Biochimie, 1987 Sep, 69(9), 939 - 48 Interaction of ribosomal proteins L25 from yeast and EL23 from E . coli with yeast 26S and mouse 28S rRNA; el-Baradi TT et al.; The interaction of ribosomal protein EL23 from E . coli and L25 from yeast with yeast 26S rRNA was analysed by nitrocellulose filter binding and RNase protection experiments using both intact rRNA and various fragments prepared by in vitro transcription of cloned yeast rDNA regions in the SP6 system . The results show that EL23 efficiently and specifically interacts with the region of 26S rRNA previously identified as the binding site for the yeast ribosomal protein L25 . A comparison of the oligonucleotides resulting from limited RNase T1 digestion of the heterologous EL23/26S rRNA complex with those obtained by the same treatment of the homologous L25/26S rRNA complex showed that the molecular details of the two r-protein/rRNA interactions are highly similar if not identical . Using the synthetic 26S rRNA fragments we could demonstrate that all information for the formation of a biologically active binding site is located within the region of the rRNA delimited by the sequences protected by L25 against RNase T1 digestion . Part of the sequence at the 3' end of the 5'-distal protected region, however, was found not to be essential for r-protein binding although it does enhance the efficiency of this binding . Binding experiments using synthetic mouse 28S rRNA fragments showed that neither EL23 nor L25 interact with the structural equivalent of their respective cognate binding sites present in this mammalian rRNA . We argue that the structure of the expansion sequence present in this region of mouse 28S rRNA is a major cause of this failure. Mol Microbiol, 1987 Sep, 1(2), 195 - 201 An E . coli promoter induced by the cessation of growth; Connell N et al.; The production of the bacterial DNA replication inhibitor Microcin B17 is induced as cultures enter stationary phase . Using S1 nuclease protection assays we have shown that this induction is the result of increased levels of transcription initiation from a promoter located upstream from mcbA, the structural gene for Microcin B17 . Upstream from the start site of transcription there is a rather typical -35 region . However, there is no good homology to the consensus -10 region . While most of the cell's transcription is shut off as a result of the cessation of growth, transcription from the mcbA promoter continues for several hours in stationary phase . A single-copy gene fusion between mcbA and lacZ was used to monitor the response of the promoter to different nutritional conditions and in different host backgrounds altered in metabolic regulatory loci . Starvation for nitrogen, phosphate or carbon sources all induced transcription from the promoter . Levels of transcription were reduced in ompR backgrounds . In contrast, mutations in other global regulatory loci, fnr, relA and cya had little or no effect. Mol Gen Mikrobiol Virusol, 1987 Sep, (9), 10 - 6 {Weakening of type I restriction in E . coli: the effect of dam mutation}; Belogurov AA et al.; The host-controlled EcoK-restriction of unmodified phages lambda.0 and T7ocr . is 100-fold alleviated in dam- mutants of E . coli . In addition the EcoK modification activity is considerably decreased in dam- strains . The I and III types restriction (EcoB, EcoD, EcoK and EcoP1) were relieved in dam- mutants, but no alleviation of EcoRI restriction occurred in dam- strains . We interpret the alleviation of the I type restriction in dam- mutants as consequence of induction of the function, which interferes with the I type restriction systems. FEBS Lett, 1987 Aug 17, 220(2), 347 - 52 Tryptophan 54 and phenylalanine 60 are involved synergistically in the binding of E . coli SSB protein to single-stranded polynucleotides; Casas-Finet JR et al.; The binding of both wild-type and point-mutated E . coli single-stranded DNA-binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy . Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies . Investigation of a point mutation in the E . coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe-60 is replaced by alanine strongly suggests the participation of Phe-60 in the binding process, possibly by the formation of an extended stacking structure by Trp-54, thymine and Phe-60 . This hypothesis is supported by results on the point mutations in which His-55 is replaced by either leucine or tyrosine. FEBS Lett, 1987 Aug 17, 220(2), 283 - 7 Similarities between a predicted secondary structure for the M1 RNA ribozyme and the tRNA binding center of 16 S rRNA from E . coli; Boehm S; We propose a new model for the secondary structure of the M1 RNA component of E . coli RNase P which is based on significant sequence homologies with parts of the E . coli 16 S rRNA . A large domain of the new model resembles closely the secondary structure of the tRNA binding center of 16 S rRNA . We suggest that this domain of M1 RNA when functioning as a ribozyme binds the mature part of the precursor tRNA. FEBS Lett, 1987 Aug 10, 220(1), 167 - 76 The GATATC-modification enzyme EcoRV is closely related to the GATC-recognizing methyltransferases DpnII and dam from E . coli and phage T4; Lauster R et al.; The amino acid sequence of EcoRV DNA methyltransferase which methylates the amino group of the 5'-adenine residue of the target sequence GATATC has been found to be closely related to that of three other adenine methyltransferases, DpnII, dam and damT4, the target sequence of which is GATC . Despite large differences on the DNA level, the four sequences show four blocks of homologies . One of these blocks has the sequence DVYXDPPY and is found with little modification in numerous other DNA methyltransferases . It is speculated that it could be the binding site of the methyl donor, S-adenosylmethionine . On the other hand, the identification of a DNA-binding region is more tenuous . As expected, no analogies with (dimeric) repressors and cro proteins which have the characteristic helix-turn-helix motif have been observed. FEBS Lett, 1987 Aug 10, 220(1), 136 - 42 Selectivity for maltose and maltodextrins of maltoporin, a pore-forming protein of E . coli outer membrane; Dargent B et al.; Homogenous maltoporin (lamB protein), an Escherichia coli outer membrane spanning protein, was incorporated in phospholipid planar bilayers . It generates aqueous channels distinct from those formed by the non-specific porin (OmpF) or by phosphoporin (phoE protein) . The single conductance, 150 pS in 1 M NaCl, is much smaller than that of the porins . The channels, which are poorly selective for cations and voltage independent, are specifically inhibited by maltose and maltodextrins . This inhibition, observed in the absence of maltose binding protein, demonstrates that the selectivity of maltoporin for maltose and maltodextrins is an intrinsic property of the protein. Mutat Res, 1987 Aug, 179(2), 143 - 9 Thermal resistance to photoreactivation of ultraviolet light induced mutations in the lacI gene of E . coli ung; Fix DF et al.; Ultraviolet light (UV) induced mutations in the lacI gene of Escherichia coli are thought to be targeted by DNA photoproducts . A number of reports suggest that both cyclobutyl pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts may be involved . To investigate the potential contribution of each of these DNA photoproducts to mutagenesis in the lacI gene, we held UV-irradiated cells at a temperature of 44 degrees C for 75 min and then exposed them to photoreactivating light (PR) . This protocol is expected to preferentially deaminate specifically those cytosines that are contained in cyclobutyl dimers and subsequently monomerize the dimers to yield uracils in the DNA . In a strain deficient for uracil-DNA glycosylase (Ung-), these uracils would not be removed and a G:C----A:T transition would result at the site of the dimer . This protocol resulted in the enhancement of amber nonsense mutations that result from transitions at potential cytosine-containing dimer sites . The enhanced mutation frequencies resulting from this procedure were used to estimate the probability of dimer formation at the individual sites . A comparison of the dimer distribution with the mutation frequencies following UV alone suggests that both cyclobutyl dimers and (6-4) photoproducts contribute to UV-mutagenesis in the lacI gene . In addition, we conclude that the frequency of mutation at any particular site not only reflects the occurrence of DNA damage, but also the action of metabolic processes that are responsible for DNA repair and mutagenesis. Biochem Biophys Res Commun, 1987 Jul 31, 146(2), 470 - 7 Expression of cDNA encoding human basic fibroblast growth factor in E . coli; Iwane M et al.; The cDNA encoding human basic fibroblast growth factor was expressed in E . coli under the control of trp promoter . Bacterially synthesized hbFGF was highly purified using a heparin affinity HPLC column . By this chromatography, hbFGF was eluted as four distinct forms, which were indistinguishable by SDS polyacrylamide gel electrophoresis, amino acid composition, and partial terminal sequence analysis . These molecules stimulated the growth of fibroblasts and endothelial cells although their specific activities varied . The angiogenesis activity of these molecules was also confirmed. Cell, 1987 Jul 31, 50(3), 485 - 94 A bacterial gene involved in transcription antitermination: regulation at a rho-independent terminator in the bgl operon of E . coli; Mahadevan S et al.; We have investigated the mechanism of regulation of the bgl operon in Escherichia coli K-12 . A regulatory region has been located downstream of the bgl promoter, revealing a 130 base leader containing a sequence from +64 to +112 characteristic of a rho-independent terminator . In vitro, over 90% of the transcripts initiated at the bgl promoter terminate within the leader, at the 3' end of the terminator . Transcriptional fusions containing the terminator require the bglC gene in trans for expression; fusions deleted for the sequence show bglC-independent expression . A mutation resulting in partially constitutive expression of the fusion maps within the terminator . We propose that the bglC gene product mediates positive regulation of the bgl operon by functioning as an antiterminator at the rho-independent terminator located within the leader. Cell, 1987 Jul 31, 50(3), 495 - 508 The physical map of the whole E . coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library; Kohara Y et al.; Thirty-four hundred lambda phage clones containing segments of the E . coli chromosome were isolated and used to construct a 4700 kb long integrated restriction map for eight six-base-recognizing enzymes by a rapid mass-analysis method . Our strategy was to measure the sizes of partial restriction enzyme digests by hybridization with a vector probe in a manner analogous to nucleotide sequencing . The data were sorted into groups by a computer program and the boundary clones were further correlated with each other using a mass hybridization method . These clones can be exploited for the isolation of any desired E . coli genes if their map positions are known . Also, the strategy is applicable to analyses of the genomes of other organisms. Cell, 1987 Jul 17, 50(2), 259 - 65 ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E . coli chromosome; Sekimizu K et al.; ATP is bound to dnaA protein with high affinity (KD = 0.03 microM) and hydrolyzed slowly to ADP in the presence of DNA . ADP is also bound tightly to dnaA protein and exchanges with ATP very slowly . The ATP form is active in replication; the ADP form is not . A unique conformation of oriC, formed in an early initiation stage, depends on dnaA protein being in the ATP form . The subsequent entry of dnaB protein to form a prepriming complex also requires ATP binding and is blocked by bound ADP . Inasmuch as hydrolysis of ATP is far slower than these initiation reactions and since the poorly hydrolyzable analogue ATP gamma S can replace ATP, the ATP function appears to be allosteric . The extraordinary affinity of ATP for dnaA protein, its slow hydrolysis to ADP, the profound inhibition of dnaA functions by ADP, and the very slow exchange of ADP all point to a possible regulatory role for these nucleotides in the cell cycle. Nucleic Acids Res, 1987 Jul 10, 15(13), 5241 - 50 Specific and cooperative binding of E . coli single-stranded DNA binding protein to mRNA; Shimamoto N et al.; Fluorometric titration of E . coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA . The binding inhibited in vitro expression of ssb and bla but not nusA . This inhibition takes place at a physiological concentration of SSB . The function of the protein in gene regulation is discussed. Bioorg Khim, 1987 Jul, 13(7), 992 - 5 {Localization of a histidine residue in the binding site for the initiating substrate of E . coli RNA-polymerase}; Grachev MA et al.; E . coli RNA polymerase was selectively labelled in the presence of promoters at a histidine residue of the beta-subunit by treatment with GDP beta-imidazolide and then with {alpha-32P}UTP (or {alpha-33P}UTP) . Partial cyanogen bromide cleavage of the labelled polypeptide afforded a series of "single-hit" labelled peptides, the electrophoretic pattern of which suggested that the labelling site was His1237 . This conclusion was confirmed by a similar pattern obtained with products of the cyanogen bromide cleavage of a radioactive peptide obtained by the limited trypsinolysis (C-terminal peptide consisting of 423 amino acid residues) . Interpretation of our earlier results in favour of His1116 as the labelling point (Dokl . Acad . nauk SSSR, 1985, v . 281, p . 723) was incorrect due to the electrophoretic "compression" of three labelled peptide bands. Biofizika, 1987 Jul-Aug, 32(4), 647 - 51 {Theoretical analysis of H+/O stoichiometry during aerobic oxidation in E . coli in the stationary state}; Drozdov-Tikhomirov LN et al.; A general scheme of E . coli respiratory chain under aerobic oxidation with NAD.H2 is considered . The ratio H+/O is calculated by the currents method for the respiratory chain in a stationary state . The maximal possible stoichiometry is shown to equal 8 . The origin of different H+/O values in the respiratory chains is discussed. Prikl Biokhim Mikrobiol, 1987 Jul-Aug, 23(4), 530 - 5 {Behavior of the large fragment of DNA polymerase I (the Klenow fragment) during fractionation of a cell-free extract of E . coli MRE-600}; Khomov VV et al.; Distribution of the DNA polymerase I large fragment (Klenow fragment) was studied during fractionation of the E . coli MRE-600 cell-free extract with polyethylenimine . On the basis of the results obtained a simple procedure is proposed that enables the Klenow fragment to be obtained as a coproduct of DNA polymerase I, RNA polymerase, polynucleotide phosphorylase, nucleotide kinases with acetokinase and nucleoside deoxy-ribosyltransferase in the framework of a combined technological scheme. Acta Physiol Scand, 1987 Jul, 130(3), 359 - 66 The role of prostanoids in the feline intestinal vascular and central haemodynamic responses to i.v . infusion of live E . coli; Schutzer KM et al.; Bacterial infusion in the cat, causing experimental septic shock, induces an early vascular response mainly characterized by pulmonary hypertension and intestinal vasoconstriction . Prostanoids are held to be important mediators of the pulmonary vascular reaction . This study was performed to explore the involvement of prostanoids in the central haemodynamics and the small intestinal vascular reactions in experimental septic shock . Aortic blood pressure was continuously monitored, as were aortic blood flow, the pressure in a . pulmonalis and the small intestinal venous outflow . All cats (n = 24) were given live E . coli (10(10) ml-1) as a continuous intravenous infusion . One series was pretreated with indomethacin, another with UK-38,485, a specific thromboxane A2 synthetase inhibitor, and a third series served as untreated control . The pulmonary hypertensive response was clearly attenuated in the two pretreated series, in fact abolished in the one given UK-38,485 . The early intestinal vasoconstriction was eliminated in the two pretreated series . Later during bacteraemia, when untreated and indomethacin-pretreated cats showed intestinal vasoconstriction, UK-38-485-pretreated animals kept intestinal blood flow within the preseptic range . These data suggest that in the cat, thromboxane A2 is the prostanoid mediating the vascular reactions, not only in the lung but also in the small intestine. Nucleic Acids Res, 1987 Jun 25, 15(12), 4973 - 85 A common structural feature in promoter sequences of E . coli; Tung CS et al.; We have searched promoter regions of E . coli, structural genes of the same organism, and computer-generated random sequence DNA for the occurrence of common structural features . This is done by converting the base sequence to a series of numbers representing the sequence of helix twist angles and examining these numerical sequences statistically . Common structural features are shared by the promoter regions with a much higher frequency than are found in structural genes or in random sequences . These structures appear to be scattered randomly throughout the promoters, both in terms of the number of such structures per promoter and in terms of location within each promoter . One particular structure consisting of five successive helix twist angles is reported, along with a list of 60 different hexanucleotide sequences that share this structure . The locations of these structural elements in 61 E . coli promoters are also tabulated. J Biol Chem, 1987 Jun 25, 262(18), 8574 - 83 Optically detected magnetic resonance of tryptophan residues in Escherichia coli ssb gene product and E . coli plasmid-encoded single-stranded DNA-binding proteins and their complexes with poly(deoxythymidylic) acid; Casas-Finet JR et al.; Optically detected magnetic resonance (ODMR) spectroscopy has been applied to several single-stranded DNA-binding (SSB) proteins encoded by conjugative plasmids of enteric bacteria . Fluorimetric equilibrium binding isotherms confirm their preferential binding to single-stranded DNA and polynucleotides and reveal a limited protein solubility at low ionic strength . The plasmid SSB-like proteins show the highest affinity for polydeoxythymidylic acid; these complexes are the least sensitive to disruption by salt . ODMR data on these complexes suggest the existence of stacking interactions between tryptophan residue(s) and thymine bases, as evidenced by spectral red shifts of the tryptophan phosphorescence 0,0 band, reduction of the magnitude of D zero field splitting parameter, and a dramatic reversal of the polarity of the ODMR signals . Wavelength-selected ODMR results point to the existence of two distinct tryptophan sites in these complexes . The triplet state properties of the red-shifted site are drastically altered by its interaction with the thymine bases . The chromosomal Escherichia coli SSB protein-poly(dT) complex shows an additional tryptophan site with zero field splitting parameters similar to those of the free protein . This site can be attributed to Trp-135, which is missing in each of the other plasmid SSB proteins, suggesting that this particular residue is not involved in the interaction with polynucleotides. Biosci Rep, 1987 Jun, 7(6), 517 - 23 Effect of E . coli endotoxin on temperature, oxygen consumption and brown adipose tissue thermogenesis in rats and mice; Jennings G et al.; The effects of E . coli endotoxin 0127 B8 on oxygen consumption, temperature, and on the activity of the proton conductance pathway in brown adipose tissue (BAT) were investigated in rats and mice . In rats an increase was observed in rectal and skin temperature, whole body oxygen consumption and GDP binding in BAT . In mice only the rise in rectal and skin temperature were significantly changed by endotoxin administration . These findings suggest that in some species BAT is involved in the production of endotoxin induced fever and increased energy expenditure. Prostaglandins, 1987 Jun, 33(6), 879 - 902 Dose-dependent effects of a pyridoquinazoline thromboxane synthetase inhibitor on arachidonic acid metabolites and hemodynamics during E . coli endotoxemia in anesthetized sheep; Morel DR et al.; We investigated the effects of a new pyridoquinazoline thromboxane synthetase inhibitor infused before administering Escherichia Coli endotoxin into 18 anesthetized sheep with lung lymph fistulas . In normal sheep increasing plasma Ro 23-3423 concentrations were associated with increased plasma levels of 6-keto-PGF1 alpha, a reduced systemic vascular resistance (SVR, r = -0.80) and systemic arterial pressure (SAP, r = -0.92), the mean SAP falling from 80 to 50 mm Hg at the 20 and 30 mg/kg doses . Endotoxin infused into normal sheep caused transient pulmonary vasoconstriction associated with increased TxB2 and 6-keto-PGF1 alpha levels while vasoconstriction and TxB2 increase were significantly inhibited by pretreatment with Ro 23-3423 in a dose-dependent manner . When compared to controls, plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 after endotoxin infusion were increased several-fold by administering Ro 23-3423 up to plasma levels of 10 micrograms/ml . Doses over 30 mg/kg with blood levels above 10 micrograms/ml reduced plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, suggesting cyclooxygenase blockade at this dose . The peak 6-keto-PGF1 alpha levels at 60 min after endotoxin infusion in sheep with Ro-23-3423 levels below 10 micrograms/ml were associated with the greatest systemic hypotension due to a reduced SVR (r = -0.86) . After endotoxin infusion the leukotrienes B4, C4, D4 and E4 in lung lymph were assayed by radioimmunoassay and high pressure liquid chromatography and remained at baseline values. Vet Immunol Immunopathol, 1987 Jun, 15(3), 223 - 37 Bovine monoclonal antibodies to the F5 (K99) pilus antigen of E . coli, produced by murine/bovine hybridomas; Anderson DV et al.; Lymph node cells from calves immunized with purified pilus antigen of K99+ enterotoxigenic E . coli (ETEC) were fused with mouse myeloma (NSO) cells, and with non-Ig producing mouse/calf hybridomas or with a bovine Ig-producing mouse/calf/calf secondary hybridoma . Lines secreting bovine monoclonal IgG1 specific for K99 pilus antigen in an ELISA were obtained in each case . The two lines derived from xenohybridoma fusion partners have been secreting anti-K99 bovine monoclonal antibody for over one year in continual passage . None of the antibodies cross-reacted with other pilus types including K88, CFAI, CFAII, 987P or CP; they all inhibited agglutination of horse RBC (which have a K99 receptor) in the presence of K99 antigen; they showed positive fluorescence in an indirect binding assay on K99+ ETEC and inhibited K99+ ETEC adhesion to piglet enterocytes . These antibodies have potential prophylactic and therapeutic use in control and treatment of diarrhoea. Biochem Int, 1987 Jun, 14(6), 1073 - 7 Altered localisation of the precursor of a secreted protein in E . coli by a carboxyl-deletion; Burns DM et al.; The effect on protein localisation of a carboxy-terminal deletion of periplasmic UDP-glucose hydrolase (5'-nucleotidase) has been determined in vivo using immunoprecipitation . The processed form of the truncated protein was found in the periplasm; however, in contrast to the situation with the normal protein, the preprotein form of the truncated protein was also found in the periplasm . This result confirms previous semi-in vitro data and supports the suggestion that a conformational change in a membrane-bound intermediate is a normal part of the secretory pathway. FEBS Lett, 1987 May 11, 215(2), 269 - 73 Localization of the cellular-fibronectin-specific epitope recognized by the monoclonal antibody IST-9 using fusion proteins expressed in E . coli; Carnemolla B et al.; Here we report on a monoclonal antibody (IST-9) which distinguishes between human cellular and plasma fibronectin . Using beta-galactosidase-fibronectin fusion proteins expressed in E . coli we have demonstrated that this monoclonal antibody is specific for a fibronectin segment (ED) which can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors . Furthermore, using the same fusion proteins we have been able to localize precisely the epitopes of two other monoclonal antibodies (IST-1 and IST-2), specific for the heparin-binding domain 5 of fibronectin. J Biochem (Tokyo), 1987 May, 101(5), 1199 - 208 Characterization of E . coli-derived recombinant human interferon-beta as compared with fibroblast human interferon-beta; Utsumi J et al.; Homogeneous E . coli-derived recombinant human interferon-beta (E . coli-rHuIFN-beta) was characterized in order to elucidate its physicochemical properties, as compared with those of fibroblast human interferon-beta (fibroblast HuIFN-beta) . Purified E . coli-rHuIFN-beta and fibroblast HuIFN-beta exhibited a single band of Mr 19,000 and 23,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The primary structure of E . coli-rHuIFN-beta was identical to the prediction from the cDNA sequence . Furthermore, both the circular dichroism (CD) spectra and the 1H nuclear magnetic resonance (NMR) spectra of E . coli-rHuIFN-beta and fibroblast HuIFN-beta at pH 6.8 were closely similar to each other . On the other hand, on reverse-phase high-performance liquid chromatography (HPLC) using a C18 column, the retention time of E . coli-rHuIFN-beta was longer than that of fibroblast HuIFN-beta . Moreover, although the isoelectric point of E . coli-rHuIFN-beta was pH 8.9, purified fibroblast HuIFN-beta exhibited multiple isoelectric points, probably due to heterogeneity of the carbohydrate moiety . These results indicate that the E . coli-rHuIFN-beta polypeptide folds similarly to fibroblast HuIFN-beta, and the carbohydrate moiety of natural HuIFN-beta has little influence on higher-order structure but does influence the hydrophobic and the electrostatic properties of the molecule. Biofizika, 1987 May-Jun, 32(3), 477 - 81 {Protein damage after treatment with ozone of protoplasts and isolated E . coli membranes}; Matus VK et al.; The features of ozone-induced damage of E . coli plasma membrane proteins are investigated . A conclusion is made that protein fluorescence quenching is connected with modification of amino acid residues in the vicinity of tryptophane residues . Such modification may be a consequence of reaction with either ozone itself or products of its interaction with membrane lipids and/or proteins . The suggestion of Goldstein and McDonagh that ozone has a predilection for more hydrophilical membrane domains is confirmed . The data obtained are in agreement with a supposition about the leading role of proteins in deleterious action of ozone on cells. Prikl Biokhim Mikrobiol, 1987 May-Jun, 23(3), 303 - 8 {Isolation and properties of immobilized alkaline phosphatase from E . coli}; Zagrebel'nyi SN et al.; Preparations of alkaline phosphatase from E . coli, immobilized on Sepharose, with a specific activity of 40-60 U/g wet weight were obtained . The immobilized enzyme was stable up to 50 degrees C; at higher temperatures it was inactivated . At 70 degrees most of the activity was lost for 1 h . The substrate (AMP) stabilized the enzyme . In the temperature range from 30 to 40 degrees C activation of the enzyme was observed, especially pronounced in the presence of the substrate . The pH optimum of the immobilized enzyme activity (7.8-8.2) is shifted towards the acid region, as compared to the soluble enzyme (8.0-8.6) . The kinetic parameters for inhibition by the reaction product were determined using the integral Michaelis-Menten equation . KmAMP was found to be higher in case of the immobilized enzyme as compared to the soluble one (5.02 X 10(-4) M and 1.85 X 10(-5) M, respectively), which seems to be associated with diffusion limitations. Bioorg Khim, 1987 May, 13(5), 599 - 605 {Functionally important lysine residues in inorganic pyrophosphatase from E . coli . II . Isolation and characteristics of modified tryptic peptide and analysis of the functional role of lysine residues controlling enzyme activity}; Komissarov AA et al.; Inactivation of inorganic pyrophosphatase from E . coli by pyridoxal-5'-phosphate is due to the modification of a lysine residue located in the tryptic peptide with the Asp-Leu-Pro-Glu N-terminal sequence . In course of the enzymatic process this lysine-residue appears to be in the protonated state and either operators as the proton donor for the product of the enzymatic reaction or is involved in stabilization of the transition state. Bioorg Khim, 1987 May, 13(5), 592 - 8 {Functionally important lysine residues in inorganic pyrophosphatase from E . coli . I . Interaction of inorganic pyrophosphatase with pyridoxal-5'-phosphate}; Komissarov AA et al.; Interaction of inorganic pyrophosphatase from E . coli with pyridoxal-5'-phosphate includes binding of the reagent at the active site through the phosphate group and then a reversible modification of one lysine residue in each of the enzyme's subunit . In the equilibrium state the protein's molecules contain both inactive modified and native subunits . A stable secondary amine is formed upon the sodium borohydride reduction of the modified protein. Mol Gen Mikrobiol Virusol, 1987 May, (5), 34 - 6 {Homology between the ribosomal and RNA-polymerase genes of E . coli and vaccine strain E of Rickettsia prowazekii}; Artem'ev MI et al.; The pIB52 plasmid contains the ribosomal rp1A, J, K, L and RNA-polymerase genes rpo C, B of Escherichia coli . No homology has been found between the plasmid used as a molecular probe and the chromosomal DNA from Rickettsia provazekii in the blot hybridization experiments. Mol Gen Mikrobiol Virusol, 1987 May, (5), 19 - 22 {Preservation of the beta-galactosidase activity in E . coli cells containing the recombinant pUC19 plasmid}; Paton EB et al.; Two BglII fragments of pJC703 cosmid were inserted into the plasmid PUC19 . E . coli cells containing the recombinant PUC19/A plasmid acquired rifampicin resistance due to inserted rpoB gene but still expressed the Lac+ phenotype. Biochimie, 1987 May, 69(5), 539 - 41 The effect of dam methylation on the expression of glnS in E . coli; Plumbridge J et al.; The wild type glnS promoter contains a dam methylation site . In dam strains, the expression of glnS is enhanced 2.6-fold . A mutated form of the promoter has been isolated in which the dam methylation site is lost . Expression of this promoter is insensitive to dam methylation. Biochem Int, 1987 May, 14(5), 911 - 9 Endonucleolytic cleavage of E . coli and pBR 322 DNAs by a placental DNA-binding protein; Premeela T et al.; A 34,000 dalton DNA-binding protein (DBP) has been purified from human placenta . The purified protein possesses endonuclease activities capable of cleaving plasmid pBR 322 and chromosomal DNAs from E . coli . Maximum endonuclease activity was observed in the pH range of 6-9 and at 30 degrees C . The nuclease activity of the DBP was completely lost at 50 degrees C . Nitrocellulose filter binding assays indicate preferential binding of the DBP to ss DNA . The protein did not bind to apurinic DNA and UV-irradiated ds DNA . Consistent with the lack of binding of the DBP to apurinic DNA, this substrate was not cleaved by the DBP . However, native and UV-irradiated E . coli DNAs which showed poor binding were also cleaved by the DBP. Somat Cell Mol Genet, 1987 May, 13(3), 253 - 65 Histochemical staining of clonal mammalian cell lines expressing E . coli beta galactosidase indicates heterogeneous expression of the bacterial gene; MacGregor GR et al.; An evaluation has been made of the E . coli beta-galactosidase (beta-gal) gene for use as a reporter gene in mammalian cells in culture . We have adopted a histochemical procedure which enables identification of those cells within a population that express the introduced bacterial gene . Data is presented concerning the sensitivity of the histochemical method relative to an immunological method of detection . It has been found that several clonal cell lines generated after transfection of human 293 cells with a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter-beta-gal construction are mosaic for expression of the introduced mini-gene . Furthermore, after treatment of these clonal cell lines with the nucleoside analog 5-aza-cytidine (5-aza-C), an increase in production of beta-gal under control of this promoter element was observed. Nucleic Acids Res, 1987 Apr 24, 15(8), 3411 - 20 Isolation of a human genomic fragment, co-amplified with c-Ki-ras, that affects plasmid supercoiling in E . coli; Heighway J et al.; Amplification of cellular proto-oncogenes has been implicated in the development of human malignancies . A library was constructed from genomic DNA extracted from a lung tumour, previously shown to carry an amplified c-Ki-ras 2 gene . Using a v-Ki-ras probe, a fragment with ras homology was isolated and shown to be amplified in the original tumour DNA to the same level as c-Ki-ras . Studies with human hamster hybrids demonstrated that it is normally located on human chromosome 12 (as is c-Ki-ras) . The restriction map of the fragment is different from that of the known Ha, Ki or N-ras genes and its sequence shows evolutionary conservation, as demonstrated by hybridisation to the genomic DNA of several mammalian species . A pUC19 subclone (pK42), carrying a 1.3kb insert, shows supercoil heterogeneity in plasmid preparations, as does a second compatible plasmid introduced into the same bacterial host with pK42 . It appears therefore that the subclone is encoding a product that affects DNA topoisomerase activity in E . coli. Cell, 1987 Apr 24, 49(2), 241 - 51 Biogenesis of E . coli Pap pili: papH, a minor pilin subunit involved in cell anchoring and length modulation; Baga M et al.; The biogenesis of Escherichia coli Pap pili, encoded by the pap gene cluster, was studied . A novel gene, papH, was identified and found to encode a weakly expressed pilin-like protein . PapH was dispensable for digalactoside-specific binding and for formation of Pap pili . However, in papH deletion mutants 50%-70% of total pilus antigen was found free of the cells . We present evidence showing coregulation of papH and the adjacent gene, papA, which encodes the major pilin subunit . A decrease in the PapA to PapH ratio resulted in a large fraction of cells producing shortened pili, whereas overproduction of PapA relative to PapH resulted in cells with lengthened pili . The data show that PapH has roles in anchoring the pilus to the cell and in modulating pilus length. Biochem Biophys Res Commun, 1987 Apr 14, 144(1), 447 - 53 Methyl iodide, a potent inducer of the adaptive response without appreciable mutagenicity in E . coli; Takahashi K et al.; Methyl iodide (MeI), a very weak mutagen, induced the adaptive response in E . coli to a similar extent to those induced by potently mutagenic methylating agents . MeI potentiated the mutagenicity of a methylating mutagen, N-methyl-N-nitrosourea, by its co-treatment . These results might give indication that MeI directly methylates O6-methylguanine-DNA methyltransferase resulting in induction of the adaptive response and depletion of the repair capacity of enzyme. Biochem Biophys Res Commun, 1987 Apr 14, 144(1), 375 - 81 3-13C-methionine-labelled E . coli alkaline phosphatase; Hines D et al.; 3-13C-methionine has been biosynthetically incorporated into E . coli alkaline phosphatase using strain CW3747 which is auxotrophic for Met . 13C NMR of the dimeric native enzyme labelled at the eight methionine residues of the primary structure shows a dispersion of resonance signals permitting resolution of at least five methionine environments, none of which coincide with the chemical shift position of free methionine . At acid pH, 13C signal intensity is shifted to a chemical shift consistent with solvent exposure . However, three discrete resonances are observed, suggesting a retention of defined structure . The labelled protein thus can serve as a probe of conformational alterations of the enzyme. DNA, 1987 Apr, 6(2), 119 - 28 Chemical mutagenesis of human interferon-beta: construction, expression in E . coli, and biological activity of sodium bisulfite-induced mutations; Stewart AG et al.; A series of modified human interferon-beta (IFN-beta) genes was produced by sodium bisulfite treatment of the IFN-beta gene cloned in M13 . A library of mutated sequences was generated from which subgenomic fragments containing one or a small number of coding alterations were isolated and substituted into the IFN-beta gene in an E . coli expression vector . A number of modified genes and their expression products were evaluated . In several instances levels of expression and biological activity profiles are altered compared to the parental gene product . A number of key amino acids can be identified, whose substitutions have marked effects on biological activity of IFN-beta. Bioorg Khim, 1987 Apr, 13(4), 565 - 7 {Highly selective affinity labeling of a promoter in a complex with E . coli RNA-polymerase by alkylating derivatives of initiating substrates}; Grachev MA et al.; The complex {promoter A2 X E . coli RNA polymerase} was treated with phosphoamides, derivatives of 4-{N-methyl, N-(2-chloroethyl)}-aminobenzylamine and guanosine-5'-mono-, di-, and triphosphates with the alkylating group attached to the terminal phosphates . After this, {alpha-32P}CTP was added . Residues of the affinity reagents bound covalently at the first stage were elongated by radioactive -pC residues due to the catalytic action of the active centre of RNA polymerase . Affinity labelled were beta-and sigma-subunits of the enzyme, and the promoter . The affinity label was localized on -pGpC residues . A guanine residue was alkylated in the promoter as suggested by radioactivity elimination kinetics . As the data obtained and the previously known length of the reagent (maximum distance between the alpha-phosphorus atom of the reagent and the point of alkylation is less than 0.6 nm) indicate, there is a direct rather than protein-mediated contact between the template and the substrate within the complex {promoter X RNA polymerase}. J Clin Pharm Ther, 1987 Apr, 12(2), 107 - 15 The effect of pH and concentration on the rates of kill of benzoic acid solutions against E . coli; Hurwitz SJ et al.; Mathematical models were developed which related benzoic acid concentrations to their D-values at room temperature . Linear regression was used to determine D-values (i.e . the time required for a particular concentration of preservative at specified pH, temperature and medium to cause a 90% reduction in the number of viable organisms, e.g . Escherichia coli . A number of concentrations of benzoic acid at either pH 3 or pH 4 were used . Linear regression of the log D-values versus the log of the concentration was used to derive power curves (a minimum of four points per pH were examined) . Concentration exponents, eta-values (the logarithmic values relating changes in rates of kill to specified changes in concentrations) and A-values (extrapolated D-values at 1% concentration), were determined . The effect of pH on the eta and A-values of benzoic acid on E . coli was investigated . Benzoic acid was found to be more sensitive to pH changes than was anticipated from the Henderson-Hasselbalch equation which relates dissociated and undissociated fractions at the two pH levels investigated . However, the sensitivity of preservative activity to dilution at both pH values (3 and 4), as given by the eta-value, remained similar. Bioorg Khim, 1987 Apr, 13(4), 552 - 5 {Localization of lysine residues in the site of initiating substrate binding of E . coli RNA-polymerase}; Grachev MA et al.; Superselective affinity labelling of E . coli RNA polymerase in a complex with the promoter-containing fragment of T7 DNA by treatment with orto-formylphenyl ester of GMP followed by addition of {alpha-33P}UTP resulted in covalent binding of the residue--pGpU (p-radioactive phosphate) with one of lysine residues of the beta-subunit, Lys1048, Lys1051, Lys1057, Lys1065 . The amino acid sequence of this region of the beta-subunit of E . coli RNA polymerase has a high extent of homology with that deduced for a region of tobacco chloroplast RNA polymerase on the basis of the nucleotide sequence of the chloroplast rpoB-like gene. Z Naturforsch {C}, 1987 Apr, 42(4), 394 - 400 E . coli maltodextrin phosphorylase: primary structure and deletion mapping of the C-terminal site; Palm D et al.; The complete 796 residue amino acid sequence of maltodextrin phosphorylase was deduced from the E . coli malP nucleotide sequence . The calculated molecular weight of 90,500, including pyridoxal phosphate, is significantly larger than experimentally determined values . Enzymatically active and inactive mutants following deletion or exchange of up to 8 codons (7 amino acids) at the 3' end (C-terminus) confirm the size of the mature native enzyme and disclose the essential functional or structural role of the highly conserved C-terminal region of phosphorylases. Nucleic Acids Res, 1987 Mar 25, 15(6), 2627 - 38 The effect of codon usage on the oligonucleotide composition of the E . coli genome and identification of over- and underrepresented sequences by Markov chain analysis; Phillips GJ et al.; As shown in the accompanying paper (5), the oligonucleotide composition of the E . coli genome is highly asymmetric for sequences up to 6 bp in length when ranked from highest to lowest abundance . We show here that this largely reflects codon usage because heavily used codons were found in the highly abundant oligomers whereas rarely used codons, with some exceptions, occurred in sequences in low abundance . Furthermore, linear regression analysis revealed a strong correlation between the frequencies of each trinucleotide and its usage as a codon . Dinucleotides are also not randomly distributed across each codon position and the dinucleotide composition of genes that are transcribed but not translated (rRNA and tRNA genes) was highly related to that seen in genes encoding polypeptides . However, 45 tetra-, 8 penta-, and 6 hexanucleotides were significantly over- or underabundant by Markov chain analysis and could not be accounted for by codon usage . Of these underrepresented sequences, many were palindromes, including the Dam methylation site. FEBS Lett, 1987 Mar 23, 213(2), 297 - 300 dam methylase from E . coli . Circular dichroism investigations of the secondary structure and influence of S-adenosylmethionine; Kriebardis A et al.; The enzyme dam methylase which recognizes and methylates the adenine in the palindromic sequence GATC in DNA was isolated and the secondary structure was determined by CD spectroscopy and various predicting methods from the amino acid sequence . The interaction of dam methylase with S-adenosylmethionine was studied by CD spectroscopy indicating a decrease of the percentage of alpha-helix as the amount of S-adenosylmethionine bound to the enzyme was increased. FEBS Lett, 1987 Mar 23, 213(2), 261 - 4 Expression of the cell-binding domain of human fibronectin in E . coli . Identification of sequences promoting full to minimal adhesive function; Obara M et al.; Two cDNA subfragments containing the cell-attachment site of human fibronectin (FN) were expressed as beta-galactosidase fusion proteins in E . coli . The products were purified to homogeneity by monoclonal antibody affinity chromatography and assayed for activity in a standard cell-adhesion assay . A fusion protein containing an 80 kDa fragment of human FN appeared functionally equivalent to intact FN purified from human plasma, whereas a truncated fusion protein of 33 kDa still containing a previously postulated cell-attachment site was approx . 50-fold less active . Our study establishes a system for analyzing adhesive protein function by DNA manipulation, rules out any major role for eukaryotic post-translational modifications in FN adhesive function, and localizes additional functional activity to a 1.3 kb region. Nucleic Acids Res, 1987 Mar 11, 15(5), 2343 - 61 Analysis of E . coli promoter sequences; Harley CB et al.; We have compiled and analyzed 263 promoters with known transcriptional start points for E . coli genes . Promoter elements (-35 hexamer, -10 hexamer, and spacing between these regions) were aligned by a program which selects the arrangement consistent with the start point and statistically most homologous to a reference list of promoters . The initial reference list was that of Hawley and McClure (Nucl . Acids Res . 11, 2237-2255, 1983) . Alignment of the complete list was used for reference until successive analyses did not alter the structure of the list . In the final compilation, all bases in the -35 (TTGACA) and -10 (TATAAT) hexamers were highly conserved, 92% of promoters had inter-region spacing of 17 +/- 1 bp, and 75% of the uniquely defined start points initiated 7 +/- 1 bases downstream of the -10 region . The consensus sequence of promoters with inter-region spacing of 16, 17 or 18 bp did not differ . This compilation and analysis should be useful for studies of promoter structure and function and for programs which identify potential promoter sequences. FEBS Lett, 1987 Mar 9, 213(1), 155 - 8 Spacer alterations which increase the expression of porcine growth hormone in E . coli; Vize PD et al.; Full-length porcine growth hormone (PGH) cDNA clones were isolated from a porcine pituitary cDNA library . When the coding portion of the PGH gene was cloned into an E . coli expression vector downstream from the powerful trc promoter, high levels of mRNA, but no protein were detected . Mutation directed by an oligodeoxynucleotide primer altered 5'-non-coding sequences and raised the level of PGH produced from undetectable to 15% of the total cellular protein . Alteration of four codons infrequently used by E . coli in the 5'-end of the gene produced no further increases. J Appl Physiol, 1987 Mar, 62(3), 1141 - 9 Pulmonary pressor responses in sheep to chemically defined precursors of E . coli endotoxin; Burhop KE et al.; The toxicity of various monosaccharide and disaccharide endotoxin precursors has now been studied in sheep . We measured the early pulmonary arterial pressure responses after injections of the monosaccharides lipid X (2,3-diacylglucosamine 1-phosphate) and MAGP (2-monoacylglucosamine 1-phosphate), of the tetraacyl disaccharide diphosphate precursor of lipid A, IV-A (Federation Proc . 43: 1567, 1984), and of Escherichia coli bacterial endotoxin (lipopolysaccharide) . We also measured the response of lipid X after prior administration of indomethacin and MAGP . Lipid X, at a total cumulative dose of 40 micrograms/kg, produced an immediate, but transient dose-dependent pulmonary arterial vasoconstrictive response . MAGP, at a total dose of 40 micrograms/kg, had no pulmonary pressure activity but did increase extravascular lung water and produce some histological changes in the lung . Disaccharide precursor IV-A, at a total dose of 40 micrograms/kg, produced an immediate dose-dependent pulmonary arterial vasoconstrictive response that was prolonged for greater than 2 h . E . coli endotoxin caused a delayed (15-min) increase in the pulmonary arterial pressure but one that also persisted for greater than 2 h . Prior administration of indomethacin blocked the pulmonary pressor activity of lipid X, whereas prior administration of MAGP increased both the magnitude and the duration of the pulmonary pressure response of lipid X . We conclude that the initial pulmonary hypertension seen after lipid X injection may involve cyclooxygenase-dependent formation of prostaglandins and that the genesis of this pulmonary pressor activity is at least in part dependent on the ester-linked hydroxymyristoyl moiety at position 3 of the lipid X molecule.(ABSTRACT TRUNCATED AT 250 WORDS) Mutat Res, 1987 Mar, 183(2), 123 - 7 Proteolytic processing of the Ada protein that repairs DNA O6-methylguanine residues in E . coli; Teo IA; In extracts of E . coli treated with an adapting regime of MNNG, the induced 39kd Ada protein having O6-MeG-DNA methyltransferase activity is processed to a 19kd active domain corresponding to the C-terminal half of the intact protein . This proteolytic processing has been followed on Western immunoblots using antisera raised against the 19kd fragment . Initial processing at 25 degrees C or 37 degrees C mainly generates a fragment of mol . wt . 24kd which then undergoes a slower second cleavage to generate the 19kd active domain . Preceding this second cleavage site is a sequence of amino acids Thr- -Gly-Met-Thr- -Lys that also occurs at another site in the N-terminal half of the 39kd methyltransferase . It is proposed that this sequence is a recognition site for proteolytic activity . On the basis of cleavage of the Ada protein at either one or both of these sites, fragments may be generated of mol . wt . 24kd and 19kd containing the active site for O6-methylguanine and O4-methylthymine repair, and 15kd and 20kd, containing the active site for methylphosphotriester repair . These observations explain previous reports by others on the existence in cell extracts of multiple methyltransferase activities of different sizes recognizing O-methyl lesions in DNA . The cellular protease involved is resistant to a wide range of protease inhibitors. Bioorg Khim, 1987 Mar, 13(3), 344 - 9 {Effectiveness of substituting E . coli DNA-polymerase for DNA-polymerase A in oligonucleotide-directed mutagenesis}; Petrenko VA et al.; The possibility to use the E . coli intact DNA polymerase I in the oligonucleotide-directed site-specific mutagenesis of DNA has been studied . Optimal conditions of the extension activity of this enzyme were found . We have shown that the substitution of the Klenow fragment of the E . coli DNA polymerase by the intact DNA polymerase I did not decrease the efficiency and fidelity of the oligonucleotide-directed mutagenesis. Biochimie, 1987 Mar, 69(3), 215 - 21 The structure of the promoter and amino terminal region of the pH 2.5 acid phosphatase structural gene (appA) of E . coli: a negative control of transcription mediated by cyclic AMP; Touati E et al.; The regulatory and promoter regions of the gene for acid phosphatase of optimum pH 2.5 (appA) of Escherichia coli has been characterized by constructing appA-lacZ protein fusions . Monitoring of beta-galactosidase activity showed that the cloned fragment harbored a region that was responsible for a negative control of transcription mediated by the cAMP-cAMP receptor (CAP) complex . The nucleotide sequence of the segment was determined . A region containing a putative CAP binding site, overlapping the -10 region of the promoter was found . Deletion analysis around the promoter region, using appA-lacZ protein fusions substantiated the hypothesis for a role of the 'consensus' sequence in the negative control mediated by cyclic AMP . In addition, the coding sequence revealed the likely existence of a signal peptide similar to the one found for other exported proteins, upstream from the phosphatase protein sequence. Biull Eksp Biol Med, 1987 Mar, 103(3), 332 - 4 {Functional properties of E . coli SSB-proteins in vivo}; Aizenberg OA et al.; An essential function of single-stranded DNA-binding (SSB) proteins--a defense from nuclease action, determined by their first and second domains, is realized in conditions of normal cell metabolism . With the inhibition of the replicative furcula growth and total protein synthesis (imbalance state), the SSB protein function associated with the third domain and responsible for certain modifications in DNA polymerase II activity is realized . This causes DNA degeneration and increases radiosensitivity of cells. Genes Dev, 1987 Mar, 1(1), 57 - 64 Induction of the heat shock response of E . coli through stabilization of sigma 32 by the phage lambda cIII protein; Bahl H et al.; The cIII protein of phage lambda favors the lysogenic response to infection by inhibiting the degradation of the lambda cII protein, which exerts the primary control on the developmental decision for lysis or lysogeny . To study the mechanism and scope of cIII-mediated regulation, we have used plasmid systems to examine the specific effect of cIII overproduction on the growth of Escherichia coli and the synthesis of bacterial proteins . We have found that maximal production of cIII prolongs the heat-induced synthesis of E . coli heat shock proteins and provokes elevated production of heat shock proteins even at low temperature . The overproduction of heat shock proteins is correlated with a rapid inhibition of cell growth, as judged by measurements of optical density . We suggest that an overactive heat shock response inhibits bacterial growth, either because excessive production of one or more of the proteins is highly deleterious or because only heat shock promoters are transcribed efficiently . To examine the effect of cIII on sigma 32, the specificity factor for the heat shock response, we have studied the stability of sigma 32 in cells carrying both cIII- and sigma 32-producing plasmids; the half-life of sigma 32 is increased fourfold in the presence of cIII . We conclude that overproduction of cIII provokes the heat shock response by increasing the steady-state level of active sigma 32 . These studies also support the concept that the rate of expression of heat shock proteins is directly correlated with the amount of active sigma 32 and that regulation of the stability of sigma 32 may be an important factor for control of the heat shock response. Z Naturforsch {C}, 1987 Mar, 42(3), 288 - 96 Acyclonucleoside analogues consisting of 5- and 5,6-substituted uracils and different acyclic chains: inhibitory properties vs purified E . coli uridine phosphorylase; Drabikowska AK et al.; Synthetic procedures are described for the preparation of a variety of pyrimidine acyclonucleoside analogues, in which the aglycones are 5- and 5,6-substituted uracils, and the ribose moiety is replaced by different acyclic chains . These were examined as potential inhibitors of purified E . coli uridine phosphorylase . None of the compounds was a substrate for uridine phosphorylase, or either a substrate or inhibitor of E . coli thymidine phosphorylase . Kinetic measurements were employed to determine inhibition constants, Ki, for inhibition of uridine phosphorylase . One of the more effective of these was 1-(1',3'-dihydroxy-2'-propoxy)methyl-5,6-tetramethyleneuracil, with Ki = 2.7 microM . The same compound was a reasonably good inhibitor of the reverse, synthetic, reaction, with Ki values of 19 microM vs uracil as the variable substrate, and 15 microM vs alpha-D-ribose-1-phosphate as the variable substrate . For one of the analogues, which was a racemate, 1-(2',3'-dihydroxypropyl)-5,6-tetramethyleneuracil, it was shown that only one of the enantiomers (R) was an inhibitor, the (S) enantiomer being totally inactive . For several of the analogues, the corresponding isomeric N(3)-acyclonucleosides were inactive as inhibitors . The results for several of the good inhibitors were compared with those of other observers for inhibition of uridine phosphorylase from mammalian sources . Preliminary measurements with several of our analogues demonstrated that some of them were indeed one to two orders of magnitude more effective against the enzyme from mammalian sources. FEBS Lett, 1987 Feb 23, 212(2), 233 - 6 trp operon induction during the expression in E . coli of two IFN-gamma sequences; Sverdlov ED et al.; Two nucleotide sequences coding for mature human immune interferon (IFN-gamma) and differing from each other by nine N-terminal nucleotides were expressed in E . coli under the control of a trp promoter . The longer gene variant after the ATG initiatory codon contained a TGT TAC TGC sequence, which was absent in the shorter gene . When expressed in E . coli under the direction of identical transcription and translation regulatory elements, these genes showed different susceptibility to induction. Biochem Biophys Res Commun, 1987 Feb 13, 142(3), 964 - 71 Characterization of the guanosine-3'-diphosphate-5'-diphosphate binding site on E . coli RNA polymerase using a photoprobe, 8-azidoguanosine-3'-5'-bisphosphate; Owens JR et al.; Nucleotide binding sites on DNA-dependent RNA polymerase from E . coli have been studied by photoaffinity labeling with a GTP analog {gamma-32P}-8-AzidoGTP and a guanosine-3'-diphosphate-5'-diphosphate analog, 8-Azidoguanosine-3'-phosphate-5'-85'-32P}phosphate . The guanosine diphosphate photoprobe labeled the beta, beta', and sigma subunits with the sigma subunit being most heavily labeled . The GTP photoprobe also labeled the beta, beta', sigma subunits but the beta' subunit was most heavily labeled . In competition experiments guanosine-3'-diphosphate-5'-diphosphate decreased photolabeling by 8-Azidoguanosine-3'-phosphate-5'-{5'-32P}phosphate better than GTP, while the opposite was true for photolabeling with {gamma-32P}8- AzidoGTP . The guanosine diphosphate photoprobe inhibited transcription on E . coli DNA with Ki of ca . 150 microM . Present studies suggest a unique ppGpp binding site distinct from substrate binding site(s) and this photoprobe may be used to localize this binding site(s). Nucleic Acids Res, 1987 Feb 11, 15(3), 1109 - 20 Binding of E . coli DNA photolyase to a defined substrate containing a single T mean value of T dimer; Husain I et al.; The E . coli DNA photolyase is a flavoprotein that catalyzes the photoreversal of pyrimidine dimers . The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and repairs the dimer upon absorbing a photon in the 300-600 nm range . The rate and equilibrium constants for the light-independent reaction were determined before, using randomly modified substrates that contained T mean value of T, T mean value of C and C mean value of C dimers in random sequence surrounding . In this paper we have determined these constants for a defined substrate (a 43 bp oligomer containing a T mean value of T dimer) using the gel retardation assay . We find that: the equilibrium constant and the off rate obtained with this substrate by this technique are similar to those obtained with randomly modified DNA using filter binding and flash photolysis techniques . the off rate with the defined substrate is heterogeneous indicating heterogeneity in the enzyme population or in the enzyme-substrate complexes, and the enzyme has 7.5 X 10(4)-fold higher affinity for pyrimidine dimer compared to non-dimer DNA nucleotides. DNA, 1987 Feb, 6(1), 41 - 6 Expression of an interferon-alpha gene variant in E . coli using tandemly repeated synthetic ribosomal binding sites; Grundstrom T et al.; A human interferon-alpha 2 gene variant was expressed in Escherichia coli under the control of the tryptophan operon promoter and of a series of synthetic partially overlapping ribosomal binding sites, which control initiation of translation . Variation in the distance between the start codon and such a set of ribosomal binding sites affected the level of interferon expression much less than previously described for single ribosomal binding sites. Cell, 1987 Jan 30, 48(2), 281 - 7 Stimulation of a Chlamydomonas chloroplast promoter by novobiocin in situ and in E . coli implies regulation by torsional stress in the chloroplast DNA; Thompson RJ et al.; We have characterized regulation of a complex Chlamydomonas reinhardtii chloroplast (PA) whose activity is stimulated by the DNA gyrase inhibitor novobiocin, both in the alga itself and in a heterologous Escherichia coli plasmid system . Since novobiocin is known to reduce torsional stress in E . coli DNA, we interpret our results to mean that PA is regulated by torsional stress in the chloroplast DNA . In E . coli, where we could readily manipulate PA, we found that this regulation depends on sequences upstream of PA . These sequences contain at least two different kinds of silencing elements that inhibit PA in the absence of novobiocin . Novobiocin stimulates PA only when the promoter-distal silencing element is present. Biochem Biophys Res Commun, 1987 Jan 30, 142(2), 302 - 8 P-chlorophenylalanine does not inhibit production of recombinant human phenylalanine hydroxylase in NIH3T3 cells or E . coli; Ledley FD et al.; P-chlorophenylalanine is an irreversible inhibitor of rat phenylalanine hydroxylase in vivo and in rat hepatoma cells and is frequently administered to rodents to create an animal model for phenylketonuria . We investigated the effect of p-chlorophenylalanine on production of human phenylalanine hydroxylase in human hepatoma cells and cells transformed with the recombinant human phenylalanine hydroxylase gene . P-chlorophenylalanine inhibited production of the human enzyme in human hepatoma cells and transformed mouse hepatoma cells but had no effect on the production of the enzyme in transformed NIH3T3 cells or in E . coli . Thus, phenylalanine hydroxylase inhibition does not result from a simple interaction between the drug and enzyme. Nucleic Acids Res, 1987 Jan 26, 15(2), 785 - 96 Sequence distributions associated with DNA curvature are found upstream of strong E . coli promoters; Plaskon RR et al.; The regions upstream from forty-three procaryotic promoters were examined for nucleotide distributions which have been associated with DNA curvature . The analysis procedure assigned a DNA curvature score based on the phasing of the 5' and 3' ends of An and Tn tracts, n greater than or equal to 3 . The weighting scheme for the curvature score was based on recent studies which showed that tracts of An and Tn periodically phased with the helix repeat cause DNA curvature . Results show that promoters which have high transcription initiation rates in vivo tend to have high curvature scores in their upstream regions . Regions downstream from the transcription start-point do not have sequences correlated with DNA curvature . Four promoters which have been shown to have upstream activation regions have curvature scores above 1.5 in their -40 to -150 regions . The correlations observed lend support to the hypothesis that DNA curvature is associated with upstream activation of transcription. Nucleic Acids Res, 1987 Jan 26, 15(2), 575 - 94 Dynamic and structural characterisation of multiple steps during complex formation between E . coli RNA polymerase and the tetR promoter from pSC101; Duval-Valentin G et al.; Kinetic, functional and structural studies of the recognition of the tetR promoter from pSC101 by E . coli RNA polymerase allowed the characterization of several steps in the specific complex formation and transcription initiation process . First, enzyme and DNA enter in a short life-time complex . An isomerization will convert this unstable complex into a closed stable one where RNA polymerase is tightly attached without establishing stable chemical contacts with the bases . In the next step, stable close contacts appear between both macromolecules involving mainly the downstream part of the promoter . A further isomerization will lead to an open complex where DNA is locally melted and the system is able to initiate transcription . This latter process is accompanied by changes in the upstream part of the promoter . Finally, in vitro transcription assays showed that the position of the major transcription start sites depends on temperature . From the reported results, it appears that the recognition event is a sequential process where different structural elements of the promoter, that can be located apart in the sequence, are involved in a concerted manner in each stage. FEBS Lett, 1987 Jan 26, 211(2), 155 - 9 Role of tryptophan 54 in the binding of E . coli single-stranded DNA-binding protein to single-stranded polynucleotides; Khamis MI et al.; Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single-stranded polynucleotides with both E . coli single-stranded DNA-binding protein and an E . coli ssb gene product in which Trp-54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis . Our results strongly suggest the involvement of Trp-54 in stabilizing the protein-nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases. Nucleic Acids Res, 1987 Jan 26, 15(2), 771 - 84 DNA sequence of the E . coli gyrB gene: application of a new sequencing strategy; Adachi T et al.; We have determined the sequence of the E . coli gyrB gene, using a new sequencing approach in which transposition from a mini-Mu plasmid into the DNA provides random start points for dideoxynucleotide sequence analysis . The gyrB sequence corresponds to a protein 804 amino acids long; a previously isolated protein fragment with partial enzymatic activity has been identified as the C-terminal half-molecule . A plausible terminator of gyrB transcription is located just beyond the structural gene. FEBS Lett, 1987 Jan 19, 211(1), 49 - 52 Loss of transforming activity of plasmid DNA (pBR322) in E . coli caused by singlet molecular oxygen; Wefers H et al.; Plasmid DNA pBR322 in aqueous solution was exposed to singlet molecular oxygen (1O2) generated by microwave discharge . DNA damage was detected as loss of transforming activity of pBR322 in E . coli (CMK) dependent on the time of exposure . DNA damage was effectively decreased by singlet-oxygen quenchers such as sodium azide and methionine . Replacement of water in the incubation buffer by D2O led to an increase in DNA damage . 9,10-Bis(2-ethylene)anthracene disulfate was used as a chemical trap for 1O2 quantitation by HPLC analysis of the endoperoxide formed. Nature, 1987 Jan 15-21, 325(6101), 281 - 4 Initiation of translation is impaired in E . coli cells deficient in 4.5S RNA; Bourgaize DB et al.; The 4.5S RNA of Escherichia coli is a small, stable RNA that is essential for cell growth but its function is not yet known . Its biosynthesis is stringently controlled, and it is processed by RNase P, a transfer RNA processing enzyme . To identify the biological role of the 4.5S species, we have characterized the physiological changes that occur when the bacterial cell is depleted of this RNA . We used a strain of E . coli in which synthesis of the 4.5S RNA can be turned off by removing an inducer of the Iac operon, resulting in cell death . We report here that an early consequence of depriving the cell of 4.5S RNA is the accumulation of translationally-defective ribosomes, which maintain their ability to elongate polypeptide chains, but can no longer participate in the initiation of protein synthesis. FEBS Lett, 1987 Jan 5, 210(2), 147 - 52 The expression in E . coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p-nitrophenyl esters; Bulow L et al.; We have prepared a hybrid protein consisting of seven esterase units, Glu-Ala-His-Ala-Ser-Phe-Phe-Phe, fused to the N-terminal of galactokinase (E . coli) . The structural gene for this bifunctional protein was obtained by cloning a polymer made up of three chemically synthesized oligonucleotides to which the galactokinase gene was fused in frame . The hybrid protein was purified to homogeneity with the aid of the galactokinase moiety and showed an Mr of 51,000-53,000 . The preparation could catalyze the hydrolysis of p-nitrophenyl esters and, due to the inbuilt hydrophobic spacers, Phe-Phe-Phe, improved catalysis of more hydrophobic substrates was obtained. Biomed Biochim Acta, 1987, 46(1), 59 - 66 {In vitro effects of E . coli endotoxin on the membrane permeability and substrate transport of isolated rat liver mitochondria}; Kopprasch S et al.; Incubation of freshly isolated rat liver mitochondria with E . coli endotoxin resulted in an increased inner membrane permeability for K+ and Cl- ions . This effect was not prevented by addition of the synthetic antioxidant butylated hydroxytoluene . The carrier-mediated transport rates of phosphate, pyruvate, citrate and reducing equivalents via the malate-aspartate shuttle were not altered significantly by endotoxin . Therefore, the endotoxin-mediated impairment of mitochondrial respiration and oxidative phosphorylation could not be attributed to a decrease in transport capacities through the inner mitochondrial membrane. Circ Shock, 1987, 21(1), 59 - 64 Protein and energy metabolism in liver tissue following intravenous infusion of live E . coli bacteria in rats; Pedersen P et al.; In the present study protein synthesis and energy level in liver tissue were studied in bacteremic rats following intravenous infusion of 8 +/- 4 X 10(9) live E . coli bacteria and in control animals receiving a corresponding infusion of sterile saline . For the study of protein synthesis, liver slices were incubated in a medium containing 14C-leucine and incorporation rate of amino acid into protein was determined . Hepatic concentrations of ATP, ADP, and AMP were measured and energy charge (EC) was calculated . Twenty-four hours after infusion of E . coli, hepatic protein synthesis rate was 55% higher than in control animals . Liver weight and hepatic protein content were also increased in bacteremic animals . There were no significant differences in adenine nucleotide levels or EC in liver tissue between control and bacteremic animals . Since impairment of various other liver functions has been reported during sepsis, the present results suggest that hepatic protein synthesis has high priority in this condition. Mutat Res, 1987 Jan, 183(1), 39 - 44 Effect of pKM101 plasmid on lethal and mutagenic damage in UV-irradiated E . coli strains; Nunoshiba T et al.; Introduction of the R-factor plasmid pKM101 increased resistance to UV-killing in uvr lexA(Ind-) recA+ strains of E . coli K12 as well as B, while their UV mutability was not affected . Similar effects were also observed in those strains when the 18-B plasmid (a pBR322 derivative carrying the region (about 5 kb) of the 35.4 kb pKM101 plasmid) was introduced . The muc genes which are considered to be involved in error-prone repair are contained in 18-B . These results suggest the possibility that the pKM101 effect requires the host recA gene and a common genetic region, including the muc genes, in both plasmids and is associated with some unmutable repair systems. Mutat Res, 1987 Jan, 183(1), 21 - 9 Further characterization of an E . coli strain resistant to the toxic and mutagenic action of cis-diamminedichloroplatinum(II); Villani G et al.; An increased resistance to the toxic and mutagenic activity of the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) in the E . coli strain BS21 compared to its wild-type parent, F26, has been reported . This resistance was neither due to different binding of cis-DDP to DNA nor to adaptive DNA repair (Germanier et al., 1984) . In the present work, we found that mutation of the uvrA, recA and polA genes did not abolish the resistance of BS21 to the toxic action of cis-DDP . The lower mutability of BS21 was not influenced by the polA mutation, while uvrA greatly reduced and recA eliminated the mutagenic activity of cis-DDP in both strains . Treatment of BS21 and F26 with equal doses of cis-DDP produced the same initial number of platinum-DNA lesions . Little excision repair was detected in vivo in either strain during 6-h post-treatment incubation, the F26 strain being the most efficient of the two for this process . In contrast, F26 and BS21 were transformed identically by pBR322 DNA which had been treated with cis-DDP in vitro . Analysis of the platinum-DNA adducts which were formed between cis-DDP and salmon sperm DNA in the buffer conditions of this experiment suggests that plasmid DNA contains 80% monofunctional adducts and 20% bifunctional bis-guanine adducts . These data indicate that the selective toxicity and mutagenicity of these two strains in vivo are neither a result of different numbers of Pt-DNA lesions nor of their repair . The selectivity disappeared when the two bacterial strains were transformed by pBR322 DNA containing identical platinum-DNA lesions, suggesting that the biochemical events which process platinum-DNA lesions are the same in both strains . Hence, it appears that cis-DDP may form qualitatively different platinum-DNA adducts in the BS21 and F26 strains which are responsible for the different toxicity and mutagenicity. Viral Immunol, 1987, 1(2), 97 - 109 Expression and characterization of hepatitis B virus precore-core antigen in E . coli; Lanford RE et al.; The hepatitis B virus core antigen, including the precore sequence (HBcAg-p25), was expressed at very high levels in bacteria . Three expression vectors were constructed in which the synthesis of HBcAg-p25 was controlled by the tac promoter, and the number of nucleotides between the bacterial ribosome binding site and the precore initiation codon was varied in order to maximize HBcAg-p25 synthesis . The relative amount of HBcAg-p25 polypeptide expressed by the different vectors was estimated by SDS-polyacrylamide gel electrophoresis and immunoblot . HBcAg-p25 was associated with an insoluble fraction of bacterial extracts and required ionic detergents for solubilization . Comparison by ELISA of the immunoreactivity of HBcAg with and without the precore sequence suggested that human anti-HBcAg IgG preferentially recognizes HBcAg lacking the precore sequence. Curr Genet, 1987, 12(4), 241 - 6 Functional in vivo verification in E . coli of promoter activities from the rDNA/tDNA(Val)(GAC) leader region of Zea mays chloroplasts; Delp G et al.; Restriction fragments containing upstream sequences of the rRNA operon from Zea mays chloroplasts were tested for promoter activity in vivo by insertion into an E . coli promoter-probe vector . The expression of this vector's reporter gene, which codes for alkaline phosphatase, was stimulated more than 1,500-fold upon linkage with the chloroplast rRNA promoter . Site specific mutagenesis of the invariant T of the -10 sequence of this promoter reduced the expression of the reporter gene to 2% of the wild type . This indicates that the chloroplast rRNA promoter, which directs transcriptional initiation 117 bp upstream of the 16S rRNA gene, is also active in the bacterial system . A restriction fragment further upstream containing the gene for tRNA(Val) (GAC) also showed strong promoter activity (29% as compared with the rRNA promoter) . This promoter activity probably reflects the chloroplast promoter directing the synthesis of the tRNA(Val) (GAC) primary transcript . Surprisingly, this restriction fragment also displayed promoter activity (13% compared with the rRNA promoter) in reverse orientation. Proteins, 1987, 2(1), 42 - 53 Subunit assembly and metabolic stability of E . coli RNA polymerase; Ishihama A et al.; Immunological cross-reaction was employed for identification of proteolytic fragments of E . coli RNA polymerase generated both in vitro and in vivo . Several species of partially denatured but assembled RNA polymerase were isolated, which were composed of fragments of the two large subunits, beta and beta', and the two small and intact subunits, alpha and sigma . Comparison of the rate and pathway of proteolytic cleavage in vitro of unassembled subunits, subassemblies, and intact enzymes indicated that the susceptibility of RNA polymerase subunits to proteolytic degradation was dependent on the assembly state . Using this method, degradation in vivo was found for some, but not all, of the amber fragments of beta subunit in merodiploid cells carrying both wild-type and mutant rpoB genes . Although the RNA polymerase is a metabolically stable component in exponentially growing cells of E . coli, degradation of the full-sized subunits was found in two cases, i.e., several temperature-sensitive E . coli mutants with a defect in the assembly of RNA polymerase and the stationary-phase cells of a wild-type E . coli . The in vivo degradation of RNA polymerase was indicated to be initiated by alteration of the enzyme structure. Padiatr Padol, 1987, 22(3), 293 - 303 {E . coli dyspepsia}; Lachmann D; The incidence of EPEC infections has decreased dramatically in industrialized countries since the 1960s, 1970s, but EPEC remains an important of acute gastroenteritis in developing countries. CRC Crit Rev Biochem, 1987, 22(3), 181 - 219 Structure and function of E . coli promoter DNA; Travers AA; The process of transcription initiation requires both the recognition of a promoter site by RNA polymerase and the melting of a short stretch of DNA . In this review I discuss the properties of promoters that are relevant to sequence recognition and to the ability of the polymerase to act as a melting protein . The regulation of promoter activity is thus dependent on both factors interacting with RNA polymerase and so altering its affinity for promoter sites and also modulations of DNA structure. Int Urol Nephrol, 1987, 19(1), 27 - 32 The effect of E . coli infection on the prostaglandin synthesizing capacity of postobstructive rat kidney; Sallai J et al.; The PGE2, PGI2, PGF2 alpha and TxA2 synthesizing activities were studied in an isolated microsomal fraction of rat kidney after temporary, unilateral ureter obstruction and E . coli infection . In the early phase of regeneration the synthesis of vasodilatory PGI2 was increased, whereas that of vasoconstrictory PGF2 alpha was decreased . An increased PGE2 synthesizing activity was observed when renal obstruction was associated with infection . The role of these changes in regenerating the haemodynamics and function of postobstructive kidney is discussed. Nahrung, 1987, 31(5-6), 625 - 9 {Intestinal beta-galactosidase in gnotobiotic animals following monoassociation with various E . coli strains}; Schulze J et al.; Germ-free rats were monoassociated by E . coli germs not utilizing (L-) or utilizing lactose (L+) on endo-medium . There was no influence of germ status on the beta-galactosidase activity in the mucosa of the small and large intestine . With standard food, beta-galactosidase activity were to be measured in the chymus of all intestinal segments and in the faeces of germ-free as well as of monoassociated rats . In the chymus of caecum and colon and in the faeces of E . coli(L+)-animals, only, the short-time (12-16 days) application of lactose containing food resulted in an increase of the enzyme activity . Compared with the E . coli(L-)-animals, the lactose content in the chymus of all intestinal segments of this test group was decreased. Int J Biochem, 1987, 19(1), 47 - 52 The inactivation of beta-galactosidase (E . coli) by the carbodiimide reaction; Gaunt MT et al.; When beta-galactosidase reacted with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC), activity was lost . The inhibitor, isopropyl-beta-D-galactopyranoside (IPTG), decreased inactivation . Of 3 nucleophiles tested, incorporation was only decreased in the protected (IPTG added) enzyme when sulfanilic acid was the nucleophile but HPLC profiles of tryptic peptides were identical in protected and unprotected enzyme (except for magnitude) . There were also no differences (except for magnitude) of HPLC profiles after 10 and 90 min of reaction and between active (soluble) and inactive (precipitated) enzyme . The data indicate that inactivation is not caused by reaction with a specific active site group . Inactivation probably occurs when a combination of groups are reacted. FEBS Lett, 1987 Jan 1, 210(1), 56 - 60 Chemical synthesis and expression in E . coli of a human Val8-calcitonin gene by fusion to a synthetic human interferon-gamma gene; Ivanov I et al.; A gene coding for human Val8-calcitonin (Val8-hCT) was synthesized by the solid-phase phosphite approach and fused to a synthetic human immune interferon-gamma (IFN-gamma) gene . The IFN gene was previously shown to be expressed at a very high level in E . coli {(1986) Gene, in press} due to the control of a strong synthetic promoter and strong ribosome binding site . The cells harboring the fused gene produced 100-150 micrograms per l of bacterial suspension of immunoreactive calcitonin in the form of hybrid IFN-gamma-Val8-hCT protein consisting of 140 amino acids . The Val8-hCT can be released from this protein by CNBr treatment. Biull Eksp Biol Med, 1987 Jan, 103(1), 80 - 3 {Characteristics of the B subunit of a thermolabile Escherichia coli enterotoxin produced by the A-B+ E . coli strain}; Voronov SE et al.; The preparation and identification of B subunit of thermolabile enterotoxin produced by A-B+ gene-containing strain are described . The E . coli strain studied is shown to produce protein identical in its molecular properties and antigenic specificity to B subunit obtained from the whole thermolabile enterotoxin . Partial antigenic affinity between B subunits of thermolabile enterotoxin obtained from different sources and B subunit from cholera enterotoxin has been established in immunochemical studies . Electrophoretic and immunochemical analysis has confirmed the absence of A-subunit admixtures in B-subunit preparation obtained from /A-B+/E . coli strain. Circ Shock, 1987, 21(1), 23 - 9 Effects of E . coli endotoxin on rat plasma angiotensin converting enzyme activity in vitro and in vivo; Deitz DM et al.; Angiotensin converting enzyme (ACE), a glycoprotein, is found in high concentration in pulmonary capillary endothelial cells . Several investigators have studied the relationship between various direct and indirect pulmonary insults and ACE activity and in some cases have found conflict . In an attempt to clarify this relationship we have examined the effect of endotoxin on rat plasma ACE activity in vitro and in vivo . We used the synthetic ACE substrate 3H-BPAP and the assay described by Catravas and Gillis {J Pharmacol Exp Ther 217:263-270, 1981} . In vitro, a statistically significant concentration-dependent reduction in ACE activity was demonstrated (p less than .005) . In vivo an intravenous dose of endotoxin (20 mg/kg) alone resulted in no significant change in plasma ACE activity . However, the combination of intravenous endotoxin (20mg/kg) and mild hemorrhage (5-10% of blood volume) caused a statistically significant reduction in plasma ACE activity by 15 min as compared to control rats with hemorrhage only (39% vs . 66%, p less than .005) . This reduction persisted at 30 and 60 min . However, by 180 min ACE activity was no longer statistically different from control values . We have demonstrated an acute reduction of plasma ACE activity in the endotoxemic rat that appears to be dependent on the amount of circulating endotoxin and the presence of mild blood loss. FEBS Lett, 1987 Jan 1, 210(1), 11 - 6 cDNA cloning and expression in E . coli of a plasminogen activator inhibitor (PAI) related to a PAI produced by Hep G2 hepatoma cell; Wun TC et al.; The human hepatoma line Hep G2 produces an acid- and SDS-sensitive plasminogen activator inhibitor (PAI) . This protein has been previously purified and used to raise polyclonal antibodies . This antiserum has been used to isolate cDNA clones from a human placental lambda gt11 cDNA library . The immunologically positive clones were screened for expression of recombinant proteins which inhibit urokinase activity and form an inhibitor-enzyme complex with 125I-urokinase . Two positives (lambda PAI 11.1 and lambda PAI 14.1) have been obtained . The cDNA insert of the longer isolate (lambda PAI 14.1) consists of 1962 base pairs encoding the entire mature Hep G2 PAI and a 3'-noncoding region of 801 base pairs . The clone apparently lacks portions of 5'- and 3'-untranslated sequences . The translated amino acid sequence matches the sequence obtained for the mature Hep G2 PAI and consists of 379 amino acids with a molecular mass of 42 770 Da . Interestingly, this PAI clone is quite different from the placental-type PAI-2 sequence as expected, but matches the sequence of the endothelial-type PAI (PAI-1) reported to be acid-insensitive and SDS-enhancible. Z Naturforsch {C}, 1987 Jan-Feb, 42(1-2), 129 - 33 Evolution of E . coli tRNA(Ile): evidence of derivation from other tRNAs; Staves MP et al.; Two E . coli tRNA(Ile) sequences were compared against those of 36 other E . coli tRNAs . tRNA(Ile) 1 was found to bear high similarity with tRNA(Val) 1 (E = 1.11 X 10(-18} while tRNA(Ile) 2 had the greatest match (E = 3.40 X 10(-19} with tRNA(Lysl) (E is the expected number of such matches, per search, based on coincidence) . These matches, which we consider to represent homologies, extend from base 7 to base 67 in the former and base 7 to the end (76) in the latter pair . These results coupled with others on the lower activity of isoleucine in reactions postulated to be important in primitive protein synthesis (i.e., esterification reactions and non-enzymatic activation by ATP {1-3}) lead us to propose that isoleucine was included among the proteinaceous amino acids, and received its anticodonic assignment, relatively late in evolution through mutation of tRNAs previously employed for other amino acids. Oncogene Res, 1987, 2(1), 95 - 101 Expression in E . coli of erg: a novel gene in humans related to the ets oncogene; Rao VN et al.; We have recently cloned a novel human gene named erg related to the ets oncogene . In this report we describe the expression of the erg gene product in E . coli under the control of the tightly-regulated phage lambda PL promoter . Two expression vectors were utilized for the synthesis of the cII-erg and erg-fusion proteins . All these products were recognized by peptide antibodies prepared against the predicted amino acid sequences of erg and the Hu-ets-2 domain which shares homology with erg. Biol Cell, 1987, 61(1-2), 1 - 4 Inclusion bodies in recombinant E . coli producing human calcitonin tetramer, as visualized by immuno-gold electron microscopy; Petrov P et al.; Inclusion bodies are described in recombinant E . coli cells harboring plasmid for the expression of a synthetic gene coding for human calcitonin tetramer . The inclusion bodies are visualized by electron microscopy and the protein is identified by immuno-gold technique, using antibodies against synthetic human calcitonin . The diameter of the inclusion bodies is 1 micron on the average. Arch Virol, 1987, 97(3-4), 365 - 72 Expression of an early Epstein-Barr virus antigen (EA-D) in E . coli . Brief report; Roeckel D et al.; The 1.34 kb BcII-BgIII-fragment of the BamHI-M region of Epstein-Barr virus genome, comprising the complete BMRF1 open reading frame, was cloned into the tryptophan regulated E . coli expression vector pATH1 . The resulting fusion protein, having a molecular weight of 80 kd, is recognized not only by anti-early antigen (EA)-positive human sera but also by the monoclonal antibody R3 directed against the diffuse component of EA (EA-D) . A possible use for this fusion protein as an indicator protein in diagnosis of IgA antibodies against EA-D is presented. Vet Med Nauki, 1987, 24(6), 19 - 24 {Phenotypic characteristics of a new modification-restriction system in E . coli}; Popovski B; It has been demonstrated phenotypically that there exists a new modificational-and-restrictional system as synthesized by the recombinant Escherichia coli tF strain . A series of passages of the T3 and T7 phages and their mutants in E . coli tF has made it possible to ascertain a specific modification of the phage DNA, with was shown to be induced by the host strain . The high level of adsorption of these phages on the cell surface of E . coli tF has ruled out the possibility of existing of a 'nonclassical' modification and restriction of DNA . In view of the further characterizing of this modificational-and-restrictional system of E . coli tF it has been comparatively studied with the already known modificational-and-restrictional systems isolated from various Escherichia coli strains . Results have shown that no identity exists between the tested systems and the one in E . coli tF . It is stated that the new modificational-and-restrictional system of E . coli tF belongs to none of the three known types of restrictional endonucleases. Biochem Biophys Res Commun, 1986 Dec 30, 141(3), 1138 - 44 Inhibition of E . coli adenylate cyclase activity by inorganic orthophosphate is dependent on IIIglc of the phosphoenolpyruvate:glycose phosphotransferase system; Liberman E et al.; The relationship of adenylate cyclase, inorganic orthophosphate and the proteins of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was studied . A strain deleted for the genes for Enzyme I and IIIglc of the PTS was transformed with plasmids expressing either Enzyme I and HPr, IIIglc or all three proteins . The fully reconstituted strain showed a Pi-dependent stimulation of adenylate cyclase activity; in contrast, the strain expressing only IIIglc showed a Pi-dependent inhibition of adenylate cyclase activity. Cell, 1986 Dec 26, 47(6), 995 - 1005 The DNA binding domain and bending angle of E . coli CAP protein; Liu-Johnson HN et al.; We use a new gel electrophoretic analysis to map the thermodynamically defined DNA binding domain of Escherichia coli CAP protein in the lac promoter . Strong binding interactions span a 28-30 bp duplex DNA region, substantially larger than that found for typical repressors . Sequence changes outside the central 28 bp of the binding site are found to affect the electrophoretically observed extent of bending . We also report a study of the DNA bending induced at a symmetrized CAP binding site, compared with the wild-type site; binding and bending are stronger at the upstream than at the downstream half of the wild-type site . Bends of the estimated 90 degrees - 180 degrees magnitude could play a vital regulatory role by producing tertiary structure in a local DNA domain, and by storing elastic energy for subsequent use in transcription or replication. Carbohydr Res, 1986 Dec 15, 158, 91 - 9 Synthesis of 2,6-anhydro-S-{ethylmercury(II)}-1-thio-D-glycero-L-manno-heptitol+ ++ and bis(2,6-anhydro-1-thio-D-glycero-L-manno-heptitol)mercury(II), and the study of their interaction with beta-D-galactosidase from E . coli; John C et al.; Two competitive inhibitors of beta-D-galactosidase activity, namely, 2,6-anhydro-S-{ethylmercury(II)}-1-thio-D-glycero-L-manno-heptitol (4) and bis(2,6-anhydro-1-thio-D-glycero-L-manno-heptitol)mercury(II) (6), with inhibition constants of 8.0 X 10(-4) M and 1.9 X 10(-4) M, were synthesized . Compound 6 was incorporated into the crystalline enzyme by cocrystallization . The stoichiometry of the enzyme-inhibitor complex was 1:4, corresponding to one molecule of inhibitor per active site of the enzyme . Compound 4 was found to be unstable against X-ray irradiation, whereas compound 6 was submitted to X-rays for several days without any radiation damage. Biochem Biophys Res Commun, 1986 Dec 15, 141(2), 804 - 11 Expression of human interleukin-2 receptor cDNA in E . coli; Chanda PK et al.; cDNAs for human interleukin-2 receptor were recently cloned and sequenced (Leonard et al., 1984, Nature 311, 626-631; Nikaido et al., 1984, Nature 311, 631-635; Cosman et al., Nature 312, 768-771) . In the studies reported here, we describe the expression of a cDNA clone for the human interleukin-2 receptor in E . coli using an "open reading frame" expression vector pMR100 . The inserted cDNA was expressed in E . coli transformants as a tripartite fusion polypeptide fused to the lambda cI protein at its amino terminus and to beta-galactosidase at its carboxy terminus . We demonstrate that the bacterially produced IL-2 receptor protein can bind to IL-2. FEBS Lett, 1986 Dec 15, 209(2), 325 - 9 Absence of a unique relationship between active transport of lactose and protonmotive force in E . coli; Ghazi A et al.; The relationship between active transport of lactose via the lactose permease and the protonmotive force has been determined in E . coli cells using either the respiratory chain inhibitor cyanide or protonophores to decrease the protonmotive force progressively . In contradiction with the prediction of the delocalized chemiosmotic theory, two different relationships were obtained depending on the method used. Science, 1986 Dec 12, 234(4782), 1392 - 5 HTLV-III/LAV-neutralizing antibodies to an E . coli-produced fragment of the virus envelope; Putney SD et al.; Immunization with either an Escherichia coli recombinant segment of the human T-cell lymphotropic virus (HTLV-III/LAV) envelope protein (gp 120) or with deglycosylated gp 120 envelope protein produced antibodies that neutralize HTLV-III/LAV infection in vitro . Virus neutralization titers of these antisera were equivalent to those obtained with purified native gp120 as immunogen . This localizes at least one class of neutralizing epitopes to the carboxyl-terminal half of the molecule . In addition, native gp120 prevented HTLV-III/LAV--mediated cell fusion, whereas the recombinant gp120 fragment did not . This shows that although glycosylation is not required for induction of neutralizing antibodies, it may be important for interaction with CD4, the virus receptor . A segment of the HTLV-III/LAV envelope produced in E . coli may be an important ingredient of a vaccine for acquired immune deficiency syndrome. J Clin Lab Immunol, 1986 Dec, 21(4), 177 - 81 Effect of intravenous immunoglobulin on E . coli opsonization in multiple myeloma; Hertler AA et al.; Infections are a major source of morbidity in multiple myeloma; E . coli is now the leading pathogen . Intravenous IgG may be a modality which could ameliorate the opsinopathy of multiple myeloma . We infused 6 patients with multiple myeloma with IgG (100 mg/kg) and compared E . coli opsonophagocytosis pre- and post-IgG infusions . Log phase, broth grown E . coli K12 (5 X 10(6)/ml) and normal, dextran-sedimented human neutrophils (5 X 10(6)/ml) were combined in 10% heat inactivated, pre- or post-IgG infusion multiple myeloma serum with 5% agammaglobulinemic serum as a complement source and incubated at 37 degrees C for 30 min . Phagocytosis was quantified as percentage survival of the inoculum . Control survival in heat inactivated normal serum + complement + neutrophils was 1.7 +/- 0.8% . Pre- and post-IgG infusion sera were equally abnormal opsonin sources with 14.3 +/- 6.5% and 17.5 +/- 3.0% survivals . Individually, patients with poor opsonophagocytosis improved with IgG (e.g., pre = 45.2 +/- 3.7%; post = 29.5 +/- 2.3% survival), whereas patients with good opsonophagocytosis showed a deleterious effect (e.g., pre = 2.3 +/- 0.9%; post 23.3 +/- 6.3% survival) . To explain these data, we measured deposited IgM and IgG on E . coli by pre- and post-IgG infusion sera in a fluorescence immunoassay . Pre- and post-IgG infusion sera had equally depressed IgM deposition (pre = 13.7 +/- 2.1%; post = 14.5 +/- 2.6% of normal serum), and also equal IgG deposition (pre = 96.8 +/- 6.5%; post = 94.6 +/- 4.8% of normal serum).(ABSTRACT TRUNCATED AT 250 WORDS) Bioorg Khim, 1986 Dec, 12(12), 1678 - 81 {Affinity modification of E . coli RNA-polymerase in a complex with the promoter by phosphorylating derivatives of primer oligonucleotides}; Grachev MA et al.; Oligonucleotides 2 to 7 nucleotide residues long, complementary to the codogenic strand of T7 promoter A2, have been synthesized; all of them contained a ribo-unit at the 3'-end . They were converted into 5'-(N-methyl)phosphoimidazolides, and the affinity reagents obtained were allowed to bind covalently to RNA polymerase in the presence of a promoter . Some of the nucleotide residues covalently attached occupied proper positions relative to the active centre of the phosphodiester bond synthesis and on addition of {alpha-32P}UTP were elongated, so that highly selective affinity labelling occurred . With oligonucleotides of various lengths, different distribution of the label between beta, beta' and sigma subunits of RNA polymerase took place . Most efficient was labelling of beta-subunit by the residue--pCpGpCpU, and of sigma-subunit by the residue--pApApApTp-CpGpCpU (p--radioactive phosphorus atom) . In both cases, the amino acid residues labelled were histidines. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1986 Dec, 183(1), 58 - 66 {Estimating the biological effect of detrimental substances on E . coli with flow microcalorimetry . III . Studies using m-cresol in glucose phosphate buffer}; Hartung J; Investigations are carried out to characterize the influence of the phenolic compound m-cresol on the heat production of glucose-respiring E . coli cells suspended in glucose phosphate buffer in a flow microcalorimeter according to the method of Perry et al . (5) The results are usually obtained within one hour . When testing m-cresol by this method thermic irritations occurred . A modified test procedure which largely avoids these troubles is described . The height (Q = heat flow) of the power-time-curves is evaluated 30 min and 60 min after the addition of the test substance . These values are compared to the values obtained from control-curves without any test substance . The 60 min-values seem less variable than the 30 min-values . The minimum effective m-cresol concentrations come to 7.50 mmol/l after 30 min and to 9.37 mmol/l after 60 min . The coefficients of variation are distinctly lower than +/- 10% at concentrations of less than 11.25 mmol/l . The coefficients of variation from 3 to 5 tests can extend +/- 20% both for the 30 min-values and the 60 min-values at higher concentrations (e.g . 15 mmol/l) . Under the influence of increasing m-cresol concentrations the germ content in the solution and the heat flow decrease in a similar way . The results indicate that the used flow microcalorimetric technique is suitable to check the effect of a test substance on a test culture within only one hour's time . Possibly, the method can be helpful for pretesting disinfectants . The gathering of further experience with this system seems advisable. EMBO J, 1986 Dec 1, 5(12), 3219 - 25 A synthetic operon containing 14 bovine pancreatic trypsin inhibitor genes is expressed in E . coli; von Wilcken-Bergmann B et al.; A synthetic gene encoding the protein sequence of mature bovine pancreatic trypsin inhibitor (BPTI) has been cloned into a novel E . coli expression vector . After in vitro gene amplification by successive DNA duplications, more than 600 000 mostly inactive inhibitor molecules may be recovered from a single cell . After purification the inhibitory activity can be reconstituted almost completely . The specificity of BPTI for trypsin is abolished by a single amino acid exchange from lysine to isoleucine at position 15 . The altered protein is shown to be an efficient inhibitor of human leukocyte elastase. Immunology, 1986 Dec, 59(4), 541 - 8 Induction of inflammatory mediators from human polymorphonuclear granulocytes and rat mast cells by haemolysin-positive and -negative E . coli strains with different adhesins; Scheffer J et al.; We investigated the role of various E . coli strains that expressed different adhesins and/or generated haemolysin with regard to the induction of inflammatory mediators, e.g . histamine release from rat mast cells as well as the chemiluminescence response and the release of lipoxygenase transformation products from human polymorphonuclear neutrophils . Our data show that the degree of haemagglutination did not parallel the induction of the chemiluminescence response . Haemolysin-negative bacteria with different adhesins induced more 5-hydroxyeicosatetraenoic acid as compared to haemolysin-positive bacteria, which generated more leukotriene B4 as compared to 5-hydroxyeicosatetraenoic acid . Among the leukotrienes, more leukotriene B4 as compared to leukotriene C4 was released from peripheral leucocytes . Studies with rat mast cells showed that histamine release was dependent on the haemolysin activity expressed by washed bacteria or present within the bacterial culture supernatant . Histamine release was markedly diminished when haemolysin activity decayed . Several haemolysin-negative bacteria with defined adhesins also released histamine, suggesting that, in addition to haemolysin, other factors contribute to mediator release . Thus, various properties of bacteria (e.g . adhesins, haemolysin) may participate to varying degrees in the induction of inflammatory mediators, e.g . oxygen radicals, lipoxygenase transformation products, leucotrienes and histamine. FEBS Lett, 1986 Nov 24, 208(2), 211 - 6 Sequence-imposed structural constraints in the TonB protein of E . coli; Evans JS et al.; The solution conformation of a 33-residue peptide segment, derived from the TonB protein which is implicated in bacterial membrane transport processes, has been investigated using high-resolution proton magnetic resonance techniques . This proline-rich peptide possesses sequence-imposed sections of elongated secondary structure that must be retained in the native protein configuration . These structural constraints provide elements of stiffness that imply a purely structural role for TonB and are relevant to the subcellular location and biological role of the protein . On the basis of these data we suggest that this protein spans the periplasmic space, linking the inner and outer membrane components of TonB-dependent transport systems. Eur J Biochem, 1986 Nov 3, 160(3), 441 - 9 Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA . Its use for E . coli ribosome structural analysis; Favre A et al.; In vivo incorporation of the uridine-photoactivable analogue, 4-thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated . It is highly dependent on the exogenous uridine and 4-thiouridine concentrations as well as on temperature . We have defined conditions allowing the substitution of 13 +/- 2% of the uridine residues in bulk RNA by 4-thiouridine . On a high-Mg2+ sucrose gradient, 33 +/- 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non-associated 50S and 30S particles containing immature rRNA . The thiolated 70S ribosomes tolerate a 4-5% substitution level (40 thiouridine molecules/particle) . Surprisingly, 3-4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4-thiouridine-substituted rRNA . Specific 366-nm photoactivation increased this proportion to 10-12%, i.e . up to six or seven ribosomal protein molecules/particle . The photochemical cross-linking proceeds with apparent first-order kinetics with a quantum yield close to 5 X 10(-3) . Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross-linking were independent of oxygenation conditions . The thiolated (4.5%) 70S ribosomes allowed the poly(U)-directed poly(Phe)synthesis at 48% the control rate . Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively. J Urol, 1986 Nov, 136(5), 1117 - 22 Immunology of pyelonephritis . VIII . E . coli causes granulocytic aggregation and renal ischemia; Kaack MB et al.; We studied renal venous blood after renal infection for its concentrations of leukocytes, complement and renin . In addition, we evaluated this blood for granulocytic aggregation and chemiluminescence of granulocytes . We found that very rapid activation of serum complement occurred with associated granulocytic aggregation and evident vascular occlusion and ischemia since renin rose rapidly . It appears that this early sequence of events will cause renal damage by ischemic change, as well as that known to occur from the inflammatory reaction. Proteins, 1986 Nov, 1(3), 247 - 55 Kinetic characterization of early intermediates in the folding of E . coli tryptophan-synthase beta 2 subunit; Blond S et al.; This report describes the use of fluorescence energy transfer between an intrinsic energy donor (tryptophan 177) and two chemically added acceptors to study intermediates in the folding of the beta 2 subunit of E . coli tryptophan-synthase . Two early folding steps are thus identified and characterized . One is very rapid (its rate constant at 12 degrees C is 0.02 sec-1) and corresponds to the folding of the N-terminal domain into a structure whose overall features approximate well those of the native domain . The second step is somewhat slower (its rate constant at 12 degrees C is 0.008 sec-1) and involves a conformational rearrangement of the N-terminal domain brought about by the interactions between the N- and C-terminal domains within a monomeric beta chain . This brings to five the number of intermediates which have been identified and ordered on the folding pathway of the dimeric beta 2 subunit. Int J Radiat Biol Relat Stud Phys Chem Med, 1986 Nov, 50(5), 861 - 9 Radiation protection of E . coli strains by cysteamine in the presence of oxygen; Hulsewede JW et al.; The survival of various E . coli K12 strains with defects in the rec system have been measured after gamma-irradiation in air in the presence (0.1 mol dm-3) or in the absence of cysteamine . The results confirm those of Bresler et al . (1978) indicating that the protection by cysteamine in the presence of oxygen is due to an influence on enzymatic repair . The low protection by cysteamine of wild-type cells pretreated with chloramphenicol which prevents protein synthesis, supports the above conclusion . The reason for the absence of a protective effect by OH radical scavenging and H-atom donation is discussed . It is proposed that DNA peroxyl radicals are formed during irradiation in the presence of oxygen and that they are transformed into hydroperoxides by H-atom donation from the intracellular glutathione and the added cysteamine . These hydroperoxides are still dangerous for the cell as indicated by the protective action of glutathione peroxidase observed by Marklund et al . (1984). Mutat Res, 1986 Nov, 166(3), 299 - 42 Mutagenesis and repair of DNA damage caused by nitrogen mustard, N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), streptozotocin, and mitomycin C in E . coli; Fram RJ et al.; Cytotoxicity and mutagenesis by streptozotocin, BCNU, nitrogen mustard, and mitomycin C were evaluated in E . coli mutants deficient in SOS repair, SOS-mediated mutagenesis, the adaptive response, and mutants that engage in aberrant mismatch repair . The results demonstrate that premutagenic lesions are caused by nitrogen mustard, BCNU and streptozotocin that are not repaired by ada or recognized by umuDC . Further, recA mutants were hypomutable after exposure to nitrogen mustard, BCNU, and streptozotocin compared to wild type . With the exception of the monofunctional nitrosourea, streptozotocin, both recA and uvrA gene products contribute to the repair of DNA damage caused by the alkylating agents tested . In the case of streptozotocin, although recA mutants were more sensitive than wild type, uvrA mutants were not . Moreover, while ada and alkA E . coli mutants showed increased sensitivity to streptozotocin, they were not more sensitive to the other alkylating agents evaluated. Mutat Res, 1986 Nov, 166(3), 243 - 54 Cytotoxicity and SOS-inducing ability of ethidium and photoactivable analogs on E . coli ethidium-bromide-sensitive (Ebs) strains; Lambert B et al.; In a recently-characterized ethidium-bromide-sensitive E . coli strain, DNA appears to be much more accessible to DNA-binding agents . This strain therefore appears to be of interest for studying the mutagenic properties of chemicals . For this purpose, a series of ethidium-sensitive E . coli strains (Ebs) with normal and defective DNA-repair capacity was constructed and made lysogenic for lambda (sfiA::lacZ) . These strains were used to study the cytotoxicity and SOS-inducing ability of ethidium and its two photoactivable analogs 8-azido- and 3,8-diazido-ethidium . When non-covalent DNA complexes are formed, these dyes elicit only a bacteriostatic effect in the Ebs strains, which is almost independent of the strain's DNA-repair capacity . The SOS system is not induced . When covalent DNA adducts are formed after photoactivation of ethidium azido analogs, the effects are quite different . The formation of about 5 DNA monoadducts per cell induces a lethal hit in the Ebs uvrB recA strain and measurable SOS induction in the Ebs uvrB (lambda (sfiA::lacZ) strain . The formation of more than 1000 DNA adducts in the Ebs strain with normal DNA-repair capacity does not induce any measurable cytotoxic effect. Nucleic Acids Res, 1986 Oct 24, 14(20), 7851 - 60 High level production and rapid purification of the E . coli trp repressor; Paluh JL et al.; Two small, multicopy, expression plasmids were constructed that permit convenient insertion of trpR, the structural gene for the trp repressor of Escherichia coli, with its natural ribosome binding site or adjacent to the ribosome binding site for the trp leader peptide . In these plasmids trpR is positioned between the strong regulated tac promoter and the rpoC transcription terminator . IPTG induction of lacIq strains bearing these plasmids results in the production of 25-50% of the soluble cell protein as trp repressor . Mutant and wild type repressors overproduced in this manner have been purified by simple procedures. Nucleic Acids Res, 1986 Oct 10, 14(19), 7529 - 39 The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E . coli and yeast; Englisch S et al.; Three analogues each of leucine and isoleucine carrying hydroxy groups in gamma- or delta- or gamma- and delta-position have been synthesized, and tested in the aminoacylation by leucyl-tRNA synthetases from E . coli and yeast . Hydrolytic proofreading, as proposed in the chemical proofreading model, of these analogues and of homocysteine should result in a lactonisation of these compounds and therefore provide information regarding the proofreading mechanism of the two leucyl-tRNA synthetases . Leucyl-tRNA synthetase from E . coli shows a high initial substrate discrimination . Only two analogues, gamma-hydroxyleucine and homocysteine are activated and transferred to tRNALeu where a post-transfer proofreading occurs . Lactonisation of gamma-hydroxyleucine and homocysteine could be detected . Leucyl-tRNA synthetase from yeast has a relatively poor initial discrimination of these substrates, which is compensated by a very effective pre-transfer proofreading on the aminoacyl-adenylate level . No lactonisation nor mischarged tRNALeu is detectable. Nucleic Acids Res, 1986 Oct 10, 14(19), 7705 - 11 Nucleotide sequences of the gal E gene and the gal T gene of E . coli; Lemaire HG et al.; The nucleotide sequences of the gal E gene coding for UDP-galactose-4-epimerase and the gal T gene coding for galactose-1-P uridyltransferase of Escherichia coli have been determined . UDP-galactose-4-epimerase and galactose-1-P uridyltransferase are predicted to consist of 338 and 347 residues, respectively, NH2-terminal methionines included. FEBS Lett, 1986 Oct 6, 206(2), 323 - 8 Palindromic units from E . coli as binding sites for a chromoid-associated protein; Gilson E et al.; Several hundred copies of a highly conserved extragenic palindromic sequence, 20-40 nucleotides long, exist along the chromosome of E . coli and S . typhimurium . These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP) . No general function for PUs has been identified . In the present work, we provide data showing that a protein associated with a chromoid extract of E . coli protects PU DNA against exonuclease III digestion . This provides the first experimental evidence that PU constitutes binding sites for a chromoid-associated protein . This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid. J Biomol Struct Dyn, 1986 Oct, 4(2), 291 - 307 Distribution and evolution of sequence characteristics in the E . coli genome; Blake RD et al.; The mean (G + C) composition (51.0%) and standard deviation (+/- 3.8%) of published DNA sequences accounting for 10% of the E . coli genome is in excellent agreement with the principal overall distribution determined by high resolution melting . While differences in base and neighbor characteristics are small and uniform throughout all regions of the genome, it is found that the (G + C) content of sequences varies in segmented fashion within boundaries corresponding to coding (53% G + C) and noncoding (46% G + C) regions; with variances in the latter being six-fold greater than in coding regions . The variance in different regions shows a strong negative dependence on (G + C) content of the region, reflecting the condition that A-T and G-C base pairs are preferred neighbors of A-T and C-G pairs, respectively; with the bias increasing with decreasing (G + C) content . Neighbor analysis indicates the most extreme positive biases occur in AA, TT, GC and CG throughout all regions, but particularly in noncoding regions . Extraordinary numbers of oligomeric strings of (A)n, etc., are the further consequence of this bias . These and other characteristics point to the existence of inherent biases in neighbor frequencies levied during replication or repair, and which reflect, in turn, neighbor influences during mutation . The bias in codon usage noted by Grantham and others is seen here as due, in part, to the adaptation of coding sequences to this microenvironment through selection among synonymous codons so as to preserve inherent neighbor