J Pept Res, 1998 Feb, 51(2), 103 - 9 Interaction of alpha-helical peptides with phospholipid membrane: effects of chain length and hydrophobicity of peptides; Ohmori N et al.; To investigate the interaction of amphiphilic alpha-helical peptides with phospholipid membranes, we synthesized Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4{3}) and three derivatives, in which the chain length and the size of the hydrophobic region of the peptides were different from each other . These peptides formed an alpha-helical structure in the presence of vesicles . In the membrane-perturbation measurement, only 43 showed a strong membrane-perturbation activity below phase-transition temperature (25 degrees C), but above phase-transition temperature (50 degrees C), most peptides showed similar strong activities . On the other hand, in membrane-fusion measurement the long peptides, e.g., Ac-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)3-NHCH3, had strong activities at low peptide concentrations at 25 degrees C . The present study indicated a parallel relationship did not always exist between membrane fusion and perturbation caused by peptides. J Bacteriol, 1998 Mar, 180(6), 1603 - 6 Trehalose is not relevant for in vivo activity of sigmaS-containing RNA polymerase in Escherichia coli; Germer J et al.; The sigmaS- and sigma70-associated forms of RNA polymerase core enzyme (E) of Escherichia coli have very similar promoter recognition specificities in vitro . Nevertheless, the in vivo expression of many stress response genes is strongly dependent on sigmaS . Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of EsigmaS and thereby contributes to promoter selectivity (S . Kusano and A . Ishihama, J . Bacteriol . 179:3649-3654, 1997) . However, we demonstrate here that a trehalose-free otsA mutant exhibits growth phase-related and osmotic induction of various sigmaS-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced) . We conclude that in vivo trehalose does not play a role in the expression of sigmaS-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes. J Bacteriol, 1998 Mar, 180(6), 1567 - 9 Hybrid Bordetella pertussis-Escherichia coli RNA polymerases: selectivity of promoter activation; Steffen P et al.; We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system . Virulence-associated genes of B . pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E . coli lac promoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E . coli alpha subunit . This suggests that activator contact sites involve a high degree of selectivity. J Bacteriol, 1998 Mar, 180(6), 1563 - 6 In vivo protein interactions within the Escherichia coli DNA polymerase III core; Jonczyk P et al.; The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies . Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication . It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia coli could be essential for maintaining the optimal fidelity of DNA replication (H . Maki and A . Kornberg, Proc . Natl . Acad . Sci . USA 84:4389-4392, 1987) . We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (alpha subunit {possessing DNA polymerase activity}) and DnaQ (epsilon subunit {possessing 3'-->5' exonucleolytic proofreading activity}) proteins . In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions . In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (theta subunit) proteins . We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype . Our results show that the mutD5 and dnaQ926 mutations do not affect the epsilon subunit-alpha subunit and epsilon subunit-theta subunit interactions . However, the dnaQ49 mutation greatly reduces the strength of interaction of the epsilon subunit with both the alpha and the theta subunits . Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the epsilon protein, which leads to altered interactions within the Pol III core. J Bacteriol, 1998 Mar, 180(6), 1525 - 32 Activation of Escherichia coli rRNA transcription by FIS during a growth cycle; Appleman JA et al.; rRNA transcription in Escherichia coli is activated by the FIS protein, which binds upstream of rrnp1 promoters and interacts directly with RNA polymerase . Analysis of the contribution of FIS to rrn transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of fis mutations . In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the rrnBp1 FIS sites correlates with cellular levels of FIS . We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made from rrnBp1), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels . FIS activates transcription throughout exponential growth at low culture density, but rrnp1 transcription increases independently of FIS immediately following upshift, before FIS accumulates . These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription from rrnp1 promoters during steady-state growth. J Bacteriol, 1998 Mar, 180(6), 1425 - 30 A conserved histidine is essential for glycerolipid acyltransferase catalysis; Heath RJ et al.; Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration . We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli . Both the PlsB{H306A} and Aas{H36A} mutants lacked acyltransferase activity . However, the Aas{H36A} mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution . The invariant aspartic acid residue in the HX4D pattern was also important . The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity . Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB{D311A} mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases . These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor. J Bacteriol, 1998 Mar, 180(6), 1411 - 7 The LysR-type transcriptional regulator CbbR controlling autotrophic CO2 fixation by Xanthobacter flavus is an NADPH sensor; van Keulen G et al.; Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy . Maximal induction of the cbb and gap-pgk operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate . The LysR-type transcriptional regulator CbbR regulates the expression of the cbb and gap-pgk operons, but it is unknown to what cellular signal CbbR responds . In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in Escherichia coli and subsequently purified to homogeneity . CbbR of X . flavus is a dimer of 36-kDa subunits . DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located . The addition of 200 microM NADPH, but not NADH, resulted in a threefold increase in DNA binding . The apparent K(dNADPH) of CbbR was determined to be 75 microM . By using circular permutated DNA fragments, it was shown that CbbR introduces a 64 degree bend in the DNA . The presence of NADPH in the DNA-bending assay resulted in a relaxation of the DNA bend by 9 degree . From the results of these in vitro experiments, we conclude that CbbR responds to NADPH . The in vivo regulation of the cbb and gap-pgk operons may therefore be regulated by the intracellular concentration of NADPH. J Bacteriol, 1998 Mar, 180(6), 1396 - 401 Identification of a sequence motif that confers SecB dependence on a SecB-independent secretory protein in vivo; Kim J et al.; SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in Escherichia coli . However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains . The characteristics which confer SecB dependence on some proteins but not others have not been defined . To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA) . The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo . Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence . We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB . Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif . The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex . Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus. J Bacteriol, 1998 Mar, 180(6), 1389 - 95 An rne-1 pnp-7 double mutation suppresses the temperature-sensitive defect of lacZ gene expression in a divE mutant; Aiso T et al.; A divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, exhibits differential loss of the synthesis of certain proteins, such as beta-galactosidase and succinate dehydrogenase, at nonpermissive temperatures . In Escherichia coli, the UCA codon is recognized only by tRNA1Ser . Several genes containing UCA codons are normally expressed after a temperature shift to 42 degrees C in the divE mutant . Therefore, it is unlikely that the defect in protein synthesis at 42 degrees C is simply caused by a defect in the decoding function of the mutant tRNA1Ser . In this study, we sought to determine the cause of the defect in lacZ gene expression in the divE mutant . It has also been shown that the defect in lacZ gene expression is accompanied by a decrease in the amount of lacZ mRNA . To examine whether inactivation of mRNA degradation pathways restores the defect in lacZ gene expression, we constructed divE mutants containing rne-1, rnb-500, and pnp-7 mutations in various combinations . We found that the defect was almost completely restored by introducing an rne-1 pnp-7 double mutation into the divE mutant . Northern hybridization analysis showed that the rne-1 mutation stabilized lacZ mRNA, whereas the pnp-7 mutation stabilized mutant tRNA1Ser, at 44 degrees C . We present a mechanism that may explain these results. J Bacteriol, 1998 Mar, 180(6), 1368 - 74 Contribution of the disulfide bond of the A subunit to the action of Escherichia coli heat-labile enterotoxin; Okamoto K et al.; Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits . These subunits oligomerize into an assembled holotoxin within the periplasm . Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus . This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm . However, it is not known whether other regions of the A subunit contribute to the assembly . The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199 . It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein . We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit . We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes . The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly . A reconstitution experiment in vitro supported the notion . Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs . These results might be useful for the analysis of the interaction of LT with target cells at the molecular level. FEBS Lett, 1998 Feb 27, 423(3), 347 - 50 Conformational independence of N- and C-domains in ribosomal protein L7/L12 and in the complex with protein L10; Bocharov EV et al.; Isolated N- (1-37) and C-terminal (47-120) fragments of L7 protein, and pentameric (L7)4L10 complex were studied by NMR spectroscopy in solution . The results indicate that the dimer state of the 1-37 fragment with a helical hairpin conformation is identical to the N-terminal structure of the intact L7 dimer . The C-terminal domain of the L7 protein does not participate in (L7)4L10 complex formation . The overall motions of the L7 C-domains are essentially independent both in the L7 dimer and in the (L7)4L10 complex . Conformational motions on a millisecond time scale are detected in the (L7)4L10 complex . The possible relevance of these motions to the biological function of L7/L12 is discussed. Mol Microbiol, 1998 Feb, 27(4), 787 - 95 Regulation of RssB-dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator-controlled process; Bouche S et al.; Sigma(S) (RpoS) is a highly unstable global regulatory protein in Escherichia coli, whose degradation is inhibited by various stress signals, such as carbon starvation, high osmolarity and heat shock . As a consequence, these stresses result in the induction of sigma(S)-regulated stress-protective proteins . The two-component-type response regulator, RssB, is essential for the rapid proteolysis of sigma(S) and is probably involved in the transduction of some of these stress signals . Acetyl phosphate can be used as a phosphodonor for the phosphorylation of various response regulators in vitro and, in the absence of the cognate sensor kinases, acetyl phosphate can also modulate the activities of several response regulators in vivo . Here, we demonstrate increased in vivo half-lives of sigma(S) and the RpoS742::LacZ hybrid protein (also a substrate for RssB-dependent proteolysis) in acetyl phosphate-free (pta-ackA) deletion mutants, even though no sensor kinase was eliminated . The in vivo data indicate that acetyl phosphate acts through the response regulator, RssB . In vitro, efficient phosphotransfer from radiolabelled acetyl phosphate to the Asp-58 residue of RssB (the expected site of phosphorylation in the RssB receiver domain) was observed . Via such phosphorylation, acetyl phosphate may thus modulate RssB activity even in an otherwise wild-type background . While acetyl phosphate is not essential for the transduction of specific environmental stress signals, it could play the role of a modulator of RssB-dependent proteolysis that responds to the metabolic status of the cells reflected in the highly variable cellular acetyl phosphate concentration. Mol Microbiol, 1998 Feb, 27(4), 751 - 61 Leucine alters the interaction of the leucine-responsive regulatory protein (Lrp) with the fim switch to stimulate site-specific recombination in Escherichia coli; Roesch PL et al.; The leucine-responsive regulatory protein (Lrp) is a global regulator that controls the expression of numerous operons in Escherichia coli . Lrp can act as a repressor or as an activator of transcription with its effects being potentiated, repressed or unaffected by the presence of exogenous leucine . The phase variation of type 1 fimbria in E . coli provides a unique system in which to investigate the effects of leucine on Lrp, as it is the only known example in which Lrp is a positive regulator and leucine potentiates this effect . Previous studies determined that Lrp binds with high affinity to two sites within the fim switch (fim sites 1 and 2), and binding to these sites stimulates recombination . Here, it is shown that, even though leucine stimulates the fim switch in vivo, it nevertheless causes a slight decrease in Lrp binding to the fim switch in vitro . These contradictory results are explicable by the finding that Lrp binding to a third region adjacent to fim sites 1 and 2 inhibits recombination . According to this model, leucine stimulates recombination by selectively disrupting Lrp binding to this newly characterized region, while having little or no effect on Lrp binding to fim sites 1 and 2. Mol Microbiol, 1998 Feb, 27(4), 739 - 50 Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli; Britton RA et al.; Era is a low-molecular-weight GTPase essential for Escherichia coli viability . The gene encoding Era is found in the rnc operon, and the synthesis of both RNase III and Era increases with growth rate . Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild-type Era become arrested in the cell cycle at the predivisional two-cell stage . The partially defective Era GTPase mutation (era1) suppresses several temperature-sensitive lethal alleles that affect chromosome replication and chromosome partitioning but not cell division . Our results suggest that Era plays an important role in cell cycle progression at a specific point in the cycle, after chromosome partitioning but before cytokinesis . Possible functions for Era in cell cycle progression and the initiation of cell division are discussed. Protein Sci, 1998 Jan, 7(1), 211 - 5 Cloning, overexpression, purification, and spectroscopic characterization of human S100P; Gribenko A et al.; The calcium-binding protein S100P has been found to be associated with human prostate cancer . We have overexpressed S100P in Escherichia coli using a T7 expression system . A rapid two-step procedure for the isolation of overexpressed S100P leads to a preparation of >95% pure protein with a yield of approximately 150 mg per liter of culture . The structural integrity of recombinant S100P was analyzed using CD and fluorescence spectroscopic techniques . The far-UV CD shows that secondary structure of recombinant S100P consists predominantly of a-helical structure . Both near-UV CD and tyrosine fluorescence spectra show that aromatic residues are involved in the formation of a specific, well packed structure, indicating that the recombinant S100P protein adopts a compact folded conformation . Ca2+ has a profound effect on S100P structure . Near-UV CD and fluorescence intensity of both internal (tyrosine) and external (ANS) probes suggest significant structural rearrangements in the tertiary structure of the molecule . The similarity of far-UV CD spectrum of S100P in the presence and in the absence of Ca2+ suggests that Ca2+ binding has only minor effects on secondary structure. Protein Sci, 1998 Jan, 7(1), 193 - 200 Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy; Vohnik S et al.; The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction . In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values . A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity . pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations . The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin . Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions . The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein . Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation . Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site . We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site. J Cell Biochem, 1998 Apr 1, 69(1), 1 - 12 L7 protein is a coregulator of vitamin D receptor-retinoid X receptor-mediated transactivation; Berghofer-Hochheimer Y et al.; The vitamin D receptor (VDR) heterodimerizes with the retinoid X receptor (RXR) and requires additional protein-protein interactions to regulate the expression of target genes . Using the yeast two-hybrid system, we identified the previously described protein L7, that specifically interacted with the VDR in the presence of vitamin D . Deletion analysis indicated, that the N-terminus of L7, which harbours a basic region leucine zipper like domain, mediated interaction with the VDR . Binding assays with purified GST-L7 demonstrated, that L7 specifically pulled down the VDR, that was either expressed in yeast or endogenously contained in the cell line U937 . Interestingly, L7 inhibited ligand-dependent VDR-RXR heterodimerization, when constitutively expressed in yeast . We also demonstrate that L7 repressed binding of VDR-RXR heterodimers to a vitamin D response element . Surprisingly, L7 recruited RXR to the same response element in the presence of 9-cis retinoic acid . Ligand-dependent protein-protein interaction in the yeast two-hybrid system confirmed, that binding of L7 also was targeted at the RXR . Our data suggest, that protein L7 is a coregulator of VDR-RXR mediated transactivation of genes, that modulates transcriptional activity by interfering with binding of the receptors to genomic enhancer elements. Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 573 - 7 Functional analysis of conserved histidines in ADP-glucose pyrophosphorylase from Escherichia coli; Hill MA et al.; Two absolutely conserved histidines and a third highly conserved histidine are noted in 11 bacterial and plant ADP-glucose pyrophosphorylases . These histidines were individually mutagenized in the E . coli enzyme to glutamine in order to determine their function . Glutamine mutations at residues 143 and 156 produced functional enzymes in cell extracts with slightly lower than wild-type specific catalytic activities and with same heat stability characteristics of the wild-type enzyme . Substitution of residue 83 with glutamine however produced an enzyme having decreased thermal stability . Additional mutageneses at residue 83 with asparagine, arginine, or aspartate gave rise to enzymes having a progressively decreasing trend in thermal stability . These mutants are more susceptible to proteolysis than wild-type enzyme . Kinetic analysis of H83Q and H83N indicates that histidine 83 is not involved in the catalytic mechanism or in substrate binding but possibly in maintenance of the active catalytic structure. Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 360 - 3 Antibody to a Helicobacter pylori species specific antigen in patients with adenocarcinoma of the stomach; Wang JT et al.; This study attempted to identify a possible antibody response to Helicobacter pylori, which is associated with patients with adeno-carcinoma of the stomach . By using proteins of H . pylori as the antigen, pooled sera from gastric cancer and non-cancer patients were used as the first antibody for Western blot analysis . Antibody responses to a 26 kD secreted protein were observed in pooled cancer sera, but not in pooled sera from non-cancer patients . The protein was purified, while amino acid sequences revealed that it was a H . pylori species specific protein . The gene of this protein was cloned and a recombinant protein was expressed in E . coli . In addition, an antibody to the recombinant protein was tested in each individual patient using Western blot analysis . None of the forty non-gastric cancer patients were positive for the antibody to the recombinantly expressed 26 kD species specific protein . Meanwhile, six of the twenty four cancer patients tested positive (0/40 vs 6/24, p < 0.01) . Results presented herein demonstrate that the species specific protein of H . pylori can be useful in detecting H . pylori associated with adenocarcinoma of the stomach. Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 131 - 7 Characterization, cloning, and expression of porcine alpha B crystallin; Liao JH et al.; alpha-Crystallin is a major lens protein present in the lenses of all vertebrate species . Recent studies have revealed that bovine alpha-crystallins possess genuine chaperone activity similar to small heat-shock proteins . In order to compare this chaperone-like structural protein from the eye lenses of different mammalian species, we have cloned and expressed one of the main alpha-crystallin subunits, i.e., alpha B crystallin, from the porcine lenses in order to facilitate the structure-function evaluation and comparison of this chaperonin protein . cDNA encoding alpha B subunit chain was obtained using a new "Marathon cDNA amplification" protocol of Polymerase Chain Reaction (PCR) . PCR-amplified product corresponding to alpha B subunit was then ligated into pGEM-T plasmid and prepared for nucleotide sequencing by the dideoxy-nucleotide chain-termination method . Sequencing several positive clones containing DNA inserts coding for alpha B-crystallin subunit constructed only one complete full-length reading frame of 525 base pairs similar to human and bovine alpha B subunits, covering a deduced protein sequence of 175 amino acids including the universal translation-initiating methionine . The porcine alpha B crystallin shows only 3 and 7 residues difference to bovine and human alpha B crystallins respectively, revealing the close relatedness among mammalian eye lens proteins . The sequence differences between porcine and sub-mammalian species such as chicken and bullfrog are much greater, especially at the N- and C-terminal regions of these alpha B crystallins . Expression of alpha B subunit chain in E . coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified alpha B subunit from the native porcine lenses albeit with a much lower activity. Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 75 - 8 Induction of UCP2 gene expression by LPS: a potential mechanism for increased thermogenesis during infection; Faggioni R et al.; UCP2 has been proposed to regulate thermogenesis and energy expenditure . To identify potential mechanisms underlying the increased energy expenditure and heat production during infection, we investigated whether LPS and cytokines might increase UCP2 mRNA levels in mice . LPS (100 micrograms, i.p.) increased the expression of UCP2 mRNA in liver (28-fold) and muscle and white adipose tissue (5-fold) . In liver, both IL-1 beta (1 microgram, i.p.) and TNF (5 micrograms, i.p.) increased UCP2 mRNA levels, 4- and 3-fold respectively, whereas in muscle and fat tissue, an increase was detectable after TNF, but not IL-1 beta . Indomethacin (10 mg/kg, i.p.) administered immediately before LPS markedly reduced (70%) the ability of LPS to increase UCP2 mRNA in liver, but not in muscle or adipose tissue . These results suggest a role for UCP2 in the heat production and increased energy expenditure that occurs during infection. Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 79 - 84 Alteration of the fatty acid substrate specificity of lysophosphatidate acyltransferase by site-directed mutagenesis; Morand LZ et al.; The JC201 strain of Eschericia coli contains a temperature-sensitive lesion in lysophosphatidate acyltransferase (LPAT) activity . The LPAT gene from JC201 was isolated by PCR and a single mutant nucleotide, adenine-440, was identified by DNA sequence analysis . Site-directed mutagenesis converted the mutant adenine-440 back to the native guanine-440 nucleotide . The restored LPAT gene rescued JC201 cells at the non-permissive temperature . The fatty acid substrate specificity of LPAT from Eschericia coli was altered by site-directed mutagenesis of a single amino acid in the restored LPAT gene . Threonine-122 of LPAT was changed to alanine or leucine . A change from threonine-122 to alanine increased the substrate specificity in vitro for oleoyl-CoA and linoleoyl-CoA; whereas a change to leucine increased the substrate specificity for lignoceroyl-CoA. Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 110 - 4 Glyphosate is an inhibitor of plant cytochrome P450: functional expression of Thlaspi arvensae cytochrome P45071B1/reductase fusion protein in Escherichia coli; Lamb DC et al.; Glyphosate (Roundup) is an herbicide used extensively worldwide which acts as an inhibitor of 5'enolpyruvylshikimate-3-phosphate synthase and for which transgenic herbicide resistant plants have been developed . Here we report for the first time that glyphosate is an inhibitor of cytochrome P450 using a functional expression system for Thlaspi arvensae CYP71B1 in Escherichia coli . CYP71B1 was fused to the soluble domain of a plant cytochrome P450 reductase (CPR) from Catharanthus roseus . CYP71B1 could obtain reducing equivalents in this fusion construct and metabolised the polycyclic aromatic hydrocarbon, benzo(a)pyrene . The fusion protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum . Addition of the herbicide resulted in a Type II spectrum indicative of binding via the nitrogen group to haem as a sixth ligand . A Ks of 60 microM was observed and an IC50 of 12 microM was observed for glyphosate inhibition of CYP71B1 activity . The implications of these results are discussed. Anal Biochem, 1998 Mar 15, 257(2), 203 - 9 A positive selection vector for cloning of long polymerase chain reaction fragments based on a lethal mutant of the crp gene of Escherichia coli; Schlieper D et al.; We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli . The positive selection pressure results from a lethal mutation within the E . coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted . Here, we show that this "suicide" vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the lethal gene. Plasmid, 1998, 39(2), 114 - 22 New shuttle vectors for the introduction of cloned DNA in Desulfovibrio; Rousset M et al.; The pBG1 replicon from the cryptic plasmid of Desulfovibrio desulfuricans G100A was inserted into pTZ18U derivatives to generate a new family of shuttle vectors . These plasmids are stable both in Escherichia coli and in Desulfovibrio, they present a large number of unique restriction sites, and colonies of recombinant clones can be identified by blue/white screening in E . coli . The pBMC, pBMK, and pBMS series carry the cat, npt, or strAB genes as selectable markers, respectively . The pBMC6, pBMK6, and pBMS6 plasmids can be introduced both in D . desulfuricans and in Desulfovibrio fructosovorans by electrotransformation, and the pBMC7, pBMK7, and pBMS7 plasmids contain additional mobilization functions which makes them suitable for conjugation. Alcohol Clin Exp Res, 1998 Feb, 22(1), 135 - 41 Acute ethanol intoxication inhibits neutrophil beta2-integrin expression in rats during endotoxemia; Zhang P et al.; The effects of acute ethanol intoxication on neutrophil {polymorphonuclear leukocyte (PMN)} adhesion molecule expression and certain other functional properties during endotoxemia were studied in rats to elucidate the mechanisms underlying the immunosuppressive effects of ethanol . Acute ethanol intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg . Control animals received an intraperitoneal injection of saline . Thirty minutes after intraperitoneal injection, animals were given a 90-min intravenous infusion of Escherichia coli endotoxin (total dose of 112.5 microg/rat in 2.5 ml of saline) or saline . Certain rats received granulocyte colony-stimulating factor (G-CSF; 50 microg/kg in 5% dextrose, subcutaneous injection twice daily) or vehicle pretreatment for 2 days before intravenous endotoxin infusion . Endotoxemia significantly upregulated CD11b/c and CD18 expression on PMNs when compared with those of saline-infused rats . Acute ethanol intoxication inhibited this endotoxin-induced upregulation of CD11b/c and CD18 expression on PMNs . Ethanol intoxication also suppressed the phagocytic activities of PMNs in saline-infused rats, but this suppression failed to reach statistical significance in endotoxin-infused rats . Hydrogen peroxide generation by PMNs in saline- or endotoxin-infused rats was not affected by ethanol intoxication . Histological examination showed extensive PMN sequestration in the liver after endotoxin infusion, and ethanol intoxication significantly attenuated this hepatic sequestration of PMNs . G-CSF pretreatment enhanced neutrophil phagocytosis, CD11b/c and CD18 expression in endotoxin-infused rats, and prevented the ethanol-induced inhibition of neutrophil CD18 expression and phagocytosis . The impairment of beta2-integrin expression on PMNs may be one mechanism underlying ethanol-induced defects of neutrophil delivery into tissue sites of infection . G-CSF may be of benefit to the infected alcoholic host by enhancing leukocyte defense functions. Protein Sci, 1998 Jan, 7(1), 216 - 9 Fragment of GABA(A) receptor containing key ligand-binding residues overexpressed in Escherichia coli; Xue H et al.; GABA(A) receptor plays a major role in inhibitory synaptic transmission in the central nervous system and is the target of drugs such as the benzodiazepine tranquilizers . The polymeric membrane protein nature of GABA(A) receptor has rendered structural elucidation of the receptor a formidable task, greatly hampering structure-based drug design . We report here the first expression in Escherichia coli of a fragment of GABA(A) receptor . This 131-residue fragment, spanning Cys166 to Leu296 of human GABAA receptor alpha1 subunit, contains residues previously suggested to be involved in benzodiazepine binding . The overexpressed non-fusion recombinant protein was purified to near homogeneity and characterized by circular dichroism (CD), which showed that the recombinant protein has well defined secondary structures where beta-strands are dominant . The stability of the secondary structures was demonstrated by CD spectra at high pH and elevated temperature . Excluding part of the sequences from the carboxyl terminal of the fragment resulted in dramatic changes in the secondary structures comparable to the effects caused by SDS denaturation . Our results therefore suggest that the 131-residue fragment harbors an integral structural domain of the receptor . The overexpression of the recombinant protein fragment thus opens the way to the biochemical and structural studies of a functionally important region of the receptor, and exemplifies an effective approach of expression and characterization that potentially may be extended to other members of the ligand gated channel receptor superfamily, to which the GABA(A) receptor belongs. J Biomol Struct Dyn, 1998 Feb, 15(4), 689 - 701 Statistical and structural analysis of trp binding sites: comparison of natural and in vitro selected sequences; Haran TE; Two different modes can be used when the trp repressor binds to trp binding sites . In the "full-site mode" each repressor molecule is bound to a DNA target containing at least two conserved five base pair tracts separated by eight base pairs . The binding of the repressor to natural trp operators is of this kind . In the "half-site mode" two repressor molecules are sequence-specifically bound, with infinite cooperativity, to two abutting DNA pentamers . We present evidence suggesting that the sequences obtained by a recent in vitro selection assay (Czernik et al . J . Biol . Chem . 269, 27869-27875, 1994) were selected by the binding of two repressor molecules, and that the repressor is bound to most of these sequences using the half-site mode . Using the results of the selection assay, and the set of natural trp binding sites, we characterize the different sequence requirements of the "full-site" versus the "half-site" binding modes . A statistical analysis of the information content of these binding sites shows that functional information on protein binding modes can be extracted from a set of DNA binding sites by comparing the information content of two different DNA populations, or sub-populations . Furthermore, it shows that the binding of proteins to sequences selected by a functional in vitro assay do not necessarily mimic the binding of the protein to the natural targets, even if the information content is similar in the two DNA target populations, i.e., even if the stringency of the selection assay is adequate for locating natural-like sequences . In addition, we show that the structural requirements for protein-DNA interactions can be achieved by different conformations at the base-pair level . Differences in the structural characteristics of different base-pair steps can be used to determine the binding mode and differential binding affinity, which can be utilized in the regulation of several binding sites by a single specific protein. Protein Eng, 1997 Nov, 10(11), 1333 - 8 Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser; Mine S et al.; When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)-lysozyme) . The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid . A Met(-2)Pro(-1) or Met(-2)Ser(-1) sequence was introduced at the N-terminus of hen egg-white lysozyme . The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine . From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(-1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N-terminus of hen egg-white lysozyme (Pro(-1)-lysozyme) . Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg-white lysozyme. Protein Eng, 1997 Nov, 10(11), 1327 - 31 Expression, purification and characterization of the homeodomain of rat ISL-1 protein; Behravan G et al.; Isl-1 is a member of a family of Homeodomains containing proteins that possess an N-terminal pair of zinc binding LIM domains . The Isl-1 gene in rat codes for a protein that binds to the insulin gene enhancer and is also involved in regulation of amylin and proglucagon genes . A DNA sequence coding for 66 amino acid residues containing the C-terminal homeodomain fragment of Isl-1 was expressed as a soluble protein in Escherichia coli . Here, we describe a procedure which allows the rapid native purification of recombinant homeodomain protein fused to an N-terminal tag of six histidines . The purified homeodomain showed DNA-binding activity to its cognate DNA sequence . An enhanced binding activity is observed in the presence of a reducing agent in electrophoretic mobility shift assays . The DNA binding was further characterized by circular dichroism spectroscopy . Addition of DNA to the homeodomain did not change the overall secondary structure content, but the thermal and chemical denaturing profiles were altered . A stabilization of the secondary structure was observed upon DNA binding . The free energy of unfolding at 23 degrees C was 7 kJ mol(-1) in absence of DNA and 29 kJ mol(-1) in the presence of DNA. Protein Eng, 1997 Nov, 10(11), 1289 - 94 Synthesis and characterization of supramolecular protein aggregates: self-assembled, molecularly-ordered, tubes from electrostatic complementation of glutamine synthetase dodecamers; Chen JP et al.; Dodecameric Escherichia coli glutamine synthetase (GS) is formed from identical subunits arranged in face-to-face hexameric rings . In the presence of Zn2+ and other transition metal ions the individual dodecamers 'stack' to form protein tubes . Previous results have suggested that six binuclear intermolecular metal binding sites are generated at each dodecamer-dodecamer interface by juxtaposition of the N-terminal helices of each subunit adjacent to an analogous helix from a docked dodecamer . In principle, replacement of one of the metal binding sites within each pair of helices with charged amino acids could generate electrostatic interactions that would provide the basis for heterospecific protein-protein interactions . In turn, this would allow for ordered assembly of protein tubes with alternating, chemically distinguishable, components . This hypothesis was tested by replacement of one of the metalligating histidines (His12) with aspartic acid, arginine or cysteine . The H12C mutant was further elaborated by selective thiol modification, with either of the charged reagents 2-iodo-acetic acid or 2-chloro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12 . Light scattering and electron microscopy were used to monitor the 'stacking ability' of these variants in the presence of Zn2+ . No, or few, GS 'tubes' were observed in solutions containing only H12D, H12R, H12C-CA or H12C-IA, in the presence or absence of Zn2+ . In contrast, in mixtures containing H12C-CA and either H12D or H12C-IA, the complementary GS variants stack in the presence of 100 microM Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers . Fluorescence energy transfer experiments with fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA (acceptor) were performed and compared with the energy transfer efficiency with mixtures containing variable ratios of acceptor-labeled and donor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangements of donor and acceptor . The efficiency of metal-dependent energy transfer in mixtures containing the acceptor-labeled H12C-CA and the donor-labeled H12C-IA was 3.2-fold greater than expected for a random distribution of charged variants . Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction between chemically distinguishable dodecamers that align in an ordered one-dimensional array. Protein Eng, 1997 Nov, 10(11), 1281 - 8 Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments; Villbrandt B et al.; The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity . Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion) . Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase . In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates . Consequently two variants were designed and constructed using site-directed mutagenesis . The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones . Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability . After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank . A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model. Br J Cancer, 1998 Mar, 77(5), 709 - 19 NAD(P)H:quinone oxidoreductase 1 reduces the mutagenicity of DNA caused by NADPH:P450 reductase-activated metabolites of benzo(a)pyrene quinones; Joseph P et al.; The role of microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase) in the mutagenicity of benzo(a)pyrene-3,6-quinone (BP-3,6-Q) was studied using supF tRNA gene as the mutational target . pUB3 carrying the supF tRNA gene upon transformation into the Escherichia coli ES87 cells exhibited a spontaneous mutation frequency of 0.62 x 10(-6) . Chemical modification of the pUB3 DNA with BP-3,6-Q caused a fourfold increase in the mutation frequency, compared with the spontaneous mutations . P450 reductase catalysed metabolic activation of BP-3,6-Q into reactive products (semiquinone and reactive oxygen species), which caused a further increase in the mutation frequency to eightfold over spontaneous mutations . Oxygen radical scavengers (SOD and catalase) blocked the P450 reductase-activated BP-3,6-Q-induced stimulation of mutations . This indicates that redox cycling of the semiquinone leading to the generation of reactive oxygen species (ROS) was directly responsible for the increased mutation frequency of P450 reductase-activated BP-3,6-Q . Analysis of the mutation spectra revealed that P450 reductase-activated BP-3,6-Q showed a significantly higher preference for frameshift mutations, particularly deletions, compared with the spontaneous mutations and the mutations generated by benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) . The single most frequently observed mutation by P450 reductase-activated quinone (semiquinone + ROS) was deletion of a single guanosine . Among the base substitutions, G:C --> T:A, G:C --> A:T and G:C --> C:G were also noticed . Interestingly, NQO1 competed with P450 reductase and specifically prevented the P450 reductase-activated BP-3,6-Q-induced mutations . However, BP-hydroquinone (BP-3,6-HQ) generated during the metabolic reduction of BP-3,6-Q catalysed by NQO1 caused specific mutations involving the deletion of a single cytosine from the DNA sequence 5'-CCCCC-3' in supF tRNA gene at a significantly high frequency . A similar cytosine deletion was also observed with benzoquinone hydroquinone (HQ), indicating that the deletion of cytosine is associated with a hydroquinone class of compounds . These results suggest that: (1) quinones and P450 reductase-activated products of quinones (semiquinones and ROS) are mutagenic compounds; (2) the mutational spectra of quinones, semiquinones and hydroquinones differ from each other with respect to their mutational frequency and specificity; (3) NQO1 competes with P450 reductase and protects the cells from quinone mutagenicity; and (4) the NQO1 -metabolized quinones (hydroquinones), if not eliminated, cause specific mutations that are not observed with quinones and P450 reductase-activated quinones (semiquinones and ROS). FEMS Microbiol Lett, 1997 Nov 15, 156(2), 217 - 22 Analysis of the G93E mutant allele of KpsM, the membrane component of an ABC transporter involved in polysialic acid translocation in Escherichia coli K1; Pigeon RP et al.; KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1 . The kpsMG93E allele is a point mutation in the first cytoplasmic loop (Cl) of KpsM which partially disrupts translocation of the capsule . While producing polymer of wild-type length, strains harboring the G93E allele exhibit a decreased production of capsular polymer and a reduced rate of polymer translocation to the cell surface. J Mol Biol, 1998 Feb 20, 276(2), 505 - 15 Thermodynamic stability and folding of GroEL minichaperones; Golbik R et al.; The apical domain of GroEL (residues 191 to 376) and its C-terminally truncated fragment GroEL(191-345) are expressed with high yield in Escherichia coli to give functional monomeric minichaperones . Owing to the reversible folding behaviour of the minichaperones we can analyse the folding of the polypeptide binding domain of the multidomain GroEL protein, the folding of which is known to be irreversible . The apical domain shows two reversible temperature transitions with transition midpoints at 35 degrees C and at 67 degrees C that can be attributed to the unfolding of the C-terminal helices and the domain core, respectively . The native state of the domain core is stabilized by 5.5 kcal mol-1 relative to the unfolded state . The rate constant of folding of the apical domain core is independent of the minichaperone concentration and the presence of the C-terminal alpha-helices . A folding intermediate on the folding pathway is destabilized relative to the native state by 1.6 kcal mol-1, which is also detected by equilibrium and kinetic binding of the dye bis-ANS . Reversible folding of the polypeptide domain of GroEL guarantees highly efficient chaperonin activity within the GroEL toroid. J Mol Biol, 1998 Feb 20, 276(2), 405 - 15 Differential cleavage of LexA and UmuD mediated by recA Pro67 mutants: implications for common LexA and UmuD binding sites on RecA; Konola JT et al.; In Escherichia coli, RecA-mediated cleavage of LexA repressor is a key regulatory event required for expression of SOS genes involved in the repair of DNA damage . RecA also mediates the cleavage of UmuD protein to UmuD, a form active in SOS mutagenesis . To determine whether LexA and UmuD have common binding determinants on RecA, we have compared the ability of several recA mutants to function in the cleavage of LexA versus UmuD in vivo . The data reveal that while some recA mutations at Pro67 have a similar effect on LexA and UmuD cleavage, others have striking differential effects . For example, a Pro67-->Trp mutation results in a high level of constitutive cleavage of both proteins . However, Pro67-->Asp and Glu mutations promote constitutive cleavage of LexA and reduce induction of UmuD cleavage to just 5 to 10% of wild-type activity . In contrast, Pro67-->Arg prevents LexA cleavage while allowing nearly 50% of wild-type induction of UmuD cleavage . These results are consistent with the idea that Pro67 is located at a site in the nucleoprotein filament where both LexA and UmuD contact RecA. J Mol Biol, 1998 Feb 20, 276(2), 355 - 65 FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli; Negre D et al.; The start site of transcription of the ppsA gene, whose expression is controlled by the regulatory protein FruR in Escherichia coli, was determined by primer extension of in vivo transcripts . The interactions of the ppsA promoter with either RNA polymerase or FruR factor were analysed by the base removal method . Our results indicate that: (i) the RNA polymerase binding site has a -10 extended module but lacks its -35 hexamer; (ii) FruR binds to a target DNA region centered around position -45.5 upstream of the ppsA gene . In addition, circular permutation analysis showed that, upon binding to its site, FruR induces a sharp bend of 120 degrees in the DNA helix, which suggests a crucial involvement of FruR-induced bending in ppsA promoter activation . Direct contacts between the upstream activating DNA and RNA polymerase were studied in an in vitro transcription assay by using reconstituted RNA polymerase mutants containing Ala substitutions in C-terminal domain of their alpha subunit . The alpha{L262A}, alpha{R265A} and alpha{N268A} substitutions, which caused the most drastic reduction in the FruR-mediated activation of the ppsA promoter, had previously been shown to inhibit the upstream element-mediated activation at the rrnBP1 promoter. J Mol Biol, 1998 Feb 20, 276(2), 339 - 53 Molecular analysis of the regulation of csiD, a carbon starvation-inducible gene in Escherichia coli that is exclusively dependent on sigma s and requires activation by cAMP-CRP; Marschall C et al.; The general stress-induced sigma subunit sigma s of Escherichia coli RNA polymerase is closely related to the vegetative sigma factor sigma 70 . In view of their very similar promoter specificity in vitro, it is unclear how sigma factor selectivity in the expression of sigma s-dependent genes is generated in vivo . The csiD gene is such a strongly sigma s-dependent gene . In contrast to sigma s, which is induced in response to many different stresses, csiD, whose expression is driven from a single promoter, is induced by carbon starvation only . To our knowledge, the csiD promoter is the first characterized promoter which is not only exclusively dependent on sigma s-containing RNA polymerase (E sigma s), but also requires an activator, cAMP-CRP . In addition, leucine-responsive regulatory protein (Lrp) acts as a positive modulator of csiD expression . Also in vitro, E sigma s is more efficient than E sigma 70 in csiD promoter binding, open complex formation and run-off transcription, which might be due to the poor match of the csiD -35 region to the sigma 70 consensus and to transcription by E sigma s being less dependent on contacts in this region . By DNase I protection experiments, a cAMP-CRP binding site centered at -68.5 nucleotides upstream of the csiD transcriptional start site was identified . While cAMP-CRP stimulates E sigma 70 binding, it does not promote open complex formation by E sigma 70, but does so in conjunction with E sigma s . With linear templates, cAMP-CRP significantly stimulates E sigma s-mediated in vitro transcription, whereas transcription by E sigma 70 is negligible and hardly stimulated by cAMP-CRP . These findings may reflect different or less stringent positional requirements for an activator site for E sigma s than for E sigma 70, and indicate that cAMP-CRP contributes to sigma factor selectivity at the csiD promoter . In vitro transcription experiments with super-coiled templates, however, revealed significant cAMP-CRP-stimulated transcription also by E sigma 70 . Yet, under these conditions, H-NS was found to restore E sigma s specificity by strongly interfering with cAMP-CRP/E sigma 70-dependent transcription . Lrp strongly and cooperatively binds to multiple sites located between positions -14 and -102 (in a way that suggests DNA wrapping around multiple Lrp molecules) and moderately stimulates in vitro transcription, especially with E sigma s . In summary, we conclude that the csiD promoter has an intrinsic preference for E sigma s, but that also protein factors such as cAMP-CRP, Lrp and probably H-NS as well as DNA conformation contribute to its strong E sigma s selectivity . Furthermore, this strong E sigma s preference in combination with a requirement for high concentrations of the essential activator cAMP-CRP ensures csiD expression under conditions of carbon starvation, but not other stress conditions. J Mol Biol, 1998 Feb 20, 276(2), 313 - 8 Stepwise selection of TetR variants recognizing tet operator 4C with high affinity and specificity; Helbl V et al.; The TetR PQ39 mutant exhibits a new recognition specificity for the tetO-4C operator, but the affinity is not sufficiently high for use in vivo . A stepwise selection of additional mutations by cassette mutagenesis with randomization of residues in the TetR alpha-helix-turn-alpha-helix motif (HTH) yielded mutant TetR EA37PQ39YM42 showing a similar affinity and increased specificity for tetO-4C as wild-type TetR for tetO . A set of mutants obtained by that approach revealed that the fourth residue of the HTH (Leu41), which points towards the core of the DNA binding domain in TetR, alters the recognition of base-pair 4, e.g . the mutant TetR LV41YM42 exhibits a new recognition specificity for tetO-4G . A small residue at the last position in the turn of the HTH increases the affinity and specificity of DNA binding of TetR mutants containing the PQ39 exchange . Thus, cooperation between residues at positions 37, 39, 41 and 42 in the HTH of TetR is necessary to optimize recognition of base-pair 4 . We conclude that creating a new DNA recognition specificity in the HTH of TetR with high affinity for the tetO-4C operator variant requires exchanges altering flexibility and/or adjustment of the recognition alpha-helix to the target DNA in addition to the contacting residue. Biochim Biophys Acta, 1998 Feb 11, 1395(3), 309 - 14 Molecular cloning and characterization of the gene encoding glutathione reductase in Brassica campestris; Lee H et al.; We have isolated the Brassica campestris cDNA encoding glutathione reductase of 502 amino acid residues with molecular mass of 54.5 kDa . The deduced amino acid sequences were 92.2%, and 79.5% identical to those of Arabidopsis thaliana, and pea, respectively . As expected, it exhibited a high degree of conservation within the region responsible for the redox reaction and for the binding of GSSG or NADPH . The gene was highly inducible by ozone fumigation or by paraquat treatment. Biochim Biophys Acta, 1998 Feb 11, 1395(3), 259 - 65 Cloning and expression of the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA; Jehan F et al.; Using a cDNA probe from the rat 24-hydrovitamin D3 24-hydroxylase, the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA has been isolated from a chicken kidney lambda gt11 library . The high degree of similarity with the mammalian 24-hydroxylase cDNAs strongly supports the belief that it is the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA . The deduced amino acid sequences are also very well conserved and 325 of them are identical among the four known 25-hydroxyvitamin D3 24-hydroxylases . This cDNA expressed in E . coli produces 24-hydroxylase activity. Antisense Nucleic Acid Drug Dev, 1998 Feb, 8(1), 53 - 61 Molecular cloning and expression of cDNA for human RNase H; Wu H et al.; We have cloned, expressed, and purified to electrophoretic homogeneity a human RNase H . The enzyme has a molecular weight of 32 kDa, is Mg2+ dependent, and is inhibited by Mn2+ and N-ethylmaleimide . Its molecular weight and cleavage characteristics are consistent with type 2 human RNase H . The human RNase H we have cloned is highly homologous to Escherichia coli RNase HI (33.6% amino acid identity) and to other RNase H enzymes homologous to E . coli RNase HI . The enzyme is encoded by a single gene that is at least 10 kb in length and is expressed ubiquitously in human cells and tissues. Acta Biochim Pol, 1997, 44(3), 565 - 78 Expression of Lupinus luteus cDNA coding for PR10 protein in Escherichia coli: purification of the recombinant protein for structural and functional studies; Sikorski MM; The cDNA clones coding for two pathogenesis-related protein homologues of PR10 class, LlPR10.1A and LlPR10.1B, were identified in yellow lupin expression library of uninfected roots . The contribution of PR10 proteins to the overall mechanism of plant defence still remains unknown . In order to elucidate the structure and function of lupin PR10.1A protein, a substantial quantity of the protein was produced in an E . coli expression system using plasmids of pET-series: pET-3a and pET-15b, carrying the T7 promoter . Both plasmids with subcloned Llpr10.1a gene were overexpressed in E . coli, strain BL21(DE3)pLysS . The recombinant LlPR10.1A protein, overproduced in bacterial cells transformed with the pET-3a/Llpr10.1a plasmid, was purified to homogeneity from the insoluble "inclusion bodies" by ammonium sulphate fractionation and two sequential chromatographic steps: ion-exchange chromatography on DE 52 cellulose followed by size exclusion chromatography on Superdex 75 FPLC column . The (His)6 LlPR10.1A protein overproduced in E . coli cells harbouring the pET-15b/Llpr10.1a plasmid was purified by chromatography on Ni2+-charged His.Bind Resin . Western blot analysis with rabbit serum containing anti-LlPR10.1AN antibody revealed identical immunochemical properties of the two recombinant polypeptides and native LlPR10.1A protein . The recombinant protein produced in pET-3a plasmid was renatured from its insoluble form, concentrated up to 22 mg/ml and submitted to crystallisation . However, the LlPR10.1A protein expressed in pET-15b plasmid precipitated from the solution when at a higher concentration (10 mg/ml) . This preparation was used at a lower concentration as an antigen for the preparation of polyclonal antibodies for immunochemical studies. J Med Microbiol, 1997 Aug, 46(8), 669 - 74 Studies on the lysozyme independence of immune immobilisation of Treponema pallidum and the frequency of lysozyme autoantibodies in syphilitic sera; Zielinski S et al.; The role of lysozyme in the immune immobilisation of Treponema pallidum is not yet fully understood . The T . pallidum immobilisation assay was used to demonstrate that the immobilisation and lysis of T . pallidum in vitro by antibodies (serum, IgG fraction or IgM fraction) and complement proceed in a lysozyme-independent mode . In the presence of lysozyme the rate of immobilisation increased . In contrast with its effect on Escherichia coli, the effect of lysozyme on T . pallidum was governed exclusively by its enzymic activity rather than by the cationic protein nature of the molecule . Lysozyme, released from stimulated phagocytes, induced formation of lysozyme antibodies in 59.6% of syphilis patients as determined by lysozyme antibody ELISA . The highest frequency was found in patients with untreated secondary syphilis, whereas untreated primary syphilis was only rarely accompanied by the presence of lysozyme antibodies . Cross-reactivities between lysozyme and treponemal antigens were excluded by immunoblotting . The autoantibodies did not influence the lysozyme activity . It was concluded that the formation of lysozyme antibodies is only an epiphenomenon in the host defence against treponemal infection. Biochim Biophys Acta, 1998 Jan 27, 1363(1), 85 - 93 Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis; Schmitz S et al.; The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E . coli . Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies . The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR . Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin . Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR . A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation . In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor. Biochim Biophys Acta, 1998 Jan 27, 1363(1), 35 - 46 Reconstitution of cytochrome b-560 (QPs1) of bovine heart mitochondrial succinate-ubiquinone reductase; Lee GY et al.; The QPs1 subunit of bovine heart mitochondrial succinate-ubiquinone reductase was overexpressed in Escherichia coli DH5 alpha cells as a glutathione S-transferase fusion protein (GST-QPs1) using the expression vector, pGEX/QPs1 . The yield of soluble active recombinant GST-QPs1 fusion protein depends on the IPTG concentration, induction growth time, temperature, and medium . Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth with 0.5 mM IPTG at 27 degrees C in an enriched medium containing betaine and sorbitol . QPs1 is released from the fusion protein by proteolytic cleavage with thrombin . Isolated recombinant QPs1 shows one protein band in SDS-polyacrylamide gel electrophoresis corresponding to subunit III of mitochondrial succinate-ubiquinone reductase . However, partial N-terminal amino acid sequence analysis of recombinant QPs1 shows two extra amino acid residues, glycine and serine, at the N-terminus of mature QPs1, resulting from the recombinant manipulation . When isolated recombinant QPs1 is dispersed in 0.01% dodecyl maltoside, it is in a highly aggregated form with an apparent molecular mass of over 1 million . Recombinant GST-QPs1 contains little cytochrome b-560 heme . However, addition of hemin chloride restores the spectral characteristics of cytochrome b-560 . Cytochrome b-560 restoration varies with the amount of hemin used . Maximum reconstitution is obtained when the molar ratio of heme to fusion protein used in the system is 0.6 . Reconstituted cytochrome b-560 shows a EPR signal at g = 2.91 which corresponds to one of the EPR signals of cytochrome b-560 in a QPs preparation . When GST-QPs1 with reconstituted cytochrome b-560 is treated with thrombin to cleave GST from QPs1, no change in the absorption and EPR characteristics of cytochrome b-560 is observed, indicating that the bis-histidine ligands of reconstituted cytochrome b-560 are provided by QPs1. Gene, 1998 Jan 30, 207(2), 241 - 9 A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines; Hegedus DD et al.; A series of shuttle vectors have been constructed that allow expression of heterologous proteins in either dipteran or lepidopteran insect cell lines . Constitutive expression in a broad range of host cells is mediated by the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus (OpMNPV) immediate-early 2 (ie2) promoter . Alternatively, if inducible expression is required, for example to express cytotoxic proteins, a vector has been constructed that uses the Drosophila metallothionein (Mtn) promoter for metal-inducible protein expression in dipteran cell lines . A chimeric synthetic bacterial-OpMNPV ie promoter-Zeocin resistance gene cassette has been included to facilitate cloning in E . coli as well as the generation of stably transformed insect cell lines . The utility of the system is demonstrated by the constitutive and inducible expression of the highly processed glycosylphosphatidylinositol-anchored glycoprotein, human melanotransferrin, in transformed insect cell lines. Gene, 1998 Jan 30, 207(2), 197 - 207 Protein SRP54 of human signal recognition particle: cloning, expression, and comparative analysis of functional sites; Gowda K et al.; Signal recognition particle (SRP) plays a critical role in the targeting of secretory proteins to cellular membranes . An essential component of SRP is the protein SRP54, which interacts not only with the nascent signal peptide, but also with the SRP RNA . To understand better how protein targeting occurs in the human system, the human SRP54 gene was cloned, sequenced, and the protein was expressed in bacteria and insect cells . Recombinant SRP54 was purified from both sources . The protein bound to SRP RNA in the presence of protein SRP19, and associated with the signal peptide of in vitro translated pre-prolactin . Comparative sequence analysis of human SRP54 with homologs from all three phylogenetic domains was combined with high-stringency protein secondary structure prediction . A conserved RNA-binding loop was predicted in the largely helical M-domain of SRP54 . Contrary to general belief, the unusually high number of methionine residues clustered outside the predicted helices, thus indicating a mechanism of signal peptide recognition that may involve methionine-rich loops. Gene, 1998 Jan 30, 207(2), 187 - 95 Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments; Shingledecker K et al.; Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases . In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected . The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E . coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively . One type of genetic alteration of the RecA intein involved deletion of the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain . The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each . Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region . The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans . The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each. Gene, 1998 Jan 19, 207(1), 53 - 60 Identification of the Dictyostelium discoideum homolog of the N-ethylmaleimide-sensitive fusion protein; Weidenhaupt M et al.; The N-ethylmaleimide-sensitive fusion protein (NSF) is required for vesicular membrane fusion in multiple cellular functions . We have cloned a cDNA encoding the Dictyostelium discoideum homolog of the NSF protein . This cDNA hybridizes with a single fragment in Southern blots suggesting that NSF is encoded by a single gene in the amoeba . It is expressed constitutively during vegetative growth and throughout the differentiation cycle . The encoded gene product comprises 738 aa with a predicted molecular mass of 82 kDa . It shows the characteristic three-domain structure of NSF proteins . A more divergent amino-terminal part is followed by two highly conserved ATP-binding domains featuring Walker A and B signature sequences . The D . discoideum protein presents an overall aa sequence identity of 44% when compared to known NSF homologs . The monoclonal antibody 2E5 directed against Cricetellus griseus NSF recognizes a protein with a molecular weight of approx . 80 000 in a D . discoideum crude extract and the recombinant D . discoideum His6-NSF expressed in Escherichia coli. J Antimicrob Chemother, 1998 Jan, 41(1), 111 - 4 Association of organic solvent tolerance and fluoroquinolone resistance in clinical isolates of Escherichia coli; Oethinger M et al.; Fluoroquinolone resistance in Escherichia coli is principally caused by two kinds of mutation: those affecting the target proteins of the drugs, i.e . DNA gyrase and topoisomerase IV, and those affecting regulatory genes such as marA, soxS or robA . Recently, overexpression of the latter genes was linked to increased organic solvent tolerance in E . coli . Among 138 clinical fluoroquinolone-resistant and -susceptible clinical isolates of E . coli we found a high association between fluoroquinolone resistance and organic solvent tolerance . This finding suggests that E . coli may undergo an adaptive response to extrinsic substances other than quinolones, while mutating to fluoroquinolone resistance. Nature, 1998 Mar 5, 392(6671), 101 - 5 Structure of the Sec7 domain of the Arf exchange factor ARNO; Cherfils J et al.; Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state . As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo . Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins . In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic . An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 and GPS1, have been identified . These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain . The Sec7 domain contains the exchange-factor activity . It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Geal and Gea2 . Here we report the crystal structure of the Sec7 domain of human ARNO at 2 A resolution and the identification of the site of interaction of ARNO with Arf. Tissue Antigens, 1998 Feb, 51(2), 156 - 63 Identification of a human heavy chain antibody fragment directed against human platelet alloantigen 1a by phage display library; Okamoto N et al.; The human platelet alloantigen HPA-1a (PlA1) is responsible for most cases of post-transfusion purpura and neonatal alloimmune thrombocytopenia in the Caucasian population . HPA-1a and HPA-1b are two allelic forms of the platelet membrane glycoprotein IIIa (GPIIIa) gene that differ by a single amino acid . In this report, we describe the development of a recombinant heavy chain antibody fragment capable of distinguishing between the homozygous forms of HPA-1a and HPA-1b . This antibody fragment was isolated from the lymphocytes of an immunized individual through the use of a phage display library system . The recombinant antibody fragment reacted with human platelet lysates from HPA-1a homozygous donors, the HPA-1a form of recombinant N-terminal GPIIIa and intact HPA-1a platelets, but did not react with platelet lysate from HPA-1b homozygous donors, reduced HPA-1a form of platelet GPIIIa or other platelet glycoproteins . This HPA-1a specific human antibody fragment works well in common laboratory assays such as ELISA and flow cytometry, which can assist in identifying HPA-1b homozygous individuals who are known to have a higher risk for developing neonatal alloimmmune thrombocytopenia and post-transfusion purpura . Thus, selection of recombinant antibody fragment using phage display offers a promising alternative to hybridoma technology for the production of human antibodies against human alloantigens and holds potential as a technique in therapeutic applications. RNA, 1998 Mar, 4(3), 319 - 30 mRNA stabilization by the ompA 5' untranslated region: two protective elements hinder distinct pathways for mRNA degradation; Arnold TE et al.; The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer that can prolong the cytoplasmic lifetimes of a variety of messages to which it is fused . Previous studies have identified two domains of this 5' UTR that together are responsible for its stabilizing effect . One is a 5'-terminal stem-loop . The other is a single-stranded RNA segment (ss2) that contains a ribosome binding site highly complementary to 16S ribosomal RNA . Here we report a detailed investigation of the function of these two stabilizing elements . Our data indicate that mRNA protection by a 5' stem-loop requires no sequence features or thermodynamic stability beyond the minimum necessary for stem-loop formation . Stabilization by ss2 appears to result not from a high frequency of translation initiation, but rather from a high degree of occupancy of this 5' UTR segment by bound ribosomes . Although close spacing of translating ribosomes is not critical for message stabilization by the ompA 5' UTR, mRNA longevity does require the periodic passage of ribosomes through the protein-coding region . Unlike bound ribosomes, which hinder mRNA cleavage by RNase E, the 5' stem-loop appears to impede degradation of ompA mRNA via a distinct pathway that is RNase E-independent . These findings imply that the ompA 5' UTR prolongs mRNA longevity by impeding multiple pathways for mRNA degradation. Zhonghua Yi Xue Za Zhi (Taipei), 1998 Jan, 61(1), 39 - 43 Bilateral idiopathic infantile pyoceles: a case report; Hsieh DS et al.; A 15-day-old infant was admitted to the 803 Military Hospital with swelling, bilateral erythematous changes, a tender firm mass in the scrotum and fever of six hours' duration . Scrotal sonography showed accumulated fluid with internal echoes in both hydrocele sacs . Aspirated fluid was found to contain Escherichia coli and bilateral scrotal pyoceles was diagnosed . The most common etiologic factor for this is meconium peritonitis, with surgical drainage being the necessary treatment . The case is reported, with a review of the literature. Mol Cells, 1997 Dec 31, 7(6), 807 - 15 Expression of a cDNA encoding Phytolacca insularis antiviral protein confers virus resistance on transgenic potato plants; Moon YH et al.; To develop an antiviral agent and virus-resistant plants, a cDNA clone encoding Phytolacca insularis antiviral protein (PIP) was isolated from a cDNA library constructed with poly(A)+ RNA purified from leaves of P . insularis . The PIP cDNA contains an open reading frame encoding 307 amino acids . The deduced amino acid sequence includes a putative signal sequence of 22 amino acids at the N-terminus . The amino acid sequence of PIP shares 84% homology with that of the pokeweed antiviral protein (PAP) . In addition, the mature PIP exhibits the conserved putative active site found in other ribosome-inactivating proteins (RIPs) . Recombinant PIP (rPIP) synthesized in Escherichia coli inhibits protein synthesis in vitro in rabbit reticulocyte lysate through the N-glycosidase activity in a similar manner with other RIPs . Local lesion assays with purified rPIP revealed that it inhibits infection of various viruses to plants . Transgenic potato plants expressing the PIP cDNA under the control of the cauliflower mosaic virus 35S promoter are resistant to viruses, such as potato virus X, potato virus Y, and potato leafroll virus . These results suggest that the PIP cDNA could be used for the development of an antiviral agent and transgenic plants resistant against a broad spectrum of plant viruses infecting through both mechanical and aphid transmission. Mol Cells, 1997 Dec 31, 7(6), 769 - 76 Cloning and analysis of the DNA polymerase-encoding gene from Thermus filiformis; Jung SE et al.; The gene encoding Thermus filiformis (Tfi) DNA polymerase was cloned and its nucleotide sequence was determined . The primary structure of Tfi DNA polymerase was deduced from its nucleotide sequence . Tfi DNA polymerase is comprised of 833 amino acid residues and its molecular mass was determined to be 93,890 Da . The deduced amino acid sequence of Tfi DNA polymerase showed a high sequence homology to E . coli DNA polymerase I-like DNA polymerases: 78.5% homology to Taq DNA polymerase, 78.4% to Tca DNA polymerase, and 41.8% to E . coli DNA polymerase I . An extremely high sequence identity was observed in the region containing polymerase activity . The G + C content of the coding region for the Tfi DNA polymerase gene was 68.5%, which was higher than that of the chromosomal DNA (65%) . The G + C contents in the first, second, and third positions of the codons used were 71.8%, 40.9%, and 92.7% respectively . Codon usage in Tfi DNA polymerase was heavily biased towards the use of G + C in the third position . Rare codons with U or A as the third base were sometimes used to avoid using GA(A/T) TC and TCGA sequences, as they are recognition sites for the restriction endonucleases TfiI and TaqI. Jpn J Ophthalmol, 1997 Nov-Dec, 41(6), 355 - 61 Synthetic lipid A-induced uveitis and endotoxin-induced uveitis--a comparative study; Hanashiro RK et al.; Endotoxin-induced uveitis (EIU) is an animal model of ocular inflammation induced by lipopolysaccharide (LPS) . The lipid A (LA) region of the LPS chemical structure is believed to be responsible for virtually all the biological activities induced by LPS . The aim of this study was to perform a more detailed investigation of the potency of LA in reproducing EIU . Various doses of either LPS or LA were injected into the footpad of an inbred strain of Lewis rat and the inflammation patterns were compared by assessing the protein concentration, by cytological study, and by determining the inflammatory cell content in samples of aqueous humor obtained during 96-hour follow-up . Evaluation of the cell number and protein concentration ratio of both groups showed the LA-stimulated group presented a higher ratio than the LPS group (Welch's t-test, (P < 0.00001) . It was noteworthy that even the injection of high doses of LPS could not reproduce the level of cellular infiltration induced by LA . Histological study confirmed the enhanced cellularity in the LA group, neutrophils being predominant in both the LPS- and the LA-stimulated groups . The divergent findings in these two models of uveitis may be valuable to further investigations of the process of inflammatory cell migration into the anterior chamber of the eye. Mol Reprod Dev, 1998 Apr, 49(4), 426 - 34 Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins; Crosby JA et al.; During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida . To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs) . The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody . The recombinant truncated beta-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation . Three of the five recombinant forms (truncated beta-acrosin, Ser/Ala222-truncated beta-acrosin, and truncated beta-acrosin "heavy chain") had the ability to bind ZPGPs . The two shorter forms (the amino and carboxy termini of truncated beta-acrosin) failed to bind . The catalytic site mutant (Ser/Ala222) of truncated beta-acrosin does not differ from the recombinant truncated beta-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process . Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site. Neuroendocrinology, 1998 Feb, 67(2), 109 - 16 Endotoxin induces interleukin-1beta and nitric oxide synthase mRNA in rat hypothalamus and pituitary; Satta MA et al.; The gases nitric oxide (NO) and carbon monoxide (CO) may be involved in hypothalamo-pituitary-adrenal axis (HPA) modulation . In the brain, NO is synthesized by two forms of NO synthase (NOS), a constitutive neuronal form (nNOS) and an inducible form (iNOS) . There are also a constitutive heme oxygenase (HO2) and an inducible form (HO1) which generate CO . We have therefore investigated the effect of peripheral lipopolysaccharide (LPS) administration on the gene expression of these enzymes along with interleukin-1beta (IL-1beta) gene expression in the hypothalamus, pituitary and liver . Male Wistar rats (200-250 g body weight) were injected intraperitoneally with endotoxin (Escherichia coli, 055 B5) dissolved in sterile normal saline {250 microg/kg first group, 2.5 mg/kg (second group) and 6.25 mg/kg (third group)} in a final volume of 0.5 ml, or saline alone in the control group . The first and the second groups were studied 1, 3, 8 and 24 h after LPS (n = 4 per group); the third group was studied at 3 h . Total RNA was extracted from the hypothalamus, pituitary and liver, and cDNA was made using standard reverse transcriptase methods . Duplex polymerase chain reaction (PCR) was standardised in order to quantify the expression of a specific gene in relation to the 'house-keeping' gene beta-actin . The specific genes studied were iNOS, nNOS, HO1, HO2 and IL-1beta . The PCR products were separated on agarose gel and densitometric analysis of the bands allowed semi-quantification . In the second group, iNOS and IL-1beta were induced in hypothalamus, pituitary and liver, showing a peak at 3 h (p < 0.001), returning to baseline levels at 24 h . Neuronal NOS was not expressed in the liver under basal conditions or after LPS; in the hypothalamus and pituitary, nNOS was expressed basally but there was no change after LPS . In the first group, iNOS and IL-1beta were again induced in all three tissues studied, but with a delayed time course compared to the second and third groups; the peak change for IL-1beta occurred at 8 h (p < 0.05), again returning to baseline levels at 24 h . The peak for iNOS occurred at 24 h . HO1 and HO2 were expressed in all three tissues under basal conditions; HO1 was increased at 1 h in the liver in the second group, and at 3 h in the pituitary in the third group . There was no change in either HO1 or HO2 in the hypothalamus at any dose at any time point . We conclude that IL-1beta and iNOS are induced in rat hypothalamus and pituitary following various doses of endotoxin . We speculate that while IL-1beta may mediate stimulation of the HPA by endotoxin, NO generation may be involved in the counter-regulation of this response. Arch Biochem Biophys, 1998 Feb 15, 350(2), 283 - 90 Truncation of human squalene synthase yields active, crystallizable protein; Thompson JF et al.; Squalene synthase catalyzes the first committed step in cholesterol biosynthesis and thus is important as a potential target for therapeutic intervention . In order to determine the important functional domains of the protein, the amino and carboxyl terminal regions thought to be involved in membrane association of the enzyme were removed genetically . The 30 N-terminal amino acids were deleted with no apparent effect on activity . Additional deletion of 81 or 97 amino acids from the C-terminus completely ablated activity . However, a protein with a C-terminal deletion of 47 amino acids retained full activity . The latter enzyme was readily overexpressed in Escherichia coli and purified to homogeneity . The pure, doubly truncated enzyme exhibited a specific activity similar to that reported for the protease-solubilized rat liver enzyme, had a KM for farnesyl diphosphate similar to that observed for native enzyme, and was inhibited by anionic compounds to the same degree as native enzyme . Using the vapor diffusion method, the protein was crystallized as an enzyme-inhibitor complex, yielding orthorhombic crystals which diffracted to 2.2 A . Mutat Res, 1998 Jan 13, 412(1), 63 - 7 Bone marrow and liver mutagenesis in lacZ transgenic mice treated with hexavalent chromium; Itoh S et al.; The mutagenic effects of the hexavalent chromium compound K2CrO4 in lacZ transgenic mice (Muta Mouse) were investigated at two sampling times . K2CrO4 was administered intraperitoneally to five male mice per treatment group at a single dose of 40 mg/kg . The animals were sacrificed on days 1 and 7 after the treatment . Mutant frequencies in the bone marrow and liver were analyzed by the positive selection method using Escherichia coli C (galE-) strain and phenyl beta-D-galactoside . K2CrO4 induced a significant increase in mutant frequency in the bone marrow on day 1, but not on day 7 after the treatment . In the liver, on the other hand, a significant induction in the mutant frequency was seen on day 7, whereas no induction was observed on day 1 . The reason for the different responses to the mutagenic activity of K2CrO4 between these organs may be related to their cell turnover rates . The mutations induced by K2CrO4 in the bone marrow may have occurred in more differentiated cells than stem cells, and the rapid proliferative activity may have caused a rapid decrease in mutated cells by day 7 . These results suggest that experiments on mutagenesis should be done with more than one sampling point, a short expression time in addition to a longer one, so as to detect mutations induced in organ with high cell proliferation. J Clin Microbiol, 1998 Mar, 36(3), 840 - 2 Enteroaggregative, Shiga toxin-producing Escherichia coli O111:H2 associated with an outbreak of hemolytic-uremic syndrome; Morabito S et al.; Shiga toxin-producing Escherichia coli O111:H2 strains from an outbreak of hemolytic-uremic syndrome showed aggregative adhesion to HEp-2 cells and harbored large plasmids which hybridized with the enteroaggregative E . coli probe PCVD432 . These strains present a novel combination of virulence factors and might be as pathogenic to humans as the classic enterohemorrhagic E . coli. J Clin Microbiol, 1998 Mar, 36(3), 652 - 6 Epidemiological study of a food-borne outbreak of enterotoxigenic Escherichia coli O25:NM by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis; Mitsuda T et al.; This study investigated the applicability of molecular epidemiological techniques to the identification of the causal agent of an outbreak of diarrhea caused by ingestion of food contaminated with enterotoxigenic Escherichia coli (ETEC) . The outbreak occurred at four elementary schools in July 1996 and affected more than 800 people . Illness was most strongly associated with eating tuna paste (relative risk, 1.79; 95% confidence interval = 1.16 to 2.79; P = 0.0001) . To evaluate the epidemiological characteristics of the pathogen, the DNAs from numerous isolated ETEC strains were subjected to randomly amplified polymorphic DNA analysis, pulsed-field gel electrophoresis of nuclease S1-treated plasmid DNA, and analysis of genomic DNA restriction fragment length polymorphisms . All ETEC isolates were of the O25:NM (nonmotile) serotype, which carries a heat-stable enterotoxin Ib gene . Genotypic analysis demonstrated that the strains isolated from the patients at all four schools were identical . The isolates of ETEC O25:NM obtained from the tuna paste that had been served for lunch at these schools were genetically indistinguishable from those isolated from the patients . Results suggest that this outbreak was food borne . The molecular biology-based epidemiological techniques used in this study were useful in characterizing the causal agent in this food-borne epidemic. Oncol Res, 1997, 9(10), 553 - 63 Expression of the verotoxin receptor glycolipid, globotriaosylceramide, in ovarian hyperplasias; Arab S et al.; The presence of cell surface receptor glycolipid, globotriaosylceramide (Gb3), is essential to confer susceptibility to the E . coli-derived verotoxin (VT) . Our earlier studies showed that Gb3 is expressed in ovarian carcinoma cell lines . The Gb3 content of normal ovary, benign and malignant primary ovarian tumors, and their metastases have now been compared by verotoxin thin-layer chromatogram (TLC) overlay of the glycolipid tissue extracts . FITC-labeled VT1 B subunit binding to frozen tumor sections was also monitored histochemically . Low to undetectable levels of Gb3 were found in "normal" ovarian tissue . Gb3 was markedly increased in both benign and malignant tumors, suggesting that increased Gb3 may be related to proliferation, rather than malignancy per se . Mucinous tumors showed the least Gb3 elevation; serous tumors were variable, showing higher levels of Gb3 in less differentiated malignant tumors . By far the highest Gb3 content was observed for secondary ovarian metastases and tumors refractory to chemotherapy . Frozen sections of neoplastic ovarian tissue overlaid with fluorescein-conjugated VT1 B subunit show extensive binding to tumor cells, particularly in poorly differentiated samples and blood vessels adjacent to, and within, the tumor mass . Tumor foci were stained but stromal tissue was consistently negative both in primary tumors and metastases . VT staining of well-differentiated primary ovarian tumor sections was weak, corresponding to their low Gb3 content, but strong staining was observed in sections from a highly differentiated primary tumor from a patient who was unexpectedly refractory to clinical chemotherapy . These studies suggest that verotoxin/Gb3 targeting may provide the basis for new treatments for ovarian cancer. Biochim Biophys Acta, 1998 Jan 15, 1382(1), 47 - 54 Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica; Saavedra-Lira E et al.; The parasite Entamoeba histolytica is an organism whose main energetic source comes from glycolysis . It has the singularity that several of its glycolytic enzymes use pyrophosphate as an alternative phosphate donor . Thus, pyruvate phosphate dikinase (PPDK), an inorganic pyrophosphate (PPi)-dependent enzyme, substitutes pyruvate kinase present in humans . We previously cloned and sequenced the gene that codifies for PPDK in E . histolytica . We now report its expression in a bacterial system and its purification to 98% homogeneity . We determined its K(m) for phosphoenolpyruvate, AMP and PPi (21, < 5 and 100 microM, respectively) . Unlike PPDK from maize and bacteria and pyruvate kinase from other cells, EhPPDk is dependent on divalent cations but does not require monovalent cations for activity . The enzyme has an optimum pH of 6.0, it is labile to low temperatures and has a tetrameric structure . Since EhPPDK is a PPi-dependent enzyme, we also tested the effect of some pyrophosphate analogs as inhibitors of activity . Studies on the function and structure of this enzyme may be important for therapeutic research in several parasitic diseases, since it has no counterpart in humans. Electrophoresis, 1997 Dec, 18(15), 2852 - 6 Variability in the pattern of random amplified polymorphic DNA; Khandka DK et al.; The random amplified polymorphic DNA (RAPD) technique is a simple method to detect DNA polymorphism . It is sensitive to reaction conditions . Small changes in the reactants' concentration cause variations in amplification products . Using DNA from Asparagus officinalis, Dactylis glomerata, Mercurialis annua and Escherichia coli, we examined variability in the amplification pattern associated with reaction constituents . An increase in the ratio of Taq DNA polymerase to DNA in the reaction increased the number of amplified fragments . Increasing the concentration of primer resulted in the amplification of low molecular weight DNA fragments, while lowering the concentration resulted in high molecular weight fragments . Subsets of amplified fragments required different concentrations of magnesium for their highest intensity . Mechanical shearing of DNA obtained by sonication led to reduction in amplification of a subset of products . Enzymatic fragmentation of DNA by restriction enzymes led to loss or gain of specific fragments, depending on the DNA, primer, and restriction enzyme . RAPD markers of pooled DNA of anonymous pedigree should be critically evaluated for frequent 'false positive' markers. Electrophoresis, 1997 Dec, 18(15), 2703 - 8 Unique identification of proteins from small genome organisms: theoretical feasibility of high throughput proteome analysis; Cavalcoli JD et al.; We evaluate current levels of accuracy for estimation of molecular weight (Mr) and isoelectric point (pI) to proteins on two-dimensional (2-D) gels as well as the distribution and clustering of proteins in the predicted proteome of E . coli . We also examine the ability to find single candidates within the predicted proteome for matching to a protein seen on 2-D gels, based on the current level of accuracy . We discuss the levels of accuracy needed to match predicted proteins to observed proteins based solely on Mr and pI criteria obtained from genomic information, and propose methodology to achieve this level of accuracy . In addition, we will address the future goals of this work since the small genomes of bacteria provide a foundation and stepping stone to similar studies in higher organisms. J Virol Methods, 1997 Dec, 69(1-2), 147 - 58 Expression of Coxsackievirus B4 proteins VP0 and 2C in Escherichia coli and generation of virus protein recognizing antisera; Harkonen T et al.; Coxsackievirus B4 (CBV-4) capsid protein VP0 and non-structural 2C protein were expressed and purified using a glutathione-S-transferase (GST) fusion protein expression system . We used a full-size CBV-4 cDNA as a template to amplify the genes by polymerase chain reaction (PCR) . The genes were cloned into expression vector pGEX-2T and expressed as a fusion protein with GST . The GST-fusion proteins (GST-2C and GST-VP0) were purified in denatured and native forms and used to generate antibodies in rabbits . The antisera raised against GST-VP0 fusion protein recognized the corresponding structural proteins (VP0, VP2 and VP4) from purified CBV-4 preparations and infected cell lysates . In addition, cross-reactivity with CAV-9 and CBV-5 capsid proteins was observed . Anti-GST-2C antisera precipitated viral 2C protein in CBV-4-infected GMK cells, showing that the antibodies recognize the corresponding natural antigen. Biol Chem, 1998 Jan, 379(1), 33 - 8 RNase E, the major player in mRNA degradation, is down-regulated in Escherichia coli during a transient growth retardation (diauxic lag); Barlow T et al.; The endoribonuclease RNase E plays a major part in mRNA degradation in Escherichia coli in addition to its role in processing rRNA . RNase E is encoded by an essential gene, rne, also known as ams and hmp, which is autoregulated post-transcriptionally . Here we report a transient decrease in the steady state level of the full-length rne transcript and a corresponding decline in the amount of the protein and enzymatic activity . During this period an mRNA fragment, lacking an intact 5' end, accumulates . This down-regulation of RNase E occurs under aerobic growth conditions in rich medium during a short diauxic lag in mid-exponential phase; it most likely reflects an exhaustion of a not yet identified medium compound which is followed by switching on a new metabolic pathway . During this lag, the levels of bulk protein are maintained . Our results suggest that a transient drop in the intracellular RNase E level is a means of cells to retard mRNA turnover in a period of adjustment to medium utilization . Furthermore, the here described regulation of the rne transcript and its cognate gene product seems to occur by an RNase E-independent mechanism responsive to changes in growth conditions. J Biochem (Tokyo), 1998 Jan, 123(1), 128 - 35 Mechanism of mitochondrial import of adenylate kinase isozymes; Nobumoto M et al.; Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of the cellular adenine nucleotide composition . Three isozymes, AK1, AK2, and AK3, have so far been characterized in vertebrates . They are located in different tissues, while their primary and tertiary structures are similar . Among them, AK2 and AK3 are located in mitochondria, but unlike most mitochondrial proteins, both proteins lack a cleavable presequence . In this study, we first confirmed that AK2 is distributed in liver cells in both the cytosol and the intermembrane space of mitochondria, while AK3 is localized exclusively in the mitochondrial matrix . Next, we analyzed the process of import of AK2 and AK3 by incubating isolated rat mitochondria with proteins that were synthesized in a reticulocyte lysate translation system . The results indicated that both AK2 (an intermembrane-space-targeting protein) and AK3 (a matrix-targeting protein) require an inner membrane electrochemical potential for their import . This finding for AK2 is in contrast with those of other noncleavable intermembrane-space-targeting proteins such as cytochrome c and cytochrome c heme lyase, which do not require the membrane potential for their import . In the transport process, AK2 and AK3 competed with the adrenodoxin precursor, which is imported into the matrix through a mechanism common to other mitochondrial matrix proteins . Thus, AK2 and AK3 were thought to be translocated into mitochondria through the same pathway as that for most mitochondrial protein precursors . Neither AK2, that was previously synthesized in reticulocyte lysates, nor AK2, that was purified from an Escherichia coli overexpression system, was imported into mitochondria in a post-translational import manner . In contrast, AK3 was imported into mitochondria after completion of protein synthesis . Thus, the import of AK2 is likely to be co-translational, and the co-translational import mechanism might contribute to the bi-topological distribution of AK2 in both the cytosol and mitochondria. J Biochem (Tokyo), 1998 Jan, 123(1), 47 - 54 Cloning and expression of human adenylate kinase 2 isozymes: differential expression of adenylate kinase 1 and 2 in human muscle tissues; Lee Y et al.; A cDNA clone coding for adenylate kinase 2B was isolated from fetal liver, and the expression of AK2 was investigated in human tissues . The ORF in the cDNA clone for human AK2B predicted a protein comprising 232 amino acids (25.6 kDa) . The features of AK2A and AK2B sequences in human were the same as those in the bovine system . Each of the recombinant proteins, AK2A and AK2B, was expressed in Escherichia coli cells, and the purified recombinant proteins were enzymatically active . The distribution of AK2 transcripts in various human tissues was examined by Northern analysis . Unlike in the bovine system, it was found that the AK2A transcript was the major form of AK2 mRNA species in all human tissues . The transcripts of AK2 isozymes were relatively abundant in heart, liver, and also in skeletal muscle, where the expression level of AK2 was known to be low . Western blot analysis of AK isozymes in human heart and skeletal muscle revealed that AK2 protein was found only in heart, whereas AK1 was detected in both tissues . These tissue-specific expressions of the AK isozymes in human might suggest the presence of organ-specific regulation of the AK2 gene including a post-transcriptional control in skeletal muscle. J Biochem (Tokyo), 1998 Jan, 123(1), 33 - 41 Nonadditive effects of double mutations at the flexible loops, glycine-67 and glycine-121, of Escherichia coli dihydrofolate reductase on its stability and function; Ohmae E et al.; The structure, stability, and enzymatic function of dihydrofolate reductase (DHFR) from Escherichia coli are influenced by point mutations at sites 67 and 121 in two flexible loops {Gekko et al . (1994) J . Biochem . 116, 34-41; Ohmae et al . (1996) J . Biochem . 119, 703-710} . In the present study, eight double mutants at sites 67 and 121 (G67V/G121S, G67V/G121A, G67V/G121C, G67V/G121D, G67V/G121V, G67V/G121H, G67V/G121L, and G67V/G121Y) were constructed in order to identify interactions between the two sites of DHFR . The far-ultraviolet circular dichroism spectra of double mutants were clearly different from those of the respective single mutants, with significant changes being observed for three mutants, G67V/G121A, G67V/G121L, and G67V/G121S . The Gibbs free energy change of urea unfolding of double mutants could not be expressed by the sum of those of the respective single mutants except for G67V/G121H . The steady-state kinetic experiments showed that the effect of double mutations manifests itself not in Km but in k(cat), and the transition-state stabilization energy for G67V/G121A, G67V/G121C, and G67V/G121L is not equal to the sum of those for the single mutants . These results indicate that the additivity rule essentially does not hold for these double mutants, and that long-range interactions occur between sites 67 and 121, even though they are separated by 27.7 A . This is evidence that the flexible loops play important roles in the stability and function of this enzyme through structural perturbations, in which a small alteration in local atomic packing due to amino acid substitution is cooperatively magnified over almost the whole molecule. Gac Med Mex, 1997 Nov-Dec, 133(6), 511 - 25 {Continuous and common epitopes present in fimbriae of enterotoxigenic Escherichia coli (ETEC)}; Lopez-Vidal Y; Colonization factor antigens (CFAs and PCFs) are important virulence factors of Escherichia coli (ETEC) diarrhea . Antibodies to CFAs produced after ETEC infection are protective; however, the CFA epitopes which induce protective antibodies have not yet been characterized . This study is the characterization of the immune response to CFAs at molecular level and identification of the epitopes associated with inhibition of cell-adherence and protection that will lead to the development of methods to prevent ETEC infection and disease . The aim of this study was the characterization of the linear epitopes of CFA/I that react with sera from acute and convalescent phase of ETEC-in-fected children, with adult sera from endemic and non-endemic areas, with monoclonal antibodies (Mabs) and with hyperimmune antiserum to CFAs and PCFs different from CFA/I . Three linear and common epitopes were recognized among the CFA/I in child sera and adult sera from endemic areas and with hyperimmune sera against other known CFAs and putative ETEC colonization factors. FEMS Microbiol Lett, 1998 Feb 15, 159(2), 343 - 7 Modification of topA in Synechococcus sp . PCC 7942 resulted in mutants capable of growing under low but not high concentration of CO2; Gabay C et al.; Insertion of a cartridge encoding kanamycin resistance within an open reading frame, ORF839, in the cyanobacterium Synechococcus sp . PCC 7942 resulted in merodiploids bearing both the normal and the modified ORF839, suggesting that its gene product is essential for growth . In the absence of kanamycin the mutants were able to grow like the wild type, but in its presence the mutants grew under 0.015% CO2 in air but not under 5% CO2 in air . ORF839, identified in this study, is highly homologous to topA encoding topoisomerase I in several organisms, but it does not contain the zinc-binding motif identified in the C-terminal region of the enzyme from Escherichia coli. FEMS Microbiol Lett, 1998 Feb 15, 159(2), 201 - 7 Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei; Schupp T et al.; The 54-kbp Type I polyketide synthase gene cluster, most probably involved in rifamycin biosynthesis by Amycolatopsis mediterranei, was cloned in E . coli and completely sequenced . The DNA encodes five closely packed, very large open reading frames reading in one direction . As expected from the chemical structure of rifamycins, ten polyketide synthase modules and a CoA ligase domain were identified in the five open reading frames which contain one to three polyketide synthase modules each . The order of the functional domains on the DNA probably reflects the order in which they are used because each of the modules contains the predicted acetate or propionate transferase, dehydratase, and beta-ketoacyl-ACP reductase functions, required for the respective step in rifamycin biosynthesis. Vopr Med Khim, 1997 Nov-Dec, 43(6), 440 - 56 {Isolation and characterization of an evolutionary precursor of human monoamine oxidases A and B}; Singer TP et al.; An interesting flavoprotein-type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned by Schilling and Lerch (1995a,b) The properties of this MAO, as well as a substantial part of its amino acid sequence resemble those of both MAO A and B from higher animals, raising the possibility that it may be an evolutionary precursor of these mitochondrial enzymes . It differs from MAO A and B in several respect, however, including the fact that it is soluble and of peroxisomal localization and that the FAD is non-covalently attached . We have overexpressed the fungal enzyme (MAO-N) in Escherichia coli, isolated it for the first time in pure form, and, in collaboration with Dr . Elena Sablin, crystallized it . Since several of the observations of previous workers on MAO-N could not be reproduced and seem to be erroneous, we have reexamined its, substrate specificity, interaction with reversible and irreversible inhibitors and other catalytic and molecular properties . MAO-N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme . Some aspects of the substrate specificity resemble those of MAO B, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots . Contrary to the report of Schilling and Lerch (1995a), however