHPS that allow it to function normally . In many pathogenic bacteria sulfonamide resistance is mediated by the horizontal transfer of foreign folP or parts of it . Clinical resistance in gram-negative enteric bacteria is plasmid-borne and is effected by genes encoding alternative drug-resistance variants of the DHPS enzymes . Two such genes, sul1 and sul2, have been sequenced and are found at roughly the same frequency among clinical isolates . Remarkably, the corresponding DHPS enzymes show pronounced insensitivity to sulfonamides but normal binding to the p -aminobenzoic acid substrate, despite the close structural similarity between substrate and inhibitor .

Cell Mol Life Sci, 2001 Jun, 58(7), 960 - 77
Regulatory RNAs; Erdmann VA et al.; In addition to mRNA, rRNA and tRNA, which play central roles within cells, there are a number of regulatory, non-coding RNAs (ncRNAs) . Of varying lengths, ncRNAs have no long open reading frame . While not encoding proteins, they may act as riboregulators, and their main function is posttranscriptional regulation of gene expression . Many ncRNAs have been identified and characterized both in prokaryotes and eukaryotes, and are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing, controlling cell growth and differentiation . Some are associated with the abnormalities in imprinted inheritance that occur in several well-known developmental and neurobehavioral disorders . Other ncRNAs accomplish regulation by modulating the activity of proteins . Several rRNAs are able to sustain enzymatic reactions implicated in the translation process including synthesis of peptide bonds within the ribosome . The different roles played by widely distributed RNAs acting in diverse ways, suggest the flexibility and versality of these molecules in regulatory processes . This review summarizes the available biochemical and structural data on known regulatory RNAs.

Microbiology, 2001 Aug, 147(Pt 8), 2113 - 26
p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase; Chang Z et al.; Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products . One matched pabAB, a locus previously identified in S . venezuelae . The second closely resembled the conserved pabB sequence consensus and hybridized with a 3.8 kb NcoI fragment of S . venezuelae ISP5230 genomic DNA . Cloning and sequence analysis of the 3.8 kb fragment detected three ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database . The ORF1 product was similar to PabB in other bacteria and to the PabB domain encoded by S . venezuelae pabAB . The ORF2 product resembled PabA of other bacteria . ORF3 was incomplete; its deduced partial amino acid sequence placed it in the MocR group of GntR-type transcriptional regulators . Introducing vectors containing the 3.8 kb NcoI fragment of S . venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into the Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains . The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosynthesis genes (designated pabB/pabA) cloned in the 3.8 kb fragment . These represent a second set of genes encoding 4-amino-4-deoxychorismate synthase in S . venezuelae ISP5230 . In contrast to the fused pabAB set previously isolated from this species, they do not participate in chloramphenicol biosynthesis, but like pabAB they can be disrupted without affecting growth on minimal medium . The gene disruption results suggest that S . venezuelae may have a third set of genes encoding PABA synthase.

Artif Cells Blood Substit Immobil Biotechnol, 2001 Jul, 29(4), 297 - 316
Beneficial effects of Pluronic F-68 and artificial oxygen carriers on the post-thaw recovery of cryopreserved plant cells; Lowe KC et al.; The storage of prokaryotic and eukaryotic cells at ultra-low temperature in liquid nitrogen (-196 degrees C) is a procedure that has assumed an increasingly important role in underpinning many aspects of biotechnology . For eukaryotic cells, the transition from a cryopreserved state to physiologically normal temperatures and oxygen tensions, induces respiratory imbalances that may stimulate the production of toxic oxygen radicals causing impaired cellular functions . Novel treatments, that focus specifically on enhancing oxygen delivery to cells, are important in maximising post-thaw recovery . Recently, several approaches have been evaluated with suspension cultured plant cells as a model, yet biotechnologically-important, totipotent eukaryotic cell system . Such treatments include non-ionic surfactants, primarily Pluronic F-68, and artificial oxygen carriers, the latter based on inert perfluorochemical liquids or chemically-modifed haemoglobin, as supplements to culture medium used during the post-thaw recovery phase of cell growth . When used either alone or in combination, such novel treatments stimulate significantly the post-thaw viability and biomass production of cultured plant cells . Many of these technologies will be exploitable in cryopreservation protocols for eukaryotic cells in general.

Bioessays, 2001 Aug, 23(8), 708 - 15
Intrinsic DNA bends: an organizer of local chromatin structure for transcription; Ohyama T; DNA with a curved trajectory of its helix axis is called bent DNA, or curved DNA . Interestingly, biologically important DNA regions often contain this structure, irrespective of the origin of DNA . In the last decade, considerable progress has been made in clarifying one role of bent DNA in prokaryotic transcription and its mechanism of action . However, the role of bent DNA in eukaryotic transcription remains unclear . Our recent study raises the possibility that bent DNA is implicated in the "functional packaging" of transcriptional regulatory regions into chromatin . In this article, I review recent progress in bent DNA research in eukaryotic transcription, and summarize the history of bent DNA research and several subjects relevant to this theme . Finally, I propose a hypothesis that bent DNA structures that mimic a negative supercoil, or have a right-handed superhelical writhe, organize local chromatin infrastructure to help the very first interaction between cis-acting DNA elements and activators that trigger transcription .

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9593 - 8 Epub 2001 Aug 07.
A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity; Blindauer CA et al.; Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic . A zinc-induced gene, smtA, is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942 . Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn(4)Cys(9)His(2) cluster . The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys(4) and two ZnCys(3)His sites, giving two fused six-membered rings with distorted boat conformations . The inorganic core strongly resembles the Zn(4)Cys(11) cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands . Also, SmtA contains elements of secondary structure not found in metallothioneins . One of the two Cys(4)-coordinated zinc ions in SmtA readily exchanges with exogenous metal ((111)Cd), whereas the other is inert . The thiolate sulfur ligands bound to zinc in this site are buried within the protein . Regions of beta-strand and alpha-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins . Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers . SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes.

J Biol Chem, 2001 Nov 2, 276(44), 41182 - 90 Epub 2001 Aug 07.
Structural and mutational analysis of the PhoQ histidine kinase catalytic domain . Insight into the reaction mechanism; Marina A et al.; PhoQ is a transmembrane histidine kinase belonging to the family of two-component signal transducing systems common in prokaryotes and lower eukaryotes . In response to changes in environmental Mg(2+) concentration, PhoQ regulates the level of phosphorylated PhoP, its cognate transcriptional response-regulator . The PhoQ cytoplasmic region comprises two independently folding domains: the histidine-containing phosphotransfer domain and the ATP-binding kinase domain . We have determined the structure of the kinase domain of Escherichia coli PhoQ complexed with the non-hydrolyzable ATP analog adenosine 5'-(beta,gamma-imino)triphosphate and Mg(2+) . Nucleotide binding appears to be accompanied by conformational changes in the loop that surrounds the ATP analog (ATP-lid) and has implications for interactions with the substrate phosphotransfer domain . The high resolution (1.6 A) structure reveals a detailed view of the nucleotide-binding site, allowing us to identify potential catalytic residues . Mutagenic analyses of these residues provide new insights into the catalytic mechanism of histidine phosphorylation in the histidine kinase family . Comparison with the active site of the related GHL ATPase family reveals differences that are proposed to account for the distinct functions of these proteins.

J Biol Chem, 2001 Oct 19, 276(42), 38995 - 9001 Epub 2001 Aug 06.
The mechanism of superoxide scavenging by Archaeoglobus fulgidus neelaredoxin; Abreu IA et al.; Neelaredoxin is a mononuclear iron protein widespread among prokaryotic anaerobes and facultative aerobes, including human pathogens . It has superoxide scavenging activity, but the exact mechanism by which this process occurs has been controversial . In this report, we present the study of the reaction of superoxide with the reduced form of neelaredoxin from the hyperthermophilic archaeon Archaeoglobus fulgidus by pulse radiolysis . This protein reduces superoxide very efficiently (k = 1.5 x 10(9) m(-1)s(-1)), and the dismutation activity is rate-limited, in steady-state conditions, by the much slower superoxide oxidation step . These data show unambiguously that the superfamily of neelaredoxin-like proteins (including desulfoferrodoxin) presents a novel type of reactivity toward superoxide, a result of particular relevance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced by the defense cells in eukaryotes . The actual in vivo functioning of these enzymes will depend strongly on the cell redox status . Further insight on the catalytic mechanism was obtained by the detection of a transient intermediate ferric species upon oxidation of neelaredoxin by superoxide, detectable by visible spectroscopy with an absorption maximum at 610 nm, blue-shifted approximately 50 nm from the absorption of the resting ferric state . The role of the iron sixth ligand, glutamate-12, in the reactivity of neelaredoxin toward superoxide was assessed by studying two site-directed mutants: E12Q and E12V.

Plant Cell, 2001 Aug, 13(8), 1919 - 28
Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation; Papa CM et al.; A cytosine DNA methyltransferase containing a chromodomain, Zea methyltransferase2 (Zmet2), was cloned from maize . The sequence of ZMET2 is similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with C-terminal motifs characteristic of eukaryotic and prokaryotic DNA methyltransferases . We used a reverse genetics approach to determine the function of the Zmet2 gene . Plants homozygous for a Mutator transposable element insertion into motif IX had a 13% reduction in methylated cytosines . DNA gel blot analysis of these plants with methylation-sensitive restriction enzymes and bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only at CpNpG sites . No reductions in methylation were observed at CpG or asymmetric sites in heterozygous or homozygous mutant plants . Our research shows that chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequences.

Trends Genet, 2001 Aug, 17(8), 465 - 72
Intein spread and extinction in evolution; Pietrokovski S; Inteins are selfish DNA elements found within coding regions . They are translated with their host protein, but then catalyze their own excision and the formation of a peptide bond between their flanking protein regions . Understanding what drives and selects inteins is relevant for assessing whether they have unidentified biological functions and whether they can invade and become established in new genes and organisms . Inteins are suggested to have been present and more common in the progenitors of eukaryotes and prokaryotes . In these cells, inteins had some beneficial function or had evolved from an unknown beneficial protein . Since then, this putative benefit has been lost and inteins are gradually becoming extinct . The proteins in which inteins are currently found are proposed to be proteins vital for the survival of the organism, where intein removal is most difficult.

Nat Cell Biol, 2001 Aug, 3(8), 767 - 70
Reproducible but dynamic positioning of DNA in chromosomes during mitosis; Dietzel S et al.; How DNA is folded into chromosomes is unknown . Mitotic chromosome banding shows reproducibility in longitudinal compaction at a resolution of several megabase pairs, but it is less clear whether DNA sequences are targeted laterally to specific locations . The in vitro chromosome assembly of prokaryotic DNA suggests that there is a lack of sequence requirements for chromosome condensation, implying an absence of DNA targeting . Protein extraction experiments indicate, however, that specific DNA sequences may bind to a chromosome scaffold . Chromosome banding patterns, using dyes with differential sequence specificity, have been interpreted to result from the alignment of AT-rich sequences in a partially helically folded chromosome scaffold . But fluorescence in situ hybridization experiments, perhaps owing to technical limitations, have shown at best only slight deviation from a random, lateral sequence distribution . Here we show that there is highly reproducible targeting of specific chromosome segments to the metaphase chromatid axis, but that these segments localize to the periphery of prophase and telophase chromosomes . Unfolding intermediates during anaphase and telophase suggest that sequence repositioning occurs through the global uncoiling of an underlying chromatid structure.

J Biol Chem, 2001 Oct 19, 276(42), 38837 - 43 Epub 2001 Aug 01.
Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening; Grozinger CM et al.; The yeast transcriptional repressor Sir2p silences gene expression from the telomeric, rDNA, and silent mating-type loci and may play a role in higher order processes such as aging . Sir2p is the founding member of a large family of NAD-dependent deacetylase enzymes, named the sirtuins . These proteins are conserved from prokaryotes to eukaryotes, but most remain uncharacterized, including all seven human sirtuins . A reverse chemical genetic approach would be useful in identifying the biological function of sirtuins in a wide variety of experimental systems, but no cell-permeable small molecule inhibitors of sirtuins have been reported previously . Herein we describe a high throughput, phenotypic screen in cells that led to the discovery of a class of sirtuin inhibitors . All three compounds inhibited yeast Sir2p transcriptional silencing activity in vivo, and yeast Sir2p and human SIRT2 deacetylase activity in vitro . Such specific results demonstrate the utility and robustness of this screening methodology . Structure-activity relationship analysis of the compounds identified a key hydroxy-napthaldehyde moiety that is necessary and sufficient for inhibitory activity . Preliminary studies using one of these compounds suggest that inhibition of sirtuins interferes with body axis formation in Arabidopsis.

J Biol Chem, 2001 Oct 5, 276(40), 37443 - 50 Epub 2001 Aug 01.
Structure of pyruvate dehydrogenase kinase . Novel folding pattern for a serine protein kinase; Steussy CN et al.; The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is of interest because it represents a family of serine-specific protein kinases that lack sequence similarity with all other eukaryotic protein kinases . Similarity exists instead with key motifs of prokaryotic histidine protein kinases and a family of eukaryotic ATPases . The 2.5-A crystal structure reported here reveals that pyruvate dehydrogenase kinase isozyme 2 has two domains of about the same size . The N-terminal half is dominated by a bundle of four amphipathic alpha-helices, whereas the C-terminal half is folded into an alpha/beta sandwich that contains the nucleotide-binding site . Analysis of the structure reveals this C-terminal domain to be very similar to the nucleotide-binding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.

Genome Res, 2001 Aug, 11(8), 1365 - 74
Prokaryotic homologs of the eukaryotic DNA-end-binding protein Ku, novel domains in the Ku protein and prediction of a prokaryotic double-strand break repair system; Aravind L et al.; Homologs of the eukaryotic DNA-end-binding protein Ku were identified in several bacterial and one archeal genome using iterative database searches with sequence profiles . Identification of prokaryotic Ku homologs allowed the dissection of the Ku protein sequences into three distinct domains, the Ku core that is conserved in eukaryotes and prokaryotes, a derived von Willebrand A domain that is fused to the amino terminus of the core in eukaryotic Ku proteins, and the newly recognized helix-extension-helix (HEH) domain that is fused to the carboxyl terminus of the core in eukaryotes and in one of the Ku homologs from the Actinomycete Streptomyces coelicolor . The version of the HEH domain present in eukaryotic Ku proteins represents the previously described DNA-binding domain called SAP . The Ku homolog from S . coelicolor contains a distinct version of the HEH domain that belongs to a previously unnoticed family of nucleic-acid-binding domains, which also includes HEH domains from the bacterial transcription termination factor Rho, bacterial and eukaryotic lysyl-tRNA synthetases, bacteriophage T4 endonuclease VII, and several uncharacterized proteins . The distribution of the Ku homologs in bacteria coincides with that of the archeal-eukaryotic-type DNA primase and genes for prokaryotic Ku homologs form predicted operons with genes coding for an ATP-dependent DNA ligase and/or archeal-eukaryotic-type DNA primase . Some of these operons additionally encode an uncharacterized protein that may function as nuclease or an Slx1p-like predicted nuclease containing a URI domain . A hypothesis is proposed that the Ku homolog, together with the associated gene products, comprise a previously unrecognized prokaryotic system for repair of double-strand breaks in DNA.

Protein Expr Purif, 2001 Aug, 22(3), 489 - 96
Large-scale preparation of recombinant ovine prolactin and determination of its in vitro and in vivo activity; Leibovich H et al.; Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No . V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No . M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid . The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of approximately 23 kDa . The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb(2) cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes . In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes .

Proc Natl Acad Sci U S A, 2001 Jul 31, 98(16), 9353 - 8
Globin-coupled sensors: a class of heme-containing sensors in Archaea and Bacteria; Hou S et al.; The recently discovered prokaryotic signal transducer HemAT, which has been described in both Archaea and Bacteria, mediates aerotactic responses . The N-terminal regions of HemAT from the archaeon Halobacterium salinarum (HemAT-Hs) and from the Gram-positive bacterium Bacillus subtilis (HemAT-Bs) contain a myoglobin-like motif, display characteristic heme-protein absorption spectra, and bind oxygen reversibly . Recombinant HemAT-Hs and HemAT-Bs shorter than 195 and 176 residues, respectively, do not bind heme effectively . Sequence homology comparisons and three-dimensional modeling predict that His-123 is the proximal heme-binding residue in HemAT from both species . The work described here used site-specific mutagenesis and spectroscopy to confirm this prediction, thereby providing direct evidence for a functional domain of prokaryotic signal transducers that bind heme in a globin fold . We postulate that this domain is part of a globin-coupled sensor (GCS) motif that exists as a two-domain transducer having no similarity to the PER-ARNT-SIM (PAS)-domain superfamily transducers . Using the GCS motif, we have identified several two-domain sensors in a variety of prokaryotes . We have cloned, expressed, and purified two potential globin-coupled sensors and performed spectral analysis on them . Both bind heme and show myoglobin-like spectra . This observation suggests that the general function of GCS-type transducers is to bind diatomic oxygen and perhaps other gaseous ligands, and to transmit a conformational signal through a linked signaling domain.

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9533 - 8 Epub 2001 Jul 31.
Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme; Sandalova T et al.; Thioredoxin reductases (TrxRs) from mammalian cells contain an essential selenocysteine residue in the conserved C-terminal sequence Gly-Cys-SeCys-Gly forming a selenenylsulfide in the oxidized enzyme . Reduction by NADPH generates a selenolthiol, which is the active site in reduction of Trx . The three-dimensional structure of the SeCys498Cys mutant of rat TrxR in complex with NADP(+) has been determined to 3.0-A resolution by x-ray crystallography . The overall structure is similar to that of glutathione reductase (GR), including conserved amino acid residues binding the cofactors FAD and NADPH . Surprisingly, all residues directly interacting with the substrate glutathione disulfide in GR are conserved despite the failure of glutathione disulfide to act as a substrate for TrxR . The 16-residue C-terminal tail, which is unique to mammalian TrxR, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer . A model of the complex of TrxR with Trx suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes . The C-terminal extension typical of mammalian TrxRs has two functions: (i) it extends the electron transport chain from the catalytic disulfide to the enzyme surface, where it can react with Trx, and (ii) it prevents the enzyme from acting as a GR by blocking the redox-active disulfide . Our results suggest that mammalian TrxR evolved from the GR scaffold rather than from its prokaryotic counterpart . This evolutionary switch renders cell growth dependent on selenium.

J Biol Chem, 2001 Oct 5, 276(40), 37194 - 8 Epub 2001 Jul 31.
Identification of the human methylmalonyl-CoA racemase gene based on the analysis of prokaryotic gene arrangements . Implications for decoding the human genome; Bobik TA et al.; In this report, we identify the human DL-methylmalonyl-CoA racemase gene by analyzing prokaryotic gene arrangements and extrapolating the information obtained to human genes by homology searches . Sequence similarity searches were used to identify two groups of homologues that were frequently arranged with prokaryotic methylmalonyl-CoA mutase genes, and that were of unknown function . Both gene groups had homologues in the human genome . Because methylmalonyl-CoA mutases are involved in the metabolism of propionyl-CoA, we inferred that conserved neighbors of methylmalonyl-CoA mutase genes and their human homologues were also involved in this process . Subsequent biochemical studies confirmed this inference by showing that the prokaryotic gene PH0272 and its human homologue both encode DL-methylmalonyl-CoA racemases . To our knowledge this is the first report in which the function of a eukaryotic gene was determined based on the analysis of prokaryotic gene arrangements . Importantly, such analyses are rapid and may be generally applicable for the identification of human genes that lack homologues of known function or that have been misidentified on the basis of sequence similarity searches.

Neoplasma, 2001, 48(2), 85 - 93
Repair of oxidative DNA damage--an important factor reducing cancer risk . Minireview; Brozmanova J et al.; Oxygen free radicals formed during normal aerobic cellular metabolism generate a variety of DNA lesions including modified bases, abasic sites and single strand breaks with blocked 3' termini . If left unrepaired, these damages may contribute to a number of degenerative processes, including cancer and aging . In most organisms, the repair of oxidative DNA lesions is supposed to be handled by the base excision repair (BER) pathway . BER is a multistep process that involves the sequential activity of several proteins, many of them were isolated and functionally characterized using the simple prokaryotic and lower eukaryotic model systems, Escherichia coli and Saccharomyces cerevisiae, respectively . As the amino acid sequence of DNA repair proteins is often well conserved from bacteria to man, our understanding of BER in higher eukaryotes drives extensively from the microbial models, namely from the yeast S . cerevisiae . Thus, results obtained on a simple yeast model are a source of new information, which can be used as a paradigm for all eukaryotic cells.

Biotech Histochem, 2001 May, 76(3), 111 - 8
Use of the gram stain in microbiology; Beveridge TJ; The Gram stain differentiates bacteria into two fundamental varieties of cells . Bacteria that retain the initial crystal violet stain (purple) are said to be "gram-positive," whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be "gram-negative." This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria . Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers . Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization . Some bacteria have walls of intermediate structure and, although they are officially classified as gram-positives because of their linage, they stain in a variable manner . One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.

Mol Med, 2001 May, 7(5), 320 - 8
Expression and characterization of six mutations in the protoporphyrinogen oxidase gene among Finnish variegate porphyria patients; von und zu Fraunberg M et al.; BACKGROUND: Variegate porphyria (VP) is an inherited disorder of heme biosynthesis that results from a partial deficiency of protoporphyrinogen oxidase (PPOX) . Patients with VP may experience acute neurovisceral attacks and cutaneous photosensitivity . To date we have characterized 109 VP patients representing 19 VP families in the Finnish population of 5 million, both biochemically and clinically . MATERIALS AND METHODS: Mutations were identified by direct sequencing of the patients' genomic DNA . The effect of the mutations was determined by sequencing the reverse transcriptase polymerase chain reaction (RT-PCR) product amplified from total RNA extracted from the patients' lymphoblast cell lines and expressing the mutations in E . coli and COS-1 cells . RESULTS: Of the six mutations identified in the PPOX gene, three mutations (IVS2-2a-->c, 338G-->C, and 470A-->4C) caused splicing defects, one produced a frameshift (78insC) and two mutations (R152C and L401F) caused amino acid substitutions . In RT-PCR, the IVS2-2a-->c mutation caused a retention of a 36-bp fragment in the 3' end of intron 2, the 338G-->C mutation caused an exon 4 deletion, and the 470A-->C mutation caused an exon 5 deletion with retention of a 19-bp fragment of the 3' end of intron 5 . In both prokaryotic and eukaryotic expression systems, the PPOX activities of five mutants were decreased to 0-5% of the normal activity . CONCLUSIONS: This study describes five novel mutations and one earlier described major mutation among Finnish VP patients . All mutations produced detectable transcripts, but resulted in decreased PPOX activity confirming the causality of the mutations and the biochemical defects in these patients.

J Clin Microbiol, 2001 Aug, 39(8), 2814 - 22
Characterization and analysis of a stable serotype-associated membrane protein (P30) of Mycoplasma agalactiae; Fleury B et al.; The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli . P30 is encoded on a monocistronic operon determined by two -10 boxes and a possible -35 region constituting the potential promoter, and a transcription termination site . The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site . Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein . Moreover, Triton X-114 phase partitioning of M . agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component . Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M . agalactiae . Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M . agalactiae strains tested and its absence in the other mycoplasma species . Among 27 strains of M . agalactiae studied, 20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies . The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30 . The p30 gene was sequenced in 15 strains of M . agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not express P30 . The negative strains carried mutations in both -10 boxes of the promoters . These mutations seem to be responsible for the lack of P30 expression in these strains . Analysis of sera from sheep that were experimentally infected with M . agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection . In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.

Nature, 2001 Jul 26, 412(6845), 456 - 61
RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes; Bae SH et al.; Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join . Despite the well established in vitro roles of these two enzymes, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal, suggesting that an additional enzymatic activity may be required for the removal of RNA . One such enzyme is the Saccharomyces cerevisiae Dna2 helicase/endonuclease, which is essential for cell viability and is well suited to removing RNA primers of Okazaki fragments . In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments . Here we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA . The sequential action of these enzymes is governed by a single-stranded DNA-binding protein, replication protein-A (RPA) . Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from, and is more complicated than, that occurring in prokaryotes . We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.

Environ Microbiol, 2001 Jun, 3(6), 380 - 96
Bacterial community associated with Pfiesteria-like dinoflagellate cultures; Alavi M et al.; Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment . Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins . As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four 'Pfiesteria-like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay . A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria . The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures . The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species . Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth . Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells . These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the alpha-3 or SAR83 cluster.

Curr Issues Mol Biol, 2000 Oct, 2(4), 125 - 31
Nitrogen fixation in methanogens: the archaeal perspective; Leigh JA; The methanogenic Archaea bring a broadened perspective to the field of nitrogen fixation . Biochemical and genetic studies show that nitrogen fixation in Archaea is evolutionarily related to nitrogen fixation in Bacteria and operates by the same fundamental mechanism . At least six nif genes present in Bacteria (nif H, D, K, E, N and X) are also found in the diazotrophic methanogens . Most nitrogenases in methanogens are probably of the molybdenum type . However, differences exist in gene organization and regulation . All six known nif genes of methanogens, plus two homologues of the bacterial nitrogen sensor-regulator glnB, occur in a single operon in Methanococcus maripaludis . nif gene transcription in methanogens is regulated by what appears to be a classical prokaryotic repression mechanism . At least one aspect of regulation, post-transcriptional ammonia switch-off, involves novel members of the glnB family . Phylogenetic analysis suggests that nitrogen fixation may have originated in a common ancestor of the Bacteria and the Archaea.

Gene, 2001 Jul 11, 272(1-2), 323 - 33
Molecular cloning and characterization of the DNA mismatch repair gene class 2 from the Trypanosoma cruzi; Augusto-Pinto L et al.; Genes with homology to the bacterial mutS gene, which encodes a protein involved in post-replication DNA mismatch repair, are known in several organisms . Using a degenerate PCR strategy, we cloned a Trypanosoma cruzi genomic DNA fragment homologous to the mutS gene class two (MSH2) . This fragment was used as a probe to select the corresponding cDNAs from a T . cruzi cDNA library . The complete sequence of the gene (3304 bp), denominated TcMSH2, was obtained . The sequence contained an open reading frame of 2889 bp coding for a putative protein of 962 amino acids . Computational analyses of the amino acid sequence showed 36% identity with MSH2 proteins from other eukaryotes and revealed the presence of all functional domains of MutS proteins . Hybridization analyses indicated that the TcMSH2 gene is present as a single copy gene that is expressed in all forms of the T . cruzi life cycle . The role of the product of the TcMSH2 gene in mismatch repair was investigated by negative dominance phenotype analyses in Escherichia coli . When eukaryotic muts genes are expressed in a prokaryotic system, they increase the bacterial mutation rate . The same phenomenon was observed with the TcMSH2 cDNA, indicating that T . cruzi MSH2 interferes with the bacterial mismatch system . Phylogenetic analyses showed that the T . cruzi gene grouped with the MSH2 clade confirming the nature of the gene isolated in this work.

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 127 - 32
RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples; Bachoon DS et al.; The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared . Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content . There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage . Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage . In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly . In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.

FEBS Lett, 2001 Jul 20, 501(2-3), 127 - 30
Translational selection shapes codon usage in the GC-rich genome of Chlamydomonas reinhardtii; Naya H et al.; In unicellular species codon usage is determined by mutational biases and natural selection . Among prokaryotes, the influence of these factors is different if the genome is skewed towards AT or GC, since in AT-rich organisms translational selection is absent . On the other hand, in AT-rich unicellular eukaryotes the two factors are present . In order to understand if GC-rich genomes display a similar behavior, the case of Chlamydomonas reinhardtii was studied . Since we found that translational selection strongly influences codon usage in this species, we conclude that there is not a common pattern among unicellular organisms.

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