J Biol Chem, 2001 Dec 14, 276(50), 46770 - 8 Epub 2001 Sep 27. Dual mode recognition of two isoacceptor tRNAs by mammalian mitochondrial seryl-tRNA synthetase; Shimada N et al.; Animal mitochondrial translation systems contain two serine tRNAs, corresponding to the codons AGY (Y = U and C) and UCN (N = U, C, A, and G), each possessing an unusual secondary structure; tRNA(GCU)(Ser) (for AGY) lacks the entire D arm, whereas tRNA(UGA)(Ser) (for UCN) has an unusual cloverleaf configuration . We previously demonstrated that a single bovine mitochondrial seryl-tRNA synthetase (mt SerRS) recognizes these topologically distinct isoacceptors having no common sequence or structure . Recombinant mt SerRS clearly footprinted at the TPsiC loop of each isoacceptor, and kinetic studies revealed that mt SerRS specifically recognized the TPsiC loop sequence in each isoacceptor . However, in the case of tRNA(UGA)(Ser), TPsiC loop-D loop interaction was further required for recognition, suggesting that mt SerRS recognizes the two substrates by distinct mechanisms . mt SerRS could slightly but significantly misacylate mitochondrial tRNA(Gln), which has the same TPsiC loop sequence as tRNA(UGA)(Ser), implying that the fidelity of mitochondrial translation is maintained by kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases. J Biol Chem, 2001 Nov 30, 276(48), 44481 - 7 Epub 2001 Sep 27. Mimic of photocycle by a protein folding reaction in photoactive yellow protein; Lee BC et al.; The blue light receptor photoactive yellow protein (PYP) displays rhodopsin-like photochemistry based on the trans to cis photoisomerization of its p-coumaric acid chromophore . Here, we report that protein refolding from the acid-denatured state of PYP mimics the last photocycle transition in PYP . This implies a direct link between transient protein unfolding and photosensory signal transduction . We utilize this link to study general issues in protein folding . Chromophore trans to cis photoisomerization in the acid-denatured state strongly decelerates refolding, and converts the pH dependence of the barrier for refolding from linear to nonlinear . We propose transition state movement to explain this phenomenon . The cis chromophore significantly stabilizes the acid-denatured state, but acidification of PYP results in the accumulation of the acid-denatured state containing a trans chromophore . This provides a clear example of kinetic control in a protein unfolding reaction . These results demonstrate the power of PYP as a light-triggered model system to study protein folding. FEBS Lett, 2001 Sep 21, 505(3), 426 - 30 Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site; Kramer RA et al.; Escherichia coli outer membrane protease OmpT has previously been classified as a serine protease with Ser(99) and His(212) as active site residues . The recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed . Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants . Our results support the involvement of a nucleophilic water molecule that is activated by the Asp(210)/His(212) catalytic dyad . Activity is also strongly dependent on Asp(83) and Asp(85) . Both may function in binding of the water molecule and/or oxyanion stabilization . The proposed mechanism implies a novel proteolytic catalytic site. Plant J, 2001 Sep, 27(5), 373 - 82 Localization and targeting of the VP14 epoxy-carotenoid dioxygenase to chloroplast membranes; Tan BC et al.; Abscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges . ABA is synthesized via an oxidative cleavage of 9-cis epoxy-carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway . Vp14 of maize encodes an epoxy-carotenoid dioxygenase that is soluble when expressed in E . coli . An important goal has been to determine how the soluble VP14 protein is targeted to epoxy-carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids . Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma-targeting domain . The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes . Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N-terminal sequence . A putative amphipathic alpha-helix within this region is essential, but not sufficient, for the membrane targeting . Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S-transferase failed to associate with the thylakoid membrane . The membrane-bound VP14 was partially resistant to chaotropic washes such as 0.1 M Na2CO3 (pH 11.5) and 6 M urea . Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane. J Pept Res, 2001 Sep, 58(3), 221 - 8 Immunological evaluation of Escherichia coli-derived hepatitis C virus second envelope protein (E2) variants; Duenas-Carrera S et al.; Two variants of the hepatitis C virus (HCV) E2 envelope protein, lacking the C-terminal domain and comprising amino acids 458-650 (E2A) and 382-605 (E2C), respectively, were efficiently produced in BL21 (DE3) Escherichia coli cells . E2A and E2C were used to immunize mice . The E2C variant induced the maximal mean antibody titer . Anti-E2C mouse sera reacted mainly with E2 synthetic peptides covering the 70 amino acid N-terminal region of the E2 protein . Moreover, a panel of anti-HCV positive human sera recognized only the E2C protein (28.2%) and the synthetic peptide covering the HVR-1 of the E2 protein (23.1%) . These data indicate the existence of an immunologically relevant region in the HVR-1 of the HCV E2 protein. J Mol Biol, 2001 Sep 28, 312(4), 833 - 47 Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle alpha-tropomyosin in an engineered chimeric protein; Greenfield NJ et al.; Tropomyosin is an alpha-helical coiled-coil protein that aligns head-to-tail along the length of the actin filament and regulates its function . The solution structure of the functionally important N terminus of a short 247-residue non-muscle tropomyosin was determined in an engineered chimeric protein, GlyTM1bZip, consisting of the first 19 residues of rat short alpha-tropomyosin and the last 18 residues of the GCN4 leucine zipper . A gene encoding GlyTM1bZip was synthesized, cloned and expressed in Escherichia coli . Triple resonance NMR spectra were analyzed with the program AutoAssign to assign its backbone resonances . Multidimensional nuclear Overhauser effect spectra, X-filtered spectra and (3)J(H(N)-H(alpha)) scalar coupling were analyzed using AutoStructure . This is the first application of this new program to determine the three-dimensional structure of a symmetric homodimer and a structure not previously reported . Residues 7-35 in GlyTM1bZip form a coiled coil, but neither end is helical . Heteronuclear (15)N-(1)H nuclear Overhauser effect data showed that the non-helical N-terminal residues are flexible . The (13)C' chemical shifts of the coiled-coil backbone carbonyl groups in GlyTM1bZip showed a previously unreported periodicity, where resonances arising from residues at the coiled-coil interface in a and d positions of the heptad repeat were displaced relatively upfield and those arising from residues in c positions were displaced relatively downfield . Heteronuclear single quantum coherence spectra, collected as a function of temperature, showed that cross-peaks arising from the alpha-helical backbone and side-chains at the coiled-coil interface broadened or shifted with T(M) values approximately 20 degrees C lower than the loss of alpha-helix measured by circular dichroism, suggesting the presence of a folding intermediate . The side-chain of Ile14, a residue essential for binding interactions, exhibited multiple conformations . The conformational flexibility of the N termini of short tropomyosins may be important for their binding specificity . J Mol Biol, 2001 Sep 28, 312(4), 625 - 35 Leucine-regulated self-association of leucine-responsive regulatory protein (Lrp) from Escherichia coli; Chen S et al.; Lrp is a global regulatory protein in Escherichia coli that activates expression of more than a dozen operons and represses expression of another dozen . For some operons, exogenous leucine reduces the extent of Lrp action, for others it potentiates the effect of Lrp, and for yet other operons it has no effect . In an effort to understand how leucine affects Lrp-mediated expression, we examined Lrp self-association and the effect of leucine on self-association using light scattering, chemical cross-linking, and analytical ultracentrifugation . The following results were obtained . (i) Lrp self-associates to a hexadecamer and octamer with the predominant species being hexadecamer at microM concentrations . (ii) Lrp undergoes a leucine-induced dissociation of hexadecamer to octamer . (iii) A mutant Lrp lacking 11 amino acid residues at the C terminus does not form higher-order oligomers, suggesting that the C terminus is involved in subunit association . (iv) At nM concentrations, Lrp dissociates to a dimer . It is proposed that leucine regulates the equilibrium between Lrp oligomers and thus Lrp occupancy of sites within different operons, leading to diverse regulatory patterns . Plant Mol Biol, 2001 Aug, 46(6), 705 - 15 Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex; Drea SC et al.; We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species . Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal . Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity . PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma . In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A . thaliana . Expression of ptlpd2 was investigated by introducing ptlpd2 promoter-beta-glucuronidase (GUS) gene fusions into Nicotiana tabacum . GUS expression was observed in seeds, flowers, root tips and young leaves . GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves . Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence . Database searches on ptlpd2 sequences identified a second A . thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P . sativum. Plant Mol Biol, 2001 Aug, 46(6), 651 - 60 cDNA cloning of two isoforms of ornithine carbamoyltransferase from Canavalia lineata leaves and the effect of site-directed mutagenesis of the carbamoyl phosphate binding site; Lee Y et al.; The immunoscreening method was used to isolate cDNAs of 1323 bp (ClOCT1) and 1433 bp (ClOCT2) encoding two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) from the cDNA expression library of Canavalia lineata leaves constructed in a lambdaZAP Express vector . ClOCT1 and ClOCT2 encode 359 and 369 amino acids, respectively . The N-terminals of deduced amino acid sequences of the two cDNAs showed typical features of the transit peptide of chloroplast targeting proteins . The ornithine-binding domain (FMHCLP) and catalytic domain (HPXQ) of ClOCT1 and ClOCT2 and the carbamoyl phosphate (CP)-binding site of ClOCT1 (SMRTR) are identical to OCTs of other plant species, pea and Arabidopsis thaliana . However, the CP-binding site sequence of ClOCT2, SLRTH, has not yet been reported . Both ClOCT1 and ClOCT2 cDNAs were expressed in Escherichia coli BL21 (DE3) by using expression vector pET30a . Recombinant ClOCT1 protein showed 14 times higher ornithine-dependent OCT activity than canaline-dependent OCT activity . In contrast, recombinant ClOCT2 protein showed 13 times higher canaline-dependent OCT activity than ornithine-dependent OCT activity . The two amino acids of the CP-binding site of ClOCT2 (SLRTH) were combinatorially changed to those of the CP-binding site of ClOCT1 (SMRTR) by site-directed mutagenesis . When Leu-118 of ClOCT2 was changed to Met, ornithine-dependent activity was increased significantly . It is assumed that the substrate specificity of ClOCT1 or ClOCT2 proteins partially depends on the amino acid sequence of the CP-binding site. J Clin Microbiol, 2001 Oct, 39(10), 3712 - 7 PCR for specific detection of H7 flagellar variant of fliC among extraintestinal pathogenic Escherichia coli; Johnson JR et al.; A newly developed PCR-based assay for the H7 variant of the Escherichia coli flagellin gene, fliC, was 100% sensitive and specific in comparison with serology and probe hybridization . It revealed broad conservation of the H7 fliC variant among phylogenetically diverse lineages of extraintestinal pathogenic E . coli (ExPEC) and superseded serotyping for certain isolates with ambiguous or non-H7 serotyping results . The H7 primers functioned well when incorporated into a multiplex PCR assay for diverse virulence-associated genes of ExPEC. J Biol Chem, 2001 Dec 7, 276(49), 45694 - 703 Epub 2001 Sep 26. A novel carbamoyl-phosphate synthetase from Aquifex aeolicus; Ahuja A et al.; Aquifex aeolicus, an extreme hyperthermophile, has neither a full-length carbamoyl-phosphate synthetase (CPSase) resembling the enzyme found in all mesophilic organisms nor a carbamate kinase-like CPSase such as those present in several hyperthermophilic archaea . However, the genome has open reading frames encoding putative proteins that are homologous to the major CPSase domains . The glutaminase, CPS.A, and CPS.B homologs from A . aeolicus were cloned, overexpressed in Escherichia coli, and purified to homogeneity . The isolated proteins could catalyze several partial reactions but not the overall synthesis of carbamoyl phosphate . However, a stable 124-kDa complex could be reconstituted from stoichiometric amounts of CPS.A and CPS.B proteins that synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia . The inclusion of the glutaminase subunit resulted in the formation of a 171-kDa complex that could utilize glutamine as the nitrogen-donating substrate, although the catalytic efficiency was significantly compromised . Molecular modeling, using E . coli CPSase as a template, showed that the enzyme has a similar structural organization and interdomain interfaces and that all of the residues known to be essential for function are conserved and properly positioned . A steady state kinetic study at 78 degrees C indicated that although the substrate affinity was similar for bicarbonate, ammonia, and glutamine, the K(m) for ATP was appreciably higher than that of any known CPSase . The A . aeolicus complex, with a split gene encoding the major synthetase domains and relatively inefficient coupling of amidotransferase and synthetase functions, may be more closely related to the ancestral precursor of contemporary mesophilic CPSases. EMBO J, 2001 Oct 1, 20(19), 5521 - 31 Structure and function of the N-terminal 40 kDa fragment of human PMS2: a monomeric GHL ATPase; Guarne A et al.; Human MutLalpha, a heterodimer of hMLH1 and hPMS2, is essential for DNA mismatch repair . Inactivation of the hmlh1 or hpms2 genes by mutation or epigenesis causes genomic instability and a predisposition to hereditary non-polyposis cancer . We report here the X-ray crystal structures of the conserved N-terminal 40 kDa fragment of hPMS2, NhPMS2, and its complexes with ATPgammaS and ADP at 1.95, 2.7 and 2.7 A resolution, respectively . The NhPMS2 structures closely resemble the ATPase fragment of Escherichia coli MutL, which coordinates protein-protein interactions in mismatch repair by undergoing structural transformation upon binding of ATP . Unlike the E.coli MutL, whose ATPase activity requires protein dimerization, the monomeric form of NhPMS2 is active both in ATP hydrolysis and DNA binding . NhPMS2 is the first example of a GHL ATPase active as a monomer, suggesting that its activity may be modulated by hMLH1 in MutLalpha, and vice versa . The potential heterodimer interface revealed by crystallography provides a mutagenesis target for functional studies of MutLalpha. EMBO J, 2001 Oct 1, 20(19), 5503 - 12 Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA; Diges CM et al.; Escherichia coli DbpA is a member of the DEAD/H family of proteins which has been shown to have robust ATPase activity only in the presence of a specific region of 23S rRNA . A series of bimolecular RNA substrates were designed based on this activating region of rRNA and used to demonstrate that DbpA is also a non-processive, sequence-specific RNA helicase . The high affinity of DbpA for the RNA substrates allowed both single and multiple turnover helicase assays to be performed . Helicase activity of DbpA is dependent on the presence of ATP or dATP, the sequence of the loop of hairpin 92 of 23S rRNA and the position of the substrate helix with respect to hairpin 92 . This work indicates that certain RNA helicases require particular RNA structures in order for optimal unwinding activity to be observed. EMBO J, 2001 Oct 1, 20(19), 5392 - 9 Repression of deoP2 in Escherichia coli by CytR: conversion of a transcription activator into a repressor; Shin M et al.; In the deoP2 promoter of Escherichia coli, a transcription activator, cAMP-CRP, binds at two sites, centered at -41.5 and -93.5 from the start site of transcription, while a repressor, CytR, binds to a space between the two cAMP-CRP complexes . The mechanisms for the cAMP-CRP-mediated transcription activation and CytR-mediated transcription repression were investigated in vitro using purified components . We classified the deoP2 promoter as a class II cAMP-CRP-dependent promoter, primarily by the action of cAMP-CRP at the downstream site . Interestingly, we also found that deoP2 carries an "UP-element" immediately upstream of the downstream cAMP-CRP site . The UP-element overlaps with the DNA site for CytR . However, it was observed that CytR functions with the RNA polymerase devoid of the C-terminal domain of the alpha-subunit as well as with intact RNA polymerase . The mechanism of repression by CytR proposed in this study is that the cAMP-CRP bound at -41.5 undergoes an allosteric change upon direct interaction with CytR such that it no longer maintains a productive interaction with the N-terminal domain of alpha, but instead acts as a repressor to interfere with RNA polymerase acting on deoP2. Vet Microbiol, 2001 Nov 26, 83(3), 275 - 86 Organisation and in vitro expression of esp genes of the LEE (locus of enterocyte effacement) of bovine enteropathogenic and enterohemorrhagic Escherichia coli; Goffaux F et al.; Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli infections are characterised by the formation of attaching and effacing (AE) lesions on intestinal epithelial cells . Secretion of extracellular proteins (EspA, EspB, and EspD) via a type III secretion apparatus is necessary for the formation of the AE lesions by human EPEC . In this study, we show that bovine EPEC and EHEC are also able to secrete polypeptides homologous to the already described Esp proteins, most probably via a type III secretion system . Bovine EPEC and EHEC strains present two different secretion profiles of Esp proteins which correlate to the pathotypes of the esp genes as determined by PCR . We also demonstrate that genes encoding secreted proteins, present in the LEE of two bovine strains, are organised in the same way as in the human EPEC strain E2348/69. J Biochem (Tokyo), 2001 Oct, 130(4), 527 - 33 Functions and ATP-binding responses of the twelve histidine residues in the TF1-ATPase beta subunit; Tozawa K et al.; The C2 proton signals of all (twelve) histidine residues of the TF1 beta subunit in the 1H-NMR spectrum have been identified and assigned by means of pH change experiments and site-directed substitution of histidines by glutamines . pH and ligand titration experiments were carried out for these signals . Furthermore, the ATPase activity of the reconstituted alpha3beta3gamma complex was examined for the twelve mutant beta subunits . Two of three conserved histidines, namely, His-119 and 324, were found to be important for expression of the ATPase activity . The former fixes the N-terminal domain to the central domain . His-324 is involved in the formation of the interface essential for the alpha3beta3gamma complex assembly . The other conserved residue, His-363, showed a very low pK(a), suggesting that it is involved in the tertiary structure formation . On the binding of a nucleotide, only the signals of His-173, 179, 200, and 324 shifted . These histidines are located in the hinge region, and its proximity, of the beta subunit . This observation provided further support for the conformational change of the beta monomer from the open to the closed form on the binding of a nucleotide proposed by us {Yagi et al . (1999) Biophys . J . 77, 2175-2183} . This conformational change should be one of the essential driving forces in the rotation of the alpha3beta3gamma complex. J Biochem (Tokyo), 2001 Oct, 130(4), 471 - 4 Accelerated refolding of subtilisin BPN' by tertiary-structure-forming mutants of its propeptide; Kojima S et al.; The propeptide of subtilisin BPN', which functions as an intramolecular chaperone and a temporary inhibitor of subtilisin, is unique in that it acquires its three-dimensional structure by formation of a complex with the cognate protease . We previously showed that the successive amino acid replacements Ala47-->Phe, Gly13-->Ile, and Val65-->Ile in the propeptide to increase its hydrophobicity resulted in formation of a tertiary structure, accompanied by increased ability to bind to the protease and increased resistance to proteolysis . In this study, we examined the effects of these tertiary-structure-forming mutations on the intramolecular chaperone activity of the propeptide . The successive amino acid replacements mentioned above were introduced into pro-subtilisin*, possessing a Ser221-->Ala mutation in the catalytic residue . Refolding experiments were started by rapid dilution of the denatured pro-subtilisin*, and formation of tertiary structure in subtilisin was monitored kinetically by increase in tryptophan fluorescence . The wild-type pro-subtilisin* was found to refold with a rate constant of 4.8 x 10(-3) s(-1) in the equation describing an intramolecular process . The Ala47-->Phe replacement in the propeptide resulted in a 1.2-fold increase in the rate constant of subtilisin refolding . When the additional replacement Gly13-->Ile was introduced, refolding of subtilisin was substantially accelerated, and its kinetics could be fitted to a double exponential process composed of a fast phase with a rate constant of 2.1 x 10(-2) s(-1) and a slow phase with a rate constant of 4.5 x 10(-3) s(-1) . The rate constant of the fast phase was increased slightly by a further replacement, Val65-->Ile . Since the slow phase is considered to correspond to proline isomerization, we concluded that tertiary-structure-forming mutations in the propeptide produce positive effects on its intramolecular chaperone activity through acceleration of the propeptide-induced formation of the tertiary structure of subtilisin BPN'. Genome Biol . 2001;2(9):RESEARCH0035 . Epub 2001 Aug 20. A functional update of the Escherichia coli K-12 genome; Serres MH et al.; BACKGROUND: Since the genome of Escherichia coli K-12 was initially annotated in 1997, additional functional information based on biological characterization and functions of sequence-similar proteins has become available . On the basis of this new information, an updated version of the annotated chromosome has been generated . RESULTS: The E . coli K-12 chromosome is currently represented by 4,401 genes encoding 116 RNAs and 4,285 proteins . The boundaries of the genes identified in the GenBank Accession U00096 were used . Some protein-coding sequences are compound and encode multimodular proteins . The coding sequences (CDSs) are represented by modules (protein elements of at least 100 amino acids with biological activity and independent evolutionary history) . There are 4,616 identified modules in the 4,285 proteins . Of these, 48.9% have been characterized, 29.5% have an imputed function, 2.1% have a phenotype and 19.5% have no function assignment . Only 7% of the modules appear unique to E . coli, and this number is expected to be reduced as more genome data becomes available . The imputed functions were assigned on the basis of manual evaluation of functions predicted by BLAST and DARWIN analyses and by the MAGPIE genome annotation system . CONCLUSIONS: Much knowledge has been gained about functions encoded by the E . coli K-12 genome since the 1997 annotation was published . The data presented here should be useful for analysis of E . coli gene products as well as gene products encoded by other genomes. Exp Neurol, 2001 Oct, 171(2), 329 - 41 Hypothalamic pituitary adrenal axis and hypothalamic-neurohypophyseal responsiveness in water-deprived rats; Grinevich V et al.; The differential effects of osmotic stimulation on magnocellular and parvocellular hypothalamic neurons were studied by analysis of corticotropin-releasing hormone (CRH) and vasopressin (VP) expression in controls and 48-h water-deprived rats subjected to either restraint for 1 h or a single lipopolysaccharide injection (250 microg/100 g) . Water deprivation reduced basal CRH mRNA levels but the increments following 4 h of restraint or 6 h lipopolysaccharide (LPS) injection were similar to those in controls . In contrast, water deprivation had no effect on basal VP heteronuclear RNA (hnRNA) and mRNA levels in parvocellular neurons, but responses to restraint or LPS injection were reduced . VP expression in magnocellular paraventricular and supraoptic nuclei, and plasma sodium and vasopressin were higher in water-deprived rats, changes which were unaffected by restraint . LPS injection reduced VP mRNA but not hnRNA levels in magnocellular neurons and increased plasma vasopressin levels only in water-deprived rats independently of changes in plasma sodium . This was accompanied by an increase in vasopressin mRNA content in the posterior pituitary . The data show that the blunted ACTH responses to acute stress during chronic osmotic stimulation are correlated with the inability of parvocellular neurons to increase VP rather than CRH expression . In addition, LPS-induced endotoxemia causes disturbances of the magnocellular vasopressinergic system with an unexpected potentiation of osmotic simulated VP secretion . The lack of increase in VP transcription after LPS and changes in VP mRNA distribution suggest that endotoxemia affect the secretory process at the levels of the neurohypophyseal axon terminal . J Clin Laser Med Surg, 2000 Aug, 18(4), 209 - 13 Newly induced beta-galactosidase molecules have a higher activity than the basally expressed enzyme; Craig DB et al.; OBJECTIVE: The objective of this study was to compare the activities of individual molecules of induced and basally expressed Escherichia coli beta-galactosidase . BACKGROUND DATA: Single-molecule assays of enzymes have determined that individual molecules are not identical . They differ with respect to catalytic rate . The structural cause and cellular role of this microheterogeneity is as yet unknown . METHODS: E . coli were grown and induced to produce beta-galactosidase by treatment with isopropyl-beta-D-thiogalactopyranoside . Cells were lysed and the beta-galactosidase assayed with capillary electrophoresis instrumentation utilizing post-column, laser-induced fluorescence detection . The enzyme obtained from treated cells were compared to that from untreated cells . RESULTS: The activity of newly induced beta-galactosidase was found to be approximately 20% greater than that of the basally expressed enzyme . This measured difference is statistically significant . CONCLUSIONS: Production of beta-galactosidase in E . coli under differing conditions results in differences in the activities of the individual enzyme molecules. Biochem Soc Symp, 2001, (68), 69 - 82 Defining the structure of the substrate-free state of the DnaK molecular chaperone; Swain JF et al.; Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell . Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism . Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also . At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs . At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time . We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH . The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well . In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain. Nat Struct Biol, 2001 Oct, 8(10), 879 - 82 Quantitative protein stability measurement in vivo; Ghaemmaghami S et al.; The equilibrium between the native and denatured states of a protein can be key to its function and regulation . Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers . However, the biological environment of proteins can differ from these in vitro conditions in ways that could significantly perturb stability . Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Escherichia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX) . The results indicate that the thermodynamic stability of monomeric lambda repressor within the cell is the same as its stability measured in a simple buffer in vitro . However, when the E . coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced . The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights. Nat Struct Biol, 2001 Oct, 8(10), 858 - 63 UDP-galactopyranose mutase has a novel structure and mechanism; Sanders DA et al.; Uridine diphosphogalactofuranose (UDP-Galf ) is the precursor of the d-galactofuranose (Galf ) residues found in bacterial and parasitic cell walls, including those of many pathogens, such as Mycobacterium tuberculosis and Trypanosoma cruzi . UDP-Galf is made from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (mutase) . The mutase enzyme is essential for the viability of mycobacteria and is not found in humans, making it a viable therapeutic target . The mechanism by which mutase achieves the unprecedented ring contraction of a nonreducing sugar is unclear . We have solved the crystal structure of Escherichia coli mutase to 2.4 A resolution . The novel structure shows that the flavin nucleotide is located in a cleft lined with conserved residues . Site-directed mutagenesis studies indicate that this cleft contains the active site, with the sugar ring of the substrate UDP-galactose adjacent to the exposed isoalloxazine ring of FAD . Assay results establish that the enzyme is active only when flavin is reduced . We conclude that mutase most likely functions by transient reduction of substrate. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11388 - 93 Contribution of individual random mutations to genotype-by-environment interactions in Escherichia coli; Remold SK et al.; Numerous studies have shown genotype-by-environment (GxE) interactions for traits related to organismal fitness . However, the genetic architecture of the interaction is usually unknown because these studies used genotypes that differ from one another by many unknown mutations . These mutations were also present as standing variation in populations and hence had been subject to prior selection . Based on such studies, it is therefore impossible to say what fraction of new, random mutations contributes to GxE interactions . In this study, we measured the fitness in four environments of 26 genotypes of Escherichia coli, each containing a single random insertion mutation . Fitness was measured relative to their common progenitor, which had evolved on glucose at 37 degrees C for the preceding 10,000 generations . The four assay environments differed in limiting resource and temperature (glucose, 28 degrees C; maltose, 28 degrees C; glucose, 37 degrees C; and maltose, 37 degrees C) . A highly significant interaction between mutation and resource was found . In contrast, there was no interaction involving temperature . The resource interaction reflected much higher among mutation variation for fitness in maltose than in glucose . At least 11 mutations (42%) contributed to this GxE interaction through their differential fitness effects across resources . Beneficial mutations are generally thought to be rare but, surprisingly, at least three mutations (12%) significantly improved fitness in maltose, a resource novel to the progenitor . More generally, our findings demonstrate that GxE interactions can be quite common, even for genotypes that differ by only one mutation and in environments differing by only a single factor. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11259 - 64 Disordered to ordered folding in the regulation of diphtheria toxin repressor activity; Twigg PD et al.; Understanding how metal binding regulates the activity of the diphtheria toxin repressor protein (DtxR) requires information about the structure in solution . We have prepared a DtxR mutant construct with three additional N-terminal residues, Gly-Ser-His-DtxR(Cys-102 --> Asp), that retains metal-binding capabilities, but remains monomeric in solution and does not bind DNA under conditions that effect dimerization and DNA binding in the functional DtxR(Cys-102 --> Asp) construct . Although the interaction properties of this inactive mutant in solution are very different from that of active repressors, crystallization imposes the same dimeric structure as observed in all crystal forms of the active repressor with and without bound metal . Our solution NMR analyses of active and inactive metal-free diphtheria toxin repressors demonstrate that whereas the C-terminal one-third of the protein is well ordered, the N-terminal two-thirds exhibits conformational flexibility and exists as an ensemble of structural substates with undefined tertiary structure . Fluorescence binding assays with 1-anilino naphthalene-8-sulfonic acid (ANS) confirm that the highly alpha-helical N-terminal two-thirds of the apoprotein is molten globule-like in solution . Binding of divalent metal cations induces a substantial conformational reorganization to a more ordered state, as evidenced by changes in the NMR spectra and ANS binding . The evident disorder to order transition upon binding of metal in solution is in contrast to the minor conformational changes seen comparing apo- and holo-DtxR crystal structures . Disordered to ordered folding appears to be a general mechanism for regulating specific recognition in protein action and this mechanism provides a plausible explanation for how metal binding controls the DtxR repressor activity. J Biol Chem, 2001 Dec 21, 276(51), 48243 - 9 Epub 2001 Sep 25. In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit; Rotem D et al.; EmrE is a small multidrug transporter, 110 amino acids long that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds . Negative dominance studies and radiolabeled substrate-binding studies suggested that EmrE functions as an oligomer . Projection structure of two-dimensional crystals of the protein revealed an asymmetric dimer . To identify the functional unit of EmrE, a novel approach was developed . In this method, quantitative monomer swapping is induced in detergent-solubilized EmrE by exposure to 80 degrees C, a treatment that does not impair transport activity . Oligomer formation is highly specific as judged by several criteria, among them the fact that (35)S-EmrE can be "pulled out" from a mixture prepared from generally labeled cells . Using this technique, we show that inactive mutant subunits are functionally complemented when mixed with wild type subunits . The hetero-oligomers thus formed display a decreased affinity to substrates . In addition, sulfhydryl reagents inhibit the above hetero-oligomer even though Cys residues are present only in the inactive monomer . It is concluded that, in EmrE, the oligomer is the functional unit. J Biol Chem, 2001 Dec 14, 276(50), 47185 - 94 Epub 2001 Sep 25. Interplay of clamp loader subunits in opening the beta sliding clamp of Escherichia coli DNA polymerase III holoenzyme; Leu FP et al.; The Escherichia coli beta dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA . The gamma complex (gammadeltadelta'chipsi) clamp loader couples ATP to the opening and closing of beta in assembly of the ring onto DNA . These proteins are functionally and structurally conserved in all cells . The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp . The delta subunit of the E . coli gamma complex clamp loader is known to bind beta and open it by parting one of the dimer interfaces . This study demonstrates that other subunits of gamma complex also bind beta, although weaker than delta . The gamma subunit like delta, affects the opening of beta, but with a lower efficiency than delta . The delta' subunit regulates both gamma and delta ring opening activities in a fashion that is modulated by ATP interaction with gamma . The implications of these actions for the workings of the E . coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed. Cell, 2001 Sep 21, 106(6), 655 - 60 Opening of the clamp: an intimate view of an ATP-driven biological machine; Ellison V et al.; DNA polymerases require tethering to an accessory factor, typically a ring-shaped clamp, to remain bound to DNA during replication . Three recent structural studies provide unique insight into how these clamps are loaded onto DNA by the clamp loader machinery. Fiziol Zh, 2001, 47(4), 19 - 24 {Modulating effects of exogenous histamine, serotonin, and heparin on leukocytic reaction during inflammation}; Klymenko MO et al.; On the model of E . coli-induced acute infectious peritonitis in rats previously depleted on mast cells of peritoneal cavity the replacing effects of various doses of exogenous histamine, serotonin and heparin and their complex on leukocytic reaction of inflammatory focus and blood were studied . In was shown that all of the used substances had a meaning in the mechanisms of modulating effect of mast cells on leukocytes . The analogous on direction to mast cells ones effects on leukocytes were produced mostly by histamine and especially by the complex of substances. Gene Ther, 2001 Sep, 8(17), 1281 - 90 Development of formulations that enhance physical stability of viral vectors for gene therapy; Croyle MA et al.; This study summarizes our initial efforts to address an issue that is critical to the success of any multicenter gene therapy clinical trial - maintenance of vector viability during shipping and storage at remote test sites . We have identified formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and storage conditions . Adenovirus and adeno-associated virus expressing E . coli beta-galactosidase (lacZ) were suspended in blends of complex carbohydrates, cyclodextrins and various surfactants . X-gal stains of 293 and 84-31 cells were used to determine infectious titer of all preparations . Potassium phosphate-buffered preparations consistently maintained high viral titers after storage at -20 and 4 degrees C . Blends of sucrose, mannitol, and surfactant showed negligible loss of titer for 35 days at 4 degrees C . Formulations of sucrose and cyclodextrin were stable for 2 years at -20 degrees C . Negligible loss in titer was observed in unit-dose viral preparations lyophilized in sucrose and stored at 4 degrees C for 1 year after an initial loss of 0.5 log due to processing . Studies with lyophilized sucrose/mannitol blends have shown that viral recovery after processing is directly related to the final moisture content of the dried product . Virus concentration also plays a significant role in recovery after processing with highly concentrated preparations showing minimal loss in titer after lyophilization . In summary, lyophilized preparations that can be shipped and stored at 25 degrees C offer a solution to the current problem of distribution of viral vectors for clinical trials. J Biol Chem, 2001 Nov 30, 276(48), 44598 - 603 Epub 2001 Sep 24. Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact; Heyduk E et al.; The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence . To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E . coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization . The alpha dimer and DNA formed a 1:1 complex in solution . Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed . The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero . Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation . No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation . The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed. Appl Environ Microbiol, 2001 Oct, 67(10), 4504 - 11 Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi; Zappa S et al.; This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon . An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli . Analysis of the sequence showed conservation of the active site and structural elements of the E . coli AP . The recombinant AP was purified and characterized . Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa . Apparent optimum pH and temperature were estimated at 11.0 and 70 degrees C, respectively . Thus far, P . abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degrees C of 18 and 5 h, respectively . Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity . The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate . Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P . abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds . In addition, P . abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively. Appl Environ Microbiol, 2001 Oct, 67(10), 4426 - 31 EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R . flavefaciens protein via a dockerin domain; Rincon MT et al.; The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp . bind to cellulose are not fully understood . The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose . The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences . EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain . Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca . 130 kDa present among supernatant proteins from Avicel-grown R . flavefaciens that attach to cellulose . The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y . Ding et al., J . Bacteriol . 183:1945-1953, 2001) . It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex. Biochemistry, 2001 Oct 2, 40(39), 11955 - 64 A Streptomyces collinus thiolase with novel acetyl-CoA:acyl carrier protein transacylase activity; Lobo S et al.; Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria . Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII . Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps . The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme . The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases . The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities . Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E . coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM) . In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM) . A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity . No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate . An ACT activity with ACP has not previously been observed for thiolases and in the case of the S . collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)) . The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII. Biochemistry, 2001 Oct 2, 40(39), 11777 - 84 Translational properties of mHNA, a messenger RNA containing anhydrohexitol nucleotides; Lavrik IN et al.; Short messenger RNAs (mRNAs) with hexitol residues in two codons were constructed and their properties were studied in an Escherichia coli in vitro translation system . The replacement of the natural ribonucleotides of mRNA in the AUG start codon and the UUC second codon by hexitol nucleotides did not influence the main steps of translation, as indicated by the same level of binding of mRNA with or without hexitol residues under P-site conditions, and the same yield of tRNA binding to the P- and A-sites . Moreover, both peptide formation and translocation took place on mRNAs with hexitol residues . The presence of an A-type messenger hexitol nucleic acid (mHNA)-transfer RNA (tRNA) duplex is important for efficient translation and the 2'-OH function in mRNA is not necessary for binding and movement through the ribosome . Groove shape recognition of the codon-anticodon complex, more than hydrogen-bond interactions of ribose residues in mRNA, is an important factor for correct translation. Biochemistry, 2001 Oct 2, 40(39), 11734 - 41 Modulation of recombinant human prostate-specific antigen: activation by Hofmeister salts and inhibition by azapeptides . Appendix: thermodynamic interpretation of the activation by concentrated salts; Huang X et al.; Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease . PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions . In addition, PSA does not react readily with prototypical serine protease inactivators . We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid . Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4) . This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-) . The nature of the cation has little effect on salt activation . The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions . Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations . These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA . CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations . Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions . Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo. Protein Expr Purif, 2001 Oct, 23(1), 207 - 17 Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp . PCC 7120; Desplancq D et al.; Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes . While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp . PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources . We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp . PCC 7120 . The 24-kDa N-terminal domain of the E . coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter . The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide . The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E . coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium . Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E . coli sample, demonstrating that it was of sufficient quality for 3D-structure determination . Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp . PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies . Protein Expr Purif, 2001 Oct, 23(1), 191 - 7 Purification of a model substrate for transcription factor phosphorylation by ERK2; Waas WF et al.; ERK2 belongs to the mitogen-activated protein kinase subfamily, which plays a pivotal role in cell signal transduction, in which it mediates effects on proliferation and differentiation by growth factors and hormones . While its cellular function has been under intense scrutiny since its initial discovery nearly 15 years ago, little progress has been made in understanding its kinetic mechanism . Such an understanding is important for the development of potent and specific inhibitors . A contributory factor has been the lack of a protein substrate suitable for rigorous mechanistic studies . Here we report the expression, purification, and characterization of the N-terminus (residues 1 through 138) of the transcription factor Ets-1, an excellent model substrate for ERK2 mechanistic studies . (His(6)-tagged)Ets-1(1-138) was expressed in Escherichia coli and rapidly purified in two steps by nickel-agarose-affinity chromatography, followed by high-resolution Mono-Q anion-exchange chromatography . A yield of 60 mg of the purified protein per liter of culture was obtained and could be stored conveniently at -80 degrees C in water . Rigorous characterization demonstrated that under the assay conditions, (His(6)-tagged)Ets-1(1-138) is exclusively phosphorylated on residue Thr-38 by ERK2 with the following Michaelis parameters: k(cat) = 17 s(-1), K(ATP)(m) = 140 microM, K(ATP)(i) = 68 microM, K(Ets-1)(m) = 19 microM, and K(Ets-1)(i) = 9.3 microM . Protein Expr Purif, 2001 Oct, 23(1), 134 - 41 Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli; Jonsson CB et al.; Sin Nombre virus is a member of the Hantavirus genus, family Bunyaviridae, and is an etiologic agent of hantavirus pulmonary syndrome . The hantavirus nucleocapsid (N) protein plays an important role in the encapsidation and assembly of the viral negative-sense genomic RNA . The Sin Nombre N protein was expressed as a C-terminal hexahistidine fusion in Escherichia coli and initially purified by nickel-affinity chromatography . We developed methods to extract the soluble fraction and to solubilize the remainder of the N protein using denaturants . Maximal expression of protein from native purification was observed after a 1.5-h induction with IPTG (2.4 mg/L) . The zwitterionic detergent Chaps did not enhance the yield of native purifications, but increased the yield of protein obtained from insoluble purifications . Both soluble and insoluble materials, purified by nickel-affinity chromatography, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatography . Both soluble and insoluble proteins had a similar A(280) profile on the Sepharose FF column, and both suggested the presence of a nucleic acid contaminant . The apparent dissociation constant of the N protein, purified by nickel-affinity and SP Sepharose FF chromatography, and the 5' end of the viral S-segment genome were measured using a filter binding assay . The N protein-vRNA complex had an apparent dissociation constant of 140 nM . Protein Expr Purif, 2001 Oct, 23(1), 106 - 12 Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein; Bonifacio MJ et al.; Rat soluble catechol-O-methyltransferase cDNA was cloned into the pCAL-n-FLAG vector and expressed in Escherichia coli as a fusion protein with a calmodulin-binding peptide tag . The recombinant protein, comprising up to 30% of the total protein in the soluble fraction of E . coli, was purified by calmodulin affinity chromatography and gel filtration . Up to 16 mg of pure recombinant enzyme was recovered per liter of culture . Recombinant catechol-O-methyltransferase, in the bacterial soluble fraction, exhibited the same affinity for adrenaline as rat liver soluble catechol-O-methyltransferase (K(m) 428 {246, 609} microM and 531 {330, 732} microM, respectively), as well as the same affinity for the methyl donor, S-adenosyl-l-methionine (K(m) 27 {9, 45} microM and 38 {21, 55} microM, respectively) . In addition, both the recombinant and the liver enzymes displayed the same sensitivity to the inhibitor 3,5-dinitrocatechol (IC(50) 132 {44, 397} nM and 74 {38, 143} nM, respectively), and both had the same catalytic number, respectively, 10.1 +/- 1.5 min(-1) and 8.3 +/- 0.3 min(-1) . The purified recombinant enzyme also displayed the same affinity for the substrate as the purified rat liver catechol-O-methyltransferase (K(m) 336 {75, 597} microM and 439 {168, 711} microM, respectively) as well as the same inhibitor sensitivity (IC(50) 44 {19, 101} nM and 61 {33, 111} nM, respectively) . This recombinant form of catechol-O-methyltransferase is kinetically identical to the rat liver enzyme . This system provides an easy and quick way of obtaining large amounts of soluble catechol-O-methyltransferase for both pharmacological and structural studies . Protein Expr Purif, 2001 Oct, 23(1), 97 - 105 Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520; Christodoulou E et al.; A new procedure for the large-scale purification of the recombinant thermostable chitinase (Chi40) cloned from Streptomyces thermoviolaceus in various expression vectors in Escherichia coli is described . Chi40 was overproduced in the cytosolic and secreted forms . The cytosolic form (Chi40c) was highly overproduced and purified by metal-affinity and ion-exchange chromatography in large amounts . The protein was highly active and thermostable but not homogeneous, since a considerable proportion of the Chi40c protein was not correctly folded as determined by native polyacrylamide gel electrophoresis . The Chi40 protein secreted into the culture medium (Chi40s) was purified by hydrophobic interaction and ion-exchange chromatography and high amounts of correctly folded and active Chi40 protein could be recovered in a short time . The enzymatic activity of Chi40s on a synthetic and on its natural substrate, chitin, was studied . Thermostability measurements showed that Chi40 has a T(m) of 60.7 degrees C at neutral pH . (13)C-(15)N double-labeled recombinant Chi40s was also produced and purified from the pECHChi40-9 construct introduced into BL21trxB(DE3) cells grown in minimal medium in the presence of the paramagnetic elements {(13)C}glucose and (15)NH(4)Cl . The presented data open the possibility of an extensive structural study on Chi40s by X-ray crystallography and on enzyme-substrate interaction by NMR spectroscopy . Protein Expr Purif, 2001 Oct, 23(1), 75 - 83 Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: the alteration of the product by an affinity tag; Rumlova M et al.; The efficiencies of different procedures for purification of the capsid protein (CA) of Mason-Pfizer monkey virus are compared . Plasmids encoding both wild-type CA and two C-terminally modified sequences of CA suitable for affinity chromatography purification were prepared . CA was expressed in Escherichia coli (i) as a wild-type protein, (ii) C-terminally extended with a six-histidine tag (CA 6His), and (iii) as a protein containing a C-terminal fusion to a viral protease cleavage site followed by a six-histidine tag (CA 6aa6His) . Electron microscopy was used for comparison of the resulting proteins, as CA is a structural protein with no enzymatic activity . We have found that these C-terminal fusions dramatically influenced the properties and morphology of structures formed by CA protein in E . coli . The formation of amorphous aggregates of CA was abolished and CA 6His and CA 6aa6His proteins formed organized structures . CA and CA 6aa6His accumulated in bacteria in inclusion bodies as insoluble proteins, CA 6His was found in a soluble form . Both six-histidine-tagged proteins were purified using affinity chromatography under either native (CA 6His) or denaturing (CA 6aa6His) conditions . CA protein was purified under denaturing conditions using gel-filtration chromatography followed by refolding . All proteins were obtained at a purity >98% . Both aforementioned C-terminal extensions led to dramatic changes in behavior of the products and they also affected the tendency to form organized structures within E . coli . We show here that the widely used histidine anchor may significantly alter the properties of the protein of interest . Protein Expr Purif, 2001 Oct, 23(1), 33 - 7 High-level expression and purification of immunogenic recombinant SAG1 (P30) of Toxoplasma gondii in Escherichia coli; Chen XG et al.; Surface antigen 1 (SAG1) of Toxoplasma gondii is a good candidate for diagnosis and vaccine development, but recombinant SAG1 produced in Escheichia coli often loses its specific immunogenicity due to the incorrect folding . In the present study, a truncated SAG1 was highly expressed in E . coli as a fusion protein, about 30% of the total protein of the cell lysate . After a simple purification and refolding procedure, purified rSAG1 can be recognized by human Toxoplasma-infective serum, and ELISA kits constructed by rSAG1 can sensitively and specifically detect toxoplasma infection . Protein Expr Purif, 2001 Oct, 23(1), 14 - 21 Expression and complement d activity of porcine adipsin; Miner JL et al.; To learn how signals from adipocytes might be involved in regulation of energy intake and storage, we have begun to characterize the porcine complement protein, adipsin . Adipsin was originally identified as a protein that is produced rather specifically by adipocytes, is secreted, and is nearly absent in several obese rodent models . We now report that porcine adipsin mRNA sequence is 74% identical to rat and predicts a protein that has 82 and 68% identity to human and rat forms, respectively . Porcine adipsin has none of the asparagine glycosylation consensus sites which make recombinant expression of mouse adipsin in Escherichia coli impractical . We present a method for engineering the porcine cDNA to facilitate expression by E . coli and provide a protocol for refolding and purifying porcine adipsin protein and for immunoassay . We have found that in addition to adipose tissue, adipsin mRNA is present in gut tissues . Coupled with the fact that adipsin is required for processing of complement C3a-desArg, and that C3a-desArg is a potent stimulant of fatty acid acylation in adipocytes, the production of adipsin in the gut may be related to a mechanism for adipocyte removal of lipid from chylomicrons . Protein Expr Purif, 2001 Oct, 23(1), 8 - 13 A method for expression and purification of soluble, active Hsp47, a collagen-specific molecular chaperone; Thomson CA et al.; Hsp47 is regarded as a collagen-specific chaperone with several suggested roles in collagen biosynthesis under normal and disease conditions . We describe here a procedure for the expression and purification of Hsp47 in Escherichia coli using the IMPACT expression system (New England Biolabs) where the guest gene is fused to the adduct, intein, with a chitin-binding domain . Use of this system resulted in relatively high levels of soluble Hsp47 compared to other available protocols, especially when the bacterial cells were induced at 14 degrees C instead of 37 degrees C . The cell lysate was passed through a chitin-Sepharose affinity column and Hsp47 was cleaved from intein using beta-mercaptoethanol . Minor degradation products were subsequently removed using a hydroxylapatite column to yield milligram amounts of pure and active protein suitable for structural studies . Gel electrophoretic analysis of the purified protein indicated the presence of a small proportion of trimeric species when non-reducing conditions were used . The ability to form a trimer may be important for its role as a chaperone . The IMPACT system allows for radiolabelling of purified Hsp47 with (35)S for use in binding experiments . Illustrative data on collagen binding by (35)S-Hsp47 are shown . Protein Expr Purif, 2001 Oct, 23(1), 1 - 7 Functionally active VEGF fusion proteins; Backer MV et al.; Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) acting via endothelial cell-specific receptors, such as VEGFR-2, that are overexpressed at the sites of angiogenesis . If VEGF retains activity as a fusion protein with a large N-terminal extension, it would facilitate development of VEGF-based vehicles for receptor-mediated delivery of therapeutic and diagnostic agents to the sites of angiogenesis . We have constructed, expressed in Escherichia coli, and purified VEGF fusion proteins containing a 158-amino acid N-terminal extension fused to human VEGF(121), VEGF(165), and VEGF(189) . We report here that VEGF fusion proteins induce tyrosine autophosphorylation of VEGFR-2 and its downstream targets, as well as cell contraction in cells overexpressing VEGFR-2 . Although N-terminal extensions decrease the affinity of VEGF fusion proteins to VEGFR-2, at saturating concentrations these proteins are as efficient as correct size VEGF(165) . We hypothesize that VEGF fusion proteins may be employed for targeting endothelial cells at the sites of angiogenesis . Am J Med Sci, 2001 Sep, 322(3), 141 - 4 Nonspecific human IgG reduces survival in neonatal rats infected with Escherichia coli; Cole CW et al.; BACKGROUND: Human intravenous IgG (IVIG) containing specific antibodies protects neonatal rats from septic death . However, IVIG has immunosuppressive properties and clinical trials of IVIG in neonates at risk for sepsis have yielded conflicting results . HYPOTHESIS: This study was designed to test the hypothesis that nonspecific antibodies in IVIG reduce survival in neonatal rats infected with Escherichia coli . METHODS: Specific antibodies were adsorbed from IVIG with E . coli to produce IVIG/anti-E . coli- . After transthoracic administration of E . coli, survival was determined in neonatal rats injected intraperitoneally with phosphate-buffered saline, IVIG/anti-E . coli- (500 mg/kg) or IVIG containing anti-E . coli antibodies (IVIG/anti-E . coli+) . Complement-mediated hemolytic activity of neonatal rat serum was quantified using sensitized sheep erythrocytes . RESULTS: Compared with placebo, intraperitoneal IVIG/anti-E . coli- reduced neonatal survival after E . coli infection . In contrast, IVIG/anti-E . coli+ protected infected animals . Both IVIG/anti-E . coli- and IVIG/anti-E . coli+ impaired the complement-mediated hemolytic activity of neonatal rat serum . CONCLUSIONS: IVIG contained (1) nonspecific antibodies that reduced survival in neonatal rats infected with E . coli and (2) protective anti-E . coli antibodies that enhanced survival in neonatal rats infected with E . coli . We speculate that in clinical trials of IVIG to treat or prevent neonatal sepsis, inconsistent results may be caused, in part, by lot-to-lot variations in the ratio of immunosuppressive, nonspecific antibodies to protective, specific antibodies. Curr Genet, 2001 Aug, 40(1), 82 - 90 Group I intron lateral transfer between red and brown algal ribosomal RNA; Bhattacarya D et al.; How group I introns originate in nuclear ribosomal (r)RNA genes is an important question in evolutionary biology . Central to this issue is the multitude of group I introns present in evolutionarily distantly related plant, fungal, and protist lineages, together with an understanding of their origin and lateral transfer from one exon to another, between cell organelles, and between cells . These introns vary considerably in primary and secondary structure; and their provenance from a few or perhaps many mobile elements that have spread in rRNAs is unknown . Here we show that a novel lineage of group IC1 introns inserted at position 516 (Escherichia coli gene numbering) in the small subunit rRNA in bangiophyte red algae and a brown alga (Aureoumbra lagunensis) are specifically related, although their host cells are not . These bangiophyte and Aureoumbra introns are the only known cases that have a helical insertion in the P5b helix . The highly conserved primary and secondary structure of the extra P5b helix suggests that it is important, although its specific function is unknown . Our study attempts to understand the origin and movement of these IC1 introns. Curr Genet, 2001 Aug, 40(1), 2 - 12 Mechanisms involved in metalloid transport and tolerance acquisition; Tamas MJ et al.; Toxic metalloids such as arsenic and antimony have always been an integral part of the natural environment . To survive in such a hostile habitat, it is crucial to develop strategies to exclude toxic substances from the cell and to acquire tolerance . Cells remove metalloids from the cytosol either by active efflux or by sequestration in an internal organelle . Controlling the influx appears to be another way of maintaining a low intracellular metalloid content . Inside the cell, the metalloid can be reduced to a form that is recognised by the expulsion system(s) . In addition, metalloid complexation and compartmentalisation contributes to enhanced cellular tolerance . Finally, the presence of metalloids activates transcription of various cellular defence genes . Metalloid-containing drugs are currently used to treat protozoan infections and promyelocytic leukaemia . Since metalloid resistance hampers efficient treatment, interest in identifying the mechanisms involved in tolerance acquisition has arisen . The possibility of using genetic approaches has made the yeast Saccharomyces cerevisiae a compelling model system to investigate the basis of metalloid tolerance at a molecular level . This review describes the recent progress made in elucidating the mechanisms involved in metalloid transport and tolerance in yeast and other organisms. Biotechniques, 2001 Sep, 31(3), 560, 562, 564 - 8, passim Universal SNP genotyping assay with fluorescence polarization detection; Hsu TM et al.; The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr) . FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr . We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays . In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs) . This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions . Even without optimization, approximately 70% of all SNP markers tested yielded robust assays . The addition of an E . coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90% . Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100% . In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers) . With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays . When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes) . The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design. Biotechniques, 2001 Sep, 31(3), 534, 536, 538 - 40 Construction of recombinant vaccinia viruses using PUV-inactivated virus as a helper; Timiryasova TM et al.; Recombinant vaccinia viruses (VVs) are widely used as expression vectors in molecular biology and immunology and are now under evaluation for gene therapy . The current techniques for inserting foreign DNA into the large VV genome are based on either homologous recombination between transfer plasmids and VVgenomes or direct DNA ligation and packaging using replication-deficient poxviruses . Here, we describe efficient new versions of both methods that produce 90%-100% of the recombinant viruses . In the new homologous recombination method, VV DNA "arms" obtained by NotI digestion and intact transfer plasmids were used for co-transfection . In the direct DNA ligation method, foreign DNA was inserted into a unique NotI restriction site of the VVgenome . In both methods, the generation of recombinant viruses was carried out in cells infected with a non-replicating, psoralen-UV (PUV)-inactivated helper VV . The convenience of these new techniques is demonstrated by the construction of recombinant VVs that produce E . coli beta-galactosidase . An important feature of these strategies is that any VV strain can be used as a helper virus after PUV inactivation. Anal Chem, 2001 Sep 1, 73(17), 4154 - 61 Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry; Hernandez H et al.; Biotin synthase from Escherichia coli was analyzed by nanoflow electrospray ionization mass spectrometry . From solution conditions in which the protein is in its native state, a distribution of monomeric, dimeric, and tetrameric species was observed . The distribution of these species was sensitive to changes in ionic strength: in the positive ion spectrum, biotin synthase at low ionic strength (pH 7.0-8.5) yielded less than 10% dimer . The masses of the monomeric species were consistent with the presence of a {2Fe-2S} cluster with a mass difference of 175.3 Da from the apomonomer with one disulfide bond . Despite the molecular mass of the noncovalent dimer (77 kDa), it was possible to observe a dimeric species containing one iron-sulfur cluster in both positive and negative ion spectra . Additionally, observation of a series of charge states assigned to the apodimer indicated that binding of the iron-sulfur cluster was not required to maintain the dimer . Binding of Cu2+ to biotin synthase was also observed; in the presence of excess chelating agent, free metals were removed and the iron-sulfur cluster remained intact . Evidence for the coordination of the iron-sulfur cluster in biotin synthase was obtained in a tandem mass spectrometry experiment . A single charge state containing the cluster at m/z 2416.9 was isolated, and collision-induced dissociation resulted in sequential loss of sulfur and retention of Fe3+. Avian Dis, 2001 Jul-Sep, 45(3), 670 - 9 Genotypic and phenotypic characterization of potential virulence of intestinal avian Escherichia coli strains isolated in Algeria; Mellata M et al.; In order to characterize potential pathogenic Escherichia coli strains isolated from diarrheic hens and chickens originating from intensive battery rearing in North Algeria, the presence of a large range of virulence factors and markers was studied in 50 strains by DNA-DNA hybridization on colonies and phenotypic tests . The sequences we focused on were those coding for adhesins F5, F41, F17, Pap, Afa, and Sfa; intimin Eae; and toxins STa, STb, LT1, Stx1, Stx2, CNF1, and CNF2 . The phenotypes explored were the colicins, aerobactin, hemolysins, and hemagglutinin production and serum resistance . The genotypic and phenotypic tests enabled us to categorize the isolates into two distinct groups: those with a potential to invade the host (27 strains were serum resistant and/or produced aerobactin), among which three strains were also potentially diarrheagenic, one strain was LT1 + F17+ Afa+ Pap+ (enterotoxigenic E . coli) and the two others were Stx1 (verotoxigenic E . coli) . Twenty-three strains were colicinogenic, including 19 strains producing colicin V . This latter factor was also detected in isolates negative for the other virulence factors . On the basis of the type of erythrocytes agglutinated, we established 14 mannose-resistant hemagglutination patterns among the 37 strains tested, including 22 serum-resistant and/or aerobactin producing strains and 15 strains negative for these two characters . None of the strains produced alpha hemolysin, whereas two strains produced beta hemolysin and enterohemolysin, respectively . Congo red fixation was observed in 25 strains . No relationship could be detected between Congo red fixation and the presence of other virulence markers, such as serum resistance and aerobactin production . This study shows that among isolates originating from the feces of diarrheic chickens, the proportion of potentially diarrheagenic E . coli strains is low. Genes Genet Syst, 2001 Jun, 76(3), 189 - 98 Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally regulated expression; Yoshioka Y et al.; We screened a gene trap library of Arabidopsis thaliana and isolated a line in which a gene encoding a homologue of monofunctional aspartate kinase was trapped by the reporter gene . Aspartate kinase (AK) is a key enzyme in the biosynthsis of aspartate family amino acids such as lysine, threonine, isoleucine, and methionine . In plants, two types of AK are known: one is AK which is sensitive to feedback inhibition by threonine and carries both AK and homoserine dehydrogenase (HSD) activities . The other one is monofunctional, sensitive to lysine and synergistically S-adenosylmethionine, and has only AK activity . We concluded that the trapped gene encoded a monofunctional aspartate kinase and designated as AK-lys3, because it lacked the HSD domain and had an amino acid sequence highly similar to those of the monofunctional aspartate kinases ofA . thaliana . AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots . Significant expression of this gene was also observed in trichomes after bolting . Slight expression of AK-lys3 was detected in vascular bundles and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmas . These results indicated that this aspartate kinase gene was not expressed uniformly but in a spatially specific manner. Am J Obstet Gynecol, 2001 Sep, 185(3), 716 - 24 A high-throughput study of gene expression in preterm labor with a subtractive microarray approach; Muhle RA et al.; OBJECTIVE: We propose that elucidation of the pathophysiology of preterm labor can be achieved with genome-scale analyses of differential gene expression . STUDY DESIGN: CD-1 mice on day 14.5 of a 19- to 20-day gestation were assigned to one of 4 treatment groups modeling different clinical conditions (n = 5 per group): group A, infection with labor (intrauterine injection of 10(10) heat-killed Escherichia coli, which causes delivery within an average of 20 hours); group B, infection without labor (intrauterine injection of 10(7) heat-killed E coli, which leads to normal delivery at term); group C, labor without infection (ovariectomy, which causes delivery within an average of 27 hours); and group D, no infection and no labor (intrauterine injection of vehicle) . Total pooled myometrial RNA was prepared 3.5 hours after surgery for groups A, B, and D and 5 hours after surgery for group C . The relative expression of 4963 genes was assayed in these pools by using DNA microarrays . Transcripts specifically involved in infection-induced labor were identified by subtracting from the list of differentially regulated genes in group A those with common expression in groups B and C . RESULTS: In group A 68 differentially expressed transcripts (>or=2-fold upregulation or downregulation) were identified . Among these are 39 characterized genes . Fourteen (45%) are involved in inflammatory responses, 7 (18%) are involved in growth-differentiation-oncogenesis, and 3 (8%) are involved in apoptosis . Subtraction identified 13 gene products most likely to be important for bacterially induced labor, as opposed to labor without infection or bacterial exposure without labor . CONCLUSION: This study demonstrates the potential of the subtractive DNA microarray technique to identify transcripts important specifically for bacterially induced preterm labor. J Biosci, 2001 Sep, 26(3), 315 - 24 Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis); Anathy V et al.; A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated . Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3' region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence . The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids . The 5' and 3' untranslated regions of the cDNA are 58 bp and 456 bp long, respectively . The predicted amino acid sequence of H . fossils GH shared 98% homology with other catfishes . Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors . The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone. Biofactors, 2001, 14(1-4), 179 - 90 Cloning of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as an anti-apoptotic and growth promoting gene of Burkitt lymphoma cells; Brielmeier M et al.; Burkitt Lymphoma (BL) cells are highly sensitive to suboptimal growth conditions and undergo apoptosis when seeded at low cell density or reduced serum concentration . Irradiated fibroblasts or a mix of pruvate, alpha-thioglycerol, and bathocuproine disulfonate can protect BL cells from apoptosis induced by lowering cell density or serum concentration by promoting cystine uptake in the cells . The availability of cystine is the limiting factor for glutathione biosynthesis in BL cells and thus for the ability of the cells to cope with oxidative stress . We have set up an expression cloning strategy to clone genes that protect BL cells from apoptosis induced by low cell density and/or serum . Using this approach we have cloned among others the cDNA for Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx). Biofactors, 2001, 14(1-4), 69 - 74 Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis; Lacourciere GM et al.; Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate from selenide and ATP . Characterization of selenophosphate synthetase revealed the determined K(m) value for selenide is far above the optimal concentration needed for growth and approached levels which are toxic . Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se(0)) and alanine, were considered as candidates for the control of free selenium levels in vivo . The ability of a lyase protein to generate Se(0) in the proximity of SPS maybe an attractive solution to selenium toxicity as well as the high K(m) value for selenide . Recently, three E . coli NifS-like proteins, CsdB, CSD, and IscS, were characterized . All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine and produce sulfane sulfur, S(0), or Se(0) respectively . Each lyase can effectively mobilize Se(0) from L-selenocysteine for selenophosphate biosynthesis. Biofactors, 2001, 14(1-4), 61 - 8 An extended Escherichia coli "selenocysteine insertion sequence" (SECIS) as a multifunctional RNA structure; Engelberg-Kulka H et al.; The genetic code, once thought to be rigid, has been found to permit several alternatives in its reading . Interesting alternative relates to the function of the UGA codon . Usually, it acts as a stop codon, but it can also direct the incorporation of the amino acid selenocysteine into a polypeptide . UGA-directed selenocysteine incorporation requires a cis-acting mRNA element called the "selenocysteine insertion sequence" (SECIS) that can form a stem-loop RNA structure . Here we discuss our investigation on the E . coli SECIS . This includes the follows: 1) The nature of the minimal E . coli SECIS . We found that in E . coli only the upper-stem and loop of 17 nucleotides of the SECIS is necessary for selenocysteine incorporation on the condition that it is located in the proper distance from the UGA {34}; 2) The upper stem and loop structure carries a bulged U residue that is required for selenocysteine incorporation {34} because of its interaction with SelB; and 3) We described an extended fdhF SECIS that includes the information for an additional function: The prevention of UGA readthrough under conditions of selenium deficiency {35} . This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it . These two regions act independently and additively and probably through different mechanisms . The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes . These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites . Finally, we present a model for the E . coli fdhF SECIS as a multifunctional RNA structure containing three functional elements . Depending on the availability of selenium the SECIS enables one of two alternatives for the translational machinery: Either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain. J Biol Chem, 2001 Dec 14, 276(50), 47285 - 90 Epub 2001 Sep 21. Structure of crystalline D-Tyr-tRNA(Tyr) deacylase . A representative of a new class of tRNA-dependent hydrolases; Ferri-Fioni ML et al.; Cell growth inhibition by several d-amino acids can be explained by an in vivo production of d-aminoacyl-tRNA molecules . Escherichia coli and yeast cells express an enzyme, d-Tyr-tRNA(Tyr) deacylase, capable of recycling such d-aminoacyl-tRNA molecules into free tRNA and d-amino acid . Accordingly, upon inactivation of the genes of the above deacylases, the toxicity of d-amino acids increases . Orthologs of the deacylase are found in many cells . In this study, the crystallographic structure of dimeric E . coli d-Tyr-tRNA(Tyr) deacylase at 1.55 A resolution is reported . The structure corresponds to a beta-barrel closed on one side by a beta-sheet lid . This barrel results from the assembly of the two subunits . Analysis of the structure in relation with sequence homologies in the orthologous family suggests the location of the active sites at the carboxy end of the beta-strands . The solved structure markedly differs from those of all other documented tRNA-dependent hydrolases. J Appl Physiol, 2001 Oct, 91(4), 1701 - 7 Normovolemic hemodilution improves oxygen extraction capabilities in endotoxic shock; Creteur J et al.; We studied the effects of normovolemic hemodilution on tissue oxygen extraction capabilities in a canine model of endotoxic shock . Eighteen anesthetized and mechanically ventilated dogs underwent normovolemic hemodilution with 6% hydroxyethyl starch solution to reach hematocrit (Hct) levels around 40, 30, or 20% before the administration of 2 mg/kg of Escherichia coli endotoxin . Cardiac tamponade was then induced by repeated injections of normal saline into the pericardial sac to reduce cardiac output and study whole body oxygen extraction capabilities . Whole body critical oxygen delivery was lower in the Hct 20% and 30% groups (8.4 +/- 0.4 and 10.4 +/- 0.7 ml . kg(-1) . min(-1), respectively) than in the Hct 40% group (12.8 +/- 0.8 ml . kg(-1) . min(-1)) (both P < 0.005) . The whole body critical oxygen extraction ratio was higher in the Hct 30% and 20% groups (49.1 +/- 8.2 and 55.2 +/- 4.6%, respectively) than in the Hct 40% group (37.1 +/- 4.4 %) (both P < 0.05) . Liver critical oxygen extraction ratio was also higher in the Hct 30% and 20% groups than in the Hct 40% group . The arterial lactate concentrations and the gradient between ileum mucosal PCO(2) and arterial PCO(2) were lower in the Hct 20% and 30% groups than in the Hct 40% group . We conclude that, during an acute reduction in blood flow during endotoxic shock in dogs, normovolemic hemodilution is associated with improved tissue perfusion and increased oxygen extraction capabilities. Comp Biochem Physiol B Biochem Mol Biol, 2001 Oct, 130(3), 299 - 312 Avian air sac and plasma proteins that bind surface polysaccharides of Escherichia coli O2; Weebadda WK et al.; Some serovars of Escherichia coli, mainly O2 and O78, are responsible for air sac and systemic infections in farm-raised turkeys (Meleagris gallopavo) and chickens (Gallus gallus) . We looked in air sac surface fluid from young turkeys to identify proteins that bind surface polysaccharides of pathogenic respiratory E . coli O2 . Turkey air sac surface fluid was subjected to affinity chromatography on Toyopearl AF-Epoxy-650M, coupled with either lipopolysaccharide (LPS) or lipid-free polysaccharide (LFP) purified from an avian pathogenic E . coli O2 isolate . A multimeric protein termed lipid-free polysaccharide binding protein-40 (LFPBP-40) composed of six covalently associated subunits of approximately 40 kDa was isolated by elution from LFP by EDTA or L-rhamnose . An analogous protein in air sac fluid proteins bound to intact E . coli O2 and eluted with L-rhamnose or N-acetylglucosamine (GlcNAc) . The N-terminal amino acid sequence of LFPBP-40 DINGGGATLPQHLYLTPDV was related to the N-terminus of fragment 3 of a partially characterized human protein possessing T cell stimulation activity in synovial membrane of rheumatoid arthritis patients . However, endogenous amino acid sequences were unrelated to other known proteins . LFPBP-40 was immunoreactively distinct from pulmonary collectins and ficolins . These studies demonstrate a novel avian respiratory soluble lectin that can bind surface polysaccharides of pathogenic E . coli responsible for respiratory disease. Vaccine, 2001 Oct 12, 20(1-2), 115 - 20 Improved vectors for expression library immunization--application to Mycoplasma hyopneumoniae infection in pigs; Moore RJ et al.; Expression library immunization (ELI) has previously been used in a number of disease models in mice . Here, we describe the first example of the application of ELI to a large animal model with the immunization of pigs against enzootic pneumonia, a disease caused by Mycoplasma hyopneumoniae . The development of new plasmid vectors and library screening methods facilitated the application of ELI to this disease by allowing random libraries to be screened for clones expressing recombinant proteins . In this way the vast majority of clones in random libraries that are unproductive can be eliminated, meaning that libraries are more likely to give protection and are subsequently easier to further screen and analyze . By using this approach we have used one library screen and two animal trials to progress from an original library of 20,000 clones to a group of just 96 clones. Vaccine, 2001 Oct 12, 20(1-2), 12 - 5 Identification of a peptide capable of inducing an HIV-1 Tat-specific CTL response; Morris CB et al.; Although Tat-specific CTL responses are elicited in HIV-infected patients and in non-human primate models, specific CTL epitopes within Tat have not been identified . In this study, we mucosally immunized mice with recombinant, full-length Tat protein or individual Tat-specific, overlapping peptides to map putative H-2d-restricted, Tat-specific CTL epitopes . Standard chromium release assays from splenocytes of immunized animals identified a peptide (QPKTACTNC) capable of inducing Tat-specific CTL responses . This newly-identified epitope lies within a region of low sequence variability among HIV-1 subtypes, suggesting its potential use in a multicomponent AIDS vaccine. Acta Crystallogr D Biol Crystallogr, 2001 Oct, 57(Pt 10), 1397 - 404 Epub 2001 Sep 21. NMR trial models: experiences with the colicin immunity protein Im7 and the p85alpha C-terminal SH2-peptide complex; Pauptit RA et al.; Two cases of successful molecular replacement using NMR trial models are presented . One is the crystal structure of the Escherichia coli colicin immunity protein Im7; the other is a heretofore unreported crystal structure of a specific PDGF receptor-derived peptide complex of the carboxy-terminal SH2 domain from the p85alpha subunit of human phosphatidylinositol 3-OH kinase . In both cases, molecular replacement was non-trivial . Success was achieved using trial models that consisted of an ensemble of NMR structures from which the more flexible portions had been excised . Use of maximum-likelihood refinement proved critical to be able to refine the poor starting models . The challenges typical of the use of NMR trial models in molecular replacement are discussed. Protein Sci, 2001 Oct, 10(10), 2131 - 7 A novel approach for assessing macromolecular complexes combining soft-docking calculations with NMR data; Morelli XJ et al.; We present a novel and efficient approach for assessing protein-protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy . This method, termed "restrained soft-docking," is validated for several known protein complexes . These data demonstrate that restrained soft-docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources . The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed. Protein Sci, 2001 Oct, 10(10), 2114 - 22 Origin of fibronectin type II (FN2) modules: structural analyses of distantly-related members of the kringle family idey the kringle domain of neurotrypsin as a potential link between FN2 domains and kringles; Ozhogina OA et al.; Analysis of complete genome sequences has made it clear that fibronectin type II (FN2) modules are present only in the vertebrate lineage, raising intriguing questions about the origin of this module type . Kringle domains display many similarities to FN2 domains; therefore it was suggested previously that they are highly divergent descendants of the same ancestral protein-fold . Since kringles are present in arthropodes, nematodes, and invertebrate chordates as well as in vertebrates, it is suggested that the FN2 domain arose in the vertebrate lineage through major structural modification of the more ancestral kringle fold . To explore this structural transition, in the present work we compare key structural features of two highly divergent kringle domains (the kringle of Caenorhabditis elegans Ror receptor tyrosine kinase and the kringle of rat neurotrypsin) with those of plasminogen kringles and FN2 domains . Our NMR conformation fingerprinting analysis indicates that characteristic (1)H-NMR markers of kringle or FN2 native folding, such as the dispersion of Trp aromatic connectivities and shifts of the Leu(46)/Thr(16) methyl signals, both decrease in the order kringles > neurotrypsin kringle > FN2 domains . These results suggest that the neurotrypsin kringle may represent an intermediate form between typical kringles and FN2 domains. Protein Sci, 2001 Oct, 10(10), 1980 - 8 Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription; Schubot FD et al.; Although it is commonly accepted that binding of mitochondrial transcription factor sc-mtTFB to the mitochondrial RNA polymerase is required for specific transcription initiation in Saccharomyces cerevisiae, its precise role has remained undefined . In the present work, the crystal structure of sc-mtTFB has been determined to 2.6 A resolution . The protein consists of two domains, an N-terminal alpha/beta-domain and a smaller domain made up of four alpha-helices . Contrary to previous predictions, sc-mtTFB does not resemble Escherichia coli sigma-factors but rather is structurally homologous to rRNA methyltransferase ErmC' . This suggests that sc-mtTFB functions as an RNA-binding protein, an observation standing in contradiction to the existing model, which proposed a direct interaction of sc-mtTFB with the mitochondrial DNA promoter . Based on the structure, we propose that the promoter specificity region is located on the mitochondrial RNA polymerase and that binding of sc-mtTFB indirectly mediates interaction of the core enzyme with the promoter site. Protein Sci, 2001 Oct, 10(10), 1962 - 9 Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: function of the Asn-His interaction in the catalytic triad; Snijder HJ et al.; Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral-membrane enzyme with a unique His-Ser-Asn catalytic triad . In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate . Here the role of the uncharged asparagine in the active site of OMPLA is investigated by structural characterization of the Asn156Ala mutant . Asparagine 156 is not involved in maintaining the overall active-site configuration and does not contribute significantly to the thermal stability of OMPLA . The active-site histidine retains an active conformation in the mutant notwithstanding the loss of the hydrogen bond to the asparagine side chain . Instead, stabilization of the correct tautomeric form of the histidine can account for the observed decrease in activity of the Asn156Ala mutant. J Biol Chem, 2001 Dec 7, 276(49), 46319 - 25 Epub 2001 Sep 20. Aspartate residue 142 is important for catalysis by ADP-glucose pyrophosphorylase from Escherichia coli; Frueauf JB et al.; Structural prediction of several bacterial and plant ADP-glucose pyrophosphorylases, as well as of other sugar-nucleotide pyrophosphorylases, was used for comparison with the three-dimensional structures of two crystallized pyrophosphorylases (Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, C., and Bourne, Y . (1999) EMBO J . 18, 4096-4107; Blankenfeldt, W., Asuncion, M., Lam, J . S., and Naismith, J . H . (2000) EMBO J . 19, 6652-6663) . This comparison led to the discovery of highly conserved residues throughout the superfamily of pyrophosphorylases despite the low overall homology . One of those residues, Asp(142) in the ADP-glucose pyrophosphorylase from Escherichia coli, was predicted to be near the substrate site . To elucidate the function that Asp(142) might play in the E . coli ADP-glucose pyrophosphorylase, aspartate was replaced by alanine, asparagine, or glutamate using site-directed mutagenesis . Kinetic analysis in the direction of synthesis or pyrophosphorolysis of the purified mutants showed a decrease in specific activity of up to 4 orders of magnitude . Comparison of other kinetic parameters, i.e . the apparent affinities for substrates and allosteric effectors, showed no significant changes, excluding this residue from the specific role of ligand binding . Only the D142E mutant exhibited altered K(m) values but none as pronounced as the decrease in specific activity . These results show that residue Asp(142) is important in the catalysis of the ADP-glucose pyrophosphorylase from E . coli. J Biol Chem, 2001 Nov 30, 276(48), 44590 - 7 Epub 2001 Sep 20. B-subunit of phosphate-specific transporter from Mycobacterium tuberculosis is a thermostable ATPase; Sarin J et al.; The B-subunit of phosphate-specific transporter (PstB) is an ABC protein . pstB was polymerase chain reaction-amplified from Mycobacterium tuberculosis and overexpressed in Escherichia coli . The overexpressed protein was found to be in inclusion bodies . The protein was solubilized using 1.5% N-lauroylsarcosine and was purified by gel permeation chromatography . The molecular mass of the protein was approximately 31 kDa . The eluted protein showed ATP-binding ability and exhibited ATPase activity . Among different nucleotide triphosphates, ATP was found to be the preferred substrate for M . tuberculosis PstB-ATPase . The study of the kinetics of ATP hydrolysis yielded K(m) of approximately 72 microm and V(max) of approximately 0.12 micromol/min/mg of protein . Divalent cation like manganese was inhibitory to the ATPase activity . Magnesium or calcium, on the other hand, had no influence on the functionality of the enzyme . The classical ATPase inhibitors like sodium azide, sodium vanadate, and N-ethylmaleimide were without any effect but an ATP analogue, 5'-p-fluorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recombinant protein with a K(i) of approximately 0.40 mm . Furthermore, there was hardly any ATP hydrolyzing ability of the PstB as a result of mutation of the conserved aspartic acid residue to lysine in the Walker motif B, confirming the recombinant protein is an ATPase . Interestingly, analysis of the recombinant PstB revealed that it is a thermostable ATPase; thus, our results highlight for the first time the presence of such an enzyme in any mesophilic bacteria. J Bacteriol, 2001 Oct, 183(20), 6144 - 7 Escherichia coli minicell membranes are enriched in cardiolipin; Koppelman CM et al.; The phospholipid composition of Escherichia coli minicells has been studied as a model for the cell division site . Minicells appeared to be enriched in cardiolipin at the expense of phosphatidylglycerol . Mass spectrometry showed no differences between the gross acyl chain compositions of minicells and wild-type cells. J Bacteriol, 2001 Oct, 183(20), 6140 - 3 Novel motility mutants of synechocystis strain PCC 6803 generated by in vitro transposon mutagenesis; Bhaya D et al.; We screened for transposon-generated mutants of Synechocystis sp . strain PCC 6803 that exhibited aberrant phototactic movement . Of the 300 mutants generated, about 50 have been partially characterized; several contained transposons in genes encoding chemotaxis-related proteins, while others mapped to novel genes . These novel genes and their possible roles in motility are discussed. J Bacteriol, 2001 Oct, 183(20), 6126 - 34 Growth phase and growth rate regulation of the rapA gene, encoding the RNA polymerase-associated protein RapA in Escherichia coli; Cabrera JE et al.; The Escherichia coli rapA gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family . We have studied the rapA promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro . We have found that the expression of rapA is growth phase dependent, peaking at the early log phase . The growth phase control of rapA is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most E . coli promoters . We also found that the rapA promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro . Furthermore, we have shown that a GC-rich or discriminator sequence between the -10 and +1 positions of the rapA promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP. J Bacteriol, 2001 Oct, 183(20), 6095 - 106 Characterization of mutations in the metY-nusA-infB operon that suppress the slow growth of a DeltarimM mutant; Bylund GO et al.; The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes . A DeltarimM mutant shows a sevenfold-reduced growth rate and a reduced translational efficiency, probably as a result of aberrant assembly of the ribosomal 30S subunits . The slow growth and translational deficiency can be partially suppressed by increased synthesis of the ribosome binding factor RbfA . Here, we have identified 14 chromosomal suppressor mutations that increase the growth rate of a DeltarimM mutant by increasing the expression of rbfA . Nine of these mutations were in the nusA gene, which is located upstream from rbfA in the metY-nusA-infB operon; three mutations deleted the transcriptional terminator between infB and rbfA; one was an insertion of IS2 in infB, creating a new promoter for rbfA; and one was a duplication, placing a second copy of rbfA downstream from a promoter for the yhbM gene . Two of the nusA mutations were identical, while another mutation (nusA98) was identical to a previously isolated mutation, nusA11, shown to decrease termination of transcription . The different nusA mutations were found to increase the expression of rbfA by increasing the read-through of two internal transcriptional terminators located just downstream from the metY gene and that of the internal terminator preceding rbfA . Induced expression of the nusA(+) gene from a plasmid in a nusA(+) strain decreased the read-through of the two terminators just downstream from metY, demonstrating that one target for a previously proposed NusA-mediated feedback regulation of the metY-nusA-infB operon expression is these terminators . All of the nusA mutations produced temperature-sensitive phenotypes of rimM(+) strains . The nusA gene has previously been shown to be essential at 42 degrees C and below 32 degrees C . Here, we show that nusA is also essential at 37 degrees C. J Bacteriol, 2001 Oct, 183(20), 6065 - 73 RecA-mediated rescue of Escherichia coli strains with replication forks arrested at the terminus; Maisnier-Patin S et al.; The recombinational rescue of chromosome replication was investigated in Escherichia coli strains with the unidirectional origin oriR1, from the plasmid R1, integrated within oriC in clockwise (intR1(CW)) or counterclockwise (intR1(CC)) orientations . Only the intR1(CC) strain, with replication forks arrested at the terminus, required RecA for survival . Unlike the strains with RecA-dependent replication known so far, the intR1(CC) strain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability . The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar in oriC and intR1(CC) strains . In the intR1(CC) strain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus . This was consistent with suppression of the RecA dependence of the intR1(CC) strain by inactivating Tus, the protein needed to block replication forks at Ter sites . Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways. J Bacteriol, 2001 Oct, 183(20), 6017 - 27 Regulatory interactions of Csr components: the RNA binding protein CsrA activates csrB transcription in Escherichia coli; Gudapaty S et al.; The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions . Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively . CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity . We have further examined the regulatory interactions of CsrA and CsrB RNA . The 5' end of the CsrB transcript was mapped, and a csrB::cam null mutant was constructed . CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic csrA, csrB, rpoS, or csrA rpoS mutant strains . CsrA levels exhibited modest or negligible effects of these mutations . The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA . In contrast, CsrB levels were drastically decreased (~10-fold) in the csrA mutants . CsrB transcript stability was unaffected by csrA . The expression of a csrB-lacZ transcriptional fusion containing the region from -242 to +4 bp of the csrB gene was decreased ~20-fold by a csrA::kanR mutation in vivo but was unaffected by CsrA protein in vitro . These results reveal a significant, though most likely indirect, role for CsrA in regulating csrB transcription . Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB. J Bacteriol, 2001 Oct, 183(20), 5974 - 81 RpoS-dependent transcriptional control of sprE: regulatory feedback loop; Ruiz N et al.; The stationary-phase response exhibited by Escherichia coli upon nutrient starvation is mainly induced by a decrease of the ClpXP-dependent degradation of the alternate primary sigma factor RpoS . Although it is known that the specific regulation of this proteolysis is exercised by the orphan response regulator SprE, it remains unclear how SprE's activity is regulated in vivo . Previous studies have demonstrated that the cellular content of SprE itself is paradoxically increased in stationary-phase cells in an RpoS-dependent