Escherichia coli

Res Vet Sci, 1987 Jul, 43(1), 122 - 3
Haemagglutination patterns of the different variants of Escherichia coli K88 antigen with porcine, bovine, guinea pig, chicken, ovine and equine erythrocytes; Bijlsma IG et al.; Determination of the porcine adhesive phenotype was not achieved by haemagglutination (HA) of porcine erythrocytes, which in all cases were agglutinated by K88ab and K88ad, independent of the adhesive phenotype as determined by the brush border adhesion test . K88ac always gave negative HA results with porcine red cells . However, HA appeared to offer a method of differentiating between the K88 variants without monospecific antisera . K88ab agglutinated porcine, guinea pig and chicken erythrocytes; K88ac agglutinated only guinea pig red cells and K88ad produced haemagglutination with porcine and guinea pig erythrocytes.

Radiobiologiia, 1987 Jul-Aug, 27(4), 516 - 21
{Protective effects of cysteamine and anoxia in Escherichia coli mutants}; Korystov IuN et al.; The values of the oxygen effect (m) and maximum radioprotective effect of cysteamine (DMF*) have been determined for the following Escherichia coli strains: AB1157 (the wild type), AB1886 (uvrA), AB2463 (recA), and p3478 (polA) . Three mechanisms are shown to be responsible for the protective effect of cysteamine, they are: (1) anoxia, (2) diminution of the indirect effect, (3) an increased efficiency of enzyme repair . Anoxia is the main contributor to the protective effect in all the strains under study while mechanism (3) is only effective in the wild type strain.

Radiobiologiia, 1987 Jul-Aug, 27(4), 454 - 9
{Survival and repair of single-stranded DNA breaks after gamma-irradiation of Escherichia coli cells depending on the presence of plasmids pKN101 and COIIB-P9}; Zhestianikov VD et al.; Plasmids, pKM101 and ColIb-P9, present in an autonomous state in E . coli AB 1157, JC5519, and P3478 cells at the stationary and logarithmic phases of growth, somewhat sensitize the cells to the lethal effect of gamma-radiation and do not influence the radiosensitivity of B/r, Bs-1 gamma R, Bs-1, and W3110 cells . The efficiency of repair of gamma-ray-induced DNA single-strand breaks in AB1157 and P3478 cells containing plasmids is somewhat lower than that in the same non-plasmid strains.

J Emerg Med, 1987 Jul-Aug, 5(4), 275 - 82
Anesthetic properties and toxicity of bupivacaine and lidocaine for infiltration anesthesia; Fariss BL et al.; The purpose of this study was to measure the toxicity and anesthetic properties of two anesthetic agents, bupivacaine and lidocaine . These anesthetic agents did not damage tissue defenses or invite infection in experimental animals . In addition, the pain of subdermal injection, the onset of anesthesia, and the frequency of satisfactory anesthesia in human volunteers were remarkably similar . Because the duration of anesthesia induced by bupivacaine was nearly four times longer than that by lidocaine, bupivacaine is recommended for infiltration anesthesia of lacerations treated in the emergency department.

J Clin Pathol, 1987 Jul, 40(7), 782 - 6
Ulcerative colitis and Escherichia coli with adhesive properties; Burke DA et al.; A quantitative assessment of the mannose resistant, adhesive property of Escherichia coli from patients with ulcerative colitis and from controls was made using a buccal epithelial cell adhesion assay, which also permitted assessment of adhesion to cells from other host sources . E coli from patients with ulcerative colitis adhered to buccal epithelial cells to a greater extent than those obtained from controls (p less than 0.0001), but did not show an increased affinity for the buccal epithelial cell of their host, compared with those obtained from other sources . The mannose resistant adhesive property of E coli raises the possibility that it has a role in the pathogenesis of ulcerative colitis.

Biull Eksp Biol Med, 1987 Jul, 104(7), 91 - 3
{Proliferative activity of the hematopoietic stem cell in antigenically stimulated splenectomized mice}; Gaidul' KV et al.; Proliferative activity of hemopoietic bone-marrow stem cells has been studied in splenectomized mice in response to sheep red blood cells (2 X 10(8}, pneumococcal polysaccharide (100 micrograms) and lipopolysaccharide E . coli (100 micrograms) injections . Spleen and its lymphoid cellular elements were shown to be of great importance for the regulation of the functional activity of hemopoietic stem cells under the antigenic influence.

Photochem Photobiol, 1987 Jul, 46(1), 15 - 21
Photochemical modifications of lac repressor--II . Tryptophan photochemistry as a probe in studying the allosteric behaviour of the protein; Spodheim-Maurizot M et al.; Irradiation of lac repressor under aerobic conditions in the near UV region (295-400 nm) decreases the Trp fluorescence of the protein . A total loss of fluorescence corresponds to the destruction of all tryptophanyl residues . Irradiation with light of wavelength between 250 and 400 nm quenches fluorescence completely when only half of the Trp residues ae destroyed . An internal photodynamic effect, in which N-formylkynurenine, a principal photoproduct of Trp, sensitizes further the destruction of the other Trp residues, accounts for our results . Experiments performed in the presence of sodium azide suggest that singlet oxygen is not involved in the destruction of Trp, but may be responsible for histidine degradation . Irradiating the repressor complexed with non-operator E . coli DNA has the same effect on Trp residues as irradiating repressor alone . On the contrary, when repressor is complexed to lac operator, both tryptophanyl residues seem to be destroyed simultaneously . This indicates that binding of specific operator DNA at the DNA site induces changes in the environment of the tryptophanyl residues (mainly tor Trp 220) which cannot further transfer in excitation energy to the photoproduct of the other Trp . A prolonged irradiation destroys the complex, leading to the same result observed for non-specific complex or for repressor alone . These results are discussed in terms of the proximity of Trp from the inducer binding site and the allosteric behaviour of the repressor.

Genetics, 1987 Jul, 116(3), 349 - 58
Evolution of Escherichia coli during growth in a constant environment; Helling RB et al.; Populations of Escherichia coli, initiated with a single clone and maintained for long periods in glucose-limited continuous culture, developed extensive polymorphisms . In one population, examined after 765 generations, two majority and two minority types were identified . Stable mixed populations were reestablished from the isolated strains . Factors involved in the development of this polymorphism included differences in the maximum specific growth rate and in the transport of glucose, and excretion of metabolites by some clones which were utilized by minority clones.

Genetics, 1987 Jul, 116(3), 343 - 7
Spontaneous mutations occur near dam recognition sites in a dam- Escherichia coli host; Carraway M et al.; The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA . Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5'-GATC-3' sequences . In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand . If both strands are fully methylated, repair is inhibited . Mutant (dam-) strains of E . coli defective in the adenine methylase that recognizes 5'-GATC-3' sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam+ hosts . We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt . The majority of these mutations are A:T----G:C transitions that occur within six base pairs of the two 5'-GATC-3' sequences in the mnt gene . In contrast, the spectrum of mnt- mutations in a dam+ host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites . These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors.

Biophys J, 1987 Jul, 52(1), 23 - 8
Quenching of alkaline phosphatase phosphorescence by O2 and NO . Evidence for inflexible regions of protein structure; Strambini GB; The rate constant for quenching the phosphorescence of alkaline phosphatase by molecular oxygen was measured as a function of temperature . The results disagree with previous determinations and, contrary to fluorescence quenching, show that diffusion of O2 to this region of the macromolecule is a highly hindered process . When nitric oxide is introduced as a quencher, similarly small rate constants were found . While the activation energy for this process is identical for both quenchers, it is much smaller than for structural fluctuations at the chromophore site as manifested by the intrinsic triplet-state lifetime . These findings are analyzed in terms of a mechanism that takes into account static quenching at large distances and does not require penetration of the quencher all the way to the chromophore.

Arch Biochem Biophys, 1987 Jul, 256(1), 90 - 100
Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene; Beremand PD et al.; A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence . Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19 . These partial gene constructions were joined and inserted into the plasmid pTZ19R . DNA sequencing confirmed the accuracy of the constructions . The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter . Western blot analysis and radioimmunoassay indicated that E . coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein . The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.

Arch Biochem Biophys, 1987 Jul, 256(1), 223 - 31
Biosynthesis of o-succinylbenzoic acid . II: Properties of o-succinylbenzoic acid synthase, an enzyme involved in vitamin K2 biosynthesis; Weische A et al.; The enzyme system (OSB2 synthase) catalyzing the synthesis of o-succinylbenzoic acid from isochorismic acid and alpha-ketoglutaric acid in the presence of thiamine pyrophosphate was isolated from Escherichia coli AN 154 and characterized . The purification factor of the enzyme did not increase during column chromatography on Sephadex G-200 or chromatofocusing, suggesting that the OSB synthase is labile . Chromatography on DEAE-Sephadex A-50 or A-25 showed that an enzyme activity separated from fractions containing OSB synthase that decarboxylates alpha-ketoglutarate . This activity is provisionally referred to as the decarboxylating "subunit" or decarboxylating activity of OSB synthase . Both the "subunit" and the holoenzyme were characterized with respect to pH optimum, temperature optimum, and KM values . The OSB synthase loses all activity during treatment with EDTA and activity is most efficiently restored with Mn2+ . The activity of the decarboxylating subunit did not depend on Mn2+ . When the decarboxylating fraction was incubated with alpha-ketoglutarate and thiamine pyrophosphate, succinic semialdehyde could be isolated as its hydrazone . After treatment of the incubation mixture with phosphorylase a compound was isolated which is most likely the thiamine adduct of succinic semialdehyde.

Am J Physiol, 1987 Jul, 253(1 Pt 1), E33 - 9
Increased uptake and phosphorylation of 2-deoxyglucose by skeletal muscles in endotoxin-treated rats; Meszaros K et al.; Glucose metabolism of respiratory and nonrespiratory muscles of different fiber composition was investigated in conscious rats . The accumulation of phosphorylated 2-deoxyglucose (2DGP) was increased in skeletal muscles by 56-102% and in diaphragm by 236% at 3 h after treatment with 100 micrograms/100 g endotoxin . The increase was still marked at 24 h, whereas it diminished at 48 h in the diaphragm, abdominal muscle, and white portion of the quadriceps . In the red portion of this muscle 2DGP accumulation was less than that in time-matched controls at 24 and 48 h . Whole gastrocnemius (mixed-fiber types) showed no changes after 24 h . The high 2DGP accumulation in brain remained stable . The retention of 2DGP in tissues, studied by sequential double labeling, did not change 3 h after endotoxin . The lumped constant was similar in the isolated epitrochlear muscles of endotoxemic and control rats . Whole-body glucose utilization (Rd) was increased by 68% 3 h after endotoxin, but it was normal at 24 and 48 h . The increase of glucose utilization by the entire skeletal muscle mass was responsible for approximately 25% of the increase in Rd; therefore it appears that other tissues also contributed significantly to the endotoxin-induced alterations in carbohydrate metabolism.

Am J Clin Pathol, 1987 Jul, 88(1), 78 - 82
The histopathology of rectosigmoid biopsies from adults with bloody diarrhea due to verotoxin-producing Escherichia coli; Kelly JK et al.; The histopathology of rectosigmoid biopsies from 20 patients with bloody diarrhea resulting from verotoxin-producing Escherichia coli infection is reported . The biopsies displayed a range of appearances, from normal to mild, nonspecific inflammation to acute infectious-type colitis . Surface-adherent or invasive bacteria were not identified . The morphologic features of infectious colitis and the absence of bacteria suggest that verotoxin may be responsible for the pathologic changes.

Virology, 1987 Jul, 159(1), 67 - 75
Proteolytic activity of the cowpea mosaic virus encoded 24K protein synthesized in Escherichia coli; Garcia JA et al.; The function of the 24-kilodalton (24K) protein encoded by cowpea mosaic virus (CPMV) has been studied by constructing a bacterial expression plasmid that contained a cloned chimeric segment consisting of partial DNA copies of CPMV M-RNA (including sequences coding for both capsid proteins) and B-RNA (including sequences coding for the 24K protein) . Viral sequences were transcribed from the phage T7 promoter phi 10 of plasmid pT7-6 using T7-RNA polymerase expressed from plasmid pGP1-2 present in the same cells . Upon inducing the synthesis of T7-RNA polymerase several new polypeptides that contained CPMV-specific sequences were expressed, as demonstrated by immunoprecipitation and immunoblotting . Furthermore a proteolytic activity was detected in induced cells which cleaved the viral protein sequences specifically at two glutamine-glycine sites . One of the cleavage products represented capsid protein VP23 . The proteolytic activity was absent when an 87-bp deletion was introduced in the coding region for the 24K protein, indicating that this protein represented the protease involved in the proteolytic processing at those specific sites.

Toxicology, 1987 Jul, 45(1), 103 - 12
Cytotoxicity and induction of repairable DNA damage by photoactivated 5-nitrofurfural; Busker RW et al.; 5-Nitrofurfural (NFA) an important photodecomposition product and metabolite of medicinal nitrofurans is phototoxic in bacterial test systems . Its major photodecomposition product 5-hydroxymethylene-2(5H)-furanone (HMF) appears to be responsible for this . Furthermore HMF and photoactivated nitrofurfural can induce repairable DNA damage in Escherichia coli bacteria . These effects may be important with respect to skin and allergic reactions and carcinogenicity appearing in nitrofuran therapy.

Proc Natl Acad Sci U S A, 1987 Jul, 84(13), 4441 - 4
Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase alpha subunit; Yutani K et al.; To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, we have constructed a series of variant alpha subunits of tryptophan synthase from Escherichia coli substituted by each of 20 amino acids at position 49, which is buried in the interior of the protein . The stabilities were quantitatively examined except for the mutant protein substituted by arginine, which was not obtained in enough quantity . The Gibbs energy of unfolding in water and the activation Gibbs energy of unfolding in 3 M guanidine hydrochloride for each protein were compared at pH 7.0 and pH 9.0 . The Gibbs energy of unfolding in water at pH 7.0 varied from 0.72 to 1.92 times that of the wild-type protein by the substitutions, but the activation Gibbs energy of unfolding in 3 M guanidine hydrochloride varied only from 0.95 to 1.03 times that of the wild-type protein . Moreover, the stability of the protein substituted at this position, which is buried in the interior of the molecule, tended to increase linearly with increasing hydrophobicity of the substituted residue, unless the volume of the substituted residue was over a certain limit.

Proc Natl Acad Sci U S A, 1987 Jul, 84(13), 4428 - 31
Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells; Moonen P et al.; Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied . Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size . Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography . Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation . Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells . Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold . Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E . coli . The E . coli produced-protein--lacking any carbohydrate--had by far the highest specific activity observed for the recombinant hGM-CSFs.

Mutat Res, 1987 Jul, 184(1), 1 - 6
Kinetics of recB-dependent repair: relationship to post-UV inactivation of the prophage; Trgovcevic Z et al.; By making use of the temperature-sensitive mutant recB270, we showed that the RecBCD enzyme is needed for repair between 1 and 4 h after UV exposure . recB-dependent prophage inactivation (Petranovic et al . (1984), Mol . Gen . Genet., 196, 167-169) takes place in all dying cells during the same period of time . The kinetics of decrease in the yield of recombinants in phage-propage crosses resemble those of prophage inactivation in UV-irradiated bacteria . This indicates that recombination processes (including site-specific recombination required for prophage excision) are blocked in cells destined to die . On the basis of our results, we suggest that a large fraction of damaged cells is rescued by the RecA-RecBCD recombination pathway . If repair is unsuccessful, RecA-RecBCD recombination intermediates persist in the irradiated cells leading to prophage inactivation.

J Leukoc Biol, 1987 Jul, 42(1), 1 - 8
In vitro cultured human monocytes release fibroblast proliferation factor(s) different from interleukin 1; Austgulen R et al.; Human monocytes cultured in vitro released factors with growth stimulatory effect upon human dermal fibroblasts . Monocytes in RPMI 1640 released a growth enhancing activity in nearly constant amounts during in vitro differentiation to macrophage-like cells . The growth stimulatory effect mediated by supernatants was reduced when lipopolysaccharide (LPS) or opsonized zymosan particles (OpZ) were added to monocytes during the first 5 days of in vitro culture, thereafter reaching similar levels . The release of interleukin 1 (IL-1) from monocytes was restricted to the first 2 days of culture . IL-1 production by unstimulated monocytes was negligible, while LPS induced a high release of IL-1 from monocytes . A rabbit antibody against human IL-1 abolished the IL-1 activity of the monocyte supernatants as assessed in a mouse thymocyte proliferation assay, but caused only a small reduction of the fibroblast proliferation activity . Thus, the fibroblast growth stimulatory activity mediated by monocytes was caused by factor(s) in addition to IL-1.

J Bacteriol, 1987 Jul, 169(7), 3375 - 8
Similarities in control of mini-F plasmid and chromosomal replication in Escherichia coli; Shields MS et al.; In Escherichia coli, concentrations of a mini-F plasmid with two origins of replication (oriV and oriS) were 50% lower in fast-growing cells than in slow-growing cells . By contrast, a mini-F plasmid deleted for oriV maintained a uniform concentration in both fast- and slow-growing cells, and in this behavior the plasmid mimicked the control by the host of chromosomal origin (oriC) concentration.

J Bacteriol, 1987 Jul, 169(7), 3340 - 9
Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product; Jones HM et al.; The fumarate reductase enzyme complex, encoded by the frdABCD operon, allows Escherichia coli to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation . To analyze the expression of fumarate reductase, protein and operon fusions were constructed between the frdA and the lacZ genes and introduced onto the E . coli chromosome at the lambda attachment site . Expression of beta-galactosidase from either fusion was increased 10-fold during anaerobic versus aerobic cell growth, increased an additional 1.5-fold by the presence of fumarate, the substrate, and decreased 23-fold by nitrate, a preferred electron acceptor . The addition of trimethylamine-N-oxide as an electron acceptor did not significantly alter frdA'-'lacZ expression . Control of frd operon expression is therefore exerted at the transcriptional level in response to the availability of the electron acceptors oxygen, fumarate, and nitrate . Anaerobic induction of frdA'-'lacZ expression was impaired in an fnr mutant and was restored when the fnr+ gene was provided in trans, thus establishing that the fnr gene product, Fnr, is responsible for the anaerobic activation of frd operon expression . Nitrate repression of frdA'-'lacZ expression was observed under either aerobic or anaerobic cell growth conditions in both wild-type and fnr mutant strains, demonstrating that the mechanism for nitrate repression is independent of nitrate respiration and oxygen control imparted by Fnr . Studies performed with a fnr'-'lacZ protein fusion confirmed that the fnr gene is expressed both aerobically and anaerobically . A model is proposed for the regulation of frdABCD operon expression in response to the availability of the alternate terminal electron acceptors oxygen, nitrate, and fumarate.

J Bacteriol, 1987 Jul, 169(7), 3289 - 94
NAD-linked aldehyde dehydrogenase for aerobic utilization of L-fucose and L-rhamnose by Escherichia coli; Chen YM et al.; Mutant analysis revealed that complete utilization of L-fucose and L-rhamnose by Escherichia coli requires the activity of a common NAD-linked aldehyde dehydrogenase which converts L-lactaldehyde to L-lactate . Mutations affecting this activity mapped to the ald locus at min 31, well apart from the fuc genes (min 60) encoding the trunk pathway for L-fucose dissimilation (as well as L-1,2-propanediol oxidoreductase) and the rha genes (min 88) encoding the trunk pathway for L-rhamnose dissimilation . Mutants that grow on L-1,2-propanediol as a carbon and energy source also depend on the ald gene product for the conversion of L-lactaldehyde to L-lactate.

J Bacteriol, 1987 Jul, 169(7), 3138 - 45
Physical mapping of the K+ transport trkA gene of Escherichia coli and overproduction of the TrkA protein; Hamann A et al.; The position on the Escherichia coli chromosome of trkA, a gene coding for a membrane protein involved in K+ transport by the constitutive uptake system Trk, was determined . We observed that the gene is transcribed in a clockwise direction and that it is located at 72.4 min on the chromosome in a 1.75-kilobase NruI-EcoRV DNA fragment 1.0 kilobase upstream of rplQ . We localized an additional gene encoding a 17,000-molecular-weight protein of unknown function between the trkA and rplQ genes . A plasmid, pDB3, was constructed in which the transcription of the trkA gene was put under the control of the lambda pL promoter . pDB3-containing cells of a strain, which contained the temperature-sensitive lambda repressor cI857 in the chromosome, overproduced the 53,000-molecular-weight TrkA protein at the nonpermissive temperature to such an extent that TrkA became the major cell protein . From cell fractionation studies, we conclude that the overproduced TrkA protein forms aggregates.

J Bacteriol, 1987 Jul, 169(7), 3131 - 7
Synthesis of linear multimers of OriC and pBR322 derivatives in Escherichia coli K-12: role of recombination and replication functions; Silberstein Z et al.; Inactivation of RecBCD nuclease (exonuclease V) and SbcB nuclease (exonuclease I) in Escherichia coli K-12 diverts most of plasmid replication activity from circular monomer production to the synthesis of linear multimers . Linear multimer synthesis has been demonstrated in plasmids of diverse origins and copy numbers, including E . coli minichromosomes . The effect of dnaA, dnaB, recF, and recJ mutations on the rate of linear multimer synthesis in sbcB cells after gam inactivation of RecBCD nuclease was investigated . Results are consistent with the hypothesis that homologous recombination, but not activities at the plasmid origin of replication, is involved in initiation of linear multimer synthesis.

J Bacteriol, 1987 Jul, 169(7), 3059 - 61
Maltotriose is the inducer of the maltose regulon of Escherichia coli; Raibaud O et al.; In a cell-free system programmed with a plasmid bearing a malP'-'lacZ gene fusion under the control of malPp, beta-galactosidase synthesis was strictly dependent on the presence of both the MalT activator protein and the inducer of the Escherichia coli maltose regulon . We show that, among all maltodextrins tested (from maltose to maltoheptaose), only maltotriose was able to induce beta-galactosidase synthesis . Likewise, in an in vitro transcription system, initiation of transcription at malPp required the presence of the MalT protein and maltotriose along with the RNA polymerase holoenzyme; neither maltose nor maltotetraose could substitute for maltotriose.

J Bacteriol, 1987 Jul, 169(7), 3023 - 8
Structural role for a conserved region in the CTP synthetase glutamine amide transfer domain; Weng ML et al.; Site-directed mutations were introduced into a conserved region of the Escherichia coli CTP synthetase glutamine amide transfer domain . The amino acid replacements, valine 349 to serine, glycine 351 to alanine, glycine 352 to proline, and glycine 352 to cysteine, all increased the lability of CTP synthetase . The proline 352 replacement abolished the capacity to form the covalent glutaminyl-cysteine 379 catalytic intermediate, thus preventing glutamine amide transfer function; NH3-dependent CTP synthetase activity was retained . In CTP synthetase (serine 349), both glutamine and NH3-dependent activities were increased approximately 30% relative to that of the wild type . CTP synthetase mutants alanine 351 and cysteine 352 were not overproduced because of apparent instability and proteolytic degradation . We conclude that the conserved region between residues 346 and 355 in the CTP synthetase glutamine amide transfer domain has an important structural role.

J Bacteriol, 1987 Jul, 169(7), 2967 - 76
Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide; Imlay JA et al.; Killing of Escherichia coli by hydrogen peroxide proceeds by two modes . Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure . Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J . A . Imlay and S . Linn, J . Bacteriol . 166:519-527, 1986) . H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2 . Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide . Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair . Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2 . After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively . Exposure of E . coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2 . Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response . Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response . Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2 . Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state . gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.

Infect Immun, 1987 Jul, 55(7), 1715 - 7
Enhanced endotoxin effects in plasma fibronectin-deficient rats; Yoder MC et al.; Immunoreactive plasma fibronectin depletion has been associated with the presence of collagen-fibronectin complexes in patients after trauma and in animal models of traumatic and burn injuries . However, the role of plasma fibronectin in the development of sepsis after traumatic and burn injuries in patients is unknown . Treatment of patients and animals with purified human plasma fibronectin ameliorates some of the clinical and metabolic effects of systemic endotoxemia . We report that the induction of immunoreactive plasma fibronectin deficiency by gelatin infusion is associated with enhanced effects of intraperitoneal Escherichia coli endotoxin injection . We have observed a significant increase in the concentrations of ammonia in plasma of treated rats compared with those in control rats administered the same dose of endotoxin.

Eur J Biochem, 1987 Jul 1, 166(1), 151 - 6
Chemical synthesis, molecular cloning and expression of gene coding for a Bowman-Birk-type proteinase inhibitor; Flecker P; A gene coding for a Bowman-Birk-type proteinase inhibitor was synthesized chemically, cloned and expressed in Escherichia coli as a fusion protein with a beta-galactosidase fragment . The corresponding mutant inhibitor, carrying a P1 = Arg16 instead of Lys and an Ile27 instead of Met was obtained after cyanogen bromide cleavage, refolding and affinity chromatography on trypsin-Sepharose . Dissociation constants of complexes with trypsin of this mutant and wild-type Bowman-Birk inhibitor are identical within experimental error . This is explained by differential patterns of hydrogen bonds between side-chains of Arg or Lys in proteinase inhibitors and the primary specificity pocket of trypsin.

Crit Care Med, 1987 Jul, 15(7), 687 - 91
Hemodynamic effects of continuous norepinephrine infusion in dogs with and without hyperkinetic endotoxic shock; Melchior JC et al.; We compared, at constant preload maintained by polygeline (gelatin) infusion, the hemodynamic effects of continuous infusion of norepinephrine (0.5, 1, and 1.5 micrograms/kg X min) in anesthetized dogs with and without hyperdynamic endotoxic shock . In both groups, norepinephrine infusion increased systolic, diastolic and mean aortic BP, cardiac index, stroke index, index of myocardial contractility, and mean pulmonary artery pressure . No significant change in right atrial pressure, left ventricular end-diastolic pressure, heart rate, systemic vascular resistance, or pulmonary vascular resistance was observed . Oxygen consumption index and oxygen extraction ratio remained unchanged . Increases in systolic aortic BP were dose-related, whereas maximal effects on other variables were obtained at 0.5 to 1 microgram/kg X min . The rise in aortic pressure resulted from an increased cardiac index but not from an increased systemic vascular resistance . Stroke index increased as contractility improved . The slight alpha-adrenergic effect of continuous, low-dose norepinephrine infusion did not impede the beneficial effects of the marked beta-adrenergic stimulation on cardiac function . The combination of these two effects improved hemodynamic disturbances of hyperdynamic endotoxic canine shock.

Biochem Int, 1987 Jul, 15(1), 35 - 43
Recognition of aminoethylhomocysteine and aminopropylcysteine by aminoacid transport systems and aminoacyl tRNA synthetases; Cini C et al.; In E . coli aminoethylhomocysteine (AEHC) and aminopropylcysteine (APC) do not affect intracellular lysine transport thus showing that they cannot bind the E . coli lysine transport systems . In CHO cells AEHC and APC inhibit lysine and arginine transport, AEHC more than APC, thus indicating that they can bind the cationic aminoacid transport system . They inhibit also leucine transport, APC more than AEHC . Some possible relationships between their structure and their effects on transport systems are considered . AEHC and APC are not activated by aminoacyl-tRNA synthetase preparations from bacterial and mammalian sources.

Biochem J, 1987 Jul 1, 245(1), 59 - 65
Analysis of enzyme kinetics by using integrated rate equations . Arginine decarboxylase; Cox TT et al.; We have used an integrated rate equation to analyse the reaction catalysed by the inducible arginine decarboxylase from Escherichia coli B . The stoichiometry Arginine----agmatine + CO2 is the simplest of the multiple-substrate/multiple-product cases . Twenty-one time courses were carried out at various initial concentrations of arginine and agmatine, and were then fitted to the integrated equation by using appropriate analytical procedures . Values were obtained for six of the seven possible kinetic constants, corresponding to kcat, KArg, the terms for competitive inhibition by agmatine, by CO2 and by agmatine and CO2 together, and the term for uncompetitive inhibition by agmatine . The uncompetitive constant for CO2 was indeterminate . Our results indicate that it is both practical and experimentally economical to obtain kinetic constants from full time courses.

Can J Vet Res, 1987 Jul, 51(3), 319 - 25
Hepatic serine and alanine metabolism during endotoxin-induced fever in sheep; Southorn BG et al.; Time course changes in plasma amino acid concentrations and the hepatic metabolism of serine and alanine were measured in six mature wethers during endotoxin-induced fever . In separate trials, the animals' responses to injections of saline and endotoxin were measured . The endotoxin was from Escherichia coli serotype 055:B5 and was injected intravenously (4 micrograms/kg body weight) . Liver biopsies were obtained from the sheep at 6 h postinjection during both endotoxin and saline injection trials . Rectal temperature in the endotoxin treated animals was increased (P less than 0.05, above that in control animals from 4.25 h to 9 h postinjection, with a maximum rise of 2.43 degrees C at 5.5 h postinjection . Glucose concentration in jugular plasma decreased (P less than 0.05) by 3 h postinjection and remained depressed throughout the 24 h postinjection sampling period . Plasma serine concentration was decreased (P less than 0.05) by 3 h postinjection . Plasma alanine concentration was decreased significantly (P less than 0.05) only at 24 h postinjection . Endotoxin injection increased (P less than 0.05) hepatic oxidation of 14C-serine (162%) and the net incorporation of 14C-serine carbon into hepatic protein (173%) and glycogen (275%) . The net incorporation of 14C-alanine carbon into hepatic protein (172%) and glycogen (323%) were increased (P less than 0.05) by endotoxin injection, while alanine oxidation was not affected by endotoxin treatment (P greater than 0.05) . The increased hepatic use of serine may explain, in part, the dramatic decrease in plasma concentrations of this amino acid following endotoxin injection into sheep.

Calcif Tissue Int, 1987 Jul, 41(1), 31 - 7
Biological characterization of interleukin-1-like cytokine produced by cultured bone cells from newborn mouse calvaria; Hanazawa S et al.; We have investigated the role of interleukin-1 (IL-1) and IL-1-like factor in the regulatory mechanisms of a bone remodeling system . To determine whether the bone cell itself produces IL-1-like cytokine, we examined bone cells cultured from newborn mouse calvaria . Bone cells migrating from fragments of newborn mouse calvaria were used in this study . We also used bone cells obtained by consecutive digestion of the calvaria with enzymes . These bone cells were cultured in fetal calf serum-containing alpha-MEM . IL-1-like cytokine activity was measured by incorporation of {3H}thymidine into C3H/HeJ thymocytes stimulated with PHA . When treated with lipopolysaccharide (LPS) from Escherichia coli 0111 B4, the cultured bone cells produced a significant amount of IL-1-like cytokine . The maximum concentration of IL-1-like cytokine was observed in culture supernatants of the bone cells cultured for 24 hours with the LPS in serum-free medium . The IL-1-like cytokine closely resembles IL-1 in some of its biological characteristics: stimulation of mitogen-induced thymocyte proliferation, stimulation of fibroblast proliferation, pyrogenicity, and molecular weight . These results show that cultured bone cells from newborn mouse calvariae produce an IL-1-like cytokine that closely resembles IL-1.

J Clin Microbiol, 1987 Jul, 25(7), 1176 - 80
Expression of proteins of Mycobacterium tuberculosis in Escherichia coli and potential of recombinant genes and proteins for development of diagnostic reagents; Cohen ML et al.; Recombinant plasmids containing DNA from Mycobacterium tuberculosis were transformed into Escherichia coli, and three colonies were selected by their reactivity with polyclonal antisera to M . tuberculosis . The three recombinant vectors contained DNA inserts of different sizes flanking a common 4.7-kilobase (kb) sequence . Each recombinant produced 35- and 53-kilodalton proteins (35K and 53K proteins, respectively) which were absent in the control E . coli . In Western blotting experiments, both proteins bound several antisera to M . tuberculosis but not antisera to other commonly isolated mycobacteria . Rabbits immunized with the recombinant 35K protein produced antisera which bound to both the 35K and 53K protein bands, a single 35K protein band present in a culture filtrate of M . tuberculosis, and single protein bands with differing molecular weights in whole-cell homogenates from other Mycobacterium spp . An additional recombinant vector containing a 2.2-kb subclone of the 4.7-kb sequence was constructed and, when used as a probe, demonstrated homology with various fragments of chromosomal digests of selected mycobacteria . Reactivity of this probe to Mycobacterium bovis and M . bovis BCG was indistinguishable from reactivity to M . tuberculosis . Immunoglobulin G reactivity to the 35K antigen was detected in antisera from 8 of 20 persons with active tuberculosis, 4 of 18 persons with leprosy, and none of 14 healthy controls . In contrast, reactivity to various proteins in M . tuberculosis culture filtrate was present in 18 of 20 patients with tuberculosis, 16 to 18 patients with leprosy, and 5 of 14 controls . The production of M . tuberculosis proteins by E . coli circumvents many difficulties encountered in the growth and manipulation of M . tuberculosis and may facilitate the development of better diagnostic and immunizing reagents.

Proc Natl Acad Sci U S A, 1987 Jul, 84(14), 4949 - 53
High-frequency deletional rearrangement of immunoglobulin kappa gene segments introduced into a pre-B-cell line; Engler P et al.; We describe an immunoglobulin gene recombination indicator in which a specific rearrangement via deletion results in the acquisition of a dominant phenotype . The indicator consists of the Escherichia coli xanthine/guanine phosphoribosyltransferase (gpt) gene, whose translation is prevented by the presence of an upstream initiation codon out of frame with respect to the gpt coding sequence . Flanking this barrier initiation codon are the heptamer-spacer-nonamer recognition sequences from a kappa chain variable region (V kappa) and from a kappa chain joining region (J kappa) . A proper V-J joint results in the deletion of the translational barrier and allows expression of the selectable marker . When tested by transfection into fibroblasts, no rearrangements were detected and the presence of the barrier initiation codon was sufficient to completely abolish gpt expression in these cells . Similarly, no rearrangements were detected after transfer of the test gene into myeloma cells . However, when the construct was introduced into the pre-B-cell line 38B9, greater than 80% of the transfected cells showed evidence of a specific rearrangement . These rearrangements were associated with the translation of gpt, although no selection for its expression was needed . DNA sequence analysis of six different V-J joints revealed that the rearrangement proceeded with a high degree of accuracy . These results indicate that only very minimal DNA sequences (21 base pairs 5' of the V heptamer and 4 base pairs 3' of its nonamer; less than 45 base pairs 5' of the J nonamer and 3' of its heptamer) are required for efficient rearrangement and provide formal proof that kappa gene segments can rearrange by a deletional mechanism.

Mutat Res, 1987 Jul, 184(1), 7 - 11
Induction of umu gene expression by cross-links and other DNA lesions; Ohnishi T et al.; The induction of umu gene expression by DNA cross-links was investigated in various strains of E . coli with different DNA-repair capacities . Expression was measured by quantifying enzymatic activity of beta-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion . The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain (uvrA) . In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS . None of these chemicals induced umu expression in either lexA and recA strains . The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.

J Bacteriol, 1987 Jul, 169(7), 2938 - 44
Hybrid Escherichia coli sensory transducers with altered stimulus detection and signaling properties; Slocum MK et al.; The tar and tap loci of Escherichia coli encode methyl-accepting inner membrane proteins that mediate chemotactic responses to aspartate and maltose or to dipeptides . These genes lie adjacent to each other in the same orientation on the chromosome and have extensive sequence homology throughout the C-terminal portions of their coding regions . Many spontaneous deletions in the tar-tap region appear to be generated by recombination between these regions of homology, leading to gene fusions that produce hybrid transducer molecules in which the N terminus of Tar is joined to the C terminus of Tap . The properties of two such hybrids are described in this report . Although Tar and Tap molecules have homologous domain structures, these Tar-Tap hybrids exhibited defects in stimulus detection and flagellar signaling . Both hybrid transducers retained Tar receptor specificity, but had reduced detection sensitivity . This defect was correlated with the presence of the C-terminal methyl-accepting segment of Tap, which may have more methylation sites than its Tar counterpart, leading to elevated steady-state methylation levels in the hybrid molecules . One of the hybrids, which carried a more extensive segment from Tap, appeared to generate constitutive signals that locked the flagellar motors in a counterclockwise rotational mode . Changes in the methylation state of this transducer were ineffective in cancelling this aberrant signal . These findings implicate the conserved C-terminal domain of bacterial transducers in the generation or regulation of flagellar signals.

Infect Immun, 1987 Jul, 55(7), 1647 - 51
Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli; Barbieri JT et al.; A mutant, full-length form of diphtheria toxin was cloned into Escherichia coli K-12 and expressed under BL-1 + EK-1 containment . A gene fragment encoding the catalytic domain of the toxin was subjected to oligonucleotide-directed mutagenesis to produce a three-base mutation of an active site residue; Glu-148 was thereby replaced by Ser . Ser-148 fragment A had less than 1% of the ADP-ribosyltransferase activity of wild-type fragment A . Next, the complementary portion of the toxin structural gene was spliced with the mutated DNA fragment downstream of codon 148 to produce a construct that encoded mutant whole toxin with Ser at position 148 . The mutant toxin was indistinguishable from authentic diphtheria toxin by Western blot analysis, but was about 800-fold less cytotoxic than wild-type toxin for BS-C-1 cells . Evidence from subunit exchange experiments indicated that a substantial fraction of the mutant toxin contained a fully functional B moiety, capable of mediating the entry of wild-type fragment A into sensitive mammalian cells . This combination of approaches provides a means of applying recombinant DNA methods in E . coli to study structure-function relationships in whole diphtheria toxin.

Photochem Photobiol, 1987 Jul, 46(1), 45 - 53
Oxygen radicals mediate cell inactivation by acridine dyes, fluorescein, and lucifer yellow CH; Martin JP et al.; Acridine dyes, fluorescein and lucifer yellow CH are fluorescent photosensitizers used experimentally to selectively stain and photodynamically destroy eukaryotic cells and subcellular structures . We have determined that the mechanism of light- and oxygen-dependent inactivation of E . coli by these dyes involves oxygen radicals and hydrogen peroxide . All of the dyes oxidized NAD(P)H+ under illumination . Superoxide (O2), detected as the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c, was a major product of the dye sensitized photooxidation . Cationic acridine dyes penetrated the membranes of E . coli and were photoreduced intracellularly . Reduced dyes diffused back into the medium and mediated the reduction of extracellular ferricytochrome c . The anionic dyes fluorescein and lucifer yellow CH were unable to mediate extracellular cytochrome c reduction, indicating that these dyes were impermeable to the E . coli membrane . Acridine dyes, when illuminated, inhibited the growth of E . coli in a rich medium, and induced the synthesis of SOD . Fluorescein and lucifer yellow CH did not inhibit growth or induce SOD synthesis because they were unable to enter the cells . Superoxide (O2) and hydrogen peroxide (H2O2), generated by the enzyme xanthine oxidase were toxic to E . coli B . Inactivation by xanthine oxidase was partially inhibited by exogenous SOD and completely inhibited by exogenous catalase or SOD plus catalase . Similarly, exogenous SOD plus catalase protected against inactivation by acridines and fluorescein-NADH or lucifer yellow CH-NADH mixtures . Prior induction of superoxide dismutase and catalase in E . coli B significantly protected cells against a subsequent challenge by illuminated acridine dyes . SOD and catalases preinduction combined with additions of exogenous SOD and catalase completely protected E . coli B against photodynamic inactivation by acridine yellow . The hydroxyl radical scavengers, dimethyl sulfoxide, sodium benzoate and thiourea, protected E . coli B against photodynamic inactivation by acridine orange . The results implicate O2, H2O2, and the hydroxyl radical (OH) as underlying molecular agents of the phototoxicity mediated by acridine orange, acridine yellow, fluorescein and lucifer yellow CH.

Mol Cell Biol, 1987 Jul, 7(7), 2352 - 9
Functional organization of the Aspergillus nidulans trpC promoter; Hamer JE et al.; We investigated the functional organization of the Aspergillus nidulans trpC promoter by the sequential removal of sequences upstream of the major trpC mRNA cap site (+1) . DNA fragments containing promoter mutations were fused to the Escherichia coli lacZ gene, and a novel method was used to select for integration of the fusion gene at the Aspergillus argB locus . beta-Galactosidase assays and S1 nuclease protection experiments demonstrated that the promoter mutations affected gene expression in three ways: (i) 5' deletions up to -82 resulted in variable increases in beta-galactosidase activity, depending on the growth conditions; (ii) a deletion from -67 to -11 did not alter the level of beta-galactosidase activity, but did give rise to mRNAs with aberrant 5' ends; and (iii) a 5' deletion with an endpoint at -11 and an internal deletion from -142 to -11 abolished gene expression . These results indicate that sequences upstream of -82 reduce transcription of the trpC gene and that distinct DNA sequence elements are required for expression versus correct initiation of transcription of the trpC gene . The sequences essential for trpC expression do not include the common eucaryotic promoter elements CCAAT and TATAAA . To our knowledge, this is the first functional analysis of a promoter from a fungus other than Saccharomyces cerevisiae.

Arch Biochem Biophys, 1987 Jul, 256(1), 335 - 42
Overproduction, purification, and characterization of adenylosuccinate synthetase from Escherichia coli; Bass MB et al.; Adenylosuccinate synthetase, encoded by the purA gene of Escherichia coli, catalyzes the first committed step toward AMP in the de novo purine biosynthetic pathway and plays an important role in the interconversion of purines . A 3.2-kb DNA fragment, which carries the purA gene, was cloned into the temperature-inducible, high-copy-number plasmid vector, pMOB45 . Upon temperature induction, cells containing this plasmid produce adenylosuccinate synthetase at approximately 40 times the wild-type level . A scheme is presented for the purification of the overproduced adenylosuccinate synthetase to homogeneity in amounts sufficient for studies of its structure and mechanism . The wild-type and the overproduced adenylosuccinate synthetase enzyme preparations were judged to be identical by the following criteria . The amino acid sequence at the N-terminus of the overproduced enzyme proved identical to the corresponding sequence of the wild-type enzyme . Michaelis constants for both the wild-type and overproduced enzyme preparations were the same . And (iii) both proteins shared similar chromatographic behavior and the same mobility during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . Results from size-exclusion chromatography and SDS-polyacrylamide gel electrophoresis suggest that adenylosuccinate synthetase exists as a dimer of identical, 48,000-Da, subunits.

Proc Natl Acad Sci U S A, 1987 Jul, 84(14), 4781 - 5
RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence; Masai H et al.; RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein . The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR) . The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase) . The replication was also completely dependent on the host DnaA function . In filter binding assays in high salt (0.5 M KCl) conditions, RepA specifically binds to both supercoiled and linear plasmid DNA containing the oriR sequence, whereas it binds to nonspecific DNA in low salt . DNase I-protection studies on a linearized DNA fragment revealed that DnaA protein specifically binds to a 9-base-pair DnaA-recognition sequence ("DnaA box") within oriR only when RepA is bound to the sequence immediately downstream of the DnaA box . These results indicate that initiation of R1 plasmid replication is triggered by interaction of RepA and DnaA proteins with the oriR sequence.

Proc Natl Acad Sci U S A, 1987 Jul, 84(13), 4389 - 92
Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits; Maki H et al.; The polymerase subunit (alpha) of Escherichia coli DNA polymerase III holoenzyme and the 3'----5' exonuclease subunit (epsilon) are each less active separately than together in the holoenzyme core (an assembly of alpha, epsilon, and theta subunits) . In a complex formed from purified alpha and epsilon subunits, polymerase activity increased 2-fold, and that of the 3'----5' exonuclease increased 10- to 80-fold . The alpha-epsilon complex contains one each of the subunits as does the core . Stimulation of 3'----5' exonuclease activity is due mainly to a greatly increased affinity of the epsilon subunit for the 3'-hydroxyl terminus, resulting from DNA binding by the alpha subunit . Proofreading in the course of DNA synthesis by the alpha-epsilon complex was indistinguishable from that of the core . These findings identify the participation of the alpha subunit in proofreading by polymerase III holoenzyme and suggest that the fidelity of DNA replication may be influenced by the relative levels of the alpha and epsilon subunits in the cell.

Mutat Res, 1987 Jul, 179(1), 55 - 63
Ionizing and ultraviolet radiation-induced reversion of sequenced frameshift mutations in Escherichia coli: a new role for umuDC suggested by delayed photoreactivation; Sargentini NJ et al.; The ultraviolet (UV) and gamma radiation-induced reversion of the trpA21, trpA9813, and trpE9777 sequenced-frameshift mutations were studied in Escherichia coli K-12 with or without the plasmid pKM101 . Radiation induced the reversion of all 3 frameshifts, and pKM101 enhanced this reversion 10-50-fold . Factors influencing the differential radiation revertability of frameshifts are discussed . The two most revertable frameshifts, trpE9777 and trpA9813, were used as probes to understand the role of the umuDC genes in radiation-induced frameshift reversion . Unlike the UV radiation-induced reversion of base-substitution mutations, the reversion of these frameshifts was not enhanced in a uvrA umuC strain by photoreactivation after a post-UV-irradiation incubation . The UmuDC proteins are suggested to have functions in the radiation induction of frameshifts that are more complex than are their functions in the induction of base substitutions.

J Gen Virol, 1987 Jul, 68 ( Pt 7), 1835 - 48
The complete nucleotide sequence of coxsackievirus B4 and its comparison to other members of the Picornaviridae; Jenkins O et al.; The genome of the prototype stain of coxsackievirus B4 (J.V.B . Benschoten) has been cloned in Escherichia coli and its complete nucleotide sequence determined . Excluding the poly(A) tract, the RNA genome is 7395 nucleotides in length and appears to encode a single polyprotein of 2183 amino acids . The predicted amino acid sequence of the polyprotein shows close homology (88%) to that of the previously sequenced coxsackievirus B3 and to certain regions of the polyproteins of the polioviruses and human rhinovirus 14 . This allows identification of putative polyprotein cleavage signals, antigenic domains and other structural features likely to be important to the biological integrity of the virus.

J Bacteriol, 1987 Jul, 169(7), 3379 - 84
Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21); Heltzel A et al.; In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene . An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame . Overexpressed MerR constituted about 5% of labeled proteins . An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes . A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties.

J Bacteriol, 1987 Jul, 169(7), 3243 - 50
Site-specific methylases induce the SOS DNA repair response in Escherichia coli; Heitman J et al.; Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli . In contrast, expression of methylases indigenous to E . coli either did not induce SOS (EcoRI {GAmeATTC} or induced SOS to a lesser extent (dam {GmeATC}) . Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction) . We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response . Cytosine methylases foreign to E . coli (MspI {meCCGG}, HaeIII {GGmeCC}, BamHI {GGATmeCC}, HhaI {GmeCGC}, BsuRI {GGmeCC}, and M.Spr) also induced SOS, whereas one indigenous to E . coli (EcoRII {CmeCA/TGG}) did not . SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E . A . Raleigh and G . Wilson, Proc . Natl . Acad . Sci . USA 83:9070-9074, 1986).

J Bacteriol, 1987 Jul, 169(7), 3237 - 42
Cloning of the cyo locus encoding the cytochrome o terminal oxidase complex of Escherichia coli; Au DC et al.; The structural genes encoding the cytochrome o terminal oxidase complex (cyo) of Escherichia coli have been subcloned into the multicopy plasmid pBR322 after the Mu-mediated transposition of the gene locus from the bacterial chromosome onto the conjugative R plasmid RP4 . Introduction of cyo plasmids into strains (cyo cyd) lacking both terminal oxidases restored the ability of the strains to grow aerobically on nonfermentable substrates . Strains carrying the cyo plasmids produced 5 to 10 times more cytochrome o oxidase than did control strains . The gene products encoded by the cyo plasmids could be immunoprecipitated with monospecific antibodies raised against cytochrome o . The cloned genes will be valuable for studying the structure, function, and regulation of the cytochrome o terminal oxidase complex.

J Bacteriol, 1987 Jul, 169(7), 2990 - 4
Physical and genetic characterization of the glucitol operon in Escherichia coli; Yamada M et al.; The glucitol (gut) operon has been identified in the colony bank of Clark and Carbon (A . Sancar and W . D . Rupp, Proc . Natl . Acad . Sci . USA 76:3144-3148, 1979) . We subcloned the gut operon by using paCYC184, pACYC177, and pBR322 . The operon, which is encoded in a 3.3-kilobase nucleotide fragment, consists of the gutC, gutA, gutB, and gutD genes . The repressor of the gut operon seemed to be encoded in the region downstream from the operon . The gene products of the gut operon were identified by using maxicells . The apparent molecular weights of the glucitol-specific enzyme II (product of the gutA gene), enzyme III (product of the gutB gene), and glucitol-6-phosphate dehydrogenase (product of the gutD gene) were about 46,000, 13,500, and 27,000, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Mol Gen Genet, 1987 Jul, 208(3), 428 - 35
Direction of transcription affects the replication mode of lambda in an in vitro system; Kouhara H et al.; A set of artificial circular plasmids, named plasmoids, was constructed . They are about 1 kb in size and consist of a 178 bp lambda dv minimal DNA replication origin (ori) which has four direct repeats and the A + T-rich region conferring polarity to the ori fragment, a 775 bp DNA segment that codes for the CAT amino acid sequence and the 99 bp lac promoter (plac) . They carry no other functional genes or genetic sites . The constructions involved various combinations of relative orientations of these components . These molecules do not replicate in vivo because they lack genes coding for initiation proteins, but they do replicate in an in vitro system (Fuller et al . 1981), and can be used for studies of interactions between transcription and replication . In these plasmids, major transcription starts from the strong plac, and some weak unscheduled transcription starts from several other initiation sites . The major RNA synthesis was found to interfere with the unscheduled RNA synthesis, which was occurring on the opposite strand . The most active replication took place when the major RNA synthesis went through the lambda origin region in the direction which occurs naturally in the lambda genome . Under these conditions, DNA synthesis going against such transcription was less than that going along with the major transcription . When RNA synthesis through the lambda origin region was in the opposite direction, DNA synthesis in the same direction was about half of that observed in the above case, whereas that going against transcription was very weak.

Mol Gen Genet, 1987 Jul, 208(3), 398 - 407
Orientation of enzymic domains in tryptophan synthase of Neurospora crassa: an immunoblot analysis of TRP3 mutant products; Matchett WH et al.; Extracts of 52 TRP3 mutants of Neurospora crassa were tested for the presence of serologically cross-reacting material by the method of electrophoretic blot analysis . The test antigen was obtained by excision of lightly stained bands of denatured pure tryptophan synthase after SDS-polyacrylamide gel electrophoresis . Rabbit antisera raised against this antigen neutralized and precipitated native tryptophan synthase . Of the 52 strains, 19 exhibited banding patterns similar to wild type on electrophoretic blots, 25 strains gave no apparent bands, and 8 strains showed unique banding patterns . This evidence and related genetic fine structure mapping data indicate that strains exhibiting banding patterns similar to wild type carry missense mutations . Strains which did not exhibit any obvious bands may have resulted from certain kinds of missense or nonsense mutations or from frameshift mutations or extended deletions . Strains exhibiting unique banding patterns on electrophoretic blots were interpreted as carrying chain-terminating mutations or deletions . Genetic fine structure mapping data place the mutant lesions of these strains in a linear order corresponding to the apparent molecular weights of the crossreacting protein fragments which they exhibit . The direction of transcription and translation of the TRP3 locus in Neurospora was inferred from these relationships . The apparent organization of the Neurospora TRP3 gene is consistent with that ascribed to the Saccharomyces TRP5 system and suggests that the N-terminal portion of the protein corresponds to the "A" protein of the Escherichia coli system and that the C-terminal portion of the protein corresponds to the "B" protein of the bacterial system.

Radiobiologiia, 1987 Jul-Aug, 27(4), 563 - 4
{Reduced restriction of phage lambda in Escherichia coli K-12 cells after gamma-irradiation}; Rabinkova EV et al.; In gamma-irradiated cells of Escherichia coli K-12 restriction alleviation of an unmodified phage lambda is only observed in AB1157 strain . No restriction alleviation by gamma-rays is registered in AB1157 mutants (rec A and ssb-1).

Virology, 1987 Jul, 159(1), 183 - 6
Indications of an involvement of heat-shock proteins in restoration of repression of temperative-inducible lambda prophage; Fuerst CR; Characteristics of lambda c/ts857 prophages that can be attributed to the ability of the temperature-sensitive phage repressor to renature at low temperature are not apparent in host cells that contain a mutation in the htpR gene . Host killing by prophages that are N- or are blocked in DNA synthesis is not prevented by the return of mutant cells to low temperature, and recovery of cells in which the phage remains derepressed is not delayed if the prophage is a mutant that cannot kill . These and other findings suggest that the phage repressor protein is unusually susceptible to inactivation in cells that are unable to respond to heat shock.

J Gen Virol, 1987 Jul, 68 ( Pt 7), 1785 - 9
Altered phage P1 attachment to strains of Escherichia coli carrying the plasmid ColV,I-K94; Goodson M et al.; Phages P1vir and P1cmclrf100 failed to form plaques on or multiply in Escherichia coli strains carrying the ColV,I-K94 plasmid; with P1cmclr100, the effect occurred both with phage from the lytic cycle and with that induced from a lysogen . The effect was on attachment, these P1 phages attaching poorly to ColV,I-K94+ strains . This receptor defect appeared to result mainly from the presence of ColV-encoded transfer and colicin components in the cells carrying ColV,I-K94 and it was specific to this plasmid . Phage Mu (which uses an attachment mechanism similar to that of phage P1) in the G(+) form attached to both Col- and ColV,I-K94+ strains but the G(-) form attached to neither type.

Mol Gen Genet, 1987 Jul, 208(3), 439 - 45
Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae; Klemm P et al.; Three novel fim genes of Escherichia coli, fimF, fimG and fimH, were characterized . These genes were not necessary for the production of fimbriae but were shown to be involved in the adhesive property and longitudinal regulation of these structures . Complementation experiments indicated that both the major fimbrial subunit gene, fimA, and the fimH gene in combination with either the fimF or the fimG gene were required for mannose-specific adhesion . The fimF, fimG and fimH gene products were likewise shown to play a major role in the fimbrial morphology as longitudinal modulators . The amount of FimF, FimG and FimH proteins appeared to control the length and number of the fimbriae . The DNA sequence of a 2050 bp region containing the three genes was determined . The corresponding protein sequences all exhibited homology with the fimbrial subunit protein, FimA.

Microbiologica, 1987 Jul, 10(3), 325 - 30
Patterns of haemagglutinating activity of Escherichia coli; Chiaradia V et al.; MR and MS adhesins on 169 strains of Escherichia coli subjected to different cultural conditions were detected . Haemagglutination Test (static settling test in plastic microtiter trays) was used and several species of red blood cells were employed . The results confirm that different media can influence the expression of the adhesins and that using as many species of red blood cells as possible one can detect different adhesins.

J Bacteriol, 1987 Jul, 169(7), 3051 - 8
Nucleotide sequence and expression of the pyrC gene of Escherichia coli K-12; Wilson HR et al.; The pyrC gene of Escherichia coli K-12, which encodes the pyrimidine biosynthetic enzyme dihydroorotase, was cloned as part of a 1.6-kilobase-pair chromosomal fragment . The nucleotide sequence of this fragment was determined . An open reading frame encoding a 348-amino acid polypeptide (Mr = 38,827) was identified as the pyrC structural gene by comparing the amino acid composition predicted from the DNA sequence with that previously determined for the dihydroorotase subunit . The pyrC promoter was mapped by primer extension of in vivo transcripts . Transcriptional initiation was shown to occur within a region located 36 to 39 base pairs upstream of the pyrC structural gene . Pyrimidine availability appears to affect the use of the minor transcriptional initiation sites . The level of pyrC transcription and dihydroorotase synthesis was coordinately derepressed by pyrimidine limitation, indicating that regulation occurs, at least primarily, at the transcriptional level . Inspection of the pyrC nucleotide sequence indicates that gene expression is not regulated by an attenuation control mechanism similar to that described for the pyrBI operon and the pyrE gene . A possible mechanism of transcriptional control involving a common repressor protein is suggested by the identification of a highly conserved, operatorlike sequence in the promoter regions of pyrC and the other pyrimidine genes (i.e., pyrD and carAB) whose expression is negatively regulated by a cytidine nucleotide effector.

J Bacteriol, 1987 Jul, 169(7), 2945 - 9
Altered translation of the uncC gene coding for the epsilon subunit of the F1F0-ATPase of Escherichia coli; Cox GB et al.; The nucleotide sequence of the previously described uncC424 allele was determined and found to be the same as that of a wild-type uncC gene . However, a G----A change occurred 7 nucleotides upstream from the translation start codon, changing the putative Shine-Dalgarno sequence from GAGG to GAAG . Four revertant strains were examined . In one revertant, which had normal growth and membrane properties, a single base deletion had occurred to re-form the Shine-Dalgarno sequence GAGG 1 nucleotide closer to the translation start codon . A second revertant had a single base deletion in the preceding uncD gene, causing an extension of the beta subunit by 6 amino acids and an increase, presumably by translational coupling, in the amount of epsilon subunit . The third and fourth revertant strains were phenotypically similar and had either C----T or G----T changes 18 or 19 nucleotides, respectively, upstream from the translation start codon.

Infect Immun, 1987 Jul, 55(7), 1734 - 6
Temperature-dependent transcriptional regulation of expression of fimbriae in an Escherichia coli strain isolated from a child with severe enteritis; Williams PH et al.; Escherichia coli 469-3 synthesizes fibrillar fimbriae at 37 degrees C, but not at 18 degrees C, which promote its adherence to human colonocytes . RNA homologous with a cloned fragment of the strain 469-3 chromosome necessary for normal expression of fimbriae was virtually undetectable in bacteria grown at 18 degrees C, indicating that environmental temperature affects expression by regulating transcription.

Mol Microbiol, 1987 Jul, 1(1), 53 - 8
Activation of the lac operon of Escherichia coli by a mutant FNR protein; Spiro S et al.; The FNR protein of E . coli is a transcriptional activator required for the expression of genes involved in anaerobic respiratory pathways . Site-directed mutagenesis was used to alter three amino acids in the recognition helix of the putative DNA-binding domain of FNR, with the aim of changing its specificity to that of the cyclic AMP receptor protein (CRP) . In the presence of the mutant protein (FNR-215) expression of the lac operon was activated during anaerobiosis and unaffected by glucose . FNR-215 did not have a uniform effect on the expression of other cAMP-CRP-dependent genes, but the results demonstrate the fundamental similarity between FNR- and CRP-mediated transcriptional activation.

Mol Microbiol, 1987 Jul, 1(1), 13 - 22
Reassortment of DNA recognition domains and the evolution of new specificities; Gann AA et al.; Type I restriction enzymes comprise three subunits only one of which, the S polypeptide, dictates the specificity of the DNA sequence recognized . Recombination between two different hsdS genes, SP and SB, led to the isolation of a system, SQ, which had a different specificity from that of either parent . The finding that the nucleotide sequence recognized by SQ is a hybrid containing components from both the SP and SB target sequences suggested that DNA recognition is carried out by two separable domains within each specificity polypeptide . To test this we have made the recombinant gene of reciprocal structure and demonstrate that it encodes a polypeptide whose recognition sequence, deduced in vivo, is as predicted by this model . We also report the sequence of the SB specificity gene, so that information is now available for the five known members of this family of enzymes . All show a similar organization of conserved and variable regions . Comparisons of the predicted amino acid sequences reveal large non-conserved areas which may not even be structurally similar . This is remarkable since these different S subunits are functionally identical, except for the specificity with respect to the DNA sequence with which they interact . We discuss the correlation of the variation in polypeptide sequence with recognition specificities.

Mol Microbiol, 1987 Jul, 1(1), 117 - 24
Translation of galE and coordination of galactose operon expression in Escherichia coli: effects of insertions and deletions in the non-translated leader sequence; Bingham AH et al.; Using gene-manipulation techniques, we made a set of short insertions and deletions in the Escherichia coli galactose operon between the transcription start site and the Shine-Dalgarno sequence of the first gene of the operon, galE . Translation initiation is severely reduced when the distance between the 5' end of the message and the Shine-Dalgarno sequence drops below 15 bases . Transcription of the gal operon can start at two distinct sites, S1 and S2, separated by 5 bp, situated 16 and 21 bp upstream of the galE Shine-Dalgarno sequence, respectively . When transcription starts at S2, gal operon expression is discoordinate as the galE gene is better translated than promoter-distal genes . Here we report that gal operon expression is discoordinate even when message starting at S2 is shortened . This shows that the better translation of galE from transcripts starting at S2 is not simply due to the fact that they are longer than transcripts starting at S1.

Plasmid, 1987 Jul, 18(1), 76 - 83
The origin of transfer of P307; Goldner A et al.; The DNA fragment carrying the oriT region from the enterotoxin plasmid P307 was isolated and its polynucleotide sequence was determined . Using Southern hybridization assays with a synthetic oligonucleotide probe, the oriT region was identified on a 7.9-kb EcoRI fragment from P307 . By ligating the fragment with the cloning vector pUC119, plasmid pAG10 was obtained . The physical map of the insert was determined and oriT was located on a 540-bp BglII/SalI fragment . After this fragment was subcloned into sequencing phages, the polynucleotide sequence was established . Part of the sequence proved to be almost identical to segments of the oriT regions of the plasmids F and R1; another neighboring region was very different among all three sequences . The polynucleotide sequence proximal to traM is highly similar to that of F but different from that of R1.

Plasmid, 1987 Jul, 18(1), 54 - 69
Analysis of Escherichia coli K12 F factor transfer genes: traQ, trbA, and trbB; Wu JH et al.; The genes that encode the transfer properties of plasmid F, the fertility factor of Escherichia coli K12, are known to be clustered over a large, 33.3-kb segment of F DNA . As the central segment of the transfer region has not previously been well characterized, we constructed a detailed restriction map of the large F EcoRI DNA fragment, fl, and isolated a series of plasmid derivatives that carry various overlapping segments of this F tra operon DNA . We also analyzed the protein products of those clones that carried DNA segments extending over the region between traF and traH . This region was known to include traQ, a gene required for efficient conversion of the direct product of traA to the 7000-Da pilin polypeptide . We identified the traQ product as a polypeptide that migrates as a 12,500-Da protein on sodium dodecyl sulfate-polyacrylamide gels . We also detected the products of two other new genes that we have named trbA and trbB . These polypeptides migrate with apparent molecular weights of 14,200 and 18,400, respectively . Analysis of plasmid deletion derivatives that we constructed in vitro shows that these genes map in the order traF trbA traQ trbB traH . The presence of a plasmid carrying a small 0.43-kb fragment that expressed only the 12,500 traQ product caused the traA product of a co-resident compatible plasmid to be converted to the 7000-Da pilin polypeptide, demonstrating that TraQ is the only tra operon product required for this step of F-pilin biosynthesis.

Mol Gen Genet, 1987 Jul, 208(3), 511 - 7
Regulation of major outer membrane porin proteins of Escherichia coli K 12 by pH; Heyde M et al.; Electrophoretic analysis of total membrane proteins of Escherichia coli cells grown at neutral or acid pH showed that ompF, ompC and lamB porin gene expression was regulated by changes in extracellular pH . Growth at acid pH was correlated with a decrease in outer membrane proteins OmpF and LamB and an increase in protein OmpC . pH-induced effects were confirmed and quantitatively estimated by using strains carrying ompF-lacZ, ompC-lacZ and lamB-lacZ fusions . Our studies showed that the pH-dependent regulation acted at a transcriptional level on ompF and ompC gene expression and also at a post-transcriptional level on ompF gene expression . Similar studies carried out with envZ strains showed that the pH-controlled transducing signal was mediated via the EnvZ protein, although other pH-dependent pathways were also involved.

Mol Gen Genet, 1987 Jul, 208(3), 499 - 505
Cloning and characterization of the pH 2.5 acid phosphatase gene, appA: cyclic AMP mediated negative regulation; Touati E et al.; The structural gene for an acid phosphatase coded for by the gene appA of Escherichia coli K12 was cloned from a cosmid library into pBR322 and the restriction map determined . Several appA deletion plasmids and a smaller appA+ plasmid were constructed by in vitro recombination techniques and tested for their ability to complement an appA1 mutation . The appA gene was localized within a 2.1 kb segment . Its orientation was determined by construction of a hybrid plasmid carrying an appA-lacZ fusion . beta-galactosidase synthesized from the appA promoter was negatively regulated by cyclic AMP.

Mol Gen Genet, 1987 Jul, 208(3), 490 - 8
Transcriptional and translational signals of the uidA gene in Escherichia coli K12; Blanco C; The expression of uidA is negatively controlled by the products of the uidR and uxuR genes and is sensitive to catabolite repression . The locations of the transcriptional and translational signals of uidA were determined using lac gene fusions and S1 mapping experiments . The promoter structure of uidA resembles that of a promoter activated by cAMP receptor protein (CRP); putative control regions are located at positions -10 and -35 (relative to the transcription start site), are separated by more than 17 bp and exhibit poor homology with the normally recognized consensus sequences . Moreover, 80 bp separate the promoter from the translational signals . No CRP binding site was detected in the promoter region of uidA . Two operator sites, 01 and 02, were identified: 01 has a greater affinity for the UidR repressor, whereas 02 has a greater affinity for the UxuR repressor, but the two repressor molecules are able to bind at both the 01 and 02 sites . Analysis of two operator constitutive mutations allowed the location of one of the two UidR repressor binding sites; it contains palindromic units spanning the TaqI site of the uidA control region.

Mol Gen Genet, 1987 Jul, 208(3), 485 - 9
Effects of linker insertions on the biogenesis and functioning of the Escherichia coli outer membrane pore protein PhoE; Bosch D et al.; To study the effects of small insertions on the biogenesis and functioning of outer membrane pore protein PhoE of Escherichia coli K12, oligonucleotides were inserted at five different sites in the phoE gene . The proteins encoded by the mutant alleles all appeared to be incorporated into the outer membrane since cells producing these proteins bound either phages specific for the PhoE protein or monoclonal antibodies or both . However, one mutant protein was apparently not incorporated normally since expression of this protein was lethal and the protein did not function as a pore . Amino acid residues 75 and 280 of the PhoE protein appear to be exposed on the cell surface because insertions at these sites interfere with the binding of PhoE-specific phages or monoclonal antibodies to whole cells, respectively.

Mol Gen Genet, 1987 Jul, 208(3), 420 - 7
Genetic analysis of constitutive stable DNA replication in rnh mutants of Escherichia coli K12; Torrey TA et al.; Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis . This constitutive stable DNA replication (SDR) is dependent upon the recA+ gene product . The requirement of SDR for recA+ can be suppressed by rin mutations (for recA+-independent), or by lexA(Def) mutations which inactivate the LexA repressor . Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA+-dependent SDR seen in rnh- rin+ lexA+ strains, recA+-independent in rnh- rin- lexA+, and recA+-independent in rnh- rin+ lexA(Def) . The expression of SDR in rin- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H . The suppression of the recA+ requirement by rin mutations was shown to depend on some new function of the recF+ gene product . In contrast, the suppression by lexA-(Def) mutations was not dependent on recF+ . The lexA3 mutation inhibited recA+-dependent SDR via reducing the amount of recA+ activity available, and was suppressed by the recAo254 mutation . The SDR in rnh- rin- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA+-regulated gene product in the recA+-independent SDR process . A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA+, rin+ and recF+ genes.

Mol Gen Genet, 1987 Jul, 208(3), 390 - 7
The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro; Lin LS et al.; The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance . This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility . The sequence also inhibits replication of R1162 DNA in a cell-free system . The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence . Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number . The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.

Mol Gen Genet, 1987 Jul, 208(3), 365 - 72
Partitioning of the F plasmid: overproduction of an essential protein for partition inhibits plasmid maintenance; Kusukawa N et al.; Multicopy plasmids carrying the sopB gene of the F plasmid inhibit stable inheritance of a coexisting mini-F plasmid . This incompatibility, termed IncG, is found to be caused by excess amounts of the SopB protein, which is essential for accurate partitioning of plasmid DNA molecules into daughter cells . A sopB-carrying multicopy plasmid that shows the IncG+ phenotype was mutagenized in vitro and IncG negative mutant plasmids were isolated . Among these amber and missense mutants of sopB, mutants with a low plasmid copy number and a mutant in the Shine-Dalgarno sequence for translation of the SopB protein were obtained . These results demonstrate that the IncG phenotype is caused by the SopB protein, and that the incompatibility is expressed only when the protein is overproduced . This suggests that the protein must be kept at appropriate concentrations to ensure stable maintenance of the plasmid.

Mol Biol (Mosk), 1987 Jul-Aug, 21(4), 936 - 41
{The SOS and catabolytic repression systems can play a key role in the regulation of infection of Escherichia coli cells with F-specific filamentous phages M13, f1 and fd}; Shumilov VIu; Theoretical analysis of DNA sequences revealed recognition sites for two global E . coli cellular regulons in M13, fd and fl phage's genomes . Both Px and Pv promoters have SOS operator sequences and therefore must be repressed by the lexA protein . PIII and PIV contains CRP-cAMP recognition sequences in activating positions and hence will be activated by the cAMP receptor protein . The model is proposed for the phage life cycle's control in the persistent infection of E . coli cells by F-specific filamentous phages.

Prostaglandins Leukot Med, 1987 Jul, 28(2), 203 - 14
Assessment of bronchoalveolar lavage fluid and plasma for sulfidopeptide leukotrienes during endotoxemia in pigs; Olson NC et al.; We hypothesized that sulfidopeptide leukotrienes (LTC4, LTD4, LTE4) might be important to the pathophysiology of endotoxin-induced acute respiratory failure (ARF) observed in young pigs . We used radioimmunoassay (RIA), reverse-phase high performance liquid chromatography (RP-HPLC) and guinea pig ileum bioassay techniques to determine the presence of sulfidopeptide leukotrienes in bronchoalveolar lavage fluid (BALF) and plasma of saline (n = 12)- and endotoxin (n = 12)-treated pigs . Endotoxin, infused at 5 micrograms/kg for 1 hr followed by 2 micrograms/kg/hr for 3.5 hrs, caused pulmonary hypertension, a biphasic increase in systemic and pulmonary vascular resistances, hypoxemia, bronchoconstriction and hemoconcentration . The levels of immunoreactive sulfidopeptide leukotrienes were not significantly increased in BALF recovered from endotoxemic pigs . Arterial plasma samples (collected at 0.5 hr intervals for 4.5 hrs) were below the detectable limits of the RIA . During RP-HPLC, ethanol extracted BALF failed to show an ultraviolet (UV) absorbance peak (280 nm) that was coincident with authentic standards . Concentrated BALF samples and BALF eluate fractions (collected at a retention time consistent with authentic LTC4) failed to cause a sustained contraction of guinea pig ileum . We conclude that sulfidopeptide leukotrienes are not increased in BALF or plasma recovered from endotoxemic pigs and that these lipoxygenase metabolites might not be important factors contributing to the pathophysiology of endotoxin-induced ARF . An alternate explanation is that the sulfidopeptide leukotrienes are rapidly metabolized so as to be undetectable by the methods employed.

Plasmid, 1987 Jul, 18(1), 24 - 34
Suppression of ColE1 RNA-RNA mismatch mutations in vivo by the ColE1 Rop protein; Dooley TP et al.; In the bacterial plasmid ColE1 the control of initiation of DNA replication is mediated by the interaction of two complementary RNA molecules, the replication primer and RNA1 . The rate of interaction between RNA1 and the primer RNA in vitro can be increased by the product of the ColE1 rop gene, a 63-amino-acid polypeptide . We have investigated the role of the Rop protein in suppressing the incompatibility defects of 13 RNA1-mutant alleles . These RNA1 mutants are defective due to single nucleotide mismatches with their target, the primer RNA . The rop gene suppresses the defective behavior of most of the RNA1 point mismatch mutants in vivo . However, certain mutations that map in stems I and III of RNA1 are not suppressed by rop . The interaction of wild-type and mutant species of RNA1 with ColE1 replication primer transcripts was studied in vitro in the presence or absence of purified Rop protein . The Rop protein is known to increase the rate of wild-type RNA1-primer interaction about twofold in vitro . This enhancement was also observed for mutant RNA1 species having point alterations or a deletion of the 5' terminus of RNA1, which is consistent with the in vivo suppression results . The implications of these results on the mechanism of Rop activity are considered.

Biokhimiia, 1987 Jul, 52(7), 1133 - 7
{Specific for apurine-apyrimidine DNA endodeoxyribonuclease from the rat brain}; Ivanov VA; Endodeoxyribonuclease was detected in rat neocortex chromatin . The partly purified enzyme was found to influence the superhelical apurine-apyrimidine DNA 50 times as effectively as compared to the native substrate . The enzyme hydrolyzes the phosphodiester bond with the formation of 3'-OH- and 5'-phosphate terminal groups . The enzyme-hydrolyzed DNA is an effective primer for DNA-polymerase I from E . coli . It was assumed that DNAase from rat brain chromatin is an apurine-apyrimidine endonuclease II.

Mol Biol (Mosk), 1987 Jul-Aug, 21(4), 1130 - 6
{Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-{4-2-chloroethyl)-N-methylamino}benzylidene derivatives of deoxyribooligonucleotides . II . The nature of rapid interaction at 0 degrees C}; Zenkova MA et al.; Fast non-covalent interactions of 16S rRNA Escherichia coli with 14C labeled 2',3'-O-{4-N-(2-chloroethyl)-N-methylamino}benzylidene derivatives of deoxyribooligonucleotides d(pACCTTGTT)rA, d{pTTACGATC)rU, d(pTTTGCTCCCC)rA (less than{14C}CHRCl-reagents) observed at 0 degrees C were investigated . It was shown, that 16S rRNA and {14C}CHRCl-reagents at 0 degrees C form stable complexes which can not be disrupted under mild acidic conditions (pH 4, 40 degrees C) and under denaturing conditions (7 M urea, 50 degrees C), but are completely disrupted in the course of centrifugation in sucrose density gradient in the presence of SDS . Formation of such complexes of 16S rRNA with greater than{14C}CHRCl-reagents at 0 degrees C was observed due to the presence in the reagent preparation of a number of unidentified products, formed in the course of the synthesis of benzylidene derivatives, and having a hydrophobicity larger, than those for greater than CHRCl-derivatives of deoxyribooligonucleotide . Preparation of {14C}CHRCl-reagents, subjected for purification by reverse-phase chromatography, were unable to form such a complex with 16S rRNA at 0 degrees C . Studies on the complementary addressed modification at 0 degrees C (or incubation at 0 degrees C) with the use of the oligonucleotide benzylidene derivatives not purified from hydrophobic contaminations may lead to alkylation within these complexes during subsequent treatments and in such a way give incorrect information about the level of alkylation within the complex under investigation.

Mol Biol (Mosk), 1987 Jul-Aug, 21(4), 1080 - 91
{5'-C-methylnucleoside triphosphates in the reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase}; Aivazashvili VA et al.; It was shown that RNA-polymerase is able to discriminate diastereoisomers of 5'-methyl-substituted analogs of ribonucleoside triphosphates (rNTP) . Under conditions of soil substrate reactions when the analog is added to the presynthesized ternary complexes, D-allo- and L-talo-stereoisomers incorporate into RNA 100 and 1000 times, respectively, less effectively, then the natural rNTP . The effectivities of incorporation of other 2'- and 3'-substituted analogs of rNTP were measured under the same conditions and compared with that for 5'-Me-rNTP . It was shown also that RNA-polymerase does not support long-chain RNA synthesis from 5'-Me-rNTP in the absence of natural rNTP . No more then two analog residues can be attached to the 3'-end of the presynthesized RNA under such conditions . Addition of one natural rNTP to this reaction mixture results in the synthesis of long alternating RNA containing D-allo-stereoisomer and natural rNTP residues . In the case of L-talo-stereoisomer RNA elongation is not inhibited, if the distance between the analog residues in the RNA chain is not shorter then five nucleotide residues . The rate of pyrophosphorolysis from the RNA of the analogs studied was the same as for the natural rNTP residues.

J Pharmacol Exp Ther, 1987 Jul, 242(1), 372 - 7
Effect of morpholinyladriamycin analogs and adriamycin on the activities of DNA polymerase alpha and RNA polymerase II of chicken leukemia cells; Chuang LF et al.; The new adriamycin (ADR) derivatives, 3'-deamino-3'-(4"-morpholinyl)adriamycin (MRA) and 3'-deamino-3'-(3"-cyano-4"-morpholinyl)-adriamycin (MRA-CN), were studied, in comparison with ADR, for their inhibitory effects on DNA and RNA syntheses in vitro using isolated DNA and RNA polymerases from both Escherichia coli and chicken (myeloblastosis) leukemia cells . Under standard assay conditions, MRA and ADR demonstrated a similar inhibitory effect on the enzymes, whereas MRA-CN showed a slightly greater inhibitory activity than ADR or MRA at low drug concentrations, but with the inhibitory effect plateauing when drug concentration reached 10 microM . Both the A and B forms of the MRA-CN diastereoisomers were effective as inhibitors . Kinetic studies of the inhibition showed that unlike ADR, MRA-CN inhibition of DNA or RNA synthesis could not be reversed by increasing DNA-template concentration in the reaction mixture . Whereas ADR or MRA relaxed completely 1 microgram of pBR322 supercoiled DNA at a drug concentration of 15 microM, MRA-CN, at 60 microM, produced a mixture of intermediate relaxation forms of the DNA . A complete relaxation was achieved at 300 microM MRA-CN . Both DNA relaxation and fluorescence spectroscopic studies indicated that DNA-drug interactions occurred in the following order: ADR (or MRA) greater than MRA-CN greater than N-trifluoroacetyl-ADR-14-O-hemiadipate (a DNA-nonbinding anthracycline).

Proc Natl Acad Sci U S A, 1987 Jul, 84(14), 4890 - 4
Effect of premature termination of translation on mRNA stability depends on the site of ribosome release; Nilsson G et al.; Translational stop codons were introduced at various locations in the protein-coding regions of the monocistronic bla and ompA gene transcripts of Escherichia coli, and the decay characteristics of the upstream and downstream mRNA segments were analyzed . Premature termination of translation at codon position 26 reduced the stability of both the translated and ribosome-free segments of bla mRNA, whereas release of ribosomes just 30 codons further downstream resulted in normal stability for both segments . Normal stability of an untranslated bla gene mRNA segment required its linkage to a ribosome-bound segment of bla gene mRNA . These findings indicate that depriving an mRNA segment of ribosomes does not necessarily render it more susceptible to degradation . However, premature termination of translation at a location that allows ribosomes to traverse only a short segment of bla mRNA can lead to destabilization of the entire transcript.

Proc Natl Acad Sci U S A, 1987 Jul, 84(14), 4762 - 6
Specialized ribosome system: preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli; Hui A et al.; In Escherichia coli, all mRNAs are translated by one pool of functionally identical ribosomes . Here, we describe a system in which a subpopulation of modified ribosomes are directed to a single mutated mRNA species . This was accomplished by changing the Shine-Dalgarno sequence that precedes the heterologous human growth hormone gene from 5' GGAGG to 5' CCTCC or 5' GTGTG . Translation of these modified mRNAs by wild-type ribosomes is very inefficient . When the anti-Shine-Dalgarno region (i.e., the region complementary to the Shine-Dalgarno sequence) at the 3' end of the gene encoding 16S rRNA (rrnB) was altered from 5' CCTCC to 5' GGAGG or 5' CACAC, thus restoring its potential to base-pair with the mutated human growth hormone mRNA, significant expression of this mRNA occurred . Growth hormone synthesis was dependent on induction of the mutated rrnB operon . Subsequently, these specialized ribosomes were made spectinomycin-resistant by the introduction of a C----U substitution at position 1192 of the 16S rRNA . Thus, host protein synthesis could be shut off by the addition of spectinomycin and the specificity and efficiency of the specialized ribosomes could be assessed . Since the specialized ribosomes represent a nonessential subpopulation in the cell, this system offers an approach to the study of mutations elsewhere in the 16S-rRNA gene that otherwise would be lethal to the cell.

J Bacteriol, 1987 Jul, 169(7), 3350 - 7
Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors; Schramm E et al.; Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation . The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined . A polypeptide consisting of 511 amino acids was deduced from the open reading frame . The active colicin had a molecular weight of 54,742 . The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A . Of 216 amino acids, 57% were identical and an additional 19% were homologous . In this part 66% of the nucleotides were identical in the colicin A and B genes . This region contained a sequence of 48 hydrophobic amino acids . Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced . A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function . The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12 . Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions . Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.

J Bacteriol, 1987 Jul, 169(7), 3007 - 12
Function of micF as an antisense RNA in osmoregulatory expression of the ompF gene in Escherichia coli; Aiba H et al.; To analyze the function of micF as an antisera RNA in the osmoregulatory expression of the ompF gene in Escherichia coli, we performed two experiments . In the first experiment, two strains were constructed in which the transcription initiation site of the ompF gene and the transcription termination site of the micF gene were separated by 186 and 4,100 base pairs, respectively, on the chromosome . These two strains showed almost the same profile of ompF expression as the wild-type strain in which the two genes are separated by 10(6) base pairs . When a high-copy-number plasmid carrying the micF gene was introduced into these strains, ompF expression was completely repressed, whereas no repression was observed with a low-copy-number plasmid carrying the micF gene . These results indicate that the distance between the two genes on the chromosome is not critical for the function of micF . In the second experiment, expression of the ompF gene was examined by pulse-labeling in both the micF+ and the micF deletion strains . Upon a shift from a low- to a high-osmolarity medium, suppression of OmpF protein synthesis occurred more quickly and more extensively in the micF+ strain than in the micF deletion strain . The steady-state synthesis of the OmpF protein was also completely suppressed in the micF+ strain in the high-osmolarity medium, whereas the suppression was incomplete in the micF deletion strain . From these results we conclude that (i) the micF gene contributes to the fast and complete response of the OmpF synthesis to the medium osmolarity, and that (ii) the distance between the micF and ompF genes on the chromosomes is not critical for the function of the micF gene . The results suggest, rather, that the ratio of the copy numbers of the two genes is critical for the function of the micF gene.

Carcinogenesis, 1987 Jul, 8(7), 955 - 8
Nitrosation of amines by stimulated macrophages; Miwa M et al.; Rats and mice treated in vivo with Escherichia coli lipopolysaccharide (LPS) synthesize and excrete large quantities of nitrate . Murine peritoneal macrophages, elicited in vivo with thioglycolate and stimulated in vitro with LPS and/or gamma-interferon (IFN), produce copious amounts of nitrate and nitrite . We report here experiments showing N-nitrosamine formation by macrophages immunostimulated in vitro . Macrophage cell lines J774.1, PU5-1.8, WEHI-3 and RAW 264 and freshly isolated macrophages from C3H/He mice were used . Macrophages were cultured in Dulbecco's modified Eagle's medium (pH 7.5) supplemented with calf serum (10%) . Supernatant NO2- and NO3- were measured . N-Nitrosamines were extracted with dichloromethane and the extracts analyzed by a gas chromatography--thermal energy analyzer . Cells (1.5 X 10(6)/ml) were incubated with LPS (10 micrograms/ml) and morpholine (15 mM) for 72 h at 37 degrees C . Under these conditions, all of the cell types listed above produced nitrite (40-70 microM) and N-nitrosomorpholine (NMOR; 114-940 nM) . LPS was required for both processes, and this effect was enhanced by IFN . Nitrite (150 microM) incubated with morpholine in cell-free medium did not form NMOR nor did cells plus morpholine and NO2- . The rate of NMOR formation in the J774.1 cell line was highest in the middle incubation period (24-36 h) although {NO2-} was highest in the final incubation period (48-72 h) . Thus, the cells do not catalyze nitrosamine formation per se, rather the amine traps out a reactive nitrosating species prior to the formation of NO2- and NO3- . These results suggest that immunostimulated macrophages may be capable of nitrosamine formation under physiological conditions.

J Virol, 1987 Jul, 61(7), 2217 - 24
Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA; Akkina RK et al.; The biosynthesis, nuclear transport, and formation of a complex among the influenza polymerase proteins were studied in influenza virus-infected MDBK cells by using monospecific antisera . To obtain these monospecific antisera, portions of cloned cDNAs encoding the individual polymerase proteins (PB1, PB2, or PA) of A/WSN/33 influenza virus were expressed as fusion proteins in Escherichia coli, and the purified fusion proteins were injected into rabbits . Studies using indirect immunofluorescence showed that early in the infectious cycle (4 h postinfection) of influenza virus, PB1 and PB2 are present mainly in the nucleus, whereas PA is predominantly present in the cytoplasm of the virus-infected cells . Later, at 6 to 8 h postinfection, all three polymerase proteins are apparent both in the cytoplasm as well as the nucleus . Radiolabeling and immunoprecipitation analyses showed that the three polymerase proteins remain physically associated as a complex in either the presence or the absence of ribonucleoproteins . In the cytoplasm, the majority of the polymerase proteins remain unassociated, whereas in the nucleus they are present as a complex of three polymerase proteins . To determine whether a polymerase protein is transported into the nucleus individually, PB1 was expressed from the cloned cDNA by using the simian virus 40 late promoter expression vector . PB1 alone, in the absence of the other polymerase proteins or the nucleoprotein, accumulates in the nucleus . This suggests that the formation of a complex with other viral protein(s) is not required for either nuclear transport or nuclear accumulation of PB1 protein and that the PB1 protein may contain an intrinsic signal(s) for nuclear transport.

Biochemistry, 1987 Jun 30, 26(13), 3938 - 43
Interaction of mono- and dianions with cyanase: evidence for apparent half-site binding; Anderson PM et al.; Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent hydrolysis of cyanate . The dianions oxalate, oxalacetate, and malonate are slow-binding inhibitors of cyanase, and some monoanions such as azide and chloride also inhibit cyanase activity {Anderson, P . M., & Little, R . M . (1986) Biochemistry 25, 1621-1626} . The purpose of this study was to investigate the interaction of selected dianions and monoanions by kinetic and equilibrium dialysis binding studies in an effort to obtain information about the active site and catalytic mechanism . Measurement of the effectiveness of 30 different dianions as inhibitors of cyanase showed a significant degree of structural and/or isomeric specificity and considerable variation with respect to the slow-binding nature of the inhibition . Oxalate and oxalacetate both show extreme slow-binding inhibition at very low concentrations . Kinetic studies of the rate of inhibition of cyanase by oxalate showed that the reaction is pseudo first order with respect to oxalate concentration and the results are consistent with a pathway in which oxalate forms a complex with the enzyme in a rapid initial reversible step followed by a slow isomerization step leading to a complex with a very low dissociation constant . The rate of inhibition is significantly reduced by the presence of relatively low concentrations of either azide (analogue of cyanate) or bicarbonate . Equilibrium dialysis binding studies showed that the stoichiometry of binding at saturation for oxalate, malonate, chloride, and bicarbonate is about 0.5 mol of ligand bound/mol of subunit for each compound.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Jun 30, 26(13), 3902 - 7
Interaction of a fluorescent analogue of GDP with elongation factor Tu: steady-state and time-resolved fluorescence studies; Eccleston JF et al.; A fluorescent derivative of GDP was prepared by the reaction of 2'-amino-2'-deoxy-GDP with fluorescamine . This derivative binds tightly (KD approximately 4.5 X 10(-8) M) to elongation factor Tu (EF-Tu) from Escherichia coli . The emission properties, including spectra, polarizations, and lifetimes, for fluorescamine-GDP free in solution and bound to EF-Tu are presented . Emission data on the fluorescamine-ethylamine conjugate are also given . A multifrequency phase and modulation lifetime study (using nine modulation frequencies over the range of 2-80 MHz) indicated that the emissions of these three systems were well characterized by single exponential decays corresponding to 1.45 ns for the fluorescamine-ethylamine derivative in buffer and to 7.74 and 11.03 ns for the fluorescamine-GDP derivative free in buffer and bound to EF-Tu, respectively . Multifrequency differential polarized phase fluorometry results indicated a rotational relaxation time of the protein-probe complex of approximately 88 ns; these data also indicated the lack of significant local motion for the probe . Addition of excess GDP to the EF-Tu-probe complex led to displacement of the fluorescamine-GDP derivative as evidenced by the change in both the steady-state and dynamic polarization values . The observed increase in fluorescence intensity upon displacement allowed us to follow the kinetics of the dissociation reaction; a dissociation rate constant of 5.0 X 10(-3) S-1 was determined . These results demonstrate the utility of this 2'-amino-2'-deoxy-GDP analogue as a probe of guanosine nucleotide dependent systems.

Biochemistry, 1987 Jun 30, 26(13), 4093 - 100
Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis; Chen JT et al.; The role of Phe-31 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution . Phe-31, a strictly conserved residue located in a hydrophobic pocket and interacting with the pteroyl moiety of dihydrofolate (H2F), was replaced by Tyr and Val . The kinetic behavior of the mutant enzymes in general is similar to that of the wild type . The rate-limiting step for both mutant enzymes is the release of tetrahydrofolate (H4F) from the E X NADPH X H4F ternary complex as determined for the wild type . The 2-fold increase in V for the two mutant enzymes arises from faster dissociation of H4F from the enzyme-product complex . The quantitative effect of these mutations is to decrease the rate of hydride transfer, although not to the extent that this step becomes partially rate limiting, but to accelerate the dissociation rates of tetrahydrofolate from product complexes so that the opposing effects are nearly compensating.

Biochemistry, 1987 Jun 30, 26(13), 4085 - 92
Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli; Fierke CA et al.; A kinetic scheme is presented for Escherichia coli dihydrofolate reductase that predicts steady-state kinetic parameters and full time course kinetics under a variety of substrate concentrations and pHs . This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy . The binding kinetics suggest that during steady-state turnover product dissociation follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex . This step, H4F dissociation from the E X NADPH X H4F ternary complex, is proposed to be the rate-limiting step for steady-state turnover at low pH because koff = VM . The rate constant for hydride transfer from NADPH to dihydrofolate (H2F), measured by pre-steady-state transients, has a deuterium isotope effect of 3 and is rapid, khyd = 950 s-1, essentially irreversible, Keq = 1700, and pH dependent, pKa = 6.5, reflecting ionization of a single group in the active site . This scheme accounts for the apparent pKa = 8.4 observed in the steady state as due to a change in the rate-determining step from product release at low pH to hydride transfer above pH 8.4 . This kinetic scheme is a necessary background to analyze the effects of single amino acid substitutions on individual rate constants.

Biochemistry, 1987 Jun 30, 26(13), 4076 - 81
Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate; Yang H et al.; 4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6% . The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions . The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column . At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein) . The pH optimum for Vmax is between 7.5 and 7.7 . Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme . The enzyme contains covalently bound pyruvate as suggested by the isolation of {3H}lactate and pyruvate from {3H}NaBH4-reduced enzyme and native enzyme, respectively . One mole of {3H}lactate was isolated per 39,000 g of {3H}NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme . Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond . These findings are in accord with similar results obtained with the horse liver enzyme (R . Scandurra, personal communication) . The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.

Biochemistry, 1987 Jun 30, 26(13), 4143 - 8
Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase; Lau FT et al.; A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene . Two mutants in the effector site of the enzyme were produced by site-specific mutagenesis of residue Tyr-55 to assess the role of its side chain in binding an allosteric inhibitor, phosphoenolpyruvate (PEP), and an activator, guanosine 5'-diphosphate (GDP): Tyr-55----Phe-55 and Try-55----Gly-55 . The dissociation constant of PEP from the T state is unaffected by the mutations . Mutation of Tyr-55----Phe-55 only slightly increases the dissociation constant of GDP from the R state, indicating a minimal involvement of the hydroxyl group in binding . A 5.5-fold increase in the dissociation constant of GDP on the mutation of Tyr-55----Gly-55 suggests a small hydrophobic interaction of the aromatic ring of the tyrosine residue with guanine of GDP.

Biochem Biophys Res Commun, 1987 Jun 30, 145(3), 1158 - 63
Essentiality of Glu-48 of ribulose bisphosphate carboxylase/oxygenase as demonstrated by site-directed mutagenesis; Hartman FC et al.; Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum . This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis . This single amino acid substitution does not prevent subunit association or ligand binding . However, the Glu-48 mutant is severely deficient in catalytic activity, exhibiting a kcat only 0.05% that of wild-type enzyme . These results demonstrate that Glu-48 is positioned at the active site and suggest that it serves a functional role . In conjunction with previous studies, the discovery of essentiality of Glu-48 argues that the active site is located at an interface between subunits.

Biochemistry, 1987 Jun 30, 26(13), 4022 - 7
Phospholipids chiral at phosphorus . Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast; Raetz CR et al.; The steric courses of the reactions catalyzed by phosphatidylserine (PS) synthase from Escherichia coli and yeast were elucidated by the following procedure . RP and SP isomers of 1,2-dipalmitoyl-sn-glycero-3-{17O,18O}phosphoethanolamine ({17O,18O}DPPE) were synthesized with slight modification of the previous procedure {Bruzik, K., & Tsai, M.-D . (1984) J . Am . Chem . Soc . 106, 747-754} and converted to (RP)- and (SP)-1,2-dipalmitoyl-sn-glycero-3-{16O,17O,18O}phosphoric acid ({16O,17O18O}DPPA), respectively, by incubating with phospholipase D . Condensation of {16O,17O,18O}DPPA with cytidine 5'-monophosphomorpholidate in pyridine gave the desired substrate for PS synthase, {17O,18O}cytidine 5'-diphospho-1,2-dipalmitoyl-sn-glycerol ({17O,18O}CDP-DPG), as a mixture of several isotopic and configurational isomers . Incubation of {17O,18O}CDP-DPG with a mixture of L-serine, PS synthase (which converted {17O,18O}CDP-DPG to phosphatidylserine), and PS decarboxylase (which catalyzes decarboxylation of phosphatidylserine) gave {17O,18O}DPPE . The configuration and isotopic enrichments of the starting {17O,18O}DPPE and the product were analyzed by 31P NMR following trimethylsilylation of the DPPE . The results indicate that the reaction of E . coli PS synthase proceeds with retention of configuration at phosphorus, which suggests a two-step mechanism involving a phosphatidyl-enzyme intermediate, while the yeast PS synthase catalyzes the reaction with inversion of configuration, which suggests a single-displacement mechanism . Such results lend strong support to the ping-pong mechanism proposed for the E . coli enzyme and the sequential Bi-Bi mechanism proposed for the yeast enzyme, both based on previous isotopic exchange experiments.

FEBS Lett, 1987 Jun 29, 218(2), 227 - 30
Comparative EPR studies on the nitrite reductases from Escherichia coli and Wolinella succinogenes; Liu MC et al.; Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K-12 and Wolinella succinogenes were studied by low-temperature EPR spectroscopy . In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K . Both high-spin and low-spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed . These signals disappeared upon reduction by dithionite . Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme-NO complexes with distinct EPR spectral characteristics . The heme-NO complexes formed with the two enzymes differed, however, in g values and line-shapes . When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme-NO complexes . These results suggested the involvement of an enzyme-bound NO intermediate during the six-electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases . Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate . The different ferrous heme-NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.

FEBS Lett, 1987 Jun 29, 218(2), 222 - 6
Beta-subunit of Escherichia coli F1-ATPase . An amino acid replacement within a conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins; Hsu SY et al.; A mutant strain KF87 of E . coli with a defective beta-subunit (Ala-151----Val) of F1-ATPase was isolated . The mutation is within the conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins . The mutant F1-ATPase had a much higher rate of uni-site hydrolysis of ATP than the wild type, and about 6% of the wild-type multi-site activity . The mutant enzyme showed defective transmission of conformational change(s) between the ligand- and aurovertin-binding sites.

J Chromatogr, 1987 Jun 26, 397, 355 - 64
Rapid dual-column chromatographic assay for recombinant leukocyte interferon alpha-2; Rybacek L et al.; A rapid dual-column chromatographic assay for determining recombinant leukocyte interferon alpha-2 in complex mixtures is described . The assay relies on the use of a high-performance monoclonal antibody affinity column connected in tandem with a reversed-phase column . The high specificity and selectivity of these columns permits the quantitation of subcomponent species, such as interferon oligomers that may be present in assay samples . The assay has a limit of sensitivity equal to 1 microgram/g over a range of 1-20 micrograms/g . The precision of the assay was estimated to be about 5%.

Nucleic Acids Res, 1987 Jun 25, 15(12), 4957 - 71
The repair of psoralen monoadducts by the Escherichia coli UvrABC endonuclease; Yeung AT et al.; We have examined the interactions of UvrABC endonuclease with DNA containing the monoadducts of 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP) . The UvrA and UvrB proteins were found to form a stable complex on DNA that contains the psoralen monoadducts . Subsequent binding of UvrC protein to this complex activates the UvrABC endonuclease activity . As in the case of incision at pyrimidine dimers, a stable protein-DNA complex was observed after the incision events . For both 8-MOP and TMP, the UvrABC endonuclease incised the monoadduct-containing strand of DNA on the two sides of the monoadduct with 12 bases included between the two cuts . One incision was at the 8th phosphodiester bond on the 5' side of the modified base . The other incision was at the 5th phosphodiester bond 3' to the modified base . The UvrABC endonuclease incision data revealed that the reactivity of psoralens is 5'TpA greater than 5'ApT greater than 5'TpG.

J Biol Chem, 1987 Jun 25, 262(18), 8826 - 33
Incorporation of six additional proteins to complete the assembly map of the 50 S subunit from Escherichia coli ribosomes; Herold M et al.; In the course of 50 S ribosome assembly in vitro, the L-proteins associate consecutively with the rRNA in two main groups . The first assembly group is found on the reconstitution intermediate particle RI50(1), the second group being added to form the RI50(2) particle . The analysis presented here allows for the first time all the ribosomal proteins to be assigned to one or the other of these groups . The following 22 proteins are found in the RI50(1) particle: L1, (L2), L3, L4, (L5), L7/12, L9, L10, L11, (L13), L15, L17, (L18), L20, L21, L22, L23, L24, (L26), L29, (L33), (L34) . The assembly map has been arranged accordingly . The assembly interdependences of six proteins (L28, L30, L31, L32, L33, and L34) were assessed and that of L14 revised by means of a large number of assembly mapping experiments, the final decisive steps of which are documented here . The interactions of these latter seven proteins and the incorporation of five additional assembly interdependences are integrated into the 50 S assembly map, which now contains all the components of the 50 S subunit, with the exception of protein L26; this protein is known to be the same as protein S20 and is predominantly associated with the 30 S subunit.

J Biol Chem, 1987 Jun 25, 262(18), 8648 - 57
Structural studies on aspartate aminotransferase from Escherichia coli . Covalent structure; Kondo K et al.; The amino acid sequence of aspartate aminotransferase from Escherichia coli was established by sequence analysis and alignment of 39 tryptic peptides and 7 cyanogen bromide peptides . The total number of amino acid residues of the subunit was 396, and the molecular weight was calculated to be 43,573 . A comparison of the primary structure of the E . coli enzyme with all known sequences of the two types of isoenzyme (mitochondrial and cytosolic enzymes) in vertebrates revealed that approximately 25% of all residues are invariant . The amino acid residues which were proposed from crystallographic studies on the vertebrate enzymes to be essential for the enzymic action are well conserved in the E . coli enzyme . The E . coli enzyme shows a similar degree of sequence homology to both the mitochondrial and cytosolic isoenzymes (close to 40%) . The finding that the positions of deletions introduced into the sequence of E . coli enzyme to give the maximum homology agree well with those of the mitochondrial enzymes supports the endosymbiotic hypothesis of mitochondrial origin.

J Biol Chem, 1987 Jun 25, 262(18), 8488 - 95
Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities; Hagervall TG et al.; The tRNA(5-methylaminomethyl-2-thiouridine)-methyltransferase, which is involved in the biosynthesis of the modified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) present in the wobble position of some tRNAs, was purified close to homogeneity (95% purity) . The molecular mass of the enzyme is 79,000 daltons . The enzyme activity has a pH optimum of 8.0-8.5, is inhibited by magnesium ions, and stimulated by ammonium ions . Two different intermediates in the biosynthesis of mnm5s2U34 are present in tRNA from the mutants trmC1 and trmC2 . Unexpectedly, the product present in tRNA from trmC1 cells was identified by mass spectrometric and chromatographic analyses as 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), i.e . a more complex derivative than the final product mnm5s2U . The product present in tRNA from trmC2 cells was identified as 5-aminomethyl-2-thiouridine (nm5s2U) . In the presence of S-adenosylmethionine the most purified enzyme fraction converts both cmnm5s2U34 and nm5s2U34 into mnm5s2U34 . In the absence of S-adenosylmethionine, however, cmnm5s2U34 is converted into nm5s2U by this enzyme fraction . We conclude that the purified polypeptide has two enzymatic activities; one actually demodifies cmnm5s2U to nm5s2U and the other catalyzes the transfer of a methyl group from S-adenosylmethionine to nm5s2U, thus forming mnm5s2U . The sequential order of the biosynthesis of mnm5s2U34 is suggested to be: (Formula: see text) . The molecular activity of the methyltransferase activity (nm5s2U34----mnm5s2U34) is 74 min-1, and the steady state concentration of the enzyme is only 78 molecules/genome equivalent in cells growing at a specific growth rate of 1.0/h.

Nucleic Acids Res, 1987 Jun 25, 15(12), 4901 - 14
Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin; de Wind N et al.; Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E . coli K12 . This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E . coli B/r-derived region of the replication origin of these strains . Cloning and DNA sequence analysis of the E . coli B/r replication origin revealed several base differences as compared to the E . coli K12 sequence . The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned . Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids . Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.

J Biol Chem, 1987 Jun 25, 262(18), 8806 - 13
A small hydrophobic domain anchors leader peptidase to the cytoplasmic membrane of Escherichia coli; Moore KE et al.; Leader peptidase is an enzyme of the Escherichia coli cytoplasmic membrane which removes amino-terminal leader sequences from many secreted and membrane proteins . Three potential membrane-spanning segments exist in the first 98 amino acids of leader peptidase . We have characterized the topology of leader peptidase based on its sensitivity to protease digestion . Proteinase K and trypsin treatment of right-side-out inner membrane vesicles and spheroplasts yields protected fragments of approximately 80 and 105 amino acid residues, respectively . We have shown that both fragments are derived from the amino terminus of the protein and that the smaller protected peptide can be derived from the larger . Removal of the third potential membrane-spanning segment (residues 82-98) does not affect the size of the proteinase K-protected fragment but does reduce the size of the trypsin-protected peptide . Because the proteinase K-protected fragment is about 9000 daltons, is derived from the amino terminus of leader peptidase, and its size is not affected when amino acids 82-98 are removed from the protein, it must extend from the amino terminus to approximately residue 80 . Likewise, the trypsin-protected fragment must extend from the amino terminus to about residue 105 . These data suggest a model for the orientation of leader peptidase in which the second hydrophobic stretch (residues 62-76) spans the cytoplasmic membrane and the third hydrophobic stretch resides in the periplasmic space.

J Biol Chem, 1987 Jun 25, 262(18), 8690 - 5
Cascade control of Escherichia coli glutamine synthetase . Purification and properties of PII protein and nucleotide sequence of its structural gene; Son HS et al.; A procedure was developed to purify large quantities of PII protein from an Escherichia coli strain which contains a multicopy plasmid harboring the structural gene of PII (the glnB gene) . Ultraviolet spectra of uridylylated and unuridylylated PII were obtained using the purified PII and empirical formulas to calculate the concentration of protein and the average number of uridylylated subunits per molecule were derived . A continuous fluorometric assay for the measurement of uridylylated PII (PIID) and adenylyltransferase (ATase) was also established . Rate measurements at various concentrations of PIID and at a fixed concentration of ATase showed that a tetrameric PIID molecule interacts with only one ATase molecule at a time . The complete nucleotide sequence of the glnB gene was determined and parts of the deduced amino acid sequence were confirmed by the results of amino acid sequence analysis of peptides . The PII subunit consists of 103 amino acids (Mr = 11,580) . Two tyrosines reside at positions 46 and 51, where Tyr51 is the site of uridylylation . Nucleotide sequence analysis of the upstream region showed no obvious sites for the binding of RNA polymerase, indicating that the glnB gene is a part of an as yet unidentified operon.

FEBS Lett, 1987 Jun 22, 218(1), 22 - 6
The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of the respiratory chain in everted membrane vesicles of Escherichia coli; Sedgwick EG et al.; N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to metabolic changes in everted inner membrane vesicles from this organism . NPN was bound to the vesicles to produce a steady-state level of fluorescence intensity . Addition of substrate or ATP did not alter the fluorescence . However, following complete removal of oxygen from the medium by oxidation of substrate through the respiratory chain, there was an increase in the fluorescence of NPN . Reoxidation of the components of the respiratory chain by the addition of oxygen, ferricyanide, fumarate or nitrate decreased fluorescence to the steady-state level until the oxidant had been completely reduced . The fluorescence changes were insensitive to the state of energization of the membrane . It is proposed that NPN responds to the state of reduction of components of the respiratory chain either directly by reacting with a component of the chain or indirectly through an effect transmitted to the membrane by a change in the conformation of respiratory chain components.

J Mol Biol, 1987 Jun 20, 195(4), 897 - 907
Limited co-operativity in protein-nucleic acid interactions . A thermodynamic model for the interactions of Escherichia coli single strand binding protein with single-stranded nucleic acids in the "beaded", (SSB)65 mode; Bujalowski W et al.; We present a statistical thermodynamic model ("tetramer/octamer" model) that describes the equilibrium binding of the Escherichia coli single strand binding (SSB) protein to single-stranded nucleic acids in its "beaded" binding mode, which seems to be equivalent to the high site size, (SSB)65 binding mode . The method of sequence-generating functions is used to derive the model, which accounts for the observation that clustering of bound SSB tetramers is limited to the formation of octamers, which have been observed as "beads" in the electron microscope . The model also accounts for the overlap of potential protein binding sites on the nucleic acid . The "tetramer/octamer" model is fully described by only three parameters: the site size, n; the intrinsic equilibrium constant, K; and the co-operativity parameter, omega, and we obtain exact, closed form expressions for the binding isotherm as well as the distribution of DNA-bound SSB tetramers and octamers . The closed form expressions allow one to calculate easily average binding properties and analyze experimental binding isotherms to obtain estimates of K and omega . In order to test the tetramer/octamer model, we have determined the equilibrium binding isotherm for the E . coli SSB protein-poly(U) interaction in 0.2 M-NaCl over a wide range of binding densities . These are conditions in which the low co-operativity (SSB)65 binding mode solely exists . The tetramer/octamer model provides a much better description of the experimental isotherm over the entire binding density range than a model that assumes the formation of clusters of unlimited size . A co-operativity parameter of omega = 420 +/- 80 provides a good fit to data for SSB binding to poly(dA) and poly(U), corresponding to an interaction free energy of -3.6 kcal/mol of SSB octamer formed . On the basis of this moderate value of omega, the tetramer/octamer model predicts that at low to intermediate binding densities, a significant fraction of bound SSB exists in the form of tetramers co-existing with octamers . In the case of E . coli SSB protein binding in the "beaded", (SSB)65 mode this model provides a significant improvement over previous treatments which assume unlimited nearest-neighbor interactions, since the binding parameters, K and omega, represent physically meaningful interaction constants rather than fitting parameters.

J Mol Biol, 1987 Jun 20, 195(4), 847 - 53
Novel rpoA mutation that interferes with the function of OmpR and EnvZ, positive regulators of the ompF and ompC genes that code for outer-membrane proteins in Escherichia coli K12; Matsuyama S et al.; The expression of the ompF and ompC genes that code for major outer-membrane proteins of Escherichia coli is positively regulated by the products of the ompR and envZ genes . Recently, we isolated the ompR77 mutation, which suppresses the envZ11 mutation . In this work, a novel mutation that interferes with the suppression of the envZ11 mutation by ompR77 was isolated . The mutation was located in the rpoA gene that codes for the alpha subunit of DNA-dependent RNA polymerase . These results suggest that an interaction between the positive regulators and RNA polymerase is involved in the initiation of transcription of the ompF and ompC genes . In addition, the results suggest that during transcription the RNA polymerase migrates along DNA strands with the alpha subunit facing backward and the beta beta' subunits facing forward.

J Mol Biol, 1987 Jun 20, 195(4), 795 - 808
Topological unwinding of strong and weak promoters by RNA polymerase . A comparison between the lac wild-type and the UV5 sites of Escherichia coli; Amouyal M et al.; Two Escherichia coli control regions have been compared in their ability to be unwound by RNA polymerase during formation of the transcriptionally active ("open") complex: the wild-type control region, consisting of two overlapping binding sites P1 and P2, both weakly transcribed, and an "up" P1 mutant, the strong lac L8UV5 promoter . The final complexes were characterized by their topological unwinding, by DNase I and orthophenanthroline footprints, as well as by methylation of unpaired cytosine residues . At the wild-type control region, the RNA polymerase footprint is weak, and single-strand formation is incomplete and slow . The same signals are strong, complete and quickly established at lac L8UV5; yet the final complexes were found to be equally unwound (by 1.7 turns) in the absence of nucleotide substrates as well as during an abortive initiation cycle . At the lac wild-type region, open complex formation occurs slowly enough to permit the measurement of the extent of a single-stranded region and of topological unwinding during the latency period . Not all the final species are active and unwinding appears to precede, in time, full open-complex formation . At the lac UV5 promoter the same conclusion was reached by a different method involving those changes in the various parameters that characterize open-complex formation monitored by an abortive initiation assay, conducted at increasing levels of template superhelicity . From both approaches we conclude that, at these promoters, the formation of the single-stranded region occurs at the expense of a negative change in linking number, initially stored in a closed intermediate, perhaps as negative writhing . Furthermore, abortive transcription assays indicate that the specific initiation efficiency of the species stored at both promoters, P1 and P2, on the wild-type template is increased as a whole with increasing superhelicity (conversion of inactive species to active ones, increased efficiency of active ones) . We conclude that negative supercoiling is not an extra-regulatory element of the lactose system, allowing modulation of expression of the wild-type promoter to the profit of P1 . Instead, P2 and P1, in the absence of active catabolite receptor protein (CRP-cAMP), appear to be equally weak and to be equally affected by negative supercoiling in the range of superhelical densities examined . The physiological importance of the P1-P2 competition in the regulation of expression in this region is thus questioned . The major effect of CRP-cAMP stimulation appears to be the direct activation at the P1 promoter.

J Mol Biol, 1987 Jun 20, 195(4), 949 - 52
Specific destruction of the second lac operator decreases repression of the lac operon in Escherichia coli fivefold; Eismann E et al.; The second operator of the lac operon, located within the 5'-coding region of the lacZ gene, was specifically destroyed by means of oligonucleotide-directed mutagenesis . Eight of its bases were exchanged without altering the wild-type amino acid sequence of beta-galactosidase . The mutation was transferred onto an F'lac+I+O+Z+pro+ episome . We observed a fivefold decrease in repression of beta-galactosidase expression compared to that in the wild-type.

J Mol Biol, 1987 Jun 20, 195(4), 929 - 37
RNA polymerase II of Drosophila . Relation of its 140,000 Mr subunit to the beta subunit of Escherichia coli RNA polymerase; Falkenburg D et al.; We have determined the nucleotide sequence of the gene coding for the 140,000 Mr subunit of the DNA-dependent RNA polymerase II from Drosophila melanogaster . This analysis revealed features that are typical for a household-function gene . The codon usage is relaxed and there is no apparent TATA box upstream from the transcription start site . A comparison of the deduced amino acid sequence with that of the Escherichia coli RNA polymerase beta subunit shows a total of nine regions of homology . These regions are also conserved in chloroplast DNA of tobacco . This supports the notion that the two large subunits of the eukaryotic RNA polymerases are the structural and functional equivalents of E . coli beta' and beta subunits.

J Chromatogr, 1987 Jun 19, 396, 209 - 15
Micropreparative purification of recombinant human interleukin-2; Weir MP et al.; Recombinant Interleukin-2 (IL-2) is expressed in E . coli as insoluble aggregates; a protocol has been developed for solubilization, renaturation and purification of IL-2 from such aggregates at the 5-10-mg level . IL-2 aggregates were isolated from soluble proteins by centrifugation, subjected to a 1 M guanidine hydrochloride wash and a butan-1-ol wash (the latter to remove lipid), dissolved in 8 M guanidine hydrochloride-10 mM dithiothreitol and partly purified by gel permeation chromatography . Refolding/oxidation was then performed by dilution into Tris-HCl, pH 8.5 containing 1.5 microM copper sulphate to accelerate autoxidation . Final purification was by successive cation-exchange and reversed-phase high-performance liquid chromatographic steps, yielding over 99.5% pure IL-2 with an overall recovery of 20%.

Science, 1987 Jun 19, 236(4808), 1532 - 9
The chemistry of self-splicing RNA and RNA enzymes; Cech TR; Proteins are not the only catalysts of cellular reactions; there is a growing list of RNA molecules that catalyze RNA cleavage and joining reactions . The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA . Wherever appropriate, catalysis by RNA is compared to catalysis by protein enzymes.

Nature, 1987 Jun 18-24, 327(6123), 591 - 7
The crystal structure of trp aporepressor at 1.8 A shows how binding tryptophan enhances DNA affinity; Zhang RG et al.; Comparison of the crystal structure of inactive unliganded trp aporepressor with that of trp repressor shows that binding tryptophan activates the dimer a thousandfold by moving two symmetrically-disposed flexible bihelical motifs . These flexible 'DNA-reading heads' flank a highly inflexible core domain formed by an unusual arrangement of interlocking alpha-helices from both subunits.

Biochim Biophys Acta, 1987 Jun 17, 913(2), 245 - 55
The tertiary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by proton magnetic resonance spectroscopy and limited proteolysis experiments; Littlechild J et al.; Ribosomal proteins from Escherichia coli have been isolated by a mild purification procedure . Their tertiary structure has been explored by two techniques, proton magnetic resonance and limited proteolysis . A number of proteins when subjected to limited proteolysis produce resistant fragments in good yields . In most cases this does not depend on the specificity of the enzyme used . The proteins S15, S16, S17 and L30 are not degraded at all, whereas a few proteins are very susceptible to proteolysis . 1H-NMR experiments show that the majority of the ribosomal proteins have a uniquely folded tertiary structure . This is particularly pronounced in the four proteins mentioned above which resist proteolysis . In general, a good agreement is observed between the degree of proteolytic resistance and the amount of folding indicated by NMR spectroscopy . Similar studies on a few ribosomal proteins purified under denaturing conditions show that, in contrast, these protein preparations are not structurally homogeneous and that they contain a mixture of denatured and renatured molecules . The results are interpreted in terms of a compactly folded tertiary structure for the four proteinase-resistant proteins while the majority of the other proteins appear to have two domains, one compactly folded and resistant to proteinase and the other flexible and susceptible to proteolysis . A few proteins seem to have a completely flexible structure and can therefore be easily degraded.

Biochim Biophys Acta, 1987 Jun 17, 913(2), 179 - 84
Proline isomerization and the slow folding reactions of the alpha subunit of tryptophan synthase from Escherichia coli; Hurle MR et al.; Previous studies on the refolding of the alpha subunit of tryptophan synthase from Escherichia coli assigned two slow refolding phases to rate-limiting isomerizations of two 'essential' proline residues, one in each of the two domains of the protein (Matthews, C.R., Crisanti, M.M., Manz, J.T . and Gepner, G.L . (1983) Biochemistry 22, 1445-1452) . The double-jump experiment (Brandts, J.F., Halvorson, H.R . and Brennan, M . (1975) Biochemistry 14, 4953-4963) was used to further investigate this phenomenon . The reaction assigned to the carboxyl domain is consistent with the proline isomerization hypothesis . The amino domain process is more rapid than expected for proline isomerization and may reflect another type of slow folding reaction . The results permit a further refinement of the folding model for the alpha subunit and demonstrate the existence of a third unfolded species whose folding is not limited by either of these two reactions.

Biochim Biophys Acta, 1987 Jun 17, 913(2), 117 - 21
A mutant pyruvate dehydrogenase complex of Escherichia coli deleted in the (alanine + proline)-rich region of the acetyltransferase component; Miles JS et al.; The acetyltransferase chains of the pyruvate dehydrogenase complex of Escherichia coli contain conformationally mobile (alanine + proline)-rich segments that link the lipoyl domains to each other and to the subunit-binding and catalytic domain, and facilitate the intramolecular coupling of active sites in the complex . A deletion of 12 of the 32 residues of the (Ala + Pro)-rich segment of an acetyltransferase containing only one lipoyl domain was made by deleting the corresponding segment of the aceF gene . A pyruvate dehydrogenase complex was still produced and the catalytic activity of the restructured complex, including active-site coupling, was not detectably impaired.

Biochim Biophys Acta, 1987 Jun 17, 913(2), 238 - 44
Nature of the interactions involved in the lipid-protein complexes of the Escherichia coli N-acetylmuramoyl-L-alanine amidase; Vanderwinkel E et al.; Depending on its concentration, phosphatidylglycerol, one of the three main Escherichia coli phospholipid species, is able to activate or inactivate the E . coli murein amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28) (Vanderwinkel, E . and De Vlieghere, M . (1985) Biochim . Biophys . Acta 838, 54-59) . The mechanisms underlying the modulation of this enzyme activity were studied by analyzing the effects of cations, polycationic molecules, various surfactants and amphiphilic water-soluble compounds . K+, Mg2+ and polyamines were all able to prevent completely the enzyme inactivation produced by millimolar order concentration of phosphatidylglycerol . The efficiencies of the ionic species tested were in the order K+ less than Mg2+ = putrescine less than spermidine less than spermine . The kinetics of the counteraction processes were all sigmoidal . By contrast, the activation of the murein amidase produced by phosphatidylglycerol in micromolar concentration appeared to be insensitive to the ionic strength of the medium . Surfactants and amphiphilic molecules differing in their polar head and hydrophobic tail were found to activate the enzyme at various degrees for concentrations below their critical micellar concentration . The non-ionic surfactants were the most potent activators and remarkably mimicked the phosphatidylglycerol activation . The enzyme activation process appeared to require only a hydrophobic solvation shell around the protein . All kinetic data supported our previous interpretation of the phosphatidylglycerol-enzyme interactions in terms of multisite non-allosteric theory.

Biochemistry, 1987 Jun 16, 26(12), 3626 - 34
Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts; Henry GD et al.; Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique . The locations of the more rapidly exchanging amides have been determined . In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom . M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method . This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments . In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine) . With corrections for sequence-related inductive effects {Molday, R . S., Englander, S . W., & Kallen, R . G . (1972) Biochemistry 11, 150-158}, the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine) . The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Jun 16, 26(12), 3493 - 500
Structural comparison of acyl carrier protein in acylated and sulfhydryl forms by two-dimensional 1H NMR spectroscopy; Jones PJ et al.; Sequence-specific assignments of 1H NMR resonances are obtained for the backbone protons of Escherichia coli acyl carrier protein, acylated with an eight-carbon chain covalently attached to the prosthetic group thiol (octanoyl-ACP) . Comparison of 1H-1H sequential connectivities in the NOESY spectra of octanoyl-ACP and the unacylated protein (ACPSH) indicates that secondary structure is largely conserved on acylation . Changes in resonance positions observed for certain groups of residues are interpreted in terms of a model that describes the spatial reorientation of secondary structural elements in the protein resulting from introduction of the acyl chain.

Biochemistry, 1987 Jun 16, 26(12), 3408 - 16
Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate; Salowe SP et al.; Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-deoxyuridine 5'-diphosphate (N3UDP) . Incubation of RDPR with {3'-3H}N3UDP resulted in 0.2 mol of 3H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction . Incubation of RDPR with {beta-32P}N3UDP resulted in stoichiometric production of inorganic pyrophosphate . One equivalent of uracil was eliminated from N3UDP, but no azide release was detected . Analysis of the reaction of RDPR with {15N3}N3UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated . The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N3UDP as reported previously {Sjoberg, B.-M., Graslund, A., & Eckstein, F . (1983) J . Biol . Chem . 258, 8060-8067}, while the specific activity of the B1 subunit was reduced by half . Incubation of {5'-3H}N3UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme . Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.

Biochemistry, 1987 Jun 16, 26(12), 3322 - 30
Salt-dependent binding of Escherichia coli RNA polymerase to DNA and specific transcription by the core enzyme and holoenzyme; Wheeler AR et al.; The interaction of the Escherichia coli RNA polymerase with several forms of DNA has been studied by difference absorption spectroscopy, protection against endonucleases, and limited, specific initiation . The core enzyme is able to open duplex poly{d(A-T)} in 10 mM KCl . The core enzyme binds to promoters in linear DNA in a salt-dependent manner, but it does not bind to the same promoters in supercoiled DNA . The binding of the core enzyme is not as tight as that of the holoenzyme . The holoenzyme initiates specific transcription from promoters in a salt-dependent manner . The core enzyme also initiates specific transcription from the same promoters at approximately one-fifth the level of the holoenzyme with a different salt dependence . The profile of the salt dependence of specific transcription initiation varies with the promoter . The origin of differences between holoenzyme-DNA and core enzyme-DNA interactions and the mechanism whereby sigma improves transcriptional specificity are discussed in light of these data.

Biochemistry, 1987 Jun 16, 26(12), 3425 - 9
Phosphonate analogues of diadenosine 5',5'''-P1,P4-tetraphosphate as substrates or inhibitors of procaryotic and eucaryotic enzymes degrading dinucleoside tetraphosphates; Guranowski A et al.; The substrate specificity of procaryotic and eucaryotic AppppA-degrading enzymes was investigated with phosphonate analogues of diadenosine 5',5'''-P1,P4-tetraphosphate (AppppA) . App(CH2)ppA (I), App(CHBr)ppA (II), and Appp(CH2)pA (III), but not Ap(CH2)pp(CH2)pA (IV), are substrates for lupin AppppA hydrolase (EC 3.6.1.17) and phosphodiesterase I (EC 3.1.4.1) . None of the four analogues is hydrolyzed by bacterial AppppA hydrolase (EC 3.6.1.41), and only analogue III is degraded by yeast AppppA phosphorylase (EC 2.7.7.53) . The analogues are competitive inhibitors of all four enzymes . The affinity of analogue IV is 3-40-fold lower than that of analogues I-III for all four enzymes . Introduction of one methylene (as in I and III) {or bromomethylene (as in II)} group into AppppA results in a 3-15-fold increase of its affinity for lupin and Escherichia coli AppppA hydrolases . The same modifications only negligibly (10-30%) affect its affinity for yeast AppppA phosphorylase and decrease its affinity for lupin phosphodiesterase I about 2.5-fold . The data provide further evidence for the heterogeneity among catalytic sites of all four AppppA-degrading enzymes.

J Biol Chem, 1987 Jun 15, 262(17), 8416 - 22
The signal sequence of an Escherichia coli outer membrane protein can mediate translocation of a not normally secreted protein across the plasma membrane; MacIntyre S et al.; The distal part of the long tail fibers of the Escherichia coli phage T4 consists of a dimer of protein 37 . A fragment of the corresponding gene, encoding 253 amino acids, was inserted into several different sites within the cloned gene for the 325-residue outer membrane protein OmpA . In plasmid pTU T4-5 the fragment was inserted once and in pTU T4-10 tandemly twice between the codons for residues 153 and 154 of the OmpA protein . In pTU T4-22 two fragments were present, in tandem, between the codons for residues 45 and 46 of this protein . In pIN T4-6 one fragment was inserted into the ompA gene immediately following the part encoding the signal sequence . The corresponding mature proteins consist, in this order, of 605, 860, 835, and 279 amino acid residues . All precursor proteins were processed and translocated across the plasma membrane . Hence, not only can the OmpA protein serve as a vehicle for export of a nonsecretory protein, but the signal sequence alone can also mediate export of such a protein . Export of the pro-OmpA protein depends on the SecA protein . Export of the tail fiber fragment expressed from pIN T4-6 remained SecA dependent . Thus, the secA pathway in this case is chosen by the signal peptide . It is proposed that a signal peptide can mediate translocation of nonsecretory proteins as long as they are export-compatible . The inability of a signal sequence to mediate export of some proteins appears to be due to export incompatibility of the protein rather than to the absence of information, within the mature part of the polypeptide, which would be required for translocation.

Eur J Biochem, 1987 Jun 15, 165(3), 621 - 5
Reversible activation of hydrogenase from Escherichia coli; Hallahan DL et al.; Hydrogenase from Escherichia coli exhibited low activity when assayed for hydrogen:methyl viologen reductase activity and no activity when assayed for hydrogen-uptake activity with acceptors of high redox potential (dichloroindophenol, methylene blue) . Nor did the enzyme as isolated catalyse proton-tritium exchange activity . Incubation under hydrogen resulted in an increase in hydrogen-uptake activity with methyl viologen and the appearance of hydrogen-uptake activity with dichloroindophenol and methylene blue . Following such treatment, the enzyme also readily catalysed isotope exchange . This process is interpreted as the conversion of the hydrogenase from an inactive 'unready' state to an 'active' state . Oxidation of active hydrogenase with dichloroindophenol caused conversion to a state resembling that of the enzyme as isolated but capable of more rapid activation under reducing conditions . This form is termed the 'ready' state . Such interconversions have been reported for hydrogenases from Desulfovibrio gigas and D . desulfuricans, and the possibility that they constitute a regulatory mechanism suggested.

Eur J Biochem, 1987 Jun 15, 165(3), 537 - 41
Purification and characterization of human interleukin-1 alpha produced in Escherichia coli; Wingfield P et al.; The production of human interleukin-1 alpha (IL-1 alpha) in Escherichia coli is described together with a method for its purification . The isolated protein was shown to be pure and physically homogeneous . The in vitro biological activity of IL-1 alpha was tested with the mononuclear-cell factor and the lymphocyte-activating factor assays . The specific activity determined with both assays was about 3 X 10(7) units mg-1 and is similar to that observed with recombinant human IL-1 beta . The purified protein was resolved by chromatofocusing into two species of isoelectric points 5.45 and 5.20 (75% and 25%, respectively, of the total protein) . Both species had similar chemical properties and biological activities to the unfractionated protein . The charge difference between the species was attributed to the deamidation of a single Asn or Gln residue.

Eur J Biochem, 1987 Jun 15, 165(3), 515 - 9
Abortive and productive elongation catalysed by purified spinach chloroplast RNA polymerase; Job C et al.; Experimental conditions are reported under which purified spinach chloroplast RNA polymerase catalyses the abortive elongation reaction on a synthetic poly{d(A-T)} template . The reaction only occurs under very stringent conditions and absolutely requires Mn2+ as the metal activator . No reaction can be detected in the presence of Mg2+ . Furthermore, the rate of abortive elongation with the chloroplast enzyme is extremely sensitive to the presence of added salts, such as KCl or (NH4)2SO4, in the reaction assays . In the combined presence of Mn2+ and Mg2+, a marked inhibition of abortive elongation is associated with an activation of productive elongation and an increased length of RNA chains . Thus, whereas Mn2+ is more active than Mg2+ for phosphodiester bond formation, it appears that Mg2+ favors the stabilization of the ternary transcription complexes . These results are compared with those obtained under similar conditions for wheat germ RNA polymerase II and Escherichia coli RNA polymerase.

Life Sci, 1987 Jun 15, 40(24), 2331 - 6
Interleukin-1 (IL-1) depresses cytochrome P450 levels and activities in mice; Shedlofsky SI et al.; Endotoxin depresses cytochrome P450 levels when injected into animals . The purpose of this study was to determine whether endotoxin itself, or monokine(s) released in response to endotoxin administration are responsible for this effect . Cytochrome P450 levels and drug metabolizing activities were measured in endotoxin resistant C3H/HeJ mice 24h after single intraperitoneal injections of either lipopolysaccharide (LPS), a semipurified murine monokine preparation containing interleukin-1 (IL-1), or murine recombinant IL-1 . In endotoxin sensitive C3H/HeN mice, LPS (0.5 mg/Kg) decreased total cytochrome P450 levels, benzphetamine demethylase activities, and ethoxyresorufin-0-deethylase activities . This dose of LPS did not alter cytochrome P450 levels or activities in the C3H/HeJ mice . However, after injection of the semipurified monokine preparation or the recombinant IL-1, there were significant decreases in cytochrome P450 levels and activities similar to the decreases observed with LPS in the C3H/HeN mice . These findings suggest that the alterations in hepatic cytochrome P450 seen with endotoxin injection are mediated, at least in part, by IL-1.

Eur J Biochem, 1987 Jun 15, 165(3), 601 - 6
Molecular cloning and sequencing of a cDNA encoding the acyl carrier protein and its flanking domains in the mammalian fatty acid synthetase; Witkowski A et al.; Cloned cDNAs containing coding sequences for domains proximal to the carboxy terminus of the rat fatty acid synthetase have been isolated using an expression vector and domain-specific antibodies . The coding regions were assigned to specific domains of the multifunctional complex by identification of sequences coding for characterized peptide fragments and by recognition of sequences homologous to other monofunctional enzymes . Two clones contain the entire coding region for the acyl carrier protein domain . The sequence is flanked at the 3'-end by a region coding for the thioesterase domain and at the 5'-end by a sequence coding for a reductase, most likely the ketoreductase domain . Thus the ordering of these domain-coding regions in the fatty acid synthetase mRNA is established . The acyl carrier protein domain exhibits about 25% homology with that of the discrete monofunctional acyl carrier proteins of Escherichia coli, spinach and barley, the ketoreductase domain exhibits about 25% homology with bacterial dihydrofolate reductases and the active site of the thioesterase domain exhibits both primary and secondary structural features common to the serine proteases . These findings lend support to the hypothesis that the polyfunctional fatty acid synthetase probably arose by a complex evolutionary process involving fusion of genes coding for seven individual enzymes.

J Biol Chem, 1987 Jun 15, 262(17), 8340 - 6
F1F0-ATPase from Escherichia coli with mutant F0 subunits . Partial purification and immunoprecipitation of F1F0 complexes; Vik SB et al.; Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations . The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations . The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion . In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation . This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition . The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits . The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation . The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit . Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits . A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector . The assembly, structure, and function of the F1F0-ATPase is discussed.

J Biol Chem, 1987 Jun 15, 262(17), 8022 - 6
Directed mutagenesis of the beta-subunit of F1-ATPase from Escherichia coli; Parsonage D et al.; Oligonucleotide-directed mutagenesis was used to generate six mutant strains of Escherichia coli which had the following specific amino acid substitutions in the beta-subunit of F1-ATPase: (i) Lys-155----Gln; (ii) Lys-155----Glu; (iii) Gly-149----Ile; (iv) Gly-154----Ile; (v) Tyr-297----Phe;(vi) Tyr-354----Phe . The effects of each mutation on growth of cells on succinate plates or limiting (3 mM) glucose and on cell membrane ATPase activity and ATP-driven pH gradient formation were studied . The results showed Lys-155 to be essential for catalysis, as has been predicted previously from sequence homology and structural considerations; however, the results appear to contradict the hypothesis that Lys-155 interacts with one of the substrate phosphate groups because the Lys-155----Glu mutation was less detrimental than Lys-155----Gln . Gly-149 and Gly-154 have been predicted to be involved in essential conformational changes in F1-ATPase by virtue of their position in a putative glycine-rich flexible loop structure . The mutation of Gly-154----Ile caused strong impairment of catalysis, but the Gly-149----Ile mutation produced only moderate impairment . The two tyrosine residues chosen for mutation were residues which have previously received much attention due to their being the sites of reaction of the inactivating chemical modification reagents 4-chloro-7-nitrobenzofurazan (Tyr-297) and p-fluorosulfonylbenzoyl-5'-adenosine (Tyr-354) . We found that mutation of Tyr-297----Phe caused only minor impairment of catalysis, and mutation of Tyr-354----Phe produced no impairment . Therefore, a direct role for either of these tyrosine residues in catalysis is unlikely.

J Immunol, 1987 Jun 15, 138(12), 4236 - 42
Monoclonal antibodies to human recombinant interleukin 1 (IL 1)beta: quantitation of IL 1 beta and inhibition of biological activity; Kenney JS et al.; Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta . Four McAb have been identified that inhibit the biological activity of IL 1 beta . McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro . McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays . The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay . These McAb block the binding of recombinant {125I}IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol . Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb . H6 and H34 recognize one epitope, and H21 and H67 another . McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta . The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay . The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E . coli LPS-stimulated human monocyte culture supernatants (HMCS) . The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml . By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard . This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb . Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay . The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.

Biochim Biophys Acta, 1987 Jun 12, 900(1), 38 - 44
Large scale transfection of mouse L-cells by electropermeabilization; Stopper H et al.; Mouse L-cells were transfected by electropermeabilization using the selectable plasmid pSV2-neo which confers resistance to G-418 (Geneticin) . The DNA concentration used was 1 microgram/ml, the field strength was 10 kV/cm, the duration of the pulse was 5 microseconds . Transfection yield was optimal at a temperature of 4 degrees C when using a time in between consecutive pulses of 1 minute compared to shorter (of the order of seconds) or longer (3 minutes) time intervals . A more detailed study of the relationship between the number of pulses applied (up to 10) and transfection yield showed it to be almost linear in this range at 4 degrees C . The yield of transfectants in response to 10 pulses was up to 1000 per 10(6) cells (using 3.3 pg DNA per cell) . The influence of the growth phase of the cells on the transfection yield and/or the subpopulation of the mouse L-cell line used was shown . Furthermore the clone yield depended on the DNA per cell ratio within a very small range.

Biochim Biophys Acta, 1987 Jun 12, 900(1), 116 - 28
Lipopeptides of the N-terminus of Escherichia coli lipoprotein: synthesis, mitogenicity and properties in monolayer experiments; Prass W et al.; The N-terminal part of the lipoprotein from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-L-Cys-Ser and analogs with longer sequences, are polyclonal activators for B-lymphocytes . Triple-chain lipopeptides also constitute efficient low-molecular-weight carrier/adjuvant systems, which can be linked to antigens to yield immunogens for antibody production without further additives . This is the first report of monolayer experiments with chemically well defined, synthetic lipopeptide mitogens with the composition of the N-terminus of an important bacterial membrane protein . Various derivatives of the lipoprotein N-terminus were synthesized . These lipopeptides differed in the length of the peptide moiety, the number of fatty acid residues, and protective groups . In order to obtain the surface areas for the lipopeptides in isotherms and hysteresis isotherms, monolayer experiments with a computer-controlled film balance were performed . To get some information about the interaction of these compounds with typical membrane lipids mixed monolayers were formed from triple-chain lipopeptides with dipalmitoylphosphatidylcholine and cholesterol . A comparison of the mitogenic response of the compounds was made in an in vitro system with B-lymphocytes from Balb/c mice.

Science, 1987 Jun 12, 236(4807), 1448 - 53
A physical map of the Escherichia coli K12 genome; Smith CL et al.; A physical map of a genome is the structure of its DNA . Construction of such a map is a first step in the complete characterization of that DNA . The restriction endonuclease Not I cuts the genome of Escherichia coli K12 into 22 DNA fragments ranging from 20 kilobases (20,000 base pairs) to 1000 kilobases . These can be separated by pulsed field gel electrophoresis . The order of the fragments in the genome was determined from available E . coli genetic information and analysis of partial digest patterns . The resulting ordered set of fragments is a macrorestriction map . This map facilitates genetic and molecular studies on E . coli, and its construction serves as a model for further endeavors on larger genomes.

Biochim Biophys Acta, 1987 Jun 12, 900(1), 63 - 72
Optimal posttranslational translocation of the precursor of PhoE protein across Escherichia coli membrane vesicles requires both ATP and the protonmotive force; De Vrije T et al.; In order to reach their final destination, periplasmic and outer membrane proteins have to pass the cytoplasmic membrane of Escherichia coli cells . To study the transport of PhoE protein, we developed an in vitro transcription-translation and translocation system . In this in vitro system, the protein is synthesized as a larger precursor, which can be processed by purified leader peptidase . The precursor can be translocated into inverted inner membrane vesicles as judged by the protection against externally added protease . Only part of the translocated protein is in the processed mature form . Translocation can occur posttranslationally and requires both ATP and the protonmotive force for an optimal process . Upon incubation of vesicles with mature PhoE protein or precursor PhoE in the absence of ATP, the proteins are bound to the vesicles, but they are not translocated, since they are still sensitive to externally added protease.

Nature, 1987 Jun 11-17, 327(6122), 532 - 5
Introduction of foreign DNA into mycobacteria using a shuttle phasmid; Jacobs WR Jr et al.; Mycobacteria are major pathogens of man and animals . There are approximately 10 million cases of tuberculosis world wide with an annual mortality of three million people . Leprosy, caused by Mycobacterium leprae, afflicts over ten million people, primarily in developing countries . M . tuberculosis and mycobacteria of the M . avium-intracellulare-scrofulaceum (MAIS) group are major opportunistic pathogens of patients with acquired immune deficiency syndrome (AIDS) . M . paratuberculosis is the cause of Johne's disease in cattle . Yet, BCG (Bacille Calmette-Guerin), an avirulent strain of M . bovis, is the most widely used human vaccine in the world, having been administered to about 2.5 X 10(9) people since 1948 (ref . 4) . BCG was highly protective against tuberculosis in England, but has been found not to be effective in preventing pulmonary tuberculosis in adults in Southern India . We have initiated studies to develop the methodology for efficient gene transfer in mycobacteria . We have constructed recombinant shuttle phasmids which are chimaeras containing mycobacteriophage DNA into which an E . coli cosmid is inserted . They can replicate in E . coli as plasmids and in mycobacteria as phages, and transfer DNA across both genera . These shuttle vectors permit for the first time the introduction of foreign DNA by infection into M . smegmatis and BCG . By introducing and ultimately expressing genes for protective antigens for a variety of pathogens, it may be possible to develop cultivatable mycobacteria into useful multivaccine vehicles.

Nucleic Acids Res, 1987 Jun 11, 15(11), 4669 - 86
The bases of the tRNA anticodon loop are independent by genetic criteria; Smith D et al.; We employed two methods to study the translational role of interactions between anticodon loop nucleotides . Starting with a set of previously constructed weakly-suppressing anticodon loop mutants of Su7, we searched for second-site revertants that increase amber suppressor efficiency . Though hundreds of revertants were characterized, no second-site revertants were found in the anticodon loop . Second site reversion was detected in the D-stem, thereby demonstrating the efficacy of the search method . As a second method for detecting interactions, we used site-directed mutagenesis to construct multiple mutations in the anticodon loop . These multiple mutants are very weak suppressors and have translational activities that are equal to or lower than that predicted for the independent action of single mutations . We conclude that although the anticodon loop sequence of Su7 has an optimal structure for the translation of amber codons, we find no evidence that interactions between loop bases can enhance translational efficiency.

Nucleic Acids Res, 1987 Jun 11, 15(11), 4403 - 15
Site-directed cleavage of RNA; Shibahara S et al.; Using complementary chimeric oligonucleotides containing deoxyribonucleotides and 2'-O-methylribonucleotides (1), enzymatically synthesized RNA (90 mer) were cleaved at a single site with Escherichia coli RNaseH, either at a hairpin loop or at a stem region . Especially, site-specific cleavage occurred in even the target region being enclosed within a stable, base-paired stem . The method is proved to be generally applicable to RNA containing secondary structures.

Biochim Biophys Acta, 1987 Jun 9, 892(1), 10 - 22
Cytochrome pools in membranes of Escherichia coli grown aerobically on L-proline; Withers HK et al.; The cytochromes of membranes of the cydA mutant Escherichia coli GR19N grown on a proline-amino acid medium were examined . Reduced minus oxidized difference spectra (including fourth-order finite difference spectra) showed that cytochromes with absorption maxima at 554-555, 556-557, 560-561.5 and 563.5-564.5 nm were present . In addition, there were two components with absorption maxima at 548.5 and 551.5 nm which made a minor contribution to the alpha-band absorbance . These were not examined further . Two pools within the cytochromes were detected . One pool, which was reduced rapidly by the substrates NADH, formate and succinate, consisted of cytochromes of the cytochrome o complex . These cytochromes had absorption maxima at 555, 557 and 563.5 nm . In addition, the low-potential cytochrome associated with formate dehydrogenase was reduced rapidly by formate, and a component absorbing at 560-561.5 nm was also present in this pool . The second pool of cytochromes was reduced more slowly by substrate, although the rate was accelerated greatly in the presence of the electron mediator phenazine methosulfate . These cytochromes absorbed maximally at about 556.5 nm . A portion of the cytochrome in this pool was reoxidized by fumarate . This cytochrome may be a component of the fumarate reductase pathway, since the membranes showed high NADH-fumarate reductase activity . The respiratory chain inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide appeared to act at two sites . One site of inhibition was between the dehydrogenases and the cytochromes . A second site of inhibition was located in the cytochrome o complex between cytochrome b-564 and oxygen.

FEBS Lett, 1987 Jun 8, 217(1), 49 - 52
Proposal that the function of the membrane-bound cytochrome a1-like haemoprotein (cytochrome b-595) in Escherichia coli is a direct electron donation to cytochrome d; Poole RK et al.; The cytochrome d-containing oxidase of oxygen-limited Escherichia coli comprises cytochromes d, cytochrome b-558 and cytochrome b-595, previously called cytochrome a1 . The reaction of the fully reduced complex with oxygen involves ligand binding to the ferrous haem d to form an oxygenated species, followed by oxidation of two b-type cytochromes, whose identity is unclear . Here we report kinetic studies on cytochrome b-595 oxidation and suggest that these results, together with optical and EPR data on the oxidase complex and its reaction with oxygen, are consistent with the hypothesis that the role of cytochrome b-595 is further reduction of the oxygen bound to cytochrome d.

Biochim Biophys Acta, 1987 Jun 6, 909(1), 58 - 70
The interaction of the trp repressor from Escherichia coli with the trp operator; Lane AN et al.; We have examined the interaction of the trp repressor from Escherichia coli with a 20 base-pair synthetic operator . Nonspecific binding was relatively strong (Kd = 2 microM), but only weakly sensitive to the concentration of added salt {d log Kd)/(d log {Na}) = -1) . 1H-NMR studies indicate that the structure of the repressor is not greatly altered on forming the complex, and that few if any of the lysine and arginine residues make direct contact with the DNA . However, the mobility of one of the two tyrosine residues is significantly decreased in the complex . The repressor makes close contact with the major grooves of the operator such that the base protons are broadened much more than expected on the basis of increased correlation time . There are large, differential changes in chemical shifts of the imino protons on forming the complex, as well as changes in the rate constants for exchange . The fraying of the ends is greatly diminished, consistent with a target size of about 20 base-pairs . The effects of the repressor on the NMR spectra and relaxation rate constants can be interpreted as a change in the conformation of the operator, possibly a kinking in the centre of the molecule.

Lancet, 1987 Jun 6, 1(8545), 1277 - 81
Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine; Ballou WR et al.; A recombinant DNA Plasmodium falciparum sporozoite vaccine produced in Escherichia coli (FSV-1) was tested in doses of 10 micrograms to 800 micrograms protein in fifteen volunteers . No serious adverse reactions occurred . Antibodies that reacted with P falciparum sporozoite antigens by enzyme-linked immunoassay developed in twelve of the volunteers . The highest antibody titres induced were similar to those resulting from lifelong natural exposure to sporozoite-infected mosquitoes . Postimmunization serum samples from a majority of volunteers mediated the circumsporozoite (CS) precipitation reaction and inhibited sporozoite invasion of hepatoma cells in vitro . Serum from the three volunteers who received 800 micrograms doses reacted with the surface of sporozoites in an immunofluorescence assay . Six immunised volunteers receiving a fourth dose of FSV-1 and two non-immunised controls were challenged by bites of mosquitoes infected from cultured P falciparum gametocytes . Parasitaemia did not develop in the volunteer with the highest titre of CS antibodies, and parasitaemia was delayed in two other immunised volunteers . This study confirms that human beings can be protected by CS protein subunit vaccines and provides a framework for the further development and testing of more immunogenic sporozoite vaccines.

J Mol Biol, 1987 Jun 5, 195(3), 741 - 4
Equilibrium DNA-binding of AraC protein . Compensation for displaced ions; Martin KJ et al.; Experiments on the AraC regulatory protein of Escherichia coli suggest a mechanism that DNA-binding proteins can use to reduce potentially drastic alterations in their affinity for DNA resulting from changes in salt concentration . Measurement of the net number of ions apparently displaced as AraC protein binds DNA and of fluorescence changes in the protein lead to the following picture . About 14 ions are displaced from the DNA as the protein binds the araI site . As the protein binds the DNA, however, it undergoes a conformational change and binds about ten ions . Consequently, the net order of the reaction is reduced from 15th to about fourth order in salt concentration.

J Mol Biol, 1987 Jun 5, 195(3), 701 - 29
Refined structure of glutathione reductase at 1.54 A resolution; Karplus PA et al.; The crystal structure of human glutathione reductase has been established at 1.54 A resolution using a restrained least-squares refinement method . Based on 77,690 independent reflections of better than 10 A resolution, a final R-factor of 18.6% was obtained with a model obeying standard geometry within 0.025 A in bond lengths and 2.4 degrees in bond angles . The final 2Fo-Fc electron density map allows for the distinction of carbon, nitrogen and oxygen atoms with temperature factors below about 25 A2 . Apart from 461 amino acid residues and the prosthetic group FAD, the model contains 524 solvent molecules, about 118 of which can be considered an integral part of the enzyme . The largest solvent cluster is at the dimer interface and contains 104 interconnected solvent molecules, part of which are organized in a warped sheet-like structure . The main-chain dihedral angles are well-concentrated in the allowed regions of the Ramachandran plot . The spread of dihedral angles in beta-pleated sheets is much larger than in alpha-helices and especially in alpha-helix cores, indicating the higher plasticity of beta-structures . The analysis revealed a large amount of 3(10)-helix . The side-chain conformations cluster at the staggered positions, and show well-defined preferences . Also, a mobility gradient is observed for side-chains . Non-polar and polar side-chains show average temperature factor increases per bond of 10% and 25%, respectively . A number of alternative conformations of internal side-chains, in particular serines and methionines, have been detected . The extended FAD molecule also shows a mobility gradient between the very rigid flavin (mean value of B) = 8.7 A2) and the more mobile adenine (mean value of B = 16.2 A2) . The entire active center is particularly well ordered, with temperature factors around 10 A2 . The dimer interface consists of a rigid contact area, which is well conserved in the Escherichia coli enzyme, and a flexible area that is not . Altogether, the buried surfaces at the crystal contacts are half as large as at the dimer interface, but less specific . The refined structure shows clearly that there are no buried cations compensating the charge of the pyrophosphate moiety of FAD . The flavin deviates slightly from standard geometry, which is possibly caused by the polypeptide environment . In contrast to an earlier interpretation, atom N5 of the flavin can accommodate a proton, and it is conceivable that this proton proceeds to the redox-active disulfide.(ABSTRACT TRUNCATED AT 400 WORDS)

J Chromatogr, 1987 Jun 5, 417(1), 11 - 25
Determination of endotoxins by gas chromatography: evaluation of electron-capture and negative-ion chemical-ionization mass spectrometric detection of halogenated derivatives of beta-hydroxymyristic acid; Sonesson A et al.; The sensitivity and selectivity of gas chromatography for analysing several halogenated ester derivatives of beta-hydroxymyristic acid were studied using both selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry and electron-capture detection . Six different derivatization methods were compared with respect to yield, chemical stability and formation of by-products . Procedures for removal of excess reagents using disposable silica columns and thin-layer chromatography were elaborated . The 3-O-pentafluorobenzoyl-methyl ester was the preferred derivative since it provided high sensitivity and had the molecular ion as the base peak in the mass spectrum . The detection limit was 0.5 pg with electron-capture detection and 0.3 pg with the mass spectrometric system . Using beta-hydroxymyristic acid as a chemical marker it was possible to detect Escherichia coli endotoxin in aqueous solution at a level of 1 ng/ml.

J Biol Chem, 1987 Jun 5, 262(16), 7463 - 71
The regiochemistry and stereochemistry of the biosynthesis of vitamin B6 from triose units; Hill RE et al.; 13C and 2H NMR spectroscopy has been employed to probe the biosynthesis of vitamin B6 in Escherichia coli . The 13C NMR spectrum of a sample of pyridoxol derived biosynthetically from D-{1,2,3,4,5,6-13C6}glucose shows that the bonds, C(2)-C(3) and C(4)-C(5), of the pyridine nucleus are the only two carbon-carbon bonds of pyridoxol which are generated de novo in the course of its biosynthesis from glucose . It follows that the pyridoxol skeleton is generated from two intact triose units and a triose-derived two-carbon unit, all of which are supplied by glucose . From the 2H NMR spectra of samples of pyridoxol derived from (R)-{1,1-2H2}glycerol and (S)-{1,1-2H2}glycerol, respectively, it can be deduced that the rehydroxymethyl group of glycerol enters C-2', C-4', and C-5' of the pyridoxol skeleton . It follows that each of the three fragments is derived from glycerol in stereo-specific fashion . These results answer questions concerning the regiochemistry and the stereochemistry of pyridoxol biosynthesis.

Cell, 1987 Jun 5, 49(5), 709 - 17
The integration host factor of Escherichia coli binds to bent DNA at the origin of replication of the plasmid pSC101; Stenzel TT et al.; The integration host factor (IHF) of Escherichia coli is necessary for maintenance of pSC101 . The protein binds specifically to the replication origin of the plasmid, in the AT-rich region located immediately adjacent to the left, weak binding site for the plasmid-encoded initiator protein . DNAase I and OH- radical footprinting experiments showed that IHF protects 49 bp of the DNA at the origin region . Methylation protection analyses revealed that IHF contacts purine residues in both the major and minor grooves of the DNA . Electrophoretic analyses showed that IHF binds to bent DNA, and the protein binding further enhances the degree of DNA bending . Site-directed mutagenesis of three of the contact points not only abolished binding of the protein to the DNA but also inactivated the replication origin . Therefore, binding of IHF to the ori sequence most probably is necessary for initiation of plasmid replication.

J Mol Biol, 1987 Jun 5, 195(3), 745 - 8
Binding of Escherichia coli RNA polymerase to a promoter carrying mutations that stop transcription initiation; Johnston F et al.; The gal P2 promoter can be inactivated by point mutations located in the -10 hexamer sequence or immediately upstream from it . Mutations at either site reduce expression in vivo and prevent the formation, in vitro, of tight complexes with RNA polymerase that give a strong footprint and can initiate transcription . However, with a mutation upstream from the -10 region, RNA polymerase could still make a specific contact with gal promoter DNA as judged by interference with cleavage by restriction enzyme SfaNI at a site within the promoter . In contrast, with a mutation in the -10 hexamer sequence, RNA polymerase could not make this contact and does not interfere with restriction by SfaNI.

J Mol Biol, 1987 Jun 5, 195(3), 555 - 79
Structure of the C-terminal domain of the ribosomal protein L7/L12 from Escherichia coli at 1.7 A; Leijonmarck M et al.; The structure of a C-terminal fragment of the ribosomal protein L7/L12 from Escherichia coli has been refined using crystallographic data to 1.7 A resolution . The R-value is 17.4% . Six residues at the N terminus are too disordered in the structure to be localized . These residues are probably part of a hinge in the complete L7/L12 molecule . The possibility that a 2-fold crystallographic axis is a molecular 2-fold axis is discussed . A patch of invariant residues on the surface of the dimer is probably involved in functional interactions with elongation factors.

J Mol Biol, 1987 Jun 5, 195(3), 495 - 504
Affinities of tight-binding lactose repressors for wild-type and pseudo-operators; Betz JL; Five tight-binding (Itb) mutants of the Escherichia coli lactose (lac) repressor have been characterized with regard to their non-specific affinity for DNA and their specific affinity for the wild-type operator and several sequence-altered (pseudo-) operators . Repressor-operator association rates were determined in the presence or absence of competitor DNA, dissociation rates of repressor from various DNA fragments were measured, and equilibrium competition for repressor binding was examined for several pseudo-operator DNAs . The mutant repressors exhibited increased non-specific affinity for DNA, and variable increases in affinity for sequence-altered operators . The known positions of amino acid substitutions for three of these Itb repressors support suggestions that residues 51 to 64 are important for operator recognition in addition to residues 1 to 50.

J Biol Chem, 1987 Jun 5, 262(16), 7484 - 5
Molecular weight of recombinant human tumor necrosis factor-alpha; Arakawa T et al.; Recombinant DNA-derived human tumor necrosis factor-alpha from Escherichia coli was examined by equilibrium ultracentrifugation under conditions similar to those where gel filtration experiments suggested an oligomeric structure . Short-column equilibrium experiments at concentrations in the range 0.015-0.12% at pH 8.5 in 0.04 M Tris/Tris-HCl gave molecular weights corresponding to 3 times the sequence molecular weight both in the presence and absence of 0.1 M NaCl . Long (2.6 mm)-column experiments under the same solvent conditions indicated molecular weights of 51,900 +/- 900 in the absence of added NaCl and 52,600 +/- 700 in the presence of added 0.1 M NaCl . No evidence of any species other than the trimer was found.

J Biol Chem, 1987 Jun 5, 262(16), 7725 - 31
Malic enzyme from archaebacterium Sulfolobus solfataricus . Purification, structure, and kinetic properties; Bartolucci S et al.; An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4 . The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration . Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits . Amino acid composition of S . solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli . In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S . solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate . The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1 . The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures . At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively . The S . solfataricus malic enzyme was shown to be very thermostable.

J Biol Chem, 1987 Jun 5, 262(16), 7802 - 7
Isolation of the yeast gene encoding elongation factor 3 for protein synthesis; Qin SL et al.; The gene YEF-3 encoding the elongation factor 3 (EF-3) for peptide chain elongation in Saccharomyces cerevisiae has been isolated by immunoscreening of a yeast genomic library in the phage lambda gt11 . The identity of the EF-3 gene was confirmed by several methods . First, a clone-encoded protein could affinity purify the antibody that specifically reacted with EF-3 . Second, a recombinant fusion protein, which reacted with anti-beta-galactosidase antibody as well as with anti-EF-3 antibody, was found in the lysate of a positive clone lysogen . Third, the function of EF-3 in a yeast mutant in which the EF-3 activity is temperature-sensitive in an in vitro assay could be complemented by transformations . EF-3 protein was overproduced in a transformant which contained the EF-3 gene on a multicopy plasmid YEp-13 . Southern blot analysis shows that YEF-3 is a single copy gene . The transcript unit as mapped by S1 nuclease mapping, is consistent with the size of the message determined by Northern blot analysis and shows no evidence of introns.

J Biol Chem, 1987 Jun 5, 262(16), 7672 - 5
Expression of human angiotensinogen cDNA in Escherichia coli; Kunapuli SP et al.; Human angiotensinogen cDNA clones were isolated from a human liver library . Nucleotide sequence analysis of these cDNA clones revealed that position 1075 in the messenger RNA, which is part of a PstI recognition sequence, is different from the published sequence (Kageyama, R., Ohkubo, H., and Nakanishi, S . (1984) Biochemistry 23, 3603-3609) . This change results in an altered amino acid at this position in the corresponding protein sequence and suggests possible restriction fragment length polymorphism . The full length human angiotensinogen cDNA was constructed from partial cDNA clones and ligated into an isopropyl-1-thio-beta-D-galactopyranoside inducible bacterial expression vector pUC9 to develop expression plasmid pUCHAG27 . This plasmid permitted the synthesis of human angiotensinogen in Escherichia coli . The recombinant bacteria overproduced a 53-kDa protein which was recognized by anti-human angiotensinogen antibodies . The synthesis of this protein was greatly increased upon induction with isopropyl-1-thio-beta-D-galactopyranoside . The chimeric protein, almost identical to human angiotensinogen, was partially purified by ammonium sulfate fractionation and gel filtration on Sephadex G-100 . Human kidney renin was shown to enzymatically cleave this recombinant protein to produce des-(angiotensin I)-angiotensinogen and a small polypeptide . Thus, we provide evidence that recombinant human angiotensinogen synthesized through E . coli is biologically active and serves as a substrate for human renin.

J Biol Chem, 1987 Jun 5, 262(16), 7443 - 6
Isolation and properties of a mutant of Escherichia coli possessing defective Na+/H+ antiporter; Ishikawa T et al.; A mutant of Escherichia coli with defective Na+/H+ antiporter was isolated . The rationale for its isolation was that cells possessing defective Na+/H+ antiporter, which is essential for establishment of a Na+ gradient, could not grow with a carbon source that was taken up with Na+ . The mutant had no appreciable Na+/H+ antiporter activity, but its K+/H+ antiporter and Ca2+/H+ antiporter activities were normal . Judging from the reversion frequency, the defect seems to be due to a single mutation . The mutant could not grow at alkaline pH . Therefore, the Na+/H+ antiporter, but not the K+/H+ antiporter or the Ca2+/H+ antiporter, seems to be responsible for pH regulation in alkaline medium . This mutant will be useful for cloning the Na+/H+ antiporter gene and for detection of Na+-substrate cotransport systems.

Cell, 1987 Jun 5, 49(5), 603 - 12
lac repressor can regulate expression from a hybrid SV40 early promoter containing a lac operator in animal cells; Brown M et al.; The E . coli lac operator and repressor were adapted for function in mammalian cells . Plasmids containing an SV40 early region (pSVlacO) or a chloramphenicol acetyl transferase gene (pSVlacOCAT) linked to a hybrid SV40 early promoter bearing a lac operator were tested for function . Identical plasmids lacking an operator (pX-8 and pX-8CAT) were controls . In vitro, early transcription from pSVlacO, but not from pX-8, was inhibited by lac repressor, and repression was overcome by IPTG . Repression of large T synthesis or CAT activity occurred in vivo only when the respective operator-containing plasmid was cotransfected with a plasmid encoding lac repressor, or when the recipient cells stably synthesized lac repressor . IPTG substantially relieved repression in both cases . CAT enzyme repression was paralleled by a decrease in CAT mRNA abundance . Thus regulatory elements of the lac operon function physiologically in mammalian cells.

J Biol Chem, 1987 Jun 5, 262(16), 7639 - 45
Purification and characterization of proteolytic fragments of lipocortin I that inhibit phospholipase A2; Huang KS et al.; Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R . L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B . (1986) Nature 320, 77-80) . To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping . Five active fragments have been analyzed in detail . The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin . Three of the larger fragments contain this region . The fifth fragment is missing 83 amino acids from the amino terminus . A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B . (1986) Cell 46, 191-199) and thus presumably is important for activity . In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases . Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.

J Biol Chem, 1987 Jun 5, 262(16), 7686 - 92
Loss of unisite and multisite catalyses by Escherichia coli F1 through modification with adenosine tri- or tetraphosphopyridoxal; Noumi T et al.; Pyridoxal phosphate (PLP) and adenosine diphospho (AP2-PL)-, triphospho (AP3-PL)-, and tetraphospho (AP4-PL)-pyridoxals (Tagaya, M., and Fukui, T . (1986) Biochemistry 25, 2958-2964) were tested as potential affinity probes for F1 ATPase of Escherichia coli . Both AP3-PL and AP4-PL bound and inhibited F1 ATPase, whereas PLP and AP2-PL were weak inhibitors . The concentrations of AP3-PL and AP4-PL for half-maximal inactivations of the multisite (steady state) ATPase activity were both 18 microM . The binding of these reagents to a reactive lysyl residue(s) was confirmed from the difference absorption spectra, and the stoichiometry of binding of {3H}AP3-PL to F1 at the saturating level was about 1 mol/mol F1 . The analogue bound to both the alpha subunit (about two-thirds of the radioactivity) and the beta subunit (about one-third of the radioactivity) . No inactivation of multisite ATPase activity or binding of AP3-PL was observed in the presence of ATP . F1 modified with about one mol of AP3-PL had essentially no uni- and multisite hydrolysis of ATP . The rate of binding of ATP decreased to 10(-2) of that of unmodified F1, and the rate of release of ATP was about two times faster . The equilibrium F1 X ATP in equilibrium F1 X ADP X Pi was shifted toward F1 X ATP, and no promotion of ATP hydrolysis at unisite was observed with excess ATP . These results suggest that the AP3-PL or AP4-PL bound to an active site, and catalysis by the two remaining sites was completely abolished.

Nature, 1987 Jun 4-10, 327(6121), 437 - 9
Mutations in the active site of Escherichia coli phosphofructokinase; Hellinga HW et al.; The enzyme-catalysed transfer of a phosphoryl group from ATP is an important reaction in a wide variety of biological processes . We demonstrate here the essential function of an aspartate group in the catalysis of phosphoryl transfer by Escherichia coli phosphofructokinase, and the minor role of an arginine residue . We have used oligonucleotide-directed mutagenesis to replace two amino-acid residues which X-ray analysis has shown to be close to the transferred phosphoryl group and we have analysed the forward and back reactions of the mutant enzymes by steady-state kinetics . Changing Asp 127 to Ser reduced the turnover number by a factor of 18,000 in the forward direction and 3,100 in the back reaction, and the Michaelis constant for fructose 1,6-bisphosphate in the reverse reaction by a factor of 45 . This shows that this aspartate is a key residue in the rate enhancement by the enzyme, probably acting as a base in the reaction mechanism, and that it also destabilizes the product complex . Changing Arg 171 to Ser reduced the turnover numbers by about 3.4, showing that this arginine has only a minor effect on the catalysis.

Biochemistry, 1987 Jun 2, 26(11), 3135 - 41
Refolding of denatured thioredoxin observed by size-exclusion chromatography; Shalongo W et al.; Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes . In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change . A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0 . Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm . Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed . Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones . Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration . Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones . As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Jun 2, 26(11), 3099 - 106
A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding . Application to Escherichia coli single-strand binding protein-nucleic acid interactions; Bujalowski W et al.; We describe a general method for the analysis of ligand-macromolecule binding equilibria for cases in which the interaction is monitored by a change in a signal originating from the ligand . This method allows the absolute determination of the average degree of ligand binding per macromolecule without any assumptions concerning the number of modes or states for ligand binding or the relationship between the fractional signal change and the fraction of bound ligand . Although this method is generally applicable to any type of signal, we discuss the details of the method as it applies to the analysis of binding data monitored by a change in fluorescence of a ligand upon binding to a nucleic acid . We apply the analysis to the equilibrium binding of Escherichia coli single-strand binding (SSB) protein to single-stranded nucleic acids, which is monitored by the quenching of the intrinsic tryptophan fluorescence of the SSB protein . With this method, one can quantitatively determine the relationship between the fractional signal change of the ligand and the fraction of bound ligand, LB/LT, and rigorously test whether the signal change is directly proportional to LB/LT . For E . coli SSB protein binding to single-stranded nucleic acids in its (SSB)65 binding mode {Lohman, T . M., & Overman, L . B . (1985) J . Biol . Chem . 260, 3594; Chrysogelos, S., & Griffith, J . (1982) Proc . Natl . Acad . Sci . U.S.A . 79, 5803}, we show that the fractional quenching of the SSB fluorescence is equal to the fraction of bound SSB.

Biochemistry, 1987 Jun 2, 26(11), 3129 - 34
Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding; Tsuji T et al.; Cloned cDNA of human interleukin 2 (IL-2) was expressed in Escherichia coli cells in which IL-2 formed insoluble inclusion bodies . Human IL-2 has three Cys residues, namely, Cys-58, Cys-105, and Cys-125, and native IL-2 has an intramolecular disulfide bond between Cys-58 and Cys-105 . Since the formation of inclusion bodies was thought to be due to disorder in the oxidation state of these Cys residues, all intramolecular disulfide bond isomers of IL-2 were prepared by denaturation of native IL-2 to characterize the state of a disulfide bond in IL-2 in the inclusion bodies . These isomers can be separated from native IL-2, reduced IL-2, and IL-2's with intermolecular disulfide bonds by means of reversed-phase high-performance liquid chromatography . Human IL-2 produced in inclusion bodies in E . coli carrying a recombinant DNA was analyzed by HPLC and was proved to be a fully reduced form with no intra- and intermolecular disulfide bonds . Refolding of reduced IL-2 in the presence of reduced and oxidized glutathione and a low concentration of guanidine hydrochloride resulted in the formation of the biologically active IL-2 quantitatively . Further purification provided a practically pure IL-2 preparation without contamination of any disulfide bond isomers.

Bioorg Khim, 1987 Jun, 13(6), 845 - 8
{Addressed modification of the promoter region of DNA in a complex with RNA polymerase by alkylating oligonucleotide derivatives}; Butorin AS et al.; Addressed chemical modification of double-stranded DNA unwound with the specific protein has been demonstrated . 4(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphoamides of oligonucleotides d(CG)rC, d(ATCG)rC, d(AATCG)rC, which can serve as primers in the RNA polymerase-catalyzed transcription, alkylated A2 promoter region of pSK-A2 plasmid in its "open" complex with E . coli RNA polymerase.

Sci Sin {B}, 1987 Jun, 30(6), 625 - 9
The effect of upstream sequences to initiator on the expression of gene coding for hepatitis B core antigen; Shi CH et al.; The nucleotide sequences of cloned adw hepatitis B core antigens (HBcAg) which have different levels of expression have been determined by dideoxy chain-terminating method . The results indicate that the different levels of gene expression is primarily due to the different structures of upstream sequences before the initiator of HBcAg genes . The hepatitis B core gene in the low-expressed clone forms a secondary structure before the initiator . This secondary structure has been removed by Bal-31 exonuclease in the high-expressed clone and half of the structure removed in the mid-expressed clone . It is apparent that the transcription/translation of the hepatitis B core gene is somehow blocked because of the secondary structure . Therefore, the amount of HBcAg synthesized in E . coli is dramatically reduced.

Can J Physiol Pharmacol, 1987 Jun, 65(6), 1325 - 8
The development of a febrile response to pyrogen in the thyroid-deficient rabbit; Macari M et al.; The development of the febrile response to E . coli lipopolysaccharide (1.5 micrograms/kg, i.v.) in thyroid-deficient rabbits has been studied . Twenty-eight New Zealand White rabbits weighing 2.1-2.3 kg were used . Hypothyroidism was induced by treatment with propylthiouracil (100 or 200 mg/kg body wt./15 days) . Thyroid-deficient animals showed a reduction in the febrile response to lipopolysaccharide, but the effect was significantly different (p less than 0.01) from the control only for rabbits treated with 200 mg/kg of propylthiouracil . Propranolol (2 mg/kg, i.p.) given 30 min before lipopolysaccharide also reduced (p less than 0.01) the fever response in control rabbits . The results of this experiment are consistent with the hypothesis that the reduction in the febrile response of thyroid-deficient rabbits is due to the reduced number of beta-adrenergic receptors, or to a change in the availability of neurotransmitter in thermogenically active tissues, such as brown fat.

Am J Vet Res, 1987 Jun, 48(6), 971 - 6
Mixed venous oxygen tension as an estimate of cardiac output in anesthetized horses; Wetmore LA et al.; The relationship between mixed venous O2 tension and cardiac output was studied in six anesthetized horses breathing 100% O2 . Cardiac output, O2 consumption, mean arterial pressure, heart rate, and arterial and venous blood gases were measured after administration of xylazine or dobutamine to horses in lateral, sternal, and dorsal recumbencies . After approximately 3 hours, Escherichia coli endotoxin was administered while horses were in dorsal recumbency, and all measurements were repeated . Relationships between cardiac index (CI) and PVO2, heart rate, mean arterial pressure, jugular PVO2, and PVO2 of blood from a superficial limb vein were evaluated by linear regression analysis . Mean arterial pressure was significantly (P less than 0.05) correlated with CI in horses in all positions and after endotoxin administration . However, data points were poorly grouped . Heart rate and CI were significantly correlated in horses in all positions, but not after endotoxin administration . Correlations between jugular PVO2 and PVO2 of blood from a superficial limb vein were not significant in horses in sternal recumbency, and PVO2 of blood from a superficial limb vein was not significantly correlated with CI in horses in lateral recumbency . There was a significant and tight correlation between PVO2 and CI in horses in all positions and after endotoxin administration.

Nature, 1987 Jun 25-Jul 1, 327(6124), 717 - 20
The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex; Dinauer MC et al.; The bacteriocidal capacity of phagocytic cells is impaired in X-linked chronic granulomatous disease (X-CGD), a disorder characterized by the absence of functional plasma-membrane-associated NADPH oxidase . The components of this oxidase system, their correspondence with specific genetic loci, and the primary protein defect in X-CGD remain incompletely defined . We recently reported cloning of the putative X-CGD gene on the basis of DNA linkage . To identify the predicted protein in vivo, antibodies were raised to a synthetic peptide derived from the complementary DNA sequence and to a fusion protein produced in Escherichia coli . In Western blots antisera detect a neutrophil protein of relative molecular mass in 90,000 (90K) that is absent in X-CGD patients . Antisera also react with the larger component of cytochrome b recently purified from neutrophil plasma membranes as a complex of glycosylated 90K and non-glycosylated 22K polypeptides . Based on our identification of the X-CGD protein in vivo, we propose that one of its critical roles is to interact with the 22K species to form a functional cytochrome b complex.

Hybridoma, 1987 Jun, 6(3), 313 - 20
Monoclonal antibody to human beta interferon: characterization and application; Sugi M et al.; Seven stable mouse hybridomas secreting monoclonal antibodies to human fibroblast beta interferon (IFN-beta) were isolated, all seven of which belonged to IgGl subclass and kappa type . While neutralizing the antiviral activity of human fibroblast IFN-beta, they failed to neutralized both that of human IFN-alpha and human IFN-gamma . These monoclonal antibodies neutralized the antiviral activity of human fibroblast IFN-beta but not that of human IFN-alpha and IFN-gamma . Two of the seven monoclonal antibodies, YSB-1 and YSB-2, showed particularly high neutralization titers in the ascitic fluid . Monoclonal antibodies were purified from cultures of hybridoma grown in a serum-free medium . The purified monoclonal antibodies, YSB-1(5 micrograms/ml) and YSB-2(1 microgram/ml), neutralized IFN-beta from 10EU/ml to 1EU/ml . Human fibroblast IFN-beta was purified to 3.5 X 10(7) IU/mg protein (1.0 X 10(8) IU/mg protein in the peak fraction) by the monoclonal antibody (YSB-2) affinity column chromatography, whereas human recombinant IFN-beta obtained from E.coli was also purified to 6.5 X 10(7) IU/mg protein (1.1 X 10(8) IU/mg protein in the peak fraction) by the monoclonal antibody (YSB-1) affinity column chromatography.

Virology, 1987 Jun, 158(2), 444 - 6
Gene I products of cauliflower mosaic virus detected in extracts of infected tissue; Young MJ et al.; The product of cauliflower mosaic virus (CaMV) gene I has been characterized from extracts of infected plants . Two size classes of this protein can be identified by the use of specific antiserum . The antiserum was induced against a chimeric protein produced in E . coli from a gene fusion between a fragment of the CaMV genome and the beta-galactosidase gene . A 36-kDa form of the gene I product is associated with virus particles . A 45-kDa form of this product was found only in the insoluble fraction of tissue homogenates . This insoluble fraction, the only fraction found to contain the product of viral gene VI, contains the virally induced inclusion bodies.

J Bacteriol, 1987 Jun, 169(6), 2847 - 53
Bordetella parapertussis and Bordetella bronchiseptica contain transcriptionally silent pertussis toxin genes; Arico B et al.; Pertussis toxin, the major virulence factor of Bordetella pertussis, is not produced by the closely related species Bordetella parapertussis and Bordetella bronchiseptica . It is shown here that these two species possess but do not express the complete toxin operon . Nucleotide sequencing of an EcoRI fragment of 5 kilobases comprising the regions homologous to the pertussis toxin genes shows that in this region, B . parapertussis and B . bronchiseptica are 98.5% and 96% homologous, respectively, to B . pertussis . The changes (mostly base pair substitutions) in many cases are identical in B . parapertussis and B . bronchiseptica, suggesting that these two species derive from a common ancestor . Many of the mutations common to B . parapertussis and B . bronchiseptica involve the promoter region, which becomes very inefficient . The S1 subunits of both species, when expressed in Escherichia coli, have the same ADP-ribosylating activity as the S1 subunit from B . pertussis, indicating that the mutations in the S1 gene described here do not affect its function.

Proc Soc Exp Biol Med, 1987 Jun, 185(2), 206 - 10
Erythrocyte deformability in canine septic shock and the efficacy of pentoxifylline and a leukotriene antagonist; Puranapanda V et al.; Decreased microcirculatory flow in the case of endotoxic shock has been extensively reported . The flexibility of the red blood cell plays an important role in tissue perfusion . The present study with a canine shock model was designed to investigate the changes in erythrocyte flow properties during septic shock . Deformabilities of canine erythrocytes were found to decrease considerably 6 hr after infusion of Escherichia coli organisms (P less than 0.05) . Pretreatment of dogs with pentoxifylline reduced the effect by improving erythrocyte deformability significantly and by increasing animal survival times . The leukotriene antagonist LY171883 also increased survival time, but did not change red cell filterability . The mortality of the dogs was not affected by either pentoxifylline or the leukotriene antagonist . Both drugs aided the concentration recovery of circulating leukocytes by 6 hr post-E . coli infusion.

J Virol, 1987 Jun, 61(6), 1834 - 41
Evidence that the sigma 1 protein of reovirus serotype 3 is a multimer; Bassel-Duby R et al.; In this report, we study the reovirus serotype 3 (strain Dearing) sigma 1 protein obtained from various sources: from Escherichia coli expressing sigma 1 protein, from reovirus-infected mouse L cells, and from purified reovirions . We demonstrate that the sigma 1 protein is a multimer in its undisrupted form and present biochemical evidence suggesting that the multimer is made up of four sigma 1 subunits.

Infect Immun, 1987 Jun, 55(6), 1466 - 75
Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG expressed in escherichia coli K-12; Thole JE et al.; We report the DNA sequence of a previously cloned Mycobacterium bovis BCG gene encoding an immunogenic 64-kilodalton protein . This protein, MbaA, was purified from overproducing Escherichia coli K-12 cells, and the presence of antibodies to MbaA in human sera was determined by an enzyme-linked immunosorbent assay . In about 80% of serum samples from tuberculosis patients and in about 60% of samples from BCG-vaccinated individuals, significant levels of anti-MbaA antibodies were found . Surprisingly, in about 30% of the control serum samples obtained from children, anti-MbaA antibodies were also observed . Guinea pigs sensitized with M . bovis BCG or MbaA showed a delayed-type hypersensitivity reaction after challenge with purified MbaA, supporting the previously observed strong reactivity of human T-cell clones with this, for mycobacteria, common antigen.

Can J Physiol Pharmacol, 1987 Jun, 65(6), 1255 - 60
Role of the anteroventral third ventricle region in fever in sheep; Blatteis CM et al.; Ablation of the anteroventral third ventricle (AV3V) region, which includes the organum vasculosum laminae terminalis (OVLT), blocks the febrile response of guinea pigs to systemically injected endotoxin; by contrast, discrete lesions of the OVLT transiently enhance fever in rabbits and rats . To assess whether separate subdivisions of the AV3V may mediate these different effects, the thermal responses to Escherichia coli lipopolysaccharide (LPS, 0.25 micrograms/kg, i.v.) were measured in eight sheep before and 12-13 days after placement of lesions at various levels within the AV3V . The responses of four of these sheep to crude homologous endogenous pyrogen (EP, 1-2 mL, i.c.v.) were also evaluated . Additionally, five other sheep were tested with LPS 2-8 months postlesion . All the experiments were performed at thermoneutrality . Sheep were used because most of the frontal wall of their 3V forms an elongated OVLT consisting of an avascular body and a vascular base . The animals were classified postmortem according to the extent of tissue ablated . Lesion overlap analyses showed that (i) medial lesions which extended from the floor of the 3V to the anterior commissure and laterally into adjacent preoptic periventricular tissue were associated with significantly depressed fever after LPS (n = 2); (ii) comparable lesions, but which excluded the ventral portion of the AV3V, i.e., the base of the OVLT, did not alter the magnitude of the febrile response to LPS (n = 4); (iii) lesions of the lateral walls of the 3V and (or) of the adjacent medial preoptic and anterior hypothalamic areas but excluding the frontal 3V wall also did not affect fever height after LPS (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Invest, 1987 Jun, 79(6), 1648 - 52
Recombinant murine granulocyte macrophage colony-stimulating factor has megakaryocyte colony-stimulating activity and augments megakaryocyte colony stimulation by interleukin 3; Robinson BE et al.; Recombinant murine granulocyte macrophage colony-stimulating factor (rGM-CSF) has been produced in Escherichia coli and purified to homogeneity . GM-CSF has an established role as an in vitro regulator of granulocyte and macrophage colony formation . We have determined that rGM-CSF also has intrinsic activity as a megakaryocyte colony-stimulating factor and that rGM-CSF augments the effect of interleukin 3 (IL-3) on megakaryocyte colony formation . The dose-response curve for megakaryocyte colony induction with rGM-CSF showed plateau megakaryocyte stimulation at 9 ng/ml . When IL-3 (at a plateau dose for megakaryocyte colony induction) was added to rGM-CSF over a 0-22-ng/ml dose range, the resultant megakaryocyte colony stimulation approximated the sum of the levels of stimulation produced by either factor alone . These results establish GM-CSF as a multilineage growth factor with definite megakaryocyte colony-stimulating activity and indicate that both GM-CSF and IL-3 are important in the regulation of megakaryocytopoiesis.

Cell Immunol, 1987 Jun, 107(1), 130 - 7
In vitro effects of retinoids on murine thymus-dependent and thymus-independent mitogenesis; Dillehay DL et al.; The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated . The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids . However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited . All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses . In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation . Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR . These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol . Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells . Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.

Protein Eng, 1987 Jun, 1(3), 201 - 3
Site-directed mutants of the cAMP receptor protein--DNA binding of five mutant proteins; Gent ME et al.; Oligonucleotide-directed mutagenesis was employed to generate mutants of the cAMP receptor protein (CRP) of Escherichia coli . The mutant proteins were purified to homogeneity and tested for stability and DNA binding . It is shown that mutations at the position of Arg180 abolish specific DNA binding, whereas those at the position Arg185 have very little effect . Both positions have previously been implicated as crucial for the specific interaction between CRP and DNA . The Ser128----Ala mutant shows a slight reduction in DNA binding affinity relative to wild-type . All mutants investigated show similar stability profiles to wild-type CRP with respect to thermolysin proteolysis as a function of temperature.

Comput Appl Biosci, 1987 Jun, 3(2), 121 - 7
Repetitive palindromic sequences in Escherichia coli . Detection and characterization with a new computer program; Saurin W; We have made use of a new computer program which rapidly searches for DNA patterns irrespective of the exact sequence, in order to search systematically for repetitive palindromic structures on the Escherichia coli chromosome . By using a relatively restrictive criterion, we detected three families of palindromic structures . One is present in ribosomal RNA operons . Since seven identical ribosomal RNA operons are found on the E . coli genome, this constitutes a validation of the method . Another family is constituted by the extragenic palindromic units (PUs or REP) already described (Gilson et al., 1984) . We propose a slightly different consensus for this highly repetitive and dispersed family . The last is a subclass of symmetrical transcription termination sites which we have identified . They are present in four attenuator sites and at least one terminator for convergent operons . We suggest that these symmetrical terminator sites may play a general role in the termination of transcription for convergent operons . We discuss briefly how this type of approach could be used to analyse the structure of genomes.

Biochem Cell Biol, 1987 Jun, 65(6), 507 - 13
Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12; Wood JM et al.; The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA . Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract . The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA) . Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein . Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain . Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein . Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold . The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form . These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.

Nippon Seikeigeka Gakkai Zasshi, 1987 Jun, 61(6), 775 - 83
{Significance of the serum rheumatoid factor-like substance in the induction of arthritis in the rabbit immunized with Escherichia coli}; Takagi T; Rabbits immunized with heat-killed Escherichia coli 0:14 for 8-10 months developed lymphocyte infiltration in the synovium at a significantly (p less than 0.05) higher rate (61.1%, 22 of 36 knees) than animals immunized for 4 months (31.3%, 10 of 32) . Serum rheumatoid factor-like substance (RFLS) was positive (RAHA-titer more than 80) as early as 3 weeks after the initial treatment . After 15 weeks of immunization 90.9% (20 of 22 rabbits) of the animals were RFLS positive . Lymphocyte infiltration in the synovium of knees was observed more frequently (81.8%, 9 of 11 rabbits) (p less than 0.05) in the group with positive RFLS earlier than 8 weeks following immunization than in that with RFLS only after this period (18.2%, 2 of 11) . These observations suggest that the long viability of serum RFLS and certain factors inducing early RFLS synthesis might be important in producing arthritis in rabbit immunized with E . coli.

Prostaglandins, 1987 Jun, 33(6), 931 - 9
Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes; Pignol B et al.; PAF-acether, at doses ranging from 1pM to 0.1 microM did not induce a significative release and/or synthesis of IL1 from monocytes . In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed . PAF-acether at 1pM increased IL1 release by 120 +/- 39% and synthesis by 87 +/- 27% whereas at 0.1 microM a decrease of IL1 release of 52 +/- 9% and synthesis of 46 +/- 6% were observed . BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase of inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1 microM of the autacoid, respectively . No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted . These results indicate that PAF-acether modulates monocytes functions, possibly via specific binding sites.

J Gen Microbiol, 1987 Jun, 133 ( Pt 6), 1601 - 10
The effect of catalase on recovery of heat-injured DNA-repair mutants of Escherichia coli; Mackey BM et al.; The apparent sensitivity of Escherichia coli K12 to mild heat was increased by recA (def), recB and polA, but not by uvrA, uvrB or recF mutations . However, addition of catalase to the rich plating medium used to assess viability restored counts of heat-injured recA, recB and polA strains to wild-type levels . E . coli p3478 polA was sensitized by heat to a concentration of hydrogen peroxide similar to that measured in autoclaved recovery medium . The apparent heat sensitivity of DNA-repair mutants is thus due to heat-induced sensitivity to the low levels of peroxide present in rich recovery media . It is proposed that DNA damage in heated cells could occur indirectly by an oxidative mechanism . The increased peroxide sensitivity of heat-injured cells was not due to a decrease in total catalase activity but may be related specifically to inactivation of the inducible catalase/peroxidase (HPI).

J Gen Microbiol, 1987 Jun, 133 ( Pt 6), 1553 - 62
Nucleotide sequence of the immunity and lysis region of the ColE9-J plasmid; James R et al.; We have determined the nucleotide sequence of a 1500 bp fragment of the ColE9-J plasmid which encodes colicin E9 immunity and colicin E5 immunity and contains two lys genes . Open reading frames corresponding to the four genes have been located and their position confirmed by transposon mutagenesis of sub-clones of the ColE9-J plasmid . The E9imm gene shows 69% homology at both the nucleotide and the amino acid level to the previously sequenced E2imm gene . The E5imm gene shows little homology to any other E colicin immunity gene which has been sequenced . The lys gene distal to the 3' end of the E5imm gene shows considerable sequence homology to all other previously sequenced E colicin lys genes . The lys gene distal to the 3' end of the E9imm gene is identical to the pColE2 and pColE3 lys genes for the first 59 nucleotides but encodes a much smaller gene product than any other lys gene which has been sequenced . The two lys genes sequenced here are exceptions to Shepherd's rule concerning the number of RNY codons in the three possible reading frames.

J Cell Sci, 1987 Jun, 87 ( Pt 5), 667 - 75
Inhibition of cell adhesion by a synthetic polymer adsorbed to glass shown under defined hydrodynamic stress; Owens NF et al.; A co-polymer with hydrophobic and hydrophilic segments was allowed to adsorb from aqueous solution onto glass previously made hydrophobic by derivatization with octadecyl dimethylchlorosilane . The polymer is thought to adsorb via its hydrophobic segments, leaving the hydrophilic segments free to extend into the water . After allowing cells to settle on the treated surface, the shear stress at the chamber wall required to remove red blood cells, Dictyostelium discoideum amoebae and Escherichia coli was determined in a calibrated laminar flow chamber . On octadecyl glass a shear stress of 2-3 Nm-2 evicts 50% of adherent red cells and E . coli . No D . discoideum amoebae could be removed at 5Nm-2 . In striking contrast, the lowest experimentally obtainable shear stress of 0.03 Nm-2 removes 97.0-99.5% of cells of all three types from the polymer-treated surface, even after a cell residence time of 1 h without flow in the absence of free polymer . The minimum shear stress of 0.03Nm-2 corresponds to only approximately equal to 20 times the force of gravity on a red cell . The mechanism of action of the polymer and the implications of the results are discussed.

Isr J Med Sci, 1987 Jun, 23(6), 678 - 82
Spiroplasma plasmids; Razin S et al.; Extrachromosomal DNA, constituting plasmids or replicative forms of viruses, has been detected in a variety of spiroplasmas, particularly in Spiroplasma citri . Only a few of the S . citri plasmids were characterized by restriction enzyme mapping, and essentially nothing is known on functions encoded by the plasmids . Our studies revealed in S . citri (R8A2) an 8.0-kbp plasmid that differed from previously described plasmids in its restriction map . It was also clonable in pBR322 . The plasmid, named pRA1, was found in large quantities as free plasmid in S . citri (R8A2) subclones of low passage level . In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome, as revealed by Southern hybridization blots of digested spiroplasmal DNA with nick-translated pRA1 or its recombinant as probes . Significant quantities of integrated plasmid sequences were also observed in S . kunkelii and in Spiroplasma sp . P40 . Small quantities of free and/or integrated plasmid DNA were detected in some spiroplasmas serologically and genotypically remote from S . citri . Chromosome-integrated pRA1 sequences were cloned into the Escherichia coli plasmids pUC13 and M13 . Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of small repetitive sequences inserted into specific sites on the spiroplasma chromosome . Despite the large number of the inserts they do not appear to affect significantly gene expression in the spiroplasma . Due to the abundance of free and integrated pRA1 in S . citri, nick-translated pRA1 was effective as a DNA probe in detecting small numbers of S . citri in infected periwinkle plants and leafhoppers.

Zh Mikrobiol Epidemiol Immunobiol, 1987 Jun, (6), 36 - 8
{Combinations of pathogenicity factors in Escherichia coli isolated from children and domestic animals}; Mnatsakanov ST; The study has shown that in E . coli strains isolated from children aged up to 1 year with acute intestinal diseases of unknown etiology different combinations of pathogenicity factors (Ent . K88, K99, Vir, CFAI, CFAII, HIy, ColV, ColI) can be detected in 47.1 +/- 3.8% of cases, while in E . coli strains isolated from practically healthy children of the same age combined carriership of these factors does not exceed 5.8 +/- 2.5% . In E . coli strains isolated from swine and cattle with diarrhea the combined carriership of pathogenicity factors is 68.5 +/- 2.8% and 58.4 +/- 4.9% respectively.

Tsitologiia, 1987 Jun, 29(6), 695 - 705
{Postreplication DNA repair in Escherichia coli cells . III . Repair independent of the presence of pKM101 and COLIb-P9 plasmids}; Zhestianikov VD et al.; The presence of pKM101 or ColIb-P9 plasmids in E . coli leads to the increase in the survival of UV-irradiated cells of wild type and of polAI, recB21 recC22 and dnaGts mutants; it does not change the survival of recA13 and lex3 mutants and does not influence kinetics and efficiency of postreplication repair (PRR) of DNA in cells of all the strains examined (with the exception of PG3 dnaGts mutant whose PRR of DNA in the presence of pKM101 plasmid is somewhat lower) . The survival of both plasmid-containing and plasmid-free bacteria treated with chloramphenicol decreases in the same degree, but the survival of chloramphenicol-treated recA13, lex3 recB21 rec C22 mutants does not change . The pKM101 plasmid does not lend the dnaGts mutant a new capacity of repairing postreplication gaps with the participation of inducible component of PRR; the chloramphenicol-sensitive component of PRR is absent in this mutant . Plasmid and plasmid-free E . coli strains of wild type and of the polA1 mutant do not differ by the kinetics and level of inducible chloramphenicol-sensitive component of PRR of DNA.

Mol Biol Med, 1987 Jun, 4(3), 169 - 81
High-level expression in Escherichia coli of a soluble and fully active recombinant interleukin-1 beta; Huang JJ et al.; A complementary DNA sequence encoding monocyte interleukin-1 (IL-1), beta form/pI7, was expressed in Escherichia coli . Recombinant plasmid pDP516 was constructed by cloning and rebuilding the mature IL-1 coding sequence into an E . coli expression vector . Bacteria transformed with pDP516 constitutively produced recombinant IL-1 (r-IL-1) at 15-20% of total E . coli protein . The r-IL-1 was found to be in the soluble fraction of sonicated E . coli Bacterial r-IL-1 (DP516) has been purified to homogeneity by anion exchange and sizing column chromatography, with an apparent molecular weight of 17,500 . The identity of the purified r-IL-1 was confirmed by amino acid and DNA sequencing analyses . Purified recombinant IL-1 DP516 exhibits biological activity similar to that of native monocyte IL-1 (3 approximately 4 X 10(7) units/mg) . An amino-terminal deletion mutant completely abolishes the biological activity, indicating that the integrity of the IL-1 molecule might be important for its function.

Int J Pept Protein Res, 1987 Jun, 29(6), 685 - 91
Conformation and stability of two recombinant human interferon-alpha analogs; Davis JM et al.; Structural features of a recombinant E . coli derived interferon-alpha analog, interferon consensus1, was studied by circular dichroism and fluorescence spectroscopy . Circular dichroic spectra of the purified protein showed that it has about 70% alpha-helix and a distinct tertiary structure . These structural features are similar to those for a natural interferon-alpha subtype, interferon-alpha 2, indicating that the amino acid substitutions in interferon consensus1 apparently did not alter the protein structure . Another analog, interferon consensus5, which has Ser instead of Cys at residues 1 and 99 but is otherwise identical to interferon consensus1, was prepared to study the role of the disulfide bond between Cys 1 and 99 . Circular dichroic and fluorescence spectra indicated similarity in the structure of these two analogs . However, interferon consensus1 was significantly more stable than interferon consensus5 against denaturation . pH unfolding experiments indicated that the former protein is more stable in the transition region by about 1.6 kcal/mol, which was interpreted in terms of the increased free energy of the denatured state due to an extra disulfide bond in interferon consensus1.

Mol Gen Genet, 1987 Jun, 208(1-2), 94 - 100
The unusual translational initiation codon AUU limits the expression of the infC (initiation factor IF3) gene of Escherichia coli; Brombach M et al.; The expression of infC, the structural gene for translational initiation factor IF3, has been studied in different constructs under the control of the lambda PL and tac promoters . The amount of synthesized IF3 has been determined by a quantitative functional test and the levels of IF3-specific mRNA have been estimated . The synthesis of IF3 is strongly enhanced when the unusual AUU initiation codon is changed to AUG by site-directed mutagenesis . Removal of the sequence upstream from the start codon including most of the Shine-Dalgarno sequence, as well as part of a 10 bp region with potential complementarity to an internal region of the 16S rRNA, which is unique to the IF3 mRNA, reduced but did not completely abolish the high expression of infC obtained after introduction of the AUG initiation codon . The level of IF3 mRNA was found to be positively influenced by the presence of the rplT gene in the plasmid downstream from the infC gene . In vivo accumulation of a large excess of IF3, obtained when the infC gene was placed under the control of an incompletely repressed tac promoter, was not accompanied by any noticeable adverse phenotype.

Mol Gen Genet, 1987 Jun, 208(1-2), 88 - 93
Alkaline phosphatase which lacks its own signal sequence becomes enzymatically active when fused to N-terminal sequences of Escherichia coli haemolysin (HlyA); Erb K et al.; Fusion of the alkaline phosphatase gene (phoA) which lacks its own signal peptide sequence to the N-terminal region of hlyA, the structural gene for Escherichia coli haemolysin, leads to active alkaline phosphatase (AP) . AP activity depends on the length of the N-terminal region of hlyA . An optimum is reached when 100-200 amino acids of HlyA are fused to PhoA but fusion of as little as 13 amino acids of HlyA to PhoA is sufficient to yield appreciable AP activity . When cells are treated with lysozyme most of the AP activity is found associated with the membrane fraction but a substantial amount is also found in the soluble fraction, most of which may represent a periplasmic pool of AP . The soluble portion of AP activity is significantly increased when the cells are disrupted by ultrasonication, which indicates that the fusion proteins are only loosely associated with the membrane and that large parts are already located on the outside of the cytoplasmic membrane . The expected fusion proteins were identified in the soluble and the membrane fractions and their amounts in these fractions correlated well with AP activity.

Mol Gen Genet, 1987 Jun, 208(1-2), 70 - 5
Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12; Nakao T et al.; The nucleotide sequence of the putP gene coding for the proline carrier in Escherichia coli has been determined and the amino acid sequence of the proline carrier deduced from it . The proline carrier is predicted to consist of 502 amino acids, resulting in a molecular weight of 54,343 . The predicted protein is very hydrophobic (70% nonpolar amino acids), and its hydropathy profile suggests that it is composed of 12 hydrophobic segments with a mean length of 24.4 residues/segment . If these segments are assumed to be alpha-helical, the mean length of each domain corresponds to the thickness of the hydrophobic core of the membrane . Potential promoter, catabolite gene activator protein (CAP) binding sites and several palindromic sequences, which might be regulatory regions by the putA gene product, were also found in the 5' flanking region of the postulated putP gene . A typical rho-independent transcription termination signal was found after the terminator codon of the putP gene.

Mol Gen Genet, 1987 Jun, 208(1-2), 294 - 300
Molecular aspects of genetic instability of an artificial 68 bp perfect palindrome in Escherichia coli; Shafferman A et al.; An artificial 68 bp perfect palindrome carried on a plasmid (pAS807) is genetically unstable . An increase in the population of cells harbouring palindrome-deleted pAS807 derivatives (pAS807-V) is observed as the number of cell generations increases . The calculated frequency of palindrome excision events per cell generation and per plasmid replication round in Escherichia coli is 0.95 X 10(-4) . Sequence analysis of eight independent isolates of palindrome-deleted molecules, reveals two symmetrical deletion types (three of type I and five of type II) . The two types of pAS807-v molecules retain 19 bp of the original sequence of the 68 bp palindrome but differ in the content of the central 3 bp . The generation of the two deletion types is best explained by formation of intermediate cruciform structures . Following the fate of the palindrome in various bacterial mutants, we find that the excision events depend on functional polA1, polA(ex1), lig, texA343(recC343) and texA344(recB344) gene products . However, recB21 recC22 mutations do not affect palindrome excision.

Mol Gen Genet, 1987 Jun, 208(1-2), 263 - 70
Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins; Shafferman A et al.; The functional R6K alpha origin is composed of two DNA elements, one of 580 bp carrying the alpha origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previously identified as also required for gamma and beta origin activity . These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for alpha origin activity . The function of the alpha origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K . Activation of the alpha origin depends on the R6K replication initiation protein pi . Within the 580 bp of the alpha origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the beta replicon . Deletion of the 96 bp or 98 bp results in inactivation of the alpha and the beta origins respectively . These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the alpha and the beta origins of R6K . DNA homology analysis performed on alpha, beta and gamma origin sequences, also reveals 10-23 bp sequences in the alpha and the beta origins that are related to the family of 22 bp direct repeats in the gamma origin which were shown previously to be binding sites for the pi protein.

Mol Gen Genet, 1987 Jun, 208(1-2), 247 - 53
Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors; Scholle A et al.; The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the beta-methylgalactoside transport system . The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties . To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains . The mutation causes the replacement of Gly74 of GBP by Asp . This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains . We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg . Our sequence also included part of the mglA gene, which is immediately distal to mglB . The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.

Mol Gen Genet, 1987 Jun, 208(1-2), 242 - 6
Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production; Boulnois GJ et al.; Transposon and deletion analysis of the cloned K1 capsule biosynthesis genes of Escherichia coli revealed that approximately 17 kb of DNA, split into three functional regions, is required for capsule production . One block (region 1) is required for translocation of polysaccharide to the cell surface and mutations in this region result in the intracellular appearance of polymer indistinguishable on immunoelectrophoresis to that found on the surface of K1 encapsulated bacteria . This material was released from the cell by osmotic shock indicating that the polysaccharide was probably present in the periplasmic space . Insertions in a second block (region 2) completely abolished polymer production and this second region is believed to encode the enzymes for the biosynthesis and polymerisation of the K1 antigen . Addition of exogenous N-acetylneuraminic acid to one insertion mutant in this region restored its ability to express surface polymer as judged by K1 phage sensitivity . This insertion probably defines genes involved in biosynthesis of N-acetylneuraminic acid . Insertions in a third block (region 3) result in the intracellular appearance of polysaccharide with a very low electrophoretic mobility . The presence of the cloned K1 capsule biosynthesis genes on a multicopy plasmid in an E . coli K-12 strain did not increase the yields of capsular polysaccharide produced compared to the K1+ isolate from which the genes were cloned.

J Interferon Res, 1987 Jun, 7(3), 285 - 99
Characterization of three species of Escherichia coli-derived human leukocyte interferon A separated by reverse-phase high-performance liquid chromatography; Nakagawa S et al.; A preparation of recombinant human leukocyte interferon A (rIFN-alpha A) obtained from Escherichia coli cells was found by reverse-phase high-performance liquid chromatography (HPLC) to contain three species . The three species, named Mf-1, Mf-2, and Ms in the order of their elution, were separated and characterized to elucidate their structural differences . The amino acid compositions, the amino-terminal amino acid sequences (Cys1-Asp2-Leu3-), and the carboxy-terminal amino acids (Glu) of the three species agreed with those predicted from the cDNA sequence, although the sequence analysis yields of Mf-2 and Ms were extremely low . They were found to have the same disulfide bonds, Cys1-Cys98 and Cys29-Cys138, by amino acid analysis of the tryptic peptides, but the recovery of the peptides linked by a Cys1-Cys98 disulfide bond for Mf-2 and Ms was extremely low . The results demonstrate that although the primary structures of the three species are almost identical, small difference(s) exists among them especially at the amino-terminal portion.

Genetics, 1987 Jun, 116(2), 201 - 6
High frequency generalized transduction by miniMu plasmid phage; Wang BM et al.; Deletion derivatives of phage Mu which replicate as multicopy plasmids, and also transpose and package like Mu, have been developed for the in vivo cloning of bacterial genes . We show here that these miniMu plasmid phage are also efficient at generalized transduction and that both in vivo cloning and generalized transduction of a given gene can be accomplished in a single experiment.

Carbohydr Res, 1987 Jun 1, 163(1), 81 - 90
Structural investigation of the capsular polysaccharide from Escherichia coli O9:K37 (A 84a); Anderson AN et al.; The structure of the capsular polysaccharide from E . coli O9:K37 (A 84a) has been studied, using methylation analysis, Smith degradation, and graded acid hydrolysis . The configurations at the anomeric centres were assigned by 1H-n.m.r . spectroscopy of the polysaccharide and its derivatives and oligosaccharide fragments . The polysaccharide has the following trisaccharide repeating-unit which is unique in the E . coli series of capsular polysaccharides in possessing a 1-carboxyethylidene group as the sole acidic function . (Formula: see text) E . coli capsular polysaccharides have been classified into seventy-four serotypes . The structures of about twenty of these polysaccharides have been elucidated, one of which, K29, has been reported to contain a 1-carboxyethylidene group . In continuation of a programme aimed at establishing the structural basis for the serology and immunochemistry of the E . coli capsular antigens, we now report on the structure of the capsular polysaccharide from E . coli O9:K37.

Appl Environ Microbiol, 1987 Jun, 53(6), 1246 - 50
Improved membrane filtration media for enumeration of total coliforms and Escherichia coli from sewage and surface waters; Freier TA et al.; Two media were developed that allowed both a total coliform count and an Escherichia coli count to be determined on the same medium after 24 h of incubation at 35 degrees C . The new media were tested along with two standard media on 10 surface water and 7 sewage samples . The experimental media yielded equivalent or higher counts relative to the standard media and recovered more specifically the desired indicator groups as determined by colony identification.

J Inorg Biochem, 1987 Jun, 30(2), 77 - 85
A spectroscopic investigation of cobalt(II) substituted alkaline phosphatase; Banci L et al.; The electronic and 1H NMR spectra are reported for the cobalt(II) alkaline phosphatase (EC 3.1.3.1.) system at pH around 6 in the range 0-2 mol of cobalt per mol protein . It is shown that under the present experimental conditions cobalt(II) selectively populates the A sites . Three isotropically shifted NH signals have been detected in the A site that indicate the presence of three histidines in the coordination sphere of cobalt(II) . The electronic spectra and the nuclear relaxation properties are consistent with pentacoordination of cobalt(II) in the A site . The finding of reproducible preparation routes for the derivatives, and of appropriate experimental conditions for the observation of their 1H NMR spectra, open new possibilities for the spectroscopic investigation of alkaline phosphatase.

Fed Proc, 1987 Jun, 46(8), 2485 - 9
Reducing complexity in metabolic networks: making metabolic meshes manageable; Palsson BO et al.; The dynamics of complex systems can be effectively analyzed by judicious use of intrinsic time constants . Order of magnitude estimation based on time constants has been used successfully to examine the dynamic behavior of complicated processes . The main goal of this paper is to introduce this approach to the analysis of complex metabolic systems . Time constants and dynamic modes of motion are defined within the context of well-established linear algebra . The order of magnitude estimation is then introduced into the systemic framework . The main goals of the analysis are: to provide improved understanding of biochemical dynamics and their physiological significance, and to yield reduced dynamic models that are physiologically realistic but tractable for practical use.

Eur J Biochem, 1987 Jun 1, 165(2), 403 - 8
The peptidyltransferase centre of the Escherichia coli ribosome . The histidine of protein L16 affects the reconstitution and control of the active centre but is not essential for release-factor-mediated peptidyl-tRNA hydrolysis and peptide bond formation; Tate WP et al.; Modification of the Escherichia coli 50S ribosomal subunit with histidine-specific diethyl pyrocarbonate affects peptide bond formation and release-factor-dependent peptidyl-tRNA hydrolysis . Unmodified L16 can restore activity to a split protein fraction from the altered subunit but other proteins of the core also contain histidine residues important for the activity of the peptidyltransferase centre . When isolated and purified by centrifugation, particles reconstituted with unmodified proteins and modified L16 do not retain the altered L16 . The modified protein does mediate the partial restoration of peptide bond formation and release-factor-2 activities to these particles . It must be exerting its effect during the assembly of the peptidyltransferase centre in the reconstituted particle . A particle could be reconstituted which lacks L16 and has significant activity in peptide bond formation and peptidyl-tRNA hydrolysis . L16 stimulates these activities . A tighter ribosomal binding of the release factor 2, dependent upon the absence of protein L11, can in part compensate for the loss of activity of the peptidyltransferase centre when it is assembled with either modified L16 or in the absence of L16 . The protein and its histidine residue seem important, therefore, for the peptidyltransferase centre to be formed in the correct conformation but not essential for activity once the centre is assembled.

DNA, 1987 Jun, 6(3), 221 - 9
Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector; Libby RT et al.; The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector . A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E . coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3 . The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor . Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay . The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus . The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF . Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC . Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.

Am J Physiol, 1987 Jun, 252(6 Pt 1), G748 - 54
Splanchnic hemodynamics in portal hypertensive dogs with portal fibrosis; Sugita S et al.; Splanchnic hemodynamics and portal systemic shunting were measured in eight dogs with experimentally induced portal fibrosis and splenomegaly and in six normal dogs by the radioactive microsphere technique . Portal fibrosis and splenomegaly were produced by repeated intraportal injections of a mixture of killed nonpathogenic Escherichia coli and dog anti-E . coli serum . All E . coli-treated dogs developed intrahepatic presinusoidal portal hypertension (portal vein pressure 15.8 +/- 5.4 vs . 7.5 +/- 0.9 mmHg in controls, P less than 0.005; intrahepatic pressure 6.8 +/- 2.1 vs . 6.2 +/- 1.4 mmHg in controls, NS) within 2.5 mo, but no portal systemic shunt was demonstrated at this time (2.1 +/- 1.5 vs . 0.7 +/- 0.4%, NS) . Portal venous inflow, the total blood flow within the portal system, was increased in the treated dogs (27.6 +/- 6.6 vs . 18.2 +/- 2.5 ml X min-1 X kg body wt-1, P less than 0.005) . Total splanchnic arterial vascular resistance was reduced in these dogs (26.0 +/- 10.4 vs . 40.9 +/- 4.6 dyn X s X cm-5 X 10(3), P less than 0.01) as a result of reduced arteriolar resistance in the spleen, jejunum and ileum, colon, and omentum, in all of which blood flow increased . In these animals both portal venous flow (27.0 +/- 6.5 vs . 18.1 +/- 2.5 ml X min-1 X kg body wt-1, P less than 0.005) and intrahepatic portal vascular resistance (1.9 +/- 0.7 vs . 0.7 +/- 0.3 dyn X s X cm-5 X 10(3), P less than 0.005) were increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1987 Jun, 158(2), 431 - 4
The attachment sites of T5-host range temperate coliphages; Poon AP et al.; The attachment sites of 13 temperate coliphages were determined . Specialized transduction of proAB mutants was shown by eight isolates and of a his mutant by another two . Two isolates were concluded to integrate at atthtt and the integration site of one isolate remained undetermined.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4318 - 21
Expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants; Gershoni JM; Restriction fragments of DNA derived from a cDNA clone of the alpha subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2 . Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound alpha-bungarotoxin . Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria . This provides a new approach for gene selection via functional expression--i.e., ligand overlays of colony blots.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4195 - 9
DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis; Hagensee ME et al.; Strains of Escherichia coli possessing the pcbA1 mutation, a functional DNA polymerase I, and a temperature-sensitive mutation in DNA polymerase III can survive at the restrictive temperature (43 degrees C) for DNA polymerase III . The mutation rate of the bacterial genome of such strains after exposure to either UV light or ethyl methanesulfonate was measured by its rifampicin resistance or amino acid requirements . In addition, Weigle mutagenesis of preirradiated lambda phage was also measured . In all cases, no increase in mutagenesis was noted at the restrictive temperature for DNA polymerase III . Introduction of a cloned DNA polymerase III gene returned the mutation rate of the bacterial genome as well as the Weigle mutagenesis to normal at 43 degrees C . Using a recA-lacZ fusion, the SOS response after UV irradiation was measured and found to be normal at the restrictive and permissive temperature for DNA polymerase III, as was induction of lambda prophage . Recombination was also normal at either temperature . Our studies demonstrate that a functional DNA polymerase III is strictly required for mutagenesis at a step other than SOS induction.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4054 - 7
Separations of open-circular DNA using pulsed-field electrophoresis; Levene SD et al.; The effect of high electric fields on the gel-electrophoretic mobility of open-circular DNA in agarose differs dramatically from that on linear molecules of the same molecular weight . At high fields, sufficiently large circular forms are prevented from migrating into the gel whereas linear molecules and smaller circular DNAs migrate normally . This effect is strongly field dependent, affecting circular molecules of decreasing size with increasing field strength . We have studied this effect with a series of plasmid DNAs ranging from 2.9 to 56 kilobase pairs using continuous and reversing-pulse electric fields . Application of reversing pulses abolishes the effect under certain conditions and supports the model for the gel electrophoresis of open-circular DNA where circular forms are trapped by engaging the free end of an agarose gel fiber.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 3977 - 81
Relative roles of T7 RNA polymerase and gene 4 primase for the initiation of T7 phage DNA replication in vivo; Sugimoto K et al.; Initiation sites of T7 phage DNA replication in the presence and absence of T7 phage gene 4 primase have been analyzed by using Escherichia coli cells infected with T7 phage amber mutants, T73,6 and T73,4,6, respectively . Restriction analysis of the {3H}thymidine-labeled DNA, synthesized by the T73,4,6 phage-infected cells in the presence of 2',3'-dideoxy-3'-azidothymidine, has shown that only the light (L) strand of T7 DNA has been synthesized from the primary origin area to the right . Transition sites from RNA to DNA have been located precisely in the primary origin region of the T7 phage genome . In the gene 4- condition, greater than 20 transition sites have been detected only in the L strand . They scattered widely downstream from the phi 1.1 promoters and mostly downstream from the phi 1.3 promoter . The same transition sites have been detected in the gene 4+ condition, suggesting that the transcripts started from these promoters are used as primers of the rightward L-strand DNA synthesis in the gene 4+ condition . In addition, many heavy (H)- and L-strand transition sites have been detected at gene 4 primase sites in the gene 4+ condition . The relative roles of T7 phage RNA polymerase and primase at the primary origin have been discussed.

Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3758 - 62
Spectrum of cisplatin-induced mutations in Escherichia coli; Burnouf D et al.; Using a forward-mutation assay based on the inactivation of the tetracycline-resistance gene located on plasmid pBR322, we have determined the mutation spectrum induced in Escherichia coli by cisplatin {cis-diamminedichloroplatinum(II)}, a widely used antitumor drug . Cisplatin is known to form mainly intrastrand diadducts at ApG and GpG sites . We found that cisplatin efficiently induces mutations in an SOS-dependent way (i.e., dependent upon UV irradiation of the host bacteria) . More than 90% of the mutations are single-base-pair substitutions occurring at the potential sites of cisplatin adducts (ApG and GpG) . Taking into account the relative proportions of ApG and GpG adducts, we found that the ApG adducts are at least 5 times more mutagenic than the GpG adducts . Moreover, a strong mutation specificity was seen at the 5' side of the ApG adducts (A X T----T X A transversions) . The observation that most mutations occur at the 5' end of the adduct at both ApG and GpG sites is discussed in relation to recent structural data.

J Med Microbiol, 1987 Jun, 23(4), 335 - 8
Examination of enteropathogenic Escherichia coli strains for an adenylcyclase stimulating factor; Law D et al.; Enteropathogenic Escherichia coli strains are a common cause of infantile diarrhoea but do not produce recognised enterotoxins . Three strains of proven virulence were examined for toxins which may be missed in conventional tests . Cell lysates and concentrated culture supernates of organisms grown in five different media gave negative results when examined for adenylcyclase stimulating activity . The additions of zinc ions or lincomycin to these media or the use of iron-depleted media also gave negative results . The significance of these findings and the possible role of other toxins in diarrhoea due to enteropathogenic E . coli are discussed.

J Lab Clin Med, 1987 Jun, 109(6), 679 - 86
Differential elaboration of prostaglandin E2 by cells of the hemopoietic microenvironment in response to endotoxin; DeGowin RL et al.; Eight daily intraperitoneal injections of endotoxin (LPS) induced hematologic abnormalities in mice like those previously observed with chronic inflammation, sterile abscess, and tumor bearing . By the ninth day, anemia, leukocytosis, hypocellularity of the bone marrow, and compensatory hemopoietic hyperplasia of the spleen had occurred . The suppressed hemopoietic recovery and impaired survival of mice with these abnormalities, after receiving an ordinarily sublethal dose of total body irradiation (600 cGy T.B.), confirmed their importance to the intact mouse and suggested that splenic hyperplasia was insufficient to compensate for a total body deficit of functional hemopoietic stem cells . Atrophy of hemopoietic tissue in the marrow with hyperplasia in the spleen implicated changes in the hemopoietic microenvironment to account for the different responses to endotoxin . Prostaglandin E2 (PGE2) serves as an important mediator of the inflammatory response and profoundly affects hemopoiesis . Previous studies had shown that low concentrations of PGE2 enhanced, and high concentrations suppressed erythropoiesis in vitro; therefore, we wondered whether stromal cells from the marrow's microenvironment produced more PGE2 in response to LPS than splenic stromal cells to explain the suppression of hemopoiesis in the marrow and its enhancement in the spleen . Indeed, synthesis of PGE2 in primary short-term cultures of adherent marrow stromal cells in response to LPS proved much greater than that observed in cultures of splenic stromal cells . Extending adherence times from 3 to 24 to 48 hours did not change the relationship . We believe that the results of our studies point to a role of PGE2 in the microenvironmental modulation of hemopoiesis in mice with activation of the inflammatory response.

J Bacteriol, 1987 Jun, 169(6), 2896 - 8
Acylation of glycerol 3-phosphate is the sole pathway of de novo phospholipid synthesis in Escherichia coli; Ray TK et al.; The inhibition of phospholipid synthesis engendered by starving glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli (plsB or gpsA) for G3P is incomplete; 5 to 10% of the normal rate of phospholipid synthesis remains, even after prolonged starvation . We report that G3P starvation of a strain having lesions in both the gpsA and plsB genes resulted in essentially complete (greater than 98.5%) inhibition of phospholipid synthesis, indicating that all de novo glycerolipid synthesis in E . coli proceeds by acylation of G3P.

J Bacteriol, 1987 Jun, 169(6), 2862 - 5
Tetrahydrothiophene 1-oxide as an electron acceptor for Escherichia coli; Meganathan R et al.; Escherichia coli used tetrahydrothiophene 1-oxide (THTO) as an electron acceptor for anaerobic growth with glycerol as a carbon source; the THTO was reduced to tetrahydrothiophene . Cell extracts also reduced THTO to tetrahydrothiophene in the presence of a variety of electron donors . Chlorate-resistant (chl) mutants (chlA, chlB, chlD, and chlE) were unable to grow with THTO as the electron acceptor . However, growth and THTO reduction by the chlD mutant were restored by high concentrations of molybdate . Similarly, mutants of E . coli that are blocked in the menaquinone (vitamin K2) biosynthetic pathway, i.e., menB, menC, and menD mutants, did not grow with THTO as an electron acceptor . Growth and THTO reduction were restored in these mutants by the presence of appropriate intermediates of the vitamin K biosynthetic pathway.

J Bacteriol, 1987 Jun, 169(6), 2835 - 42
Stability and replication control of Escherichia coli minichromosomes; Lobner-Olesen A et al.; A stabilized minichromosome--a plasmid replicating from the chromosomal origin oriC--was constructed by cloning the sopA,B,C, genes from plasmid F . This minichromosome had a loss frequency of less than 10(-3), while that of the nonstabilized parental plasmid was 2 X 10(-2) to 4 X 10(-2) . Both minichromosomes had the same average copy number per chromosomal origin, and the copy numbers were constant over an eightfold range of growth rates . Different mutations in the mioC gene and promoter, from which transcription enters oriC, were constructed, and their effects on minichromosome copy number and stability were tested . The results indicated that normal replication control at oriC was independent of the MioC protein and most of the sequences between the promoter and oriC, but required both transcription from the mioC promoter and probably also the presence of the DnaA box (DnaA protein-binding site) just upstream of the mioC promoter . Transcription from the mioC promoter was shown to be efficiently repressed in vivo after overproduction of DnaA protein and to be derepressed at the nonpermissive temperature in six different dnaA(Ts) mutants.

J Bacteriol, 1987 Jun, 169(6), 2724 - 9
Suppressible base substitution mutations induced by angelicin (isopsoralen) in the Escherichia coli lacI gene: implications for the mechanism of SOS mutagenesis; Miller SS et al.; Angelicin- plus near-UV-induced mutations were umuC dependent in Escherichia coli K-12 . Angelicin, a monofunctional psoralen derivative, is believed to damage DNA almost exclusively at pyrimidine bases . To broaden our knowledge about the mutagenic specificity of SOS-dependent mutagens, we determined the mutational specificity of 233 suppressible lacI mutations induced by angelicin . More than 90% of the nonsense mutations arose via transversion substitutions . The three most frequently mutated sites were at A-T base pairs and accounted for more than one-third of all induced nonsense mutations . The two hottest sites were at the only occurrences of the 5'-TATA-3' tetranucleotide in lacI, a sequence expected to be a preferred binding site for a psoralen . Both A-T-to-T-A and A-T-to-C-G transversions were well induced by angelicin treatment, but the frequency of each transversion depended on the particular site . We also detected significant induction of transversion mutations at G-C sites . The induction of transversions by an SOS-dependent mutagen that generates lesions at pyrimidines supports the idea that DNA lesions influence the selection of bases that are incorporated via the process of SOS repair.

J Bacteriol, 1987 Jun, 169(6), 2718 - 23
An aminoacyl-tRNA synthetase complex in Escherichia coli; Harris CL; Aminoacyl-tRNA synthetases from several strains of Escherichia coli are shown to elute as a high-molecular-weight complex on 6% agarose columns (Bio-Gel A-5M) . In contrast, very little synthetase activity was observed in such complexes on Sephadex G-200 columns, suggesting that these enzymes may interact with or are dissociated during chromatography on dextran . The size of the complex observed on Bio-Gel A-5M was influenced by the method of cell breakage and the salt concentrations present in buffers . The largest complexes (greater than 1,000,000 daltons) were seen with cells broken with a freeze press, whereas with sonicated preparations the average size of the complex was about 400,000 daltons . Extraction of synthetases at 0.15 M NaCl, to mimic physiological salt concentrations, also resulted in high-molecular-weight complexes, as demonstrated by both agarose gel filtration and ultracentrifugation analysis . Evidence is presented that dissociation of some synthetases does occur in the presence of higher salt levels (0.4 M NaCl) . Partial purification of the synthetase complex on DEAE-Sephacel was accomplished with only minor dissociation of individual synthetases . These data suggest that a complex(es) of aminoacyl-tRNA synthetase does exist in bacterial cells, just as in eucaryotes, and that the complex may have escaped earlier detection due to its fragility during isolation.

J Bacteriol, 1987 Jun, 169(6), 2659 - 66
Association of thioredoxin with the inner membrane and adhesion sites in Escherichia coli; Bayer ME et al.; The intracellular localization of thioredoxin in Escherichia coli was determined by immunoelectron microscopy and correlated to previous biochemical data which had suggested that thioredoxin resides at inner-outer membrane adhesion sites . Since a considerable amount of thioredoxin was lost during preparation of cells for electron microscopy, we immobilized the protein with the heterobifunctional photoactivatable cross-linker p-azidophenacylbromide before the cells were fixed with aldehyde and embedded in Lowicryl K4M . Thin sections were labeled with affinity-purified antithioredoxin antiserum and protein A-gold complexes . Densities of immunolabel in a designated membrane-associated area and in the rest of the cytoplasm were compared and the data were statistically evaluated . Wild-type strain W3110 and strain SK3981, an overproducer of thioredoxin, exhibited increased labeling at the inner membrane and its adjacent cytoplasmic area . In contrast, the more centrally located cytoplasm of both strains showed much lower label density . This label distribution did not change with cell growth or in the stationary phase . Immunolabel was often found at bridges between the inner and outer membranes; this result is consistent with a model which places at least a portion of the thioredoxin at membrane adhesion sites, corresponding to an osmotically sensitive cytoplasmic compartment bounded by a hybrid inner-outer membrane (C.A . Lunn and V . Pigiet, J . Biol . Chem . 257:11424-11430, 1982; C.A . Lunn and V . Pigiet, J . Biol . Chem . 261:832-838, 1986) . Specific label was absent in the periplasmic space.

J Bacteriol, 1987 Jun, 169(6), 2624 - 30
Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor; de Lorenzo V et al.; The promoter region of the pColV-K30-encoded operon specifying biosynthesis and transport of the siderophore aerobactin was subjected to deletion analysis to determine the smallest DNA sequence affording iron regulation of a iucA'-'lacZ gene fusion . A 78-base-pair (bp) region containing the main (P1) promoter retained the character of inducibility under iron starvation . A 250-bp fragment carrying this sequence was examined for protection against DNase I by the Fur protein, the product of a gene (fur) required for negative control of several iron-regulated functions . The DNase I footprints, in the presence of various divalent heavy-metal ions added as corepressors, revealed two contiguous binding sites with different lengths and affinities for Fur . Increased concentrations of the protein appeared to elicit formation of repressor oligomers which bind to the upstream and downstream regions of the P1 promoter in a metal-dependent fashion, but with a presently undefined stoichiometry . The primary site for Fur binding spans 31 bp and contains two overlapping symmetry dyads which share the sequence 5'-TCATT-3' . It also contains extensive homology with a 19-bp consensus sequence for iron-regulated genes as deduced from comparison with the fhuA and fepA putative promoter sequences.

J Bacteriol, 1987 Jun, 169(6), 2570 - 8
Positive and negative regulation of the bgl operon in Escherichia coli; Mahadevan S et al.; We have analyzed the functions encoded by the bgl operon in Escherichia coli K-12 . Based on the ability of cloned regions of the operon to complement a series of Bgl- point mutations, we show that the three bgl structural genes, bglC, bglS, and bglB, are located downstream of the regulatory locus bglR in the order indicated . Using a bgl-lacZ transcriptional fusion, we show that bglC and bglS are involved in regulating operon expression . The presence of the bglC gene in trans is absolutely required for the expression of the fusion, which is constitutive when only the bglC gene is present . When the bglC and the bglS genes are both present in the cell, expression of the fusion requires a beta-glucoside inducer . From these observations, we conclude that (i) the bglC gene encodes a positive regulatory of bgl operon expression and (ii) the bglS gene encodes a negative regulator of operon expression, causing the requirement for a beta-glucoside inducer . These conclusions are supported by our observations that (i) a majority of bglC mutants exhibits a Bgl- phenotype, whereas rare trans-dominant mutations in bglC result in constitutive expression of the bgl operon and the fusion, and (ii) mutations in the bglS gene lead to constitutive expression of the fusion . Based on several lines of evidence presented, we propose that the bglS gene product has an additional role as a component of the beta-glucoside transport system.

J Bacteriol, 1987 Jun, 169(6), 2523 - 8
Characterization of the sppA gene coding for protease IV, a signal peptide peptidase of Escherichia coli; Suzuki T et al.; The sppA gene codes for protease IV, a signal peptide peptidase of Escherichia coli . Using the gene cloned on a plasmid, we constructed an E . coli strain carrying the ampicillin resistance gene near the chromosomal sppA gene and an sppA deletion strain in which the deleted portion was replaced by the kanamycin resistance gene . Using these strains, we mapped the sppA gene at 38.5 min on the chromosome, the gene order being katE-xthA-sppA-pncA . Although digestion of the signal peptide that accumulated in the cell envelope fraction was considerably slower in the deletion mutant than in the sppA+ strain, it was still significant, suggesting the participation of another envelope protease(s) in signal peptide digestion.

J Bacteriol, 1987 Jun, 169(6), 2500 - 6
Regulatory mutants of the aroF-tyrA operon of Escherichia coli K-12; Cobbett CS et al.; The regulatory region of the aroF-tyrA operon was fused to the chloramphenicol acetyltransferase (cat) gene on a plasmid vector . Expression of the cat gene was subject to repression by tyrR+ . This fusion was used to isolate regulatory mutants with increased expression of the cat gene in which repression by tyrR+ was affected . Nucleotide sequencing of these mutants has led to the identification of three sites involved in the repression of aroF by tyrR+ . The existence of a functional promoter divergently transcribing from the aroF regulatory region was also demonstrated by using the cat fusion vector . The expression of this promoter is also regulated by tyrR+.

J Bacteriol, 1987 Jun, 169(6), 2494 - 9
Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis; Silverman PM et al.; Acetohydroxyacid synthase I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate . Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate . Inactivation was accompanied by incorporation of radioactivity from 3-bromo{2-14C}pyruvate into acid-insoluble material . More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit . The stoichiometry of incorporation at nearly complete inactivation was 1 mol of 14C per mol of IlvB polypeptide . These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme . We confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I . These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site.

J Bacteriol, 1987 Jun, 169(6), 2488 - 93
Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli; Kalyananda MK et al.; When either 3H-labeled L-glyceraldehyde or 3H-labeled L-glyceraldehyde 3-phosphate (GAP) was added to cultures of Escherichia coli, the phosphoglycerides were labeled . More than 81% of the label appeared in the backbone of the phosphoglycerides . Chromatographic analyses of the labeled phosphoglycerides revealed that the label was normally distributed into phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin . These results suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the bacterial phosphoglycerides . Cell-free bacterial extracts catalyzed an NADPH-dependent reduction of L-GAP to sn-G3P . The partially purified enzyme was specific for L-GAP and recognized neither D-GAP nor dihydroxyacetone phosphate as a substrate . NADH could not replace NADPH as a coenzyme . The L-GAP:NADPH oxidoreductase had an apparent Km of 28 and 35 microM for L-GAP and NADPH, respectively . The enzyme was insensitive to sulfhydryl reagents and had a pH optimum of approximately 6.6 . The phosphonic acid analog of GAP, 3-hydroxy-4-oxobutyl-1-phosphonate, was a substrate for the reductase, with an apparent Km of 280 microM.

J Bacteriol, 1987 Jun, 169(6), 2405 - 9
Identification and expression of a copy number control gene in the IncFIII hemolytic plasmid pSU316; Andres I et al.; A DNA fragment carrying both the IncFIII determinant and a copy number control gene of the hemolytic plasmid pSU316 has been cloned in pBR322 . Deletion derivatives of the hybrid plasmid generated by Bal 31 digestion, which no longer exhibit the IncFIII phenotype, fall into two complementation groups when tested against a pSU316 miniplasmid derivative . Type 1 mutants exhibit the copy number control (Cop+) phenotype whereas type 2 mutants do not . Restriction analysis of type 1 and type 2 mutants allowed us to locate the cop gene of pSU316 in a 700-base-pair fragment adjacent to the IncFIII determinant . Plasmid expression in a minicell system suggests that the product of the cop gene of pSU316 could be a 13,000-dalton protein.

J Bacteriol, 1987 Jun, 169(6), 2367 - 72
Photoreactivation in phr mutants of Escherichia coli K-12; Husain I et al.; We have investigated the genetics of photoreactivation in Escherichia coli K-12 . We found that strains with point mutations or deletions in the phr gene showed a significant residual level of photoreactivation after exposure to large fluences of photoreactivating light . It had been previously proposed that a gene in the gal-att lambda interval is also involved in photoreactivation and that the residual photoreactivating activity might be due to this so-called phrA gene located at this interval . We found that deletions of the gal-att lambda region had no effect on either the rate or the final extent of photoreactivation observed in phr+ cells or phr mutants; however strains carrying the delta (gal-att lambda) deletions displayed increased sensitivity to near-UV radiation.

J Bacteriol, 1987 Jun, 169(6), 2352 - 9
Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells; Fikes JD et al.; The Escherichia coli maltose-binding protein (MBP) R2 signal peptide is a truncated version of the wild-type structure that still facilitates very efficient export of MBP to the periplasm . Among single amino acid substitutions in the R2 signal peptide resulting in an export-defective precursor MBP (pMBP) were two that replaced residues in the consensus Ala-X-Ala sequence (residues -3 to -1) that immediately precedes the cleavage site . It was suggested that the functional hydrophobic core and signal peptidase recognition sequence of this signal peptide substantially overlap and that these two alterations affect both pMBP translocation and processing . In this study, the export of pMBP by the mutants, designated CC15 and CC17, with these two alterations was investigated further . The pMBP of mutant CC17 has an Arg substituted for Leu at the -2 position . It was found that CC17 cells exported only a very small amount of MBP, but that which was exported appeared to be correctly processed . This result was consistent with other studies that have concluded that virtually any amino acid can occupy the -2 position . For mutant CC15, which exhibits a fully Mal+ phenotype, an Asp is substituted for the Ala at the -3 position . CC15 cells were found to export large quantities of unprocessed, soluble pMBP to the periplasm, although such export was achieved in a relatively slow, posttranslational manner . This result was also consistent with other studies that suggested that charged residues are normally excluded from the -3 position of the cleavage site . Using in vitro oligonucleotide-directed mutagenesis, we constructed a new signal sequence mutant in which Asp was substituted for Arg at the -3 position of an otherwise wild-type MBP signal peptide . This alteration had no apparent effect on pMBP translocation across the cytoplasmic membrane, but processing by signal peptidase was inhibited . This pMBP species with its full-length hydrophobic core remained anchored to the membrane, where it could still participate in maltose uptake . The implications of these results for models of protein export are discussed.

J Bacteriol, 1987 Jun, 169(6), 2345 - 51
Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide; Fikes JD et al.; The wild-type maltose-binding protein (MBP) signal peptide is 26 amino acids in length . A mutationally altered MBP signal peptide has been previously described that is missing one of the basic residues from the hydrophilic segment and seven residues from the hydrophobic core; however, it still facilitates MBP secretion to the periplasm at a rate and efficiency comparable to those of the wild-type structure . Thus, this truncated signal peptide (designated the R2 signal peptide) must retain all of the essential features required for proper export function . In this study, alterations were obtained in the R2 signal peptide that resulted in an export-defective MBP . For the first time, signal sequence mutations were obtained that resulted in the synthesis of a totally export-defective MBP . As was previously the case for the wild-type signal peptide, the introduction of either charged residues or helix-breaking proline residues adversely affected export function . Despite these similarities, the position of these alterations within the R2 signal peptide, their relative effects on MBP secretion and processing, and an analysis of the ability of various extragenic prl mutations to suppress the secretion defects provide additional insight into the minimal requirements for a functional MBP signal peptide.

Protein Eng, 1987 Jun, 1(3), 195 - 9
Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45; Engels JW et al.; By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 {(Leu27)GHRH-Gly45} was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator . Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein . After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography . The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion . The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries . Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.

Anal Biochem, 1987 Jun, 163(2), 470 - 5
A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions; Peterhans A et al.; The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described . The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing . A fusion protein was demonstrated in crude extracts of E . coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies . Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E . coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon . The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.

Pathol Biol (Paris), 1987 Jun, 35(5 Pt 2), 699 - 701
{Titration curves of beta-lactamases using pH gradient electrophoresis}; Vedel G et al.; The molecular relationships of two types of plasmid-mediated beta-lactamases, TEM-1 (R 111), TEM-2 (RP 4) and OXA-1 (RGN 238), OXA-4 (pMG 90) were analysed by combined isoelectrofocusing-electrophoresis . Titration curves of TEM-1 (pI 5.4) and TEM-2 (pI 5.6) together were consistent with the known substitution of a glutamic acid in the former by a lysin in the latter . When OXA-1 (pI 7.4) and OXA-4 (pI 7.45) were titrated, one single mobility curve was obtained reflecting their structural homogeneity . The titration curve technique will be usefull for the study of structure of beta-lactamases.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 3987 - 91
Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism; Slilaty SN et al.; LexA repressor of Escherichia coli is inactivated in vivo by a specific cleavage reaction requiring activated RecA protein . In vitro, cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH . These two cleavage reactions have similar specificities, suggesting that RecA acts indirectly to stimulate self-cleavage, rather than directly as a protease . We have studied the chemical mechanism of cleavage by using site-directed mutagenesis to change selected amino acid residues in LexA, chosen on the basis of kinetic data, homology to other cleavable repressors, and potential similarity of the mechanism to that of proteases . Serine-119 and lysine-156 were changed to alanine, a residue with an unreactive side chain, resulting in two mutant proteins that had normal repressor function and apparently normal structure, but were completely deficient in both types of cleavage reaction . Serine-119 was also changed to cysteine, another residue with a nucleophilic side chain, resulting in a protein that was cleaved at a significant rate . These and other observations suggest that hydrolysis of the scissile peptide bond proceeds by a mechanism similar to that of serine proteases, with serine-119 being a nucleophile and lysine-156 being an activator . Possible roles for RecA are discussed.

Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3787 - 91
Kinds of mutations formed when a shuttle vector containing adducts of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo{a}pyrene replicates in human cells; Yang JL et al.; We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo{a}pyrene (BPDE) replicates in human cells . A human embryonic kidney cell line, 293, was used as the eukaryotic host . The target gene for mutation analysis, supF, codes for a tyrosine suppressor tRNA and is strategically located between the origin of replication of the plasmid in Escherichia coli and the gene for a selectable marker, so that the possibility of recovering supF mutants containing gross rearrangements is low . The frequency of supF mutants obtained when untreated plasmid replicated in 293 cells was 1.4 X 10(-4) . The frequency with BPDE-treated plasmid increased linearly as a function of the number of adducts, with 16 adducts per plasmid giving 38 X 10(-4) . Polyacrylamide gel and agarose gel electrophoresis analysis of 137 plasmids with mutations in the supF gene indicated that 70% (21/30) from untreated plasmids contained deletions or insertions or showed altered gel mobility, whereas only 28% (30/107) of those derived from BPDE-treated plasmids contained such alterations . Of the 86 unequivocally independent mutants derived from BPDE-treated plasmids that were analyzed by sequencing, the majority (60/86) exhibited base substitutions . Mutants exhibiting frameshifts (insertions or deletions of one, two, or four base pairs) were also found, but they were a minority (11/86) . In the progeny of BPDE-treated plasmids 61/71 base substitutions observed were transversions, with 45/61 G X C----T X A . Examination of the location of BPDE-induced mutations among the 85 base pairs in the structure of the tRNA revealed that 30% of the base substitutions occurred at two sites and 44% of the rest occurred at five other hot spots . Only 20% of all these base changes involved a site in which a guanine containing a BPDE adduct is predicted to be labile--i.e., a guanine that has a pyrimidine to its 5' side.

J Bacteriol, 1987 Jun, 169(6), 2739 - 47
Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp . strain PCC 7002; Murphy RC et al.; The recA gene of Synechococcus sp . strain PCC 7002 was detected and cloned from a lambda gtwes genomic library by heterologous hybridization by using a gene-internal fragment of the Escherichia coli recA gene as the probe . The gene encodes a 38-kilodalton polypeptide which is antigenically related to the RecA protein of E . coli . The nucleotide sequence of a portion of the gene was determined . The translation of this region was 55% homologous to the E . coli protein; allowances for conservative amino acid replacements yield a homology value of about 74% . The cyanobacterial recA gene product was proficient in restoring homologous recombination and partial resistance to UV irradiation to recA mutants of E . coli . Heterologous hybridization experiments, in which the Synechococcus sp . strain PCC 7002 recA gene was used as the probe, indicate that a homologous gene is probably present in all cyanobacterial strains.

J Bacteriol, 1987 Jun, 169(6), 2611 - 7
Genetic characterization of a highly efficient alternate pathway of serine biosynthesis in Escherichia coli; Ravnikar PD et al.; There exists in Escherichia coli a known set of enzymes that were shown to function in an efficient and concerted way to convert threonine to serine . The sequence of reactions catalyzed by these enzymes is designated the Tut cycle (threonine utilization) . To demonstrate that the relevant genes and their protein products play essential roles in serine biosynthesis, a number of mutants were analyzed . Strains of E . coli with lesions in serA, serB, serC, or glyA grew readily on minimal medium supplemented with elevated levels of leucine, arginine, lysine, threonine, and methionine . No growth on this medium was observed upon testing double mutants with lesions in one of the known ser genes plus a second lesion in glyA (serine hydroxymethyltransferase), gcv (the glycine cleavage system), or tdh (threonine dehydrogenase) . Pseudorevertants of ser mutants capable of growth on either unsupplemented minimal medium or medium supplemented with low levels of leucine, arginine, lysine, threonine, and methionine were isolated . At least two unlinked mutations were associated with such phenotypes.

J Bacteriol, 1987 Jun, 169(6), 2385 - 90
Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii; Anderson BE et al.; DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19 . The ligation mixture was used to transform Escherichia coli . A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum . One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R . rickettsii serum that comigrated with an antigen from R . rickettsii . A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen . The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences . The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840 . The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E . coli . The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.

Crit Care Med, 1987 Jun, 15(6), 574 - 81
Thyrotropin releasing hormone: effects in monkeys and dogs subjected to experimental circulatory shock; Gurll NJ et al.; We tested the hypothesis that thyrotropin releasing hormone (TRH) would improve cardiovascular function and survival in circulatory shock by opposing the adverse effects of endogenous opioids and other pathophysiologic mediators . Cynomolgus monkeys and mongrel dogs were anesthetized and catheterized to measure mean arterial pressure (MAP) and left ventricular contractility (LV dp/dtmax) . Hemorrhagic shock was induced by bleeding into a reservoir to achieve and maintain MAP at 45 mm Hg for one hour . Endotoxic shock was produced by the iv injection of an LD80 dose of Escherichia coli lipopolysaccharide endotoxin (3 mg/kg in dogs and 5 mg/kg in monkeys) . Animals were treated iv with either TRH (2 mg/kg plus 2 mg/kg X h) or equivolume saline . TRH significantly increased MAP and LV dp/dtmax in primate hemorrhagic and endotoxic shock . In primate hemorrhagic shock, TRH significantly (p = .02) improved survival (alive/total = 4/5 vs . 0/5) . However, TRH had no effect on survival in endotoxemic primates . In contrast, TRH treatment in dogs produced only a transient hemodynamic response after endotoxemia and no significant hemodynamic effect after acute hemorrhage (even at twice the TRH dose) . TRH did not affect survival in either dog model of circulatory shock . Based on extensive evidence with the opiate receptor antagonist naloxone in other studies, endogenous opioids play a role in the cardiovascular depression in primate and canine circulatory shock . From these studies with TRH, we conclude that TRH is relatively ineffective in canine circulatory shock, and physiologic antagonism of the adverse effects of opioids and other cardiodepressant substances by TRH administration may prove to be a useful alternative treatment of primate hemorrhagic shock.

Mol Gen Mikrobiol Virusol, 1987 Jun, (6), 23 - 8
{Properties of transposon Tn2555 carrying the genes for sucrose utilization}; Doroshenko VG et al.; Tn2555, a new transposon coding for genes of sucrose utilization was studied . Tn2555 was shown to integrate into the plasmids RP4 and R6K, phage P1CmClr100 and Escherichia coli K12 chromosome . Tn2555 frequency of transposition to RP4 and R6K DNA is (2-5) X 10(-7) in Rec+-strain, (3-6) X 10(-8) in Rec--strain . Tn2555 integration site in phage P1CmClr100 Sac+-derivative studied has been localised within the C-segment of P1 DNA . In three independent cases of Tn2555 transposition to the chromosome the transposon was found to be integrated in the region between 29 and 32 min of Escherichia coli K12 linkage map . The restriction endonuclease analysis of seven independent isolates of RP4::Tn2555 has shown the grouping of Tn2555 integration sites in the Tn1 region of RP4 . Frequent rearrangements occurring within Tn2555 have been revealed by the analysis . However, an invertible DNA segment of about 6-7 kb was preserved in all transposon structures.

Mol Gen Mikrobiol Virusol, 1987 Jun, (6), 17 - 23
{Composite transposons Tn5 and Tn10 in Escherichia coli K12: precise excision and recombination}; Goryshin IIu et al.; The number of exconjugants having the transposon Tn5 excised precisely during the crosses of the Escherichia coli proA::Tn5 donor with the recipients F- rec+ or F- recA441 (tif) was 20-30 times higher for the crosses involving the latter recipient . The high recombinogenic activity is characteristic of the tif recipient . Precise excision from a tandem duplication is more efficient than from nonduplicated region of the genome . It is four orders higher, if a transposon is localized in an arm of a duplication . The effect is recA-dependent . The presented data permit us to suggest the participation of RecA protein (its synaptic function) in the formation of the intermediate "stem-loop" structure . The latter is predicted by the three mechanisms of transposon excision: "slippage", "correctional" and "recombinational" . The latter two mechanisms were formulated in the paper . The experimental proof of the postexcision transposition presented in the paper, is a good support to the version of "recombinational" excision.

Mol Gen Genet, 1987 Jun, 208(1-2), 219 - 25
The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing; Coupland GM et al.; The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization . We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion analysis and transposon mutagenesis . The nucleotide sequence of the oriT region contains 13 direct repeats of an 11 bp consensus sequence, 3 different pairs of 10 bp inverted repeats, and a segment that is extremely A-T rich . The direct repeats are within a region required for high frequency transfer and their sequence is such that their periodic alignment along the helix may induce curvature of the DNA . Analysis of Tn1725 insertions within the sequenced fragment of R46 revealed that, unlike most other transposons, transposition of Tn1725 can cause target sequence duplications of three different sizes.

Genetics, 1987 Jun, 116(2), 185 - 9
A new generalizable test for detection of mutations affecting Tn10 transposition; Huisman O et al.; We describe here a new rapid screen that allows easy detection of transposon or host mutations that affect Tn10 transposition in Escherichia coli . This test involves a new Tn10 derivative called the "mini-lacZ-kanR fusion hopper" or mini-Tn10-LK for short . This element does not direct expression of beta-galactosidase when present at its original starting location on a suitably engineered plasmid or phage genome because it lacks appropriate transcription and translation start signals . However, transposition of this element into the chromosome of E . coli lacZ- bacteria leads to productive fusions in which the lacZ gene within the transposon is expressed from external chromosomal signals . Such fusions are readily detectable on MacConkey lactose indicator plates as red (Lac+) papillae inside of white (LacZ-) colonies . The length of time required to see red papillae appearing in a white colony sensitively and accurately reflects the transposition frequency of the mini-transposon within the colonies . Differences in times for color formation are sensitive enough that 10-fold differences in transposition frequency can readily be detected . This papillation assay can be used to identify mutant clones in which the frequency of Tn10 transposition is either increased or decreased . We have successfully used the assay to identify mutations in the terminal sequences of Tn10; mutations in the Tn10 transposase gene or the bacterial host can be isolated just as easily . This screen should be readily adaptable to transposable elements other than Tn10.

EMBO J, 1987 Jun, 6(6), 1809 - 15
DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues; Zell R et al.; Derivatives of phage M13 were constructed and used for the in vitro preparation of heteroduplex DNA molecules containing base/base mismatches that mimick DNA lesions caused by hydrolytic deamination of 5-meC residues in Escherichia coli DNA (i.e . they carry a T/G mismatch in the special sequence context provided by the recognition site -CCA/TGG-of the Dcm-methyltransferase) . Upon introduction of these heteroduplex DNAs into CaCl2-treated E . coli cells, the mismatches are efficiently repaired with high bias in favour of the DNA strand containing the mismatched guanine residue . This special DNA mismatch-repair operates on fully dam-methylated DNA and is independent of gene mutH . It thus fulfills the salient requirements of a repair pathway responsible for counteracting the spontaneous hydrolytic deamination of 5-meC in vivo . The repair efficiency is boosted by a 5-methyl group present on the cytosine residue at the next-nearest position to the 5' side of the mismatched guanine . The repair is severely impaired in host strains carrying a mutation in any of the three loci dcm, mutL and mutS.

EMBO J, 1987 Jun, 6(6), 1799 - 803
Base substitutions in transposable element IS1 cause DNA duplication of variable length at the target site for plasmid co-integration; Machida C et al.; We demonstrate that base substitutions in the IS1 sequence affect the length of the nucleotide sequence which is duplicated during IS1-mediated co-integration . IS1K, an IS1 variant present in the Escherichia coli chromosome, has seven base substitutions in its sequence as compared with that of IS1R derived from the plasmid R100 . All substitutions are located in the internal region of IS1K . We have constructed plasmids containing IS1R, IS1K and hybrids between them: one contains four base substitutions causing an amino acid substitution in the insA gene and the other has three substitutions producing an amino acid substitution in the insB gene . We have isolated co-integrate plasmids formed by each IS1 and analysed nucleotide sequences of the target sites duplicated at the co-integration junctions . The results show that IS1K generates duplications of 8 or 14 bp as well as 9 bp, while IS1R exclusively generates the 9-bp duplications . Both hybrid IS1s also create 8- or 7-bp target duplications in addition to 9-bp duplications . These results indicate that the base substitutions in either insA or insB are sufficient for the occurrence of unusual target duplications, suggesting that both genes are involved in the target duplication.

Mol Cell Biol, 1987 Jun, 7(6), 2248 - 55
Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions; Brouillette S et al.; We present an intermolecular recombination assay for mammalian cells that does not involve the reconstitution of a selectable marker . It is based on the generation of a shuttle vector by recombination between a bacterial and a mammalian vector . The recombinants can thus be amplified in mammalian cells, isolated by plasmid rescue in an Escherichia coli RecA- host, and identified by in situ hybridization, by using mammalian vector sequences as probes . Since both parental molecules can share defined lengths of homology, this assay permits a direct comparison between homologous and nonhomologous intermolecular recombination . Our results indicate that the dominant intermolecular recombination mechanism is a nonhomologous one . The relative frequency of homologous to nonhomologous recombination was influenced by the length of shared homology between parental molecules and the replicative state of the parental molecules, but not by the introduction of double-strand breaks per se . Finally, almost all of the recombinants with a homologous junction did not have the reciprocal homologous junction but instead had a nonhomologous one . We propose a model to account for the generation of these recombinants.

Mol Cell Biol, 1987 Jun, 7(6), 2070 - 9
The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation; Ach RA et al.; Formation of the 3' end of U1 and U2 small nuclear RNA (snRNA) precursors is directed by a conserved sequence called the 3' box located 9 to 28 nucleotides downstream of all metazoan U1 to U4 snRNA genes sequenced so far . Deletion of part or all of the 3' box from human U1 and U2 genes drastically reduces 3'-end formation . To define the essential nucleotides within this box that direct 3'-end formation, we constructed a set of point mutations in the conserved residues of the human U1 3' box . The ability of the various mutations to direct 3'-end formation was tested by microinjection into Xenopus oocytes and transfection into HeLa cells . We found that the point mutations had diverse effects on 3'-end formation, ranging from no effect at all to severe inhibition; however, no single or double point mutation we tested completely eliminated 3'-end formation . We also showed that a rat U3 3' flank can effectively substitute for the human U1 3' flank, indicating that the 3' boxes of the different U snRNA genes are functionally equivalent.

Arch Biochem Biophys, 1987 Jun, 255(2), 329 - 36
Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation; Martin JP Jr et al.; Hematoxylin, a natural dye commonly used as a histological stain, generates superoxide upon oxidation to its quinonoid product, hematein . The parameters affecting this reaction were assessed in developing a new and versatile assay for superoxide dismutase . The autoxidation of hematoxylin to hematein was accompanied by an increase in absorbance between 400 and 670 nm . The autoxidation rate was proportional to hematoxylin concentration and increased with pH above 6.55 . Trace metals accelerated the autoxidation and this effect was eliminated by EDTA . Superoxide dismutase inhibited the autoxidation 90-95% below pH 7.8, but above pH 8.1 the rate was augmented by superoxide dismutase . The rate inhibition at low pH was proportional to the superoxide dismutase concentration up to 70% inhibition . The rate acceleration at high pH was proportional to superoxide dismutase concentration up to approximately 200% acceleration . The autoxidation rate was not significantly affected by ethanol, cyanide, azide, hydrogen peroxide, or catalase . However, the reaction was inhibited by the reducing agents NADH, reduced glutathione, ascorbate, and dithiothreitol, and by undialyzed extracts of Escherichia coli B . When cell extracts were dialyzed prior to assay, the degree of inhibition observed was proportional to the concentration of superoxide dismutase in the extract . These observations form the basis for negative and positive assays of superoxide dismutase which are inexpensive and simple to perform . The negative assay has the added advantage of being applicable at physiological pH.

Virology, 1987 Jun, 158(2), 456 - 60
Synthesis of the X-protein of hepatitis B virus in vitro and detection of anti-X antibodies in human sera; Pfaff E et al.; A protein of 154 amino acids, predicted to be encoded by the X-open reading frame of the hepatitis B virus (HBV) genome, was synthesized in an in vitro translation system from SP6 transcripts containing the X-coding sequence . As characterized by SDS-PAGE and immunoprecipitation this X-protein possesses the expected molecular weight of 17 kDa and reacts specifically with rabbit antisera directed against a fusion protein from Escherichia coli that contained 145 of the 154 amino acids from the X-sequence . The X-protein, radiolabeled with {35S}methionine, provided a sensitive and specific antigen to screen for anti-X antibodies in sera from HBV patients . Positive signals were obtained preferentially in subjects suffering from HBV-induced liver cirrhosis or primary hepatocellular carcinoma (PHC), i.e., individuals that had been exposed for an extended time period to HBV gene products . Carefully controlled experiments failed to reveal the presence of X-related proteins specific to liver specimens from HBV patients.

Virology, 1987 Jun, 158(2), 348 - 60
Partial nucleotide sequence of the Japanese encephalitis virus genome; McAda PC et al.; Approximately 10 kb of the estimated 10.9-kb genome of the Japanese encephalitis virus (JE; Nakayama strain) has been cloned as cDNA; the uncloned portion includes 430 bases at the 5'-terminus and 450 bases at the 3'-end . A map of the genome has been developed through nucleotide sequencing and in vivo expression with the Escherichia coli expression vector lambda gt11 and immunological identification . Sequence results for 4320 nucleotides suggest the JE genome organization is very similar to those of three other flaviviruses for which sequence information is available . Like the other flaviviruses, the JE proteins are encoded by a single open reading frame that continues uninterrupted throughout the region sequenced . Considerable homology exists between the JE RNA and protein sequences and those of the other characterized flaviviruses . Comparative nucleotide and (amino acid) homology values for the M-E-NS1-ns2 segment of JE are approximately MVE, 70% (80%), WN, 68% (76%), and YF, 50% (45%) . Even greater homology is suggested when the protein hydrophobicity profiles are compared . The molecular relationships are consistent with the established serological relationships among JE, MVE, and WN viruses and argue that these flaviviruses may have been derived from a common evolutionary ancestor.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4185 - 9
Modulation of transcription by DNA supercoiling: a deletion analysis of the Escherichia coli gyrA and gyrB promoters; Menzel R et al.; Expression of the genes determining the subunits of Escherichia coli DNA gyrase (gyrA and gyrB) is known to be induced by relaxation of the template DNA . In this paper we report a deletion analysis of the gyrA and gyrB promoter regions . We find that a DNA sequence 20 base pairs long that includes the -10 consensus region, the transcription start point, and the first few transcribed bases is responsible for the property of induction by DNA relaxation . We propose a model for relaxation-stimulated transcription in which promoter clearance is the rate-limiting step.

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