Biochem J, 1999 Nov 1, 343 Pt 3, 669 - 72 Properties of the 40 kDa antigen of Mycobacterium tuberculosis, a functional L-alanine dehydrogenase; Hutter B et al.; The 40 kDa antigen of Mycobacterium tuberculosis is the first antigen reported to be present in the pathogenic M . tuberculosis, but not in the vaccine strain Mycobacterium bovis BCG . It is a functional L-alanine dehydrogenase (EC 1.4.1.1) and hence one of the few antigens possessing an enzymic activity . This makes the 40 kDa antigen attractive for potential diagnostic and therapeutic interventions . Recently, we developed a strategy to purify quantities of the recombinant protein in active form, and here we describe the biochemical properties of this enzyme . In the oxidative-deamination reaction, the enzyme showed K(m) values of 13 . 8 mM and 0.31 mM for L-alanine and NAD(+), respectively, in a random-ordered mechanism . K(m, app) values in the reductive-amination reaction are 35.4 mM, 1.45 mM and 98.2 microM for ammonium, pyruvate and NADH, respectively . The enzyme is highly specific for all of its substrates in both directions . The pH profile indicates that oxidative deamination virtually may not occur at physiological pH . Hence L-alanine most likely is the product of the reaction catalysed in vivo . The enzyme is heat-stable, losing practically no activity at 60 degrees C for several hours. Biochem J, 1999 Nov 1, 343 Pt 3, 563 - 70 An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates; Buchanan CL et al.; Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 degrees C . It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff pathway, in which the reversible C(6) to C(3) aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating pyruvate and glyceraldehyde . Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with non-phosphorylated metabolites, we report here the cloning and sequencing of the S . solfataricus gene encoding KDG-aldolase, and its expression in Escherichia coli to give fully active enzyme . The recombinant enzyme was purified in a simple two-step procedure, and shown to possess kinetic properties indistinguishable from the enzyme purified from S . solfataricus cells . The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 degrees C of 2.5 h, and is equally active with both d- and l-glyceraldehyde . It exhibits sequence similarity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent aldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH(4). J Struct Biol, 1999 Sep, 127(2), 120 - 34 Gold-ATP; Hainfeld JF et al.; The synthesis, purification, and chemical analysis of two covalent conjugates between ATP and undecagold are described, one in which gold is attached to the ribose moiety of ATP and the other in which it is attached to the N-6 position of the adenine base . The former probe was then used to bind to two ATP binding proteins, the helicase DnaB and the chaperone DnaK . After purification from unbound gold by column chromatography, binding was measured by UV-Vis spectroscopy, then the protein and gold were visualized by scanning transmission electron microscopy . Binding was observed with the conjugates, and virtually no binding occurred in the control of undecagold without the ATP attached . This new probe may be useful for studying nucleotide binding sites on proteins or for labeling nucleic acids or oligonucleotides directly . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 287 - 90 Residues within the HFRIGC sequence of HIV-1 vpr involved in growth arrest activities; Berglez JM et al.; HIV-1 Vpr is a virion-associated protein that can cause growth arrest when produced inside the cell but when added externally it can cause cell death . Employing the yeast model system, the C-terminal domain, in particular the sequence HFRIGCRHSRIG (Vpr(71-82)), is essential for both the growth arrest and cytocidal activities . Conservation of this sequence in HIV-2 and SIV suggests that these residues may be functionally important . Using site-directed mutagenesis we show that the most highly conserved aa residues, His71 and Gly75, were important for the cell cycle inhibitory effects . In contrast, we show that the wild-type Vpr(71-82) peptide and three variants of this peptide with Gly75 changed to Ser, Ala, and Ile all exhibited the same cytocidal activity suggesting that the intracellular and extracellular effects are unrelated . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 225 - 9 Fragments from alpha-actinin insert into reconstituted lipid bilayers; Goldmann WH et al.; Recent experiments have indicated that alpha-actinin interacts with phospholipid membranes . Using computer analysis methods we determined two possible lipid binding sites capable of membrane attachment/insertion, residues 281-300 and 720-739 of the primary amino acid sequence on smooth muscle alpha-actinin . Having expressed these regions as fusion proteins with schistosomal GST (glutathione S-transferase), we used differential scanning calorimetry (DSC) to investigate their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers . Calorimetric measurements showed that as fusion protein concentration increased, the main chain transition enthalpy decreased and chain melting temperatures shifted, which is indicative of partial protein insertion into the hydrophobic region of the lipid membranes . Centrifugation assay and subsequent SDS/Page chromatography confirmed this finding . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 196 - 200 Evidence that histidine-163 is critical for catalytic activity, but not for substrate binding to Escherichia coli agmatinase; Carvajal N et al.; Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal . Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector complex (12 mM) was essentially equal to the K(i) value for this compound acting as a competitive inhibitor of agmatine hydrolysis . Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-type activity, without any change in K(m) for agmatine or K(i) for putrescine inhibition . The mutant was insensitive to DEPC and dye-sensitized inactivations . We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 181 - 5 Multiple domains of DFF45 bind synergistically to DFF40: roles of caspase cleavage and sequestration of activator domain of DFF40; McCarty JS et al.; CAD/DFF40, the nuclease responsible for DNA fragmentation during apoptosis, exists as a heterodimeric complex with DFF45/ICAD . The study presented here augments the accompanying inhibition and chaperone study with an analysis of specific binding strengths and locations of DFF45 binding sites within DFF40 . This allows us to show that DFF40/45 interaction is mediated by binding of three functional domains (D1, D2, and D3) of DFF45 to two domains (activator and catalytic) of DFF40 . D1 binds exclusively to the activator domain and D2 binds to the catalytic domain of DFF40 . Inhibition of DFF40 nuclease activity arises independently from D1 functional sequestration of the activator domain and D2 blockage of the catalytic domain of DFF40 . The mechanism of caspase activation of DFF40 is the disruption of the synergistic binding activity of DFF45 domains to DFF40 after caspase recognition and cleavage of DFF45 in the context of a DFF45/40 complex . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 69 - 75 Hierarchical order of critical residues on the immunity-determining region of the Im7 protein which confer specific immunity to its cognate colicin; Lu FM et al.; The directed mutagenesis study of the Im7 protein of colicin E7 revealed that three residues, D31, D35, and E39, located in the loop 1 and helix 2 regions of the protein were critical for initiating the complex formation with its cognate colicin E7 . Interestingly, the importance of these three critical residues in conferring specific immunity to its own colicin was exhibited in a hierarchical order, respectively . Moreover, we found that existence of the three critical residues was common among the DNase-type Im proteins . Most likely the three residues of the DNase-type immunity proteins are critical for initiating the unique protein-protein interactions with their cognate colicin . In addition, replacement of the helix 2 of Im7 by the corresponding region of Im8 produced a phenotype of the mutant protein very similar to that of Im8 . This result suggests that the DNase-type Im proteins indeed share a "homologous-structural framework" and evolution of the Im proteins may be engendered by minor amino acid changes in this specific immunity-determining region without causing structural alteration of the proteins . Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 51 - 4 Era, an essential Escherichia coli small G-protein, binds to the 30S ribosomal subunit; Sayed A et al.; Era is an essential G-protein in Escherichia coli identified originally as a homologue protein to Ras (E . coli Ras-like protein) . It binds to GTP/GDP and contains a low intrinsic GTPase activity . Its function remains elusive, although it has been suggested that Era is associated with the cytoplasmic membrane, cell division, energy metabolism, and cell-cycle check point . Recently, a cold-sensitive phenotype was found to be suppressed by the overexpression of 16S rRNA methyltransferase, suggesting Era association with the ribosome . Here we demonstrate that Era specifically binds to 16S rRNA and the 30S ribosomal subunit . Both GTP and GDP, but not GMP, inhibit Era binding to ribosomal component . Involvement of Era in protein synthesis is suggested by the fact that Era depletion results in the translation defect both in vitro and in vivo . Anal Biochem, 1999 Oct 15, 274(2), 235 - 40 Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis; Schmidt T et al.; Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates . Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution . Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE) . The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found . The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks . Additionally, the different isoforms can now be quantified by CGE . Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification . CGE also offers high resolution and the amount of DNA required is very low . Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation . Anal Biochem, 1999 Oct 15, 274(2), 181 - 7 The efficient removal of endotoxins from insulin using quaternized polyethyleneimine-coated porous zirconia; McNeff C et al.; The synthesis and use of a zirconia-based, alkali-stable strong anion-exchange stationary phase are described for the removal of pyrogenic lipopolysaccharides (LPS) from insulin . The strong anion-exchange material is produced by deposition of polyethyleneimine (PEI) onto porous zirconia particles, followed by cross-linking with a novel reagent, 1,2-bis-(2-iodoethoxy) ethane, and quaternization with iodomethane . Physical characterization of the chromatographic support shows that it has an ion-exchange capacity of 0.6 mmol/g, and 82% of the amine sites on the surface are in quaternized form . Isocratic elution of small benzoic acid derivatives shows good column efficiency . The two primary virtues of this material are its chemical stability under alkali conditions (up to pH 13) and its lower hydrophobicity compared to previously described alkali-stable PEI-coated zirconia supports cross-linked with 1,10-diiododecane . Using this new zirconia-based phase, a purification protocol is developed for the efficient removal of Escherichia coli 0111:B4 LPS from bovine insulin samples . An endotoxin clearance rate of up to 1.3 x 10(8) was attained . Endotoxin levels were reduced to less than 5 endotoxin units/ml even at initial contamination levels as high as 5.0 x 10(6) endotoxin units/ml . Furthermore, endotoxin adsorbed to the porous zirconia column may be easily removed (depyrogenated) using alkali for repeated purification cycles . Anal Biochem, 1999 Oct 1, 274(1), 40 - 9 Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes; Layfield R et al.; We have used solid-phase chemistry to synthesize proteins equivalent to a human ubiquitin precursor (ubiquitin-52-amino-acid ribosomal protein fusion; UBICEP52) and representative of isopeptide-linked ubiquitin-protein conjugates {ubiquitin-(epsilonN)-lysine}; these proteins were precisely cleaved by a purified recombinant Drosophila deubiquitinating enzyme (DUB), UCH-D . Along with the previously synthesized ubiquitin-(alphaN)-valine, these synthetic proteins were used as substrates to assess the catalytic capacities of a number of diverse DUBs expressed in Escherichia coli: human HAUSP; mouse Unp; and yeast Ubps 1p, 2p, 3p, 6p, 11p, and 15p and Yuh1p . Distinct specificities of these enzymes were detected; notably, in addition to UCH-D, isopeptidase activity {ubiquitin-(epsilonN)-lysine cleavage} was only associated with Yuh1p, Unp, Ubp1p, and Ubp2p . Additionally, human placental 26S proteasomes were only able to cleave UBICEP52 and ubiquitin-(epsilonN)-lysine, suggesting that 26S proteasome-associated DUBs are class II-like . This work demonstrates that the synthetic approach offers an alternative to recombinant methods for the production of small proteins in vitro . Plant Mol Biol, 1999 Aug, 40(6), 1063 - 71 Cloning and characterization of a class II DNA photolyase from Chlamydomonas; Petersen JL et al.; Damage to DNA induced by ultraviolet light can be reversed by a blue light-dependent reaction catalyzed by enzymes called DNA photolyases . Chlamydomonas has been shown to have DNA photolyase activity in both the nucleus and the chloroplast . Here we report the cloning and sequencing of a gene, PHR2, from Chlamydomonas encoding a class II DNA photolyase . The PHR2 protein, when expressed in Escherichia coli, is able to complement a DNA photolyase deficiency . The previously described Chlamydomonas mutant, phr1, which is deficient in nuclear but not chloroplast photolyase activity was shown by RFLP analysis not to be linked to the PHR2 gene . Unlike the recently reported class II DNA photolyase from Arabidopsis, the protein encoded by PHR2 is predicted to contain a chloroplast targeting sequence . This result, together with the RFLP data, suggests that PHR2 encodes the chloroplast targeted DNA photolyase. Plant Mol Biol, 1999 Aug, 40(6), 1009 - 18 Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitin-binding domains; Zhao KJ et al.; We have cloned a 1.3 kb Brassica juncea cDNA encoding BjCHI1, a novel acidic chitinase with two chitin-binding domains that shows 62% identity to Nicotiana tabacum Chia1 chitinase . BjCHI1 is structurally unlike Chia1 that has one chitin-binding domain, but resembles Chia5 chitinase UDA1, the precursor of Urtica dioica agglutinin: however there is only 36.9% identity between them . We propose that BjCHI1 should be classified under a new class, Chia7 . The spacer and the hinge region of BjCHI1 are proline-rich, like that of Beta vulgaris Ch1, a Chia6 chitinase with half a chitin-binding domain . Northern blot analysis showed that the 1.3 kb BjCHI1 mRNA is induced by wounding and methyljasmonate (MeJA) treatment but is unaffected by ethylene, salicylic acid (SA) or abscisic acid (ABA) . This is the first report on MeJA induction of chitinase gene expression and further suggests that wound-related JA-mediated signal transduction is independent of that involving SA . Western blot analysis using polyclonal antibodies against BjCHI1 showed a cross-reacting band with an apparent molecular mass of 37 kDa in wounded tissues of B . juncea, revealing that, unlike UDA1, BjCHI1 is not cleaved post-translationally at the hinge . Expression of recombinant BjCHI1 in Escherichia coli BL21(DE3) inhibited its growth while crude extracts from E . coli JM109 expressing recombinant BjCHI1 showed chitinase activity . Results from polymerase chain reaction (PCR) suggest that genes encoding chitinases with single or double chitin-binding domains exist in B . juncea. Plant Mol Biol, 1999 Aug, 40(6), 969 - 76 A cDNA from tobacco codes for an inhibitor of virus replication (IVR)-like protein; Akad F et al.; We have shown previously that localization of tobacco mosaic virus (TMV) in tobacco is associated with a ca . 23 kDa protein that inhibits replication of several plant viruses . This protein, named 'inhibitor of virus replication' (IVR), was purified from the medium of TMV-inoculated protoplasts derived from Nicotiana tabacum cv . Samsun NN . IVR was shown to be present also in induced-resistant leaf tissue of N . tabacum cv . Samsun NN . We prepared an expression cDNA library from such induced-resistant tissue and screened it with a polyclonal antibody raised against the IVR protein . A 1016 bp clone (named NC330) containing a 597 bp open reading frame, coding for a 21.6 kDa polypeptide, was isolated . The NC330 clone hybridized with RNA from induced-resistant tissue from N . tabacum cv . Samsun NN but not with RNA from non-induced tissue . Likewise, it did not hybridize with RNA from infected or uninfected tissue of N . tabacum cv . Samsun nn . Similarly, the NC330 cloned probe hybridized with the RT-PCR products from RNA of the induced-resistant tissue only . In Southern blot hybridization the NC330 DNA probe detected several genomic DNA fragments in both N . tabacum cv . Samsun NN and Samsun nn . The size of the DNA fragments differed in Samsun NN and Samsun nn . We suggest that DNA encoding the IVR-like protein is present in resistant and susceptible N . tabacum genotypes, but is expressed only in NN . We have inserted the NC330 into the expression vector pET22b and a 21.6 kDa protein was produced in Escherichia coli that reacted in immunoblots with the IVR antibody . This protein greatly reduced replication of TMV in N . tabacum cv . Samsun nn leaf disk assays. Plant Mol Biol, 1999 Aug, 40(6), 921 - 33 Elicitor- and A23187-induced expression of WCK-1, a gene encoding mitogen-activated protein kinase in wheat; Takezawa D; Wheat cultured cells were used to study the role of Ca2+ in regulating protein kinases during the induction of defense-related genes by fungal elicitor treatments . Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1, a gene encoding mitogen-activated protein (MAP) kinase . The induction of WCK-1 mRNA by A23187 did not occur when extracellular Ca2+ was chelated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) . The WCK-1 mRNA was also induced by Typhula ishikariensis-derived elicitors, suggesting a possible involvement of WCK-1 in the plant defense response against pathogens . BAPTA and a calcium channel blocker, La3+, inhibited the elicitor-induced expression of the WCK-1 mRNA . A recombinant fusion protein of WCK-1 (GST-WCK-1) autophosphorylated at the Tyr residue and exhibited an autophosphorylation-dependent protein kinase activity towards myelin basic protein . Alteration of Tyr-196 in the conserved 'TEY' motif in GST-WCK-1 to Phe by site-directed mutagenesis abolished the autophosphorylation . The GST-WCK-1 protein was activated by elicitor-treated wheat cell extracts but not by the control extract . These results suggest that fungal elicitors activate WCK-1, a specific MAP kinase in wheat . Furthermore, the results suggest a possible involvement of Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts. J Crit Care, 1999 Sep, 14(3), 125 - 32 Myocardial oxygen consumption during dobutamine infusion in endotoxemic pigs; Herbertson MJ et al.; PURPOSE: Dobutamine infusion is used to increase whole-body oxygen delivery in septic patients to satisfy unmet oxygen demand of hypoxic tissues . However, dobutamine infusion also increases myocardial work and myocardial oxygen consumption . Our goal was to determine the importance of this effect as a fraction of the increase in whole-body oxygen consumption, in a porcine model of septic shock . MATERIALS AND METHODS: Four hours after a 50 microg/kg infusion of Escherichia coli endotoxin (0111: B4, Sigma) in eight anesthetized pigs, whole-body oxygen delivery and myocardial oxygen delivery and consumption were calculated from blood flow and arterial and venous oxygen content measurements . We directly measured whole-body oxygen consumption by analysis of inhaled and exhaled gases using a metabolic cart . Then dobutamine 10 and 20 microg/kg/min was infused and measurements were repeated . RESULTS: Dobutamine infusion increased whole-body oxygen delivery but did not increase metabolic cart measured whole-body oxygen consumption . Dobutamine infusion of 10 and 20 microg/kg/min increased myocardial oxygen consumption by 7.0 +/- 0.6 (80 +/-10%) and 12.0 +/- 2.0 mL O2/min (142 +/- 30%), respectively (P < .01) . CONCLUSIONS: In this porcine model of sepsis, dobutamine infusion significantly increases myocardial oxygen consumption . Because whole-body oxygen consumption does not change, dobutamine infusion may fail to increase and may decrease oxygen consumption by other organs. Acta Biol Hung, 1998, 49(2-4), 173 - 84 Related alphaN- and epsilonN-methyltransferases methylate the large and small subunits of Rubisco; Ying Z et al.; Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is methylated at the ax amino position of the N-terminal methionyl residue of the processed and assembled form of the small subunit (SS), and is also methylated in some species at the epsilon-amino group of lysine-14 in the large subunit (LS) . The gene (rbcMT-S) and cDNAs for the SS alphaN-methyltransferase (SSMT) from spinach (Spinach oleracea) have been cloned, sequenced, and expressed . The gene is closely related to a previously characterized LS methyltransferase (Rubisco LSMT) cDNA from pea (Rubisco LSMT) and a Rubisco LSMT gene from tobacco . Sequence analysis of the cDNA and transcript mapping experiments demonstrate that the rbcMT-S pre-mRNAs experience alternative 3' splice site selection, such that mRNAs for a long form with a four amino acid insertion and a short form are expressed at approximately equal abundance . The coding sequence of spinach SSMT includes a putative targeting presequence with sequence identity at a plastid processing site . A N-terminal truncated form of spinach SSMT was expressed and purified from E . coli cells . Both long and short forms of the cDNAs were shown to catalyze methylation of the a amine of the N-terminal methionine of the SS of Rubisco. Proteins, 1999, Suppl 3, 133 - 40 Threading with explicit models for evolutionary conservation of structure and sequence; Panchenko A et al.; We have attempted to predict the three-dimensional structures of 19 proteins for the CASP3 experiment, each showing less than 25% sequence identity with known structures . Predictions were based on a threading method that aligns the target sequence with the conserved cores of structural templates, as identified from structure-structure alignments of the template with homologous neighbors . Alternative alignments were scored using contact potentials and a position-specific score matrix derived from sequence neighbors of the template . We find that this method identified the correct structural family for 11 of the 19 targets and predicted the remaining 8 targets to be similar to "none" of the templates, avoiding false positives . Threading alignments are relatively accurate for 10 of the 11 targets, including alignments for 6 of 7 identified at CASP3 as fold-recognition targets . These predictions were ranked "first place" by the CASP3 assessor when compared to fold-recognition predictions made by other methods . It appears that threading with family-specific models for structure and sequence conservation has improved threading prediction accuracy. Proteins, 1999, Suppl 3, 112 - 20 Sustained performance of knowledge-based potentials in fold recognition; Domingues FS et al.; We describe the results obtained using fold recognition techniques in our third participation in the CASP experiment . The approach relies on knowledge-based potentials for alignment production and fold identification . As indicated by the increase in alignment quality and fold identification reliability, the predictions improved from CASP1 to CASP3 . In particular, we identified structural relationships in which no known evolutionary link exists . Our predictions are based on single sequences rather than multiple sequence alignments . Additionally, we voluntarily submitted only a single model for each target because, in our view, submission of a single model is the most stringent test . We describe the methods used, the strategy adopted in the predictions, and the prediction results and discuss future work. Proteins, 1999, Suppl 3, 88 - 103 Structure classification-based assessment of CASP3 predictions for the fold recognition targets; Murzin AG; The sequences of at least 23 of the 43 CASP3 targets showed no significant similarity to the sequences of known structures . The experimental structures of all but three of these 23 targets revealed substantial similarities to known structures, with at least eleven of the target structures likely being distantly homologous to known structures . Nineteen of the 23 target structures were available at the time of the final CASP3 meeting in Asilomar in December 1998, whereas the experimental data on the protein folds of the remaining four targets were obtained afterwards . The predicted three-dimensional structures for each of the 23 targets were analyzed to select those predictions sharing with the experimental structures a similar overall fold and/or having correctly folded a substantial fraction of the target sequence . Initially, predicted models were numerically evaluated and the evaluation results aided the selection process . Each target structure was then classified to identify a minimal set of structural features characteristic to its protein fold and evolutionary superfamily . The predictions containing this set were assessed comparatively to find the best predictions for each target . The predictions of new folds were assessed separately . The total number of the selected 'correct' predictions and the quality of these predictions were used to compare the performance of different predictor teams and different prediction methods in the fold prediction/recognition category. Proteins, 1999, Suppl 3, 22 - 9 Processing and analysis of CASP3 protein structure predictions; Zemla A et al.; Livermore Prediction Center provides basic infrastructure for the CASP (Critical Assessment of Structure Prediction) experiments, including prediction processing and verification servers, a system of prediction evaluation tools, and interactive numerical and graphical displays . Here we outline the essentials of our approach, with discussion of the superposition procedures, definitions of basic measures, and descriptions of new methods developed to analyze predictions . Our primary focus is on the evaluation of three-dimensional models and secondary structure predictions . To put the results of the three prediction experiments held to date on the same footing, the latest CASP3 evaluation criteria were retrospectively applied to both CASP1 and CASP2 predictions . Finally, we give an overview of our website PredictionCenter.llnl.gov), which makes the target structures, predictions, and the evaluation system accessible to the community. Proteins, 1999, Suppl 3, 7 - 14 Automated large scale evaluation of protein structure predictions; Lackner P et al.; Evaluation and assessment are critical issues in CASP experiments . Automated procedures are necessary to compare a large number of predictions with the target folds . The evaluation has to reveal the maximum extent of similarity between predictions and targets, it should be applicable across prediction categories, and it should be transparent and accessible to a wide community . Here we present an automated evaluation scheme which is an attempt to meet these requirements . In the implementation and execution of this scheme we had to solve or circumvent problems of convergence, where algorithms fail to find optimum solutions, problems of ambiguity where no unique optimum solution exists, and problems in ranking and interpretation . Key features of this implementation are (1) the root mean square deviation of structure superimposition is kept close to a constant value throughout the evaluation and (2) all structural matches found between two folds are taken into account . We discuss these points in detail and describe the numerical criteria used in the CASP3 evaluation. Genes Cells, 1999 Sep, 4(9), 501 - 15 Intragenic suppression of trans-dominant lethal substitutions in the conserved GEME motif of the beta subunit of RNA polymerase: evidence for functional cooperativity within the C-terminus; Malik T et al.; BACKGROUND: The ubiquitous multimeric RNA polymerases contain two large, conserved subunits, of which the beta subunit has been implicated in all three stages of transcription . We have previously described a genetic system involving random, PCR-mediated mutagenesis of the region of rpoB encoding the C-terminal 116 amino acids of the beta subunit of Escherichia coli RNA polymerase and the characterization of dominant-negative mutations . This study identified the invariant motif GEME (residues 1271-->1274; Cromie et al . 1999) . Starting with three of these GEME-motif lethal mutations (G1271E, G1271V, M1273V), we have selected for intragenic suppressors, located within the same 3'-region, that prevent expression of the trans-dominant phenotype . RESULTS: We isolated a total of 24 missense mutants and a further 14 frameshift alleles (the latter generating a nested set of C-terminal deletions of the beta subunit) and studied the effect of the missense suppressors in vivo and in vitro . The majority of the second-site substitutions pinpoint highly conserved residues and were allele-specific . In contrast, one particular missense substitution (S1332P) acted on all three primary site mutations whilst not appreciably affecting assembly proficiency, suggesting motif-specific suppression . Two missense substitutions were found to perturb assembly of the beta subunit (M1232T and L1233P) and define a small conserved region (1228-->1233) adjacent to one of the active-site residues identified by affinity-labelling, H1237 . The majority of primary mutations were located in three main clusters within the 116 amino acid region . CONCLUSIONS: The importance and functional co-operativity of the three main clusters pinpointed is supported by the present isolation of suppressors of three different GEME primary mutations in the same three regions (whereas the suppressors of G1271V and M1273V are located in all three clusters, those for G1271E are all C-terminal of this residue) . Moreover, the location of the suppressors suggests that the GEME and HLVDDK regions are present as alpha-helices in holoenzyme, and that functional co-operativity is through one particular face of each helix. Genes Cells, 1999 Aug, 4(8), 475 - 85 Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNA polymerase; Honda A et al.; BACKGROUND: Influenza virus RNA polymerase with the subunit composition of PB1-PB2-PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome . Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymerase-associated capped RNA endonuclease . To identify the RNA cap 1-binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV-crosslinking with RNA which was labelled with 32P only at the cap-1 structure . RESULTS: After SDS-PAGE of UV-crosslinked cores, 32P was found to be associated only with the PB2 subunit (759 amino acid residues) . The labelled PB2 was subjected, together with PB2 expressed in E . coli, to limited digestion with V8 protease . Analysis of the amino terminal sequences of some isolated fragments with the crosslinked cap-1 indicated that two separate sequences within the PB2 were involved in RNA cap-1 binding, one (N-site) at the N-terminal proximal region approximately between amino acid residues 242-282 downstream from the PB1 subunit-binding site and the other (C-site) between residues 538-577 including the cap-binding motifs . Two lines of evidence support the prediction of the involvement of two separate PB2 sequences on the RNA cap-binding: (i) cross-linking of the capped RNA on to expressed and isolated PB2 fragments, each containing either the N-site or the C-site; and (ii) competition of capped RNA-binding to PB2 by both of the N- and C-terminal PB2 fragments . Taking together, we propose that two separate sequences within PB2 constitute the capped RNA-binding site of the RNA polymerase . CONCLUSION: Two separate sequences, one N-(242-282) and the other C-terminal (538-577) proximal segments of PB2 subunit, constitute the RNA cap-binding site of the influenza virus RNA polymerase. Mutat Res, 1999 Sep 13, 435(1), 77 - 80 A UmuD,C-dependent pathway for spontaneous G:C to C:G transversions in stationary phase Escherichia coli mut Y; Timms AR et al.; In Escherichia coli trpA23 bacteria lacking the MutY glycosylase and incubated on plates in the absence of tryptophan, tryptophan-independent mutants continue to arise during incubation over many days . Their appearance is enhanced in umuD+,C+ strains in comparison with strains carrying a deletion through the umu operon and the umuD,C-dependent mutants were greater in number in uvrA bacteria (lacking nucleotide excision repair) than in uvr+ bacteria . Sequencing of mutations occurring in uvrA bacteria revealed the presence of G:C to C:G transversions but only in umuD+,C+ strains . There is thus a pathway in starved bacteria that generates G:C to C:G transversions and requires the inducible UmuD,C proteins . The data are consistent with the occurrence of a lesion, probably 8-oxoguanine, against which guanine may be incorporated during DNA synthesis by "dNTP stabilised" misalignment against the downstream template base . Upon realignment the configuration is substrate for MutY glycosylase which can remove the unmodified guanine . It is hypothesised that UmuD,C proteins are required for primer extension from the mismatch once formed. Mutat Res, 1999 Oct 19, 429(2), 199 - 208 Plasmid-mediated expression of the UmuDC mutagenesis proteins in an Escherichia coli strain engineered for human cytochrome P450 1A2-catalyzed activation of aromatic amines; Josephy PD et al.; The mutagenic actions of many chemicals depend on the activities of bacterial "mutagenesis proteins", which allow replicative bypass of DNA lesions . Genes encoding these proteins occur on bacterial chromosomes and plasmids, often in the form of an operon (such as umuDC or mucAB) encoding two proteins . Many bacterial strains used in mutagenicity testing carry mutagenesis protein genes borne on plasmids, such as pKM101 . Our objective was to introduce mutagenesis protein function into Escherichia coli strain DJ4309 . This strain expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase and carries out the metabolic conversion of aromatic and heterocyclic amines into DNA-reactive mutagens . We discovered that many mutagenesis-protein plasmids severely inhibit the response of strain DJ4309 to 2-amino-3,4-dimethylimid-azo{4,5-f}quinoline (MeIQ), a typical heterocyclic amine mutagen . Among many plasmids examined, one, pGY8294, a pSC101 derivative carrying the umuDC operon, did not inhibit MeIQ mutagenesis . Strain DJ4309 pGY8294 expresses active mutagenesis proteins, as shown by its response to mutagens such as 1-nitropyrene and 4-nitroquinoline 1-oxide (4-NQO), and is as sensitive as the parent strain DJ4309 to P450-dependent mutagens, such as MeIQ and 1-aminopyrene. FEBS Lett, 1999 Oct 15, 459(3), 433 - 7 Cloning and characterization of two novel aldo-keto reductases (AKR1C12 and AKR1C13) from mouse stomach; Ikeda S et al.; In contrast to hepatic hydrosteroid dehydrogenases (HSDs) of the aldo-keto reductase family (AKR1C), little is known about a stomach one . From a mouse stomach cDNA library, we isolated two clones encoding proteins of 323 amino acid residues . They exhibited 93.2% amino acid sequence identity and 64-68% with any known HSDs . Recombinant proteins expressed in Escherichia coli reduced 9,10-phenanthraquinone with NAD(P)H as cofactor . The mRNAs were exclusively expressed in stomach, liver and ileum . The present study demonstrates that these proteins are new members of the HSD subfamily and they are named AKR1C12 and AKR1C13 . Immunohistochemical analysis suggests that they are involved in detoxification of xenobiotics in the stomach. FEBS Lett, 1999 Oct 15, 459(3), 411 - 4 Mössbauer studies of Escherichia coli biotin synthase: evidence for reversible interconversion between {2Fe-2S}(2+) and {4Fe-4S}(2+) clusters; Tse Sum Bui B et al.; The nature and properties of the iron-sulphur (Fe-S) cluster in as-prepared and reduced biotin synthase of Escherichia coli have been investigated by Mossbauer spectroscopy . Our data clearly demonstrate that in the as-prepared sample, the cluster is present as {2Fe-2S}(2+) with isomer shift, delta = 0.29 mm/s and quadrupole splitting, DeltaE(Q) = 0.53 mm/s, indicating incomplete cysteinyl-S coordination . Anaerobic reduction by dithionite in the presence of 55% (v/v) glycerol converts this form to {4Fe-4S}(2+) (delta = 0.45 mm/s and DeltaE(Q) = 1.11 mm/s) and is accompanied by some destruction to Fe(2+) . This cluster conversion is reversible and when exposed to air, the {4Fe-4S}(2+) cluster is quantitatively reconverted to the {2Fe-2S}(2+) cluster without any further cluster degradation. Brain Res, 1999 Sep 18, 842(1), 139 - 47 Lipopolysaccharide evokes the modulation of brain cytochrome P4501A in the rat; Renton KW et al.; The cytochrome P450 enzyme system is a multigene family of enzymes that is modulated in the liver during systemic inflammatory responses or during infection Several forms of the enzyme are expressed in discrete areas of the brain and likely play a critical role in the metabolism of drugs and endogenous chemicals in the central nervous system (CNS) . Even though the brain responds to inflammation in a manner different from most tissues, we examined the possible modification of a major cytochrome P450 form (CYP1A) in the brain during inflammation confined to that organ . Total brain CYP1A activity, as measured by ethoxyresorufin dealkylase (EROD), was downregulated 24 and 48 h following the administration of a single dose of lipopolysaccharide (LPS) . Regionally, a similar effect was determined in the cortex, hippocampus and the mid-brain but the activity in the cerebellum was unaffected . The examination of coronal brain sections using an antibody directed against CYP1A indicated that the enzyme was distributed in discrete cells of the hippocampus, thalamus and cortex and in the tanycytes surrounding the third ventricle . In each of these areas, the immunoreactivity was diminished in animals receiving LPS as compared to saline-treated animals . LPS also evoked the expression of the small molecular weight heat shock protein hsp27 throughout the brain indicating the development of an inflammatory response . These studies indicate that inflammation localized to the CNS causes an alteration in the levels and activity of a major cytochrome P450 form in the brain . This could have implications to the metabolism or activation of drugs and endogenous chemicals in the CNS during a disease state that features an inflammatory component. Arch Microbiol, 1999 Oct, 172(4), 233 - 9 The correlation of the gene csoS2 of the carboxysome operon with two polypeptides of the carboxysome in thiobacillus neapolitanus Baker SH, Lorbach SC, Rodriguez-Buey M, Williams DS, Aldrich HC, Shively JM. The carboxysomal polypeptides of Thiobacillus neapolitanus with apparent molecular masses of 85 and 130 kDa were isolated and subjected to N-terminal sequencing . The first 17 amino acids of the two peptides were identical . The sequence perfectly matched the deduced amino acid sequence of an open reading frame in the carboxysome operon . The gene was subsequently named csoS2 . Expression of the gene in Escherichia coli resulted in the production of two peptides with apparent molecular masses of 85 and 130 kDa . Immunospecific antibodies generated against the smaller peptide recognized both peptides; the peptides were named CsoS2A and CsoS2B, respectively . A digoxigenin-hydrazide glycosylation assay revealed that both CsoS2A and CsoS2B are post-translationally modified by glycosylation . CsoS2 was localized to the edges of purified carboxysomes by immunogold electron microscopy using the monospecific CsoS2A antibodies . The molecular mass of CsoS2A calculated from the nucleotide sequence was 92.3 kDa. Diabetologia, 1999 Oct, 42(10), 1175 - 86 Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis; Davis EA et al.; AIMS/HYPOTHESIS: Mutations of the glucokinase gene cause hyperglycaemia or hypoglycaemia . A quantitative understanding of these defects of glucose homeostasis linked to the glucokinase gene was lacking . Therefore a database of kinetic variables of wild-type and 20 missense mutants of glucokinase was developed and used in mathematical modelling to predict the thresholds for glucose-stimulated insulin release . METHODS: Recombinant human glucokinase was generated in E . coli . The k(cat), glucose S(0.5), ATP K(m), and Hill number of glucokinase were determined . Inhibition by Stearoyl CoA and glucokinase regulatory protein and thermal stability were assayed for all mutants kinetically similar to wild-type glucokinase . A mathematical model predicting the threshold for glucose-stimulated insulin release was constructed . This model is based on the two substrate kinetics of glucokinase and the kinetic variables of the database . It is assumed that both glucokinase gene alleles are equally expressed in beta-cells and that induction of glucokinase occurs as a function of basal blood glucose . RESULTS: Large changes, varying greatly between mutants were found in nearly all variables . Glucokinase flux at threshold for glucose-stimulated insulin release was about 25 % of total phosphorylating potential in the normal beta-cell and this was used to predict thresholds for the mutant heterozygotes . Clinical data for maturity onset diabetes of the young type linked to the glucokinase gene and familial hyperinsulinaemic hypoglycaemia linked to the glucokinase gene and the glucokinase kinetic data of this study were used to test the model . The model predicts fasting blood glucose between 3 and 7 mmol/l in these cases . CONCLUSION/INTERPRETATION: A kinetics database of wild-type and 20 mutants of glucokinase was developed . Many kinetic differences were found for the mutants . The mathematical model to calculate the threshold for glucose-stimulated insulin release predicts fasting blood glucose between 3 and 7 mmol/l in subjects with glucokinase gene mutations . {Diabetologia 42: 1175-1186} Methods, 1999 Sep, 19(1), 148 - 55 Mucosal delivery of vaccines; Del Giudice G et al.; Oral delivery represents one of the most pursued approaches for large-scale human vaccination . Due to the different characteristics of mucosal immune response, as compared with systemic response, oral immunization requires particular methods of antigen preparation and selective strategies of adjuvanticity . In this paper, we describe the preparation and use of genetically detoxified bacterial toxins as mucosal adjuvants and envisage the possibility of their future exploitation for human oral vaccines . J Mol Biol, 1999 Oct 1, 292(4), 931 - 44 Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation; Cole KB et al.; The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms . In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4 . 5S RNA . In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E . coli RNase P) in vitro for cleavage of a DNA substrate . In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation . A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA . This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates . This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E . coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates . M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding . The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule . This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available. J Mol Biol, 1999 Oct 1, 292(4), 855 - 69 Insight into odorant perception: the crystal structure and binding characteristics of antibody fragments directed against the musk odorant traseolide; Langedijk AC et al.; Monoclonal antibodies were elicited against the small hydrophobic hapten traseolide, a commercially available musk fragrance . Antibody variable region sequences were found to belong to different sequence groups, and the binding characteristics of the corresponding antibody fragments were investigated . The antibodies M02/01/01 and M02/05/01 are highly homologous and differ in the binding pocket only at position H93 . M02/05/01 (H93 Val) binds the hapten traseolide about 75-fold better than M02/01/01 (H93 Ala) . A traseolide analog, missing only one methyl group, does not have the characteristic musk odorant fragrance . The antibody M02/05/01 binds this hapten analog about tenfold less tightly than the original traseolide hapten, and mimics the odorant receptor in this respect, while the antibody M02/01/01 does not distinguish between the analog and traseolide . To elucidate the structural basis for the fine specificity of binding, we determined the crystal structure of the Fab fragment of M02/05/01 complexed with the hapten at 2.6 A resolution . The crystal structure showed that only van der Waals interactions are involved in binding . The somatic Ala H93 Val mutation in M02/05/01 fills up an empty cavity in the binding pocket . This leads to an increase in binding energy and to the ability to discriminate between the hapten traseolide and its derivatives . The structural understanding of odorant specificity in an antibody gives insight in the physical principles on how specificity for such hydrophobic molecules may be achieved. J Mol Biol, 1999 Oct 1, 292(4), 787 - 96 The DNA translocation and ATPase activities of restriction-deficient mutants of Eco KI; Davies GP et al.; Eco KI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase, endonuclease and DNA translocation activities . One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction . These activities involve ATP-dependent DNA translocation and DNA cleavage . Mutations that change amino acids within recognisable motifs in HsdR impair restriction . We have used an in vivo assay to monitor the effect of these mutations on DNA translocation . The assay follows the Eco KI-dependent entry of phage T7 DNA from the phage particle into the host cell . Earlier experiments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity of purified enzymes . We find that the mutations abolish DNA translocation in vivo . This provides evidence that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation . Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been shown to abolish DNA nicking activity . When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability to hydrolyse ATP and to translocate DNA at wild-type levels . It has been speculated that nicking may be necessary to resolve the topological problems associated with DNA translocation by type I restriction and modification systems . Our experiments show that loss of the nicking activity associated with the endonuclease motif of Eco KI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo. J Mol Biol, 1999 Oct 1, 292(4), 771 - 8 Interaction of the Escherichia coli DEAD box protein DbpA with 23 S ribosomal RNA; Pugh GE et al.; The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA . We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity . Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted . These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA . We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested. Arch Biochem Biophys, 1999 Nov 1, 371(1), 70 - 82 Soybean nodule sucrose synthase (nodulin-100): further analysis of its phosphorylation using recombinant and authentic root-nodule enzymes; Zhang XQ et al.; Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant "sink" tissues . In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli . Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized . We document that a conserved serine near the N-terminus (Ser(11)) is the primary phosphorylation site for a nodule Ca(2+)-dependent protein kinase (CDPK) in vitro . Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100 . In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala(2) and Arg(13)) were phosphorylated, albeit weakly, by the CDPK . This secondary site(s) likely resides between Glu(14) and Met(193) as evidenced by CNBr cleavage and phosphopeptide mapping . Phosphorylation of the recombinant and authentic nodule Ser(11) enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties . However, phosphorylation decreased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin-100 between the nodule symbiosome/plasma membranes and cytosol in planta . Arch Biochem Biophys, 1999 Nov 1, 371(1), 35 - 40 Oligomerization of the EK18 mutant of the trp repressor of Escherichia coli as observed by NMR spectroscopy; Chae YK et al.; The regulation of the trp repressor system of Escherichia coli is frequently modeled by a single equilibrium, that between the aporepressor (TR) and the corepressor, l-tryptophan (Trp), at their intracellular concentrations . The actual mechanism, which is much more complex and more finely tuned, involves multiple equilibria: TR and Trp association, TR oligomerization, specific and nonspecific binding of various states of TR to DNA, and interactions between these various species and ions . TR in isolation exists primarily as a homodimer, but the state of oligomerization increases as the TR concentration goes up and/or the salt concentration goes down, leading to species with lower affinity for DNA . We have used multinuclear, multidimensional NMR spectroscopy to investigate structural changes that accompany the oligomerization of TR . For these investigations, the superrepressor mutant EK18 (TR with Glu 18 replaced by Lys) was chosen because it exhibits less severe oligomerization at higher protein concentration than other known variants; this made it possible to study the dimer to tetramer oligomerization step by NMR . The NMR results suggest that the interaction between TR dimers is structurally linked to folding of the DNA binding domain and that it likely involves direct contacts between the C-terminal residues of the C-helix of one dimer with the next dimer . This implies that oligomerization can compete with DNA binding and thus serves as a factor in the fine-tuning of gene expression . Arch Biochem Biophys, 1999 Nov 1, 371(1), 15 - 23 Mutations in the charged residues of the amino terminus of rat liver fructose 6-phosphate,2-kinase:Fructose 2,6-bisphosphatase: effects on regulation; Wu RF et al.; Amino and carboxyl termini of the bifunctional enzyme Fru 6-P, 2-kinase:Fru 2,6-bisphosphatase regulate the relative activities of the kinase/phosphatase . The N-terminus of the rat liver bifunctional enzyme is highly basic, containing a protein kinase A phosphorylation site that regulates these enzyme activities in a reciprocal manner . To determine the role of charged residues in the N-terminal peptide, mutant enzymes were constructed in which these residues were altered to residues carrying opposite charges, and the effect on the catalytic properties, thermal lability, and susceptibility to trypsin digestion and phosphorylation by protein kinase A was determined . Most of these mutations decreased k(cat)/K(ATP) and/or k(cat)/K(Fru) (6-P) of the kinase and increased k(cat)/K(Fru 2,6-P2) of the phosphatase . These mutant enzymes were more susceptible to trypsin digestion, phosphorylation by protein kinase A, and thermal inactivation . In general, the effect was greater with amino acid residues located more distant from the N-terminus . The resulting changes were not as large as observed with the phosphorylated enzyme . Mutation of Ser22 to Pro produced large changes in the kinetic properties comparable to those of phosphorylation, suggesting that the flexible region of the N-terminus containing five serines (Ser20 to S24) is essential for the enzyme activities . These results indicated that the charged residues as well as Ser20-Ser24 in the N-terminus of the liver Fru 6-P,2-kinase:Fru 2,6-Pase are essential in the allosteric regulation and probably involved in interactions with the catalytic domains that induce a conformation that has high Fru 6-P,2-kinase and low Fru 2,6-Pase activities . Any disruption of this N-terminal interaction results in inhibition of the kinase and activation of the phosphatase . Chem Res Toxicol, 1999 Oct, 12(10), 917 - 23 Sequence-dependent repair of synthetic AP sites in 15-mer and 35-mer oligonucleotides: role of thermodynamic stability imposed by neighbor bases; Sagi J et al.; We previously reported that 15-mer oligonucleotides with a central 1, N(6)-epsilonA were cleaved by alkylpurine-DNA N-glycosylase as a function of T(m), modulated by neighbor bases {Hang, B., Sagi, J., and Singer, B . (1998) J . Biol . Chem . 273, 33406-33413} . This type of investigation has now been extended to cleavage by Escherichia coli endonuclease IV of a centrally placed synthetic AP site using both 15-mer and 35-mer duplexes . In 15-mers, the triplet sequences adjunct to the central AP site greatly affected the thermodynamic stability . The repair rate paralleled the thermal stability since endonuclease IV requires a double-stranded substrate . When the AP site-containing duplexes were 35-mers, there was also a general correlation between the thermostability and cleavage efficiency . However, the difference in the cleavage rates between different sequences was much less than with the 15-mers . Since the 35-mers were more than 96% annealed, this difference presumably results from local stability and structure adjacent to the AP site . These results suggest that under enzyme limiting conditions or overproduction of AP sites, sequence-dependent differential repair could occur in vivo. Nature, 1999 Oct 7, 401(6753), 610 - 3 Phytochrome signalling is mediated through nucleoside diphosphate kinase 2; Choi G et al.; Because plants are sessile, they have developed intricate strategies to adapt to changing environmental variables, including light . Their growth and development, from germination to flowering, is critically influenced by light, particularly at red (660 nm) and far-red (730 nm) wavelengths . Higher plants perceive red and far-red light by means of specific light sensors called phytochromes(A-E) . However, very little is known about how light signals are transduced to elicit responses in plants . Here we report that nucleoside diphosphate kinase 2 (NDPK2) is an upstream component in the phytochrome signalling pathway in the plant Arabidopsis thaliana . In animal and human cells, NDPK acts as a tumour suppressor . We show that recombinant NDPK2 in Arabidopsis preferentially binds to the red-light-activated form of phytochrome in vitro and that this interaction increases the activity of recombinant NDPK2 . Furthermore, a mutant lacking NDPK2 showed a partial defect in responses to both red and farred light, including cotyledon opening and greening . These results indicate that NDPK2 is a positive signalling component of the phytochrome-mediated light-signal-transduction pathway in Arabidopsis. Biochim Biophys Acta, 1999 Oct 12, 1434(2), 356 - 64 Characterization of genes encoding two manganese peroxidases from the lignin-degrading fungus Dichomitus squalens(1); Li D et al.; Genes encoding two manganese peroxidases from the white-rot basidiomycete Dichomitus squalens were cloned and sequenced . The mnp1 and mnp2 genes encode mature proteins of 369 and 365 amino acids, respectively . The amino acids involved in peroxidase function, those forming the Mn(II) binding site, and those forming the five disulfide bonds in other Mn peroxidases are conserved in these sequences . Both predicted D . squalens proteins contain multiple acidic residues in their C-terminal sequences, which may be involved in additional metal binding . Both genes contain seven small introns, the locations of which align with each other . The promoters of both D . squalens genes contain putative AP-2 sites, which may be involved in their regulation by nutrient nitrogen . Southern blot analysis of genomic PCR fragments suggests that these sequences represent separate genes rather than allelic variants. Biochim Biophys Acta, 1999 Oct 12, 1434(2), 317 - 30 Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme; Kowlessur D et al.; The first step in the biosynthesis of melatonin in the pineal gland is the hydroxylation of tryptophan to 5-hydroxytryptophan . A cDNA of human tryptophan hydroxylase (TPH) was cloned from a library of human pineal gland and expressed in Escherichia coli . This cDNA sequence is identical to the cDNA sequence published from the human carcinoid tissue {1} . This human pineal hydroxylase gene encodes a protein of 444 amino acids and a molecular mass of 51 kDa estimated for the purified enzyme . Tryptophan hydroxylase from human brainstem exhibits high sequence homology (93% identity) with the human pineal hydroxylase . The recombinant tryptophan hydroxylase exists in solution as tetramers . The expressed human pineal tryptophan hydroxylase has a specific activity of 600 nmol/min/mg when measured in the presence of tetrahydrobiopterin and L-tryptophan . The enzyme catalyzes the hydroxylation of tryptophan and phenylalanine at comparable rates . Phosphorylation of the hydroxylase by protein kinase A or calmodulin-dependent kinase II results in the incorporation of 1 mol of phosphate/mol of subunit, but this degree of phosphorylation leads to only a modest (30%) increase in BH(4)-dependent activity when assayed in the presence of 14-3-3 . Rapid scanning ultraviolet spectroscopy has revealed the formation of the transient intermediate compound, 4alpha-hydroxytetrahydrobiopterin, during the hydroxylation of either tryptophan or phenylalanine catalyzed by the recombinant pineal TPH. Biochim Biophys Acta, 1999 Oct 12, 1434(2), 284 - 95 cDNA cloning, overproduction and characterization of rat adrenodoxin reductase; Sagara Y et al.; We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR) . The precursor of rat AdR was predicted to consist of 34 amino-terminal residues of extrapeptide for transport into mitochondria and the following 460 residues of the mature peptide region . The deduced amino acid sequence was 70.8 and 61.8% homologous to those of bovine and human AdRs in the extrapeptide region, respectively, and 88.5% homologous to both the sequences of bovine and human AdRs in the mature peptide region . The predicted mature form of rat AdR was directly expressed in Escherichia coli, using cDNA, and was purified with a yield of 32 mg/l of culture . The purified recombinant rat AdR showed an absorption spectrum characteristic of a flavoprotein with peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm . The extinction coefficient was estimated to be 10.9 mM(-1) cm(-1) at 450 nm . The absorbance ratio at 270 nm/450 nm was 7.1 . From the &theta;(208) value in the circular dichroism spectrum, the alpha-helix content in the rat AdR was calculated to be 30% . In NADPH-cytochrome c reductase activity reconstituted with adrenodoxin (Ad), the apparent K(m) value of rat AdR for NADPH was 0.32 microM, a value significantly lower than that of bovine AdR (1.4 microM) . The rat AdR showed a higher affinity to the heterologous redox partner (bovine Ad, K(m)=9.3 nM) than to the native partner (rat Ad, K(m)=16.7 nM), whereas the affinity of bovine AdR was slightly higher to the native partner (bovine Ad, K(m)=37.1 nM) than to the heterologous partner (rat Ad, K(m)=46.8 nM) . The K(m) values showed a reverse correlation to the difference of pI values between the redox partners . These results indicate that AdR binds to Ad mainly by ionic interaction. Microbiol Immunol, 1999, 43(8), 765 - 71 Protective immune response to 16 kDa immunoreactive recombinant protein encoding the C-terminal VP1 portion of Foot and Mouth Disease Virus type Asia 1; Bayry J et al.; Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C-terminal half of VP1 was expressed in Escherichia coli . As an alternative to the synthetic peptide, this selected C-terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture . A primary dose of 40 microg/animal followed by a booster of the same dose was injected after a 21-day interval . The sera were collected at intervals of 21, 42 and 63 days after the booster . The humoral response to vaccine was monitored by sandwich enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (SNT) . The guinea pig sera showed high titers both in ELISA and SNT, which could be protective . Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post-vaccination in 13 of 15 animals (86%) . The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C-terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization. Microbiol Immunol, 1999, 43(8), 743 - 9 Characterization of the Coxiella burnetti sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase; Nguyen SV et al.; The Coxiella burnetii sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme was cloned by immunological screening of a lambda EMBL3 genomic library prepared from strain Nine Mile DNA and sequenced . The homology of the cloned gene product to the counterpart in Escherichia coli was 54.3%, but the homology of the N-terminal region was only 42% . The gene was expressed in E . coli as an independent unit from its own promoter, producing an immunoreactive protein of about 50 kDa on SDS-PAGE which reacted with antisera from laboratory animals and sera from human patients with acute Q fever . The study results suggest that the C . burnetii E2o enzyme may serve as a potential target antigen for diagnostic assays for Q fever. Biotechniques, 1999 Oct, 27(4), 834 - 6, 838, 840 passim RecA-mediated affinity capture: a method for full-length cDNA cloning; Zhumabayeva B et al.; We describe an improved method for rapid cloning of full-length cDNA from cDNA libraries . This approach is based on the ability of Escherichia coli RecA protein to form a stable nucleoprotein complex with a linear single-stranded DNA probe and homologous sequences in circular double-stranded DNA . Hybridization of RecA-coated biotinylated DNA probes to homologous plasmid DNA creates triple-stranded complexes, which are then captured on streptavidin-coated magnetic beads . Following magnetic separation of the hybrid molecules, the enriched plasmid population is recovered by alkaline treatment, precipitated, resuspended and used to transform bacteria . Typically, many clones can then be recovered by colony hybridization screening of a single plate of the enriched library . We have used this technology to clone full-length and alternatively spliced forms of the human bcl-xL cDNA from a human liver cDNA library. Biotechniques, 1999 Oct, 27(4), 790 - 6 Identification of ribonucleoprotein (RNP)-specific protein interactions using a yeast RNP interaction trap assay (RITA); Bouffard P et al.; We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs) . To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs . hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La) . Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled . Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA . The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones . RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA. Biotechniques, 1999 Oct, 27(4), 734 - 8 Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion; Li F et al.; DpnI can cleave fully methylated parental DNA while leaving hemi-methylated DNA intact . Based on this observation, we developed a rapid site-directed mutagenesis method using uracil-containing, double-stranded (ds)DNA templates and DpnI digestion . A 38% mutation efficiency was achieved by DpnI treatment of the mutagenic strand-extension reaction, and it increased to 70%-91% when uracil-containing dsDNA templates were used . This method compares favorably to the most efficient current methods, but is simpler and does not require the use of single-stranded templates or phage vectors. Mol Cell Biol, 1999 Nov, 19(11), 7828 - 40 The Gal3p-Gal80p-Gal4p transcription switch of yeast: Gal3p destabilizes the Gal80p-Gal4p complex in response to galactose and ATP; Sil AK et al.; The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation of GAL regulon genes . In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p . We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP . We have also found that retention of Gal80p by GSTG4AD (amino acids {aa} 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP . Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose . These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex . The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p . Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881. Mol Cell Biol, 1999 Nov, 19(11), 7461 - 72 Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA; Zebarjadian Y et al.; In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA . Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Psi) synthase . Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Psi synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Psi synthases . To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Psi synthase domain of Cbf5p . Yeast strains expressing these mutated cbf5 genes in a cbf5Delta null background are viable at 25 degrees C but display pronounced cold- and heat-sensitive growth phenotypes . Most of the mutants contain reduced levels of Psi in rRNA at extreme temperatures . Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA . Some of the mutants are temperature sensitive both for growth and for formation of Psi in the rRNA . In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects . There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks Psi in rRNA . A subset of mutations in the Psi synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction . Our results provide additional evidence that Cbf5p is the Psi synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes. J Clin Microbiol, 1999 Nov, 37(11), 3475 - 80 Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay; Ikadai H et al.; A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B . caballi merozoite . A cDNA encoding a 48-kDa protein of B . caballi was cloned and designated BC48 . The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron . Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B . caballi genome . Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa . The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B . caballi 48-kDa protein . Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA) . The ELISA was able to differentiate very clearly between B . caballi-infected horse sera and B . equi-infected horse sera or noninfected normal horse sera . These results suggest that this simple and highly sensitive test might be applicable to the detection of B . caballi-infected horses in the field. J Basic Microbiol, 1999, 39(4), 237 - 42 Transcription level of operon ftsYEX and activity of promoter P1 of rpoH during the cell cycle in Escherichia coli; Gomez-Eichelmann MC et al.; In Escherichia coli, the genes of the main heat-shock proteins are under the control of the product of gene rpoH, protein sigma 32 . The distal promoter P1 of rpoH is located within the terminator of the division operon ftsYEX which could imply some coupling between ftsYEX transcription termination, P1 transcription and cell division . To study the possibility of this coupling, the level of transcription of ftsYEX and the activity of promoter P1 of rpoH were examined in synchronous cultures . Results indicate that transcription of ftsYEX and of rpoH from P1 is continuous, suggesting that ftsYEX transcription termination and P1 activity are not coupled to the cell cycle. EMBO J, 1999 Oct 15, 18(20), 5724 - 34 FtsK-dependent and -independent pathways of Xer site-specific recombination; Recchia GD et al.; Homologous recombination between circular chromosomes generates dimers that cannot be segregated at cell division . Escherichia coli Xer site-specific recombination converts chromosomal and plasmid dimers to monomers . Two recombinases, XerC and XerD, act at the E . coli chromosomal recombination site, dif, and at related sites in plasmids . We demonstrate that Xer recombination at plasmid dif sites occurs efficiently only when FtsK is present and under conditions that allow chromosomal dimer formation, whereas recombination at the plasmid sites cer and psi is independent of these factors . We propose that the chromosome dimer- and FtsK-dependent process that activates Xer recombination at plasmid dif also activates Xer recombination at chromosomal dif . The defects in chromosome segregation that result from mutation of the FtsK C-terminus are attributable to the failure of Xer recombination to resolve chromosome dimers to monomers . Conditions that lead to FtsK-independent Xer recombination support the hypothesis that FtsK acts on Holliday junction Xer recombination intermediates. Microbiol Res, 1999 Sep, 154(2), 179 - 83 The damage-inducible (din) genes of Escherichia coli are induced by various genotoxins in a different way; Oh TJ et al.; The SOS response of Escherichia coli strains carrying the lacZ gene fused to the polB (dinA), dinB or dinD gene were investigated after treatment with several chemical agents and gamma-radiation . The induction levels of polB::lacZ reached levels between 4.0- and 9.0-fold 120 min after treatment with nalidixic acid, H2O2 or ethanol . Pentachlorophenol did not significantly induce any din genes . gamma-Irradiation is not an inducer of polB and ethanol failed to induce dinB::lacZ and dinD::lacZ . Following irradiation with a dose of 10 Gy the responses of dinB and dinD were induced about 2.5-3.0-fold above non-irradiated dinB and dinD . We found that the responses of din::lacZ fusion genes to these genotoxins are induced in a dose-dependent manner . The polB gene showed antagonistic responses to the simultaneous treatment of nalidixic acid and H2O2 or nalidixic acid and ethanol . In addition, dinB and dinD in the presence of both nalidixic acid and H2O2 at the same time showed no synergistic responses. Biochemistry (Mosc), 1999 Sep, 64(9), 1079 - 88 Comparative structural-functional characterization of recombinant and natural adrenodoxin . Interaction with cytochrome P450scc; Lepesheva GI et al.; The conditions for heterologous expression of recombinant bovine adrenodoxin in E . coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium . A highly efficient method for purification of this recombinant ferredoxin from the E . coli cells has been developed . The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria . In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues . The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc . The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin . The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed. Biochemistry (Mosc), 1999 Sep, 64(9), 1021 - 9 Interaction of mutant alkaline phosphatase precursors with membrane phospholipids in vivo and in vitro; Kalinin AE et al.; Positively charged amino acid residues in the N-terminal domain of the signal peptides of secreted proteins are thought to interact with negatively charged anionic phospholipids during the initiation of secretion . To test this hypothesis, substitutions of the uncharged Ala or the negatively charged Glu residue for the positively charged Lys-20 of the N-terminus of the signal peptide of Escherichia coli alkaline phosphatase were introduced using a modified method of oligonucleotide-directed mutagenesis . We found that Lys-20 is involved in the interaction of the signal peptide with anionic phospholipids in vivo and effects the efficiency of insertion of the signal peptide of isolated precursor into model phospholipid membranes in vitro . We also show that the efficiency of signal peptide insertion into the lipid bilayer depends on the fluidity of the bilayer. Mutat Res, 1999 Sep 15, 445(1), 127 - 35 Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue Rat2 cell line; Ryu J et al.; This paper describes the spectrum of mutations induced by 4-nitroquinoline N-oxide (4-NQO) in the lacI target gene of the transgenic Big Blue Rat2 cell line . There are only a few report for the mutational spectrum of 4-NQO in a mammalian system although its biological and genetic effects have been well studied . Big Blue Rat2 cells were treated with 0.03125, 0.0625 or 0.125 microg/ml of 4-NQO, the highest concentration giving 85% survival . Our results indicated that the mutant frequency (MF) induced by 4-NQO was dose-dependent with increases from three- to seven-fold . The DNA sequence analysis of lacI mutants from the control and 4-NQO treatment groups revealed an obvious difference in the spectra of mutations . In spontaneous mutants, transition (60%) mutations, especially G:C-->A:T transition (45%), were most frequent . However, the major type of base substitution after treatment of 4-NQO was transversions (68.8%), especially G:C-->T:A (43.8%), while only 25% of mutants were transitions . These results are consistent with those produced by 4-NQO in other systems and the transgenic assay system will be a powerful tool to postulate more accurately the mechanism of chemical carcinogenesis involved. Mutat Res, 1999 Sep 15, 445(1), 93 - 8 Delayed transfection of DNA after riboflavin mediated photosensitization increases G:C to C:G transversions of supF gene in Escherichia coli mutY strain; Takimoto K et al.; We have previously reported that the majority of base substitution mutations of the Escherichia coli supF gene induced by riboflavin mediated photosensitization were G:C to C:G changes, in addition to G:C to T:A changes which were probably caused by 8-hydroxyguanine (oh(8)Gua), in wild type and mutM mutator mutant strains . This implies that lesions other than oh(8)Gua are produced by riboflavin-photosensitization . G:C to C:G base substitutions have been found in the mutations induced by ionizing radiation and reactive oxygen species, as well as spontaneous mutation . To characterize the G:C to C:G mutation, riboflavin- photosensitized plasmid DNA carrying the supF gene was left at room temperature for 5 h in the dark before transfection . The delayed transfection gave a mutational spectrum different from that for immediate transfection . G:C to C:G transversions significantly increased in mutY mutator strain, in which the transversion was not detected in the immediate transfection . Lesions causing G:C to C:G changes increased during 5-h holding after photosensitization and MutY protein presumably takes part in this type of base change mutation. Mutat Res, 1999 Aug 18, 444(2), 405 - 11 Impact of dimethyl sulfoxide and examples of combined genotoxicity in the SOS chromotest; Gebel T et al.; The bacterial SOS chromotest with Escherichia coli PQ37 was used for the assessment of genotoxicity of combined xenobiotic treatments . The modulation of test compound genotoxicity by dimethyl sulfoxide (DMSO), a common solvent for test compounds, was assessed as well . It was shown that DMSO modulated SOS chromotest genotoxicity of several xenobiotics: in comparison to test compound dissolution in water, the commonly used addition of 3.2% (v/v) DMSO as solvent lead to a significant increase in the genotoxicity of K(2)RhCl(5) and beta-propiolactone (BPL) . However, the effects of cisplatin decreased significantly when DMSO was added . Thus, albeit DMSO is not genotoxic in this test itself, it can interfere with SOS chromotest responses . Further experiments were performed in the absence of DMSO . BPL and cisplatin in combination showed an over-additive synergism in SOS genotoxicity as well as K(2)RhCl(5) and cisplatin did . Addition of Pd(NH(3))(4)Cl(2) and NaAsO(2), which are non-genotoxic in the SOS chromotest, did not enhance the K(2)RhCl(5)- or BPL-mediated SOS sfiA induction . Nevertheless, at the highest subcytotoxic dose of NaAsO(2) tested (200 microM), a slight yet significant suppression of BPL-mediated SOS genotoxicity was observed . These results confirm that the SOS chromotest is a useful tool for the rapid evaluation of the combined genotoxicity of compound mixtures . However, the use of DMSO as test solvent has to be taken with caution. Gene, 1999 Sep 17, 237(2), 333 - 42 pET-prof, a plasmid for high-level expression of recombinant peptides fused to a birch profilin-derived hexadecapeptide tag: a system for the detection and presentation of recombinant antigens; Pandjaitan B et al.; We have previously identified a birch pollen profilin hexadecapeptide (Bp36/51), which was recognized by a monoclonal antibody (moAb 4A6) with high affinity . Here, we report the construction of a T7 RNA polymerase-driven high-level plasmid expression system, pET-prof, capable of producing proteins and peptides containing the Bp36/51 birch profilin-derived peptide fused to their N-terminus . As examples, the cDNAs coding for two major timothy grass (Phleum pratense) pollen allergens, Phl p 2 and Phl p 6, as well as for an alder (Alnus glutinosa) pollen allergen, Aln g 4, were overexpressed in Escherichia coli as BP36/51-tagged proteins . All three recombinant allergens were readily detected in nitrocellulose-blotted E . coli extracts by the Bp36/51-specific moAb 4A6 . We demonstrate comparable IgE recognition of Bp36/51-tagged and untagged recombinant allergens by immunoblotting . A sandwich ELISA was developed using plate-bound moAb 4A6 to immobilize and present Bp36/51-tagged recombinant allergens to IgE antibodies of allergic patients . Using immunoelectronmicroscopy, we demonstrate that even under harsh fixation conditions, tagged allergens can be localized simultaneously in situ by moAb 4A6 and allergen-specific antisera . We suggest the use of the pET-prof system for the high-level expression of Bp36/51-tagged polypeptides that can be rapidly detected in total protein extracts, immunolocalized in situ, immobilized and presented to other antigen-specific antibodies (e.g . IgE), even when they occur in minute concentrations. J Biol Chem, 1999 Oct 22, 274(43), 31094 - 101 The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase; Revington M et al.; In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation . The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation . b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence . Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure . A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants . These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface. J Biol Chem, 1999 Oct 22, 274(43), 31034 - 8 The role of the Escherichia coli mug protein in the removal of uracil and 3,N(4)-ethenocytosine from DNA; Lutsenko E et al.; The human thymine-DNA glycosylase has a sequence homolog in Escherichia coli that is described to excise uracils from U.G mismatches (Gallinari, P., and Jiricny, J . (1996) Nature 383, 735-738) and is named mismatched uracil glycosylase (Mug) . It has also been described to remove 3,N(4)-ethenocytosine (epsilonC) from epsilonC.G mismatches (Saparbaev, M., and Laval, J . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 8508-8513) . We used a mug mutant to clarify the role of this protein in DNA repair and mutation avoidance . We find that inactivation of mug has no effect on C to T or 5-methylcytosine to T mutations in E . coli and that this contrasts with the effect of ung defect on C to T mutations and of vsr defect on 5-methylcytosine to T mutations . Even under conditions where it is overproduced in cells, Mug has little effect on the frequency of C to T mutations . Because uracil-DNA glycosylase (Ung) and Vsr are known to repair U.G and T.G mismatches, respectively, we conclude that Mug does not repair U.G or T.G mismatches in vivo . A defect in mug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations due to endogenous damage to DNA in growing E . coli . Cell-free extracts from mug(+) ung cells show very little ability to remove uracil from DNA, but can excise epsilonC . The latter activity is missing in extracts from mug cells, suggesting that Mug may be the only enzyme in E . coli that can remove this mutagenic adduct . Thus, the principal role of Mug in E . coli may be to help repair damage to DNA caused by exogenous chemical agents such as chloroacetaldehyde. J Biol Chem, 1999 Oct 22, 274(43), 31020 - 4 Increases in acidic phospholipid contents specifically restore protein translocation in a cold-sensitive secA or secG null mutant; Suzuki H et al.; Both the secAcsR11 and DeltasecG::kan mutations cause cold-sensitive growth, although the growth defect due to the latter mutation occurs in a strain-specific manner . Overexpression of pgsA encoding phosphatidylglycerophosphate synthase suppresses the growth defects of the two mutants . We investigated the mechanism underlying the pgsA-dependent suppression of the two mutations using purified mutant SecA and inverted membrane vesicles (IMVs) prepared from pgsA-overexpressing cells . The acidic phospholipid content increased by about 10% upon pgsA overexpression . This increase resulted in the stimulation of proOmpA translocation only when mutant SecA or SecG-depleted IMVs were used . The translocation-coupled ATPase activity of SecA was significantly defective with the mutant SecA or SecG-depleted IMVs, but it recovered to a near normal level when the acidic phospholipid level was increased . The stimulation of ATPase activity was observed only at low temperature . The steady-state level of membrane-inserted SecA was low with the mutant SecA or SecG-depleted IMVs, and it decreased further upon the increase in the acidic phospholipid content . However, the level of SecA insertion markedly increased upon the inhibition of SecA deinsertion by the addition of beta,gamma-imido adenosine 5'-triphosphate (AMP-PNP), especially with IMVs containing increased levels of acidic phospholipids . These results indicate that the increase in the level of acidic phospholipids stimulates the SecA cycle in the two mutants by facilitating both the insertion and deinsertion of SecA. J Biol Chem, 1999 Oct 22, 274(43), 30995 - 9 Characterization of the LolA-LolB system as the general lipoprotein localization mechanism of Escherichia coli; Yokota N et al.; The major outer membrane lipoprotein (Lpp) of Escherichia coli requires LolA for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane . We examined the significance of the LolA-LolB system as to the outer membrane localization of other lipoproteins . All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of LolA . Some lipoproteins were released in the absence of externally added LolA, albeit at a slower rate and to a lesser extent . This LolA-independent release was also strictly dependent on the outer membrane sorting signal . A lipoprotein-LolA complex was formed when the release took place in the presence of LolA, whereas lipoproteins released in the absence of LolA existed as heterogeneous complexes, suggesting that the release and the formation of a complex with LolA are distinct events . The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-LolA complex, occurred with a trace amount of LolA, and therefore was extremely efficient . The LolA-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of LolA were nonspecifically and inefficiently incorporated into the membrane . The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane . From these results, we conclude that lipoproteins in E . coli generally utilize the LolA-LolB system for efficient release from the inner membrane and specific localization to the outer membrane. J Biol Chem, 1999 Oct 22, 274(43), 30950 - 6 Conformational change in the pheromone-binding protein from Bombyx mori induced by pH and by interaction with membranes; Wojtasek H et al.; The pheromone-binding protein (PBP) from Bombyx mori was expressed in Escherichia coli periplasm . It specifically bound radiolabeled bombykol, the natural pheromone for this species . It appeared as a single band both in native and SDS-polyacrylamide gel electrophoresis and was also homogeneous in most chromatographic systems . However, in ion-exchange chromatography, multiple forms sometimes appeared . Attempts to separate them revealed that they could be converted into one another . Analysis of the protein by circular dichroism and fluorescence spectroscopy demonstrated that its tertiary structure was sensitive to pH changes and that a dramatic conformational transition occurred between pH 6.0 and 5.0 . This high sensitivity to pH contrasted markedly with its thermal stability and resistance to denaturation by urea . There was also no significant change in CD spectra in the presence of the pheromone . The native protein isolated from male antennae displayed the same changes in its spectroscopic properties as the recombinant material, demonstrating that this phenomenon is not an artifact arising from the expression system . This conformational transition was reproduced by interaction of the protein with anionic (but not neutral) phospholipid vesicles . Unfolding of the PBP structure triggered by membranes suggests a plausible mechanism for ligand release upon interaction of the PBP-pheromone complex with the surface of olfactory neurons . This pH-linked structural flexibility also explains the heterogeneity reported previously for B . mori PBP and other members of this class of proteins. J Biol Chem, 1999 Oct 22, 274(43), 30849 - 57 Glycine insertion in the hinge region of lactose repressor protein alters DNA binding; Falcon CM et al.; Amino acid alterations were designed at the C terminus of the hinge segment (amino acids approximately 51-59) that links two functional domains within lactose repressor protein (LacI) . Gly was introduced between Gly(58) and Lys(59) to generate Gly(58+1); Gln(60) was changed to Gly or Pro, and up to three additional glycines were inserted following Gln(60) --> Gly . All mutant proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns . In contrast, significant differences were observed in DNA binding properties . Gly(58+1) exhibited a decrease of approximately 100-fold in affinity for O(1) operator, and sequential Gly insertion C-terminal to Gln(60) --> Gly resulted in progressively decreased affinity for O(1) operator, approaching nonspecific levels for insertion of >/=2 glycines . Where sufficient affinity for O(1) operator existed, decreased binding to O(1) in the presence of inducer indicated no disruption in the allosteric response for these proteins . Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose repressor protein for its wild-type target sequence. J Biol Chem, 1999 Oct 22, 274(43), 30534 - 9 Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ; Suh WC et al.; Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding . Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70 . We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ . Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis . In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK. J Biol Chem, 1999 Oct 22, 274(43), 30451 - 8 Cloning and characterization of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family; Knoops B et al.; Using two-dimensional electrophoresis, we have recently identified in human bronchoalveolar lavage fluid a novel protein, termed B166, with a molecular mass of 17 kDa . Here, we report the cloning of human and rat cDNAs encoding B166, which has been renamed AOEB166 for antioxidant enzyme B166 . Indeed, the deduced amino acid sequence reveals that AOEB166 represents a new mammalian subfamily of AhpC/TSA peroxiredoxin antioxidant enzymes . Human AOEB166 shares 63% similarity with Escherichia coli AhpC22 alkyl hydroperoxide reductase and 66% similarity with a recently identified Saccharomyces cerevisiae alkyl hydroperoxide reductase/thioredoxin peroxidase . Moreover, recombinant AOEB166 expressed in E . coli exhibits a peroxidase activity, and an antioxidant activity comparable with that of catalase was demonstrated with the glutamine synthetase protection assay against dithiothreitol/Fe3+/O(2) oxidation . The analysis of AOEB166 mRNA distribution in 30 different human tissues and in 10 cell lines shows that the gene is widely expressed in the body . Of interest, the analysis of N- and C-terminal domains of both human and rat AOEB166 reveals amino acid sequences presenting features of mitochondrial and peroxisomal targeting sequences . Furthermore, human AOEB166 expressed as a fusion protein with GFP in HepG2 cell line is sorted to these organelles . Finally, acute inflammation induced in rat lung by lipopolysaccharide is associated with an increase of AOEB166 mRNA levels in lung, suggesting a protective role for AOEB166 in oxidative and inflammatory processes. J Biol Chem, 1999 Oct 22, 274(43), 30447 - 50 Characterization of an 8-oxoguanine DNA glycosylase from Methanococcus jannaschii; Gogos A et al.; A thermostable 8-oxoguanine (oxoG) DNA glycosylase from Methanococcus jannaschii has been expressed in Escherichia coli, purified, and characterized . The enzyme, which has been named mjOgg, belongs to the same diverse DNA glycosylase superfamily as the 8-oxoguanine DNA glycosylases from yeast (yOgg1) and human (hOgg1) but is substantially different in sequence . In addition, unlike its eukaryotic counterparts, which have a strong preference for oxoG.C base pairs, mjOgg has little specificity for the base opposite oxoG . mjOgg has both DNA glycosylase and DNA lyase (beta-elimination) activity, and the combined glycosylase/lyase activity occurs at a rate comparable with the glycosylase activity alone . Mutation of Lys-129, analogous to Lys-241 of yOgg1, abolishes glycosylase activity. Genes Dev, 1999 Oct 1, 13(19), 2594 - 603 Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase; Coburn GA et al.; The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs . We have developed a method for reconstituting a minimal degradosome from purified proteins . Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components . Moreover, ATP-dependent degradation of the malEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells . The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required . Rather, Rne coordinates the activation of RhlB dependent on a 3' single-stranded extension on RNA substrates . A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli. Biochemistry, 1999 Oct 12, 38(41), 13725 - 35 tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase; Bovee ML et al.; Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs . Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments . Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-{N-(L-histidyl)sulfamoyl}adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM) . By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA . Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS {Yan, W., Augustine, J., and Francklyn, C . (1996) Biochemistry 35, 6559}, which possesses a single amino acid change in the C-terminal anticodon binding domain . Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe) . Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions . Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA. Biochemistry, 1999 Oct 12, 38(41), 13530 - 41 Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex; Eubanks S et al.; Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor . To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented . The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor . Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex . Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10 . Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein. Biochemistry, 1999 Oct 12, 38(41), 13523 - 9 Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli; Quick M et al.; The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters . To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis . Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type) . The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered . Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions . Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions . These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport . The observations confirm the functional importance of TM II described in earlier studies {M . Quick and H . Jung (1997) Biochemistry 36, 4631-4636} . In contrast to Arg40, Arg117 is apparently not important for function of the mature protein . The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane . However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments . These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i . e., by stabilizing a folding intermediate. Biochemistry, 1999 Oct 12, 38(41), 13502 - 11 Probing the two-gate mechanism of DNA gyrase using cysteine cross-linking; Williams NL et al.; Cross-linking a pair of novel cysteine residues on either side of the bottom dimer interface of DNA gyrase blocks catalytic supercoiling . Limited strand passage is allowed, but release of the transported DNA segment (T segment) via opening of the bottom dimer interface is prevented . In contrast, ATP-independent relaxation of negatively supercoiled DNA is completely abolished, suggesting that T-segment entry via the bottom gate is blocked . These findings support a two-gate model for supercoiling by DNA gyrase and suggest that relaxation by gyrase is the reverse of supercoiling . Cross-linking a truncated version of gyrase (A64(2)B2), which lacks the DNA wrapping domains, does not block ATP-dependent relaxation . This indicates that passage of DNA through the bottom dimer interface is not essential for this reaction . The mechanistic implications of these results are discussed. DNA Seq, 1998, 9(3), 183 - 8 The tdcE gene in Escherichia coli strain W3110 is separated from the rest of the tdc operon by insertion of IS5 elements; Hesslinger C et al.; Unlike other Escherichia coli K-12 strains, W3110 contains multiple copies of the insertion sequence IS5 . Some of these IS5 elements have been involved in tandem duplication of a portion of the chromosome which includes, amonst others, the tdcABC-DEFG operon genes . The nucleotide sequence and insertion site of one of these elements, IS5P, was determined . It was shown that IS5P has inserted within the coding sequence of the tdcA gene and is flanked, not by the remaining portion of the tdcA gene, but by the extreme 3' end of the tdcD gene . In other E . coli K-12 strains the tdcD gene and three other genes, tdcE, tdcF and tdcG, all form part of the tdc operon . Our results demonstrate that during the duplication event the tdcABCgenes have been amplified and separated from the remaining genes tdcE, tdcF and tdcG of the operon, which are each present in single copy. Curr Eye Res, 1999 Oct, 19(4), 313 - 22 Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers; Conley CA et al.; PURPOSE . To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy . METHODS . Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits . Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue . RESULTS . Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle . Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle . The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits . CONCLUSIONS . The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism. J Mol Biol, 1999 Sep 3, 291(5), 1191 - 206 An NMR investigation of solution aggregation reactions preceding the misassembly of acid-denatured cold shock protein A into fibrils; Alexandrescu AT et al.; At pH 2.0, acid-denatured CspA undergoes a slow self-assembly process, which results in the formation of insoluble fibrils . 1H-15N HSQC, 3D HSQC-NOESY, and 15N T2 NMR experiments have been used to characterize the soluble components of this reaction . The kinetics of self-assembly show a lag pha