Eur J Biochem, 2000 Mar, 267(6), 1805 - 12 Molecular cloning of the human and murine 2-amino-3-ketobutyrate coenzyme A ligase cDNAs; Edgar AJ et al.; The conversion of L-threonine to glycine in both prokaryotes and eukaryotes takes place through a two-step biochemical pathway involving the enzymes L-threonine dehydrogenase (EC 1.1.1103) and 2-amino-3-ketobutyrate coenzyme A ligase (KBL; EC 2.3.1.29) . The genes encoding these enzymes have been described in prokaryotes but not in eukaryotes . We report the cloning of transcripts for KBL, the second enzyme in the pathway, from human and murine lung and a partial transcript from bovine liver . Two peptide sequences from the purified bovine KBL protein, one from the N-terminus and the other from the peptide containing the pyridoxal 5'-phosphate-binding lysine residue {Tong, H . & Davis, L . (1994) J . Biol . Chem . 269, 4057-4064}, are identical with regions of the conceptual translation of the transcript obtained from bovine liver . The partial transcript from bovine liver was very similar to the human transcript, being 91% and 92% identical at the nucleotide and amino-acid levels, respectively . The human and murine KBL transcripts are 1.5 kb long, with ORFs encoding proteins of 419 and 416 residues, respectively . The mouse protein has 90% identity with the human protein . The human transcript is strongly expressed in heart, brain, liver and pancreas compared with the lung . The N-termini of both human and mouse proteins have characteristics of mitochondrial import sequences . Both human and murine proteins have 54% identity with the well-characterised prokaryote KLB protein from Escherichia coli . Database searches with the human cDNA sequence enabled us to identify the human KBL gene on chromosome 22q12-13, consisting of nine exons over 9 kb, and a hypothetical Caenorhabditis elegans KLB gene on chromosome IV, consisting of five exons over 2 kb. FEBS Lett, 2000 Mar 3, 469(1), 61 - 6 A chloroplast envelope-transfer sequence functions as an export signal in Escherichia coli; Liu YY et al.; The small subunit precursor of pea ribulose-1,5-bisphosphate carboxylase/oxygenase engineered with prokaryotic elements was expressed in Escherichia coli . This resulted in a dependable level of synthesis of the precursor protein in E . coli . The bacterially synthesised plant precursor protein was translocated from the cytoplasm and targeted to the outer membrane of the envelope zone . During the translocation step, a significant proportion of the precursor was processed to a soluble, mature SSU and found localised in the periplasm . The determined amino acid sequence of the isolated precursor showed that it had a deletion of an arginine residue at position -15 in the transit peptide . Expression of this transit peptide-appended mammalian cytochrome b(5) in E . coli displayed a targeting profile of the chromogenic chimera that was similar to that observed with the plant precursor protein. Science, 2000 Mar 10, 287(5459), 1796 - 9 An archaeal iron-oxidizing extreme acidophile important in acid mine drainage; Edwards KJ et al.; A new species of Archaea grows at pH approximately 0.5 and approximately 40 degrees C in slime streamers and attached to pyrite surfaces at a sulfide ore body, Iron Mountain, California . This iron-oxidizing Archaeon is capable of growth at pH 0 . This species represents a dominant prokaryote in the environment studied (slimes and sediments) and constituted up to 85% of the microbial community when solution concentrations were high (conductivity of 100 to 160 millisiemens per centimeter) . The presence of this and other closely related Thermoplasmales suggests that these acidophiles are important contributors to acid mine drainage and may substantially impact iron and sulfur cycles. Microbiology, 2000 Feb, 146 ( Pt 2), 465 - 75 Catalase deficiency in Staphylococcus aureus subsp . anaerobius is associated with natural loss-of-function mutations within the structural gene; Sanz R et al.; Degenerate oligonucleotide primers based on internal peptide sequences obtained by HPLC from purified Staphylococcus aureus catalase were used to locate the S . aureus and S . aureus subsp . anaerobius kat regions by PCR . Southern hybridization analysis with a probe derived from a 1.1 kb PCR-amplified fragment showed that a single copy of the putative catalase gene was present in the S . aureus and S . aureus subsp . anaerobius chromosome . The nucleotide sequence of S . aureus katA revealed a 1518 bp open reading frame for a protein with 505 amino acids and a predicted molecular mass of 58347 Da, whereas S . aureus subsp . anaerobius katB is 1368 nt long and encodes a polypeptide of 455 amino acids with a predicted molecular mass of 52 584 Da . These catalases are highly homologous to typical monofunctional catalases from prokaryotes . The active-site residues, proximal and distal haem-binding ligands and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in S . aureus KatA . Escherichia coli cells carrying cloned katA had a catalase activity approximately 1000 times that of untransformed E . coli, but no detectable increase in catalase activity was observed with E . coli carrying cloned katB . Northern blotting showed the presence of a kat-specific transcript in S . aureus subsp . anaerobius, suggesting that the lack of catalase activity in this bacterium is due to a post-transcriptional alteration . Compared to the nucleotide sequence of katA, katB showed a single base-pair deletion and six mis-sense mutations, and these alterations were present in three other S . aureus subsp . anaerobius strains analysed . The deletion, located at 1338 bp from the initiation codon, originates a shift of the nucleotide reading frame and is responsible for the premature translation termination at 1368 bp, generating a KatB polypeptide 50 amino acid residues shorter than KatA . Moreover, four of the mis-sense mutations present in katB lead to non-conservative amino acid replacements, the most significant being that located at residue 317 (Pro in KatA-->Ser in KatB) because the affected amino acid is involved in determining the proximal haem-binding site . Both the main alterations found in KatB (the deletion and the substitution in residue 317) seem to contribute to the lack of catalase activity in S . aureus subsp . anaerobius, as deduced from results obtained with chimeric catalase constructs. Int Arch Allergy Immunol, 2000 Feb, 121(2), 87 - 97 CpG DNA as a Th1 trigger; Heeg K et al.; Over the last few years, it has been recognized that along with structural components and products of bacteria, bacterial DNA is also capable of signaling infectious danger to cells of the innate immune system . Particular DNA sequences (CpG motifs), which are abundant in prokaryotic (bacterial) but not in mammalian DNA, cause the activation and stimulation of immune cells . Research has been catalyzed by the finding that certain synthetic oligodeoxynucleotides mimic the action of bacterial DNA . Immunostimulation induced by bacterial DNA or synthetic oligonucleotides not only contributes to our knowledge of the pathogen-host interrelationship during infection, but can also be used therapeutically to condition or modify ongoing immune responses of the adaptive immune system . Accordingly, CpG motifs have been used as vaccine adjuvants as well as instructing agents to selectively induce Th1-dominated immune responses . Hence, CpG motifs might be used in the future as adjuvants and/or immunomodulatory agents to treat or prevent undesired Th2-dominated immune responses, such as allergy . Microbiol Mol Biol Rev, 2000 Mar, 64(1), 153 - 79 Microbial relatives of the seed storage proteins of higher plants: conservation of structure and diversification of function during evolution of the cupin superfamily; Dunwell JM et al.; This review summarizes the recent discovery of the cupin superfamily (from the Latin term "cupa," a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein . Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic beta-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids . In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing . This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase . In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors . As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1, 2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins . Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis . Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation . Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications. Cell Stress Chaperones, 2000 Jan, 5(1), 62 - 71 In vivo and in vitro interaction of DnaK and a chloroplast transit peptide; Ivey RA 3rd et al.; Chloroplast transit peptides have been proposed to function as substrates for Hsp70 molecular chaperones . Many models of chloroplast protein import depict Hsp70s as the translocation motors that drive protein import into the organelle, but to our knowledge, no direct evidence has demonstrated that transit peptides function either in vivo or in vitro as substrates for the chaperone . In this report, we demonstrate that DnaK binds SStp (the full-length transit peptide for the precursor to the small subunit of Rubisco) in vivo when fused to either glutathione-S-transferase (GST) or to an His6-S-peptide tag (His-S) via an ATP-dependent mechanism . Three independent biophysical and biochemical assays confirm the ability of DnaK and SStp to interact in vitro . The cochaperones, DnaJ and GrpE, were also associated with the DnaK/SStp complex . Therefore, both GST-SStp and His-S-SStp can be used as affinity-tagged substrates to study prokaryotic chaperone/transit peptide interactions as well as to provide a novel functional probe to study the dynamics of DnaK/DnaJ/GrpE interactions in vivo . The combination of these results provides the first experimental support for a transit peptide-dependent interaction between a chloroplast precursor and Hsp70 . These results are discussed in light of a general mechanism for protein translocation into chloroplasts and mitochondria. Biochim Biophys Acta, 2000 Feb 28, 1495(3), 223 - 30 NADH dehydrogenase in Neurospora crassa contains myristic acid covalently linked to the ND5 subunit peptide; Plesofsky N et al.; The mitochondrial, proton-pumping NADH:ubiquinone oxidoreductase consists of at least 35 subunits whose synthesis is divided between the cytosol and mitochondria; this complex I catalyzes the first steps of mitochondrial electron transfer and proton translocation . Radiolabel from {(3)H}myristic acid was incorporated by Neurospora crassa into the mitochondrial-encoded, approximately 70 kDa ND5 subunit of NADH dehydrogenase, as shown by immunoprecipitation . This myristate apparently was linked to the peptide through an amide linkage at an invariant lysine residue (Lys546), based upon analyses of proteolysis products . The myristoylated lysine residue occurs in the predicted transmembrane helix 17 (residues 539-563) of ND5 . A consensus amino acid sequence around this conserved residue exists in homologous subunits of NADH dehydrogenase . Cytochrome c oxidase subunit 1, in all prokaryotes and eukaryotes, contains this same consensus sequence surrounding the lysine which is myristoylated in N . crassa. Appl Environ Microbiol, 2000 Mar, 66(3), 1001 - 6 Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling; Rohwer F et al.; Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.) . The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products . In this study, free 3'-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage . Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3'-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT . Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry . TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli . DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3'-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E . coli and H . volcanii, respectively . DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL . This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment. Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1501 - 5 Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I; Hajnsdorf E et al.; Current evidence suggests that the length of poly(A) tails of bacterial mRNAs result from a competition between poly(A) polymerase and exoribonucleases that attack the 3' ends of RNAs . Here, we show that host factor Hfq is also involved in poly(A) tail metabolism . Inactivation of the hfq gene reduces the length of poly(A) tails synthesized at the 3' end of the rpsO mRNA by poly(A) polymerase I in vivo . In vitro, Hfq stimulates synthesis of long tails by poly(A) polymerase I . The strong binding of Hfq to oligoadenylated RNA probably explains why it stimulates elongation of primers that already harbor tails of 20-35 A . Polyadenylation becomes processive in the presence of Hfq . The similar properties of Hfq and the PABPII poly(A) binding protein, which stimulates poly(A) tail elongation in mammals, indicates that similar mechanisms control poly(A) tail synthesis in prokaryotes and eukaryotes. J Mol Biol, 2000 Mar 10, 296(5), 1175 - 81 Site-specific recombination in human cells catalyzed by phage lambda integrase mutants; Lorbach E et al.; Phage lambda Integrase (Int) is the prototype of the so-called integrase family of conservative site-specific recombinases, which includes Cre and FLP . The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively . In contrast to Cre and FLP, however, wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination . Here, we show that two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are proficient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells . Intramolecular integrative recombination is also detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences . Recombination by wild-type Int, however, is not detectable by this method . The latter result implies that eukaryotic co-factors, which could functionally replace the prokaryotic ones normally required for wild-type Int, are most likely not present in human cells . Nature, 2000 Feb 17, 403(6771), 800 - 5 The structures of HsIU and the ATP-dependent protease HsIU-HsIV; Bochtler M et al.; The degradation of cytoplasmic proteins is an ATP-dependent process . Substrates are targeted to a single soluble protease, the 26S proteasome, in eukaryotes and to a number of unrelated proteases in prokaryotes . A surprising link emerged with the discovery of the ATP-dependent protease HslVU (heat shock locus VU) in Escherichia coli . Its protease component HslV shares approximately 20% sequence similarity and a conserved fold with 20S proteasome beta-subunits . HslU is a member of the Hsp100 (Clp) family of ATPases . Here we report the crystal structures of free HslU and an 820,000 relative molecular mass complex of HslU and HslV-the first structure of a complete set of components of an ATP-dependent protease . HslV and HslU display sixfold symmetry, ruling out mechanisms of protease activation that require a symmetry mismatch between the two components . Instead, there is conformational flexibility and domain motion in HslU and a localized order-disorder transition in HslV . Individual subunits of HslU contain two globular domains in relative orientations that correlate with nucleotide bound and unbound states . They are surprisingly similar to their counterparts in N-ethylmaleimide-sensitive fusion protein, the prototype of an AAA-ATPase . A third, mostly alpha-helical domain in HslU mediates the contact with HslV and may be the structural equivalent of the amino-terminal domains in proteasomal AAA-ATPases. Mol Microbiol, 2000 Feb, 35(4), 765 - 76 A new mechanism for the control of a prokaryotic transcriptional regulator: antagonistic binding of positive and negative effectors; Schreiber V et al.; MalT, the transcriptional activator of the Escherichia coli maltose regulon, self-associates, binds promoter DNA and activates initiation of transcription only in the presence of ATP and maltotriose, the inducer . In vivo studies have revealed that MalT action is negatively controlled by the MalY protein . Using a biochemical approach, we analyse here the mechanism whereby MalY represses MalT activity . We show that MalY inhibits transcription activation by MalT in a purified transcription system . In vitro, a constitutive MalT variant (which is partially active in the absence of maltotriose) is less sensitive than wild-type MalT to repression by MalY, as observed in vivo . We demonstrate that MalY forms a complex with MalT only in the absence of maltotriose and that, conversely, MalY inhibits maltotriose binding by MalT . Together, these results establish that MalY acts directly upon MalT without the help of any factor, and that MalY is a negative effector of MalT competing with the inducer for MalT binding. Mol Microbiol, 2000 Feb, 35(4), 756 - 64 Two roles for integration host factor at an enhancer-dependent nifA promoter; Wassem R et al.; Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase . For the sigma54 RNA polymerase, regulatory proteins bound to distally located enhancers engage the polymerase via DNA looping . The sigma54-dependent nifA promoter of Herbaspirillum seropedicae (Hs) is activated under nitrogen-limiting growth conditions . Potential enhancers for the nitrogen control activators NTRC and NIFA and binding sites for integration host factor (IHF) and sigma54-holoenzyme were identified . DNA footprinting experiments showed that these sites functioned for protein binding . Their involvement in the promoter regulation was explored . In vitro, activation of the Hs nifA promoter by NTRC is stimulated by the DNA bending protein IHF . In marked contrast, activation by NIFA is greatly reduced by IHF, thus diminishing potentially destabilizing autoactivation of the nifA promoter by NIFA . Additionally, high levels of NIFA appear to limit NTRC-dependent activation . This inhibition is IHF dependent . Therefore, IHF acts positively and negatively at the nifA promoter to restrict transcription activation to NTRC and one signal transduction pathway. Genes Dev, 2000 Feb 15, 14(4), 414 - 21 Translation by ribosome shunting on adenovirus and hsp70 mRNAs facilitated by complementarity to 18S rRNA; Yueh A et al.; Translation initiation on eukaryotic mRNAs involves 40S ribosome association with mRNA caps (m(7)GpppN), mediated by initiation factor eIF4F . 40S eukaryotic ribosomes and initiation factors undergo 5' scanning to the initiation codon, with no known role for complementarity between eukaryotic 18S rRNA and the 5' noncoding region of mRNAs . We demonstrate that the 5' noncoding region of human adenovirus late mRNAs, known as the tripartite leader, utilizes a striking complementarity to 18S rRNA to facilitate a novel form of translation initiation referred to as ribosome shunting, in which 40S ribosomes bind the cap and bypass large segments of the mRNA to reach the initiation codon . Related elements are also shown to promote ribosome shunting in adenovirus IVa2 intermediate phase mRNA during virus infection and in human heat shock protein 70 (hsp70) mRNA for selective translation during heat shock . The importance of mRNA complementarity to 18S rRNA suggests that ribosome shunting may involve either specific RNA structural features or a prokaryotic-like interaction between mRNA and rRNA. IUBMB Life, 1999 Sep, 48(3), 283 - 6 Immunochemical determination of cellular content of translation release factor RF4 in Escherichia coli; Andersen LD et al.; The biosynthesis of proteins in prokaryotes is terminated when a stop codon is present in the A-site of the 70S ribosomal complex . Four different translation termination factors are known to participate in the termination process . Release factor RF1 and RF2 are responsible for the recognition of the stop codons, and RF3 is known to accelerate the overall termination process . Release factor RF4 is a protein involved in the release of the mRNA and tRNA from the ribosomal complex . Furthermore, RF4 is involved in the proofreading in the elongation step of protein biosynthesis . The cellular contents of RF1, RF2, and RF3 were determined earlier . Here we report the cellular content of RF4 in Escherichia coli to be approximately 16,500 molecules per cell . The cells were grown in a rich medium and harvested in the beginning of the exponential growth phase . The quantifications were performed by using Western immunoblotting with radioactive iodinated streptavidin and biotinylated rabbit anti-mouse immunoglobulins plus a highly specific monoclonal antibody against RF4 as first antibody. Annu Rev Genet, 1999, 33, 533 - 64 Mammalian DNA mismatch repair; Buermeyer AB et al.; DNA mismatch repair (MMR) is one of multiple replication, repair, and recombination processes that are required to maintain genomic stability in prokaryotes and eukaryotes . In the wake of the discoveries that hereditary nonpolyposis colorectal cancer (HNPCC) and other human cancers are associated with mutations in MMR genes, intensive efforts are under way to elucidate the biochemical functions of mammalian MutS and MutL homologs, and the consequences of defects in these genes . Genetic studies in cultured mammalian cells and mice are proving to be instrumental in defining the relationship between the functions of MMR in mutation and tumor avoidance . Furthermore, these approaches have raised awareness that MMR homologs contribute to DNA damage surveillance, transcription-coupled repair, and recombinogenic and meiotic processes. Annu Rev Genet, 1999, 33, 193 - 227 Messenger RNA stability and its role in control of gene expression in bacteria and phages; Grunberg-Manago M; The stability of mRNA in prokaryotes depends on multiple factors and it has not yet been possible to describe the process of mRNA degradation in terms of a unique pathway . However, important advances have been made in the past 10 years with the characterization of the cis-acting RNA elements and the trans-acting cellular proteins that control mRNA decay . The trans-acting proteins are mainly four nucleases, two endo- (RNase E and RNase III) and two exonucleases (PNPase and RNase II), and poly(A) polymerase . RNase E and PNPase are found in a multienzyme complex called the degradosome . In addition to the host nucleases, phage T4 encodes a specific endonuclease called RegB . The cis-acting elements that protect mRNA from degradation are stable stem-loops at the 5' end of the transcript and terminators or REP sequences at their 3' end . The rate-limiting step in mRNA decay is usually an initial endonucleolytic cleavage that often occurs at the 5' extremity . This initial step is followed by directional 3' to 5' degradation by the two exonucleases . Several examples, reviewed here, indicate that mRNA degradation is an important step at which gene expression can be controlled . This regulation can be either global, as in the case of growth rate-dependent control, or specific, in response to changes in the environmental conditions. Annu Rev Genet, 1999, 33, 171 - 91 Shufflons: multiple inversion systems and integrons; Komano T; Conservative site-specific recombination functions to create biological diversity in prokaryotes . Simple site-specific recombination systems consist of two recombination sites and a recombinase gene . The plasmid R64 shufflon contains seven recombination sites, which flank and separate four DNA segments . Site-specific recombinations mediated by the product of the rci gene between any two inverted recombination sites result in the inversion of four DNA segments independently or in groups . The shufflon functions as a biological switch to select one of seven C-terminal segments of the PilV proteins, which is a minor component of R64 thin pilus . The shufflon determines the recipient specificity in liquid matings of plasmid R64 . Other multiple inversion systems as well as integrons, which are multiple insertion systems, are also described in this review. Infect Immun, 2000 Mar, 68(3), 1672 - 80 Complement activation in Mycoplasma fermentans-induced mycoplasma clearance from infected cells: probing of the organism with monoclonal antibodies against M161Ag; Kikkawa S et al.; Mycoplasma fermentans, a cell wall-less prokaryote, is capable of infecting humans and has been suggested to serve as a cofactor in AIDS development . Recently, we discovered a novel lipoprotein with a molecular mass of 43 kDa originating from M . fermentans . This protein, named M161Ag, activated human complement via the alternative pathway and efficiently induced the proinflammatory cytokines interleukin 1beta (IL-1beta), tumor necrosis factor alpha, IL-6, IL-10, and IL-12 in human peripheral blood monocytes . It is likely that M161Ag of M . fermentans affects the host immune system upon mycoplasma infection . In this study, we developed monoclonal antibodies (MAbs) against M161Ag and examined the direct role of complement in M . fermentans infection using these MAbs as probes . M . fermentans was rapidly cleared from the surfaces of infected cells by human complement, but a low-grade infection persisted in human tumor cell lines . Mycoplasma particles remaining alive in host cells may cause recurrent infection, and liberated M161Ag may serve as a biological response modifier affecting both innate and acquired immunity. Infect Immun, 2000 Mar, 68(3), 1069 - 79 The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence; Viswanathan VK et al.; Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease . Using mini-Tn10 mutagenesis, we previously isolated a L . pneumophila mutant that was hypersensitive to iron chelators . This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells . To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation . NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose . Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites . While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease . DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon . This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases . The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes . Southern hybridization analyses determined that the iraAB locus was largely limited to strains of L . pneumophila, the most pathogenic of the Legionella species . A newly derived mutant containing a targeted disruption of iraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells . These data suggest that iraA is critical for virulence of L . pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides. Nature, 2000 Feb 10, 403(6770), 680 - 4 A tripeptide 'anticodon' deciphers stop codons in messenger RNA; Ito K et al.; The two translational release factors of prokaryotes, RF1 and RF2, catalyse the termination of polypeptide synthesis at UAG/UAA and UGA/UAA stop codons, respectively . However, how these polypeptide release factors read both non-identical and identical stop codons is puzzling . Here we describe the basis of this recognition . Swaps of each of the conserved domains between RF1 and RF2 in an RF1-RF2 hybrid led to the identification of a domain that could switch recognition specificity . A genetic selection among clones encoding random variants of this domain showed that the tripeptides Pro-Ala-Thr and Ser-Pro-Phe determine release-factor specificity in vivo in RF1 and RF2, respectively . An in vitro release study of tripeptide variants indicated that the first and third amino acids independently discriminate the second and third purine bases, respectively . Analysis with stop codons containing base analogues indicated that the C2 amino group of purine may be the primary target of discrimination of G from A . These findings show that the discriminator tripeptide of bacterial release factors is functionally equivalent to that of the anticodon of transfer RNA, irrespective of the difference between protein and RNA. Int J Biochem Cell Biol, 2000 Feb, 32(2), 243 - 53 Detection of a mammalian histone H4 kinase that has yeast histidine kinase-like enzymic activity; Besant PG et al.; A well characterized histidine kinase purified from yeast has been shown to phosphorylate histone H4 on a histidine residue . This enzyme is unlike the two-component histidine kinases predominantly found in prokaryotes . Until now, a histidine kinase similar to this yeast enzyme has not been purified from a mammalian source . By using a purification scheme similar to that used to purify the yeast histidine kinase, a protein fraction with histone H4 kinase activity has been isolated from porcine thymus . The yeast histidine kinase was shown to be detectable using an in-gel kinase assay system and using this system, four major bands of histone H4 kinase activity were apparent in the porcine thymus preparation . Through the use of immunoprecipitation, alkaline hydrolysis and subsequent phosphoamino acid analysis it has been demonstrated that this partially purified kinase fraction is capable of phosphorylating histone H4 on histidine . In conclusion, an preparation has been made from porcine thymus that contains histone H4 kinase activity and at least one of the kinases present in this preparation is a histidine kinase. Biochemistry, 2000 Feb 29, 39(8), 2052 - 62 From redox flow to gene regulation: role of the PrrC protein of Rhodobacter sphaeroides 2.4.1; Eraso JM et al.; Activation of photosynthesis (PS) gene expression by the PrrBA two-component activation system in Rhodobacter sphaeroides 2.4.1 results from the interruption of an inhibitory signal originating from the cbb(3) cytochrome c oxidase via its interaction with oxygen, in conjunction with the Rdx redox proteins . The CcoQ protein, encoded by the ccoNOQP operon, which encodes the cbb(3) cytochrome c oxidase, was shown to act as a "transponder" that conveys the signal derived from reductant flow through cbb(3) to oxygen, to the Prr system . To further define the elements comprising this signal transduction pathway we considered the prrC gene product, which to date possessed no definable role in this signal transduction pathway despite its being part of the prrBCA gene cluster . Similar to mutations in cbb(3) and rdx, suitably constructed prrC deletion mutations lead to PS gene expression in the presence of high oxygen . Unlike mutations that remove cbb(3) terminal oxidase activity or Rdx function, the PrrC deletion mutant shows no effect upon cbb(3) activity, nor does it affect the ratio of the carotenoid (Crt) spheroidene (SE) to spheroidenone (SO) . Thus, the PrrC deletion mutant behaves identically to the CcoQ deletion mutant . Taking these and previous results together, we suggest that PrrC is located upstream of the two-component PrrBA activation system in the signal transduction pathway but downstream of the cbb(3) cytochrome c oxidase and its "transponder" CcoQ . The PrrC deletion mutant was also shown to lead to an increase in the DorA protein under aerobic conditions as was shown earlier for the cbb(3) mutant . Finally, PrrC is a member of a highly conserved family of proteins found in both prokaryotes and eukaryotes, and this appears to be the first instance in which a direct regulatory role has been ascribed to a member of this protein family. Biochemistry, 2000 Feb 15, 39(6), 1256 - 62 Actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor; Chen DZ et al.; Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making it an attractive target for the discovery of novel antibiotics . We have identified actinonin, a naturally occurring antibacterial agent, as a potent PDF inhibitor . The dissociation constant for this compound was 0.3 x 10(-)(9) M against Ni-PDF from Escherichia coli; the PDF from Staphylococcus aureus gave a similar value . Microbiological evaluation revealed that actinonin is a bacteriostatic agent with activity against Gram-positive and fastidious Gram-negative microorganisms . The PDF gene, def, was placed under control of P(BAD) in E . coli tolC, permitting regulation of PDF expression levels in the cell by varying the external arabinose concentration . The susceptibility of this strain to actinonin increases with decreased levels of PDF expression, indicating that actinonin inhibits bacterial growth by targeting this enzyme . Actinonin provides an excellent starting point from which to derive a more potent PDF inhibitor that has a broader spectrum of antibacterial activity. J Mol Evol, 2000 Feb, 50(2), 116 - 22 The presence of GSI-like genes in higher plants: support for the paralogous evolution of GSI and GSII genes; Mathis R et al.; Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain . Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes . The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy . In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula . This gene is part of a small gene family and is expressed in many organs of the plant . It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species . Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences . The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution. FEBS Lett, 2000 Jan 28, 466(2-3), 283 - 6 Closed loops of nearly standard size: common basic element of protein structure; Berezovsky IN et al.; By screening the crystal protein structure database for close Calpha-Calpha contacts, a size distribution of the closed loops is generated . The distribution reveals a maximum at 27+/-5 residues, the same for eukaryotic and prokaryotic proteins . This is apparently a consequence of polymer statistic properties of protein chain trajectory . That is, closure into the loops depends on the flexibility (persistence length) of the chain . The observed preferential loop size is consistent with the theoretical optimal loop closure size . The mapping of the detected unit-size loops on the sequences of major typical folds reveals an almost regular compact consecutive arrangement of the loops . Thus, a novel basic element of protein architecture is discovered; structurally diverse closed loops of the particular size. J Biol Chem, 2000 Feb 25, 275(8), 5723 - 32 Distinct physiological functions of thiol peroxidase isoenzymes in Saccharomyces cerevisiae; Park SG et al.; A new type of peroxidase ("thiol peroxidase"; TPx) having cysteine as the primary site of catalysis has been discovered from prokaryotes to eukaryotes . In addition to two yeast TPx isoforms (TSA I and TSA II/AHPC1) previously described, three additional TPx homologues were identified by analysis of the open reading frame data base for Saccharomyces cerevisiae . Three novel isoforms showed a distinct thiol peroxidase activity supported by thioredoxin, and appeared to be distinctively localized in cytoplasm, mitochondria, and nucleus . Each isoform was named after its subcellular localization such as cytoplasmic TPx I (cTPx I or TSA I), cTPx II, cTPx III (TSA II/AHPC1), mitochondrial TPx (mTPx), and nuclear TPx (nTPx) . Their transcriptional activities suggest that cTPx I and cTPx III are the most predominant isoforms among the five type isoforms . Transcriptional activities of TPx isoenzymes during yeast life span were quite different from each other . Unlike other TPx null mutants, cTPx I null mutant was hypersensitive to various oxidants except for 4-nitroquinoline N-oxide . The null mutant was more resistant toward 4-nitroquinoline N-oxide and acidic culture than its wild type . The severe growth retardation of cTPx II mutant resulted in accumulation of G(1)-phased cells . Based on kinetic properties of five isoforms, their subcellular localizations, and distinct physiology of each null mutant, we discussed the physiological functions of five types of TPx isoenzymes in yeast throughout the full growth cycle. J Biol Chem, 2000 Feb 25, 275(8), 5668 - 74 Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit; Garay-Arroyo A et al.; The late embryogenesis abundant (LEA) proteins are plant proteins that are synthesized at the onset of desiccation in maturing seeds and in vegetative organs exposed to water deficit . Here, we show that most LEA proteins are comprised in a more widespread group, which we call "hydrophilins." The defining characteristics of hydrophilins are high glycine content (>6%) and a high hydrophilicity index (>1.0) . By data base searching, we show that this criterion selectively differentiates most known LEA proteins as well as additional proteins from different taxons . We found that within the genomes of Escherichia coli and Saccharomyces cerevisiae, only 5 and 12 proteins, respectively, meet our criterion . Despite their deceivingly loose definition, hydrophilins usually represent <0.2% of the proteins of a genome . Additionally, we demonstrate that the criterion that defines hydrophilins seems to be an excellent predictor of responsiveness to hyperosmosis since most of the genes encoding these proteins in E . coli and S . cerevisiae are induced by osmotic stress . Evidence for the participation of one of the E . coli hydrophilins in the adaptive response to hyperosmotic conditions is presented . Apparently, hydrophilins represent analogous adaptations to a common problem in such diverse taxons as prokaryotes and eukaryotes. J Biol Chem, 2000 Feb 25, 275(8), 5521 - 6 X-ray structure of beta-carbonic anhydrase from the red alga, Porphyridium purpureum, reveals a novel catalytic site for CO(2) hydration; Mitsuhashi S et al.; The carbonic anhydrases (CAs) fall into three evolutionarily distinct families designated alpha-, beta-, and gamma-CAs based on their primary structure . beta-CAs are present in higher plants, algae, and prokaryotes, and are involved in inorganic carbon utilization . Here, we describe the novel x-ray structure of beta-CA from the red alga, Porphyridium purpureum, at 2.2-A resolution using intrinsic zinc multiwavelength anomalous diffraction phasing . The CA monomer is composed of two internally repeating structures, being folded as a pair of fundamentally equivalent motifs of an alpha/beta domain and three projecting alpha-helices . The motif is obviously distinct from that of either alpha- or gamma-CAs . This homodimeric CA appears like a tetramer with a pseudo 222 symmetry . The active site zinc is coordinated by a Cys-Asp-His-Cys tetrad that is strictly conserved among the beta-CAs . No water molecule is found in a zinc-liganding radius, indicating that the zinc-hydroxide mechanism in alpha-CAs, and possibly in gamma-CAs, is not directly applicable to the case in beta-CAs . Zinc coordination environments of the CAs provide an interesting example of the convergent evolution of distinct catalytic sites required for the same CO(2) hydration reaction. Gene Ther, 2000 Jan, 7(1), 70 - 4 Fas ligand gene-carrying adeno-5 AdEasy viruses can be efficiently propagated in apoptosis-sensitive human embryonic retinoblast 911 cells; Watzlik A et al.; Recent reports have pointed out the difficulties in generating recombinant adenoviral vectors expressing FasL using eukaryotic cells . In the present study we cloned the murine FasL (mFasL) gene into the pCA14 or pShuttle vectors and recombined them with the adeno-5 virus backbone pBHG10 or pAdEasy-1 in eukaryotic (911 cells) or prokaryotic (E . coli) cells, respectively . Recombination of pCA14-mFasL with pBHG10 in Fas-carrying 911 cells leads to rapid expression of mFasL and cell death by apoptosis before virus replication is initiated . The same effect is observed when 911 cells are transfected with the pCA14-mFasL shuttle vector only . If recombination (pShuttle-mFasL with pAdEasy-1) is performed first in bacteria and 911 cells are transfected thereafter with recombined AdEasy-mFasL, virus production starts immediately and the cells survive longer . The resulting AdEasy-mFasL viruses are able to infect other eukaryotic cells and induce expression of functional mFasL . This study describes a method for efficient generation of recombinant FasL adeno-5 viruses in eukaryotic cells . The method may be generally suitable for producing viruses carrying deleterious genes . Gene Therapy (2000) 7, 70-74. Curr Opin Struct Biol, 2000 Feb, 10(1), 117 - 23 Insights into transcription: structure and function of single-subunit DNA-dependent RNA polymerases; Cheetham GM et al.; Single-subunit RNA polymerases are widespread throughout prokaryotic and eukaryotic organisms, and also viruses . T7 RNA polymerase is one of the simplest DNA-dependent enzymes, capable of transcribing a complete gene without the need for additional proteins . During the past two years, three illuminating crystal structures of T7 RNA polymerase complexed to either T7 lysozyme, which is a transcription inhibitor, an open promoter DNA fragment or a promoter DNA fragment being transcribed into RNA at initiation have been determined . For the first time, these structures describe in detail the intricate mechanism of transcription initiation by T7 RNA polymerase, which is likely to be a general model for other related RNA polymerases. Curr Opin Struct Biol, 2000 Feb, 10(1), 26 - 33 Protein folding in vivo: the importance of molecular chaperones; Feldman DE et al.; The contribution of the two major cytosolic chaperone systems, Hsp70 and the cylindrical chaperonins, to cellular protein folding has been clarified by a number of recent papers . These studies found that, in vivo, a significant fraction of newly synthesized polypeptides transit through these chaperone systems in both prokaryotic and eukaryotic cells . The identification and characterization of the cellular substrates of chaperones will be instrumental in understanding how proteins fold in vivo. Curr Opin Immunol, 2000 Feb, 12(1), 35 - 43 The role of CpG motifs in innate immunity; Krieg AM; Pattern recognition receptors of the innate immune system are able to distinguish certain prokaryotic DNAs from vertebrate DNAs by detecting unmethylated CpG dinucleotides in particular base contexts ('CpG motifs') . Recent studies have begun to define the molecular mechanisms of actions of CpG motifs and have demonstrated their stimulatory effects on leukocytes from humans and vertebrates other than mice . Oligodeoxynucleotides containing CpG motifs are highly effective Th1-like vaccine adjuvants through multiple routes of immunization and show promise as immunotherapeutic agents for cancer and allergic diseases. J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 119 - 30 Purification of the membrane binding domain of cytochrome b5 by immobilised nickel chelate chromatography; Begum RR et al.; The purification of a eukaryotic membrane protein has been achieved using a prokaryotic expression system . Bovine cytochrome b5 is an integral membrane protein (Mr approximately 16500) . It comprises of a globular haem containing catalytic domain positioned at the N-terminus of the protein and a hydrophobic membrane binding segment at the C-terminus . The membrane binding domain (MBD) is resistant to purification using conventional strategies that have proved successful in isolating the soluble haem containing fragment . We report here a versatile purification method for the isolation of the MBD involving a gene fusion system . The fusion protein incorporates thioredoxin at the amino terminus and six histidines as the metal affinity binding site followed by cytochrome b5 in a pET expression system . This supports high level expression of cytochrome b5 in E . coli C43(DE3) cells . The fusion protein is effectively solubilised from lysed cells with Triton X-100 . A step gradient elution with imidazole under non-denaturing conditions on a His-Bind nickel chelate affinity column, saturated with proteins as a crude cell extract, purified the protein in a single step . Proteolytic digestion of pure fusion protein, with trypsin, yielded the MBD . This fragment was further purified by RP-HPLC to a final yield of approximately 10 mg/l. Structure Fold Des, 2000 Jan 15, 8(1), 13 - 23 Crystal structure of RNA 3'-terminal phosphate cyclase, a ubiquitous enzyme with unusual topology; Palm GJ et al.; BACKGROUND: RNA cyclases are a family of RNA-modifying enzymes that are conserved in eucarya, bacteria and archaea . They catalyze the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA, in a reaction involving formation of the covalent AMP-cyclase intermediate . These enzymes might be responsible for production of the cyclic phosphate RNA ends that are known to be required by many RNA ligases in both prokaryotes and eukaryotes . RESULTS: The high-resolution structure of the Escherichia coli RNA 3'-terminal phosphate cyclase was determined using multiwavelength anomalous diffraction . Two orthorhombic crystal forms of E . coli cyclase (space group P2(1)2(1)2(1) and P2(1)2(1)2) were used to solve and refine the structure to 2.1 A resolution (R factor 20.4%; R(free) 27.6%) . Each molecule of RNA cyclase consists of two domains . The larger domain contains three repeats of a folding unit comprising two parallel alpha helices and a four-stranded beta sheet; this fold was previously identified in translation initiation factor 3 (IF3) . The large domain is similar to one of the two domains of 5-enolpyruvylshikimate-3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvyl transferase . The smaller domain uses a similar secondary structure element with different topology, observed in many other proteins such as thioredoxin . CONCLUSIONS: The fold of RNA cyclase consists of known elements connected in a new and unique manner . Although the active site of this enzyme could not be unambiguously assigned, it can be mapped to a region surrounding His309, an adenylate acceptor, in which a number of amino acids are highly conserved in the enzyme from different sources . The structure of E . coli cyclase will be useful for interpretation of structural and mechanistic features of this and other related enzymes. Res Microbiol, 1999 Nov-Dec, 150(9-10), 779 - 99 Promoter sequences and algorithmical methods for identifying them; Vanet A et al.; This paper presents a survey of currently available mathematical models and algorithmical methods for trying to identify promoter sequences . The methods concern both searching in a genome for a previously defined consensus and extracting a consensus from a set of sequences . Such methods were often tailored for either eukaryotes or prokaryotes although this does not preclude use of the same method for both types of organisms . The survey therefore covers all methods; however, emphasis is placed on prokaryotic promoter sequence identification . Illustrative applications of the main extracting algorithms are given for three bacteria. Res Microbiol, 1999 Nov-Dec, 150(9-10), 725 - 33 Functional and evolutionary roles of long repeats in prokaryotes; Rocha EP et al.; Most recently published complete bacterial genomes have revealed unexpectedly high numbers of long strict repeats . In this article we discuss the various functional and evolutionary roles of these repeats, focusing in particular on their role in terms of genome stability, gene transfer, and antigenic variation. Res Microbiol, 1999 Nov-Dec, 150(9-10), 689 - 99 Paralogous genes encoding transport proteins in microbial genomes; Saier MH Jr et al.; The largest superfamilies of prokaryotic genes encode transport proteins, but many transporters are encoded by orphan genes or those that comprise very small families . We have analyzed eighteen completely sequenced prokaryotic genomes for paralogous transport systems and have thereby identified 76 permease families . In this short review, we present and discuss the paralogues in some of these families and interpret the most prominent results, particularly those relevant to the largest permease superfamilies. Ann N Y Acad Sci, 1999, 893, 61 - 78 The alpha-ketoglutarate dehydrogenase complex; Sheu KF et al.; The alpha-ketoglutarate dehydrogenase complex (KGDHC) is an important mitochondrial constituent, and deficiency of KGDHC is associated with a number of neurological disorders . KGDHC is composed of three proteins, each encoded on a different and well-characterized gene . The sequences of the human proteins are known . The organization of the proteins into a large, ordered multienzyme complex (a "metabolon") has been well studied in prokaryotic and eukaryotic species . KGDHC catalyzes a critical step in the Krebs tricarboxylic acid cycle, which is also a step in the metabolism of the potentially excitotoxic neurotransmitter glutamate . A number of metabolites modify the activity of KGDHC, including inactivation by 4-hydroxynonenal and other reactive oxygen species (ROS) . In human brain, the activity of KGDHC is lower than that of any other enzyme of energy metabolism, including phosphofructokinase, aconitase, and the electron transport complexes . Deficiencies of KGDHC are likely to impair brain energy metabolism and therefore brain function, and lead to manifestations of brain disease . In general, the clinical manifestations of KGDHC deficiency relate to the severity of the deficiency . Several such disorders have been recognized: infantile lactic acidosis, psychomotor retardation in childhood, intermittent neuropsychiatric disease with ataxia and other motor manifestations, Friedreich's and other spinocerebellar ataxias, Parkinson's disease, and Alzheimer's disease (AD) . A KGDHC gene has been associated with the first two and last two of these disorders . KGDHC is not uniformly distributed in human brain, and the neurons that appear selectively vulnerable in human temporal cortex in AD are enriched in KGDHC . We hypothesize that variations in KGDHC that are not deleterious during reproductive life become deleterious with aging, perhaps by predisposing this mitochondrial metabolon to oxidative damage. Mol Microbiol, 2000 Feb, 35(3), 490 - 516 A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi; Casjens S et al.; We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium . Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp . We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate . This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences) . Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids . Many of these events appear to have been mediated by recombinational processes that formed duplications . These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene . These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome . The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution . The sequence predicts 535 plasmid genes >/=300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes) . The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions. Eur J Biochem, 2000 Feb, 267(4), 1178 - 87 Specificity of the wound-induced leucine aminopeptidase (LAP-A) of tomato activity on dipeptide and tripeptide substrates; Gu YQ et al.; Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A) that preferentially hydrolyzes amino acid-p-nitroanilide and -beta-naphthylamide substrates with N-terminal Leu, Met and Arg residues . To determine the substrate specificity of LAP-A on more natural substrates, the rates of hydrolysis of 60 dipeptide and seven tripeptide substrates were determined . For comparison, the specificities of the porcine and Escherichia coli LAPs were evaluated in parallel . Several marked differences in substrate specificities for the animal, plant and prokaryotic LAP enzymes were observed . Substrates with variable N-terminal (P1) residues (Xaa) were evaluated; these substrates had Leu or Gly in the penultimate (P1') position . The plant, animal, and prokaryotic LAPs hydrolyzed dipeptides with N-terminal nonpolar aliphatic (Leu, Val, Ile, and Ala), basic (Arg), and sulfur-containing (Met) residues rapidly, while P1 Asp or Gly were cleaved inefficiently from peptides . Significant differences in the cleavage of dipeptides with P1 aromatic residues (Phe, Tyr, and Trp) were noted . To systematically evaluate the impact of the P1' residue on cleavage of dipeptides, three series of dipeptides (Leu-Xaa, Gly-Xaa, and Arg-Xaa) were evaluated . The P1' residue strongly influenced hydrolysis of dipeptides and the magnitude of its effect was dependent on the P1 residue . P1' Pro, Asp, Lys and Gly slowed the hydrolysis rates of the tomato LAP-A, porcine LAP, and E . coli PepA markedly . Analysis six Arg-Gly-Xaa tripeptides showed that more diversity was tolerated in the P2' position . P2' Arg inhibited tripeptide cleavage by all three enzymes, while P2' Asp enhanced hydrolysis rates for the porcine and prokaryotic LAPs. Philos Trans R Soc Lond B Biol Sci, 1999 Dec 29, 354(1392), 1923 - 39 Life: past, present and future; Nealson KH et al.; Molecular methods of taxonomy and phylogeny have changed the way in which life on earth is viewed; they have allowed us to transition from a eukaryote-centric (five-kingdoms) view of the planet to one that is peculiarly prokarote-centric, containing three kingdoms, two of which are prokaryotic unicells . These prokaryotes are distinguished from their eukaryotic counterparts by their toughness, tenacity and metabolic diversity . Realization of these features has, in many ways, changed the way we feel about life on earth, about the nature of life past and about the possibility of finding life elsewhere . In essence, the limits of life on this planet have expanded to such a degree that our thoughts of both past and future life have been altered . The abilities of prokaryotes to withstand many extreme conditions has led to the term extremophiles, used to describe the organisms that thrive under conditions thought just a few years ago, to be inconsistent with life . Perhaps the most extensive adaptation to extreme conditions, however, is represented by the ability of many bacteria to survive nutrient conditions not compatible with eukaryotic life . Prokaryotes have evolved to use nearly every redox couple that is in abundance on earth, filling the metabolic niches left behind by the oxygen-using, carbon-eating eukaryotes . This metabolic plasticity leads to a common feature in physically stratified environments of layered microbial communities, chemical indicators of the metabolic diversity of the prokaryotes . Such 'metabolic extremophily' forms a backdrop by which we can view the energy flow of life on this planet, think about what the evolutionary past of the planet might have been, and plan ways to look for life elsewhere, using the knowledge of energy flow on earth. Int J Biol Markers, 1999 Oct-Dec, 14(4), 263 - 7 Endostatin: a promising drug for antiangiogenic therapy; Cirri L et al.; Angiogenesis, the formation of new blood vessels from existing capillaries, is critical for tumors to grow beyond a few in size . Tumor cells produce one or more angiogenic factors including fibroblast growth factor and vascular endothelial growth factor . Surprisingly, antiangiogenic factors or angiogenesis inhibitors have been isolated from tumors . Some angiogenesis inhibitors, such as angiostatin, are associated with tumors while others, such as platelet-factor 4 and interferon-alpha are not . Endostatin, a C-terminal product of collagen XVIII, is a specific inhibitor of endothelial cell proliferation, migration and angiogenesis . The mechanism by which endostatin inhibits endothelial cell proliferation and migration is unknown . Endostatin was originally expressed in a prokaryotic system and, late, in a yeast system, thanks to which it is possible to obtain a sufficient quantity of the protein in a soluble and refolded form to be used in preclinical and clinical trials. Mol Cell Biol, 2000 Mar, 20(5), 1816 - 24 A DNA helicase required for maintenance of the functional mitochondrial genome in Saccharomyces cerevisiae; Sedman T et al.; A novel DNA helicase, a homolog of several prokaryotic helicases, including Escherichia coli Rep and UvrD proteins, is encoded by the Saccharomyces cerevisiae nuclear genome open reading frame YOL095c on the chromosome XV . Our data demonstrate that the helicase is localized in the yeast mitochondria and is loosely associated with the mitochondrial inner membrane during biochemical fractionation . The sequence of the C-terminal end of the 80-kDa helicase protein is similar to a typical N-terminal mitochondrial targeting signal; deletions and point mutations in this region abolish transport of the protein into mitochondria . The C-terminal signal sequence of the helicase targets a heterologous carrier protein into mitochondria in vivo . The purified recombinant protein can unwind duplex DNA molecules in an ATP-dependent manner . The helicase is required for the maintenance of the functional ({rho(+)}) mitochondrial genome on both fermentable and nonfermentable carbon sources . However, the helicase is not essential for the maintenance of several defective ({rho(-)}) mitochondrial genomes . We also demonstrate that the helicase is not required for transcription in mitochondria. Acta Crystallogr D Biol Crystallogr, 1999 Dec, 55 ( Pt 12), 2049 - 50 Crystallization and preliminary X-ray analysis of Thermotoga maritima ribosome recycling factor; Selmer M et al.; Thermotoga maritima ribosome recycling factor (RRF) is one of the proteins catalyzing the fourth step in prokaryotic protein synthesis, ribosome recycling . The RRF protein was crystallized with ammonium sulfate . Native diffraction data to 2.55 A resolution were obtained at the MAX II synchrotron from a flash-frozen crystal at 100 K . The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 47, c = 298 A, and probably contain one monomer per asymmetric unit. Nucleic Acids Res, 2000 Mar 1, 28(5), 1237 - 44 Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase; Carrodeguas JA et al.; Previous studies have shown that the small subunit of Xenopus DNA polymerase gamma (pol gammaB) acts as a processivity factor to stimulate the 140 kDa catalytic subunit of human DNA polymerase gamma . A putative human pol gammaB initially identified by analysis of DNA sequence had not been shown to be functional, and appeared to be an incomplete clone . In this paper, we report the cloning of full-length human and mouse pol gammaB . Both human and mouse pol gammaB proteins were expressed in their mature forms, without their apparent mitochondrial localization signals, and shown to stimulate processivity of the recombinant catalytic subunit of human pol gammaA . Deletion analysis of human pol gammaB indicated that blocks of sequence conserved with prokaryotic class II aminoacyl-tRNA synthetases are necessary for activity and inter-action with human pol gammaA . Purification of DNA pol gamma from HeLa cells indicated that both proteins are associated in vivo. Cytobios, 1998, 96(383), 151 - 6 Role of DNA-membrane interactions in prokaryote-to-eukaryote transition: an hypothesis; Zhdanov RI et al.; A model system of experiments to consider the problem of the origin of eukaryotic cells as well as the prokaryote-to-eukaryote transition was investigated, in terms of the role of nucleic acid-membrane interactions . It was thought worthwhile to consider the importance of DNA-membrane contacts for the organization of the prokaryotic nucleoid . The model for the fusion of four proto-eukaryotic cells was proposed to clarify the prokaryote-to-eukaryote transition as well as the formation of the nuclear pores of eukaryotes from the Bayer's junctions of proto-eukaryotes . The basic requirements following from the cell fusion model suggest such orientation of the cells involved . The obstacles for division of the ancestor cell were excluded by merging . Enormous advantages to the cell metabolism due to the fusion of four proto-eukaryotic cells and an intensive growth of the inner membranous structures resulted. Trends Microbiol, 2000 Feb, 8(2), 77 - 81 The origin of prokaryotic C2H2 zinc finger regulators; Bouhouche N et al.; C2H2 zinc finger bearing proteins are a large superfamily of nucleic acid binding proteins, which constitute a major subset of eukaryotic transcription factors . Although originally thought to occur only in eukaryotes, a novel C2H2 zinc finger transcription factor, Ros, which regulates both prokaryotic and eukaryotic promoters has been found in bacteria . Phylogenically, Ros is distantly related to eukaryotic zinc finger regulators. Membr Cell Biol, 1999, 13(1), 3 - 21 Bacterial outer membrane proteins: topological analyses and biotechnological perspectives; Stathopoulos C; The outer membrane proteins (OMPs) from gram-negative bacteria form a distinct group of integral membrane proteins with unusual primary, secondary and tertiary structures . Unlike typical prokaryotic and eukaryotic membrane proteins, bacterial OMPs contain primarily polar sequences, arranged in amphipathic antiparallel beta-barrels, and inclined to the plane of the membrane . Due to their unique structure, OMPs have recently become the subject of extensive study . This article reviews (i) experimental and theoretical approaches of topological analyses used in the study of OMPs, and (ii) the applications of OMPs. Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1813 - 7 Defensive extrusive ectosymbionts of Euplotidium (Ciliophora) that contain microtubule-like structures are bacteria related to Verrucomicrobia; Petroni G et al.; Epixenosomes, ectosymbionts on hypotrich ciliates (genus Euplotidium) defend their host against the ciliate predator Litonotus lamella . Although here only Euplotidium itoi and Euplotidium arenarium from tide pools along a rocky shore near Leghorn (Ligurian sea) were studied in detail, these epibionts are certainly present on specimens of E . itoi and on other Euplotidium species in similar north coastal habitats . The complex life history of epixenosomes has two main stages . In stage I, cells with typical prokaryotic structure divide by binary fission . Stage II cells show complex organization with different cytoplasmic compartments where an extrusive apparatus within a proteinaceous matrix, although not membrane-bounded, differs from the remaining cytoplasm . The ejection process is involved in defense; extrusive apparatus is surrounded by a basket consisting of bundles of tubules . These tubules, 22 +/- 3 nm in diameter, delimited by a wall made up of globular structures, are sensitive to inhibitor of tubulin polymerization (nocodazole/4 degrees C temperature) and react positively with different antitubulin antibodies, two of which are monoclonal . The prokaryotic vs . eukaryotic nature of epixenosomes was resolved by comparative sequence analysis of amplified small subunit rRNA genes and in situ hybridization with fluorescently labeled rRNA-targeted polynucleotide probes . These unique ectosymbionts are phylogenetically related to Verrucomicrobia . Epixenosomes represent marine symbionts in this recently discovered division of the Bacteria. Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1926 - 31 A novel precursor recognition element facilitates posttranslational binding to the signal recognition particle in chloroplasts; DeLille J et al.; Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively . The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences . An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins . In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP/LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids . In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation . Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex . In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration . Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined . Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts. Curr Biol, 2000 Jan 13, 10(1), R11 - 4 Signal transduction: hair brains in bacterial chemotaxis; Stock J et al.; The conserved cytoplasmic domains of bacterial chemotaxis receptors are a fibrous arrangement of alpha-helical coiled coils that look a lot like hair . Such bundles of alpha-helical filaments mediate sensory-motor responses in all prokaryotic cells . How do they work? Very nearly perfectly is probably as good an answer as any. Mol Gen Genet, 2000 Jan, 262(6), 949 - 56 Involvement of a natural transport system in the process of efflux-mediated drug resistance in Mycobacterium smegmatis; Banerjee SK et al.; The phosphate-specific transporter (Pst) in bacteria is a multi-subunit system which belongs to the ABC family of transporters . The gene forms part of an operon and it is involved in phosphate uptake in prokaryotes . Its import function is known to be operative only under conditions of phosphate starvation . However, we found overexpression of this transporter in a Mycobacterium smegmatis strain selected for ciprofloxacin resistance (CIPr) which was grown under conditions in which the phosphate-scavenging function of this operon was inoperative . In CIPr cells, active efflux of the drug plays a predominant role in conferring high levels of fluoroquinolone resistance . We therefore investigated the role of this transporter in the process of efflux-mediated drug resistance by inactivating the pst operon in the CIPr strain . Phenotypic characterization of the resulting strain, CIPrd, showed a striking reduction in the minimal inhibitory concentration (MIC) of ciprofloxacin and in the drug extrusion profile as well . Genotype analysis, on the other hand, revealed partial disruption of the pst operon in CIPrd as a consequence of transporter gene amplification . Furthermore, disruption of this operon in wild-type cells resulted in hypersensitivity to ciprofloxacin and other xenobiotics to which CIPr cells exhibited cross-resistance . Thus our results provide strong evidence that Pst is a natural membrane transport system that has the ability to promote drug efflux in addition to its phosphate-scavenging function in the CIPr strain. Nature, 2000 Jan 20, 403(6767), 332 - 5 The joining of ribosomal subunits in eukaryotes requires eIF5B; Pestova TV et al.; Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits . The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex . The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon . eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome . Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join . A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process . It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function. FASEB J, 2000 Feb, 14(2), 379 - 90 Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation; Yang CR et al.; Regulation of transcriptional responses in growth-arrested human cells under conditions that promote potentially lethal damage repair after ionizing radiation (IR) is poorly understood . Sp1/retinoblastoma control protein (RCP) DNA binding increased within 30 min and peaked at 2-4 h after IR (450-600 cGy) in confluent radioresistant human malignant melanoma (U1-Mel) cells . Increased phosphorylation of Sp1 directly corresponded to Sp1/RCP binding and immediate-early gene induction, whereas pRb remained hypophosphorylated . Transfection of U1-Mel cells with the human papillomavirus E7 gene abrogated Sp1/RCP induction and G(0)/G(1) cell cycle checkpoint arrest responses, increased apoptosis and radiosensitivity, and augmented genetic instability (i.e., increased polyploidy cells) after IR . Increased NF-kappaB DNA binding in U1-Mel cells after IR treatment lasted much longer (i.e., >20 h) . U1-Mel cells overexpressing dominant-negative IkappaBalpha S32/36A mutant protein were significantly more resistant to IR exposure and retained both G(2)/M and G(0)/G(1) cell cycle checkpoint responses without significant genetic instability (i.e., polyploid cell populations were not observed) . Nuclear p53 protein levels and DNA binding activity increased only after high doses of IR (>1200 cGy) . Disruption of p53 responses in U1-Mel cells by E6 transfection also abrogated G(0)/G(1) cell cycle checkpoint arrest responses and increased polyploidy after IR, but did not alter radiosensitivity . These data suggest that abrogation of individual components of this coordinate IR-activated transcription factor response may lead to divergent alterations in cell cycle checkpoints, genomic instability, apoptosis, and survival . Such coordinate transcription factor activation in human cancer cells is reminiscent of prokaryotic SOS responses, and further elucidation of these events should shed light on the initial molecular events in the chromosome instability phenotype.-Yang, C.-R., Wilson-Van Patten, C., Planchon, S . M., Wuerzberger-Davis, S . M., Davis, T . W., Cuthill, C., Miyamoto, S., Boothman, D . A . Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation. J Mol Biol, 2000 Feb 11, 296(1), 145 - 53 Cu,Zn superoxide dismutase structure from a microbial pathogen establishes a class with a conserved dimer interface; Forest KT et al.; Macrophages and neutrophils protect animals from microbial infection in part by issuing a burst of toxic superoxide radicals when challenged . To counteract this onslaught, many Gram-negative bacterial pathogens possess periplasmic Cu,Zn superoxide dismutases (SODs), which act on superoxide to yield molecular oxygen and hydrogen peroxide . We have solved the X-ray crystal structure of the Cu,Zn SOD from Actinobacillus pleuropneumoniae, a major porcine pathogen, by molecular replacement at 1.9 A resolution . The structure reveals that the dimeric bacterial enzymes form a structurally homologous class defined by a water-mediated dimer interface, and share with all Cu,Zn SODs the Greek-key beta-barrel subunit fold with copper and zinc ions located at the base of a deep loop-enclosed active-site channel . Our structure-based sequence alignment of the bacterial enzymes explains the monomeric nature of at least two of these, and suggests that there may be at least one additional structural class for the bacterial SODs . Two metal-mediated crystal contacts yielded our C222(1) crystals, and the geometry of these sites could be engineered into proteins recalcitrant to crystallization in their native form . This work highlights structural differences between eukaryotic and prokaryotic Cu,Zn SODs, as well as similarities and differences among prokaryotic SODs, and lays the groundwork for development of antimicrobial drugs that specifically target periplasmic Cu,Zn SODs of bacterial pathogens . Copyright 12000 Academic Press. Free Radic Biol Med, 2000 Jan 1, 28(1), 75 - 83 Fapyadenine is a moderately efficient chain terminator for prokaryotic DNA polymerases; Graziewicz MA et al.; Hypoxanthine inverted question markxanthine oxidase inverted question markFe3+ inverted question markethylenediaminetetraacetate (EDTA) was used to modify ss M13 mp18 phage DNA . The dominant base modifications found by GC/IDMS-SIM were FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol . Analysis of in vitro DNA synthesis on oxidatively modified template by three DNA polymerases revealed that T7 DNA polymerase and Klenow fragment of polymerase I from Escherichia coli were blocked mainly by oxidized pyrimidines in the template whereas some purines that were easily bypassed by the prokaryotic polymerases constituted a block for DNA polymerase beta from calf thymus . DNA synthesis by T7 polymerase on poly(dA) template, where FapyAde content increased 16-fold on oxidation, yielded a final product with a discrete ladder of premature termination bands . When DNA synthesis was performed on template from which FapyAde, FapyGua, and 8OHGua were excised by the Fpg protein new chain terminations at adenine and guanine sites appeared or existing ones were enhanced . This suggests that FapyAde, when present in DNA, is a moderately toxic lesion . Its ability to arrest DNA synthesis depends on the sequence context and DNA polymerase . FapyGua might possess similar properties. Proteins, 2000 Feb 1, 38(2), 210 - 25 Super-motifs and evolution of tandem leucine-rich repeats within the small proteoglycans--biglycan, decorin, lumican, fibromodulin, PRELP, keratocan, osteoadherin, epiphycan, and osteoglycin; Matsushima N et al.; Leucine-rich repeats (LRRs) with 20-30 amino acids in unit length are present in many proteins from prokaryotes to eukaryotes . The LRR-containing proteins include a family of nine small proteoglycans, forming three distinct subfamilies: class I contains biglycan/PG-I and decorin/PG-II; class II: lumican, fibromodulin, PRELP, keratocan, and osteoadherin; and class III: epiphycan/PG-Lb and osteoglycin or osteoinductive factor . Comparative sequence analysis of the 34 available protein sequences reveals that these proteoglycans have two types of LRRs, which we call S and T . The type S LRR is 21 residues long and has the consensus sequence of xxaPzxLPxxLxxLxLxxNxI . The type T LRR has 26 residues; its consensus sequence is zzxxaxxxxFxxaxxLxxLxLxxNxL . In both "x" indicates variable residue; "z" is frequently a gap; "a" is Val, Leu, or Ile; and I is Ile or Leu . These type S and TLRRs are ordered into two super-motifs--STT with about 73 residues in classes I and II and ST with about 47 residues in class III . The 12 LRRs in the small proteoglycans of I and II are best represented as (STT)4; the seven LRRs of class III as (ST)T(ST)2 . Our analyses indicate that classes I/II and III evolved along different paths after the establishment of the precursor ST, and classes I and II also diverged after the establishment of the precursor (STT)4. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1050 - 5 A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins; Lange H et al.; Iron-sulfur (Fe/S) cluster-containing proteins catalyze a number of electron transfer and metabolic reactions . The components and molecular mechanisms involved in the assembly of the Fe/S clusters have been identified only partially . In eukaryotes, mitochondria have been proposed to execute a crucial task in the generation of intramitochondrial and extramitochondrial Fe/S proteins . Herein, we identify the essential ferredoxin Yah1p of Saccharomyces cerevisiae mitochondria as a central component of the Fe/S protein biosynthesis machinery . Depletion of Yah1p by regulated gene expression resulted in a 30-fold accumulation of iron within mitochondria, similar to what has been reported for other components involved in Fe/S protein biogenesis . Yah1p was shown to be required for the assembly of Fe/S proteins both inside mitochondria and in the cytosol . Apparently, at least one of the steps of Fe/S cluster biogenesis within mitochondria requires reduction by ferredoxin . Our findings lend support to the idea of a primary function of mitochondria in the biosynthesis of Fe/S proteins outside the organelle . To our knowledge, Yah1p is the first member of the ferredoxin family for which a function in Fe/S cluster formation has been established . A similar role may be predicted for the bacterial homologs that are encoded within iron-sulfur cluster assembly (isc) operons of prokaryotes. EMBO J, 2000 Feb 1, 19(3), 445 - 52 A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases; Cahuzac B et al.; Aminoacyl-tRNA synthetases of higher eukaryotes possess polypeptide extensions in contrast to their prokaryotic counterparts . These extra domains of poorly understood function are believed to be involved in protein-protein or protein-RNA interactions . Here we showed by gel retardation and filter binding experiments that the repeated units that build the linker region of the bifunctional glutamyl-prolyl-tRNA synthetase had a general RNA-binding capacity . The solution structure of one of these repeated motifs was also solved by NMR spectroscopy . One repeat is built around an antiparallel coiled-coil . Strikingly, the conserved lysine and arginine residues form a basic patch on one side of the structure, presenting a suitable docking surface for nucleic acids . Therefore, this repeated motif may represent a novel type of general RNA-binding domain appended to eukaryotic aminoacyl-tRNA synthetases to serve as a cis-acting tRNA-binding cofactor. Microcirculation, 1999 Dec, 6(4), 259 - 65 A web-based research tool for functional genomics of the microcirculation: the leukocyte adhesion cascade; Ley K et al.; OBJECTIVE: Currently, microvascular data are almost exclusively deposited in scholarly journals . With the increasing availability of molecular data and the construction of genomic databases, a need arises to organize physiological data in a more accessible format . The microcirculation is a functional system that spans all organ systems and shares certain characteristics of organization with metabolic pathways, which have successfully been organized around genomic information for several prokaryotes . Here, we present a web-based research and teaching tool that covers a small aspect of microcirculatory physiology, the leukocyte adhesion cascade . METHODS: Currently, the site is organized in a flat-text mode with hypertext links to GenBank, Medline, and online journals where available . RESULTS: The web-based research and education tool is useful for graduate student education, and as a research resource for genetic researchers interested in gene function and for physiologists and biomedical engineers interested in the molecular basis of the leukocyte adhesion cascade . Our effort is intended to be a beginning toward a distributed database for the microcirculation (microcirculation physiome . see CONCLUSIONS: We invite all microvascular researchers to provide annotations and comments to make the research and teaching tool more useful to the scientific community. J Bioenerg Biomembr, 1999 Oct, 31(5), 493 - 506 Mitochondrial uncoupling: role of uncoupling protein anion carriers and relationship to thermogenesis and weight control "the benefits of losing control"; Diehl AM et al.; Uncoupling proteins, a subgroup of the mitochondrial anion transporter superfamily, have been identified in prokaryotes, plants, and mammalian cells . Evolutionary conservation of these molecules reflects their importance as regulators of two critical mitochondrial functions, i.e., ATP synthesis and the production of reactive oxygen species (ROS) . Although the amino acid sequences of the three mammalian uncoupling proteins, UCP1, UCP2 and UCP3, are very similar, each homolog is the product of a unique gene and important differences have been demonstrated in their tissue-specific expression and regulation . UCP1 and UCP3 appear to be key regulators of energy expenditure, and hence, nonshivering thermogenesis, either in brown adipose tissue (UCP1) or skeletal muscle (UCP3) . UCP2 is expressed more ubiquitously, although generally at low levels, in many tissues . There is conflicting evidence about its importance as a regulator of resting metabolic rate . However, evidence suggests that this homolog might modulate the mitochondrial generation of ROS in some cell types, including macrophages and hepatocytes . While the induction of various uncoupling protein homologs provides adaptive advantages, both to the organism (e.g., thermogenesis) and to individual cells (e.g., reduced ROS), increased uncoupling protein activity also increases cellular vulnerability to necrosis by compromising the mitochondrial membrane potential . This narrow "risk-benefit" margin necessitates tight control of uncoupling protein activity in order to preserve cellular viability and much remains to be learned about the regulatory mechanisms involved. Plant J, 1999 Dec, 20(5), 529 - 39 Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth; Espinosa-Ruiz A et al.; We have isolated two Arabidopsis thaliana genes, AtHAL3a and AtHAL3b, showing homology with HAL3, a yeast protein which regulates the cell cycle and tolerance to salt stress through inhibition of the PPZ1 type-1 protein phosphatase . Expression of AtHAL3a in yeast hal3 mutants partially complements their LiCl sensitivity, suggesting possible conserved functions between both proteins . AtHAL3a and AtHAL3b are induced by salt stress and AtHAL3a is the most expressed in non-stressed plants, particularly in seeds . In situ hybridization demonstrates enrichment of AtHAL3a mRNA in seed embryos and in the vascular phloem of different plant tissues . AtHAL3 proteins show striking homology with a group of proteins found in fungi, plants and animals and some homology with a large family of prokaryotic flavoproteins . Recombinant AtHAL3a protein purified from Escherichia coli was yellow because it contained a non-covalently bound chromophore revealed as flavin mononucleotide . Trans- genic Arabidopsis plants, with gain of AtHAL3a function, show altered growth rates and improved tolerance to salt and osmotic stress. Crit Rev Eukaryot Gene Expr, 1999, 9(3-4), 175 - 82 Evolution of transcriptional control from prokaryotic beginnings to eukaryotic complexities; Huang L et al.; Mechanisms for regulating gene transcription became increasingly complex as organisms evolved . In prokaryotes the relatively simple mechanism of repression is based on a few proteins that bind to specific DNA sequences in a ligand-dependent fashion . In eukaryotes large complexes that include ligand binding proteins regulate transcription . Lower eukaryotes developed an additional level of control based on protein complexes that include modifying enzymes . The DNA/histone complex, in combination with gene-specific transcriptional factors, is the basis of gene regulation in eukaryotes . Higher eukaryotes took regulation a level further by methylating CpGs in promoter sequences of DNA, thereby allowing binding of histone deacetylases and inhibiting transcription . Finally, long-lasting "superrepression" provides another mechanism for coordinate transcriptional regulation of large blocks of genes. Biotechniques, 2000 Jan, 28(1), 82 - 4, 86, 88-9 Green fluorescent protein as a quantitative reporter of relative promoter activity in E . coli; Lissemore JL et al.; Green fluorescent protein (GFP) has become a valuable tool for the detection of gene expression in prokaryotes and eukaryotes . To evaluate its potential for quantitation of relative promoter activity in E . coli, we have compared GFP with the commonly used reporter gene lacZ, encoding beta-galactosidase . We cloned a series of previously characterized synthetic E . coli promoters into GFP and beta-galactosidase reporter vectors . Qualitative and quantitative assessments of these constructs show that (a) both reporters display similar sensitivities in cells grown on solid or liquid media and (b) GFP is especially well suited for quantitation of promoter activity in cells grown on agar . Thus, GFP provides a simple, rapid and sensitive tool for measuring relative promoter activity in intact E . coli cells. Bioessays, 2000 Jan, 22(1), 10 - 5 The circadian clock of cyanobacteria; Kondo T et al.; A circadian clock, with physiological characteristics similar to those of eukaryotes, functions in the photosynthetic prokaryote, cyanobacteria . The molecular mechanism of this clock has been efficiently dissected using a luciferase reporter gene that reports the status of the clock . A circadian clock gene cluster, kaiABC, has been cloned via rhythm mutants of cyanobacterium, Synechococcus, and many clock mutations mapped to the three kai genes . Although kai genes do not share any homology with clock genes so far identified in eukaryotes, analysis of their expression suggests that a negative feedback control of kaiC expression by KaiC generates the circadian oscillation and that KaiA functions as a positive factor to sustain this oscillation . BioEssays 22:10-15, 2000 . J Bacteriol, 2000 Feb, 182(4), 1127 - 35 A role for the umuDC gene products of Escherichia coli in increasing resistance to DNA damage in stationary phase by inhibiting the transition to exponential growth; Murli S et al.; The umuDC gene products, whose expression is induced by DNA-damaging treatments, have been extensively characterized for their role in SOS mutagenesis . We have recently presented evidence that supports a role for the umuDC gene products in the regulation of growth after DNA damage in exponentially growing cells, analogous to a prokaryotic DNA damage checkpoint . Our further characterization of the growth inhibition at 30 degrees C associated with constitutive expression of the umuDC gene products from a multicopy plasmid has shown that the umuDC gene products specifically inhibit the transition from stationary phase to exponential growth at the restrictive temperature of 30 degrees C and that this is correlated with a rapid inhibition of DNA synthesis . These observations led to the finding that physiologically relevant levels of the umuDC gene products, expressed from a single, SOS-regulated chromosomal copy of the operon, modulate the transition to rapid growth in E . coli cells that have experienced DNA damage while in stationary phase . This activity of the umuDC gene products is correlated with an increase in survival after UV irradiation . In a distinction from SOS mutagenesis, uncleaved UmuD together with UmuC is responsible for this activity . The umuDC-dependent increase in resistance in UV-irradiated stationary-phase cells appears to involve, at least in part, counteracting a Fis-dependent activity and thereby regulating the transition to rapid growth in cells that have experienced DNA damage . Thus, the umuDC gene products appear to increase DNA damage tolerance at least partially by regulating growth after DNA damage in both exponentially growing and stationary-phase cells. J Bacteriol, 2000 Feb, 182(4), 993 - 1000 The detergent-soluble maltose transporter is activated by maltose binding protein and verapamil; Reich-Slotky R et al.; The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathione S-transferase molecular tag . In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity . This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits . The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP . Wild-type MBP increased the V(max) of the ATPase 2.7-fold but had no effect on the K(m) of the enzyme for ATP . When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible . The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN(3), as well as a mercurial, p-chloromercuribenzosulfonic acid . Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well . The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation. J Bacteriol, 2000 Feb, 182(4), 944 - 8 A novel operon encoding formaldehyde fixation: the ribulose monophosphate pathway in the gram-positive facultative methylotrophic bacterium Mycobacterium gastri MB19; Mitsui R et al.; A 4.2-kb PstI fragment harboring the gene cluster of the ribulose monophosphate (RuMP) pathway for formaldehyde fixation was identified in the chromosome of a gram-positive, facultative methylotroph, Mycobacterium gastri MB19, by using the coding region of 3-hexulose-6-phosphate synthase (HPS) as the hybridization probe . The PstI fragment contained three complete open reading frames (ORFs) which encoded from the 5' end, a DNA-binding regulatory protein (rmpR), 6-phospho-3-hexuloisomerase (PHI; rmpB), and HPS (rmpA) . Sequence analysis suggested that rmpA and rmpB constitute an operon, and Northern blot analysis of RNA extracted from bacteria grown under various conditions suggested that the expression of the two genes is similarly regulated at the transcriptional level . A similarity search revealed that the proteins encoded by rmpA and rmpB in M . gastri MB19 show high similarity to the unidentified proteins of nonmethylotrophic prokaryotes, including bacteria and anaerobic archaea . The clusters in the phylogenetic tree of the HPS protein of M . gastri MB19 and those in the phylogenetic tree of the PHI protein were nearly identical, which implies that these two formaldehyde-fixing genes evolved as a pair . These findings give new insight into the acquisition of the formaldehyde fixation pathway during the evolution of diverse microorganisms. Virology, 2000 Feb 1, 267(1), 102 - 10 Genetic engineering of herpes simplex virus and vector genomes carrying loxP sites in cells expressing Cre recombinase; Logvinoff C et al.; The prokaryotic Cre-loxP recombination system is a powerful tool that enables in vitro and in vivo site-specific manipulations of the genome of eukaryotic cells as well as of DNA viruses and their derived vectors . This system, however, has not yet been exploited in the context of herpes simplex virus type 1 (HSV-1) infected cells, perhaps because this virus encodes several functions that induce a strong shutoff of cellular protein synthesis, a fact that could preclude expression of cellular-encoded Cre recombinase . In the present study, we show that efficient site-specific recombination can take place in cell lines expressing Cre, even in the context of HSV-1 infection, as evidenced by the engineering of an HSV-1 recombinant virus and several viral vectors carrying one or two loxP sequences . More precisely, we have used this system to induce an irreversible switch in the expression of a viral complex transcription unit encoding two different open reading frames and allowing consecutive expression of two reporter genes . Furthermore Cre recombinations were also used to induce the decatenation of the genomic concatemers harbored by amplicon particles upon infection of cells under nonreplicative conditions, thus enabling the rescue of many independent plasmids corresponding to the original amplicon plasmid used to generate the vectors . Thus the Cre-loxP recombination system can successfully be used for engineering the genome of HSV-1 or HSV-1-based vectors in cultured cells . J Biochem Biophys Methods, 2000 Jan 3, 42(1-2), 15 - 29 Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems; Nath K et al.; In an in-gel polymerase chain reaction (PCR), the generation of a 1750-bp yeast DNA fragment was inhibited when yeast DNA gel-stabs or gel-slices stained with ethidium bromide (EtBr) or SYBR Green I were used . Similar inhibition occurred to a varying degree in the reamplification of PCR fragments in prokaryotic systems . Inclusion of the dyes in PCR resulted in an inhibition at about 10 microg/ml EtBr and at 10,000-20,000-fold dilution of SYBR Green I in all systems . The effect remained unchanged despite increasing the PCR cycles to 40 . However, increasing the magnesium chloride concentration did reverse the inhibitory actions, although the PCR specificity was lost . In an unusual observation, we find that, at higher dye concentrations (50 microg/ml EtBr, or thousand fold dilution of SYBR Green I), the input yeast DNA electrophoretic profile is maintained following 25 PCR cycles (despite a denaturation temperature of 94 degrees C) . It varied significantly in different DNA systems and was readily reversed by high Mg++ concentrations . It is concluded that, at low Mg++ concentrations, different PCR systems are inhibited to varying extents by intercalating dyes and, in some PCR systems, intercalating dyes at unusually high concentrations maintain input DNA electrophoretic profile. Arch Microbiol, 2000 Jan, 173(1), 1 - 9 Nickel transport systems in microorganisms; Eitinger T et al.; The transition metal Ni is an essential cofactor for a number of enzymatic reactions in both prokaryotes and eukaryotes . Molecular analyses have revealed the existence of two major types of high-affinity Ni2+ transporters in bacteria . The Nik system of Escherichia coli is a member of the ABC transporter family and provides Ni2+ ion for the anaerobic biosynthesis of hydrogenases . The periplasmic binding protein of the transporter, NikA, is likely to play a dual role . It acts as the primary binder in the uptake process and is also involved in negative chemotaxis to escape Ni overload . Expression of the nik operon is controlled by the Ni-responsive repressor NikR, which shows functional similarity to the ferric ion uptake regulator Fur . The second type of Ni2+ transporter is represented by HoxN of Ralstonia eutropha, the prototype of a novel family of transition metal permeases . Members of this family have been identified in gram-negative and gram-positive bacteria and recently also in a fission yeast . They transport Ni2+ with very high affinity, but differ with regard to specificity . Site-directed mutagenesis experiments have identified residues that are essential for transport . Besides these uptake systems, different types of metal export systems, which prevent microorganisms from the toxic effects of Ni2+ at elevated intracellular concentrations, have also been described. Structure Fold Des, 1999 Dec 15, 7(12), 1517 - 26 Structural transitions in the FixJ receiver domain; Gouet P et al.; BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants . FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti . In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism . RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions . Three conformations of the protein were found . In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis . In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes . The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred . Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region . CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site . Their movements may act as molecular switches that define the functional status of the protein . Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions. Plant Mol Biol, 1999 Nov, 41(5), 645 - 55 A conserved His-Asp signal response regulator-like gene in Heterosigma akashiwo chloroplasts; Jacobs MA et al.; Regulation of gene expression in plastids may involve molecular components conserved from cyanobacteria-like ancestors . Among prokaryotes, genes are commonly regulated at the transcriptional level by 'two-component' or 'His-Asp' signal transducers, consisting of a 'sensor kinase', which autophosphorylates at a conserved histidine residue, and a cognate response regulator, which is phosphorylated by the sensor kinase at a conserved aspartate residue . A putative His-Asp response regulator gene (trg1: transcriptional regulatory gene 1) has been identified in the estuarine raphidophytic alga Heterosigma akashiwo . The chloroplast-encoded trg1 is 693 bp in length, contains no introns, and yields a conceptual translation product of 231 amino acids, with a predicted mass of 27 kDa . Homology searches suggest that Heterosigma trgl has an omnpR-like identity within the DNA-binding His-Asp family of response regulators . trg1 contains both the phosphorylation and DNA-binding domains which are present in prokaryote response regulators . Quantitative competitive RT-PCR showed that Heterosigma trg1 is expressed at low levels (5 microg per g total RNA) . In contrast, psbA (a photosystem II component) transcript is abundant (60 mg per g total RNA) . Cell cycle analysis showed that psbA abundance oscillates in response to light but trg1 mRNA levels are invariant . We hypothesize that a His-Asp phosphorelay mechanism may affect chloroplast genome transcription in a manner similar to bacterial signal transduction pathways in which 'sensor kinase' and cognate 'response regulator' proteins interact. Tuber Lung Dis, 1998, 79(1), 43 - 53 Identification of an immunogenic histone-like protein (HLPMt) of Mycobacterium tuberculosis; Prabhakar S et al.; We report the identification of the first histone-like protein of Mycobacterium tuberculosis (MTB) (HLPMt) . The T cell blot assay was used to identify antigens of MTB associated with human immune response in healthy contacts . Fraction 21 corresponding to proteins in the molecular weight range of approximately 30 kDa were found to be immunogenic in tuberculin reactors . None of the fractions were found to be immunogenic by this assay in non-reactors to tuberculin . All sera, irrespective of the source, showed reactivity with MTB antigen(s) over a wide molecular weight range (205-->16 kDa) . In the present study fraction 21 was processed for the generation of murine polyclonal sera and amino acid sequencing . The sequence of a 16-amino acid long peptide showed a 100% homology with an open reading frame (ORF) in the translated sequence of cosmid cY349 (Sanger Centre, Cambridge, UK) . The ORF was predicted to code for a protein of 214 amino acids . Oligonucleotide primers were synthesized based on the nucleotide sequence located at the 5' and 3' regions of the gene . The gene encoding the predicted protein was PCR-amplified, cloned, sequenced and expressed in Escherichia coli as a protein of 28 kDa . The expressed HLPMt protein was shown to react with the polyclonal murine sera originally raised against fraction 21 . Human immune response to the recombinant HLPMt protein was demonstrated by its ability to induce lymphoproliferation in peripheral blood derived mononuclear cells, and the presence of anti-HLPMt antibodies in pooled patient sera by immunoblot . The recombinant HLPMt protein elicited a vigorous lymphoproliferative response especially in healthy tuberculin reactors compared to non-reactors and patients of tuberculosis, (P < 0.05) . The protein has unique dual domains with homology to both bacterial histone-like proteins (HU) and eukaryotic histone H1 . Homology to prokaryotic and eukaryotic deoxyribonucleic acid (DNA)-binding proteins suggested that HLPMt could bind DNA . DNA-binding properties were confirmed by South-Western analysis strongly suggesting an interaction between HLPMt and the MTB chromosome. Int J Oncol, 2000 Feb, 16(2), 305 - 13 Regulation of the activity and polymerization status of recombinant human cytosolic thymidine kinase by thiols and ATP; Kuroiwa N et al.; The cDNA clone encoding human thymidine kinase (hTK), was expressed in E . coli using a prokaryotic expression vector, pKK 223-3 . The kinetics of the recombinant hTK (rhTK) were similar to those of cytosolic TK but not of mitochondrial TK . rhTK was highly purified in the presence of either ATP or dithiothreitol (DTT) . The specific activity of rhTK purified in the presence of ATP {rhTK(ATP)} was lower than that of rhTK purified in the presence of DTT {rhTK(DTT)} . Activity of the purified rhTK(ATP) was enhanced by addition of thiols including DTT, cysteine, homocysteine and beta-mercaptoethanol but inhibited by various sulfhydryl reagents such as 5,5'-dithio-bis(2-nitrobenzoic acid) . Hence, it was suggested that rhTK is a thiol-type enzyme . Apparent Mr of purified rhTK(ATP) was 100 kDa, which corresponds to the size of a tetramer (25 kDa subunit), while that of purified rhTK(DTT) was 50 kDa, the size of a dimer . The tetramer form of rhTK(ATP) was converted to the dimer by replacement of ATP by DTT . On the other hand, the dimer form of rhTK(DTT) was converted to the tetramer by addition of ATP . Thus, the catalytic activity of human cytosolic TK might be regulated by thiols as well as ATP via its polymerization status. Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 652 - 6 CopA: An Escherichia coli Cu(I)-translocating P-type ATPase; Rensing C et al.; The copA gene product, a putative copper-translocating P-type ATPase, has been shown to be involved in copper resistance in Escherichia coli . The copA gene was disrupted by insertion of a kanamycin gene through homologous recombination . The mutant strain was more sensitive to copper salts but not to salts of other metals, suggesting a role in copper homeostasis . The copper-sensitive phenotype could be rescued by complementation by a plasmid carrying copA from E . coli or copB from Enterococcus hirae . Expression of copA was induced by salts of copper or silver but not zinc or cobalt . Everted membrane vesicles from cells expressing copA exhibited ATP-coupled accumulation of copper, presumably as Cu(I) . The results indicate that CopA is a Cu(I)-translocating efflux pump that is similar to the copper pumps related to Menkes and Wilson diseases and provides a useful prokaryotic model for these human diseases. Nature, 2000 Jan 6, 403(6765), 77 - 80 The Santa Barbara Basin is a symbiosis oasis; Bernhard JM et al.; It is generally agreed that the origin and initial diversification of Eucarya occurred in the late Archaean or Proterozoic Eons when atmospheric oxygen levels were low and the risk of DNA damage due to ultraviolet radiation was high . Because deep water provides refuge against ultraviolet radiation and early eukaryotes may have been aerotolerant anaerobes, deep-water dysoxic environments are likely settings for primeval eukaryotic diversification . Fossil evidence shows that deep-sea microbial mats, possibly of sulphur bacteria similar to Beggiatoa, existed during that time . Here we report on the eukaryotic community of a modern analogue, the Santa Barbara Basin (California, USA) . The Beggiatoa mats of these severely dysoxic and sulphidic sediments support a surprisingly abundant protistan and metazoan meiofaunal community, most members of which harbour prokaryotic symbionts . Many of these taxa are new to science, and both microaerophilic and anaerobic taxa appear to be represented . Compared with nearby aerated sites, the Santa Barbara Basin is a 'symbiosis oasis' offering a new source of organisms for testing symbiosis hypotheses of eukaryogenesis. Trends Biochem Sci, 2000 Jan, 25(1), 29 - 32 Intracellular copper routing: the role of copper chaperones; Harrison MD et al.; Copper is required by all living systems . Cells have a variety of mechanisms to deal with this essential, yet toxic trace element . A recently discovered facet of homeostatic mechanisms is the protein-mediated, intracellular delivery of copper to target proteins . This routing is accomplished by a novel class of proteins, the 'copper chaperones' . They are a family of conserved proteins present in prokaryotes and eukaryotes, which suggests that copper chaperones are used throughout nature for intracellular copper routing. Curr Med Chem, 2000 Jan, 7(1), 1 - 15 Targeting DNA secondary structures; Wadkins RM; DNA secondary structures containing regions of single-stranded DNA have now been identified in the genomic DNA of a number of prokaryotic and eukaryotic species, including humans . Many of these secondary structures are associated with regions of DNA involved in regulation of transcription: promoters or upstream elements . The secondary structures involved appear likely to be hairpin or cruciform structures that may be recognition sites for binding of transcription factors . In the case of the coliphage N4 virion RNA polymerase, a defined hairpin in the polymerase promoter necessary for binding of the polymerase and regulation of transcription has been shown to be extruded under physiological conditions in plasmid DNA . The presence of single-stranded DNA in the promoters of several species suggests that regulatory hairpins may be involved in transcription of a number of genes . In support of this, hairpin- or cruciform-binding proteins have been identified from several species . These results imply that secondary structures in regulatory regions may be targets for drugs that bind and either block or enhance binding of proteins involved in transcription . In this review, we discuss the evidence for DNA secondary structures, particularly hairpins and cruciforms, in genomic DNA and review the studies to date of development of small molecules that can selectively bind these structures. Nucleic Acids Res, 2000 Feb 1, 28(3), 784 - 90 cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics; Tosi LR et al.; Mos1 and other mariner / Tc1 transposons move horizon-tally during evolution, and when transplanted into heterologous species can transpose in organisms ranging from prokaryotes to protozoans and vertebrates . To further develop the Drosophila Mos1 mariner system as a genetic tool and to probe mechanisms affecting the regulation of transposition activity, we developed an in vitro system for Mos1 transposition using purified transposase and selectable Mos1 derivatives . Transposition frequencies of nearly 10(-3)/target DNA molecule were obtained, and insertions occurred at TA dinucleotides with little other sequence specificity . Mos1 elements containing only the 28 bp terminal inverted repeats were inactive in vitro, while elements containing a few additional internal bases were fully active, establishing the minimal cis -acting requirements for transposition . With increasing transposase the transposition frequency increased to a plateau value, in contrast to the predictions of the protein over-expression inhibition model and to that found recently with a reconstructed Himar1 transposase . This difference between the 'natural' Mos1 and 'reconstructed' Himar1 transposases suggests an evolutionary path for down-regulation of mariner transposition following its introduction into a naive population . The establishment of the cis and trans requirements for optimal mariner transposition in vitro provides key data for the creation of vectors for in vitro