Escherichia coli

Eur J Surg, 1991 Feb, 157(2), 117 - 20
Bile increases lipopolysaccharide release in experimental E . coli peritonitis; Andersson R et al.; Previous studies in rats showed increased mortality when bile was added to intraperitoneally injected Escherichia coli . In the present study bacterial counts and levels of lipo-polysaccharide (LPS) were determined in the peritoneal cavity and in blood 0.5, 1, 4 and 10 hours after induction of peritonitis with E . coli alone or together with bile . LPS was measured with gas chromatographic analysis of beta-hydroxymyristic acid, a characteristic component of E . coli-LPS . Bacterial counts and LPS levels in peritoneal fluid and blood rose higher in E . coli + bile peritonitis than in E . coli peritonitis . The intergroup difference in LPS levels was evident at 0.5 and 1 hour, whereas the bacterial counts began to differ at 2 hours . Presence of intraperitoneal bile in E . coli peritonitis thus produced rapid rise in LPS levels that could not be caused by bacterial numbers alone . This early load of LPS may help to explain the noxious effect of bile in E . coli peritonitis.

Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 853 - 60
Mutagenesis of human fibrinogen gamma chain 259-411 synthesized in E . coli: further characterization of the role of the disulfide bond CYS326-CYS339 in calcium binding; Bolyard MG et al.; We have produced human fibrinogen gamma 259-411 in Escherichia coli in order to study the relationship between the calcium binding activity of the polypeptide and the integrity of the disulfide bond cysteine326-cysteine339 . The polypeptide was produced from a plasmid expression vector at approximately 5 micrograms per milliliter of bacterial culture . The identity of the polypeptide was confirmed by N-terminal amino acid sequence analysis . The expression vector was modified by oligonucleotide directed mutagenesis to remove the nucleotides encoding cysteines gamma 326 and gamma 339 . The calcium binding activity of wild-type and altered polypeptides were compared using a solid phase assay . Our results indicate that removal of the two cysteine residues had no appreciable effect on calcium binding activity . We conclude that the integrity of the disulfide bond cysteine326-cysteine339 is not critical for calcium binding to gamma 259-411.

Nucleic Acids Res, 1991 Jan 25, 19(2), 265 - 9
Cysteinyl-tRNA synthetase: determination of the last E . coli aminoacyl-tRNA synthetase primary structure; Eriani G et al.; The gene coding for E . coli cysteinyl-tRNA synthetase (cysS) was isolated by complementation of a strain deficient in cysteinyl-tRNA synthetase activity at high temperature (43 degrees C) . Sequencing of a 2.1 kbp DNA fragment revealed an open reading frame of 1383 bp coding for a protein of 461 amino acid residues with a Mr of 52,280, a value in close agreement with that observed for the purified protein, which behaves as a monomer . The sequence of CysRS bears the canonical His-Ile- Gly -His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS) motifs characteristic of the group of enzymes containing a Rossmann fold; furthermore, it shows striking homologies with MetRS (an homodimer of 677 residues) and to a lesser extent with Ile-, Leu-, and ValRS (monomers of 939, 860, and 951 residues respectively) . With its monomeric state and smaller size, CysRS is probably more closely related to the primordial aminoacyl-tRNA synthetase from which all have diverged.

Nature, 1991 Jan 10, 349(6305), 178 - 80
Three-dimensional structure of the E . coli DNA-binding protein FIS; Kostrewa D et al.; The factor for inversion stimulation, FIS, is involved in several cellular processes, including site-specific recombination and transcriptional activation . In the reactions catalysed by the DNA invertases Gin, Hin and Cin, FIS stimulates recombination by binding to an enhancer sequence . Within the enhancer, two FIS dimers (each 2 x 98 amino acids) bind to two 15-base-pair consensus sequences and induce bending of the DNA . Current models propose that the enhancer-FIS complex organizes a specific synapse, either through direct interactions with Gin, or by modelling the substrate into a configuration suitable for recombination . Using X-ray analysis at 2.0 A resolution, we now show that FIS is composed of four alpha helices tightly intertwined to form a globular dimer with two protruding helix-turn-helix motifs . The 24 N-terminal amino acids are so poorly defined in the electron density map as to make interpretation doubtful, indicating that they might act as 'feelers' suitable for DNA or protein (invertase) recognition . We infer from model building that DNA has to bend for tight binding to FIS.

Free Radic Res Commun, 1991, 12-13 Pt 1, 379 - 82
The molecular genetics of superoxide dismutase in E . coli; Touati D; The biological role and the regulation of superoxide dismutase (SOD) in E . coli have been investigated using genetics . Cloning of both E . coli SOD genes permitted construction of mutants completely lacking SOD . The conditional oxygen sensitivity of those mutants, together with their increased mutation rate, demonstrated the essential biological role of SOD . SOD-deficient mutants constitute a powerful tool to assess a possible role of O-2 or SOD in biological processes . Complementation of their deficiencies by the expression of SOD originating from a different organism is used for screening libraries for SOD genes of other species . Regulation of MnSOD has been studied using protein and operon fusions with the lactose operon, and isolating regulation mutants . These studies reveal multiregulation of MnSOD including response to the superoxide mediated oxidative stress and response to variations of the intracellular redox state induced by metabolic changes.

J Biochem (Tokyo), 1991 Jan, 109(1), 49 - 54
Site-directed mutagenesis of phosphoenolpyruvate carboxylase from E . coli: the role of His579 in the catalytic and regulatory functions; Terada K et al.; Phosphoenolpyruvate carboxylases (PEPC) {EC 4.1.1.31} from a wide variety of organisms contain a unique and highly conserved sequence, 578FHGRGGSIGRGGAP591 (coordinates for the Escherichia coli enzyme), which has been presumed to participate in the binding of phosphoenolpyruvate (PEP) . Since previous chemical modification studies had suggested the importance of His for the catalytic activity, the role of His579 was investigated by constructing variants of E . coli PEPC, in which this residue was substituted to Asn (H579N) or Pro (H579P) . Kinetic studies with partially purified enzymes revealed the following: (1) The apparent maximal velocities in the presence of acetyl-CoA (CoASAc, one of the allosteric activators) were 29% and 5.4% of the wild-type enzyme, for H579N and H579P, respectively . (2) The half-saturation concentration for PEP was increased about 40-fold by the substitutions, while those for another substrate (HCO3-) and the metal cofactor (Mg2+) were increased only 2- to 4-fold . (3) The half-saturation concentrations of four kinds of allosteric activators and of dioxane, an artificial activator, were also changed to various extents . Among them the most remarkable increase was observed for CoASAc (28-fold) . (4) The concentration of an allosteric inhibitor, aspartate, required for 50% inhibition remained substantially unchanged . It was concluded that the imidazole group of His579 is not obligatory for the enzyme catalysis, but plays important roles in catalytic and regulatory functions.

EMBO J, 1991 Jan, 10(1), 183 - 93
The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E . coli; Niki H et al.; An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated . The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome . The mukB gene codes for a large protein (approximately 180 kd) . A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene . Computer analysis revealed that the predicted MukB protein has distinct domains: an amino-terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha-helical coiled-coil domains and one globular domain, and a carboxyl-terminal globular domain which is rich in Cys, Arg and Lys . A 180 kd protein detected in wild-type cell extracts by electrophoresis is absent in mukB null mutants . Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning . At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells . We conclude that the MukB protein is required for chromosome partitioning in E . coli.

Mutat Res, 1991 Jan, 246(1), 75 - 91
Specificities mediated by neighboring nucleotides appear to underlie mutation induced by antifolates in E . coli; Veigl ML et al.; The antifolate, trimethoprim (TRMP, 5 microM) caused a 10-fold increase in mutation frequency and primarily induced base substitution and deletion mutations in wild-type E . coli . Base-substitutions induced by antifolates were equally divided between transition and transversion mutations . When mutations consistent with expected antifolate-induced deoxynucleotide pool imbalances were considered, 29 out of 32 base-substitution mutations in the i-d region of the lacI gene were followed 3' by the same nucleotide substituted at the base mismatch site and all but one mutation occurred at sites consistent with next nucleotide effects resulting from antifolate-induced deoxynucleotide pool alterations . 66% of the TRMP-induced base-substitutions were also found at sites of frequent mutation identified in the spontaneous spectrum of a mutator D5 strain of E . coli which is deficient in the 3'-exonucleolytic proofreading function of DNA polymerase III holoenzyme . These results suggest that the pool imbalances induced by the antifolate trimethoprim compromise the proofreading activity of polymerase III holoenzyme and lead to mutation at specific sites . The results also imply that not all DNA sequence environments encountered by DNA polymerase III holoenzyme and its accompanying exonuclease are handled with equal facility at the level of nucleotide insertion and exonucleolytic proofreading when the enzyme is faced with an intracellular nucleotide pool imbalance . A number of small deletion and duplication mutations were also induced by the antifolate trimethoprim . In most cases these mutations were flanked by at least two A:T base pairs which could facilitate DNA-strand breakage at deoxyuridine misincorporation sites.

Mutat Res, 1991 Jan, 246(1), 139 - 49
Diverse backmutations at an ochre defect in the tyrA gene sequence of E . coli B/r; Li BH et al.; A DNA fragment including most of the tyrA gene from E . coli B/r strain WU (Tyr-, Leu-) was amplified in vitro by polymerase chain reaction . The sequence was determined, first, for essentially all of the fragment to locate an ochre nonsense defect, and second, repeatedly for a region of the fragment from several independent isolates containing backmutations at the ochre codon (spontaneous and UV-induced) . There were 20 single base differences in the tyrA gene region from the analogous wild-type E . coli K12 sequence: an ochre codon at amino acid position 161, 18 silent changes (1 at the first codon base and 17 at the third) and one replacement of valine by alanine . Different backmutations at the ochre codon encoded lysine, glutamine, glutamic acid, leucine, cysteine, phenylalanine, serine or tyrosine . The diversities of base substitutions at the ochre codon after UV mutagenesis or after mutagenesis where targeting by dimers was reduced or eliminated (after photoreversal of irradiated cells treated with nalidixic acid to induce SOS functions or after UV mutagenesis of cells containing amplified DNA photolyase) were similar (with two notable exceptions) . The overall differences between the gene sequences for E . coli K12 or B/r seemed consistent with the neutral theory of molecular evolution.

Free Radic Res Commun, 1991, 12-13 Pt 2, 697 - 702
MPP+ toxicity in E . coli under aerobic and anaerobic conditions; Haskel Y et al.; MPP+ and paraquat (PQ+2) are two structurally analogous and highly toxic pyridinium compounds . The mechanism of PQ+2 toxicity is best understood in the bacterial model system . While numerous studies, in a variety of systems, have indicated the causative role of free radicals and other oxygen-derived active species in PQ+2 toxicity, this question is yet unresolved in the case of MPP+ . In this study we have used the E . coli model and have demonstrated that MPP+ is toxic to bacterial cells in a dose and time dependent modes . Additionally, it is shown that only in the presence of molecular oxygen, bacterial inactivation occurred . The protective effects of the chemical scavenger--mannitol--and of histidine are presented . These results are in complete accord with a free radical mechanism for MPP+ toxicity.

Am J Physiol Imaging, 1991, 6(2), 81 - 4
In vivo assessment of regional microvascular albumin leakage during E . coli septic shock in the baboon model; Dormehl IC et al.; Changes in regional microvascular albumin flux during septic shock were studied noninvasively by scintigraphy in the baboon model . Use was made of an i.v . injection of 99mTc-labelled baboon serum albumin . Count ratios of lung to cardiac, liver to cardiac, and abdominal to cardiac regions were measured every 2 hr for 6 hr in control and septic shock baboons, and compared . Increased ratios obtained during shock pointed to an increase in extravascular albumin . Linear regression lines fitted to these count ratios provided regional albumin leak indices . These indices demonstrated statistically significant increases (P less than 0.05) during septic shock for the abdominal region during the 6-hr study, and for all regions, but especially the abdomen, when data were calculated over 4 hr . Increasing ratios and leak indices correlated with postmortem data and changes in neutrophil and platelet behaviour previously established during shock.

Acta Cient Venez, 1991, 42(5), 270 - 5
Selection of lacZ operon fusions in genes of gluconate metabolism in E . coli . characterization of a gntT::lacZ fusion; Porco A et al.; The initial steps involved in the utilization of gluconate by E . coli, its incorporation into the cell and subsequent phosphorylation to gluconate 6-phosphate, conform two systems that duplicate activities . These systems, GntI and GntII, are specified by two sets of genes distinctly regulated and located respectively at the malA-asd (75 min) and fdp-valS (96 min) regions of the bacterial chromosome . The presence of duplicate activities in the metabolism of gluconate of E . coli, has made difficult the study of the expression and participation of the GntI and GntII systems . In order to advance in this respect, the phage lambda placMu53 was used to select operon gnt::lacZ fusion in a E . coli strain delta (edd-zwf), delta (gnd-his), delta lac . Here we report the study of a gntT::lacZ fusion . This transductant allowed to differentiate the inducible expression of the gntT and gntU genes . Its characteristics, in agreement with previous reports, support the central role of the gntT gene in this metabolism.

Nucleic Acids Symp Ser, 1991, (24), 33 - 6
Solid phase synthesis of mRNA by the phosphoramidite approach using 2'-O-1-(2-chloroethoxy)ethyl protection and its stability in E . coli system; Sakatsume O et al.; The decaoligoribonucleotides containing initiation codon AUG of the phage O beta-A protein mRNA were synthesized on a solid phase by the phosphoramidite approach using 2'-O-1-(2-chloroethoxy)ethyl (Cee) protection . The Cee group is completely stable under the acidic conditions required to remove the 5'-terminal protecting groups in oligoribonucleotide synthesis on a solid support, and yet is easily removable at pH 2.0 for the final unblocking step . Stabilization of the synthetic mRNA to in the cell-free translation system from E . coli A19 was measured . It was found that the oligomers with selected 2'-O-methylation were degraded completely in the translation system.

Biofizika, 1991 Jan-Feb, 36(1), 102 - 4
{K+-dependent enzyme activity of H+-ATPase in the E . coli membrane}; Trchunian AA et al.; It was shown that DCCD-sensitive ATPase activity of isolated membranes and preparations of F1F0 only from anaerobically grown E . coli depended on K+ activity . F1F0 include two additional proteins which correspond to the Trk system . The data improve the possibility to form supercomplex (F1F0-Trk) functioning as the H(+)-K(+)-pump.

Cesk Epidemiol Mikrobiol Imunol, 1991 Jan, 40(1), 8 - 14
{Personal experience in testing for E . coli enterotoxigenicity}; Kerestesova A et al.; When testing the production of the thermolabile enterotoxin by strains of E . coli, using six different methods, the highest positivity (100%) was recorded when using the modified test of oedema on mouse paws and the lowest one (5%) when using the Biken test . The strains were isolated from diarrhoeal infections in young children.

Braz J Med Biol Res, 1991, 24(11), 1133 - 5
Effect of IL-3 and E . coli LPS on the proliferation of mouse bone marrow cells in vitro; Nardi NB et al.; Bone marrow cells from adult BALB/c mice were cultured at 37 degrees C, with 5% CO2 in air, in RPMI 1640 medium complemented with fetal calf serum . The addition of IL-3 (5% of WEHI-3-conditioned medium) or E . coli lipopolysaccharides (LPS, 50 micrograms/ml) to the cultures stimulated cell proliferation (1.29- and 1.22-fold, respectively, relative to control culture), whereas the simultaneous addition of the two factors reduced the number of cells recovered by 38% relative to those from control cultures (which were around 2.83 x 10(5) cells for each 10(6) plated cells) . The frequency of blasts and cells with surface Ig presented the same pattern of variation (0.07 and 0.02%, respectively, in control cultures) . The inhibitory effect of IL-3+LPS on cell proliferation was evident from the first day of culture, but more apparent on day 3 . Macrophage-colony stimulating factor (M-CSF, L929-conditioned medium) and LPS each given alone stimulated proliferation but reduced it when given together . In contrast, fetal liver cells were not affected by the simultaneous addition of IL-3 and LPS or by M-CSF and LPS . The mechanism of action of the cumulative effect of these two factors is unknown . Since crude cell-conditioned medium was used as the source of IL-3, it is possible that another factor present in this medium interacts with LPS to cause the inhibitory effect on cell proliferation.

Arch Immunol Ther Exp (Warsz), 1991, 39(4), 349 - 55
Synthesis and expression in E . coli of the gene for human tumor necrosis factor (h alpha TNF); Klysik J et al.; A gene coding for human tumor necrosis factor (h alpha TNF) has been assembled by ligating short oligodeoxyribonucleotides and cloning into plasmid vectors . These oligonucleotides were prepared by the modified phosphoramidite methodology using isopropoxyacetyl (IPA) as a protecting group for exoamino- functions of nucleosides . Gene was expressed in E . coli and the protein product was purified to homogeneity by ion-exchange chromatography.

Acta Morphol Hung, 1991, 39(3), 239 - 48
Hypoxic damage to tubules due to blockage of perfusion in acute hematogenous E . coli pyelonephritis of rats; Ivanyi B; To determine the role of hypoxia in the evolution of tubular damage in acute pyelonephritis (PN), the blockage of blood flow and injury to peritubular capillaries and tubules were studied morphologically in hematogenous acute E . coli PN of rats . Renal microvessels were stained by in situ intraarterial administration of Alcian blue . The non-occurrence of staining indicated blockage of perfusion . Injury to cortical capillaries and tubules was examined by electron microscope . In areas of inflammation, binding of Alcian blue did not occur in capillaries plugged by polymorphonuclear leukocytes (PMNL-s) and in the majority of glomerules . Ultrastructurally, severe injury to capillaries was found around endothelium-adhered, degranulated PMNL-s containing bacteria: the vessel wall was fragmented, the capillary basement membrane perivascular connective tissue matrix and collagen fibrils had disappeared, and fibrin had deposited intra- and extravascularly . Tubular changes varied from swelling to ischemic necrosis . The observations suggest that tubular damage was related to hypoxia due to preglomerular and capillary perfusion defects, and that PMNL-s injure capillaries via lysosomal enzymes discharged into the capillary fluid during the phagocytosis of bacteria . Since leukocyte plugs in capillaries, PMNL-dependent lytic injury to capillaries and mild ischemic tubular changes, but not ischemic necrosis, have been found in human acute PN previously, the preglomerular vasospasm may cause the tubular necrosis in experimental acute PN.

Biomed Biochim Acta, 1991, 50(4-6), 701 - 5
Construction and cloning of recombinant rat trypstatin variants and their expression as fusion proteins in E . coli; Dolinar M et al.; A synthetic master gene coding for rat trypstatin was designed, assembled of eight oligonucleotides, ligated into the cloning vector pUC8 and cloned in E . coli . In addition to the expected product DNA sequencing revealed the presence of several gene variants . The gene coding for (-1M F44G) rat trypstatin was mutated to a wild type form (-1M) rat trypstatin by cassette mutagenesis . For the first expression experiments the E . coli pEx31 system was used . After SDS-PAGE analysis fusion proteins were detected consisting of the N-terminal part of MS2-polymerase, a linker region and of the appropriate rat trypstatin variant . These fusion proteins were synthesized in amounts as high as 25% of total E . coli proteins.

Acta Biochim Pol, 1991, 38(1), 71 - 4
Effect of flavones and their metabolites on induction of SOS repair in the strain PQ37--E . coli K-12; Czeczot H et al.; The SOS-Chromotest was used to detect DNA damage induced by two flavones: apigenin and luteolin and/or their metabolites . It was found that the compounds tested weakly induced the SOS repair system in the strain studied.

Adv Exp Med Biol, 1991, 309B, 105 - 8
Expression of normal and variant human hypoxanthine-guanine phosphoribosyltransferase in E . coli; Davidson BL et al.; 1 . ECPR is a rapid and effective means for generating recombinant human HPRT . 2 . The Bl21 (DE3) T7 polymerase/T7 promoter system provides high level expression of human HPRT constructs after induction of the T7 polymerase gene with IPTG . 3 . Human HPRT constructs expressed in E . coli mimic the variant properties originally demonstrated in lymphoblast extracts from affected individuals . 4 . Human HPRT expressed in E . coli can be rapidly purified to near homogeneity by a two step purification scheme.

Yi Chuan Xue Bao, 1991, 18(6), 564 - 9
{Cloning and sequencing of spinach chloroplast DNA fragments with promoter activities in E . coli}; Wei L et al.; Sau3A partially digested spinach cDNA fragments were cloned in BgIII restriction site of a E . coli promoter probing vector pGA46 . The cloned fragments with promoter activities in E . coli were found by the abilities of recovering tetracycling resistance of their host cells . The promoter functions of the two fragments and their different strengths are deciphered by analyzing their DNA sequences.

Yi Chuan Xue Bao, 1991, 18(6), 552 - 8
{Through homologous recombination pathway recA gene takes part in chromosome replication of E . coli initiated by integrated plasmid}; Mao YM et al.; We have reported in our previous paper that replication of E . coli chromosome initiated by the integrated F' plasmid depends on the recA gene . Here we report on work dealing with the role played by recA, recB, recC, lexA alleles in chromosome replication . Our results show that the recA gene takes part in chromosome replication through homologous recombination rather than SOS pathway and that Chi hot spot is not concerned with . They also show that the ATP-dependent dsDNA exonuclease activity of the RecBC enzyme has nothing to do with the recA gene in chromosome replication.

Yi Chuan Xue Bao, 1991, 18(4), 370 - 7
{Subcloning and sequencing of the Streptomyces griseus DNA fragment with promoter activity in E . coli}; Chen Y et al.; A 410-bp DNA fragment of Streptomyces griseus No.45 with in vivo promoter activity in E . coli has been isolated by subcloning the cloning strain E . coli No.8-1 . The result of sequencing shows that the promoter fragment contains 50.5% G + C bases . It has some repeat sequences . It shows good homology to the E . coli promoter consensus sequences in -10 and -35 regions . There are 18 bp between the two regions . It has two regions that are similar to SD sequence in E . coli and consensus sequence in SEP (Streptomyces-E.coli-type promoter) of Streptomyces lividans respectively . There is 1 Alu I site, 1 Cla I site, 2 Mbo I sites, 3 NlaIII sites and 1 PvuII site in the promoter fragment.

Proteins, 1991, 11(4), 263 - 270
Changes in the electron density of the cofactor NADPH on binding to E . coli dihydrofolate reductase; Bajorath J et al.; Quantum-mechanical electron density calculations reveal that a significant polarization is induced in the cofactor NADPH (reduced nicotinamide adenine dinucleotide phosphate) on binding to the enzyme dihydrofolate reductase . The calculations indicate that electron density corresponding to approximately 0.7 electron charges is shifted within the molecule, extending over more than 20 A . Further calculations on proposed enzyme mutants show that the polarization of NADPH on binding to DHFR is, in large part, induced by a motif of three positively charged residues . This motif was also identified to be directly responsible for the positive electrostatic potential surrounding the cofactor binding site in the enzyme . The possibility of this long-range polarization of NADPH was originally proposed based on a previous study of ligand binding to DHFR where a conserved structural motif of three positively charged residues was found to play a major role in polarizing the substrate folate over its entire length of 18 A.

Nucleic Acids Symp Ser, 1991, (25), 153 - 4
Identity determinants of E . coli tRNAs; Hasegawa T et al.; Identity determinants of E . coli tRNA(Val) and three class II tRNAs, tRNA(Ser), tRNA(Tyr) and tRNA(Leu), are studied by using various variants of tRNA transcripts . Anticodon, discriminator base and acceptor stem are involved in the identity elements for tRNA(Val) . Discrimination among class II tRNAs are considered to be dependent on the bases at positions 2, 71 and 73 as well as their different tertiary structures including the long variable arm.

Zentralbl Bakteriol, 1991 Jan, 274(4), 514 - 8
Binding of cloned S-fimbriated E . coli to human buccal epithelial cells--different inhibition of binding by neonatal saliva and adult saliva; Schroten H et al.; Investigations were carried out on the adhesion of cloned S-fimbriated E . coli, labelled with fluoresceinisothiocyanate (FITC) to human buccal epithelial cells . Fluorescence microscopic analysis revealed binding of bacteria to 75-95% of epithelial cells . Inhibition experiments with fetuin, alpha 1-acid glycoprotein and N-acetyl neuraminic acid confirmed the specificity of bacterial binding to sialoglycoproteins . Further studies using saliva as an inhibitor resulted in a 4-5 times stronger binding inhibition by newborn saliva in comparison to adult saliva coinciding with a 4-5 times higher content of total N-acetyl neuraminic acid in samples of newborn saliva . In Western blot analysis sialoglycoprotein bands with a molecular weight greater than 200 kD reacting with wheat germ agglutinin (WGA), were only identified in samples of newborn saliva . These bands are classified as mucins on account of molecular weight and staining . These data suggest that saliva mucins could represent a major defense mechanism against bacterial infections at a stage of ontogeny where the secretory IgA-system is not yet developed.

APMIS, 1991 Jan, 99(1), 30 - 2
Two new Escherichia coli O groups: O172 from "Shiga-like" toxin II-producing strains (EHEC) and O173 from enteroinvasive E . coli (EIEC); Orskov I et al.; Two Escherichia coli strains were established as antigenic test strains for two new O groups, O172 and O173 . The O172 strain (EHEC) which produces "Shiga-like" toxin II (verocytotoxin 2) was isolated from a case of haemorrhagic colitis while the enteroinvasive O173 strain (EIEC) originated from a child with diarrhoea.

Gen Pharmacol, 1991, 22(5), 923 - 7
The effect of doxazosin, urapidil and indoramin pretreatment on fever produced by E . coli lipopolysaccharide in rabbits; Gagalo I et al.; 1 . Thermal responses to i.v . administration of doxazosin (0.75 or 1.50 mg/kg), urapidil (5.0 or 10.0 mg/kg), or indoramin (0.75 or 1.50 mg/kg) were investigated in febrile rabbits (treated with Escherichia coli lipopolysaccharide) at an ambient temperature of 19 +/- 1 degree C . 2 . All these alpha 1-adrenoceptor blockers produced statistically significant antipyresis which developed as a result of inhibition of metabolic heat production and/or stimulation of heat elimination from the ear skin area or respiratory tract . 3 . It appears that the antipyretic effect is a general feature of alpha 1-adrenergic receptor blockers . The possible mechanisms by which antipyresis is produced are being considered.

Int J Radiat Biol, 1991 Jan, 59(1), 153 - 64
Do intracellular thiol or peroxidase levels block radiation sensitization by nitrous oxide in some E . coli strains?
Ewing D, Guilfoil DS, Ohm MB.
Although nitrous oxide (N2O) is often a radiation sensitizer in procaryotic cells, it fails to sensitize some strains of bacteria, some yeast strains, and most eucaryotic cell lines . At present this inconsistency cannot be satisfactorily explained . The experiments here use eight strains of E . coli, some of which are not sensitized by N2O, to test the hypothesis that N2O's failure to sensitize might be based on high thiol content or on low peroxidase activity . Our data contradict those hypotheses . In addition, further data show that the strains not sensitized by N2O contain no unique cellular component or compound which blocks damage from N2O.

Nucleic Acids Res, 1990 Dec 25, 18(24), 7389 - 96
Transcription regulates oxolinic acid-induced DNA gyrase cleavage at specific sites on the E . coli chromosome; Condemine G et al.; Prominent DNA gyrase-mediated cleavage sites, induced by oxolinic acid, occur at specific, but infrequent, locations on the Escherichia coli chromosome . These sites, which we call toposites, may represent high affinity DNA gyrase binding sites or may mark chromosomal regions that accumulate superhelical stress . Toposites are usually grouped in 5 to 10 kb clusters that are mostly 50 to 100 kb apart . The total number of clusters on the chromosome is between 50 and 100 . The location of sites depends on the local sequence . The extent of DNA gyrase cleavage at toposites can be strongly modulated by transcription occurring at as far as 35 kb away.

FEBS Lett, 1990 Dec 17, 277(1-2), 109 - 11
NaF and mononucleotides as inhibitors of 3'-5'-exonuclease activity and stimulators of polymerase activity of E . coli DNA polymerase I Klenow fragment; Potapova IA et al.; It has been shown that, in the absence of dATP in the poly(dT).oligo(dA) template-primer complex, the rate of primer cleavage by the E . coli DNA polymerase I Klenow fragment equals 4% of polymerization rate, while in the presence of dATP it equals as much as 50-60% . NaF and NMP taken separately inhibit exonuclease cleavage of oligo(dA) both with and without dATP . The addition of NaF (5-10 mM) or NMP (5-20 mM) increases the absolute increment of polymerization rate 5-9-fold relative to the absolute decrement of the rate of nuclease hydrolysis of primer . This proves the assumption that not more than 10-20% of primer molecules, interacting with the exonuclease center of polymerase, are cleaved by the enzyme . Presumably, NaF and nucleotides disturb the coupling of the 3'-end of oligonucleotide primer to the exonuclease center of the enzyme . As the primers mostly form complexes with the polymerizing center, the reaction of polymerization is activated.

FEBS Lett, 1990 Dec 17, 277(1-2), 194 - 6
Interaction of dNTP, pyrophosphate and their analogs with the dNTP-binding sites of E . coli DNA polymerase I Klenow fragment and human DNA polymerase alpha; Potapova IA et al.; The 3',5'-exonuclease center of the Klenow fragment of E . coli DNA polymerase I (FK) was selectively blocked by NaF . The latter was shown to forbid the binding of nucleotides and their analogs to the enzyme exonuclease center . In the presence of poly(dT).r(pA)10 template.primer complex and NaF, we observed AMP, ADP, ATP, PPi and dATP to be competitive inhibitors of the FK-catalyzed DNA polymerization . The interactions of the nucleotides with FK and human DNA polymerase alpha were compared to reveal similarity of binding to the DNA polymerizing centers . Structural components of dNTP and PPi playing key roles in forming complexes with pro- and eukaryotic DNA polymerases were identified.

Carbohydr Res, 1990 Dec 15, 208, 139 - 44
Structure of the K24 antigen of E . coli O83:K24:H, a polymer that consists of alpha-Kdop and glycerol phosphate; Lenter MC et al.; The structure of the K24 antigen of Escherichia coli O83:K24:H31 was elucidated by determination of composition and by 1H-, 13C-, and 31P-n.m.r . spectroscopy of the polymer and of a Kdo-glycerol (Gro) glycoside, obtained by mild alkaline hydrolysis and subsequent incubation with alkaline phosphatase . The K24 antigen has the repeating unit----7)-alpha-Kdop-(2----1)-Gro-(3-P . In the polymer, 56% of the repeating units are O-acetylated at C-4 of Kdo, approximately 28% at C-5 of Kdo, and approximately 16% are not acetylated.

Biochem Biophys Res Commun, 1990 Dec 14, 173(2), 680 - 8
Replica filter assay of human beta-adrenergic receptors expressed in E.coli; Galli G et al.; We have developed a replica filter assay that permits the direct identification of bacterial colonies expressing membrane receptors . E.coli transformed with appropriate phage or plasmid vectors containing human beta-adrenergic receptor cDNAs were grown on LB/agar plates . Bacterial colonies transferred onto nitrocellulose filters showed specific {125I}-iodocyanopindolol binding . beta-adrenergic receptors expressed in bacteria retained their pharmacological properties when transferred onto filters . This strategy, which is considerably simplified and more rapid compared to similar methods based upon expression of receptor genes in eukaryotic cells, may be a useful tool for cloning membrane receptors.

Neurosci Lett, 1990 Dec 11, 120(2), 263 - 6
Retrograde neuronal labelling by E . coli enterotoxin subunit B; Okado N et al.; The present communication reports that subunit B of Escherichia coli heat-labile enterotoxin (LTB) can be utilized as a powerful tracer in neuroanatomical studies . LTB was injected into the limb muscle of the chick, or into the superior cervical ganglion of the rat . Sections were processed with an immunohistochemical technique using an antibody against LTB . After the LTB injection into the muscle, retrogradely labelled motoneurons were found: the entire extent of dendritic arborizations appeared labeled . After the LTB injection into the ganglion, virtually all of the preganglionic neurons were retrogradely labelled in the rat spinal cord.

Nucleic Acids Res, 1990 Dec 11, 18(23), 6761 - 6
Mutagenesis of selC, the gene for the selenocysteine-inserting tRNA-species in E . coli: effects on in vivo function; Baron C et al.; The selenocysteine-inserting tRNA (tRNA(Sec)) of E . coli differs in a number of structural features from all other elongator tRNA species . To analyse the functional implications of the deviations from the consensus, these positions have been reverted to the canonical configuration . The following results were obtained: (i) inversion of the purine/pyrimidine pair at position 11/24 and change of the purine at position 8 into the universally conserved U had no functional consequence whereas replacements of U9 by G9 and of U14 by A14 decreased the efficiency of selenocysteine insertion as measured by translation of the fdhF message; (ii) deleting one basepair in the aminoacyl acceptor stem, thus creating the canonical 7 bp configuration, inactivated tRNA(Sec); (iii) replacement of the extra arm by that of a serine-inserting tRNA abolished the activity whereas reduction by 1 base or the insertion of three bases partially reduced function; (iv) change of the anticodon to that of a serine inserter abolished the capacity to decode UGA140 whereas the alteration to a cysteine codon permitted 30% read-through . However, the variant with the serine-specific anticodon efficiently inserted selenocysteine into a gene product when the UGA140 of the fdhF mRNA was replaced by a serine codon (UCA) . Significantly, none of these changes resulted in the non-specific incorporation of selenocysteine into protein, indicating that the mRNA context also plays a major role in directing insertion . Taken together, the results demonstrate that the 8-basepair acceptor stem and the long extra arm are crucial determinants of tRNA(Sec) which enable decoding of UGA140 in the fdhF message.

EMBO J, 1990 Dec, 9(13), 4527 - 33
Significance of the third tRNA binding site, the E site, on E . coli ribosomes for the accuracy of translation: an occupied E site prevents the binding of non-cognate aminoacyl-tRNA to the A site; Geigenmuller U et al.; The E site (exit site for deacyl-tRNA) has been shown to be allosterically linked to the A site (aminoacyl-tRNA binding site), in that occupation of the E site reduces the affinity of the A site, and vice versa, whereas the intervening peptidyl-tRNA binding site (P site) keeps its high affinity . Here the question is analysed of whether or not the low affinity state of the A site caused by an occupied E site is of importance for the ribosomal accuracy of the aminoacyl-tRNA selection . In a poly(U) dependent system with high accuracy in poly(Phe) synthesis, the acceptance of the cognate ternary complex Phe-tRNA--EF-Tu--GTP (which has the correct anticodon with respect to the codon at the A site) was compared with the competing acceptance of ternary complexes with near-cognate Leu-tRNA(Leu) (which has a similar anticodon) or non-cognate Asp-tRNA(Asp) (which has a dissimilar anticodon), by monitoring the formation of AcPhePhe, AcPheLeu or AcPheAsp, respectively . Cognate (but not near-cognate) occupation of the E site reduced synthesis of the 'wrong' dipeptide AcPheLeu only marginally relative to that of the cognate AcPhe2, whereas the formation of AcPheAsp was decreased as much as 14-fold, thereby reducing it to the background level . It follows that the allosteric interplay between E and A sites, i.e . the low affinity of the A site induced by the occupation of the E site, excludes the interference of non-cognate complexes in the decoding process and thus reduces the number of aminoacyl-tRNA species competing for A site binding by an order of magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1990 Dec, 9(12), 4065 - 72
The AT richness and gid transcription determine the left border of the replication origin of the E . coli chromosome; Asai T et al.; We have identified novel, cis-acting elements which enhance in vivo the replication activity of plasmids carrying the minimal oriC of Escherichia coli . These are (i) the AT rich sequence ('AT-cluster') which exists immediately left of the 13mer repeats and (ii) the gid transcriptional unit . The 'AT-cluster' was functionally replaced by an unrelated AT rich sequence . This was also the case for the left and middle 13mers; they were substituted by the AT rich fragment from mini-F plasmid . The left 13mer was replaced by the AT rich sequence which did not show the 'reduced helical stability' known as the important character of the 13mer region . In contrast to these results, the right 13mer sequence was strictly required . As to the effect of the transcription from the gid promoter, the minimal oriC was activated only when the transcription was directed away from the left side of it . mioC transcription proceeding toward the oriC had no effect on the activation . Mutations in the DnaA boxes were partially suppressed by gid transcription leaving oriC from the left side . From these results, we propose that the AT richness is a determinant to identify the left border of oriC . It is presumed that gid transcription introduces negative superhelicity at the AT rich region and facilitates DnaA dependent duplex opening.

EMBO J, 1990 Dec, 9(12), 4027 - 36
Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E . coli mutants lacking the DnaK chaperone; Bukau B et al.; An Escherichia coli mutant lacking HSP70 function, delta dnaK52, is unable to grow at both high and low temperatures and, at intermediate temperature (30 degrees C), displays defects in major cellular processes such as cell division, chromosome segregation and regulation of heat shock gene expression that lead to poor growth and genetic instability of the cells . In an effort to understand the roles of molecular chaperones such as DnaK in cellular metabolism, we analyzed secondary mutations (sid) that suppress the growth defects of delta dnaK52 mutants at 30 degrees C and also permit growth at low temperature . Of the five suppressors we analyzed, four were of the sidB class and mapped within rpoH, which encodes the heat shock specific sigma subunit (sigma 32) of RNA polymerase . The sidB mutations affected four different regions of the sigma 32 protein and, in one case, resulted in a several fold reduction in the cellular concentration of sigma 32 . Presence of any of the sidB mutations in delta dnaK52 mutants as well as in dnaK+ cells caused down-regulation of heat shock gene expression at 30 degrees C and decreased induction of the heat shock response after shift to 43.5 degrees C . These findings suggest that the physiologically most significant function of DnaK in the metabolism of unstressed cells is its function in heat shock gene regulation.

J Leukoc Biol, 1990 Dec, 48(6), 488 - 94
Eicosanoid production in nonparenchymal liver cells isolated from rats infused with E . coli endotoxin; Rodriguez de Turco EB et al.; Continuous i.v . infusion of a nonlethal dose of Escherichia coli endotoxin induced an early (3-h) accumulation of neutrophils in the rat liver followed by a later (30-h) greater extravasation of mononuclear phagocytes (MNP) . These inflammatory cells, recovered together by centrifugal elutriation, were analyzed for their potential capacity to metabolize {1-14C}-AA . Ca2+ ionophore A23187 (5 microM) stimulated the release of {1-14C}-AA from PC and PI both in cells from saline- and ET-infused rats, the latter showing a higher capacity to further metabolize AA to eicosanoids . LTB4 and 5-HETE were the major metabolites accumulated in cells from rats infused with ET for 3 h, while PGD2 played the main role in cells from saline-infused rats . This could reflect {1-14C}-AA metabolism by PMNP and Kupffer cells, respectively . By 30 h of ET-infusion, a shift from PGD2 to PGE2 release was observed . These results suggest that eicosanoids released by nonparenchymal cells (i.e., Kupffer and endothelial cells) and PMNP in the liver of ET-infused rats may alter the normal intercellular information flow between parenchymal and nonparenchymal cells, contributing to the severe impairment in liver function and metabolism during endotoxicosis and sepsis.

Bioorg Khim, 1990 Dec, 16(12), 1661 - 9
{Model polycistron operon for studying conjugated translation in E . coli cells . The role of a stream of ribosomes}; Kravchenko VV et al.; The role of "stream" of ribosomes upon translation of polycistronic mRNAs has been studied using an artificial polycistron . It has been found that the levels of activation of cistron Ci + 1 out of two adjacent cistrons (Ci and Ci + 1) depends, in addition to earlier described effects of mutual arrangement of initiation and termination signals, also on efficiency of translation of the foregoing cistron Ci . The results obtained lead to the conclusion that in polycistronic systems the levels of translation of cistron Ci + 1 can be regulated by "stream" of ribosomes resulted from translation of the proximal cistron Ci.

Mol Endocrinol, 1990 Dec, 4(12), 1841 - 9
Overexpression of a partial human androgen receptor in E . coli: characterization of steroid binding, DNA binding, and immunological properties; Young CY et al.; The recent cloning of human androgen receptor (AR) cDNAs in this and other laboratories has provided valuable probes for investigating the structure and function of the AR at the molecular level . We now report the overexpression of a region of the human AR containing both the DNA- and hormone-binding domains in E . coli, which provides a means to produce large amounts of AR for analysis and use in functional studies . Under isopropyl-beta-D-thiogalactopyranoside induction, a tripartite protein, consisting of beta-galactosidase, a collagenase recognition site, and AR polypeptide, was produced in E . coli JM109 using pSS20 a as a vector . About 1 mg of the fused AR could be recovered per liter bacterial culture . The induced protein could readily be detected in a sodium dodecyl sulfate-polyacrylamide gel by Coomassie blue staining . Its identity was confirmed by Western blot analysis using antibodies to both beta-galactosidase and the AR . Scatchard analysis of the androgen-binding activity of the hybrid AR revealed high affinity binding to the synthetic androgen, Mibolerone (Kd, approximately 1.2 nM) . Competition studies demonstrated the fusion protein's specificity for androgens . The hybrid receptor formed immune complexes with human anti-AR serum that sedimented at about 19S in 10-50% linear sucrose gradients containing 0.4 M KCl . Gel band shift assays revealed that the hybrid receptor protein forms specific complexes with a synthetic steroid response element derived from the mouse mammary tumor virus long terminal repeat region . These results demonstrate that the recombinant AR expressed in E . coli possesses many of the functional properties characteristic of DNA- and steroid-binding domains of the native AR.

Behring Inst Mitt, 1990 Dec, (87), 48 - 55
Properties of FV and Fab fragments of the antibody McPC603 expressed in E . coli; Pluckthun A et al.; The FV and Fab fragments of the phosphorylcholine binding antibody McPC603 were functionally expressed in E . coli . This was achieved by the co-expression and co-secretion of both chains to the periplasm, where correct processing, folding and assembly occurred . Interestingly, the fraction of correctly folded Fab fragment is smaller than that of the Fv fragment in E . coli . The intrinsic hapten binding affinity was shown to be identical for the recombinant FV or Fab fragment, the whole antibody and the Fab fragment obtained by proteolysis from the mouse antibody . Fluorescence and crosslinking analyses showed that the FV fragment dissociates at high dilution, but that it is stabilized by hapten binding . The recombinant FV fragment was shown to have catalytic activity to hydrolyze choline-p-nitrophenyl carbonate and constitutes therefore a promising model system with which the structural requirements of catalytic antibodies can be studied by altering the protein itself.

Br J Pharmacol, 1990 Dec, 101(4), 937 - 43
The effect of E . coli STa enterotoxin on the absorption of weakly dissociable drugs from rat proximal jejunum in vivo; McEwan GT et al.; 1 . The effect of E . coli heat stable (STa) enterotoxin on the absorption of radio-labelled weak electrolytes and their appearance in peripheral blood was assessed in vivo by use of an intestinal recirculation procedure . 2 . STa reduced the luminal disappearance (P less than 0.02) and peripheral blood appearance (P less than 0.02) of label from salicylic acid as well as the luminal disappearance (P less than 0.02) of diphenylhydantoin . 3 . In contrast, STa increased the appearance in peripheral blood and disappearance from the lumen of label from morphine (P less than 0.05), amphetamine (P less than 0.01) and lignocaine (P less than 0.01) . 4 . Increased weak base (lignocaine) absorption can also be achieved by a combination of forskolin and theophylline which resembles STa in its ability to neutralise the usually acid surface pH of the proximal jejunum . 5 . Increased weak base absorption and hindered weak acid absorption occurs despite a uniform reduction in net fluid absorption after STs exposure, making it unlikely that variations in fluid absorption account for the variations in drug absorption . 6 . The ability of STa to elevate the mucosal surface pH (or acid microclimate) to neutral values, thereby altering the proportion of uncharged weak-electrolyte, may explain its different effects on weak acids and bases: neutralisation of the acid microclimate would increase the amount of undissociate weak base available for uptake.

AIDS Res Hum Retroviruses, 1990 Dec, 6(12), 1399 - 408
Expression of HIV-1 integrase in E . coli: immunological analysis of the recombinant protein; Marcus-Sekura CJ et al.; Sequences encoding the human immunodeficiency virus type 1 (HIV-1) integrase gene have been cloned and expressed in Escherichia coli . The expressed protein is a lambda cII fusion protein of 37 kD containing the carboxyl-terminal 23 {corrected} amino acids of reverse transcriptase fused to the entire integrase sequence and is insoluble, a feature which allows partial purification away from soluble bacterial proteins . As judged by its reactivity with HIV positive sera in Western blot and in enzyme-linked immunosorbent assay (ELISA), the recombinant integrase retains antigenicity similar to native protein . Additionally, ELISA data obtained with the cloned protein indicate that patients infected with HIV-1 who are at different stages of progression to AIDS have antibodies reactive with the cloned integrase . HIV-2 positive human sera are also reactive with the cloned integrase . Rabbit antibodies produced against the recombinant protein react both by ELISA and Western blot with the homologous bacterially expressed protein, recognize both virion HIV-1 integrase and reverse transcriptase in Western blots, and immunoprecipitate an HIV-1 virion protein of 34 kD . Unlike human antisera from patients infected with HIV-1 or HIV-2 which are frequently reactive with both HIV-1 and HIV-2 integrase, the rabbit antibodies are type specific, reacting with HIV-1, but not with HIV-2 integrase by Western blot.

Zentralbl Veterinarmed B, 1990 Dec, 37(10), 728 - 38
Iscom (immunostimulating complex) vaccines containing mono- or polyvalent pili of enterotoxigenic E . coli; immune response of rabbit and swine; Nagy B et al.; Iscom (immunostimulating complex) vaccines were prepared to contain K88ab, K88ac, K99 and 987P pili (fimbriae) of enterotoxigenic E . coli bacteria as monovalent or quadrivalent preparations . The iscoms injected into rabbits and into pigs elicited similar or higher immune response in both animal species than the oil adjuvanted vaccine containing about 5 times more of the same pilus protein . It is concluded that inclusion of pili into iscoms results in immunogenic preparations likely worth pursuing for vaccine production against enterotoxic colibacillosis of newborn pigs . The iscoms did not induce local reaction at the injection sites in contrast to the oil adjuvanted vaccines.

Enzyme Microb Technol, 1990 Dec, 12(12), 933 - 9
Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E . coli; Huang J et al.; Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity . These parameters were studied in continuous cultures for free and immobilized cells, respectively . Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate . Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth . Higher enzyme expression of catechol 2-3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells . Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.

Cell, 1990 Nov 30, 63(5), 1053 - 60
The E . coli cell surface specifically prevents the initiation of DNA replication at oriC on hemimethylated DNA templates; Landoulsi A et al.; A particular outer membrane fraction previously defined as possessing specific affinity for the hemimethylated form of the origin of replication of the E . coli chromosome (oriC) is shown to inhibit the initiation of DNA synthesis at this site on hemimethylated DNA templates in vitro . The replication of fully methylated or unmethylated DNA templates is not affected . Also, no inhibition is observed if initiation takes place at random sites on the hemimethylated template . The key inactivation step appears to be membrane inhibition of DnaA initiator protein binding to oriC . Remethylation of the membrane-bound hemimethylated DNA results in reactivation . Our results demonstrate direct involvement of the membrane in the control of DNA replication . We propose that association/dissociation of the origin from the cell membrane is one of the control elements governing interinitiation times in E . coli.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6665 - 72
De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the trifunctional glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase by functional complementation in E . coli; Aimi J et al.; The trifunctional enzyme encoding glycinamide ribonucleotide synthetase (GARS)-aminoimidazole ribonucleotide synthetase (AIRS)-glycinamide ribonucleotide transformylase (GART) was cloned by functional complementation of an E . coli mutant using an avian liver cDNA expression library . In E . coli, genes encoding these separate activities (purD, purM, and purN, respectively) produce three proteins . The avian cDNA, in contrast, encodes a single polypeptide with all three enzyme activities . Using the avian DNA as a probe, a cDNA encoding the complete coding sequence of the trifunctional human enzyme was also isolated and sequenced . The deduced amino acid sequence of the human and avian polyproteins show extensive sequence homologies to the bacterial purD, purM, and purN encoded proteins . Avian and human liver RNAs appear to encode both a trifunctional enzyme (G-ARS-AIRS-GART) as well as an RNA which encodes only GARS . The trifunctional protein has been implicated in the pathology of Downs Syndrome and molecular tools are now available to explore this hypothesis . Initial efforts to compare the expression of GARS-AIRS-GART between a normal fibroblast cell line and a Downs Syndrome cell line indicate that the levels of RNA are similar.

Nature, 1990 Nov 15, 348(6298), 261 - 3
Mechanosensitive ion channels of E . coli activated by amphipaths; Martinac B et al.; Mechanosensitive channels have been found in more than 30 cell types, including bacterial, yeast, plant and animal cells . Whether tension is transferred to the channel through the lipid bilayer and/or underlying cytoskeleton is not clear . Using the patch-clamp method, we found that amphipathic compounds, which are molecules having hydrophobic and hydrophilic character with positive, negative or no net electric charge at pH 7, could slowly activate the mechanosensitive channels of giant Escherichia coli spheroplasts, with effectiveness proportional to their lipid solubility . The cationic or anionic amphipaths were able to compensate for each other's effect . After a channel was activated by an amphipath of one charge, if that amphipath was gradually replaced by one with the opposite charge, the channel first inactivated before reactivating . These findings support the view that the mechanical gating force can come from the surrounding lipids.

FEBS Lett, 1990 Nov 12, 274(1-2), 39 - 42
Protonated triplex DNA in E . coli cells as detected by chemical probing; Karlovsky P et al.; The triplex structure in vitro is well established; however, no direct evidence has been available concerning its existence in the cell . Using the direct chemical probing here we show that the triplex H structure can exist in E . coli cells at acidic intracellular pH values; this structure differs in some details from that observed in vitro.

Nucleic Acids Res, 1990 Nov 11, 18(21), 6271 - 5
The signal for growth rate control and stringent sensitivity in E . coli is not restricted to a particular sequence motif within the promoter region; Zacharias M et al.; Hybrid promoter constructs were used to determine the DNA sequence requirements for stringent and growth rate control within a promoter region . The promoters were obtained by fusing complementing sequence regions located upstream and downstream from the GCGC discriminator motif of the growth rate regulated rRNA P1 promoter and a non-regulated tac promoter variant . The activities and the regulatory response of the hybrid promoters were determined in vivo using a promoter test vector system with the chloramphenicol acetyltransferase (CAT) reporter gene . Measurements were made at different growth rates and after starvation for isoleucine to induce the stringent response . Neither the upstream nor the downstream sequence of P1 relative to the GCGC discriminator motif conferred comparable regulatory features when fused to the complementing sequences of the non-regulated mutant tac promoter . A minor response to amino acid deprivation or changes in the growth rate was noted for the hybrid promoter with the rrnB P1 upstream segment and the tac downstream element, pointing to a slightly different importance of the two sequence elements for regulation . The parallel effects for stringent as well as growth rate regulation of the hybrid promoters supports the view of a common mechanism for both types of control . However, none of the promoter sequence elements on its own was able to restore the complete regulatory behaviour of their 'parent' promoters.

Cell, 1990 Nov 2, 63(3), 591 - 600
E . coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle; Ribes V et al.; E . coli 4.5S RNA and P48 have been shown to be homologous to SRP7S RNA and SRP54, respectively . Here we report that expression of human SRP7S in E . coli can suppress the lethality caused by depletion of 4.5S RNA . In E . coli, both RNAs are associated with P48 . In vitro, both E . coli P48 and SRP54 specifically bind to 4.5S RNA . Strains depleted of 4.5S RNA strongly accumulate pre-beta-lactamase and fail to accumulate maltose binding protein . These effects commence well before any growth defect is observed and are suppressed by expression of human SRP7S . Strains overproducing P48 also accumulate pre-beta-lactamase . 4.5S RNA and P48 are components of a ribonucleoprotein particle that we propose to be required for the secretion of some proteins.

Zhong Xi Yi Jie He Za Zhi, 1990 Nov, 10(11), 675 - 6, 645
{Comparison between the therapeutic effects of ginseng-aconitum-bupleurum injection and dexamethasone on septic shock complicated with disseminated intravascular coagulation induced by E . coli in dogs}; Liu YY; In this paper, the authors report the therapeutic effects of Ginseng-Aconitum-Bupleurum (GAB) injection on septic shock complicated with DIC induced by intravenous injection of live E . Coli in dogs . The experimental results indicated that the survival rate at 48 h after intravenous injection of live E . Coli was 30% in saline group, 80% in GAB, 90% in dexamethasone (Dex) group . The BPC, WBC, FG, AT-III, beta-GC, PHb were 82 +/- 28, 9.1 +/- 5.9, 1.85 +/- 0.41, 112 +/- 43, 0.37 +/- 0.17, 0.11 +/- 0.07 respectively for GAB, 70 +/- 37, 6.7 +/- 3.7, 1.55 +/- 0.36, 59 +/- 29, 0.91 +/- 0.28, 0.12 +/- 0.06 respectively for the saline group, 58 +/- 33, 6.3 +/- 2.9, 1.95 +/- 0.21, 103 +/- 43, 0.50 +/- 0.16, 0.13 +/- 0.06 respectively for Dex . The BPC, WBC, FG, AT-III levels of the GAB group were significantly higher than those in saline group and were not significantly difference from the Dex group . The beta-GC and PHb levels of the GAB group were significantly reductive than the saline group and were not significantly different from the Dex group . The results showed that the survival rate was higher, the DIC was inhibited and normal biomembranes were maintained in the GAB group.

Berl Munch Tierarztl Wochenschr, 1990 Nov 1, 103(11), 380 - 4
{Detection of verotoxin-producing E . coli (VTEC) in healthy cattle and swine with the DNA-DNA colony hybridization method}; Bulte M et al.; With the DNA-DNA colony hybridization technique using specific gene probes for Verotoxin 1 (VT 1) and Verotoxin 2 (VT 2) 2100 E . coli strains from healthy animals were tested . Ten out of 82 milk cows (21.2%), 20 out of 212 beef cattle (9.4%) and five out of 75 pigs (6.7%) were found to carry genes for VT 1, VT 2 or both toxins, respectively . Among these strains the biotypes 5 and 6 were predominant . Some of the serotyped isolates have been described to be pathogenic for humans, like O157:H7, 082:H8, 0116, 0113, 0126 and 091, respectively . The unexpected high incidence of VTEC positive healthy animals possibly indicates a health hazard for human beings . Further investigations on the incidence of VTEC in food are necessary.

EMBO J, 1990 Nov, 9(11), 3733 - 42
E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo; Ross W et al.; An upstream activation region (UAR) contributes to the extremely high activity of the Escherichia coli ribosomal RNA promoter, rrnB P1, increasing its activity 20- to 30-fold over that of the same promoter lacking the UAR . We have used DNase footprinting to define three specific sites in the rrnB P1 UAR that bind Fis, a protein identified previously by its role in recombinational enhancer function in other systems . We find that purified Fis activates transcription from promoters containing these sites 10- to 20-fold in vitro at concentrations correlating with the filling of these sites . Three approaches indicate that Fis contributes to the function of the UAR in vivo . First, there is a progressive loss in the activity of rrnB P1-lacZ fusions as Fis binding sites are deleted . Second, an rrnB P1 promoter with a mutation in a Fis binding site has 5-fold reduced transcription activity in vivo, dramatically reduced Fis binding in vitro, and shows no Fis dependent transcription activation in vitro . Third, upstream activation is reduced 5-fold in a Fis- strain . We show that rRNA promoters derepress in response to the loss of Fis in vivo in accord with the predictions of the negative feedback model for rRNA regulation . We find that fis is not essential for the function of two control systems known to regulate rRNA, growth rate dependent control and stringent control . On the basis of these results, we propose roles for Fis and the upstream activation system in rRNA synthesis.

Tsitol Genet, 1990 Nov-Dec, 24(6), 25 - 31
{Potential for suppression of E . coli rplj gene expression by antisense RNA}; Paton EB et al.; Construction of the recombinant plasmids, containing the antisense sequence of the E . coli rplJ gene under control of lac promoter has shown their effect on the level of the detector rplJ-lacZ gene expression.

Biochimie, 1990 Nov, 72(11), 779 - 90
Protection from chemical modification of nucleotides in complexes of M1 RNA, the catalytic subunit of RNase P from E coli, and tRNA precursors; Knap AK et al.; Certain nucleotides in M1 RNA, the catalytic RNA subunit of RNase P from E coli, are protected from chemical modification when M1 RNA forms complexes with tRNA precursor molecules (ES complexes) . Many of these nucleotides are important in the formation of the Michaelis complex . In the presence of tRNA precursor molecules, the pattern of protection from chemical modification of a region in M1 RNA that resembles the E site in 23S rRNA is similar to the pattern of protection of the E site in the presence of deacylated tRNA . In the complex with the RNA enzyme, more nucleotides in the substrate become accessible to modification, an indication that the substrate is in an unfolded conformation under these conditions.

Zentralbl Bakteriol, 1990 Nov, 274(2), 155 - 73
Isolation and characterization of monoclonal antibodies directed against different epitopes of type-1-like fimbriae from a multifimbriated E . coli strain; Biggemann B et al.; Monoclonal antibodies (MAbs) were raised against purified fimbriae isolated from the uropathogenic E . coli strain WF96 (O7:K1:H6:F11rel,F10) . This strain expresses at least four different types of fimbriae . 11 MAbs were selected for further characterization . They are directed against epitopes of a fimbrial type which is composed of 19.5 kDa subunits . It resembles type 1 fimbriae with regard to its high resistance to disruption by SDS . The MAbs were tested for crossreactivity to type 1 fimbriae and other fimbriae with known F-serotypes by ELISA . Two of these MAbs, Pili III 2F7 and Pili III 68C5, were directed against an epitope which was also found on MS fimbriae (type 1) . Thus type 1 like fimbriae of E . coli WF96 share at least one epitope with MS fimbriae . Nevertheless, the antigenic properties of these two fimbrial types were found not to be completely identical, since all the other 9 MAbs were not crossreactive . The MAbs were not able to inhibit haemagglutination of erythrocytes of different species and thus not directed against adhesive sites of the fimbriae . All the epitopes detected by MAbs were accessible on native fimbriae; some of them were also detectable on denatured fimbrial subunits . Electron micrographs revealed that these epitopes were evenly distributed on the fimbrial organelle.

Arzneimittelforschung, 1990 Nov, 40(11), 1284 - 7
Cloning and expression of the complete SIVagm pol region in E . coli . Purification and partial characterization of the reverse transcriptase; Baier M et al.; The complete pol region of the simian immunodeficiency virus from African green monkeys was cloned and expressed in E . coli . The reverse transcriptase was purified to high specific activity and could be shown to contain both reverse transcriptase activity as well as an associated RNase H activity . As is observed with other reverse transcriptases the enzyme is composed of two subunits which cannot be separated by conventional techniques . When comparing the recombinant enzyme with the authentic enzyme isolated from virus no differences were found by biochemical, enzymological, or immunological criteria . Moreover, the action of inhibitors against this enzyme did not show significant differences when compared to reverse transcriptases from HIV-1 and HIV-2.

Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 804 - 10
Functional interactions between phage T4 and E . coli DNA-binding proteins during the presynapsis phase of homologous recombination; Kodadek T; The "protein machine" for phage T4 homologous recombination has begun to be assembled in vitro . A particularly heavily studied reaction has been the uvsX protein (a RecA-like strand transferase)-mediated homologous pairing reaction between single and double-stranded DNAs, a key step in the recombination cycle in vivo . A necessary prerequisite for uvsX protein-mediated pairing is the polymerization of this factor along the invading single strand, a process known as presynapsis . Recent work has indicated that at least two other T4 recombination factors are involved in this process as well, the uvsY and gene 32 products . These proteins are also ssDNA-binding factors and exhibit an affinity for UvsX and each other . In order to begin to sort out the potential functional roles played by these protein-protein interactions in presynapsis, I have examined the ability of the uvsX protein to form stable filaments along ssDNA in the presence of these proteins . It is shown that the uvsY protein relieves the inhibition to filament formation due to the presence of the gene 32 protein, but experiments with the E . coli SSB protein (the bacterial analogue of gp32) suggest that this effect does not involve a direct interaction between UvsY and gp32.

Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 511 - 6
A structural protein of hepatitis C virus expressed in E . coli facilitates accurate detection of hepatitis C virus; Muraiso K et al.; A putative core protein derived from hepatitis C virus was expressed in E . coli . More than 5% of the total protein expressed in the bacteria after induction by isopropylthio-beta-D-galactoside was shown to be the expected protein . Western blotting with this E . coli lysate proved to be more efficient than ELISA with a non-structural viral protein, C100, to detect infection of hepatitis C virus in the sera of patients with non-A, non-B chronic hepatitis, hepatocellular carcinoma as well as in sera from healthy persons.

Cell, 1990 Oct 19, 63(2), 325 - 31
A novel protein binds a key origin sequence to block replication of an E . coli minichromosome; Hwang DS et al.; A sequence of three tandem repeats of a 13-mer in the replication origin (oriC) of E . coli is the highly conserved site of opening of the duplex for initiation of DNA synthesis . A protein that binds this sequence has been discovered in E . coli and purified to homogeneity . This novel 33 kd polypeptide behaves as a dimer . Binding to the 13-mers is specific and limited to this region . At a ratio of 10-20 monomers per oriC plasmid, the binding blocks initiation by preventing the opening of the 13-mer region by dnaA protein . Once the 13-mers are opened by dnaA protein action, the 33 kd protein is without effect on the subsequent stages of replication . The specificity of binding and profound inhibitory effect suggest a regulatory role for this protein at an early stage of chromosome initiation.

Cell, 1990 Oct 19, 63(2), 393 - 404
New topoisomerase essential for chromosome segregation in E . coli; Kato J et al.; The nucleotide sequence of the parC gene essential for chromosome partition in E . coli was determined . The deduced amino acid sequence was homologous to that of the A subunit of gyrase . We found another new gene coding for about 70 kd protein . The gene was sequenced, and the deduced amino acid sequence revealed that the gene product was homologous to the gyrase B subunit . Mutants of this gene were isolated and showed the typical Par phenotype at nonpermissive temperature; thus the gene was named parE . Enhanced relaxation activity of supercoiled plasmid molecules was detected in the combined crude cell lysates prepared from the ParC and ParE overproducers . A topA mutation defective in topoisomerase I could be compensated by increasing both the parC and the parE gene dosage . It is suggested that the parC and parE genes code for the subunits of a new topoisomerase, named topo IV.

Cell, 1990 Oct 19, 63(2), 269 - 79
The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E . coli plasma membrane; Hartl FU et al.; The export of many E . coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase . Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain . SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites . The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein . SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway . SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids . The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.

FEBS Lett, 1990 Oct 15, 272(1-2), 181 - 3
Highly selective affinity labeling of the primer-binding site of E . coli DNA polymerase I; Mitina RL et al.; Highly selective affinity labeling of the primer site of E . coli DNA polymerase I was performed with the 5'-reactive derivatives of oligothymidylate in the presence of poly(dA) template . Subtilysine cleavage proved that the site of affinity modification belonged to the 'Klenow' part of DNA polymerase I . If taken separately, Klenow fragment was not labeled by these oligonucleotide derivatives . The site of affinity labeling were tested in the structure of DNA polymerase I by hydroxylamine cleavage . At least two sites of labeling were revealed . The main one was localized between Gly-833 and His-928.

Trends Genet, 1990 Oct, 6(10), 329 - 34
The genetics of protein secretion in E . coli; Bieker KL et al.; Genetic studies have identified six genes whose products comprise the general protein secretion machinery of Escherichia coli . Insights from mutant analysis and the biochemical properties of the purified components allows the secretion pathway to be described in some detail . The picture emerging provides a useful paradigm for similar pathways in other organisms.

Mutat Res, 1990 Oct, 245(2), 67 - 74
Preventive action of thioethers towards in vitro DNA binding and mutagenesis in E . coli K12 by alkylating agents; Kroese ED et al.; Thioethers are effective scavengers of electrophilic metabolites derived from the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (van den Goorbergh et al., 1987) . In this study 2 of these thioethers, 4-(methylthio)benzoic acid (MTB) and its methylester, methyl 4-(methylthio)benzoate (MMTB), have been tested for their ability to prevent in vitro DNA binding and mutation induction in E . coli K12 by the direct alkylating agents ethylnitrosourea (ENU), methylnitrosourea (MNU), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) . In addition to MTB and MMTB, the thioether L-methionine (Met), and the thiols glutathione (GSH) and L-cysteine (Cys) were included for reasons of comparison . MTB was able to (partially) prevent DNA binding and mutation induction by ENU . However, this thioether was ineffective with EMS . DNA binding and mutagenesis by EMS were (partially) prevented by GSH and Cys, while these thiols could not prevent DNA binding and mutation induction by ENU . MMTB was unable to prevent mutation induction by these ethylating agents . With the methylating agents, similar effects of MTB were observed: MTB effectively prevented mutation induction by MNU while it was much less effective towards MMS . GSH and Cys were comparably effective as antimutagenic agents towards both methylating agents . Met was unable to prevent either DNA binding or mutation induction by these agents . Taken together, the results show that aromatic thioethers are able to trap genotoxic electrophiles derived from the nitrosoureas ENU and MNU, and may therefore act as potential anticarcinogens towards these agents, which are only poorly detoxified by GSH.

Chin Med J (Engl), 1990 Oct, 103(10), 811 - 6
Effect of obstructive jaundice on renal Na-K ATPase activity and ATP contents, their response to E . coli or endotoxin and effect of 1-6 fructose diphosphate; Gu Y et al.; A two-week bile duct ligation (BDL) in Sprague-Dawley (SD) rats raised the serum billirubin level and decreased the mean arterial blood pressure and renal cortical ATP contents compared with those in sham-operated (SO) rats (3.6 +/- 1.15 mg% vs 0.54 +/- 0.36, P less than 0.001; 69 +/- 24 mmHg vs 86 +/- 21, P less than 0.05; 3.72 +/- 0.86 x 10(-10) mol/mg tissue vs 7.27 +/- 0.18, P less than 0.05) . No difference could be found in the medullary ATP contents (8.42 +/- 2.20 vs 8.70 +/- 2.80, P = NS) . In SO rats, injection of endotoxin (0.7 mg/kg BW) and E . coli (3.1 x 10(5) bacteria/100 g BW) reduced cortical ATP content to 1.86 +/- 0.97 and 1.30 +/- 0.47 (P less than 0.001), and medullar ATP to 1.33 +/- 0.31 and 2.12 +/- 0.46 (P less than 0.001) respectively . In BDL rats, the same treatment led to further decrease in cortical ATP to 1.25 +/- 0.40 and 0.62 +/- 0.20, medullary ATP to 0.97 +/- 0.41 and 1.64 +/- 0.83 (P less than 0.001) . Basal Na-K ATPase activity in BDL is the same compared with that in SO both in the cortex (2.85 +/- 2.2 mumol/mgpr/h vs 2.19 +/- 0.75; P = NS) and medulla (2.79 +/- 1.83 vs 3.05 +/- 1.38; P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Jpn J Genet, 1990 Oct, 65(5), 381 - 6
An E . coli promoter that is sensitive to visible light; Nakahigashi K et al.; It has been discovered that expression of promoter activity can be inhibited by visible light when specific fragments of E . coli DNA are inserted in a vector system designed to assay for promoter activity . These fragments have been located on regions of the E . coli chromosome to which no gene has been assigned to date . The effective wavelength of light that produces this phenomenon has been determined.

EMBO J, 1990 Oct, 9(10), 3209 - 16
The secD locus of E.coli codes for two membrane proteins required for protein export; Gardel C et al.; Cold-sensitive mutations in the secD locus of Escherichia coli result in severe defects in protein export at the non-permissive temperature of 23 degrees C . DNA sequence of a cloned fragment that includes the secD locus reveals open reading frames for seven polypeptide chains . Both deletions and TnphoA insertions in this clone have been used in maxicell and complementation studies to define the secD locus and its products . The secD mutations fall into two complementation groups, defining genes we have named secD and secF . These two genes comprise an operon, the first case of two genes involved in the export process being co-transcribed . The DNA sequence of the two genes along with alkaline phosphatase fusion analysis indicates that they code for integral proteins of the cytoplasmic membrane . We suggest that these two proteins may form a complex in the membrane which acts at late steps in the export process.

Bioorg Khim, 1990 Oct, 16(10), 1339 - 47
{Plasmid expression vectors with elements of the E . coli alkaline phosphatase gene}; Zozulia SA et al.; A family of plasmid expression vectors, containing fragments of the E . coli alkaline phosphatase gene (phoA), has been constructed for the lambda PR promoter-directed thermoinducible superproduction of fusions of heterologous polypeptides to the N- or C-terminus of E . coli alkaline phosphatase and its leader peptide . Effective expression and export to periplasm of resulting fusion proteins, which may retain enzymatic activity of the phosphatase, has been shown.

J Trop Pediatr, 1990 Oct, 36(5), 247 - 50
Use of human colostrum in the management of chronic infantile diarrhoea due to enteropathogenic E . coli infection with associated intestinal parasite infestations and undernutrition; Saha K et al.; This study reports the management of infants with chronic diarrhoea by colostrum feeding . Eight children with chronic diarrhoea, ranging from 9 months to 3 years of age and all from low socio-economic families, formed the basis of this study . They were undernourished and marasmic . Stool examination showed enteropathogenic E . coli in all eight cases, Ascaris lambricoidis in four, and Giardia lamblia in one . Patients with chronic diarrhoea, in whom no cause was found were excluded from this study . All eight patients were administered 20 ml fresh human colostrum daily for 7 days . In addition, those patients, who had giardiasis, received metronidazole treatment, while cases with ascariasis were given antihelminthic therapy irrespective of the groups they belonged to . Our results indicated effective antidiarrhoea action of colostrum in some patients with chronic diarrhoea of infective origin.

Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 1312 - 8
Allosteric control of quaternary states in E . coli aspartate transcarbamylase; Stevens RC et al.; Changes in the molecular dimensions of ATCase in the unligated T-state are an increase of 0.4 A in the separation of catalytic trimers when ATP binds . When the R-state is produced by binding of phosphonoacetamide and malonate, addition of CTP or CTP + UTP decreases the separation of catalytic trimers by 0.5 A . In the unliganded Glu239----Gln mutant, in which the T-state is destabilized so that the enzyme exists in an intermediate quaternary state, ligation of ATP transforms the mutant enzyme to the R-state, whereas CTP converts this enzyme to the T-state . Thus, this mutant is much more sensitive to heterotropic allosteric control than is the native enzyme . In this communication we propose a preliminary model based on new crystallographic results that heterotropic regulation occurs partly through control of the quaternary structure by these effectors, thus regulating catalysis.

Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 963 - 71
High-level production of human acidic fibroblast growth factor in E . coli cells: inhibition of DNA synthesis in rat mammary fibroblasts at high concentrations of growth factor; Ke YQ et al.; Recombinant human acidic fibroblast growth factor has been produced in E . coli cells at a level of at least 50 mg/l culture . The recombinant and natural acidic fibroblast growth factors are almost identical to one another when tested on rat mammary fibroblasts for their ability to stimulate DNA synthesis, to bind to the high-affinity surface receptors of the cells and to inhibit DNA synthesis when present in the culture medium at high concentrations . The recombinant acidic fibroblast growth factor binds to two cell-surface polypeptides of molecular masses 160 kDa and 140 kDa, which are the same size as the receptors for basic fibroblast growth factor, and it binds preferentially to the smaller polypeptide.

Cell, 1990 Sep 21, 62(6), 1153 - 63
Transcriptional antitermination in the bgl operon of E . coli is modulated by a specific RNA binding protein; Houman F et al.; Regulation of bgl operon expression in E . coli occurs by a mechanism involving antitermination of transcription at two termination sites within the operon . The bglG gene product is absolutely required for this process . Here we provide evidence that BglG is an RNA binding protein that recognizes a specific sequence located just upstream of each of the terminators . The sequence was delimited using a series of specific oligonucleotide probes . Mutational analysis of this sequence indicates that the protein requires a specific RNA secondary structure for recognition . We propose that BglG prevents transcription termination by binding to nascent RNA and blocking formation of the terminator structure.

Nature, 1990 Sep 20, 347(6290), 306 - 9
Three-dimensional structure of ribonuclease H from E . coli; Katayanagi K et al.; The three-dimensional structure of RNase H from Escherichia coli was determined at 1.8 A resolution by X-ray crystallography . The enzyme was found to belong to the alpha + beta class of structures, consisting of two distinct domains . The structure implies a possible region interacting with a DNA-RNA hybrid . The Mg2(+)-binding site essential for activity is located near a cluster of four acidic amino acids--one glutamic and three aspartic acid residues . These residues are completely conserved in the homology alignment of sequences of RNase H and reverse transcriptases from retroviruses and retrovirus-like entities . The structural motif of beta strands around the Mg2(+)-binding site has similarities to that in DNase I.

FEBS Lett, 1990 Sep 17, 270(1-2), 119 - 22
In vivo and in vitro mutants of FNR the anaerobic transcriptional regulator of E . coli; Sharrocks AD et al.; FNR regulates the expression of target genes in response to anaerobiosis . It resembles the catabolite gene activator or cAMP-receptor protein (CRP) except for the presence of an N-terminal cysteine cluster, which may form a redox-sensing iron-binding site . Site-directed mutagenesis has shown that 3 of the 4 cysteine residues in the N-terminal cluster (Cys-20, -23 and -29, but not Cys-16) and the only other cysteine residue (Cys-122), are essential for the normal activation and repression of FNR-dependent promoters . Deletion of residues Pro-3-Arg-9 (inclusive) had no effect, but FNR was inactivated by a frameshift extending through the C-terminal DNA-binding domain . Four independent in vivo mutants contained identical Gly-96----Asp substitutions, which may inactivate FNR by distorting a sharp turn between beta-strands in the predicted structure.

Nucleic Acids Res, 1990 Sep 11, 18(17), 5051 - 3
The repair patch of E . coli (A)BC excinuclease; Sibghat-Ullah et al.; The size of repair patch made by E . coli DNA polymerase I (Poll) following the removal of a thymine-psoralen monoadduct by E . coli (A)BC excinuclease was determined by using an M13mp19 DNA with a single psoralen monoadduct at the polylinker region . Incubation of this substrate with (A)BC excinuclease, Poll and a combination of 3 dnTP plus 1 dNTP(alpha S) for each nucleotide, and DNA ligase resulted in a repair patch with phosphorothioate linkages . The preferential hydrolysis of phosphorothioate bonds by heating in iodoethanol revealed a patch size--with minimal nick translation--equal in length to the 12 nucleotide gap generated by this excision nuclease.

Nucleic Acids Res, 1990 Sep 11, 18(17), 5127 - 32
Characterization of the binding of cAMP and cGMP to the CRP*598 mutant of the E . coli cAMP receptor protein; Ren YL et al.; Wild type cAMP receptor protein (CRP) activates in vitro lac transcription only in the presence of cAMP . In contrast the mutant CRP*598 (Arg-142 to His, Ala-144 to Thr) can activate lac transcription in the absence of cyclic nucleotide or at concentrations of cAMP below that required by CRP . To further characterize the properties of CRP*598, the binding of cAMP and cGMP to CRP and CRP*598 has been determined . The intrinsic binding constant (K) values obtained for cAMP binding are: CRP, 1.9 x 10(4) M-1; CRP*598, 3.8 x 10(5) M-1 . The K values obtained for cGMP binding are: CRP, 2.9 x 10(4) M-1; CRP*598, 2.7 x 10(4) M-1 . The results indicate that the affinity of CRP and CRP*598 for cGMP is relatively unchanged while the affinity of CRP*598 for cAMP is approximately twenty times greater than that shown by CRP . Binding of cAMP by CRP and cGMP by CRP or CRP*598 exhibits slight negative cooperativity . The major difference seen is that CRP*598 binds cAMP with strong positive cooperativity . The importance of the unsubstituted N6 position of the adenine moiety is also shown by the similar affinity of both forms of CRP for N6-butyryl cAMP . The cAMP binding properties evinced by CRP*598 suggest that its intrinsically altered conformation may be related to that assumed by CRP in a CRP-DNA or a cAMP-CRP-DNA complex.

Cell, 1990 Sep 7, 62(5), 981 - 9
The role of dam methyltransferase in the control of DNA replication in E . coli; Boye E et al.; The timing and control of initiation of DNA replication in E . coli was studied under conditions where the cellular level of dam methyltransferase was controlled by a temperature-inducible promoter . Flow cytometry was used to demonstrate that the synchrony of initiation at the several origins within each cell was critically dependent on the level of dam methyltransferase . Initiations were shown to be synchronous only in a narrow temperature range . The data are explained by a model where a newly replicated and therefore hemimethylated oriC is inert for reinitiation . Such a model may be applicable to eukaryotic cells, where classes of origins are initiated in synchrony and only once per cell cycle.

Cell, 1990 Sep 7, 62(5), 939 - 44
The E . coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner; Skowyra D et al.; Pelham previously proposed that the hsp70 family of heat shock proteins could prevent the formation and/or allow the dissolution of protein aggregates created during stress conditions . We confirmed this hypothesis by showing that the E . coli hsp70 homolog, the dnaK gene product, protects the host RNA polymerase enzyme from heat inactivation in an ATP-independent reaction . In addition, we show that heat-inactivated and aggregated RNA polymerase is both disaggregated and reactivated following simultaneous incubation with DnaK protein and hydrolyzable ATP . The DnaK756 mutant protein has lost the ability to disaggregate the inactivated RNA polymerase enzyme . Our results demonstrate that the DnaK protein contributes to E . coli's growth not only by protecting some enzymes from denaturation but also by reactivating some once they are misfolded or aggregated.

Cell, 1990 Sep 7, 62(5), 967 - 79
E . coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork; Campbell JL et al.; We have examined individual GATC sites throughout the E . coli genome for their kinetics of remethylation by dam methyltransferase following the passage of the chromosomal replication fork . We present evidence for three major conclusions: that oriC is a single function unit that is specifically sequestered from dam methyltransferase for a significant period of time and then released; that the dnaA promoter region is subject to sequestration analogous to that observed at oriC and thus that hemimethylation-dependent sequestration is a general phenomenon; and that each round of replication initiation triggers a transient, temporally coordinate block in both reinitiation at oriC and expression of the dnaA gene . These and other observations are all consistent with the notion that hemimethylation in these two regions acts coordinately to ensure that every origin undergoes initiation once and only once per cell cycle; other possible roles for sequestration at dnaA are also considered.

J Biochem Biophys Methods, 1990 Sep-Oct, 21(3), 247 - 66
A new method for stoichiometric analysis of proteins in complex mixture--reevaluation of the stoichiometry of E . coli ribosomal proteins; Tal M et al.; A novel way is presented for determination of the stoichiometry of ribosomal proteins in the ribosome . The 70S E . coli r-proteins, completely separated on a two-dimensional gel system, were used throughout our experiments . The method is based on our previous observation that the amount of Coomassie R bound to a protein molecule is directly proportional to the number of positive charges on that protein . By plotting the amount of bound Coomassie as a function of the number of positive charges of each r-protein, and relating the experimental amount of the dye bound to each r-protein to the value obtained from the linear regression line based on all (a total of some 50 proteins), one can obtain the molar concentration of every protein in the ribosome . A parallel experiment can be carried out, which relates the radioactivity contributed by 3H-labeled amino acid in each r-protein to its amino acid content in that molecule . The two sets of data, which are completely independent of each other, are well correlated . Further verification of the validity of our procedure is provided by the fact that we found the known proportions of four copies of L7/L12 and one copy of S6 per ribosome . The rationale behind the present study was our finding that recalculation of Hardy's data (Hardy, S.J.S . (1975) Mol . Gen . Genet . 140, 253-274), with the accurate molecular weight value of the r-proteins provided by Giri et al . (Adv . Protein Chem . (1984) 36, 1-78), raises some doubt with regard to his experimental results, although we agree with his final conclusion that E . coli ribosome is homogeneous with respect to its proteins.

Zhonghua Bing Li Xue Za Zhi, 1990 Sep, 19(3), 197 - 9
{Pathological changes in an animal model of acute pulmonary injury induced by hemorrhagic shock and E . coli infection}; Zhou XD et al.; An animal model of API similar to RDS was established by feeding the rabbits with live E . Coli (10(11)/kg B.W.) and bleeding them till a mean arterial pressure of 6.0 kPa was maintained for 90 minutes . After resuscitation, there were decrease of PaO2, increase of respiratory rate as well as lung edema . Histological and electron microscopic examination showed interstitial and alveolar edema . The tight junctions both in alveolar epithelium and capillary endothelium showed alterations including discontinuity, irregularity, and free-ended strands being present as early as the 4th hour by freeze-fracture technique . Impairment of the tight junctions is considered to be the main route of fluid leakage from pulmonary capillaries inducing pulmonary edema.

Biull Eksp Biol Med, 1990 Sep, 110(9), 303 - 6
{Systems of surface exclusion of F-like plasmids of E . coli and their genetic control}; Shchipkov VP et al.; The F-like plasmids belonging to 5 different Sfx-groups were discovered . The existence of atypical plasmids belonging to different Sfx-groups was shown . The molecular cloning of plasmid DNA fragment (3.9 mD) determining SfxII phenotype was performed.

EMBO J, 1990 Sep, 9(9), 2731 - 41
Cleavages in the 5' region of the ompA and bla mRNA control stability: studies with an E . coli mutant altering mRNA stability and a novel endoribonuclease; Lundberg U et al.; We describe here the partial purification of a novel Escherichia coli endoribonuclease, RNase K . This protein catalyses site-specific cleavages in the 5' region of in vitro transcribed ompA and bla transcripts . Some of the resulting cleavage products are also found in cellular ompA mRNA, defining the in vivo activity of RNase K . The following evidence suggests that RNase K initiates mRNA degradation . First, RNase K cleavages are suppressed in the ams mutant, which has a generally prolonged mRNA half-life . Secondly, RNase K cleavage products seem to have very short half-lives in vivo, indicating that they are decay intermediates rather than processing products . Thirdly, the differences in in vivo half-life between the ompA and bla mRNAs are mimicked in in vitro decay reactions with purified RNase K . The relationship between RNase K and the ams locus might point to a more general role of RNase K in mRNA degradation . We discuss the influence of mRNA secondary structure on RNase K cleavage specificity.

Biophys Chem, 1990 Aug 31, 37(1-3), 31 - 41
Differential scanning calorimetric studies of E . coli aspartate transcarbamylase . III . The denaturational thermodynamics of the holoenzyme with single-site mutations in the catalytic chain; Burz DS et al.; Aspartate transcarbamylase (EC 2.1.3.2) from E . coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions . Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J . Biol . Chem . 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081) . We have examined the effects of nine single-site mutations in the catalytic chains . Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit . These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J . Mol . Biol . 160 (1982) 219; Krause et al., J . Mol . Biol . 193 (1987) 527) . The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state . DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks . At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations . The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized . Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al . (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe.

Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 60 - 6
Affinity purification of HIV-1 and HIV-2 proteases from recombinant E . coli strains using pepstatin-agarose; Rittenhouse J et al.; A procedure is described which employs pepstatin-agarose for the affinity purification of either HIV-1 or HIV-2 protease from two similar recombinant E . coli constructs that were developed for the expression of these enzymes . HIV-2 protease was routinely expressed at much higher levels than the HIV-1 enzyme and pepstatin-agarose was the only chromatography step required to isolate pure HIV-2 protease from crude bacterial lysates . A Mono S ionic exchange step following pepstatin-agarose chromatography was sufficient to bring the HIV-1 protease to homogeneity . Purification of either enzyme can be completed in several days yielding homogeneous preparations suitable for crystallization and other physical characterization.

Biophys Chem, 1990 Aug 31, 37(1-3), 239 - 50
Thermodynamic analysis of the transcription cycle in E . coli; Gill SC et al.; The E . coli RNA transcription cycle can be divided into three major phases, which are generally called initiation, elongation, and termination . In this paper, we review recent biophysical studies of the interactions of the transcriptional regulatory proteins, sigma 70 and NusA, with themselves and with core RNA polymerase in solution, as well as with core polymerase within the transcription complex . The different affinities of sigma 70 and NusA for core RNA polymerase at various stages in the transcription cycle, together with other quantitative data, are then used to construct a partial free energy diagram for the overall transcription process . This thermodynamic framework, which is interrupted by at least two irreversible steps, can be used to rationalize physiological aspects of the transcription cycle and its regulation, as well as to identify crucial points at which our knowledge is still incomplete.

J Immunol Methods, 1990 Aug 28, 132(1), 13 - 24
The use of E . coli exonuclease III to generate single stranded DNA in BrdUrd cell-cycle analysis permits simultaneous detection of cell surface antigens; Bayer JA et al.; An immunocytochemical method for the simultaneous flow cytometric quantitation of total cellular DNA, incorporated 5-bromo-2'-deoxyuridine (BrdUrd) and one or more cell surface antigens has been developed . Biotin labeling of cell surface antigens, critically tuned fixation techniques and an enzymatic denaturation of cellular DNA are the essential features of this method . Enzymatic denaturation of cellular DNA was shown to prevent loss of cell surface antigen-bound biotin moieties, and thus to preserve cell surface immunofluorescence distribution . After a mild protein extraction and the introduction of breaks into the chromatin using restriction endonucleases, E . coli exonuclease III was used to generate stretches of single stranded DNA . This approach permits detection of the incorporated BrdUrd using anti-BrdUrd monoclonal antibodies . The enzymatic denaturation protocol was optimized using in vitro BrdUrd-labeled L1210 murine leukemia cells, and applied to both in vivo and ex vivo BrdUrd-labeled murine bone marrow cells . With this new method it is possible to study DNA content, cell cycle kinetics and cell surface antigen expression simultaneously, and hence functional relationships between these parameters can be investigated.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 312 - 6
Regulatory elements downstream of the promoter of an rRNA gene of E . coli; Csiszar K et al.; Previously we have shown that plasmid constructs carrying a reporter gene fused to the P2 promoter of the E . coli rrnB gene exhibited a strange two-phase kinetics of expression depending on the physiological conditions of the cell if a short DNA region downstream of the promoter was present between the promoter and the reporter gene (Lukacsovich et al . (1987) J . Bacteriol . 169, 272-277) . Insertion of a synthetic oligonucleotide corresponding to the first half of this region into constructs where the reporter directly follows the promoter, leads to a complete block of expression in vivo, while in vitro--in a purified system--transcription is not inhibited . Band-shift experiments indicate that the putative regulatory region downstream of the promoter specifically binds protein(s) present in total bacterial extracts.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 203 - 8
Structural determination of the functional sites of E . coli elongation factor Tu; Clark BF et al.; Recently, we have made significant progress in solving the structure of a nicked form of elongation factor (EF)-Tu complexed with GDP . The structure has been refined to an R factor of 19.2% at 2.6 A resolution, so that most of the structure is clearly visible in the electron density map . Here we describe what is known about functional sites of EF-Tu in terms of the structure, which still lacks amino acids 40-60.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 18 - 26
Site-specific mutation of the conserved m6(2)A m6(2)A residues of E . coli 16S ribosomal RNA . Effects on ribosome function and activity of the ksgA methyltransferase; Cunningham PR et al.; In vitro synthesis of mutant 16S RNA and reconstitution with ribosomal proteins into a mutant 30S ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues . All of the mutant RNAs could be assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins . A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity . The amount varied according to the assay and mutant, but was never less than 30% and was generally above 50% . Therefore, neither the conserved A1518 nor A1519 residues are essential for ribosome function . The mutant ribosomes could also be methylated by the ksgA methyltransferase to 70-120% of the expected amount . Thus, neither of the A residues is required for methylation of the other, ruling out any obligate order of methylation of A1518 and A1519.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 323 - 7
Single base alterations upstream of the E . coli 16S rRNA coding region result in temperature-sensitive 16S rRNA expression; Mori H et al.; We have isolated three new temperature-sensitive mutants of 16S rRNA, using the U1192 spectinomycin resistance as a selectable marker . These differ from our previously characterized ts mutants in that they map in the upstream leader region of the rRNA precursor (at positions -13, -30 and -59) . The relative distribution of plasmid and chromosome-derived 16S rRNA is similar between 30S subunits, 70S ribosomes and polysomes at the permissive and restrictive temperatures . Processing of the 5' end of the RNA does not appear to be affected by the mutations . Second-site suppressors were found, and all of these except one (which is within 16S rRNA) were also due to point mutations in the upstream leader.

Cell, 1990 Aug 24, 62(4), 649 - 57
The purified E . coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation; Brundage L et al.; We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles . We now report the isolation of the SecY/E protein, the integral membrane protein component of the E . coli preprotein translocase . The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A . This translocation requires ATP and is strongly stimulated by the protonmotive force . The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable . The SecY/E protein consists of SecY, SecE, and an additional polypeptide . Antiserum against SecY immunoprecipitates all three components of the SecY/E protein.

FEBS Lett, 1990 Aug 20, 269(1), 244 - 6
Influence of the second and third codon on the expression of recombinant hirudin in E . coli; Degryse E; The effect of all possible codons corresponding to the second and third amino acid (isoleucine and threonine) on the expression level of hirudin in E . coli has been analysed . These levels could not be correlated with changes in primary and secondary mRNA structure . A decrease in the rate of synthesis and of product accumulation follows the introduction for ile of the ATA codon which is of very low usage, and for thr of the ACC codon, which results in homology of the mRNA with the 3'-end of 16S rRNA . The results are discussed according to current concepts of protein expression in E . coli.

Biochem Biophys Res Commun, 1990 Aug 16, 170(3), 1061 - 6
Characterization of an internally initiated integrase protein of HIV-1 produced in E . coli; Zervos P et al.; In E . coli cells transformed by an expression vector for the production of the protease (PR) integrase (IN) of HIV-1, three vitally encoded proteins were produced: an 11-kDa protein and a 32-kDa protein identified by immunoassays as the mature PR and IN protein, respectively, and an additional protein 15-kDa in size that reacted strongly with an antiserum recognizing a region in the carboxyl half of the IN protein . The kinetics of its synthesis indicated that it was not a degradation product of p32-IN, rather it probably arose from internal initiation at an AUG codon in the middle of the IN gene . Amino terminal sequence analysis of the first 70 residues demonstrated a perfect match with those predicted from the nucleotide sequence, beginning with the methionine codon at position 154 of the integrase gene.

Nucleic Acids Res, 1990 Aug 11, 18(15), 4369 - 75
The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E . coli; Bergerat A et al.; The turnover of DNA-adenine-methylase of E . coli strongly decreases when the temperature is lowered . This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place . The enzyme can bind non-specific DNA with low affinity . Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor . The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met . This suggests that Ado-Met could bind to Dam methylase in two different environments . In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site . The analogues can not compete for such binding . In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues . We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.

Nucleic Acids Res, 1990 Aug 11, 18(15), 4325 - 33
Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E . coli: a model for the tertiary structure of the tRNA binding domain in 23S RNA; Mitchell P et al.; Intramolecular RNA cross-links were induced within the large ribosomal subunit of E . coli by mild ultraviolet irradiation . Regions of the 23S RNA previously implicated in interactions with ribosomal-bound tRNA were then specifically excised by addressed cleavage using ribonuclease H, in conjunction with synthetic complementary decadeoxyribonucleotides . Individual cross-linked fragments within these regions released by such 'directed digests' were isolated by two-dimensional gel electrophoresis and the sites involved in the cross-links determined using classical oligonucleotide analysis techniques . Using this approach, seven 'new' cross-links could be precisely localised, between positions 1782 and 2608-2609, 1940 and 2554, 1941-1942 and 1964-1965, 1955 and 2552-2553, 2145-2146 and 2202, 2518-2519 and 2544-2545, and between positions 2790-2791 and 2892-2895 in the 23S RNA sequence . These data, in conjunction with data from RNA-protein cross-linking studies carried out in our laboratory, were used to define a model for the tertiary organisation of the tRNA binding domain of 23S RNA 'in situ', in which the specific nucleotides associated with tRNA binding in the 'A' and 'P' sites are clustered at the base of the 'central protuberance' of the 50S subunit.

Biochim Biophys Acta, 1990 Aug 9, 1019(1), 67 - 72
Uncoupler resistance in E . coli Tuv and Cuv is due to the exclusion of uncoupler by the outer membrane; Haworth RS et al.; The uncoupler resistant bacterial strains E . coli Tuv and Cuv share the high deoxycholate sensitivity of the parent strain, Doc S . However, both Tuv and Cuv show greater resistance than Doc S to other detergents . Measurement of the periplasmic volume indicates that the outer membrane of Doc S is freely permeable to both TPP+ and hydroxymethylinulin . Tuv and Cuv are able to exclude these compounds . EDTA treatment was necessary prior to measuring membrane potential in Tuv and Cuv . Under conditions where delta phi could be measured, uncouplers acted to dissipate delta phi with equal potency in all strains . Uncoupler resistant proline uptake in Tuv and Cuv was abolished by EDTA treatment . Transduction experiments with phage P1 showed that uncoupler resistance could be transferred from Tuv to Doc S . Such transductants were no longer sensitive to novabiocin . The gene for uncoupler resistance cotransduced with the gene pyrE (82 min) . Plating efficiency experiments with P1 suggests that detergent sensitivity in Doc S arises from an rfa (81 min) mutation . This mutation is no longer present in Tuv.

Bioorg Khim, 1990 Aug, 16(8), 1019 - 23
{Catalytic properties of E . coli tryptophanase in a 50% aqueous solution of methanol and dimethylformamide}; Faleev NG et al.; Tryptophanase from E . coli retains its ability to form quinonoid intermediate with L-alanine in water--methanol and water--dimethylformamide (1:1 v/v) solutions . Under these conditions the enzyme catalyzes decomposition of S-o-nitrophenyl-L-cysteine (SOPC) to o-nitrophenylthiol, pyruvate and ammonium ion . The enzyme's affinity for this substrate increases on going from water to water-organic solvents whereas the reaction rate decreases . In 50% methanol tryptophanase catalyzes the formation of L-tryptophan from indole and SOPC; in the mixture of 2H2O and C2H3O2H (1:1) the enzymatic isotope exchange of alpha-proton of L-phenylalanine with complete retention of configuration was observed.

J Biochem (Tokyo), 1990 Aug, 108(2), 153 - 7
Human aldolase A of a hemolytic anemia patient with Asp-128----Gly substitution: characteristics of an enzyme generated in E . coli transfected with the expression plasmid pHAAD128G; Takasaki Y et al.; Aldolase A derived from a hemolytic anemia patient with aldolase A deficiency was shown to have an amino acid substitution of glycine for aspartic acid at the 128th position (Asp-128) in the enzyme {Kishi et al . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 8623-8627} . We constructed an Escherichia coli expression plasmid, pHAAD128G, which carries the mutant aldolase A {aldolase A(D-G)} cDNA, and the enzyme generated in E . coli transfected with the expression plasmid was purified and characterized . Conversion of Asp to Gly at the 128th position in the enzyme rendered the enzyme thermolabile and susceptible to tryptic digestion . CD spectra analysis also revealed that the mutant enzyme had a remarkable conformation change with a decrease of regular form in the molecule . Addition of glycerol or some other polyalcohols during thermal treatment protected this altered enzyme (but not the normal enzyme) against denaturation and activity decrease . In order to determine the function of the amino acid residue at the 128th position, two artificial mutant enzymes with the substitutions of Glu for Asp {aldolase A(D-E)} and Ser for Asp {aldolase A(D-S)}, respectively, at the position were constructed by site-directed mutagenesis and characterized . These analyses demonstrated the necessity for Asp to be present at the 128th residue in order for this enzyme to be thermally stable.

Clin Invest Med, 1990 Aug, 13(4), 145 - 51
Effect of E . coli pyelonephritis on the intracortical accumulation kinetics of gentamicin and netilmicin in rats; Beauchamp D et al.; The role of serum levels on the intrarenal accumulation kinetics of gentamicin and netilmicin in normal and infected kidneys was evaluated in a short-term infusion model in conscious rats . Female Sprague-Dawley rats were infused over a period of 6 h with gentamicin and netilmicin achieving individual steady-state serum levels ranging from 0.5 to 120 micrograms/ml . The model of pyelonephritis used resulted in severe left pyelonephritis and mild right pyelonephritis . Only the right infected kidneys were studied . Gentamicin and netilmicin cortical concentrations were analysed as a function of serum levels by linear (least-squares regression analysis) and non-linear regression . For the non-linear regression analysis, the Michaelis-Menten kinetic was the best fitting curve . Steady-state elevation of serum concentrations of gentamicin and netilmicin was associated with a non-linear increase of cortical concentrations in normal kidneys, suggesting a saturable process . By contrast, in the mildly-infected right kidneys, the steady-state elevation of serum concentrations of gentamicin was associated with a linear increase of cortical concentrations while the accumulation kinetic of netilmicin showed a saturable process . At lower serum levels (therapeutic range, from 0.5 to 15 micrograms/ml) both gentamicin and netilmicin showed a first order kinetics of accumulation and netilmicin accumulated less than gentamicin in normal kidneys (p = 0.0004) . By contrast, the uptake of netilmicin was higher in the right infected kidneys, as compared to the uptake of netilmicin in the normal kidneys, (p = 0.00005), and as compared to gentamicin in the respective kidneys . We conclude that renal infection modifies the intrarenal accumulation of aminoglycosides.

Trends Biochem Sci, 1990 Aug, 15(8), 309 - 14
Beta-galactoside transport in E . coli: a functional dissection of lac permease; Kaback HR et al.; The polytopic membrane protein lac permease harnesses energy from the electrochemical H+ gradient to transport beta-galactosidases against a concentration gradient . Although high-resolution structural information is still lacking, the permease is thought to possess 12 membrane-spanning alpha-helical segments . Various experimental strategies, including site-directed mutagenesis, have been employed to probe the function of this membrane protein at the molecular level.

EMBO J, 1990 Aug, 9(8), 2649 - 55
A single base mutation at position 2661 in E . coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome; Tapprich WE et al.; A single base substitution mutation from guanine to cytosine was constructed at position 2661 of Escherichia coli 23S rRNA and cloned into the rrnB operon of the multi-copy plasmid pKK3535 . The mutant plasmid was transformed into E.coli to determine the effect of the mutation on cell growth as well as the structural and functional properties of the mutant ribosomes in vivo and in vitro . The results show that the mutant ribosomes have a slower elongation rate and an altered affinity for EF-Tu-tRNA-GTP ternary complex . This supports previous findings which indicated that position 2661 is part of a region of 23S rRNA that forms a recognition site for binding of the ternary complex in the ribosomal A site . Combinations of the 2661 mutation with various mutations in ribosomal protein S12 also demonstrate that elements of both ribosomal subunits work in concert to form this binding site.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1990 Aug, 12(4), 306 - 10
{A preliminary study of the inhibiting mechanism of anisodamine on rabbit platelets activated by E . coli endotoxin}; Xu S; Bacterial endotoxin binds to platelets and causes platelet aggregation, thrombocytopenia, increase in thromboxane A2 production, serotonin release and increase in the generation of platelet factor 3 procoagulant activity . These effects have been demonstrated in a variety of animal species . Much evidence has shown that such changes may participate in the pathogenesis of DIC and RDS in septic shock . The anisodamine (654) antishock effect has been studied from different angles for more than 20 years, with its effect on the cardiovascular system, including the microcirculation, being demonstrated . But few reports concerning its effect on platelet function have emerged . In this study, the effect of anisodamine on rabbit platelet aggregation, release, morphological changes, cellular cAMP, and membrane lipid fluidity induced by E . coli endotoxin were studied both in vivo and in vitro . After anisodamine intervention, all of these parameters improved to a certain extent . The possible protective mechanisms of anisodamine on platelets both in vivo and in vitro are discussed.

Biochimie, 1990 Aug, 72(8), 625 - 32
Methionyl-tRNA synthetase from E . coli--a review; Meinnel T et al.; Methionyl-tRNA synthetase (MetRS) from E coli is a dimer composed of 2 identical subunits of Mr 76 kDa . A fully active monomeric fragment (64 kDa) could be obtained by mild proteolysis of the native dimer . Earlier studies reviewed in Blanquet et al (1979) have compared the catalytic mechanisms of native and trypsin-modified MetRS . Moreover, the truncated form of the enzyme was crystallized and its 3-D structure solved at low resolution . In the last few years, the availability of the corresponding metG gene has facilitated the development of studies using affinity labelling and site-directed mutagenesis techniques . In parallel, the 3-D structure has been solved at a resolution of 2.5 A . These convergent approaches have allowed significant progress in the understanding of the structure-function relationships of this enzyme, and, in particular, of the rules governing the recognition of tRNA.

Biochimie, 1990 Aug, 72(8), 617 - 24
Modelling allosteric processes in E coli aspartate transcarbamylase; Cherfils J et al.; The allosteric properties of aspartate transcarbamylase from E coli have been investigated by a combination of genetic, biochemical and structural studies . Based on the X-ray structures of the enzyme in T and R state established by Lipscomb et al, we have analyzed the interactions between the 12 polypeptide chains and have identified subunit interfaces that play a major part in the allosteric mechanism: the c1c4 interface between the 2 catalytic trimers, and one of 2 different interfaces between catalytic and regulatory chains, the c1r4 interface, which exists only in T state . We have modelled mutations affecting these interfaces: mutation pAR5 in the gene coding for r chains concerns the c1r4 interface, mutation Tyr----Phe 240 in the gene coding for c chains, the c1c4 interface . Both mutant proteins have reduced cooperativity and/or allosteric regulation by CTP and ATP . Molecular mechanic simulations lead to specific proposals for the structural origin of these effects, and some of the proposals can be checked by site-directed mutagenesis . Finally, we have modelled substrates bound at the active site of the T state, which binds aspartate less tightly than the R state and for which X-ray structures of bound substrate analogs were not available.

Carbohydr Res, 1990 Jul 15, 202, 225 - 38
Use of the methylsulfenyl cation as an activator for glycosylation reactions with alkyl (aryl) 1-thioglycopyranosides: synthesis of methyl O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-(1----6)-O-alpha-D-glucopyranosyl-(1----2) -alpha-D -glucopyranoside, a derivative of the core trisaccharide of E . coli K12; Dasgupta F et al.; Methylsulfenyl bromide (MSB) and methylsulfenyl trifluoromethanesulfonate (MST) have been used to prepare 1,2-cis-linked disaccharides . Ethyl (phenyl) 1-thio-beta-D-gluco- and galacto-pyranosides having non-participating (benzyloxy) protecting groups were used as the donors . The alpha beta-ratio of the products depended on the promoter and conditions of reaction . Intimate ion-pairs, formed initially, may be responsible for the steric outcome of the glycosylations . Thus, with ethyl 2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranoside as a donor, moderate quantities of the beta-linked disaccharide could be produced using MSB as the activator . The synthesis of the title trisaccharide glycoside that contains 1,2-cis and 1,2-trans-linkages is described.

Carbohydr Res, 1990 Jul 15, 202, 131 - 49
Derivatives of methyl beta-lactoside as substrates for and inhibitors of beta-D-galactosidase from E . coli; Bock K et al.; The 2'-,4'-, and 6'-deoxy derivatives of methyl beta-lactoside have been synthesised by deoxygenation at positions 2', 4', and 6', and the 3'-deoxy derivative was obtained by a glycosylation reaction . The 2'-O-methyl, 2'-O-benzyl, 2'-amino-2'-deoxy, and 1'-deuterio derivatives have been synthesized also . Only the 6'-deoxy and 1'-deuterio derivatives were substrates for the beta-D-galactosidase from E . coli, and the 2'-deoxy- and 2'-amino-2'-deoxy derivatives were potent inhibitors for the hydrolysis of methyl beta-lactoside by the enzyme.

Nucleic Acids Res, 1990 Jul 11, 18(13), 3961 - 6
Evidence that covalent complex formation between BCNU-treated oligonucleotides and E . coli alkyltransferases requires the O6-alkylguanine function; Gonzaga PE et al.; Chloroethylnitrosoureas (CENUs) are thought to induce cytotoxic DNA interstrand cross-links via an initial reaction at O6-position of guanine, yielding a rearranged intermediate, O6,N1-ethanoguanine . Repair of these adducts by mammalian and bacterial DNA alkyltransferases blocks the formation of cross-links . Human alkyltransferase can form a covalent complex with DNA containing BCNU-induced cross-link precursors, but the nature of the DNA-protein linkage remains unknown . Using E . coli alkyltransferases expressed by the ada and ogt genes, we now demonstrate that both enzymes can form such complexes with CENU-treated DNA . We attribute this reaction to the O6-alkylguanine repair function, because an N-terminal fragment of the ada protein, which has only alkylphosphotriester repair activity, failed to form a similar complex . This result is consistent with the idea that complex formation requires an alkyltransferase reaction with a guanine adduct, such as O6,N1-ethanoguanine . It tends to exclude the possibility that such reactions simply involve alkylation of the enzyme by reactive DNA adducts such as chloroethylphosphate or chloroethylguanine.

Nucleic Acids Res, 1990 Jul 11, 18(13), 3941 - 52
DNA polymerase I and a protein complex bind specifically to E . coli palindromic unit highly repetitive DNA: implications for bacterial chromosome organization; Gilson E et al.; Starting from a crude E . coli extract, two activities which specifically protect highly repetitive bacterial DNA sequences (called PU for Palindromic Unit or REP for Repetitive Extragenic Palindromic sequence) against a digestion with Exonuclease III have been purified . We show that one of these activities is due to the DNA polymerase I (Pol I) . This constitutes the first indication for a specific interaction between Pol I and a duplex DNA . This interaction requires the presence of PU . It was confirmed and analyzed by native gel electrophoresis and DNase I footprinting experiments . The other activity contained at least five polypeptides . Its binding to PU DNA sequences was confirmed by native gel electrophoresis . Implications for the possible origin and functions of PU are discussed.

Biofizika, 1990 Jul-Aug, 35(4), 624 - 7
{Electrochemical potential difference for H+-ions as a regulator of redox profile of membrane during ATP-dependent ion transport in E . coli}; Bagramian KA et al.; The importance of delta mu H+ for transport of K+ via K(+)-ionophore and H(+)-K(+)-pump was studied . It was shown that the operation of the pump was decelerated by oxidant ferrycyanide, whereas sulfhydryl reagent dithiothreitol (DTT) drastically accelerated ATP driven ion exchange . Introduction of protonophore CCCP into the medium completely blocked the pump operation . However, the addition of DTT after CCCP restored the high level activity of the pump . At the same time DTT was unable to restore K+ accumulation after CCCP in aerobically grown bacteria for which the K+ uptake was performed across the electrical field gradient . Thus it was established that delta mu H+ was necessary for ATP driven ionic systems as a regulator of the membrane redox state.

EMBO J, 1990 Jul, 9(7), 2215 - 20
Topography of intermediates in transcription initiation of E.coli; Schickor P et al.; Three characteristic footprinting patterns resulted from probing the Escherichia coli RNA polymerase T7 A1 promoter complex by hydroxyl radicals in the temperature range between 4 degrees C and 37 degrees C . These were attributed to the closed complex, the intermediate complex and the open complex . In the closed complex, the RNA polymerase protects the DNA only at one side over five helical turns . In the intermediate complex, the range of the protected area is extended further downstream by two helical turns . This region of the DNA helix is fully protected, indicating that the RNA polymerase wraps around the DNA between base positions -13 and +20 . In the open complex, a stretch between base positions -7 and +2, which was fully protected in the intermediate complex, becomes accessible towards hydroxyl radicals but only in the codogenic strand, indicating that the DNA strands are unwound . Our data suggest that only the DNA downstream of the promoter is involved in this unwinding process.

J Virol Methods, 1990 Jul, 29(1), 105 - 13
HIV-1 envelope protein gp120 expression by secretion in E . coli: assessment of CD4 binding and use in epitope mapping; Morikawa Y et al.; A non-glycosylated form of the HIV-1 envelope protein gp120 and four truncated derivatives have been expressed as non-fused secreted products in the periplasmic space of E . coli . We show that the full length molecule, whilst folded and soluble, fails to bind to CD4 consistent with other work that suggests an essential role for carbohydrate in gp120 function . In addition, when used in conjunction with the truncated derivatives, rapid epitope mapping of anti-gp120 monoclonal antibodies is achieved using both Western-blot and ELISA formats.

EMBO J, 1990 Jul, 9(7), 2341 - 8
Strand separation required for initiation of replication at the chromosomal origin of E.coli is facilitated by a distant RNA--DNA hybrid; Skarstad K et al.; The Escherichia coli initiator protein, dnaA complexed with ATP, binds to the replication origin (oriC) and melts the duplex in the AT-rich part of oriC . Opening of the duplex requires a minimum temperature and superhelix density . A low level of HU protein (5 molecules/oriC) is stimulatory, probably by facilitating DNA bending . High levels of HU protein (136 molecules/oriC) restrain the free superhelicity . Under adverse conditions for oriC melting (i.e . absence of HU, high levels of HU, low temperature or low superhelicity), transcription can activate initiation of replication by generating an R-loop located even a large distance from oriC . The strand-separation stage at oriC prior to the entry of the dnaB helicase was specifically stimulated . Activation by the R-loop was blocked when a stretch of 14 GC base pairs was inserted between the loop and oriC . Thus, activation of oriC by the distantly located R-loop appears to require propagation of DNA melting through the intervening sequence.

Nucleic Acids Res, 1990 Jun 25, 18(12), 3597 - 603
E . coli promoter spacer regions contain nonrandom sequences which correlate to spacer length; Beutel BA et al.; The -10 and -35 regions of E . coli promoter sequences are separated by a spacer region which has a consensus length of 17 base-pairs . This region is thought to contribute to promoter function by correctly positioning the two conserved regions . We have performed a statistical evaluation of 224 spacer sequences and found that spacers which deviate from the 17 base-pair consensus length have nonrandom sequences in their upstream ends . Spacer regions which are shorter than 17 base-pairs in length have a significantly higher than expected frequency of purine-purine and pyrimidine-pyrimidine homo-dinucleotides at the six upstream positions . Spacer regions which are longer than 17 base-pairs in length have a significantly higher than expected frequency of purine-pyrimidine and pyrimidine-purine hetero-dinucleotides at these positions . This suggests that the nature of the purine-pyrimidine sequence at the upstream end of spacer regions affect promoter function in a manner which is related to the spacer length . We examine the spacer sequences as a function of spacer length and discuss some possible explanations for the observed relationship between sequence and length.

FEBS Lett, 1990 Jun 18, 266(1-2), 1 - 3
In vivo processing of N-terminal methionine in E . coli; Dalboge H et al.; The processing of amino-terminal methionine from cytosolic proteins in E . coli has been investigated in vivo, using amino-terminal-extended human growth hormone (hGH) as a model system . Twenty different hGH-genes with the sequence Met-Xxx-Glu-Glu-hGH where Xxx denotes each of the 20 different amino acids, were constructed and expressed in E . coli . Following purification of the products, the N-terminal amino acid sequences (10 cycles) were determined . The results demonstrate that the removal of methionine is dependent on the amino acid adjacent to methionine, and that the processing is strongly correlated to the radius of gyration of this amino acid . In addition, measurement of the hGH expression level from the 20 clones demonstrated that the small difference in the amino acid extension leads to a change in the specific hGH expression rate.

Bioorg Khim, 1990 Jun, 16(6), 765 - 79
{Highly selective labeling of the E . coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity}; Tsarev IG et al.; A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E . coli RNA polymerase . Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling . The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation . Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.

Jpn J Exp Med, 1990 Jun, 60(3), 97 - 103
Synthesis of hepatitis B virus core antigen polypeptide in E . coli using pKK223-3 plasmid, a vector for expression, with tac promoter; Shirai M et al.; A hybrid plasmid was constructed by insertion of the HBc gene encoding HBcAg into the pKK223-3 plasmid at the SmaI cleavage site in the correct direction just downstream from the tac promoter and upstream from the rrnB terminator . The recombinant plasmid carrying the HBc gene was introduced into E . coli and cloned . HBcAg was synthesized in E . coli by using the expression plasmid under the regulation of the tac promoter and rrnB terminator . The tac promoter, derived from sequences of trp and lac UV5 promoters, has identical sequences in two domains (-35 and -10 regions) with optimal distance, and the Shine-Dalgarno sequence, which enables protein synthesis to start at the ATG of the adjacent HBc gene . The nucleotide sequence of the HBc gene and its predicted amino acid sequence were almost identical to those previously reported . Purified HBcAg has a molecular weight of 21,500 . This polypeptide gave a positive reaction with anti-HBcAg and anti-HBe antibodies, and was assembled into spherical particles 37 nm in diameter . The recombinant plasmid, carrying the HBc gene between the tac promoter (trp-lac hybrid promoter) and the rrnB terminator in expression plasmid pKK223-3, was useful for efficient expression of the HBc gene and production of HBcAg particles in E . coli.

Biochimie, 1990 Jun-Jul, 72(6-7), 385 - 95
A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli; Bolla JM et al.; Inducible hybrid genes encoding two large domains, a periplasmic domain consisting of the PhoS sequence and an outer membrane domain corresponding to various lengths of the OmpF mature sequence were constructed . The synthesized hybrid polypeptides are correctly processed during the early times of induction, their precursor forms being accumulated at later times . These hybrids restore sensitivity toward colicin A to ompF E coli B strain which suggests an outer membrane location . At least 2 of them are indeed localized in the outer membrane after immunogold labelling on ultrathin cryosections . Insertion of a hydrophobic sequence between PhoS and OmpF improves the trimerization and the assembly of the OmpF part . Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimeric conformation which is recognized by specific antibodies . Moreover, the deletion of the C-terminal region impairs the functional insertion of the OmpF domain; this indicates that the last membrane-spanning region of OmpF is necessary for the correct folding and orientation of the protein in the outer membrane.

J Bioenerg Biomembr, 1990 Jun, 22(3), 311 - 36
SecA protein: autoregulated initiator of secretory precursor protein translocation across the E . coli plasma membrane; Oliver DB et al.; Several classes of secA mutants have been isolated which reveal the essential role of this gene product for E . coli cell envelope protein secretion . SecA-dependent, in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process . Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states . These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis . The protein secretion capability of the cell has been shown to translationally regulate secA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.

Biochimie, 1990 Jun-Jul, 72(6-7), 397 - 402
Progress in the identification of interaction sites on the periplasmic maltose binding protein from E coli; Martineau P et al.; The periplasmic maltose binding protein (MBP) is required for the high affinity transport of maltose and maltodextrins and for chemotaxis towards these sugars . In these functions, MBP interacts with proteins of the cytoplasmic membrane: MalF and MalG for transport, Tar for chemotaxis . A large number of MBP mutations have been isolated by us and other laboratories . We grouped these mutations into classes depending on the interactions affected and we represented the corresponding residues on the 3-D model for MBP so as to further identify the sites of MBP interacting with the MalF-MalG complex and with the Tar protein . MBP (like the other binding proteins) is composed of 2 lobes enclosing a cleft where the substrate binds . The face of the protein opposite the cleft seems to interact neither with MalF-MalG nor with Tar . The other face, corresponding to the cleft, contains sites for interactions with MalF-MalG and Tar . These sites appear to cover both sides of the cleft and may overlap in part . The present definition of the interaction sites suggests further that MBP has different in vivo orientations when it interacts with MalF-MalG or with Tar . This work constitutes an additional step in combining the use of genetic and structural analysis to define the interaction sites on MBP . Because of the structural similarities between periplasmic binding proteins, the regions of interaction defined could be relevant for other members of this family.

Cell, 1990 Jun 1, 61(5), 833 - 42
PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E . coli; Bieker KL et al.; Three strategies for genetic analysis show that two inner membrane components of the export machinery, PrlA (SecY) and PrlG (SecE), interact directly while catalyzing the translocation of secreted proteins across the cytoplasmic membrane of E . coli . The first, suppressor-directed inactivation (SDI), exploits the specific interaction between dominant prl suppressors of signal sequence mutations and mutant LacZ hybrid proteins . The second, Sec titration, extends SDI to allow the identification of various Sec proteins that are present in the translocation complex . The third uses the synthetic lethality of certain double-mutant strains to infer physical interactions between gene products . Biochemical data obtained with SDI strains allow the identification of two different secretory intermediates and indicate that PrlG functions before PrlA in the secretion pathway.

Biochimie, 1990 Jun-Jul, 72(6-7), 485 - 94
The relation between catalytic activity and gene regulation in the case of E coli threonyl-tRNA synthetase; Romby P et al.; The expression of the gene for threonyl-tRNA synthetase (thrS) has previously been shown as being negatively autoregulated at the translational level . The region of the thrS leader mRNA responsible for that control is located immediately upstream of the ribosomal binding site, and was proposed to fold in a tRNA(Thr) anticodon arm-like structure . The present paper reviews experiments using enzymatic and chemical probes that prove the existence of a tRNA(Thr) anticodon-like structure in the thrS mRNA . These structural studies have also shown the presence of another arm upstream in the leader mRNA that has striking similarities with the acceptor arm of the tRNA(Thr) isoacceptors . This second arm was shown, by mutational analysis, to also be involved in thrS regulation . Footprinting experiments have shown that both the anticodon-like and the acceptor-like arms interact with the synthetase . Finally, the similarity of the interaction of the synthetase with its 2 RNA ligands (mRNA and tRNA) has been investigated by selecting and studying mutants of the synthetase itself . The observed correlation between regulatory and aminoacylation defects in these mutants strongly suggests that the synthetase recognizes similar regions of its 2 RNA ligands in an analogous manner.

Mol Endocrinol, 1990 Jun, 4(6), 869 - 79
Molecular characterization of recombinant human acidic fibroblast growth factor produced in E . coli: comparative studies with human basic fibroblast growth factor; Watanabe T et al.; Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E . coli under the control of the T7 promoter . The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns . The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin . This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF) . Circular dichroism spectra of haFGF was not affected by the presence of heparin . The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF . These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule . The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column . In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.

Biochim Biophys Acta, 1990 May 8, 1038(3), 338 - 45
Conservation of functional residues between yeast and E . coli inorganic pyrophosphatases; Lahti R et al.; The alignments of the amino acid sequences of inorganic pyrophosphatase (PPase) from Saccharomyces cerevisiae (Y1-PPase, 286 amino acids) and Escherichia coli (E-PPase, 175 amino acids) are examined in the light of crystallographic and chemical modification results placing specific amino acid residues at the active site of the yeast enzyme . The major results are: (1) the full E-PPase sequence aligns within residues 28-225 of Y1-PPase, raising the possibility that the N-terminal and C-terminal portions of Y1-PPase may not be essential for activity, and (2) that whereas the overall identity between the two sequences is only modest (22-27% depending on the choice of alignment parameters), of some 17 putative active site residues, 14-16 are identical between Y-PPase and E-PPase . PPase thus appears to be an example of enzymes from widely divergent species that conserve common functional elements within the context of substantial overall sequence variation.

FEBS Lett, 1990 May 7, 264(1), 81 - 3
Expression in E . coli of the catalytic domain of rat poly(ADP-ribose)polymerase; Thibodeau J et al.; A 2 kilobase pair cDNA coding for the entire C-terminal catalytic domain of rat poly(ADP-ribose)polymerase has been expressed in E . coli . The overproduced 55 kDa polypeptide is active in synthesizing poly(ADP-ribose) and the 4 kDa N-terminal region of this domain is recognized by the monoclonal antibody C I,2 directed against the calf enzyme . Also, the minor alpha-chymotrypsin cleavage site found in the human catalytic domain is not present in the rat enzyme as revealed by the absence of the 40 kDa specific degradation product in the E . coli cells expressing the rat domain . The expression of this partial rat cDNA should thus permit the rapid purification and subsequent crystallization of the catalytic domain of the enzyme.

Vopr Virusol, 1990 May-Jun, 35(3), 206 - 9
{The properties of HIV protease synthesized in E . coli cells}; Shulenin SV et al.; A hybrid plasmid pPR6 was constructed containing BgII-EcoRI fragment of the pol region of HIV (strain IIIB) genome which determined the synthesis of virus-specific protease . Extracts of E . coli DN5/pPR6 bacteria provided for specific hydrolysis of hybrid protein p165 (the N-terminus of which is presented by complete beta-galactosidase and the C-terminus by duplicated area of virus-specific precursor p55 containing a site for virus-specific protease located at the border of proteins p17 and p24) with formation of products having molecular weights of 19, 42, 28, 23, and 19 kD . Polypeptides 119K, 23K, and 19K are products of complete hydrolysis, and 42K a result of partial cleavage . The kinetics of hydrolysis in relation to pH values of the reaction mixture was analysed . It is suggested that the reported system of HIV protease activity determination be used for screening of potential inhibitors of this enzyme.

Biofizika, 1990 May-Jun, 35(3), 440 - 3
{Relation between H2 production and energy-dependent metabolism of H+ and K+ ions in E.coli}; Bagramian KA et al.; Anaerobically grown E . coli escape H2 into the medium during the operation of H(+)-K(+)-pump exchanging 2H+ from a cell for one K+ of the medium . Anaerobic cells grown in the nitrate medium as well as the aerobically grown bacteria possessed neither 2H+/K+ exchange system, nor the ability for H2 production . Introduction of N,N'-dicyclohexylcarbodiimide into the medium, the removal of external K+ or the decrease of external osmotic pressure blocked both the functioning of H(+)-K(+)-pump and H2 production . The substitution of glucose by lactate reduced the activity of bacteria without change in pump operation and H2 production . It is assumed that formate-hydrogen lease and H(+)-K(+)-pump are working in collaboration.

Cell Tissue Res, 1990 May, 260(2), 387 - 94
Autoradiographic demonstration of specific binding sites for E . coli enterotoxin in various epithelia of the North American opossum; Krause WJ et al.; In the North American opossum, heat-stable specific binding sites for E . coli enterotoxin are observed (i) in epithelial cells lining the small intestine, colon, gall bladder, cystic duct, common bile duct and trachea, and (ii) in epithelial cells forming the duodenal (Brunner's) glands, liver, kidneys (metanephros, mesonephros) and testis, as demonstrated by autoradiography . Enterotoxin-specific binding sites in the intestinal tract are only found in intestinal epithelial cells with the highest concentration in the microvillus border . Enterotoxin-specific binding sites also occur in epithelial cells comprising the secretory tubules and ducts of the duodenal glands . In the kidneys (metanephros and mesonephros), enterotoxin-specific binding sites are confirmed primarily to the proximal tubules, whereas in the testis they are localized in seminiferous tubules . In the liver, enterotoxin-specific binding sites are confined primarily to hepatocytes . E . coli enterotoxin caused a 7-fold increase of cGMP in the liver and a 30-fold increase in the duodenal glands . The liver responded in about half of the animals studied, whereas the duodenal glands gave a consistent response in each case . Likewise, the duodenal glands consistently showed strong labelling for 125I-enterotoxin, whereas receptor labelling of hepatocytes was inconsistent in nearly half the incubations and corresponds to the observed cGMP measurements.

Int J Radiat Biol, 1990 May, 57(5), 993 - 1005
Lambda-prophage induction in repair-deficient and wild type E . coli strains by gamma-rays and heavy ions; Bonev MN et al.; Lambda-prophage induction in repair-deficient and wild-type E . coli strains by heavy ions and gamma-rays has been investigated . The dose dependence of the fraction of induced cells has been measured and its initial slope (lambda-induction potency) determined . The induction by gamma-rays was found to be more efficient in a polA-repair-deficient strain; the value of lambda-induction potency is zero in lexA- and recA- strains . The lambda-induction potency increased with LET for wild-type cells but remained constant in the case of polA- mutant cells . It is suggested that the DNA damage triggering the lambda-prophage induction in the case of ionizing radiation could be a type of DNA single-strand break with complex structure which cannot be repaired by fast repair processes, and which requires a substantial level of energy deposition for induction in a DNA molecule.

EMBO J, 1990 May, 9(5), 1383 - 9
The optional E . coli prr locus encodes a latent form of phage T4-induced anticodon nuclease; Levitz R et al.; The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase . Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys . We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA . The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents . They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme . The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E . coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.

Biochem Biophys Res Commun, 1990 Apr 30, 168(2), 756 - 62
Chemical nuclease activity of 5-phenyl-1,10-phenanthroline-copper ion detects intermediates in transcription initiation by E . Coli RNA polymerase; Thederahn T et al.; The nuclease activity of the copper complex of 5-phenyl-1,10-phenanthroline (5-phi-OP-Cu) detects conformational changes in the lac UV-5 promoter caused by E . Coli RNA polymerase . The template strand in melted regions of initiation complexes upstream of the site of nucleotide triphosphate incorporation is very reactive . In open and elongation complexes, downstream scission sites (e.g . +4 and +5 for the open complex) on both strands are observed . The patterns of both downstream cutting sites suggest an atypical double helix at the leading edge of the transcription bubble.

Nature, 1990 Apr 26, 344(6269), 882 - 4
Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E . coli; Phillips GJ et al.; The use of lacZ gene fusions, producing a hybrid protein containing an amino terminus specified by a target gene fused to the functional carboxy terminus of beta-galactosidase, has facilitated the study of protein targeting in various organisms . One of the best characterized fusions in Escherichia coli is phi(lamB-lacZ)42-1(Hyb), which produces a hybrid protein with the signal sequence and 181 N-terminal amino acids of the exported protein LamB, attached to LacZ . In common with other LacZ hybrids, the LamB-LacZ(42-1) protein is poorly exported from E . coli, conferring a Lac+ phenotype . beta-Galactosidase activity decreases markedly when cells producing the LamB-LacZ protein are grown at 42 degrees C or when a heat-shock response is induced at lower temperatures by overproducing heat-shock factor RpoH3, indicating the LacZ hybrids are being efficiently targeted to the cell envelope . We now report that the heat-shock proteins DnaK and GroEL can, in sufficient amounts, decrease beta-galactosidase activity and facilitate the export of lacZ-hybrid proteins.

Nucleic Acids Res, 1990 Apr 11, 18(7), 1847 - 52
Application of a new method of pattern recognition in DNA sequence analysis: a study of E . coli promoters; Alexandrov NN et al.; An algorithm from the pattern recognition theory 'generalized portrait' was used to find a distinguishing vector (scoring matrix) for E . coli promoters . We have attempted to solve three closely linked problems: (i) the selection of significant features of the signal; (ii) subsequent multiple alignment and (iii) calculation of the vector coordinates . Promoters with known strength have been successfully ranked in the correct order using this vector . We demonstrate the use of this method in predicting the location of promoters . A revised consensus promoter sequence is also presented.

Nucleic Acids Res, 1990 Apr 11, 18(7), 1711 - 8
The use of two-cistron constructions in improving the expression of a heterologous gene in E . coli; Makoff AJ et al.; Many heterologous genes when cloned into bacterial expression vectors are poorly expressed because of an inefficient ribosome binding site (RBS) . We have constructed a plasmid which expresses human gamma-interferon (gamma-IF), where the level of expression is limited by the RBS . Expression was increased by placing the gamma-IF sequence immediately downstream of a small translated sequence . The production of gamma-IF was dependent upon the efficiency of translation of this upstream cistron and could be increased to very high levels . The same upstream cistron would greatly improve the expression of gamma-IF in a plasmid where the RBS was very poor due to inhibitory secondary structure at the 5' end of its mRNA . However, it would not improve the efficiency of a poor RBS containing a weak Shine-Dalgarno sequence . The general utility of the two-cistron expression strategy to diagnose a weak RBS is discussed.

Biochim Biophys Acta, 1990 Apr 6, 1048(2-3), 178 - 86
The expression of a synthetic rainbow trout metallothionein gene in E . coli; Kille P et al.; A synthetic gene for rainbow trout metallothionein was constructed and inserted into a dual origin plasmid where expression was induced by a temperature shift in a proteinase-deficient strain of Escherichia coli . The recombinant protein was purified to homogeneity, and a partial amino acid sequence was determined to confirm its identity . Its immunochemical characteristics were similar to those of native metallothionein from rainbow trout . The amounts of recombinant metallothionein produced were quantified in soluble cell extracts by ELISA . Low concentrations were detected when growth was performed either in L-broth or defined (GMM-II) medium . Supplementation of the medium with zinc or copper had no effect on the amount of metallothionein produced . By contrast, when cadmium was included in either L-broth or GMM-II medium, much higher concentrations of the protein within the cells (approx . 13 micrograms/mg soluble cell protein) were detected . This stabilisation of the protein by metal reconstitution in vivo is considered in relation to the selective uptake/exclusion of metals by the cells and its significance for the scavenging of certain precious or toxic heavy metals is discussed.

Bratisl Lek Listy, 1990 Apr, 91(4), 292 - 7
{Killing of seroresistant plasmid bearing E . coli strains in polymorphonuclear leukocytes}; Siegfried L et al.; Killing of 61 seroresistant E . coli strains equipped with virulence plasmids (F, Ent, Hly, Col) and resistance plasmids (R) in polymorphonuclear leukocytes (PMNL) of healthy donors was determined by the cultivation plate and fluorescence methods of phagocytosis . Killing of the control E . coli strain K12 which does not possess any plasmids was studied by the fluorescent method . Compared to the fluorescent method, the cultivation plate method yielded a significantly higher killing rate of the bacteria after opsonization by 5% (p less than 0.05) and concentrated serum (p less than 0.001) . Compared to the control E . coli strain K12, the killing rate of seroresistant E . coli strains in PMNL of healthy donors was significantly lower (p less than 0.001).

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 711 - 5
SecY protein, a membrane-embedded secretion factor of E . coli, is cleaved by the ompT protease in vitro; Akiyama Y et al.; SecY is an integral membrane protein, spanning the cytoplasmic membrane of E . coli probably 10 times and required for efficient translocation of other proteins across the membrane . We report here that this protein can be specifically cleaved at the central region of the polypeptide after cell disruption, and cytoplasmic membrane preparations often contain a degradation product of SecY . This cleavage was ascribed to the action of the outer membrane-associated protease specified by the ompT gene, since the cleavage was not observed in ompT-defective mutants . Thus, we propose that an ompT mutant should be used for in vitro analysis of protein translocation and the SecY protein . We mutated the ompT gene by insertion of a kanamycin resistance determinant to facilitate strain construction by P1 transduction.

FEBS Lett, 1990 Mar 12, 262(1), 39 - 41
Construction of expression plasmids for the fusion protein of Sendai virus, and their expression in E . coli cells and eucaryotic cells; Taira H et al.; To examine the properties and the role of the fusion protein (F) of Sendai virus at the molecular level, a plasmid, pUC-F, was constructed by inserting cDNA for the F protein into a pUC vector . Upon induction of E . coli cells transformed with pUC-F, a new protein was obtained, which was identified as Fo on Western blot analysis . The cDNA fragment for the F gene was excised from pUC-F and inserted into an eucaryotic expression vector, pSVL, to yield pSVL-F . COS-1 cells transfected with pSVL-F gave a band on SDS-gel electrophoresis which corresponded to the size of the Fo proteins.

Nucleic Acids Res, 1990 Mar 11, 18(5), 1137 - 43
Transcription in vivo directed by consensus sequences of E.coli promoters: their context heavily affects efficiencies and start sites; Jacquet MA et al.; We studied in vivo transcription and gene expression directed by a series of synthetic sequences, bearing the consensus hexamer (CH) pair of E.coli promoters in various contexts . The results demonstrate that, for the contexts tested, the CH pair supports transcription activity and gene expression, whether the spacer linking them is AT or GC rich, or is as short as 14 bp or as large as 26 bp (standard size 17 bp) . However, we find that the context influences transcription efficiency by as much as an order of magnitude, and is able to scatter transcription start sites over a region of as much as 30 bp, including start sites within a CH or even between the two sequences of the CH pair . The results demonstrate that, although the CH pair can be sufficient for directing transcription by E.coli RNAP, important determinants for promoter activity are at least in part contained in the context of the consensus sequences; they advocate a synergic interplay of signals borne by the CH pair and its context, extending over all parts of the promoter sequence . A two-step model is proposed, in which properly located consensus sequences provide RNAP with facilities required for stereospecific docking along the promoter sequence; the result would be a sharp change in the local environment of the double helix inducing local isothermal unwinding . The size of the loop (related to the AT constraint in the promoter) and the extend of the environmental change required for unwinding would determine the rate of transcriptionally competent complex formation, positioning and grouping of start sites.

Nature, 1990 Mar 8, 344(6262), 173 - 5
New data on excisions of Mu from E . coli MCS2 cast doubt on directed mutation hypothesis; Mittler JE et al.; According to the directed mutation hypothesis, certain mutations in bacteria occur more frequently in environments in which the resulting phenotype is selectively favoured than in non-selective environments . This hypothesis therefore challenges the fundamental tenet that mutations occur spontaneously, irrespective of effects on the organism's fitness . One purported case of directed mutation is the excision of a Mu sequence from Escherichia coli strain MCS2 in minimal lactose-arabinose medium . Here, we show that this case can be more simply explained by an accelerated rate of excision mutation in response to non-specific physiological stresses of starvation and by slight growth of MCS2 on minimal lactose-arabinose medium.

Mutat Res, 1990 Mar, 243(3), 179 - 86
DNA-replication recovery inhibition and subsequent reinitiation in UV-radiation-damaged E . coli: a strategy for survival; Doudney CO; Using the incorporation of {14C}thymine to measure DNA accumulation, it was shown that exposure of the B/r strain of Escherichia coli to 10 J/m2 of ultraviolet radiation (UV) inhibits replication for about 20 min, but then resumption of replication occurs . Pulse-labelling with {3H}thymidine after exposure of the WT strain to this fluence confirmed the transient inhibition and recovery of DNA replication . After recovery, the rate of accumulation of DNA in the culture increases, to exceed that of the exponentially growing culture, so that eventually the amount of DNA almost equals that of the unirradiated culture . After a higher fluence (20 J/m2), an inhibition of replication recovery was revealed . This fluence delays the reinitiation of DNA accumulation in the culture, measured by {14C}thymine incorporation, for 25 min more, in addition to the 20-min recovery period . This finding was confirmed with pulse-labelling studies, which revealed that the higher exposure represses the rates of replication for 45 min before replication at the normal rate reinitiates in the culture . It was proposed that the inhibition of recovery revealed by these investigations is effected by the UV-induction of an active DNA-replication recovery-inhibition process . With the uvrA strain, rate studies revealed that 1.5 J/m2 of UV (a reduced fluence necessary because of the greater sensitivity of the strain) induces a transient inhibition of DNA replication, with considerable recovery following . Exposure to 3.0 J/m2 induces the transient inhibition of replication, followed by massive recovery inhibition after 20 min of incubation . With uvrA recA, both the lower and the higher fluence resulted in an immediate block of replication with no recovery, confirming the recA gene dependency of the recovery process . The decrease in rate of replication comparable to that seen in the uvrA strain after 20 min, and taken as evidence of the function of the recovery-inhibition process, was not seen . The evidence supports the concept that a process somehow triggered by higher UV fluences functions to repress replication temporarily, presumably allowing time for repair processes to take place before replication overruns closely linked pyrimidine dimers on opposite strands to create lethal lesions.

New Biol, 1990 Mar, 2(3), 265 - 71
Bending of the origin of replication of E . coli by binding of IHF at a specific site; Polaczek P; In studies of DNA replication in Escherichia coli, an important question concerns the role of the initiator protein DnaA . This protein is known to bind to a specific 9-bp sequence in the origin of replication, but it is not understood how it can recognize another, relatively distant, 13-bp sequence that has no homology to the binding site but is where the DnaA protein serves its catalytic function in the initiation of DNA replication . This effect of DnaA might be achieved by bending of DNA in this region . I have searched for putative binding sites for integration host factor (IHF), a protein known to bend DNA . Here I report the finding of an IHF binding site in the E . coli origin and present direct evidence that IHF binds and causes DNA bending in this region . On the basis of these results I propose a model wherein formation of a higher-order nucleoprotein structure would facilitate the action of DnaA protein in the initiation events.

Radiobiologiia, 1990 Mar-Apr, 30(2), 190 - 3
{The modifying effect of glycerine and cysteamine on prophage lambda induction in E . coli cells under gamma irradiation}; Bartsevich VV et al.; In studying the modifying influence of glycerol and cysteamine of gamma radiation induction of lambda prophage it was shown that dose curves for lambda prophage induced upon irradiation in normal conditions and in the presence of glycerol are those with a maximum . In the presence of cysteamine, the prophage induction is inhibited significantly.

Rev Sanid Hig Publica (Madr), 1990 Mar-Apr, 64(3-4), 203 - 9
{Genotoxic study of commercial dyes with tartrazine base in S . typhimurium his- and E . coli trp-}; Pollastrini MT et al.; We study the genotoxic character of 21 food dyes samples with tartrazine in different trade-marks . Two homologated assays "in vitro" of reverse mutation in S . typhimurium and E . coli are used, with metabolic activation . There is no evidence of mutagenic character in any sample.

EMBO J, 1990 Mar, 9(3), 727 - 34
The role of FIS in trans activation of stable RNA operons of E . coli; Nilsson L et al.; The thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence . A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS . It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter . All three protein-DNA complexes contain one and the same protein . Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range . The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator . We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes . A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium.

FEBS Lett, 1990 Feb 26, 261(2), 389 - 91
Energy transfer in complexes of E . coli single-stranded DNA-binding protein with single-stranded poly-(2-thiouridylic acid); Tsao DH et al.; The complexes of point-mutated Escherichia coli single-stranded DNA-binding protein (Eco SSB) with poly-(2-thiouridylic acid) (poly S2U) have been studied by optical detection of magnetic resonance spectroscopy (ODMR) . Previous work has determined that two of four tryptophan (Trp) residues in Eco SSB undergo stacking interactions with nucleic acid bases . Selective photoexcitation of S2U bases was performed and subsequent triplet----triplet energy transfer from S2U to nearby Trp residues in the protein took place . The zero-field splitting (ZFS) parameters and sublevel kinetics were determined for each Trp residue sensitized by S2U . The sublevel lifetimes of the two sensitized residues are similar to those of normal Trp . The ZFS parameters, on the other hand, show a dramatic reduction relative to those of the uncomplexed protein, implying a more polarizable environment for the sensitized Trp residues and/or charge transfer interactions with the S2U bases.

Experientia, 1990 Feb 15, 46(2), 167 - 73
Structural and functional properties of porin channels in E . coli outer membranes; Rosenbusch JP; Porin is a channel-forming, voltage-dependent protein of E . coli outer membranes . It exhibits relatively unspecific molecular sieve properties (exclusion size 600 Da) . The trimer (110 kDa) consists of three identical polypeptides . Its secondary structure is mostly beta-structure, part of which can be visualized by electron microscopy to form a single beta-pleated sheet near the protein-lipid interface of the trimer . This folding pattern is significantly different from those of the reaction centers and of bacteriorhodopsin . Moreover, it contains many polar and ionizable side chains . It is argued that local as well as global electroneutrality, and complete saturation of the entire hydrogen bonding potential not only allow the protein to reside in the hydrophobic membrane core, but also confer upon it its unusual stability.

Nucleic Acids Res, 1990 Feb 11, 18(3), 547 - 52
Reiterative copying by E.coli RNA polymerase during transcription initiation of mutant pBR322 tet promoters; Harley CB et al.; The major in vitro transcripts from the tet promoter of pBR322 derivatives pTA22 and pTA33 have heterogeneous 5' ends consisting of variable lengths of oligo(A) . Their structure is 5'pppAnU..., where n ranges from 1 to greater than 12, but the template strand can encode at most four A residues at the site of transcription initiation . The abundance of additional A residues at the 5' end of the pTA22 and pTA33 tet transcripts could be reduced by elevating the concentration of UTP, but even at high concentrations (greater than 1 mM) non-cognate A residues were still observed . Aberrant initiation was not artifactual since the major and minor transcripts of the pBR322 tet promoter region, and other transcripts arising from minor promoters on pTA22 or pTA33 DNA all had unique 5' termini . Mixing experiments showed that RNA polymerase did not utilize pppA2-4-OH produced by abortive initiation as primers . The data suggest that the initial nascent RNA chain 'slips' in the 5' direction during elongation opposite T4 on the template strand causing RNA polymerase to reiteratively add A residues to the 5' end of the transcript . The generality and possible significance of this mechanism is discussed.

Nucleic Acids Res, 1990 Feb 11, 18(3), 487 - 91
Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu from E.coli; Forster C et al.; Selenocysteine-incorporating tRNA(Sec)(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine . The equilibrium dissociation constant for the interaction of Ser-tRNA(Sec)(UCA) with elongation factor Tu.GTP was determined to be 5.0 +/- 2.5 x 10(-8) M . Compared with the dissociation constants of the two elongator Ser-tRNA(Ser) species (Kd = 7 x 10(-10) M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu.GTP . This suggests a mechanism by which the Ser-tRNA(Sec) is prevented in recognition of UGA codons . This tRNA is not bound to EF-Tu.GTP and is converted to selenocysteinyl-tRNA(Sec) . We also demonstrate the lack of an efficient interaction of Sec-tRNA(Sec)(UCA) with EF-Tu.GTP . The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu.GTP.

Cell, 1990 Feb 9, 60(3), 365 - 74
Chromosome replication does not trigger cell division in E . coli; Bernander R et al.; An essential part of the chromosome replication origin of E . coli K-12 and B/r was replaced by the plasmid pOU71 . The average initiation mass of replication for pOU71 decreases with increasing temperature . The constructed strains were grown exponentially at different temperatures, and cell sizes and DNA content were measured by flow cytometry . The average DNA content increased with increasing temperature, but the cell size distribution was largely unaffected . Furthermore, cells in which DNA replication had not yet initiated (cells in the B period) became less abundant with increasing temperature . The increased DNA content could not be explained by an increase in the length of the C period . It is concluded that chromosome replication does not trigger cell division in E . coli, but that the chromosome replication and cell division cycles of E . coli run in parallel independently of each other.

Cell, 1990 Feb 9, 60(3), 439 - 49
The MotA protein of E . coli is a proton-conducting component of the flagellar motor; Blair DF et al.; A number of mutants of motA, a gene necessary for flagellar rotation in E . coli, were isolated and characterized . Many mutations were dominant, owing to competition between functional and nonfunctional MotA for a limited number of sites on the flagellar motor . A new class of mutant was discovered in which flagellar torque is normal at low speeds but reduced at high speeds . Hydrogen isotope effects on these mutants indicate that MotA catalyzes proton transfer . We confirmed an earlier observation that overproduction of MotA leads to accumulation of the protein in the cytoplasmic membrane and to significant decreases in growth rate . When nonfunctional mutant variants of MotA were overproduced instead, they accumulated in the cytoplasmic membrane, but growth was not impaired . These results also suggest that MotA conducts protons . This was confirmed by measuring the proton permeabilities of vesicles containing wild-type or mutant MotA proteins.

Nature, 1990 Feb 8, 343(6258), 575 - 6
Active-site mutants altering the cooperativity of E . coli phosphofructokinase; Berger SA et al.; Crystal structures of the high- and low-activity states of the allosteric enzyme phosphofructokinase implicate three arginines in substrate binding, catalysis and cooperativity . Arginines 162 and 243 reach into the active site from an adjacent subunit and interact with the cooperative substrate fructose 6-phosphate . Mutation of these arginines to serine results in mutant enzymes with reduced substrate binding and lowered cooperativity, but with little change in their catalytic ability (kcat) . Arg 72 bridges the two substrates fructose 6-phosphate and ATP, and interacts with the 1-phosphate of the product fructose 1,6-biphosphate . Mutation of this residue to serine reduces the catalytic activity, cooperativity and binding of fructose 6-phosphate and fructose 1,6-bisphosphate . In the reverse reaction, the kinetics of wild-type and the Ser 72 mutant with respect to fructose 1,6-bisphosphate are hyperbolic, whereas those of the Ser 162 and Ser 243 mutants are sigmoidal . These results show that each of the three arginines contributes to cooperativity and to the transmission of allosteric signals between the four subunit of the enzyme.

Mutat Res, 1990 Feb, 228(2), 177 - 85
Genotoxic potency of monofunctional alkylating agents in E . coli: comparison with carcinogenic potency in rodents; Quinto I et al.; A quantitative correlation between carcinogenicity and genotoxicity was investigated by a comparison between the carcinogenic potency in rodents and the mutagenic (M), recombinogenic (R) and SOS-inducing (I) potencies in a bacterial test (E . coli multitest) for 9 monofunctional alkylating agents: N-nitroso-N-methylurethane, N-nitroso-N-ethylurea, epichlorohydrin, N-nitroso-N-methylurea, N-nitroso-N-methyl-N'-nitroguanidine, methyl methanesulfonate, diethylsulfate, dimethylsulfate, ethyl methanesulfonate . A significant positive correlation between the carcinogenic potency and the product of the mutagenic and recombinogenic potencies was found for all tested compounds . Thus, the E . coli multitest may be used as a simple test to search for correlations between carcinogenicity and genotoxicity of DNA-damaging agents.

J Bacteriol, 1990 Feb, 172(2), 1085 - 91
Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E . coli; Kroncke KD et al.; The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I . S . Roberts, R . Mountford, R . Hodge, K . B . Jann, and G . J . Boulnois, J . Bacteriol . 170:1305-1310, 1988) . In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide . Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants . Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm . With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria . Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide . The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid . The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid . These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.

Tierarztl Prax, 1990 Feb, 18(1), 65 - 8
{Clinical experience with kennel-specific E . coli oral vaccines in dogs}; Baljer G et al.; Kennel-specific oral E . coli vaccines were tested for efficacy and side effects at breeding and boarding kennels with severe diarrhoea problems . Oral vaccines contained heat inactivated E . coli bacteria from specific kennels and were given once daily for 14 days . Oral vaccines were administered directly orally to puppies while for adult dogs vaccines were mixed with daily food rations . In breeding kennels with 4 to 10 week old puppies suffering from non-fatal diarrhoea, the oral immunization led to a decrease in morbidity from 86.5% to 0% . In kennels with some cases of fatal diarrhoea the rate of morbidity decreased from 45% to 21% and the mortality rate from 25% to 10.3% after using the vaccination . By vaccinating adult dogs in boarding kennels the morbidity rate dropped from 83.5% to 6.5% and the mortality rate from 4.1% to 0.5% . The kennel specific oral E . coli vaccine was found to be free of side effects . No adverse effects were observed in either puppies or adult dogs.

Biochimie, 1990 Feb-Mar, 72(2-3), 183 - 9
Creation of targets for proteolytic cleavage in the LamB protein of E coli K12 by genetic insertion of foreign sequences: implications for topological studies; Ronco J et al.; LamB, an integral outer membrane protein of E coli K12, is highly resistant to protease digestion . We had previously genetically inserted a foreign sequence corresponding to an epitope from the poliovirus next to amino acids 146, 153, 189, and 374 of LamB . In 3 cases (sites 146, 153, 374), insertion of the foreign peptide did not extensively affect the functions of LamB (and therefore folding) . In 2 cases (sites 146 and 374) the polio virus epitope was detectable on the bacterial surface with a specific monoclonal antibody . We show here that the 4 modified proteins are sensitive to trypsin, including on intact cells . The sizes of the major cleavage products is that expected for proteolysis at or near the sequences inserted . In 1 case (site 153), this was directly demonstrated by protein sequencing . The results confirm the cell surface exposure of the regions of residues 153 and 374 and provide information on the regions around residues 146 and 189 . Perspectives and limitations of this approach for fine studies on the mode of insertion of membrane proteins are briefly discussed.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1990 Feb, 12(1), 25 - 30
{Enhancing effect of chuangxinmycin on synthesis of enzymes in tryptophan synthesis pathway of chuangxinmycin-resistant mutant strain of E . coli}; Xu S et al.; Chuangxinmycin (CXM) and L-trp cause repression of trp-operon in wild strain of E . coli and indolepropanic acid (IPA) causes derepression of this strain . In CXM-resistant mutant strain, CXM and IPA both cause derepression, and interaction of CXM and IPA is competitive . However, L-trp results in neither repression nor derepression in the mutant, but it can counteract the activity of CXM or IPA . These results suggest that there are repression and derepression sites on repressor of E . coli . CXM can bind to both sites, but mainly to repression site . The structure of repression site of the CXM-resistant mutant is altered, so that CXM can not bind to the repression site and binds mainly to the derepression site, thus leading to the derepression of trp-operon.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1990 Feb, 12(1), 1 - 8
{A study of protein engineering for human cardionatrin . I . Synthesis, cloning and expression of a gene analog of human atrial natriuretic polypeptide in E . coli}; Ren H; An analog of the alpha-human atrial natriuretic polypeptide (alpha-hANP) gene, articulated with a peptidase inhibitor SQ20881 at its N-terminal and two prolines at the C-terminal was expressed in E . coli by cloning the reconstituted plasmid pRHL-1 in vivo . This gene analog, RH-1, comprising 154 base pairs in total, was designed to contain an equivalent of the alpha-hANP gene, capping the peptidase inhibitor SQ20881 at its 5' end with a glutamic acid codon GAA to facilitate enzymatic cleavage of the expressed end product by endoproteinase Glu-C, wedging in two proline codons CCG & CCG before the double terminal codons TGA TAG at the 3' end to retard hydrolysis of the expressed product by exopeptidase, and adding 3 restriction sites to both ends . Synthesis of the RH-1 gene was effected enzymatically by joining in predicted order the ten segments of oligodeoxynucleotides which had been chemically synthesized by the solid-phase phosphite-triester method . The synthetic gene was cloned into vector M13mp18 . Phage bearing the gene analog was identified by dot blotting and restriction endonuclease mapping . Nucleotide sequence of the gene was determined by the dideoxynucleotide chain termination method.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochimie, 1990 Feb-Mar, 72(2-3), 131 - 41
Haemolysin secretion from E coli; Holland IB et al.; Haemolysin (HlyA) secretion from E coli is directed by a specific C-terminal targeting signal, located within the last 27-50 amino acids, with quite novel characteristics . The HlyA molecule is secreted directly to the medium without a periplasmic intermediate or detectable proteolytic processing . The C-terminal domain of HlyA can also be used to promote the secretion of several other E coli and mammalian proteins . HlyD and HlyB are essential for translocation of HlyA to the medium and we propose that these proteins form a transenvelope complex which initially binds the HlyA signal followed by transport of HlyA to the medium . HlyB is a member of a family of membrane proteins engaged in ATP dependent secretion mechanisms conserved in many organisms including man (P-glycoprotein and the CF protein) . In this review we discuss the structure, function and regulation of the secretion mechanism.

J Clin Microbiol, 1990 Feb, 28(2), 293 - 5
Serotypes of Escherichia coli that hybridized with DNA probes for genes encoding Shiga-like toxin I, Shiga-like toxin II, and serogroup O157 enterhemorrhagic E . coli fimbriae isolated from adults with diarrhea in Thailand; Bettelheim KA et al.; Escherichia coli strains isolated from adults with diarrhea in Bangkok, Thailand, were examined for hybridization with DNA probes for genes that code for Shiga-like toxin (SLT)-I, SLT-II, and serogroup O157 enterhemorrhagic E . coli (EHEC) fimbriae . Seven isolates that hybridized with the SLT-I, SLT-II, and O157 EHEC fimbria probes and produced verocytotoxin (VT; group A) were isolated from two patients with diarrhea . Two strains that hybridized with only the SLT-II probe and the O157 EHEC fimbria probe and were VT+ (group B) were isolated from two patients with diarrhea, 7 strains that hybridized with only the SLT-II probe and were VT+ (group C) were isolated from two patients with diarrhea, and 26 strains that hybridized with only the O157 EHEC fimbria probe and were VT- (group D) were isolated from four patients with diarrhea . Seven strains in group A had serotypes O2:H1 (n = 1), O110:H19 (n = 1), and Ont:H8 (n = 5); 2 strains in group B were O112ab:H21 (n = 1) and O113:H21 (n = 1); 7 strains in group C were O6:H28 (n = 1), O22:H16 (n = 1), O52:H25 (n = 1), O112ab:H21 (n = 1), OR:H45 (n = 2), and OR:H11 (n = 1); and 26 strains in group D were O76:H7 (n = 18), O146:H3 (n = 2), O146:H10 (n = 1), O146:Hnt (n = 1), OR:H16 (n = 1), Ont:H2 (n = 1), Ont:H8 (n = 1) and Ont:H16 (n = 1) . In Thailand, E . coli strains that hybridized with SLT-I, SLT-II, and O157 EHEC fimbria probes were of a variety of serotypes, none of which were O157:H7.

Biochimie, 1990 Feb-Mar, 72(2-3), 123 - 30
Uptake across the cell envelope and insertion into the inner membrane of ion channel-forming colicins in E coli; Baty D et al.; Pore-forming colicins exert their lethal effect on E coli through formation of a voltage-dependent channel in the inner (cytoplasmic-membrane) thus destroying the energy potential of sensitive cells . Their mode of action appears to involve 3 steps: i) binding to a specific receptor located in the outer membrane; ii) translocation across this membrane; iii) insertion into the inner membrane . Colicin A has been used as a prototype of pore-forming colicins . In this review, the 3 functional domains of colicin A respectively involved in receptor binding, translocation and pore formation, are defined . The components of sensitive cells implicated in colicin uptake and their interactions with the various colicin A domains are described . The 3-dimensional structure of the pore-forming domain of colicin A has been determined recently . This structure suggests a model of insertion into the cytoplasmic membrane which is supported by model membrane studies . The role of the membrane potential in channel functioning is also discussed.

Biochim Biophys Acta, 1990 Jan 30, 1048(1), 19 - 23
Bleomycin-treated DNA is specifically cleaved only by endonuclease IV in E . coli; Hagensee ME et al.; We have isolated an endonuclease from E . coli active on bleomycin-treated DNA . Purification on DEAE-cellulose separated this activity in strains lacking endonuclease I, endonuclease III or exonuclease III . After DEAE chromatography, the enzyme was active in the absence of divalent cations and was not inhibited by tRNA or harmane . In addition, this enzyme was stable at 45 degrees C for 20 min . These properties are consistent with this activity being endonuclease IV . This was supported by our finding no activity in a strain lacking endonuclease IV.

Nucleic Acids Res, 1990 Jan 25, 18(2), 331 - 5
Expression of the E.coli ada gene in yeast protects against the toxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine; Brozmanova J et al.; The E.coli ada gene protein coding region has been ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase gene promoter region to produce pADH06C . The yeast strains SX46A, 7799-4B and VV-6 are deficient in endogenous O6-alkylguanine-DNA-alkyltransferase and transformation of these strains with this shuttle vector resulted in the expression of 1730, 1260 and 374 fmoles ada-encoded ATase/mg protein in stationary phase yeast: transformation with the parent vector had no effect on endogenous ATase activity which remained less than 2 fm/mg . In comparison with parent vector transformed yeast, all of the pADH06C-transformed strains showed an increase in the resistance to the toxic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . In addition, 7799-4B and VV-6 were more resistant to the mutagenic effects of this agent . These results indicate that the toxic and mutagenic effects of MNNG in yeast are mediated, at least in part, by DNA lesions than can be repaired by the E.coli ada gene product.

Nucleic Acids Res, 1990 Jan 25, 18(2), 285 - 9
An anticodon change switches the identity of E . coli tRNA(mMet) from methionine to threonine; Schulman LH et al.; Recent evidence indicates that the anticodon may often play a crucial role in selection of tRNAs by aminoacyl-tRNA synthetases . In order to quantitate the contribution of the anticodon to discrimination between cognate and noncognate tRNAs by E . coli threonyl-tRNA synthetase, derivatives of the E . coli elongator methionine tRNA (tRNA(mMet)) containing wild type and threonine anticodons have been synthesized in vitro and assayed for threonine acceptor activity . Substitution of the threonine anticodon GGU for the methionine anticodon CAU increased the threonine acceptor activity of tRNA(mMet) by four orders of magnitude while reducing methionine acceptor activity by an even greater amount . These results indicate that the anticodon is the major element which determines the identity of both threonine and methionine tRNAs.

Biochem Biophys Res Commun, 1990 Jan 15, 166(1), 90 - 100
Expression of epidermal growth factor receptor sequences as E . coli fusion proteins: applications in the study of tyrosine kinase function; Koland JG et al.; To investigate the functions of key domains of the epidermal growth factor receptor (EGFR), various EGFR-derived peptide sequences were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins . The purified fusion proteins (GST-TK0-8) were tested as substrates for the tyrosine kinase activities of the EGFR and c-src . Both the GST-TK4 fusion protein, which contains the major C-terminal tyrosine autophosphorylation sites of the EGFR, and GST-TK7, which contains the connecting sequence between the EGFR kinase domain and the C-terminal autophosphorylation domain, were strongly phosphorylated by the EGFR and c-src . Hence the candidate tyrosine phosphorylation sites present in the connecting sequences of the EGFR, as well as the known autophosphorylation sites of the EGFR, can be phosphorylated by the two tyrosine kinases . The protein GST-TK7 was phosphorylated by c-src with a KM of 5-10 microM, which indicated a potential interaction between the connecting segment of the EGFR and the c-src kinase . The GST fusion proteins were also used to map the sites recognized by two anti-EGFR monoclonal antibodies and a polyclonal serum raised against an EGFR tyrosine kinase domain fragment . The recognition site of one monoclonal antibody was determined to be in a short sequence surrounding tyr1068, a primary site of autophosphorylation in the C-terminal domain of the receptor . The anti-peptide polyclonal serum recognized only sequences in the GST-TK7 fusion protein, and hence binds to the connecting sequence between the kinase core and the C-terminal domain . These antibodies will therefore be useful reagents for studying the function of two key structural elements of the EGFR tyrosine kinase . The GST-TK fusion proteins should have many other applications in the study of EGFR catalysis and mitogenic signalling.

FEMS Microbiol Lett, 1990 Jan 15, 55(1-2), 5 - 8
Dihydroxybenzoylserine--a siderophore for E . coli; Hantke K; Dihydroxybenzoylserine, the breakdown product of enterochelin, was able to stimulate growth of Escherichia coli under iron limiting conditions by acting as a siderophore . The dihydroxybenzoylserine-iron complex was taken up via the outer membrane receptor proteins Fiu, FepA and to a minor extent via Cir . Transport of Fe3(+)-dihydroxybenzoylserine across the cytoplasmic membrane was only dependent on genes from the fep region . In addition, it was shown that dihydroxybenzoate was taken up via Fiu and Cir and less efficiently by FepA.

FEMS Microbiol Lett, 1990 Jan 15, 55(1-2), 39 - 43
Characterization of adhesion zones in E . coli cells; Leduc M et al.; After plasmolysis of Escherichia coli cells, the adhesion zones were characterized using the cytochemical PTA and SP procedures which stain peptidoglycan and lipopolysaccharides (LPS) respectively . A PTA-stained layer was detected at the adhesion sites . This layer was visualized irrespective of the electron microscopy procedure used . Also, using SP staining an outer membrane in which LPS molecules were asymmetrically distributed, was observed.

Adv Biochem Eng Biotechnol, 1990, 43, 31 - 42
Impact of genetic engineering on downstream processing of proteins produced in E . coli; Enfors SO et al.; Genetic engineering can be used to give a protein properties that are advantageous for downstream processing . Many heterologous proteins are degraded at high rates by proteases . Depending on which type of proteolytic degradation is encountered the strategy may be different: induction of inclusion bodies, change of the amino acid sequence in the sensitive site of the product, or protection by fusion of the product with other proteins . The number of unit operations needed to purify a protein may be reduced by addition of other polypeptides or amino acids to the product . Affinity chromatography, immobilized metal ion affinity chromatography, and extraction in aqueous two-phase systems are unit operations which can be made more versatile by the fusion technique.

Yi Chuan Xue Bao, 1990, 17(5), 390 - 7
{Cloning and expression of E . coli glucose isomerase gene in Streptomyces lividans}; Tan HR et al.; E . coli glucose isomerase gene was cloned into Streptomyces lividans using shuttle plasmid vector pSE-3 . First plasmid pXI203 was constructed in E . coli using plasmids pXI200 (containing 1.6 kb glucose isomerase gene) and pGEM-3 digested with EcoR1 . Then, recombinant plasmid pSEX100 was also constructed in E . coli using plasmids pXI203 and pSE-3 digested with HindIII . When the pSEX100 was transformed into Streptomyces lividans protoplasts, recombinants were obtained on R5YE medium containing 50 micrograms/ml neomycin and 50 micrograms/ml thiostrepton . The results showed that the E . coli glucose isomerase gene cloned and expressed in Streptomyces lividans via the analysis of restriction enzyme digestion as well as the detection of the glucose isomerase activity.

Yi Chuan Xue Bao, 1990, 17(3), 202 - 10
{Study on antifreeze protein in fishes . II . The cloning of antifreeze protein gene cDNA of Pseudopleuronectes yokohamae and its expression in E . coli}; Jiang YQ et al.; Pseudopleuronectes yokohamae, existing in the Yellow Sea of China, contains antifreeze peptide (AFP) . This protein could depress the serum freezing point . On the basis of purification and characterization of this protein, we synthesized a segment of Oligo-nucleotides of antifreeze protein gene as a primer and hybridized with the mRNA of Pseudopleuronectes yokohamae . The cDNA of antifreeze protein gene has specifically been reversely transcribed . This segment has been cloned onto pUC19 with Ecor I linker method . After confirmation of the insert segment of the cDNA of antifreeze protein gene, the zymogram was detected and the sequence of the nucleotides was determined . This recombinated clone was transformed into E . coli JM83 and expressed well.

Adv Exp Med Biol, 1990, 264, 217 - 22
Zinc--a redox-inactive metal provides a novel approach for protection against metal-mediated free radical induced injury: study of paraquat toxicity in E . coli; Chevion M et al.; The essential mediatory role of copper and iron in a variety of free radical-induced injuries, including paraquat-induced biological damage has been recently demonstrated . It was postulated that these transition metals undergo cyclic redox reactions, and serve as centers for repeated production of hydroxyl radical, which are the ultimate deleterious agents . Additionally, we had presented evidence indicating efficient protection against paraquat toxicity by agents commonly employed (chelators, chemical scavengers and protecting enzymes) . In this study we have used the E . coli model in order to develop a new approach for protection against paraquat-induced metal-mediated cellular injury . It entails the administration of excess zinc (up to 50 fold over copper), which results in an inhibition of the toxic effect of paraquat . Lineweaver- Burk analysis demonstrates the competitive mode of this inhibition . The suggested mechanism involves the displacement of the redox-active copper (or iron) from its binding site and by this diverting the site of repeated production of free radicals . Thus, use of redox-inactive metals, which possess high similarity of their ligand chemistry, to that of iron and copper but are of relative low toxicity by themselves, should be considered for intervention in paraquat toxicity and in other metal-mediated free radical-induced injurious processes.

Arch Virol, 1990, 113(3-4), 267 - 77
Characterisation of an avian influenza virus nucleoprotein expressed in E . coli and in insect cells; Harley VR et al.; The nucleoprotein (NP) gene from influenza virus A/Shearwater/Australia/72 has been expressed intracellularly in both E . coli and insect cells . E . coli-derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP . This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive- and negative-sense influenza neuraminidase RNA transcribed in vitro . ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector . Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.

Haematologia (Budap), 1990, 23(1), 3 - 7
E . coli antibodies do not cause false-positivity in recombinant anti-HIV assays; Ujhelyi E et al.; 129 sera with known antibody titres against E . coli 026 and E . coli 055 strains were tested with the Abbot second generation anti-HTLV III recombinant screening assay . No difference in the O.D . values was found between sera with high, normal and low anti-E . coli titres . In addition, no false-positive reactions were observed with the anti-HIV negative sera containing E . coli antibodies in high titres in a Western blot assay in which recombinant env antigen was applied . These results suggest that E . coli assays in which E . coli-produced recombinant antigens are used.

Prog Clin Biol Res, 1990, 340A, 179 - 93
Mechanisms of caffeine inhibition of DNA repair in E . coli; Selby CP et al.; Caffeine inhibits excision repair and photoreactivation in E . coli in vivo . We used purified E . coli enzymes and DNase I footprinting to study the mechanism of inhibition in vitro . Photolyase binds to pyrimidine dimers in DNA in a radiation-independent process . Upon irradiation of this enzyme-substrate complex with photoreactivating light, pyrimidine dimers are reverted to their constituent pyrimidine monomers . Using an oligonucleotide containing a thymine dimer at a unique site, we found that caffeine associates with the substrate and inhibits photoreactivation by blocking the binding of photolyase to the dimer . ABC excinuclease catalyses early events of excision repair; recognition of covalently modified DNA and incision of the phosphodiester backbone on both sides of the modification . The UvrA subunit is involved in the damage recognition process, which we studied using an oligonucleotide containing a unique psoralen adduct . UvrA binds to the adduct and protects 33 base pairs surrounding the adduct from DNase I digestion . In the presence of caffeine, the DNaseI footprint of UvrA covers the entire oligonucleotide; thus, caffeine promotes the binding of UvrA to undamaged DNA . UvrA subunits "trapped" by caffeine would be unable to catalyze repair . The intercalators ethidium bromide and chloroquine also promoted UvrA binding to DNA, so it may be caffeine's ability to intercalate into DNA that results in the trapping of UvrA . Thus, as a consequence of its interaction with DNA, caffeine inhibits these repair systems in E . coli by two entirely different mechanisms, by promoting the nonspecific binding of the nucleotide excision repair enzyme and by interfering with specific binding of the photoreactivating enzyme.

Proteins, 1990, 7(3), 280 - 90
The characterization of recombinant mouse glandular kallikreins from E . coli; Blaber M et al.; A system has been developed for the expression in E . coli of 12 of the 14 expressed mouse submandibular gland kallikreins as cassettes subcloned directly from cDNA . Using the epidermal growth factor binding protein (mGK-9) and the gamma-subunit of nerve growth factor (mGK-3), as test cases, mature processed forms, obtained as functionally active proteins, as well as various precursor forms, were isolated . The expression system described allows rapid isolation of kallikrein protein from corresponding cDNA with yields of approximately 1.0 mg of purified protein from 10 g of initial cell paste . This expression system will facilitate structure/function studies of the mouse glandular kallikrein gene family and help elucidate the regions of the mature proteins responsible for the diverse catalytic behavior and growth factor interactions observed in this family of proteins.

Izv Akad Nauk SSSR Biol, 1990 Jan-Feb, (1), 97 - 102
{The selective reduction of nitro compounds by E . coli cells}; Davidenko TI et al.; Nitroreduction of 2,4-dinitro-5H-11-p-R-phenyl-{b,f}-1,4-diazepines and 4-(2'-R-3',5'-dinitro)benzoyl-3,4-dihydroquinoxalinones-2 by E . coli with formation of 2-nitro-4-amino-11-p-R-phenyldibenzo-{b,f}-1,4-diazepines and 4-(2'-R-3'-nitro-5'-amino)-benzoyl-3,4-dihydroquinoxalinones-2 has been demonstrated using a set of physical and chemical methods.

Antibiot Khimioter, 1990 Jan, 35(1), 32 - 5
{Effect of interferon on the course of experimental E coli-induced pyelonephritis}; Rudenko AV et al.; The effect of the type I interferon on the development and process of experimental pyelonephritis caused by E . coli was studied on mice weighing 12 to 14 g . Interferon was administered intraperitoneally in a dose of 1000 units on days 3 and 7 of the disease . It was shown that the administration of the type I interferon to the mice with experimental pyelonephritis promoted rapid elimination of bacteria from the kidneys, prevented their penetration to the contralateral (intact) kidney, prevented marked macro- and microscopic damages in the kidneys, lowered the intensity of the inflammatory reaction, and increased the phagocytic activity of neutrophils and the number of the E-rosette-forming lymphocytes in the thymus . The data provided experimental grounding for clinical trials of interferon preparations in treatment of bacterial pyelonephritis.

Trends Genet, 1990 Jan, 6(1), 22 - 5
Regulation of cell division in E . coli; Lutkenhaus J; Recent investigation of some old cell division mutants of E . coli suggests that genes playing central roles in the regulation of division have been identified . The results suggest that cell division is triggered when a critical level of a single protein, FtsZ, is attained . The activity of this protein is channelled to the new division site by the activity of the min locus, which blocks access to old sites . Continued study of these genes should yield further insights into the cell division process.

J Biochem Biophys Methods, 1990 Jan, 20(2), 143 - 56
Use of analytical gel chromatography to analyze tertiary and quaternary structural changes in E . coli aspartate transcarbamylase; Bromberg S et al.; E . coli aspartate transcarbamylase (ATCase) is a large (310 kDa) protein that undergoes major changes in quaternary structure when substrates and regulatory nucleotides bind . We have used analytical gel chromatography to detect quaternary structure changes in both the holoenzyme and its catalytic subunit (c3), to characterize the quaternary structure of single site mutant proteins and to monitor urea-induced dissociation and unfolding of c3 . Binding of the bisubstrate analog PALA (N-(phosphonacetyl)-L-aspartate) to ATCase and c3 has been shown to alter s20.w by -3.3% and + 1.4%, respectively {Howlett, G.J . and Schachman, H.K . (1977), Biochemistry 23, 5077-5083} . The corresponding changes in the chromatographic partition coefficient (sigma) are -2.6 +/- 0.3% and 5.5 +/- 1.9% on Sephacryl S400HR and S200, respectively . Partition coefficients of mutant ATCases with single site mutations in the c chain differ from those of the wild-type protein by +/- 0.5% in small zone experiments; for example, mutations Arg 269----Gly and Glu 239----Gln alter the partition coefficient by 0.4% and -0.5%, respectively . The partition coefficient of mutant Glu 50----Gln is identical to the wild type enzyme . In the presence of saturating PALA, partition coefficients of Glu 50----Gln and Arg 269----Gly, but not Glu 239----Gln are identical to those of the wild type . Results for Glu 239----Gln are consistent with measurements of activity, small angle X-ray scattering and sedimentation coefficient that indicate that mutations at this site shift the quaternary structure towards the R state {Ladjimi and Kantrowitz (1988), Biochemistry 27, 276-83; Vachette and Herve, cited by Kantrowitz and Lipscomb (1988), Science 241, 669-674; Newell and Schachman (1988), FASEB J . 2, A551} . Results for Glu 50----Gln are also consistent with measurements of activity (Ladjimi et al . (1988), Biochemistry 27, 268-276) . The changes in tertiary and quaternary structure that result from urea-induced denaturation of c3 result in larger changes in the partition coefficient . Dissociation into folded monomers in 1-1.75 M urea is accompanied by a 4.6% increase in partition coefficient, while denaturation at greater than 5 M urea gives rise to a 43% decrease on S-300 Sephacryl . The bisubstrate analog PALA suppresses dissociation and increases the cooperativity of the unfolding reaction.

Acta Oncol, 1990, 29(8), 1055 - 8
Radiation response of E . coli after combined treatment with misonidazole and WR-2721 at various oxygen concentrations; Dam AM et al.; It has been reported that the aminothiol compound WR-2721 is a promising radioprotective agent and in combination with misonidazole (MISO) seems to be of therapeutic benefit . Since the radiomodification is oxygen-dependent, the actual oxygen status of cells and the surrounding media is an important factor influencing their effectiveness . Escherichia coli B/r radioresponse was studied either alone or in combination with these compounds at various oxygen concentrations ranging from anoxia to high oxygen content . WR-2721 had a protective effect under anoxic conditions and gave overall protection when oxygen was present . The maximum protection was seen at 3.2% O2 in N2 (PF 2.08) . In combination with MISO the hypoxic sensitization of MISO was completely abolished by WR-2721, resulting in radioprotection under hypoxic conditions as well . Under euoxic conditions MISO was able to reduce the protective effect of WR-2721 by about 21% . According to our results MISO and WR-2721 influence each other in their radiomodifying effect in either fixation or repair of the radiation-induced damage.

Appl Biochem Biotechnol, 1990 Spring-Summer, 24-25, 843 - 55
Analysis and molecular cloning of genes involved in thiophene and furan oxidation by E . coli; Alam KY et al.; Escherichia coli NAR30 can oxidize thiophenes, furans, and various other aromatic compounds as a result of successive mutations in three novel genes (thdA, C, and D) . Some of the mutant genes involved in thiophene degradation have been cloned into pUC19 . One of the resulting plasmids, pKA10, which carries a 3.8-kb chromosomal fragment from NAR30, conferred the ability to partially degrade furfuryl alcohol and thiophene methanol on a fadR atoC host strain . The location of the thd gene(s) on the plasmid pKA10 was found by insertion of a kanamycin resistance cassette . One such insertion into a BglII site inactivated the thd gene(s) . Disappearance of two proteins correlated with loss of oxidation ability vs thiophenes and furans . Strains carrying the thdA mutation oxidize these substrates by an oxidase that can be coupled to methylene blue . However, degradation of thiophenes by NAR30 is incomplete, with no organic sulfur being released.

Oncogene Res, 1990, 5(3), 161 - 73
An E . coli expression system for the rapid purification and characterization of a v-abl tyrosine protein kinase; Lydon NB et al.; A bacterial expression vector containing a segment of the v-abl gene from Abelson murine leukemia virus (A-MuLV) was constructed such that the gag region of v-abl was replaced by a sequence encoding the IgG-binding domain of the S . aureus protein A . pabl HP, a fusion protein encoded by this vector was rapidly purified to near homogeneity by affinity chromatography on IgG-Affigel and Mono Q FPLC . The Km of the pabl HP kinase for ATP varied with {Val5}-angiotensin II concentration and was 21.2 microM at saturating concentrations of {Val5}-angiotensin II . The Km for {Val5}-angiotensin II at saturating concentrations of ATP was 3.8 mM . The turnover number, at 20 degrees C, was 62 mumol min-1 mumol-1 . Initial rate studies support a ternary complex kinetic mechanism for phosphoryl transferase . The substrate specificity of the pabl HP kinase was further characterized using synthetic peptides . This expression system, which enables the rapid purification of recombinant v-abl kinase is suitable for the comparative enzymological study of mutant v-abl enzymes generated by site-directed mutagenesis.

Int J Biochem, 1990, 22(11), 1341 - 9
Expression of biologically active human pre-procorticotropin releasing hormone in E . coli: characterization and purification; Castro MG et al.; 1 . Human pre-procorticotropin releasing hormone (CRH) was expressed in E . coli strain TG2 as a fusion protein with beta-galactosidase . 2 . A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) {ph PPC (10-196)} after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining . The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay . 3 . Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea . 4 . When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).

Chin J Biotechnol, 1990, 6(3), 157 - 64
High expression of tumor necrosis factor alpha cDNA in E . coli using site-specific deletion mutagenesis; Zhang DZ et al.; Using oligonucleotide-directed site specific mutagenesis technique, a TNF-alpha cDNA coding 157 amino acids without signal sequence and initiated with ATG was obtained . Sequencing data showed the precise deletion and insertion of the DNA fragment as designed . The modified TNF gene was expressed in E . coli with a expression vector pBV220 . The expression level reached 10(8) units per liter culture . The scanning of the SDS-PAGE gel showed that the amount of the expressed TNF is about 22.8% of total soluble bacterial protein . The TNF activity can be neutralized with TNF monoclonal antibody.

Toxicon, 1990, 28(5), 477 - 91
Pore-forming toxins: experiments with S . aureus alpha-toxin, C . perfringens theta-toxin and E . coli haemolysin in lipid bilayers, liposomes and intact cells; Menestrina G et al.; Three quite different bacterial toxins (S . aureus alpha-toxin, C . perfringens theta-toxin and E . coli haemolysin) induce the leakage of phosphorylated metabolites from Lettre cells and of calcein from liposomes; in each case leakage is inhibited by Zn2+ greater than Ca2+ greater than Mg2+ . Inhibition is not due to displacement of toxin from the membrane, since divalent cations inhibit leakage through pre-formed pores . Electrical conductivity across phospholipid bilayers is induced by each of the three toxins; in each case the probability of channels being in the open state is reduced by divalent cations . Although the pores induced in phospholipid bilayers and liposomes vary greatly in size (theta-toxin much greater than haemolysin greater than alpha-toxin), in Lettre cells the lesions appear more uniform, suggestive of a limiting effect in cells.

Folia Microbiol (Praha), 1990, 35(2), 155 - 62
Local and systemic antibody response in infants after oral administration of inactivated enteropathogenic E . coli serotype O111 and O55; Lodinova-Zadnikova R et al.; In ten infants divided into two groups (up to one month of age and at 2-7 months of age) the dynamics and formation of different antibody isotypes produced locally in the intestine and in serum after orally administered inactivated enteropathogenic E . coli strains O111 and O55 was followed during 30 d after the first and booster dose by using an indirect immunofluorescence method . Infants up to one month of age produced antibodies of IgM isotype in stool together with the IgA isotype after the first and booster dose of the vaccine against both antigens . Serum IgG antibody increased after 2 d following the first and second dose of antigens and remained higher during 5 d . The infants aged 2-7 months expressed predominantly the IgA isotype response in stool after the first and booster dose of antigens . The serum immunoglobulin levels did not change after oral antigen administration.

Lett Appl Microbiol, 1990 Jan, 10(1), 31 - 4
Detection and identification of E . coli producing heat-labile enterotoxin type I by enzymatic amplification of a specific DNA fragment; Furrer B et al.; The polymerase chain reaction (PCR) was used to identify strains of Escherichia coli which produce heat-labile toxin type I (LTI) . Amplification primers were designed to detect E . coli strains of human as well as porcine origin . This assay was used to test the ATCC 37218 strain, which carries a recombinant plasmid with the genetic information for production of porcine LTI (pLTI) . In addition, three clinical E . coli isolates of human and one of porcine origin were tested . All clinical isolates were reported to produce heat-labile enterotoxin (hLTI and pLTI, respectively) when tested by the Y1 adrenal cell method and/or by the CHO cell method . All strains yielded the expected 275 bp DNA fragment after enzymatic amplification . This fragment was further identified by allele specific oligonucleotide hybridization . Alternatively, the fragment was identified by a SmaI restriction enzyme site which is present in the genes of both the E . coli isolated from humans and pigs . The detection limit determined in water with the ATCC 37218 strain was 20 bacteria . The amplified sequence included a CfoI polymorphism which allowed to distinguish between the genes coding for pLTI and hLTI . All of the strains tested showed this polymorphism as expected . Depending on the identification method chosen, SmaI digestion or oligonucleotide hybridization, pure water can be analysed within 8 h or 12 h, respectively . This method may be adapted to environmental and food samples.

Nucleic Acids Res, 1989 Dec 25, 17(24), 10295 - 305
Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E . coli lac DNA site; Ebright RH et al.; We have synthesized two 40 base pair DNA fragments; one fragment contains the consensus DNA site for CAP (fragment 'ICAP'); the other fragment contains the E . coli lac promoter DNA site for CAP (fragment 'LCAP') . We have investigated the binding of CAP to the two DNA fragments using the nitrocellulose filter binding assay . Under standard conditions {( NaCl} = 200 mM, pH = 7.3), CAP exhibits a 450-fold higher affinity for ICAP than for LCAP . The salt dependence of the binding equilibrium indicates that CAP makes eight ion pairs with ICAP, but only six ion pairs with LCAP . Approximately half of the difference in binding free energy for interaction of CAP with ICAP vs . LCAP is attributable to this difference in ion-pair formation . The pH dependence of the binding equilibrium indicates that the eight CAP-ICAP ion pairs and the six CAP-LCAP ion pairs do not involve His residues of CAP.

FEBS Lett, 1989 Dec 18, 259(1), 83 - 5
The efficiency of interaction of deoxyribonucleoside-5'-mono-, di- and triphosphates with the active centre of E . coli DNA polymerase I Klenow fragment; Doronin SV et al.; The interaction of deoxyribonucleoside-5'-mono-, di- and triphosphates with E . coli DNA polymerase I Klenow fragments was examined . Dissociation constants of the enzyme complex with nucleotides were determined from the data on the enzyme inactivation by adenosine 2',3'-riboepoxide 5'-triphosphate . The role of nucleotide bases, phosphate groups and sugar moieties in the complex formation of nucleotides with the enzyme was elucidated . The necessity of complementary interaction of nucleotides with templates for template-controlled 'adjusting' of complementary dNTP to its reactive state was found . The crucial role of the interaction of dNTP gamma-phosphate with the enzyme in this process is discussed.

FEBS Lett, 1989 Dec 18, 259(1), 27 - 32
A patch-clamp study of ion channels of inner and outer membranes and of contact zones of E . coli, fused into giant liposomes . Pressure-activated channels are localized in the inner membrane; Berrier C et al.; Inner and outer membranes of Escherichia coli and contact zones were isolated and fused separately with giant liposomes amenable to patch-clamp recording . Different types of large pressure-activated channels were localized in the inner membrane fraction which also contained smaller, pressure-insensitive channels . The outer membrane contained pressure-insensitive channels with large conductances and long opening and closing times which are likely to be porins . Large channels were also observed in contact zones.

Nucleic Acids Res, 1989 Dec 11, 17(23), 9811 - 22
Ribosome associated protein(s) specifically bind(s) to the upstream activator sequence of the E . coli rrnA P1 promoter; Nachaliel N et al.; A sequence located upstream to the E . coli rrnA P1 promoter is required for optimal promoter activity . Deletion of this sequence reduces in vivo transcription by 90% . Substitution of this upstream activating sequence with the unrelated bent DNA sequence of the kinetoplast of Crithidia fasciculata, restores in vivo expression to high levels . Cellular proteins which are present only in exponentially growing cells bind specifically to intact rrnA P1, but do not bind to the promoter missing the upstream activating sequence . These proteins are associated with the 30S ribosomal subunits but can be washed off with concentrated salt . The correlation between the binding activity and cell growth rate suggests a role for these proteins in the transcriptional control of rRNA synthesis.

Cesk Epidemiol Mikrobiol Imunol, 1989 Dec, 38(6), 362 - 5
{New findings on enteroinvasive strains of E . coli}; Durovicova J et al.; The group of enteroinvasive strains of E . coli (EIEC) comprises at present eleven serogroups . Diarrhoeal diseases caused by EIEC strains are found sporadically or epidemically . It was found that the pathogenicity of EIEC strains is coded in plasmids . So far the plasmid was proved in serogroups 028 a, c, 029, 0124 and 0143 . The incidence of EIEC strains is recorded also in the CSSR.

Science, 1989 Dec 1, 246(4934), 1152 - 4
Structural basis for misaminoacylation by mutant E . coli glutaminyl-tRNA synthetase enzymes; Perona JJ et al.; A single-site mutant of Escherichia coli glutaminyl-synthetase (D235N, GlnRS7) that incorrectly acylates in vivo the amber suppressor supF tyrosine transfer RNA (tRNA(Tyr} with glutamine has been described . Two additional mutant forms of the enzyme showing this misacylation property have now been isolated in vivo (D235G, GlnRS10; I129T, GlnRS15) . All three mischarging mutant enzymes still retain a certain degree of tRNA specificity; in vivo they acylate supE glutaminyl tRNA (tRNA(Gln} and supF tRNA(Tyr) but not a number of other suppressor tRNA's . These genetic experiments define two positions in GlnRS where amino acid substitution results in a relaxed specificity of tRNA discrimination . The crystal structure of the GlnRS:tRNA(Gln) complex provides a structural basis for interpreting these data . In the wild-type enzyme Asp235 makes sequence-specific hydrogen bonds through its side chain carboxylate group with base pair G3.C70 in the minor groove of the acceptor stem of the tRNA . This observation implicates base pair 3.70 as one of the identity determinants of tRNA(Gln) . Isoleucine 129 is positioned adjacent to the phosphate of nucleotide C74, which forms part of a hairpin structure adopted by the acceptor end of the complexed tRNA molecule . These results identify specific areas in the structure of the complex that are critical to accurate tRNA discrimination by GlnRS.

DNA, 1989 Dec, 8(10), 779 - 89
A rapid procedure for cloning genes from lambda libraries by complementation of E . coli defective mutants: application to the fabE region of the E . coli chromosome; Alix JH; I describe a general and rapid procedure allowing the isolation of specialized lambda transducing phages from a lambda library by lysogenic complementation of defective mutants of Escherichia coli . As an example, the cloning of the E . coli fabE gene and of two other adjacent genetic determinants is presented . Subcloning and determination of its nucleotide sequence reveals that fabE codes for the biotin carboxyl carrier protein (BCCP), one of the three subunits of acetyl coenzyme A carboxylase.

Biochem Int, 1989 Dec, 19(6), 1195 - 203
Thialysine utilization for protein synthesis by an exponentially growing E . coli culture; Di Girolamo M et al.; The extent of protein lysine substitution by thialysine in E . coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium . By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine . Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog . The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.

Science, 1989 Dec 1, 246(4934), 1135 - 42
Structure of E . coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution; Rould MA et al.; The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln} and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences . The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1 . Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA . The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe} with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures . Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3; (ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences . The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is

Nucleic Acids Res, 1989 Nov 25, 17(22), 9447 - 68
Influence of the sequence-dependent flexure of DNA on transcription in E . coli; Collis CM et al.; In order to study the effects of DNA structure on cellular processes such as transcription, we have made a series of plasmids that locate several different kinds of DNA structure (stiff, flexible or curved) near the sites of cleavage by commonly-used restriction enzymes . One can use these plasmids to place any DNA region of interest (e.g., promoter, operator or enhancer) close to certain kinds of DNA structure that may influence its ability to work in a living cell . In the present example, we have placed a promoter from T7 virus next to the special DNA structures; the T7 promoter is then linked to a gene for a marker protein (chloramphenicol acetyl transferase) . When plasmids bearing the T7 promoter are grown in cells of E . coli that contain T7 RNA polymerase, the special DNA structures seem to have little or no influence over the activity of the T7 promoter, contrary to our expectations . Yet when the same plasmids are grown in cells of E . coli that do not contain T7 RNA polymerase, some of the DNA structures show a surprising promoter activity of their own . In particular, the favourable flexibility or curvature of DNA, in the close vicinity of potential -35 and -10 promoter regions, seems to be a significant factor in determining where E . coli RNA polymerase starts RNA chains . We show directly, in one example, that loss of curvature between -35 and -10 regions is associated with a nearly-complete loss of promoter activity . These results, and others of their kind, show that the structural and/or vibrational properties of DNA play a much more important role in determining E . coli promoter activity than has previously been supposed.

FEBS Lett, 1989 Nov 20, 258(1), 159 - 62
Expression of the E . coli Lac Z gene from a defective HSV-1 vector in various human normal, cancer-prone and tumor cells; Boothman DA et al.; Introducing foreign genetic material into human cells is essential for the elucidation of the function of various human genes and has potential use in the treatment of human diseases by gene therapy . In this study we demonstrate that a defective herpes simplex virus type 1 vector, pHSVlac, can effectively transfer and express the Escherichia coli Lac Z gene in a variety of exponential and quiescent human cells . The human cells tested included representative cells derived from cancer-prone patients that presumably have various DNA repair deficiencies.

FEBS Lett, 1989 Nov 20, 258(1), 166 - 70
The algorithm of estimation of the Km values for primers of various structure and length in the polymerization reaction catalyzed by Klenow fragment of DNA polymerase I from E . coli; Nevinsky GA et al.; DNA synthesis at primers d(pT)n, d(pA)n, d(pC)n, and d(pG)n in the presence of corresponding complementary templates and at hetero-oligoprimers complementary to M13 phage DNA was investigated . The values of both -log Km and log Vmax increased linearly if homo-oligoprimers contained less than 10 nucleotides . The lengthening of d(pT)n and d(pA)n primers by one mononucleotide unit (n = 1-10) resulted in the 1.82-fold decrease of the Km values . The incremental decreases of Km for d(pC)n and d(pG)n were equal to about 2.46 . The enhancement of the homo- and hetero-oligonucleotide primers' affinity to the enzyme due to one Watson-Crick hydrogen bond between complementary template and primer is about 1.35 times . This allows to calculate the Km values for primers of various structure and length up to 10 units . The objective laws of the Km and Vmax values changes for primers containing more than 10 nucleotides were analyzed.

Cell, 1989 Nov 17, 59(4), 667 - 74
The replication terminator protein of E . coli is a DNA sequence-specific contra-helicase; Khatri GS et al.; We have cloned the tus gene that encodes the replication terminator protein of Escherichia coli and have efficiently expressed its gene product . The overproducer strain has been used to purify the terminator (ter) protein in high yield to near homogeneity . The protein is a single 36 kd polypeptide . Using the ter protein and highly purified dnaB helicase, we show that the terminator protein is a DNA sequence-specific contra-helicase, i.e., the protein when bound to its recognition sequence (tau) strongly impedes the ATP-dependent unwinding of double-stranded DNA . This contra-helicase activity is polar, i.e., the impedance to unwinding takes place in only one orientation of the tau sequence . The results illuminate the mechanism of replication termination specifically at tau.

Wiad Lek, 1989 Nov 15-Dec 15, 42(22-24), 1152 - 5
{Jaundice of the subcortical ganglia in a newborn caused by E . coli infection}; Maszkiewicz W et al.; A case is reported of icterus of the subcortical ganglia in a premature newborn with total bilirubin level 7.42 mg% . The role of various risk factors in the aetiology of this icterus is discussed.

Wiad Lek, 1989 Nov 15-Dec 15, 42(22-24), 1115 - 8
{Clinical analysis of 18 cases of E . coli septicemia in children}; Szymborski J et al.; A clinical analysis of 18 cases of E . coli septicaemia in children showed that 90% of cases were in infants aged up to 6 months, mainly in newborns . Half the cases developed in hospital . All strains with one exception were sensitive to gentamicin . In the whole complex of non-characteristic clinical manifestations loose stools were most frequent . In the laboratory investigations thrombocytopenia was the most characteristic abnormality . The mortality was 16.7%.

Biochem Pharmacol, 1989 Nov 15, 38(22), 3903 - 7
Mechanistic aspects of paraquat toxicity in E . coli . A spin trapping study; Sion A et al.; Mechanistic aspects of paraquat monocation radical (PQ.+) and copper involvement in paraquat toxicity have been examined using E . coli B cells . Electron spin resonance (ESR) spectrometry combined with cell survival studies were used to explore the correlation between radical production and biological damage . The line broadening agent oxalato-chromiate (CrOx) was used to characterize the anoxic partition of PQ.+ inside and outside the cell . In the presence of CrOx the ESR signal was totally eliminated, indicating that intracellular species were undetectable and that, contrary to previous reports, PQ.+ exclusively accumulates outside the cell . The PQ.+ radical does not react with H2O2 but disappears in the presence of H2O2 when catalytic traces of Cu(II) are present . Spin-trapping studies using DMPO showed that in aerobic environment paraquat-induced O2 radicals are detectable exclusively in the extracellular compartment . The correlation between PQ.+ appearance and the biological damage is not simple . PQ.+ non-toxically accumulates, in the absence of oxygen and either Cu(II) or H2O2 . By contrast, with both H2O2 and Cu(II) the cells are rapidly killed but PQ.+ was undetectable . These results reconfirm the key catalytic mediatory function of transition metals in paraquat toxicity.

Nucleic Acids Res, 1989 Nov 11, 17(21), 8767 - 82
Ribosomal proteins S7 and L1 are located close to the decoding site of E . coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3'-end of the anticodon; Podkowinski J et al.; Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon . The maximum distance between the purine ring and the azido group estimated for the two probes is 16-17 and 23-24A, respectively . Binding and cross-linking of the uncharged, modified tRNAs to E . coli ribosomes have been studied with and without poly(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site . The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields . Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A,U,G) . Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe . Cross-linking to 168 rRNA reaches significant levels only in the absence of the message.

Nucleic Acids Res, 1989 Nov 11, 17(21), 8475 - 84
Purification of the E . coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides; Wilkinson MC et al.; The E . coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase) . The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography . Good correlation was found between the determined and predicted amino acid compositions . The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein . With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.

Biochim Biophys Acta, 1989 Nov 9, 999(1), 36 - 41
On the role of histidine and tyrosine residues in E . coli asparaginase . Chemical modification and 1H-nuclear magnetic resonance studies; Bagert U et al.; The relative importance of tyrosine and histidine residues for the catalytic action of Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) was studied by chemical modification and 1H-NMR spectroscopy . We show that, under appropriate reaction conditions, N-bromosuccinimide (NBS) as well as diazonium-1H-tetrazole (DHT) inactivate by selectively modifying two tyrosine residues per asparaginase subunit without affecting histidyl moieties . We further show that diethyl pyrocarbonate (DEP), a reagent considered specific for histidine, also modifies tyrosine residues in asparaginase . Thus, inactivation of the enzyme by DEP is not indicative of histidine residues being involved in catalysis . In 1H-nuclear magnetic resonance (NMR) spectra of asparaginase signals from all three histidine residues were identified . By measuring the pH dependencies of these resonances, pKa values of 7.0 and 5.8 were derived for two of the histidines . Titration with aspartate which tightly binds to the enzyme at low pH strongly reduced the signal amplitude of the pKa 7 histidyl moiety as well as those of resonances of one or more tyrosine residues . This suggests that tyrosine and histidine are indeed constituents of the active site.

J Chromatogr, 1989 Nov 3, 481, 343 - 51
Application of free flow electrophoresis to the preparative purification of basic proteins from an E . coli cell extract; Kuhn R et al.; The application of free flow electrophoresis (FFE) to the purification of a basic protein from a complex protein mixture was investigated . For this purpose lysozyme (E.C.3.2.1.17) from hen egg white, serving as a model for a basic protein, was added to a crude E . coli cell extract and reisolated . For three techniques of FFE (zone electrophoresis, isoelectric focusing and field step electrophoresis), suitable electrolyte systems were developed . The purity, purification factor, recovery and throughput were determined for the optimized experiments . A combination of field step electrophoresis and zone electrophoresis gave the best purification factor (9.5) and the highest recovery (95%) . The purification factors achieved in zone electrophoresis and isoelectric focusing were comparable to each other and ranged from 3.5 to 4.75 . In isoelectric focusing, 94% of the enzyme activity was recovered . Zone electrophoresis gave recoveries of 82% and 87%, respectively . Purities of more than 95% were achieved with all the techniques described . With the exception of zone electrophoresis, all the techniques effected a concentration of the enzyme during the separation . Zone electrophoresis and field step electrophoresis were very simple in application.

Z Naturforsch {C}, 1989 Nov-Dec, 44(11-12), 1036 - 48
Heat production and growth kinetics of E . coli K12 from flow calorimetric measurements on chemostat cultures; Leiseifer HP; The heat production of E . coli K12 growing aerobically in glucose limited chemostat cultures is determined in the range of specific growth rates mu (= dilution rates D) from 0.058 h-1 to 0.852 h-1 for two different glucose concentrations Se in the instream of the chemostat, namely Se1 = 0.3182 g.l-1 and Se2 = 0.6364 g.l-1 . Heat production Q and biomass production P per unit of culture volume show well correlated patterns for Se1 and Se2 . For Se1 the highest value Q actually measured is 443.10(-3) W.l-1 at D = 0.74 h-1 with P = 0.068 g.l-1.h-1; and for Se2 593.10(-3) W.l-1 at D = 0.497 h-1 with P = 0.108 g.l-1.h-1 . Heat production QB per unit of biomass appears to be independent of Se at least up to D = 0.5 h-1 . At higher D there is strong indication that QB possesses a real maximum . The highest value of QB actually measured is 4.8 W.g-1 at D = 0.74 h-1 . For Se1 and Se2 there were significantly higher specific growth rates verified in chemostat culture than mu Batchmax = 0.717 h-1 which is the maximum specific growth rate in comparable, unlimited batch cultures . The real maximum of QB is estimated to be in the vicinity of mu Batchmax . This suggests the hypothesis of a maximum principle for the growth in batch culture . For Se1 a closed analytical expression is derived for the relationship between mu and the substrate concentration S . mu{S} features a S-shaped characteristic with mu Chemostatmax = 0.905 h-1; 1/2 mu Chemostatmax is reached at S = 2.85.10(-3) g.l-1 . Three basic parameters which characterize the overall metabolism of the cells, namely the heat released per unit of substrate consumed, Qs, the effective yield of biomass, Yeff, and mu Chemostatmax are identified to depend on Se.

Appl Biochem Biotechnol, 1989 Nov, 22(2), 151 - 68
Immunization procedures for E . coli proteins; Anicetti VR et al.; Three immunization procedures were compared for the production of antibodies to the minor components of a complex E . coli protein (ECP) mixture: a conventional protocol and two methods that allow for the selective in vitro (cascade) or in vivo (passive) depletion of highly immunogenic proteins . An indirect ELISA showed that a maximum ELISA antibody titer was obtained with all the procedures 60 d after immunization . Analysis of these antisera by two-dimensional SDS-PAGE immunoblots, however, demonstrated that antibody reactivity to minor components in the mixture was not achieved until 112 d . This analysis also showed that a marked improvement in antibody response to minor components was obtained with the cascade immunization procedure . The mean titer and spectrum of antibody reactivity was similar for each group, and suggested that, although some individual variation was noted, the improvements observed were the result of the protocol used . Thus, for these ECPs, and multiple antigen mixtures in general, the preferred immunization protocol should employ at least three hosts and utilize the cascade immunization of Thalhamer and Freund . Characterization of the resulting antisera is best performed by use of silver stained two-dimensional SDS-PAGE and immunoblotting.

Protein Eng, 1989 Nov, 3(2), 145 - 51
Biosynthesis of winter flounder antifreeze proprotein in E.coli; Peters ID et al.; A semisynthetic winter flounder antifreeze proprotein (proAFP) coding region was constructed and inserted into a lacZ expression vector . ProAFP was produced from the vector in Escherichia coli as a C-terminal fusion to the first 289 amino acids of beta-galactosidase (beta-gal) . The proAFP and beta-gal domains of the beta-gal-proAFP fusion protein were separated by the recognition signal for the blood coagulation protease, factor Xa . Upon induction with isopropylthio-beta-D-galactoside the fusion protein accumulated to levels of 15% of the total protein . The beta-gal-proAFP fusion protein was partially purified by differential centrifugation, but required solubilization prior to factor Xa digestion . The solubilized fusion protein was efficiently and correctly cleaved by factor Xa, after which the proAFP was purified by gel permeation . Bacterial proAFP was indistinguishable from natural proAFP by the criteria of antifreeze activity, amino-terminal sequence (15 cycles), reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis . Circular dichroism measurements showed that proAFP is a composite of random coil and alpha-helical secondary structure, with an alpha-helix content of 44% at 0 degrees C . It seems probable that the C-terminal region of proAFP, which corresponds to the mature AFP protein, is mainly alpha-helical, and that the N-terminal pro-segment is random coiled.

Protein Eng, 1989 Nov, 3(2), 139 - 43
Direct expression in E . coli of a functionally active protein A--snake toxin fusion protein; Ducancel F et al.; We constructed a recombinant expression plasmid encoding a protein A--neurotoxin fusion protein . The fused toxin is directly expressed in the periplasmic space of Escherichia coli and can be purified in the milligram range by a single immuno-affinity step . The LD50 values of the fused toxin and native toxin are 130 and 20 nmol/kg mouse respectively . The Kd values characterizing their binding to the nicotinic acetylcholine receptor (AcChoR) are respectively 4.8 +/- 0.8 and 0.07 +/- 0.03 nM . In contrast, the fused and native toxins are equally well recognized by a toxin-specific monoclonal antibody which recognizes the AcChoR binding site . The lower toxicity of the fused toxin might result, therefore, from a steric hindrance, due to the presence of the bulky protein A moiety (mol . wt = 31 kd) rather than to a direct alteration of the 'toxic' site . The fused toxin is more immunogenic than native toxin, since 1 nmol of hybrid toxin and 14 nmol of native toxin give rise to comparable titers of antitoxin antibodies which, furthermore, are equally potent at neutralizing neurotoxicity . The work described in this paper shows that the use of fused toxins may be of paramount importance for future development of serotherapy against envenomation by snake bites.

New Biol, 1989 Nov, 1(2), 159 - 69
E . coli ribosomes re-phase on retroviral frameshift signals at rates ranging from 2 to 50 percent; Weiss RB et al.; Many retroviruses express gag-pol or gag-pro-pol polypeptides by coupling their translation from overlapping reading frames with -1 ribosomal frameshifts . Here, we show that the well-known ribosomal frameshift signals found in retroviral mRNA will provoke Escherichia coli ribosomes to shift frame in the same manner as their eukaryotic counterparts . Ribosomes of E . coli respond in vivo to both the tandem slippery codons present at the retroviral frameshift site and the 3' flanking sequence . Slight alteration of the mouse mammary tumor virus gag-pro frameshift site from A-AAA-AAC to A-AAA-AAG boosts the level of frameshifting in E . coli to over 50% . This suggests that A-AAA-AAG, and its slippery relatives, may be utilized by E . coli genes as sites of high-level ribosomal frameshifting . This observed conservation of response to retroviral frameshift signals affords new avenues to dissect the mechanism of ribosomal frameshifting evoked by these mRNA sequences.

Z Naturforsch {C}, 1989 Nov-Dec, 44(11-12), 1053 - 7
{Protective effect of D20 in bacteria (E . coli)}; Papadimitriou C et al.; E . coli suspended in D2O showed a better survival than in H2O when the bacteria were treated with various damaging agents like UV, heat and freezing . On the contrary E . coli grown in D2O-containing media - therefore being fully deuterated - were more sensitive than normally grown bacteria.

Mutat Res, 1989 Nov, 218(3), 197 - 206
Enhanced O6-methylguanine-DNA methyltransferase activity in transgenic mice containing an integrated E . coli ada repair gene; Matsukuma S et al.; The E . coli ada gene encodes O6-methylguanine DNA methyltransferase (O6MTase) which repairs the methylation of guanine at the O6 position in DNA . After recombination with a Chinese hamster metallothionein I gene promoter, the ada gene was microinjected into C3H/HeN mouse zygotes . Eventually, transgenic mice containing the ada fusion DNA were generated . The integrated ada DNA complex was transmitted to the progeny in a mode conforming to tandem integration at a single chromosome site, and homozygotes were also obtained from an inter-transgenic mouse cross . RNA transcripts of the chimeric ada gene were identified in the livers of these transgenic mice using dot and Northern blot analyses . O6MTase activity was increased in the liver of transgenic mice of line No . 708, and was more than 3 times the activity found in non-transgenic mice, especially in the transgenic homozygotes . The ada gene product was detected in the liver of a transgenic homozygote by immunoblot analysis . These transgenic mice have great potential for analysis of the role played by O6MTase in chemical carcinogenesis.

Chin Med J (Engl), 1989 Nov, 102(11), 879 - 84
A preliminary study of the inhibiting mechanism of anisodamine on rabbit platelets activated by E . coli endotoxin; Xu SH et al.; In this study, we observed the endotoxin induced platelet aggregation, 5-HT and beta-g releases, cellular cAMP decrease, and also the changes of morphology and membrane fluidity of platelet . These changes play an important role in septic shock, especially in disseminated intravascular coagulation (DIC) and respiratory distress syndrome (RDS) . After anisodamine (654) intervention all the parameters mentioned above were improved to a certain extent . This preliminary study of the inhibiting mechanism of 654 on rabbit platelets activated by ET supports the clinical possibility of using 654 for relieving DIC and RDS.

Nucleic Acids Res, 1989 Oct 25, 17(20), 8047 - 60
Expression of the ogt gene in wild-type and ada mutants of E . coli; Potter PM et al.; O6-alkylguanine (O6-AlkG) DNA alkyltransferase (ATase) and alkylphosphotriester (AlkP) ATase activity have been quantitated individually in extracts of various E . coli strains by means of ATase specific DNA substrates . O6-AlkG ATase activity was higher than AlkP ATase activity in the wild-type strains F26, AB1157 and SB229 and in the ada- mutants PJ1, PJ3, PJ5 and PJ6 indicating a 5-70 times higher level of expression of the ogt gene than the ada gene . The ada- mutant strains BS23, BS73 and GW5352 expressed O6-AlkG ATase but not AlkP ATase activity indicating expression only of the ogt gene . Southern analysis of DNA from F26, BS23, BS73, PJ1 and GW5352 showed a consistent pattern of hybridisation to an ogt probe but not to an ada probe . Exposure of E . coli to adaptive doses of N-methyl-N-nitro-N-nitroso-guanidine (MeNNG) caused an increase in AlkP ATase activity in F26, AB1156, SB229, PJ1, PJ3, PJ5 and PJ6 . O6-AlkG ATase activity also increased in F26, AB1157 and SB229 but decreased to almost undetectable levels in all other strains examined except PJ3 where it remained constant . MeNNG increased ada mRNA abundance in F26 but no ada mRNA was detected in BS23, BS73 or GW5352: there was no evidence for increased ogt mRNA in any of the strains examined . In a limited survey, other bacterial strains have been shown to possess an ogt-like ATase activity.

Rev Ig Bacteriol Virusol Parazitol Epidemiol Pneumoftiziol Bacteriol Virusol Parazitol Epidemiol, 1989 Oct-Dec, 34(4), 337 - 44
{The incidence of E . coli O157H7 strains in causing hemorrhagic enterocolitis}; Gancevici C et al.; The authors describe the evidencing of the O157H7 E . coli serotype--an enterohaemorrhagic strain of E . coli--for the first time in our country . This germ is incriminated in haemorrhagic enteritis of children and adults, accompanied or not by an uremic haemolytic syndrome . A total of 717 strains were investigated, obtained from cases of haemorrhagic enteritis (283), nonhemorrhagic enteritis (174), food poisoning (27), and a control lot of subjects without signs of enteritis (233) . Identification of the strains was done with a screening method (McConkey) with D-sorbitol in place of lactose, and with anti-O157, and anti-H7 sera . The results obtained have indicated a total of 37 positive strains in haemorrhagic enteritis (69.8%), 12 strains in nonhemorrhagic enteritis (22.65%), and 4 strains in food poisoning (7.55%) . No strain was isolated from the control group . The percentage of isolated O157H7 E . coli strains in our county is of 7.55% and this contributes to additional knowledge in the definition of the still unknown etiology of diarrhoeic disease in our country.

Bioorg Khim, 1989 Oct, 15(10), 1356 - 61
{Covalent labelling of the Klenow fragment of DNA-polymerase I from E . coli}; Degtiarev SKh et al.; Incubation of the Klenow fragment of E . coli DNA polymerase I with {alpha-32P} dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such as beta-mercaptoethylamine, efficiently inhibiting the labelling . It is suggested that radiolabelling of the enzyme is the result of formation of chemically active products of the radiolysis of {alpha-32P}NTP (which are likely to be radicals) . Non-radioactive NTP hinder the labelling, whereas Mg2+ and polynucleotide do not affect it . Cleavage of the enzyme by hydroxylamine and cyanogen bromide and analysis of gel-electrophoretic patterns of the cleavage products led to conclusion that 32P-label is located between Gly-544 and Met-647.

J Biochem Biophys Methods, 1989 Oct, 19(4), 301 - 8
A recycling assay for alkaline phosphatase applied to studies on its transport in E . coli K12; Rao NM et al.; A recycling assay for alkaline phosphatase, based on its ability to hydrolyse NADP to NAD+, is presented . The product NAD+ is recycled in a coupled assay consisting of NADH regeneration and reduction of a nitroblue tetrazolium salt . This assay is 10-12 times more sensitive than the conventional assay . We demonstrate the role of energy poisons in transport of this protein into the periplasm by combining the improved detection with phase separation of the periplasmic and cytoplasmic alkaline phosphatase pools.

Protein Eng, 1989 Oct, 3(1), 61 - 6
Recombinant rabbit uteroglobin expressed at high levels in E . coli forms stable dimers and binds progesterone; Peter W et al.; In order to express uteroglobin in Escherichia coli we have constructed a DNA coding for complete mature rabbit uteroglobin by fusing genomic sequences from the second exon of the gene to an incomplete cDNA . This DNA was inserted into various positions of the polylinker cloning region of pDS expression vectors and the uteroglobin gene was expressed in E . coli by IPTG induction . Four different uteroglobin-derived proteins were produced containing 1, 3, 5 and 7 more N-terminal amino acids than the naturally occurring mature protein . The yield of soluble protein strongly increased with increasing length of the N-terminal additions . Protein and RNA analysis showed that this variation is most likely due to progressively higher translation efficiencies of the larger recombinants . UG7, the most efficiently synthesized recombinant protein, carrying seven additional N-terminal amino acids, was purified and further characterized . Like natural uteroglobin, UG7 forms a dimer and binds progesterone with an affinity indistinguishable to the natural protein . This bacterially produced protein can be used for detailed structure-function investigations of uteroglobin.

Proc Natl Sci Counc Repub China B, 1989 Oct, 13(4), 276 - 83
Involvement of E . coli dcm methylase in Tn3 transposition; Yang MK et al.; The effects of DNA methyltransferases on Tn3 transposition were investigated . The E . coli dam (deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition . In contrast, Tn3 was found to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants . When the EcoRII methylase gene was introduced into dcm- cells (E . coli strain GM208), the frequency of Tn3 transposition in GM208 was dramatically increased . The EcoRII methylase recognizes and methylates the same sequence as does the dcm methylase . These results suggest that deoxycytosine methylase modified DNA may be a preferred target for Tn3 transposition . Experiments were also performed to determine whether the Tn3 transposase was involved in DNA modification . Plasmid DNA isolated from dcm- E . coli containing the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion, suggesting that Tn3 transposase modified the dcm recognition sequence . In addition, restriction enzymes TaqI, AvaII, BglI and HpaII did not digest this DNA completely, suggesting that the recognition sequences of TaqI, AvaII, BglI and HpaII were modified by Tn3 transposase to a certain degree . The type(s), the extent and mechanism(s) of this modification remain to be investigated.

J Bacteriol, 1989 Oct, 171(10), 5262 - 7
Release of outer membrane fragments from wild-type Escherichia coli and from several E . coli lipopolysaccharide mutants by EDTA and heat shock treatments; Marvin HJ et al.; EDTA-induced outer membrane losses from whole cells of wild-type Escherichia coli (O111:B4) and several lipopolysaccharide (LPS) mutants derived from E . coli K-12 D21 were analyzed . EDTA treatment induced losses of LPS (up to 40%), outer membrane proteins OmpA, OmpF/C, and lipoprotein, periplasmic proteins, and phosphatidylethanolamine . The extent of these releases was strain specific . Successively more EDTA was necessary to induce these losses from strains containing LPS with increasing polysaccharide chain length . An additional heat shock immediately following the EDTA treatment had no effect on LPS release, but it decreased the release of outer membrane proteins and reduced the leakage of periplasmic proteins, suggesting that the temporary increase in outer membrane "permeability" caused by Ca2+-EDTA treatment was rapidly reversed by the redistribution of outer membrane components, a process which is favored by a mild heat shock . The fact that the material released from E . coli C600 showed a constant ratio of lipoprotein, OmpA, and phosphatidylethanolamine at all EDTA concentrations tested suggests that the material is lost as specific outer membrane patches . The envelope alterations caused by EDTA did not result in cell lysis.

Vet Rec, 1989 Sep 30, 125(14), 365 - 8
Occurrence of 'attaching and effacing' lesions in the small intestine of calves experimentally infected with bovine isolates of verocytotoxic E coli; Wray C et al.; Nine calves (five colostrum-fed, four colostrum-deprived) were challenged with two field strains of Escherichia coli which produced either verocytotoxin 1 (VT1) or verocytotoxin 2 (VT2) . Although three colostrum-fed calves had blood and mucus in their faeces, no diarrhoea was observed . Three of the four colostrum-deprived calves had diarrhoea and in two of them severe lesions were detected in the small intestine . Focal changes were detected in the colon of three calves . E coli were associated with the lesions in the small and large intestine and were shown by transmission electron microscopy to be intimately attached to the enterocyte surface with effacement of microvilli (attaching and effacing lesions) . This is the first report of E coli which produce VT2 being associated with disease in calves.

Nucleic Acids Res, 1989 Sep 25, 17(18), 7229 - 39
The effect of E . coli host strain on the consensus sequence of regions of the human L1 transposon; Crowther PJ et al.; We have used highly methylation tolerant host strains to clone hyper- and hypo-methylated genomic elements from different regions of the same family of long interspersed repetitive elements from human DNA, specifically the 1.8 kilobase (kb) and 1.2kb KpnI fragments from members of the L1 family of transposable elements in which respectively some 18% and 2.7% of cytosines are methylated in vivo in human spleen DNA . The consensus of the DNA sequences of the ends of 13 clones from the hypomethylated region of human L1 agreed exactly with the consensus derived previously from clones made using conventional host strains . However the sequences of 18 of our clones from the 5' end of the hypermethylated region differed significantly from the sequences of clones made using conventional hosts (P less than 0.0001) . The 5' region of the 1.8kb L1 region is a CpG island which, in human somatic tissue, appears to be maintained in a highly methylated state, including methylation at sites other than CpG dinucleotides . The consensus sequence of this region also has features suggestive of a previously unrecognized open reading frame.

Nucleic Acids Res, 1989 Sep 25, 17(18), 7159 - 65
The selenocysteine-inserting opal suppressor serine tRNA from E . coli is highly unusual in structure and modification; Schon A et al.; Selenocysteine is cotranslationally incorporated into selenoproteins in a unique pathway involving tRNA mediated suppression of a UGA nonsense codon (1-3) . The DNA sequence of the gene for this suppressor tRNA from Escherichia coli predicts unusual features of the gene product (4) . We determined the sequence of this serine tRNA (tRNA(UCASer} . It is the longest tRNA (95 nt) known to date with an acceptor stem of 8 base pairs and lacks some of the 'invariant' nucleotides found in other tRNAs . It is the first E . coli tRNA that contains the hypermodified nucleotide i6A, adjacent to the UGA-recognizing anticodon UCA . The implications of the unusual structure and modification of this tRNA on recognition by seryl-tRNA synthetase, by tRNA modifying enzymes, and on codon recognition are discussed.

Cell, 1989 Sep 8, 58(5), 847 - 55
Protein phosphorylation regulates transcription of the beta-glucoside utilization operon in E . coli; Amster-Choder O et al.; We have investigated the interaction between BgIF and BgIG, two proteins that regulate expression of the E . coli bgI operon . BgIF is both a negative regulator of operon expression and a phosphotransferase involved in uptake of beta-glucosides . BgIG is a positive regulator that functions as a transcriptional antiterminator . We show here that BgIF is phosphorylated by the soluble components of the phosphotransferase system: Enzyme I, HPr, and the phosphate donor phosphoenolpyruvate . Phosphorylated BgIF can then transfer phosphate either to beta-glucosides or to wild-type BgIG . Mutant BgIG derivatives, which give constitutive expression of the bgI operon, show little or no phosphorylation by BgIF . Hence BgIF exerts its negative effect on operon expression by phosphorylating BgIG, blocking its action as an antiterminator . BgIG is dephosphorylated only in the presence of both BgIF and beta-glucosides . Based on these results, we propose the following mechanism: In the absence of beta-glucosides, BgIG is phosphorylated by BgIF and is inactive in antitermination . Addition of inducer stimulates BgIF to dephosphorylate BgIG, allowing BgIG to function as a positive regulator of operon expression . Beta-Glucosides are then phosphorylated and transported into the cell by BgIF.

J Appl Bacteriol, 1989 Sep, 67(3), 343 - 6
Membrane filtration differentiation of E . coli from coliforms in the examination of water; Mates A et al.; A membrane filter-Endo agar method for enumerating Escherichia coli as distinct from other coliforms in drinking water was developed . Membranes containing coliform colonies are transferred to nutrient agar containing 4-methyl umbelliferyl-beta-D-glucuronide (MUG) and incubated at 35 degrees C for 4 h . The MUG is hydrolyzed by the glucuronidase of E . coli and the fluorogenic product is visualized . The method recovered 98% of E . coli without false positives and is proposed as an additional test in routine water examination for the detection of pollution.

Biokhimiia, 1989 Sep, 54(9), 1520 - 5
{Membrane potential of E . coli recipient cells determines the rate of linear transport of DNA during conjugation}; Berzhinskene IaA et al.; The rate of conjugal DNA transport from donor to recipient cells has been shown to depend on the membrane potential (delta psi) value in the DNA recipient cell . On the other hand, delta psi in the DNA donor cells is required for the formation of stable aggregates of conjugating cells, but not for the RNA transport . Both components of the electrochemical proton gradient on the cytoplasmic membrane of the recipient cells, the delta psi and the pH gradient are equivalent in the conjugal process.

Z Naturforsch {C}, 1989 Sep-Oct, 44(9-10), 819 - 23
Singlet oxygen mediated photobinding of 8-methoxypsoralen to DNA and genotoxicity in E . coli; Beijersbergen van Henegouwen GM et al.; Although oxygen dependent photoreactions of 8-methoxypsoralen (8-MOP) are known, damage to DNA is mostly considered to proceed via photocycloaddition to this biomacromolecule . In this study the survival of colony forming ability of E . coli K12/343, which is deficient in DNA-repair capability, appeared to be lower in D2O than in H2O after exposure to the combination of 8-MOP and UV-A . Photobinding to bacterial DNA was approximately 40% higher in D2O than in H2O . However this last difference was found only when the bacteria were kept in the reaction medium for 1 h at 37 degrees C after the irradiation was stopped and not when they were plated out immediately afterwards . The results indicate that in this bacterial test system 1O2 mediated photobinding of 8-MOP to DNA contributes to the genotoxic effect observed.

Virus Res, 1989 Sep, 14(1), 27 - 47
Synthesis of hepatitis B virus e antigen in E . coli; Inada T et al.; Hepatitis B virus core antigen (HBcAg) gene was deleted at some unique restriction enzyme sites, or at random, and inserted into the expression plasmids of E . coli which had the tryptophan promoter . E . coli transformants with the plasmids, synthesized materials with many kinds of antigenicity of HBcAg, HBeAg, or both HBcAg and HBeAg . HBeAg-specific material smaller than native HBeAg was produced in a stable condition.

Z Naturforsch {C}, 1989 Sep-Oct, 44(9-10), 791 - 6
{Effect of neocarzinostatin on E . coli mutants deficient in DNA repair}; Denklau D et al.; The colony forming ability of E . coli mutants defective in DNA repair was compared to that of the parent strain AB 1157 after neocarzinostatin treatment . A recA and a recB mutant were most sensitive . The suppression of the recB mutation in the recBC sbcBC mutant, which is as sensitive as the parent strain, indicates that recB is not the primary pathway by which lesions after NCS treatment are repaired . The survival curve of the recBC recF sbcBC mutant, corresponding to that of the recF mutant, further supports this interpretation . The relative resistance of the recBC recF sbcBC mutant suggests that NCS lesions are not only repaired by the recF and recB pathway . An alternative pathway could be the SOS induction, as a lexA mutant also is sensitive to NCS . The sensitivity of the uvrA and polA xthA mutants, however is explained by the involvement of the uvrA and polA gen products in rec repair.

Toxicol Lett, 1989 Sep, 48(3), 225 - 34
Induction of sfiA SOS function by peroxides using three different E . coli strains; Eder E et al.; Five peroxides and two related compounds were tested for genotoxicity by the SOS Chromotest using 3 different E . coli strains (PQ37, PM21, GC4798) . All tested hydroperoxides (hydrogen peroxide, tert-butylhydroperoxide, cumene hydroperoxide) were clearly positive in all strains . From a comparison of results obtained from the different strains it can be concluded that neither DNA lesions leading to the induction of excision repair nor covalent binding of radicals to DNA is responsible for the induction of sfiA-SOS function by hydroperoxides . Among the remaining compounds tested, only dibenzoylperoxide gave a clearly positive result in strain PQ37 whereas di-tert-butylperoxide and azobisisobutyronitrile showed only borderline activity . When using strains PM21 and GC4798, none of the latter compounds was positive . Paraquat was inactive in all strains.

Mutat Res, 1989 Sep, 218(2), 125 - 33
DNA repair mechanisms affecting cytotoxicity by streptozotocin in E . coli; Fram RJ et al.; Mechanisms underlying cytotoxicity by the monofunctional nitrosourea streptozotocin (STZ) were evaluated in DNA repair-deficient E . coli mutants . Strains not proficient in recombinational repair which lack either RecA protein or RecBC gene products were highly sensitive to STZ . In contrast, cells that constitutively synthesize RecA protein and cannot initiate SOS repair mechanisms because of uncleavable LexA repressor (recAo98 lexA3) were resistant to this drug compared to a lexA3 strain . Further, E . coli cells lacking both 3-methyladenine DNA glycosylases I (tag) and II (alkA) also were highly sensitive to STZ . DNA synthesis was most inhibited by STZ in recA and alkA tag E . coli mutants, but was suppressed less markedly in wild-type and recBC cells . DNA degradation was most extensive in recA E . coli after STZ treatment, while comparable in recBC, alkA tag, and wild-type cells . Although increased single-stranded DNA breaks were present after STZ treatment in recA and recBC mutants compared to the wild type, no significant increase in DNA single-stranded breaks was noted in alkA tag E . coli . Further, DNA breaks in recBC cells were repaired, while those present in recA cells were not . These findings establish the critical importance of both recombinational repair and 3-methyladenine DNA glycosylase in ameliorating cytotoxic effects and DNA damage caused by STZ in E . coli.

Nucleic Acids Res, 1989 Aug 11, 17(15), 5901 - 12
Completion of the detailed restriction map of the E . coli genome by the isolation of overlapping cosmid clones; Knott V et al.; Ordered sets of cosmids derived from E . coli K-12 803 overlap the 6 remaining gaps left in the physical map of strain W3110 . We present detailed restriction maps of the gaps and surrounding regions, thus providing a comparison of about 30% of the genome of the two E . coli strains . Our analysis shows that there is a high degree of homology between the strains, with only occasional restriction fragment differences . However, the large inversion occurring between rrnD (72.1') and rrnE (90.4') in strain W3110 is absent in strain 803 . Instead, a new inversion and adjacent deletion near argF is present in strain 803 . The distribution of cosmid clones at, and adjacent to, the gaps shows that all gaps except one were difficult to clone in both lambda and cosmid clones . A low copy number cosmid vector, pOU61cos, developed previously, was essential for cloning 3 of the 8 gaps.

Nature, 1989 Aug 10, 340(6233), 478 - 82
Homology of 54K protein of signal-recognition particle, docking protein and two E . coli proteins with putative GTP-binding domains; Romisch K et al.; Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER) . Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane . SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein . SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref . 1) . The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane . The 54K protein binds to the signal sequence when it emerges from the ribosome . Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta) . No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli . To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP . Significant sequence homology was found to part of DP alpha and two E . coli proteins of unknown function . The homologous region includes a putative GTP-binding domain.

Biosci Rep, 1989 Aug, 9(4), 465 - 73
Voltage-dependent gating properties of the channel formed by E . coli hemolysin in planar lipid membranes; Menestrina G et al.; Escherichia coli hemolysin forms cation selective, ion-permeable channels of large conductance in planar phospholipid bilayer membranes . The pore formation mechanism is voltage dependent resembling that of some colicins and of diphtheria toxin: pores open when negative voltages are applied and close with positive potentials . The pH dependence of this gating process suggests that it is mediated by a negative fixed charge present in the lumen of the pore . A simple physical model of how the channel opens and closes in response to the applied voltage is given.

Bioorg Khim, 1989 Aug, 15(8), 1091 - 9
{Inclusion of biotinylated analogs of dUTP and dCTP in DNA by DNA-polymerases . Cloning DNA fragments, containing biotinylated deoxyribouridine in E . coli}; Oshevskii SI et al.; The synthesis of a biotinylated derivative of dCTP, viz . N4-{(N-biotinyl)-4-amino-butoxyl}-2'-deoxycytidine 5'-triphosphate (I), is described . DNA polymerase I (Klenow fragment) incorporates (I) in DNA chains instead of thymidine, although with a lower efficiency than previously described biotinylated dUTP derivative (II), whereas highly purified DNA polymerase alpha from human placenta uses as substrate derivative (II) but not (I) . A DNA fragment bearing biotin residues in one of strands was synthesized with the use of DNA polymerase alpha and dUTP derivative (II); its cloning in the plasmid vector pBR322 revealed that the DNA nucleotide sequence remained intact.

Biokhimiia, 1989 Aug, 54(8), 1308 - 14
{Protease from a strain of bacteria E . coli A2, specifically cleaving actin}; Usmanova AM et al.; A novel bacterial protease specifically hydrolyzing actin with the formation of a stable fragment with Mr of 36 kDa was obtained . This protease was shown to be synthesized at the stationary phase of bacterial culture growth . The actin hydrolysis by bacterial protease was inhibited by o-phenanthroline, EDTA and p-chloromercuribenzoate but not by N-ethyl-maleimide, phenylmethylsulfonylfluoride, Leu-peptin, pepstatin and other serine proteinase inhibitors . The protease was stable within the pH range of 4.5-8.5 and had an activity optimum at pH 7.0-8.0 . The protease activity was maintained for 40 min at 45 degrees C and for 30 min at 50 degrees C; at 65 degrees C the enzyme was fully inactivated by 5 min heating . The protease preparations causing quantitative conversion of actin into a 36 kDa fragment did not hydrolyze casein, albumin, ovalbumin, lysozyme, DNAase I, RNAase, myosin, alpha-actinin, tropomyosin and troponin . It was assumed that the protease under consideration is a neutral metalloprotease specifically hydrolyzing actin.

J Biochem (Tokyo), 1989 Aug, 106(2), 323 - 30
Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E . coli; Shinkai A et al.; The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied . Both the intact OmpC and central domain-deleted OmpC were examined . The hydrophobic segment was derived from the signal peptide of OmpF . Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment . Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments . Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane . Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane . It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain . The secretion was dependent on the amino-terminal signal peptide . Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation . Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.

Br J Dermatol, 1989 Aug, 121(2), 229 - 33
Preventive effect of an E . coli-filtrate (Colibiogen) in polymorphous light eruption; Przybilla B et al.; During the administration of Colibiogen, a commercially available E . coli-filtrate, there was a significant (P less than 0.01) increase of the test dose of UVA necessary to elicit skin lesions in nine patients with polymorphous light eruptions . Control tests performed in the same manner without the drug indicate that this was due to treatment . Colibiogen appears to be of value in the treatment of polymorphous light eruption (PLE).

Zentralbl Hyg Umweltmed, 1989 Aug, 188(5), 481 - 5
A single-tube confirmatory test for E . coli using brilliant green mannitol methylumbelliferyl-beta-d-glucuronide tryptophane tryptone broth (BMMTT-medium); Schubert RH et al.; The paper briefly describes the development of a third marker in the single tube technique used in the confirmation of E . coli in water . It has been shown that the third marker, is totally compatible with the markers of gas production from mannitol and indole from tryptophane . The principle of the third marker is to utilise the E . coli specific enzyme, beta-d-glucuronidase, which produces the photoluminescent methylumbelliferone.

Mutat Res, 1989 Aug, 226(4), 259 - 62
DNA photolyase in E . coli: effects on UV mutagenesis by plasmids expressing the phr gene; Yamamoto K et al.; Derivatives of an E . coli plasmid pKY33 are described having specific insertions or deletions that effect or do not effect the phr gene (for DNA photolyase) carried in this plasmid . The various plasmids are tested to determine which cause an inhibition of UV mutagenesis producing glutamine tRNA ochre suppressor mutations . The inhibition is found to require a functional phr gene, which substantiates our earlier report that amplified DNA photolyase interferes specifically with a category of mutagenesis involving targeting by a pyrimidine dimer.

Mutat Res, 1989 Aug, 216(4), 189 - 96
Investigations on the use of EDTA-permeabilized E . coli cells in liquid suspension and animal-mediated genotoxicity assays; Knasmuller S et al.; The potential use of EDTA-permeabilized E . coli cells for the investigation of genotoxic effects of compounds with a large molecular configuration in vitro and in animal-mediated differential DNA-repair assays was studied . The indicator for the induction of (repairable) DNA damage was a pair of E . coli K-12 strains (343/765 and 343/753) differing vastly in DNA-repair capacity (uvr+/rec+ vs . uvrB/recA) . Investigations on the influence of EDTA treatment on the viability of these strains show that during short-term exposure (3 min), the EDTA level should not exceed 0.5 mmole/l in the pretreatment mix, since at higher concentrations a marginal titer reduction of the repair-deficient strain occurs, thus indicating a weak genotoxic activity of this chelating agent . Comparisons of the results gained in vitro with permeabilized and untreated cells demonstrate that EDTA exposure leads to a substantial enhancement of the sensitivity of the indicator bacteria towards DNA damage induced by B(a)P and N-Ac-2AAF which is essential for the detection of genotoxic activities of these polycyclic aromatic compounds . Experiments to elucidate the possibility of employing EDTA-treated cells in vivo show that following intravenous and oral administration the recovery rates of permeabilized indicator strains from various mouse organs are substantially lower than those found under identical conditions (exposure time 150 min) with untreated strains . Nevertheless enough viable cells can be recovered from liver, spleen, kidneys, lungs and stomach to allow the investigation of organ-specific genotoxicity . It is furthermore noteworthy that exposure of permeabilized indicator cells in control animals (for 150 min) resulted in a marginal reduction of the relative survival of the repair-deficient strain in all organs investigated, whereas with non-treated strains such effects are only detectable after extended exposure periods . The observation of a slightly elevated genotoxic background under in vivo conditions does not prevent the assessment of the organ distribution of genotoxic effects induced by mutagens and/or carcinogens: in the case of B(a)P, intraperitoneal administration to mice in the dose range of 10-50 mg/kg body weight resulted in a pronounced dose-dependent inactivation of the uvrB/recA cells in the liver . Also in the lungs differential killing effects occurred at the highest dose tested, whereas no genotoxic activities were detectable in stomach, kidneys and spleen of the host animals.

Biochem Biophys Res Commun, 1989 Jul 31, 162(2), 715 - 23
Stereoselective, strong inhibition of ribonucleotide reductase from E . coli by cisplatin; Smith SL et al.; Using ribonucleotide reductase (EC 1.17.4.1) purified from E . coli clones with overproducing plasmids for the B1 and B2 subunits, respectively, studies have been carried out of the inhibition of this enzyme by cisplatin . Under anaerobic conditions, using the dithiol, reduced form of the enzyme, it was found that ribonucleotide reductase is extremely sensitive to cisplatin: greater than 90% inhibition was achieved with 2-fold molar excess of platinum reagent even at 10(-8)M enzyme . Inhibition was essentially instantaneous and irreversible to G-25 gel filtration . The site of inhibition was found to be the B1 subunit . Transplatin was much less effective . Inhibition of the enzyme by cisplatin (molar ratio cisplatin:B1 = 4.3) led to a decrease in thiol titre corresponding to approximately 1 thiol group per dimer of B1 subunits under conditions leading to 94% inactivation of the ribonucleotide reductase activity.

Nucleic Acids Res, 1989 Jul 11, 17(13), 5097 - 105
NMR study of the structural changes induced in the E . coli lac promoter by the specific binding of the CAP protein; Schumacher R et al.; We have studied the binding of the CAP protein to an 18 base pair lac promoter sequence comprising the core of the CAP recognition sequence . Specific binding of this sequence was established by competition binding assays and comparison of the relative affinities of a number of lac promoter, lac operator, and unspecific sequences of different lengths . The effect of the binding of CAP to the 18 base pair promoter sequence and, for comparison, to an 18 base pair symmetric operator and an oligonucleotide of unrelated sequence have been studied by 1H NMR . Binding of CAP does not bring about any changes in the chemical shift values of the imino proton resonances of the DNA, but causes the selective line broadening of two of the resonances . The comparison of these data with results of gel retardation assays published previously (1) allows the identification and localization of a kink induced in the DNA by the CAP binding to its specific site on the lac promoter.

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