Escherichia coli

Nucleic Acids Res, 1989 Aug 11, 17(15), 6299 - 317
DNA primase and the replication of the telomeres in Oxytricha nova; Zahler AM et al.; An enzymatic activity in crude extracts of macronuclei from the hypotrichous ciliate Oxytricha nova catalyzes the synthesis of RNA consisting of (C4A4)n using an oligodeoxynucleotide template of the telomeric sequence (dG4T4)n . Single-stranded (dG4T4)n is an effective template if it has a random sequence at its 5' end . The enzyme will not use a (dG4T4)n template of any length (up to 64 bases) if it lacks a random sequence at the 5' end . With a random, single-stranded sequence at the 5' end, the (dG4T4)n oligodeoxynucleotide must be at least 36 bases long to work as a template . A 16-base, single-stranded region of (dG4T4)2 is an effective template when joined to a 20-base double-stranded region of (dG4T4)n/(dA4dC4)n, a structural arrangement that is the same as the native telomere of Oxytricha macronuclear DNA . The RNA-synthesizing activity is unaffected by 1.0 mg/ml of alpha-amanitin . Macronuclear extracts have an alpha-amanitin-insensitive, RNA-polymerizing activity that can use a random 55mer oligodeoxynucleotide as a template . This enzyme activity may be the same one that uses (dG4T4)n templates to make (C4A4)n RNA . The (C4A4)n RNA made in the reaction can prime DNA synthesis by the E . coli DNA polymerase I Klenow fragment . Therefore, the RNA polymerase activity fulfills the requirements of the telomere DNA primase that we postulated for replication of telomeres in hypotrichs (Zahler and Prescott, 1988, Nucleic Acids Research 16, 6953-6972).

Nucleic Acids Res, 1989 Aug 11, 17(15), 6269 - 82
The use of thioglycolate to distinguish between 3' AP (apurinic/apyrimidinic) endonucleases and AP lyases; Bricteux-Gregoire S et al.; Addition of thioglycolate and DEAE-Sephadex chromatography were used to analyze the cleavage of the C(3')-O-P bond 3' to AP (apurinic/apyrimidinic) sites in DNA and to distinguish between a mechanism of hydrolysis (which would allow the nicking enzyme to be called 3' AP endonuclease) or beta-elimination (so that the nicking enzyme should be called AP lyase) . For this purpose, DNA labelled in the AP sites was first cleaved by rat-liver AP endonuclease, then with the 3' nicking catalyst in the presence of thioglycolate and the reaction products were analyzed on DEAE-Sephadex: deoxyribose-5-phosphate (indicating a 3' cleavage by hydrolysis) and the thioglycolate:unsaturated sugar-5-phosphate adduct (indicating a cleavage by beta-elimination) are well separated allowing to eventually easily discard the hypothesis of a hydrolytic process and the appellation of 3' AP endonuclease . We have shown that addition of thioglycolate to the unsaturated sugar resulting from nicking the C(3')-O-P bond 3' to AP sites by beta-elimination is an irreversible reaction . We have also shown that the thioglycolate must be present from the beginning of the reaction with the nicking catalyst to prevent the primary 5' product of the beta-elimination reaction from undergoing other modifications that complicate the interpretation of the results.

Nature, 1989 Aug 10, 340(6233), 482 - 6
Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle; Bernstein HD et al.; Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP) . Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome . Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain . The properties of this latter domain suggest that it contains the signal sequence binding site . A previously uncharacterized Escherichia coli protein has strong homology to both domains . Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E . coli protein, ftsY, which resembles SR alpha . Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane . We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.

Biochemistry, 1989 Aug 8, 28(16), 6611 - 8
Effects of distal point-site mutations on the binding and catalysis of dihydrofolate reductase from Escherichia coli; Adams J et al.; The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis . The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C . Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected . Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively) . Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L . The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 A away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to greater than 45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 microM for H45Q (similar to wild type) but from 8 to 5000 microM for R44L.

J Biol Chem, 1989 Aug 5, 264(22), 13114 - 21
Isolation and molecular characterization of a cDNA encoding the 23-kDa subunit of human RNA polymerase II; Pati UK et al.; We have shown that antibodies against native calf thymus RNA polymerase II and antibodies against its 23-kDa subunit cross-reacted with the 23-kDa subunit of human RNA polymerase II . Immunoglobin G (IgG) against the 23-kDa subunit of calf thymus RNA polymerase II inhibited transcription in vitro from the adenovirus major late promoter . By immunoscreening of a human placenta lambda gt11 cDNA library with IgG against native CT RNA polymerase II and with IgG against its 23-kDa subunit, we isolated and characterized a full length 1.2-kilobase cDNA . We also generated oligonucleotide probes from a sequence of amino acid residues obtained by a modified peptide microsequencing procedure . The cDNAs isolated both from oligoscreening and immunoscreening were identical . The amino acid sequence deduced from the nucleotide sequence analysis indicates a polypeptide of 197 amino acid (23 kDa) . The in vitro translation product of human cDNA HP-23 was precipitated by IgG against the 23-kDa subunit of CT RNA polymerase II . The amino acid sequence deduced from HP-23 showed no obvious homology with Escherichia coli RNA polymerase subunits or with any of its sigma factors.

J Biol Chem, 1989 Aug 5, 264(22), 12902 - 8
Formation of a photoreversible phycocyanobilin-apophytochrome adduct in vitro; Elich TD et al.; Avena seedlings grown in the presence of the plant tetrapyrrole synthesis inhibitor 4-amino-5-hexynoic acid contain less than 10% of the spectrally detectable phytochrome levels found in untreated seedlings, but continue to accumulate phytochrome apoprotein (Elich, T . D., and Lagarias, J . C . (1988) Plant Physiol . 88, 747-751) . Using such tetrapyrrole-deficient seedlings, we have previously reported that phycocyanobilin, the cleaved prosthetic group of C-phycocyanin, can be incorporated into phytochrome in vivo to yield spectrally active holoprotein (Elich, T . D., McDonagh, A . F., Palma, L . A., and Lagarias, J . C . (1988) J . Biol . Chem . 264, 183-189) . Here we show that addition of phycocyanobilin to soluble extracts of inhibitor-treated seedlings results in a rapid increase in spectrally active phytochrome holoprotein . The newly formed photoactive species displays a blue-shifted absorbance difference spectrum similar to that observed in the previous in vivo studies . The increase in spectral activity is consistent with conversion of all of the preexisting phytochrome apoprotein to functionally active holoprotein . The formation of a covalent phycocyanobilin-apophytochrome adduct is shown by an increase in Zn2+-dependent bilin fluorescence of the phytochrome polypeptide . A photoreversible, covalent adduct with a similar optical spectrum also forms when immunopurified apophytochrome is incubated with phycocyanobilin . ATP, reduced pyridine nucleotides, or other cofactors are not required for adduct formation . When biliverdin IX alpha is substituted for phycocyanobilin, no spectrally active covalent adduct is produced . These results indicate that an A-ring ethylidene-containing bilatriene is required for post-translational covalent attachment of bilin to apophytochrome and that apophytochrome may be the bilin C-S lyase which catalyzes bilin attachment.

J Mol Biol, 1989 Aug 5, 208(3), 509 - 10
First crystallization of a phosphoenolpyruvate carboxylase from Escherichia coli; Inoue M et al.; Two different forms of crystal for a phosphoenolpyruvate carboxylase from Escherichia coli were obtained by the hanging-drop vapor diffusion technique, using polyethylene glycol 4000 as precipitant . The hexagonal crystal in space group P6(2)22 (or P6(4)22) has cell dimensions of a = 131 A and c = 325 A, whereas the orthorhombic crystal in space group I222 has a = 119 A, b = 252 A and c = 83 A . A tetrameric molecule (396,244 Mr), a subunit of which contains 883 amino residues, has a crystallographic 2 symmetry in the hexagonal crystal or 222 symmetry in the orthorhombic crystal, respectively.

J Mol Biol, 1989 Aug 5, 208(3), 501 - 5
Unidirectional transfer of broad host-range plasmid R1162 during conjugative mobilization . Evidence for genetically distinct events at oriT; Kim K et al.; A segment of R1162 DNA containing genes for conjugative mobilization (Mob) and the origin of transfer (oriT) was integrated into the Escherichia coli chromosome . Bacterial genes were transferred in a polar fashion during conjugative mobilization of the integrated segment by a self-transmissible plasmid vector . The direction of transfer, together with the properties of mutated derivatives of oriT, indicate that initial cleavage of oriT, and subsequent religation after transfer, entail different mechanisms that can be distinguished genetically.

J Biol Chem, 1989 Aug 5, 264(22), 13012 - 17
Translational frameshifts induced by mutant species of the polypeptide chain elongation factor Tu of Escherichia coli; Vijgenboom E et al.; Translational frameshifts, both +1 and -1, are promoted by mutations in tufA and tufB, the two genes encoding the polypeptide chain elongation factor (EF) Tu of Escherichia coli . Strains harboring the mutant EF-Tu(Ala375----Thr) encoded by either tufA or tufB or by both, display a linear relationship between the frequency of frameshifting and the concentration of mutant EF-Tu, relative to the total amount of EF-Tu . A second mutant species, EF-TuB(Gly222----Asp), also promotes frameshifting . The frequency is strikingly enhanced by the combined action of EF-TuA(Ala375----Thr) and EF-TuB(Gly222----Asp) and exceeds by far the total contribution of the two mutant EF-Tus studied separately . These observations raise the question whether the formation of each peptide bond under conditions that no frameshifting occurs also requires the combined action of two EF-Tu molecules, in this case not differing functionally.

J Biol Chem, 1989 Aug 5, 264(22), 12752 - 3
Crystallization and preliminary x-ray characterization of thioredoxin reductase from Escherichia coli; Kuriyan J et al.; Single crystals of thioredoxin reductase, suitable for x-ray diffraction studies, have been obtained at room temperature by vapor diffusion of 10-20 mg/ml protein solution against 35% polyethylene glycol containing 200 mM ammonium sulfate . Good quality crystals appear spontaneously only from a protein solution that had been stored for more than a year at 4 degrees C, although large single crystals are reproducibly obtained from fresh protein solutions by micro-seeding . The space group is P6(3)22 (a = b = 123.8 A, c = 81.6 A), with one monomer of the enzyme (34.5 kDa) in the crystallographic asymmetric unit . The crystals are well ordered and diffract to beyond 2 A resolution.

J Biol Chem, 1989 Aug 5, 264(22), 13321 - 8
Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase . The effect of defective cAMP binding on holoenzyme activation; Woodford TA et al.; The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses . The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites . In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated . All of the mutant RI subunits which bound {3H}cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit . Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional . The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed . Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM . The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites . Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit . The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.

J Biol Chem, 1989 Aug 5, 264(22), 13086 - 92
C-terminal truncation of p21H preserves crucial kinetic and structural properties; John J et al.; The human c-Ha-ras protooncogene product p21C was truncated at the C terminus by 23 amino acids . The resulting G-binding domain, p21 (1-166) = p21C', can be crystallized as a complex with the slowly hydrolyzing GTP analogues guanosin-5'-{beta,gamma-imido}triphosphate, guanosin-5'-{beta,gamma-methylene}triphosphate, and guanosin-5'-O-(3-thiotriphosphate) . We show here that this protein has biochemical properties very similar to those of the intact protein . Activating mutations in position 12 (Gly12----Val; Gly12----Arg) have the same effect on the properties of the truncated protein as on intact protein . Nuclear magnetic resonance (NMR) measurements show no apparent effect of the C-terminal deletion on the solution structure of p21 . This suggests that neither the structure of the G-binding domain nor any of its biochemical properties are markedly influenced by the truncation.

J Mol Biol, 1989 Aug 5, 208(3), 429 - 43
Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition; Mellot P et al.; Site-directed nuclease digestion and nonsense mutations of the Escherichia coli metG gene were used to produce a series of C-terminal truncated methionyl-tRNA synthetases . Genetic complementation studies and characterization of the truncated enzymes establish that the methionyl-tRNA synthetase polypeptide (676 residues) can be reduced to 547 residues without significant effect on either the activity or the stability of the enzyme . The truncated enzyme (M547) appears to be similar to a previously described fully active monomeric from of 64,000 Mr derived from the native homodimeric methionyl-tRNA synthetase (2 x 76,000 Mr) by limited trypsinolysis in vitro . According to the crystallographic three-dimensional structure at 2.5 A resolution of this trypsin-modified enzyme, the polypeptide backbone folds into two domains . The former, the N-domain, contain a crevice that is believed to bind ATP . The latter, the C-domain, has a 28 C-residue extension (520 to 547), which folds back, toward the N-domain and forms an arm linking the two domains . This study shows that upon progressive shortening of this C-terminal extension, the enzyme thermostability decreases . This observation, combined with the study of several point mutations, allows us to propose that the link made by the C-terminal arm of M547 between its N and C-terminal domains is essential to sustain an active enzyme conformation . Moreover, directing point mutations in the 528-533 region, which overhangs the putative ATP-binding site, demonstrates that this part of the C-terminal arm participates also in the specific complexation of methionyl-tRNA synthetase with its cognate tRNAs.

J Chromatogr, 1989 Aug 4, 476, 245 - 55
Isolation of recombinant hirudin by preparative high-performance liquid chromatography; Bischoff R et al.; The purification of recombinant hirudin variant 2-Lys47 (rHV2-Lys47), produced by a genetically engineered yeast strain, is described . rHV2-Lys47 expressed and secreted into the culture medium was the starting material for the purification process of hirudin from the culture broth after cell harvesting by centrifugation . Initial purification of the product by preparative reversed-phase high-performance liquid chromatography (HPLC) using step-gradient elution, followed by precipitation of rHV2-Lys47 in the presence of acetone, removed most of the contaminants from the culture medium . The pure product was obtained by successive preparative anion-exchange and reversed-phase HPLC on silica based stationary phases . Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants . Liquid secondary ion mass spectrometry was used to confirm its primary structure . The isolated product was tested in an inhibition assay of human alpha-thrombin and proved to be fully active.

Science, 1989 Aug 4, 245(4917), 522 - 4
Triggering of allostery in an enzyme by a point mutation: ornithine transcarbamoylase; Kuo LC et al.; The origin of allostery is an unanswered question in the evolution of complex regulatory proteins . Anabolic ornithine transcarbamoylase, a trimer of identical subunits, is not an allosteric enzyme per se . However, when the active-site residue arginine-106 of the Escherichia coli enzyme is replaced with a glycine through site-directed mutagenesis, the resultant mutant enzyme manifests substrate cooperativity that is absent in the wild-type enzyme . Both homotropic and heterotropic interactions occur in the mutant enzyme . The initial velocity saturation curves of the substrates, carbamoyl phosphate and L-ornithine, conform to the Hill equation . The observed cooperativity depends on substrate but not enzyme concentration . The finding underscores the possibility that a single mutation of the enzyme in the cell could turn transcarbamoylation into a regulatory junction in the biosynthesis of L-arginine and urea.

J Chromatogr, 1989 Aug 4, 476, 147 - 58
Sodium dodecyl sulphate-protein complexes . Changes in size or shape below the critical micelle concentration, as monitored by high-performance agarose gel chromatography; Mascher E et al.; We have determined the sodium dodecyl sulphate (SDS) concentration needed to complete the formation of SDS-protein complexes . A Superose-6 column was equilibrated with SDS for 7 h . A sample of a native protein or an SDS-protein complex was applied, and the elution volume, Ve, was determined . Then the SDS concentration, CSDS, was changed, etc., i.e., Ve was determined as a function of CSDS . The critical micelle concentration of SDS (cmcSDS) was 1.8 mM in the eluent (ionic strength 0.10 M) . Native bovine carbonic anhydrase (BCA) formed an SDS complex above 0.2 mM SDS . As CSDS was increased, Ve decreased gradually in two main transitions, (TI) at 0.2-1.0 mM and (TII) at 1.2-2.0 mM SDS . These concentrations are corrected for a lag in the column equilibration with SDS . SDS-BCA, pre-equilibrated at 1.6 mM SDS, showed transitions similar to those observed with native BCA, except that transition TII included a minor transition at 2.0-2.2 mM SDS . The SDS complexes of reduced and carboxamidomethylated bovine serum albumin, of N-5'-phosphoribosylanthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli (PRAI-IGPS) and of two tryptic fragments of this enzyme behaved similarly . For SDS-PRAI-IGPS the major part of transition TII was completed at 1.6-1.7 mM SDS, as shown by analyses after 20-h column equilibrations with increasing as well as decreasing CSDS . The SDS complex of an integral membrane protein, the glucose transporter from human red cells, was smaller or less elongated than the SDS complexes of water-soluble proteins of the same polypeptide length . The formation of all five SDS-protein complexes investigated was practically completed at cmcSDS.

Nature, 1989 Aug 3, 340(6232), 397 - 400
Complete mutagenesis of the HIV-1 protease; Loeb DD et al.; Retroviruses encode a protease which needs to be active for the production of infectious virions . A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease . Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein . Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation . These results show that random mutagenesis can be used to identify functionally important regions within a protein . Mutants with conditional phenotypes have also been identified within this collection.

N Engl J Med, 1989 Aug 3, 321(5), 280 - 7
The cardiovascular response of normal humans to the administration of endotoxin; Suffredini AF et al.; Marked abnormalities in cardiovascular function accompany septic shock, and bacterial endotoxin is believed to be one of the principal mediators of these abnormalities . To evaluate the cardiovascular effects of endotoxemia in humans, we measured hemodynamic variables in nine normal subjects given an intravenous bolus dose of endotoxin (Escherichia coli, 4 ng per kilogram of body weight) and in six normal subjects given a bolus dose of saline, before and three hours after administration . All the subjects then underwent volume loading with normal saline (mean, 2217 ml) during the fourth and fifth hours after administration of the bolus, and the measurements were repeated . Three hours after the administration of endotoxin and before volume loading, the cardiac index had increased by 53 percent and the heart rate by 36 percent (both changes were significant; P less than or equal to 0.008), and the systemic vascular-resistance index had decreased by 46 percent (P = 0.004) . After volume loading (five hours after the administration of endotoxin), the left ventricular ejection fraction decreased by 1 percent of the base-line value in the subjects given endotoxin, but increased by 14 percent in the controls (P = 0.008) . The left ventricular end-diastolic and end-systolic volume indexes increased by 18 percent (P = 0.07) and 24 percent (P = 0.042), respectively . Left ventricular performance, as measured by the ratio of the peak systolic pressure to the end-systolic volume index, was depressed (a decrease of 0.90 in the subjects given endotoxin vs . an increase of 0.76 in the controls; P = 0.024) . We conclude that the administration of endotoxin to normal subjects causes a depression of left ventricular function that is independent of changes in left ventricular volume or vascular resistance . The changes in function are similar to those observed in septic shock and suggest that endotoxin is a major mediator of the cardiovascular dysfunction in this condition.

Gene, 1989 Aug 1, 80(1), 171 - 6
A high efficiency transformation system for the basidiomycete Ustilago violacea employing hygromycin resistance and lithium-acetate treatment; Bej AK et al.; A basidiomycete phytopathogenic fungus, Ustilago violacea, was transformed with pUCH1, a bacterial plasmid containing the hygromycin (Hyg)-resistance hygB gene fused to a promoter from the ascomycete Cochliobolus heterostrophus . After lithium acetate/polyethylene glycol treatment of whole sporidial cells, U . violacea transformants appeared on Hyg-agar at a frequency of 60-80 per microgram pUCH1 DNA . The Hyg phenotype was 100% stable in these transformants for at least 30 generations of mitotic growth under non-selective conditions . Southern DNA-DNA hybridization revealed multiple integrations of the pUCH1 plasmid into the U . violacea nuclear DNA . In addition, Escherichia coli transformants appeared at a frequency of 12 per microgram nuclear fraction DNA from Hyg U . violacea transformants; these E . coli consistently contained a deleted pUCH1 plasmid . This latter result suggested the low-frequency production of circular molecules by recombination within the integrated sequences.

Hybridoma, 1989 Aug, 8(4), 435 - 48
Specific, high affinity colchicine binding monoclonal antibodies: development and characterization of the antibodies; Edmond Rouan SK et al.; Nine colchicine specific monoclonal antibodies have been developed by immunizing BALB/c mice with a colchicine-keyhole limpet hemocyanin (Col-KLH) conjugate prepared using a bishydroxysuccinimide coupling reagent . Of four immunization procedures examined, intraperitoneal injection of the antigen attached to acid treated E . coli resulted in the maximum antigen specific antibody titers . A colchicine bovine serum albumin (Col-BSA) conjugate, prepared using a water soluble carbodiimide coupling technique, formed the basis of an enzyme linked immunosorbent assay used for screening hybridomas for colchicine specific antibody secretion and for determining the relative affinity and specificity profile of the monoclonal antibodies . All antibodies demonstrated high affinity, saturable binding to colchicine and low cross-reactivity with a panel of compounds structurally related to colchicine . The IC50 for the highest affinity antibody, C44, was 3.6 +/- 0.84 nM colchicine in the competitive enzyme immunoassay . The affinity of this antibody determined from Scatchard analysis of antibody binding to tritiated colchicine was 0.66 +/- 0.11 nM . Antibody C44 has the level of specificity and affinity suitable for a sensitive and selective immunoassay of colchicine for monitoring therapeutic drug levels . In addition, this antibody provides a specific pharmacologic antagonist for studies of colchicine's therapeutic mechanism and has the potential to reverse colchicine toxicity.

Zentralbl Hyg Umweltmed, 1989 Aug, 188(5), 421 - 38
{Nitrated polycyclic aromatic hydrocarbons (nitro-PAH) in the suspended substances of the atmosphere . 2 . Comparison of the mutagenicity of nitro-PAH and dust extracts of the air in the Ames, SOS repair induction and SCE test}; Schleibinger H et al.; Extracts of airborne suspended particulate matter were assayed in the Ames-test with strain TA 98, in the SOS chromotest (test on induction of the SOS repair) with strains E . coli PQ37 and S . typhimurium TA 1535 and in the sister chromatid exchange test . Samples from Berlin-Wedding were extracted by dichloromethane and tested up to the limits of solubility . All three test systems showed biologic activity . In the Ames- and the SOS chromotest these extracts revealed higher activity with S9-mix . 4 nitro-PAH, which are known to be present in ambient urban air, were assayed in these test systems, checking them additionally with strains TA 1538, TA 100 and TA 1535 in the Ames-test . In this test these compounds appeared to be direct acting frameshift mutagens and their activity is diminished by addition of S9-mix . On the contrary these compounds were either active at all or more active in the SOS chromotest in the presence of S9-mix . In the SCE-test only with exogenous activation a raise of the SCE frequency could be detected.

J Gen Virol, 1989 Aug, 70 ( Pt 8), 2087 - 95
Protection of woodchucks from infection with woodchuck hepatitis virus by immunization with recombinant core protein; Roos S et al.; Woodchucks were immunized with recombinant woodchuck hepatitis virus (WHV) core antigen (WHcAg) to investigate whether such immunization protects against WHV infection . The C gene was cloned into a pUC12 vector and expressed in Escherichia coli . Core particles purified by sucrose and CsCl gradient centrifugation had a buoyant density of 1.37 g/ml which corresponded to the density of WHcAg particles present in chronically infected liver . Two animals immunized with the recombinant antigen developed high antibody titres and were protected against infection after challenge with WHV . The surface antigen (WHsAg) and WHV DNA were not detected in the sera of immunized animals after challenge and these animals did not develop anti-WHs . Three control animals developed a typical WHV infection . The protection from WHV infection may depend not on the presence of antibodies against the core protein but on a cellular immune response to WHcAg.

Virology, 1989 Aug, 171(2), 579 - 87
Mapping and insertional mutagenesis of a vaccinia virus gene encoding a 13,800-Da secreted protein; Kotwal GJ et al.; The objective of this study was to identify and characterize the gene encoding a protein of approximately 12 kDa that is secreted from cells infected with the vaccinia virus . The absence of this protein from the medium of cells infected with a spontaneous deletion mutant (6/2) suggested that the open reading frame (ORF) was located within a 12,800-base pair segment near the left end of the genome (G . Kotwal and B . Moss, Nature (London) 335, 176-178, 1988) . Antibody to the 12-kDa protein immunoprecipitated an appropriate size in vitro translation product of mRNA that hybridized to a DNA segment containing an ORF (N1L) that could encode a 13.8-kDa polypeptide . The similarity in the sizes of the in vitro translation product and the secreted protein was consistent with the absence of processing . Transcriptional analysis revealed major and minor early RNA start sites preceding the N1L ORF as well as a late RNA start site with an atypical TAAAAT sequence . The N1L gene was interrupted by replacing a segment of the ORF with the Escherichia coli beta-galactosidase gene . When two-dimensional polyacrylamide gel electrophoretic patterns of {35S}methionine-labeled proteins secreted from cells infected with parental and recombinant viruses were compared, a spot missing from the latter corresponded in molecular weigh and isoelectric point with that predicted from the N1L ORF . The latter analysis revealed the presence of other secreted proteins of similar molecular weight but different isoelectric points that also appear to map within the left end of the vaccinia genome . The recombinant virus was attenuated as judged by the increased intracranial LD50 for mice but nevertheless induced antibody and cytotoxic responses after intradermal and intraperitoneal injections . Relative to the parental virus, the recombinant was also more attenuated for immunodeficient nude mice, based on their survival time after infection.

Arch Biochem Biophys, 1989 Aug 1, 272(2), 281 - 9
Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications; Berthiaume L et al.; Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle . Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture . The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively . Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence . Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases . Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases . Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme . Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology . The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.

Ryumachi, 1989 Aug, 29(4), 259 - 67
{Pathological changes in cartilage of the knees in rabbits immunized with Escherichia coli 0:14}; Sakai N; Pathological changes in the articular cartilage were found in the knees of rabbits immunized with heat-killed Escherichia coli for 8-10 months at significantly higher rate (33.3%, 12 of 36 knees, p less than 0.05) than in those immunized for 1-4 months (17.6%, 6 of 34) . Cartilage degeneration showed two types which were pannus invasion at the cartilage-synovial junction, and degeneration at superficial layer of the articular cartilage as seen in rheumatoid arthritis of human . Depositions of immunoglobulin were found in 10 out of 16 knees (62.5%) with the pannus invaded lesion and 5 out of 8 knees (75%) with the degenerated superficial layer of articular cartilage.

Acta Chir Scand, 1989 Aug, 155(8), 409 - 12
Psoas abscess complicating Crohn's disease; Shokouh-Amiri MH et al.; Six cases of psoas abscess complicating Crohn's disease are presented . The most common manifestations were weight loss, pain, fever and a palpable mass in the flank or iliac fossa . CT-scan confirmed the diagnosis . The treatment of choice in this condition is either laparotomy with drainage and primary resection of affected bowel or initial ultrasound-guided percutaneous drainage followed by early resection of the affected bowel.

Biochem Cell Biol, 1989 Aug, 67(8), 468 - 72
Production and interconversion of 1,2-propanediol and hydroxyacetone by Escherichia coli; Commodari F et al.; The anaerobic metabolism of marginally lethal levels of {13C}formaldehyde by Escherichia coli (K12, MU352, CRB, and CR63) was followed in vivo by 13C NMR . The products include 1,2-propanediol . Under aeration, the 1,2-propanediol is converted to hydroxyacetone . The hydroxyacetone is reconverted to 1,2-propanediol when aeration is stopped . The process can be cycled by varying the rate of aeration.

Biochem Cell Biol, 1989 Aug, 67(8), 404 - 10
Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli; Brisson A et al.; The charging of glutamate on tRNA(Glu) is catalyzed by glutamyl-tRNA synthetase, a monomer of 53.8 kilodaltons in Escherichia coli . To obtain the large amounts of enzyme necessary for the identification of structural domains, we have inserted the structural gene gltX in the conditional runaway-replication plasmid pOU61, which led to a 350-fold overproduction of glutamyl-tRNA synthetase . Partial proteolysis of this enzyme revealed the existence of preferential sites of attack that, according to their N-terminal sequences, delimit regions of 12.9, 2.3, 12.1, and 26.5 kilodaltons from the N- to C-terminal of the enzyme . Their sizes suggest that the 2.3-kilodalton fragment is a hinge structure, and that those of 12.9, 12.1, and 26.5 kilodaltons are domain structures . The 12.9-kilodalton domain of the glutamyl-tRNA synthetase of E . coli is the only long region of this enzyme displaying a good amino acid sequence similarity with the glutaminyl-tRNA synthetase of Escherichia coli.

Bioorg Khim, 1989 Aug, 15(8), 1078 - 90
{Synthesis of human proinsulin in Escherichia coli cells}; Efimov VA et al.; Expression of the synthetic gene for human proinsulin in E . coli has been investigated . The proinsulin gene has been expressed directly under the control of a synthetic promoter of phage fd DNA and a promoter of tryptophan operon, or using fusions with fragments of some bacterial proteins . These fusions gave insoluble polypeptide products amounting to 20-30% of total cellular protein . The scheme for isolating proinsulin from bacterial cells was developed . Proinsulin was cleaved from leader polypeptides by treatment with cyanogen bromide and converted into human insulin.

Bioorg Khim, 1989 Aug, 15(8), 1070 - 7
{Construction of a family of artificial genes of human insulin}; Efimov VA et al.; Using oligonucleotide-directed mutagenesis and chemical-enzymatic DNA synthesis, genes for A and B insulin chains, C-peptide and Tyr-C-peptide have been constructed starting from synthetic gene for human proinsulin synthesized earlier . The genes for human preproinsulin, mini-proinsulin, single-chain insulin and their modifications were also synthesized . The constructions obtained were cloned in plasmid vectors.

Bioessays, 1989 Aug-Sep, 11(2-3), 62 - 7
Invertebrate cytokines: the phylogenetic emergence of interleukin-1; Beck G et al.; Cytokines are polypeptides released by activated vertebrate blood cells which have profound effects on other blood cells and which have hormone-like properties affecting other organ systems as well . In recent years a wide variety of these mediators has been isolated and characterized . Many of these molecules have subsequently been cloned and expressed in E . coli . The tremendous importance of these proteins to host immune and non-specific defense systems along with the striking similarities of their properties among different species suggested to us that cytokines may have been proteins that have been conserved through evolution . Investigations of the evolution of cytokines will help us decipher the complex cellular, humoral and molecular interactions that regulate host defenses . Studies of the invertebrates will shed light on the phylogenetic emergence of these molecules as well.

Genetika, 1989 Aug, 25(8), 1384 - 90
{Integration of phage Mu into the RP4::D3112A-plasmid in Escherichia coli cells}; Kaplan AM et al.; Hybrid plasmids obtained as a result of Mu phage insertions into the RP4::D3112 plasmid in Escherichia coli cells were studied . Stable maintenance of RP4::D3112 plasmid in E . coli cells was provided by using the D3112 phage genome with a point polar mutation in the A gene which prevented early genes' expression . The presence of D3112A- in the RP4 plasmid has been shown to have no effect on efficiency of phage Mu transposition into this plasmid . Moreover, RP4 and D3112 genomes were equivalent targets for Mu integration . The integration of transposable phage into genome of nonrelated phage can be used as one of the approaches to construct recombinant phage genomes in vivo in the absence of DNA homology.

FEMS Microbiol Lett, 1989 Aug, 51(3), 267 - 71
Partial complementation of pyruvate dehydrogenase deficiency by independently expressed lipoyl and catalytic domains of the dihydrolipoamide acetyltransferase component; Russell GC et al.; Two compatible plasmids encoding a hybrid lipoyl domain and a defective pyruvate dehydrogenase (PDH) complex which lacks lipoyl domains, were co-expressed in a strain of Escherichia coli deleted for the PDH complex genes . In vivo complementation between the mutant complexes and the independent lipoyl domains was observed using growth tests in liquid and solid media . However, no PDH complex activity could be detected in the corresponding cell-free extracts . This suggests that untethered lipoyl domains can interact productively with the three types of active site in the multienzyme complex, but this association is disrupted in cell-free extracts.

Can J Microbiol, 1989 Aug, 35(8), 779 - 85
Nonspecific inhibition of proline dehydrogenase synthesis in Escherichia coli during osmotic stress; Deutch CE et al.; L-Proline, which is accumulated by Escherichia coli during growth in media of high osmolality, also induces the synthesis of the enzyme degrading it to glutamate . To determine if proline catabolism is inhibited during osmotic stress, proline utilization and the formation of proline dehydrogenase were examined in varying concentrations of NaCl and sucrose . Although the specific growth rate of E . coli with proline as the sole nitrogen source diminished as the solute osmolality increased, a comparable reduction in growth rate occurred with ammonium as the primary nitrogen source . Proline catabolism, as measured in whole cells by the conversion of {14C}proline to {14C}glutamate, was only slightly inhibited by solute osmolalities up to 1.0 osmol/kg; more than 50% of the initial activity was still found at 2.0 osmol/kg . By contrast, the specific activity of proline dehydrogenase in bacteria grown in the presence of added solutes decreased to less than 20% of the control level . This reduction was related to a lower rate of synthesis, but was independent of genes currently known to be involved in osmoregulation or proline metabolism . The specific activities of tryptophanase, beta-galactosidase, and histidinol dehydrogenase were also reduced under similar growth conditions . These results indicate that while proline catabolism is not directly inhibited by high solute concentrations, prolonged exposure to osmotic stress leads to its reduction as part of a more general metabolic response.

Biokhimiia, 1989 Aug, 54(8), 1247 - 53
{The effect of mutations in ribosomal proteins S4, S12 and L7/L12 on EF-Tu-dependent expenditure of GTP in the process of codon-specific elongation and misreading of poly(U)}; Kakhniashvili DG et al.; The effect of mutations in ribosomal proteins S4 (rpsD12), S12 (rpsL282) and L7/L12 (rplL265) of Escherichia coli K12 on the EF-Tu-dependent expenditure of GTP during codon-specific elongation (poly(Phe) synthesis on poly(U} and misreading (poly(Leu) synthesis on poly(U}, was studied . Under the conditions used the mutations in proteins S4 and L7/L12 did not practically affect the EF-Tu-dependent expenditure of GTR during the poly(Phe) synthesis on poly(U): the GTP/Phe ratio was about 1, as in the case of the wild strain . Under the same conditions, the ribosomes with a mutant S12 protein tended to discard some amount of Phe-tRNA, as a result of which the GTP/Phe ratio increased to about 3 . The marked inhibition of misreading by ribosomes with a mutant S12 protein was accompanied by a significant increase of GTP expenditure at the stage of EF-Tu-dependent non-cognate aminoacyl-tRNA binding . In mutant S 12 proteins the GTP/Leu ratio was about 30-40, whereas in the wild type it was about 12 . In contrast, stimulation of misreading by ribosomes with mutant S4 and L7/L12 proteins was accompanied by a decrease of the EF-Tu-dependent expenditure of GTP by 2-3 GTP molecules per one Leu residue included into the peptide.

J Biomol Struct Dyn, 1989 Aug, 7(1), 181 - 6
Additional Watson-Crick interactions suggest a structural core in large subunit ribosomal RNA; Haselman T et al.; Two new Watson-Crick type interactions in 23S-like ribosomal RNA have been identified by comparative sequence analysis . These interactions, A1269/U2011 and C1270/G2010 (E . coli numbering) along with the previously proposed A1262/U2017 suggest an anti-parallel helical arrangement characteristic of secondary structure in the 1265/2015 region of 23S rRNA . Nested within these three interactions are three universal juxtapositions which in principle allow the formation of an irregular helix containing two additional A-G interactions and a universal A-U pair . Whether or not this extended helix is biologically significant is uncertain . The proponderance of interactions in the 1265/2015 region and its location relative to the known structural domains of 23S rRNA suggest that this region may be part of a central structural core similar to that already known in 16S rRNA.

Protein Eng, 1989 Aug, 2(8), 597 - 604
Sequence alignment of citrate synthase proteins using a multiple sequence alignment algorithm and multiple scoring matrices; Henneke CM et al.; The alignment of Escherichia coli citrate synthase to pig heart citrate synthase and the multiple alignment of the known sequences of the citrate synthase family of enzymes have been performed using six different amino acid similarity scoring matrices and a large range of gap penalty ratios for insertions and deletions of amino acids . The alignment studies have been performed as the first step in a project aimed at homology modelling E . coli citrate synthase (a hexamer) from pig heart citrate synthase (a dimer) in a molecular modelling approach to the study of multi-subunit enzymes . The effects of several important variables in producing realistic alignments have been investigated . The difference between multiple alignment of the family of enzymes versus simple pairwise alignment of the pig heart and E . coli proteins was explored . The effects of initial separate multiple alignments of the most highly related or most homologous species of the family of enzymes upon a subsequent pairwise alignment between species was evaluated . The value of 'fingerprinting' certain residues to bias the alignment in favour of matching those residues, as well as the worth of the computerized approach compared to an intuitive alignment technique, were assessed.

Mol Gen Mikrobiol Virusol, 1989 Aug, (8), 17 - 20
{Cloning of chloroplast psbA and rbcL-genes from cotton Gossipium hirsutum}; Ul'masov TN et al.; The fragments of cotton Gossipium hirsutum c.v . 108-f chloroplast genome were cloned in Escherichia coli cells . The cloned psbA and rbcL genes have been selected using the heterologous probes from spinach . The preliminary attempts to clone the complete psbA gene in pUC19 vector failed, probably, due to the toxicity of its product to Escherichia coli cells, and its 5'- and 3'-ends were cloned separately . Reconstruction experiments revealed that while the complete psbA gene was unable to be stably inherited by Escherichia coli cells, its structural part lacking the promoter region could be readily cloned in the bacterial cells.

Yakugaku Zasshi, 1989 Aug, 109(8), 574 - 81
{Biological characteristics of plasmid carrying a repeated deoxyribonucleic acid sequence}; Fujii T et al.; It was found that a plasmid which had a foreign deoxyribonucleic acid (DNA) between two repeated sequences did not multiply in E . coli recBCsbcB, even if it multiplied in wild-type E . coli, E . coli recBC or E . coli recBCsbcBrecF when the insert was longer than 351 base pair . The multiplication of these plasmids were, however, inhibited when a plasmid expressing recF gene was introduced into E . coli recBCsbcBrecF . The inviability of the plasmid carrying the repeated sequence in E . coli recBCsbcB was discussed by the mechanism of recombination, and the functions of recF, recBC and sbcB were speculated . When E . coli recBC was transformed with pDR1 which was a derivative of pBR322 carrying a directly repeated sequence between which a DNA fragment derived from plasmid R6K with its origin was inserted, the intramolecular recombinant appeared . The recombinant recovered was, however, only the plasmid which had the replication origin of pBR322 . The result suggests that pBR322 is compatible with pDR1 but R6K is not . The replication origin of R6K seems to be preferrentially used by pDR1.

Acta Med Okayama, 1989 Aug, 43(4), 197 - 202
Non-radioactive hybridization probes prepared using M13 phage vector and the universal sequencing primer; Ikeda S et al.; Non-radioactive hybridization probes were prepared using the M13 phage vector and the universal sequencing primer . The probe sequence to be used was first cloned into the M13 vector, and the minus strand of the template DNA was then synthesized with the Klenow fragment of E . coli DNA polymerase I in the presence of the biotinylated nucleotide, biotin-11-dUTP, as a label . Resultant DNA was heavily biotinylated, and made up of the entire minus strand of the template DNA . The long tag sequence derived from the M13 vector may increase the sensitivity of the detection . The biotinylated hybrids were visualized with the streptavidin-alkaline phosphatase conjugate and chromogenic substrates . As shown by Southern hybridization, the probe prepared in this way could be used to detect less than 1 pg of target sequence and a single copy gene sequence in human genomic DNA within several hours of signal development.

Vet Microbiol, 1989 Aug, 20(4), 357 - 68
In vitro adhesion of K88ac+ Escherichia coli to Peyer's patch and peripheral blood lymphocytes, buccal and rectal epithelial cells or intestinal epithelial brush borders of weaned pigs; Valpotic I et al.; Escherichia coli adhesion assays were conducted using isolated porcine peripheral blood lymphocytes, Peyer's patch lymphocytes, rectal epithelial cells or brush borders, buccal epithelial cells and brush borders from small intestinal epithelial cells . The cells and brush borders were tested for their ability to bind K88-piliated enterotoxigenic E . coli Strain M1823B (K88ac) and E . coli Strain 1476 (K-12, K88ac) . Comparison of adhesive phenotypes of 37 weaned pigs as determined by the adhesion assay with small intestinal brush borders and the adherence of K88ac+ enterotoxigenic E . coli to peripheral blood lymphocytes, Peyer's patch lymphocytes and rectal epithelial cells or brush borders, revealed no correlation . In vitro adhesion of K88ac-bearing E . coli was always negative with buccal epithelial cells . K88ac strains varied in their ability to adhere to lymphocytes and rectal epithelial cells or brush borders, indicating that the mechanism of adherence is unrelated to K88-mediated adhesion observed in animals that had the receptors on small-intestinal epithelial-cell brush borders . The non-piliated control E . coli Strain 123 adhered to fresh peripheral blood lymphocytes, and less intensively to frozen-thawed peripheral blood lymphocytes or Peyer's patch lymphocytes . It was concluded that none of the cell types or brush borders, except small-intestinal epithelial-cell brush borders, could be used as targets for phenotyping pigs for the presence of the K88 receptors that have been associated with adhesion and colonization of K88+ enterotoxigenic E . coli in the porcine small intestine.

Gene, 1989 Aug 1, 80(1), 109 - 18
Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone; Redmond TM et al.; We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector . The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined . These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP . This cDNA sequence validates the intron: exon boundaries predicted from the gene . A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1 . Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination . This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots . This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP . Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.

Genes Dev, 1989 Aug, 3(8), 1226 - 32
Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation; Parsell DA et al.; To test the idea that unfolded protein might act as an intracellular signal for induction of the heat shock response in Escherichia coli, we examined the synthesis of several heat shock proteins after expression of an unfolded variant of the amino-terminal domain of lambda repressor . These experiments show that expression of a single mutant protein, and not its wild-type counterpart, is sufficient to induce a heat shock-like response . In addition, by measuring the abilities of unfolded variants of differing proteolytic susceptibilities to induce heat shock protein synthesis and by monitoring heat shock protein synthesis as a function of the amount of a single unfolded protein, we show that it is the concentration of unfolded protein in the cell, and not its degradation, that is important for inducing the heat shock-like response.

EMBO J, 1989 Aug, 8(8), 2417 - 24
tRNA-like structures and gene regulation at the translational level: a case of molecular mimicry in Escherichia coli; Springer M et al.; Escherichia coli threonyl-tRNA synthetase regulates the translation of its own mRNA by binding to it in a region, called the operator, located in front of the ribosomal binding site . The primary and secondary structures of the operator resemble those of the anticodon arm of several tRNA(Thr) isoacceptor species . We reasoned that if the interaction between the synthetase and its two partially analogous ligands, the tRNA and the mRNA, had some common features, single mutations in the enzyme should affect both interactions in a very similar way . We thus isolated synthetase mutants (called super-repressors) that repress the translation of their mRNA in trans to an extreme level, and other mutants that are completely unable to perform any repression . The super-repressors, which are suspected to bind their mRNA with high affinity, are shown to bind the tRNA with an increased affinity . The non-repressing mutants, which are suspected to have lost their capacity to bind the mRNA, are shown to bind their tRNA with less affinity . The binding properties of the mutant enzymes for the other substrates, ATP and threonine, are unchanged . The observed correlation between regulatory and aminoacylation defects strongly suggests that the synthetase recognizes the similar parts of its two RNA ligands--the anticodon-like arm of the mRNA and the true anticodon arm of the tRNA--in an analogous way.

EMBO J, 1989 Aug, 8(8), 2411 - 5
In vitro reconstitution of anticodon nuclease from components encoded by phage T4 and Escherichia coli CTr5X; Amitsur M et al.; During phage T4 infection of Escherichia coli strains containing the prr locus the host tRNALys undergoes cleavage-ligation in reactions catalyzed by anticodon nuclease, polynucleotide kinase and RNA ligase . Known genetic determinants of anticodon nuclease are prr, which restricts T4 mutants lacking polynucleotide kinase or RNA ligase, and stp, the T4 suppressor of prr encoded restriction . The present communication describes an in vitro anticodon nuclease assay in which the specific cleavage of tRNALys is driven by an extract from E . coli prrr (restrictive) cells infected by phage T4 . The in vitro anticodon nuclease reaction requires factor(s) encoded by prr, is stimulated by a synthetic Stp polypeptide and appears to require additional T4 induced factor(s) distinct from Stp.

Berl Munch Tierarztl Wochenschr, 1989 Aug 1, 102(8), 266 - 72
{The administration of homeopathic drugs for the treatment of acute mastitis in cattle}; Merck CC et al.; The general principles of homeopathic therapy are described together with a number of homeopathic drugs used for the treatment of acute bovine mastitis . Fifty cows with acute mastitis were used in the study . The initial treatment comprised aconitum D 4, phytolacca D 1 and bryonia D 4 . In subsequent treatments phytolacca D 1, bryonia D 4 and lachesis D 8 either singly or in combination were used; mercurius solubilis D 4 was also used . Encouraging results, especially in the treatment of cases of E.coli mastitis, were achieved.

Am J Vet Res, 1989 Aug, 50(8), 1294 - 6
Cytotoxin activity on Vero cells among Escherichia coli strains associated with diarrhea in cats; Abaas S et al.; The role of Escherichia coli as a causative agent of diarrhea in cats was investigated . Isolates of E coli from healthy and diarrheic cats were serotyped and investigated for their biochemical characters, production of cytotoxin activity on Vero cells, heat-labile enterotoxin, heat-stable enterotoxin, and hemagglutination of erythrocytes from other animal species . None of 48 investigated strains produced heat-labile enterotoxin or heat-stable enterotoxin, nor did they agglutinate erythrocytes . Most strains were hemolytic and belonged to O-serotypes 2 and 6 . Cytotoxin activity on Vero cells was significantly more common and produced in greater amounts among E coli strains isolated from diarrheic cats, and was neutralized by anti-Shiga-like toxin I serum.

Mol Gen Genet, 1989 Aug, 218(2), 284 - 8
Ars region TL-DNA on octopine type Ti plasmids; Suzuki Y et al.; In the TL-DNA region of the octopine type Ti plasmids, an ars region was assigned as the DNA segment conferring the replicational ability to YIp5 in Saccharomyces cerevisiae . T-DNA: YIp5 hybrid plasmids containing a particular T-DNA region could transform yeast cells at a frequency of 10(3)-10(4) transformants per microgram plasmid DNA and they were rescued in Escherichia coli, although the transformed phenotype was mitotically unstable . The instability was inferred to be caused by segregation of the plasmids due to their low efficiency of replication . The ars region was mapped on the noncoding region between the coding regions corresponding to no . 5 and no . 7 mRNA, and its minimal length determined in this experiment was about 150 bp.

Mol Gen Genet, 1989 Aug, 218(2), 249 - 56
Nitrate reductase of Escherichia coli: completion of the nucleotide sequence of the nar operon and reassessment of the role of the alpha and beta subunits in iron binding and electron transfer; Blasco F et al.; The nucleotide sequence of the narGHJI operon that encodes the nitrate reductase of Escherichia coli was completed . It encodes four polypeptides NarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5 and 25.5 kDa, respectively . The analysis of deduced amino acid sequence failed to reveal any structure capable of binding iron within the NarG polypeptide . In contrast, cysteine arrangements typical of iron-sulfur centers were found in the NarH polypeptide . This suggested that the latter is an electron transfer unit of the nitrate reductase complex . Such a view is opposite to the current description of the nitrate reductase . The findings allowed us to propose a model for the electron transfer steps that occur during nitrate reduction . The NarG polypeptide was found to display a high degree of homology with numerous E . coli molybdoproteins . Moreover, the same genetic and functional organizations as well as the presence of highly conserved stretches of amino acids were noted between both NarG/NarH and DmsA/DmsB (encoding the dimethyl sulfoxide reductase) pairs.

Mol Gen Genet, 1989 Aug, 218(2), 190 - 8
Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86; Maas R et al.; Many plasmids belonging to the F incompatibility groups contain more than one basic replicon . The chimeric plasmid pCG86 is an example of such a multireplicon plasmid . The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied . The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication . In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility . This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated . We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.

Mol Gen Genet, 1989 Aug, 218(2), 183 - 9
Involvement of DnaK protein in mini-F plasmid replication: temperature-sensitive seg mutations are located in the dnaK gene; Ezaki B et al.; The seg mutants (seg-1 and seg-2) of Escherichia coli cannot support the replication of the F factor and mini-F plasmids at 42 degrees C . We cloned the wild-type E . coli chromosomal DNA fragment complementing the seg-1 and seg-2 mutations and found that both mutations were complemented by the wild-type dnaK gene coding for a heat shock protein . Transduction with phage P1 indicated that the seg-2 mutation is located at about 0.3 min in the region containing the dnaK gene in the order trpR--thrA--seg-2--leuB, consistent with the locus of the dnaK gene . Cloning and sequencing of the dnaK gene of the seg mutants showed that there was one base substitution within the dnaK gene in each mutant causing an amino acid substitution . These results indicate that the seg gene in which the seg-1 and seg-2 mutations occurred is identical to the dnaK gene . The mini-F plasmid pXX325 did not transform a dnaK null mutant to ampicillin resistance at 30 degrees C in contrast to plasmids pBR322, pACYC184 and pSC101, which did . The active dnaK (seg) gene product is therefore essential for replication of the mini-F plasmid at both 30 degrees and 42 degrees C.

Epidemiol Infect, 1989 Aug, 103(1), 211 - 5
Necrobacillosis and immunity in mice; Smith GR et al.; The study arose from the recent finding that sub-lethal numbers of certain bacterial species greatly enhanced the infectivity of Fusobacterium necrophorum . A severe F . necrophorum infection in mice, cured with metronidazole, produced significant though slight resistance, which was demonstrable by challenge with a minute dose of F . necrophorum (less than 20 organisms) suspended in a sub-lethal dose of Escherichia coli (300 x 10(6) organisms) to enhance fusobacterial infectivity . In an earlier comparable experiment, challenge with F . necrophorum alone, in necessarily large doses (greater than or equal to 3 x 10(6) organisms), failed to demonstrate that a single cured fusobacterial infection gave rise to resistance; such an infection neither protected against the fatal necrobacillosis produced by challenge nor prolonged survival . A sub-lethal E . coli infection was also shown by challenge with a minute dose of F . necrophorum (less than 10 organisms), suspended in a sub-lethal dose of E . coli (152 x 10(6) organisms), to produce significant though slight protection against necrobacillosis . The degrees of resistance demonstrated were too slight to give any encouragement to the prospect of an effective necrobacillosis vaccine.

Circ Shock, 1989 Aug, 28(4), 385 - 94
Temporal response of lipoprotein lipase-suppressing mediator and tumor necrosis factor in lipopolysaccharide -injected and -infused rats; Bagby GJ et al.; This study was initiated to compare the temporal response of serum lipoprotein lipase-suppressing mediator (LSM) and tumor necrosis factor (TNF) in lipopolysaccharide (LPS) -infused or -injected rats . Serial blood samples were obtained over a 5-day period from rats implanted with vascular catheters . Control rats infused with saline exhibited no detectable LSM activity during the 5-day observation period . LPS administered either by injection or infusion (6 h or 5 d) resulted in detection of serum LSM and TNF activities during the early period of observation with the LSM temporal response (8 h) outlasting the TNF response (3 h) . The LSM response lasted a little longer in LPS-infused rats than it did in injected rats, but in each case LSM activity was not detected in serum samples collected at or after 12 h post-LPS . Neither the duration nor the magnitude of the TNF response differed between LPS-infused vs . -injected rats . Despite similarities in the LSM and TNF pattern in all rats receiving LPS, lethality was greater in LPS-infused animals than it was in LPS-injected rats . The results indicate that differences in lethality between LPS-injected and -infused rats cannot be explained solely by a differential response of serum TNF.

Biotechnol Appl Biochem, 1989 Aug, 11(4), 401 - 12
Expression of repetitive human calcitonin genes in Escherichia coli; Gigova L et al.; In order to stabilize recombinant human calcitonin (rhCT) against Escherichia coli proteases a series of concatemeric hCT genes with varying degrees of repetition were synthesized and expressed in E . coli under the control of a constitutive synthetic phage promoter . The series of expression vectors thus constructed was used as a model to study the effect of gene repetition on the efficiency of expression (both transcription and translation), stability of mRNA, proteolytic stability of recombinant protein, genetic stability of expression plasmids, etc . The oligomerization of the hCT gene resulted in stabilization of the mRNA increasing its half-life from 60-70 s (as in the hCT monomer, dimer, and trimer genes) to 100-120 s (for the hCT tetramer gene) . This effect held true as well for the proteins coded by the corresponding repetitive hCT genes . The genetic stability (segregation and recombination) of the expression plasmids containing hCT oligomeric genes also depended on the number of hCT gene repeats . The expression plasmid containing the hCT tetramer gene segregated from one of the best producers of rhCT (E . coli LE392) up to 100% after 100 cell generations in nonselective media (free of antibiotics) . One of the plasmids most sensitive to recombination events was that containing the hCT pentamer gene . The series of expression plasmids bearing hCT oligomeric genes was used for transformation of various E . coli strains in order to find the optimal host for production of rhCT . The highest yield (44-100 mg rhCT per 1 liter of bacterial culture) was obtained with the strains LE392, JM107, and DH1.

Pediatr Res, 1989 Aug, 26(2), 158 - 61
Direct long-term effects of L-asparaginase on rat and human pancreatic islets; Clausen N et al.; L-Asparaginase, an effective agent in the treatment of acute lymphoblastic leukemia, may induce a diabetic state . The pathogenesis of the diabetogenic effect was studied in cultured pancreatic islets . Mean serum concentrations in three children with acute lymphoblastic leukemia were 2.4 U/mL (range 1.4-4.5) before and 31.5 U/mL (range 18.6-51.8) immediately after an intravenous injection of 1000 U/kg L-asparaginase . Glucose-induced insulin release from pancreatic islets of rat and man was measured after 3 and 7 days of culture in media with or without clinically relevant concentrations of Escherichia coli L-asparaginase (0.01-100 U/mL) . After culture, the remaining insulin, glucagon, and DNA in the islets were determined . After 7 days of culture of adult rat or human islets, both the accumulation of insulin in the medium and the content of insulin and glucagon in the islets were significantly reduced in the presence of 100 U/mL L-asparaginase compared with controls . Addition of 10(-6) M hydrocortisone to the culture medium enhanced this effect . In newborn rat islets a significant reduction in insulin release and content was observed already in the presence of 0.1 U/mL asparaginase, whereas the glucagon content was unchanged . Removal of the drug resulted in partial recovery of the insulin secretion . To elucidate the mechanisms of of action of the drug, insulin biosynthesis was studied in islets cultured in asparagine-free medium with or without asparaginase . No difference in biosynthesis was seen between media with or without asparagine, whereas 0.1 U/mL asparaginase caused about a 50% reduction under both conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Virol, 1989 Aug, 70 ( Pt 8), 1961 - 74
Molecular cloning, sequencing and expression in Escherichia coli of the bean yellow mosaic virus coat protein gene; Hammond J et al.; The sequence of 1015 nucleotides from the 3' poly(A) tract of the potyvirus bean yellow mosaic virus (BYMV) RNA has been determined from two cDNA clones . This sequence contained a single long open reading frame (ORF) starting upstream of the cloned region . The ORF was expressed as a fusion protein in Escherichia coli, and the product was detected by antibodies specific for the coat protein of BYMV . The predicted length of the coat protein gene was 822 nucleotides, corresponding to a 273 amino acid coat protein of Mr 30910 . The deduced amino acid sequence of the BYMV coat protein was compared to the chemically determined amino acid composition of purified virion protein, and of protein prepared from trypsin-treated virions . The nucleotide and deduced amino acid sequences were compared to the sequences of the coat protein genes of other potyviruses . The BYMV coat protein gene was found to be 50 to 61% homologous to those of other potyviruses at both the nucleotide and amino acid levels; the greatest variation was between the 5'-proximal one-fifth of the genes . Amino acid sequences and hydrophilicity plots of the different potyvirus coat proteins showed similarities which indicated that the structure of the coat protein is highly conserved; a non-terminal region of variability was predicted to be exposed on the exterior of the virion . A putative cleavage site at a glutamine-serine dipeptide was identified by similarity in context to the cleavage sites of tobacco etch virus and tobacco vein mottling virus coat proteins from the viral polyproteins . The BYMV 3'-terminal non-coding region of 166 nucleotides is followed by a poly(A) tract.

Arthritis Rheum, 1989 Aug, 32(8), 1041 - 4
An adsorption procedure to increase the specificity of enzyme-linked immunosorbent assays for Lyme disease without decreasing sensitivity; Fawcett PT et al.; Previous efforts to use adsorption techniques to enhance the specificity of enzyme-linked immunosorbent assays for antibodies to the spirochete that causes Lyme disease resulted in unacceptable reductions in assay sensitivity . We present here evidence that preadsorption of serum with Escherichia coli can enhance test specificity without significantly reducing test sensitivity.

Anesth Analg, 1989 Aug, 69(2), 163 - 8
Alterations in host defense associated with inhalation anesthesia and blood transfusion; Waymack JP et al.; Anesthesia and blood transfusions have been demonstrated to impair immune function . The present study evaluated whether these impairments led to increased susceptibility to infectious complications in two animal models . Both transfusions and anesthesia (methoxyflurane) were found to increase susceptibility to peritoneal Escherichia coli infections . This susceptibility increased with time after the transfusions but decreased with time after anesthesia . Neither transfusions nor anesthesia altered susceptibility to an intravenous Escherichia coli challenge in rats.

Surgery, 1989 Aug, 106(2), 439 - 43
Failure of tumor necrosis factor to produce hypotensive shock in the absence of endotoxin; Neilson IR et al.; Tumor necrosis factor (TNF) is reported to cause a shock syndrome similar to that produced by endotoxin (LPS) . The purpose of this study was to determine the relationship between TNF and LPS in causing shock . Eighty rats received infusions of either TNF, LPS, or TNF plus LPS, as compared with saline solution . Temperature, blood, and tissue specimens were obtained at 2 hours . Blood pressure was measured over 4 hours in a separate group of awake rats . Mortality was assessed over 24 hours . Neither TNF (1 mg/kg) nor LPS (1 mg/kg) altered hematocrit, blood gases, temperature, or caused hypotension or mortality . If the same dose of TNF was combined with LPS, however, there was significant (p less than 0.05) hemoconcentration and metabolic acidosis associated with hypotension and 100% mortality by 4 hours . Pathologic changes were restricted to the small intestine and occurred in this group only . It was concluded that TNF does not cause hypotension or shock in the rat . TNF will cause lethal shock, however, if combined with a sublethal dose of endotoxin . This suggests that synergy between TNF and endotoxin is important in septic shock.

Surgery, 1989 Aug, 106(2), 216 - 22; discussion 222-3
Protein kinase C: a potential pathway of endothelial cell activation by endotoxin, tumor necrosis factor, and interleukin-1; Magnuson DK et al.; Human endothelial cells exposed to lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin-1 (IL-1) in vitro acquire a cell surface property that promotes the adherence of neutrophils (PMNs) . The common mechanism by which endothelial cells are activated by these agents is unknown . We examined adherence of PMNs to cultured human umbilical vein endothelium (HUVE) pretreated with LPS (100 ng/ml), TNF (100 U/ml), and IL-1 (1 U/ml) in medium alone or medium containing protein kinase inhibitors H-7 or HA-1004 . Both compounds inhibit a similar spectrum of protein kinases, but H-7 is an effective inhibitor of protein kinase C, whereas HA-1004 is not . We found that H-7 (25 mumol/L) reduced the adherence of PMNs to LPS-, TNF-, and IL-1-stimulated HUVE monolayers to 16.7% +/- 3.0%, 12.1% +/- 2.5%, and 18.3% +/- 2.9% of control, respectively (mean plus or minus standard error of three experiments); HA-1004 (25 mumol/L) did not inhibit endothelial adhesiveness . Cytotoxicity of H-7 was less than 10% in LPS-, TNF-, and IL-1-treated HUVE . Protein synthesis, as measured by the incorporation of tritiated amino acids, was not significantly impaired in LPS-treated HUVE concurrently exposed to H-7 . We conclude that protein kinase C appears to be a necessary common mediator of endothelial cell activation by LPS, TNF, and IL-1.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6087 - 91
Characterization and functional reconstitution of a soluble form of the hydrophobic membrane protein lac permease from Escherichia coli; Roepe PD et al.; Lac permease, a polytopic membrane protein from Escherichia coli, has been purified in soluble form by overexpressing the lacY gene by means of the T7 RNA polymerase system . Soluble permease is dissociated from membranes with urea or other chaotropes and appears after the membrane is saturated with newly synthesized permease . Remarkably, this form of the permease appears to remain soluble in phosphate buffer at neutral pH after removal of urea, although it aggregates in a time- and concentration-dependent manner . Importantly, soluble permease behaves as a monomer during size-exclusion chromatography with or without urea, contains less than 3 mol of organic phosphate per mol of protein, and is largely helical . Soluble permease binds p-nitrophenyl alpha-D-galactopyranoside approximately 40% as well as permease in the native environment of the membrane and can be reconstituted into phospholipid vesicles that catalyze lactose counterflow or active transport in response to a membrane potential (interior negative) . The results suggest that lac permease can assume a nondenatured conformation in aqueous solution.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6052 - 6
Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli; Forst S et al.; EnvZ and OmpR, the regulatory proteins for ompF and ompC expression in Escherichia coli, belong to a modulator-effector family of regulatory proteins which are essential for the response to environmental signals . We show that the soluble cytoplasmic domain of the transmembrane modulator protein EnvZ is phosphorylated in vitro by {gamma-32P}-ATP . We also demonstrate that the phosphate group can, in turn, be transferred to the transcription activator protein OmpR . The pH stability properties of the phosphate groups linked to EnvZ indicate that this molecule contains histidyl phosphate . The invariant His-243 of EnvZ corresponds to the phosphorylated His-48 of the chemotactic modulator protein CheA . Substitution of His-243 with valine produces an EnvZ that is refractory to phosphorylation and can no longer catalyze the transfer of phosphate to OmpR . Furthermore, in a delta envZ strain of E . coli, containing the envZ Val-243 plasmid, ompC expression is elevated 7-fold relative to that found in cells carrying the wild-type envZ plasmid . Based on these results we propose a model in which the phosphorylated state of OmpR modulates the expression of the ompF and ompC genes.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6023 - 7
Homology of aspartyl- and lysyl-tRNA synthetases; Gampel A et al.; The yeast nuclear gene MSD1 coding for mitochondrial aspartyl-tRNA synthetase has been cloned and sequenced . The identity of the gene is confirmed by the following evidence . (i) The primary structure of the protein derived from the gene sequence is similar to that of the yeast cytoplasmic aspartyl-tRNA synthetase . (ii) In situ disruption of MSD1 in a respiratory-competent haploid strain of yeast induces a pleiotropic phenotype consistent with a lesion in mitochondrial protein synthesis . (iii) Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA . The primary structures of the cytoplasmic and mitochondrial aspartyl-tRNA synthetases are similar to the yeast cytoplasmic lysyl-tRNA synthetase, suggesting that the two types of synthetases may have a common evolutionary origin . Searches of the current protein banks also have revealed a high degree of sequence similarity of the lysyl-tRNA synthetase to the product of the Escherichia coli herC gene and to the partial sequence of a protein encoded by an unidentified reading frame located adjacent to the E . coli frdA gene . Based on the sequence similarities and the map positions of the herC and frdA loci, we propose herC to be the structural gene of the constitutively expressed lysyl-tRNA synthetase of E . coli and the unidentified reading frame to be the structural gene of the heat-inducible lysyl-tRNA synthetase.

Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5898 - 902
Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences; Traver CN et al.; A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA . A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells . A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate . This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters . Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence . An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene.

Mutat Res, 1989 Aug, 213(2), 165 - 73
RecA protein inhibits in vitro replication of single-stranded DNA with DNA polymerase III holoenzyme of Escherichia coli; Shwartz H et al.; Purified RecA protein from Escherichia coli inhibited 5-10-fold the rate of in vitro replication of both unirradiated and UV-irradiated single-stranded DNA (ssDNA) with DNA polymerase III holoenzyme . Maximal inhibition occurred at a ratio of 1 molecule of RecA per 2-4 nucleotides of DNA, and it affected primarily the initiation of elongation on primed ssDNA . Adding single-strand DNA-binding protein (SSB) caused a relief of inhibition . Under conditions when there was enough SSB to cover the ssDNA completely, RecA protein had no effect on initiation, elongation or dissociation steps of replication . These observations together with data from in vivo studies suggest a role for RecA protein in the arrest of DNA replication observed in cells exposed to UV-radiation and a variety of chemical carcinogens.

Metabolism, 1989 Aug, 38(8 Suppl 1), 6 - 13
Muscle glutamine concentration and protein turnover in vivo in malnutrition and in endotoxemia; Millward DJ et al.; A comparison of the changes in the concentration of glutamine {Gln} in skeletal muscle in a variety of catabolic states with the attendant changes in rates of protein synthesis and degradation indicates a number of substantial correlations which provide insight into both the way in which {Gln} is regulated in muscle and possible regulatory influences of {Gln} on protein balance . There is a striking direct correlation between {Gln} and the rate of protein synthesis in the whole data set . Further examination of this relationship in protein deficiency shows that the changes in {Gln} correlate mainly with the reductions in ribosomal concentration (RNA/protein) and with the decrease in the rate of protein degradation . Because the fall in {Gln} in protein deficiency is also correlated with the decrease in free T3 concentrations, it is suggested that in this case the correlations of {Gln} with rates of protein turnover may be incidental, reflecting thyroidal influences on both protein turnover and glutamine transport . In contrast, in endotoxemia the changes in {Gln} were highly correlated with the ribosomal activity, kRNA, and in this case {Gln} was inversely correlated with the rate of protein degradation . Similar correlated changes occur in starvation and in response to glucocorticoids, and it is suggested that the reductions in {Gln} in endotoxemia could be causally related to the development of insulin resistance and the inhibition of the translational phase of protein synthesis which occurs in these circumstances . The mechanism of the reduction in {Gln} and any linked inhibition of protein synthesis is unknown, but it is shown to be independent of prostaglandin production.(ABSTRACT TRUNCATED AT 250 WORDS)

J Trauma, 1989 Aug, 29(8), 1076 - 84; discussion 1084-5
Complement depletion with Naje haje cobra venom factor limits prostaglandin release and improves visceral perfusion in porcine endotoxic shock; Fink MP et al.; We tested the hypothesis that complement (C')-dependent release of prostaglandin (PG) I2 is an important factor contributing to the development of hypotension and low systemic vascular resistance index (SVRI) in endotoxic shock . Two groups (n = 7) of pentobarbital-anesthetized pigs (12-15 kg) were infused over 40 min with Escherichia coli lipopolysaccharide (LPS; 200 micrograms/kg) and continuously resuscitated with normal saline (1 ml/kg min): LPS-Control (no pretreatment) and LPS-Decomplemented (pretreatment 18 hr before study with 500-1,500 units of Naje haje cobra venom factor, CVF) . Prior treatment with CVF: i) decreased the mean titer of total hemolytic C' to 15.9% of pretreatment levels; ii) significantly decreased post-LPS plasma concentrations of immunoreactive TxB2 (TxA2 metabolite) and 6-keto-PGF1 alpha (PGI2 metabolite); iii) abrogated the early transient decrease in cardiac index observed in the LPS-Control group; iv) tended to improve post-LPS visceral perfusion assessed using radioactive microspheres; and v) had no discernible effect on the late sustained decrease in SVRI observed following infusion of LPS . We conclude that C' activation is a major determinant of LPS-induced prostanoid release in vivo, although our results do not support the view that C'-dependent release of PGI2 is an important factor contributing to low SVRI in resuscitated endotoxic shock.

J Infect Dis, 1989 Aug, 160(2), 243 - 7
Escherichia coli O114:nonmotile as a pathogen in an outbreak of severe diarrhea associated with a day care center; Bower JR et al.; Five infants from a day care center developed severe diarrhea associated with enteropathogenic Escherichia coli O114:nonmotile (EPEC O114:NM) and required hospitalization . Five additional cases of diarrhea associated with EPEC O114:NM subsequently occurred, four in hospital contacts of the patients and one in a household contact . Biochemically, all EPEC O114:NM isolates were sorbitol nonfermenters . All isolates produced low concentrations of cytotoxin with a mean of 10(1.23) CD50/mg of protein . Cytotoxin was not neutralized with antibody to Shiga-like toxin I or II . Heat-labile and heat-stable enterotoxins were not present by gene probe analysis . Stool isolates from 9 of 10 hospitalized infants were positive for EPEC adherence factor by colony blot DNA probe analysis . The severity of the disease, sorbitol nonfermentation, and presence of enteroadherence are unusual features of this organism.

Eur J Biochem, 1989 Aug 1, 183(2), 371 - 9
Iron metabolism of Escherichia coli studied by Mössbauer spectroscopy and biochemical methods; Matzanke BF et al.; To date it has barely been recognized that the nature of about 75% of the Escherichia coli iron pool is unknown . Here we report the isolation of two iron species representing major components of iron metabolism in various growth states of E . coli . In vivo Mossbauer spectroscopy was applied to obtain information on the intracellular distribution pattern of iron in E . coli K12 W3110 . Only two types of iron could be detected in the cell spectra: hexacoordinated Fe2+ and Fe3+ high-spin complexes . Other iron-requiring compounds are at least one order of magnitude less abundant in E . coli . The Mossbauer parameters of these complexes fit neither cytochromes nor iron-sulfur proteins nor ferric holo-bacterioferritin . They are sensitive to metabolic changes and inhibitors . The ratio of Fe/subunit, Fe2+/Fe3+ interconversion, chromatographic and electrophoretic data exclude bacterioferritin as the main iron metabolite in E . coli . Bacterioferritin can be observed only at very high ferric ion concentrations in the medium . The 55Fe fluorograms of both cytoplasmic and membrane fractions exhibit two exclusive bands with apparent molecular masses of 17 and 15 kDa, respectively . The two bands comprised 70% of the applied radioactivity . In gel filtration the main iron peak elutes at 155 kDa yielding two bands with apparent molecular masses of 17 and 15 kDa on SDS/PAGE . We therefore conclude that the iron species form a protein with an apparent molecular mass of 155 kDa containing 17-kDa and 15-kDa subunits . The iron content of the protein is 44 micrograms Fe/mg protein which corresponds to approximately 13 iron ions/subunit . No iron protein exhibiting the observed features has been described so far . Additional Mossbauer experiments suggest that these novel iron proteins are not restricted to E . coli but that similar components are detectable in several bacterial and fungal systems, thus pointing to a general occurrence.

Eur J Biochem, 1989 Aug 1, 183(2), 339 - 45
Modulation of arginine decarboxylase activity from Mycobacterium smegmatis . Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme; Balasundaram D et al.; Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity . The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa . The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation . The enzyme is specific for arginine (Km = 1.6 mM) . The holoenzyme is completely resolved in dialysis against hydroxylamine . Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature . The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths . A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations . The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme . Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function . Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively . These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.

Eur J Biochem, 1989 Aug 1, 183(2), 311 - 6
Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene; Harford S et al.; In vitro mutagenesis of the LTA gene, encoding the A subunit of the Escherichia coli heat-labile enterotoxin, has been used to obtain A subunits deficient in enzymic activity . One inactive A-subunit mutant which contained two amino acid substitutions, was shown to associate with native B subunits to form a holotoxoid lacking toxin activity . A serine to phenylalanine mutation appears to be responsible for the loss of toxicity.

Eur J Biochem, 1989 Aug 1, 183(2), 281 - 4
The third ribosomal tRNA-binding site, the E site, is occupied in native polysomes; Remme J et al.; The nucleic acids of Escherichia coli cells were uniformly labelled with 32P by growing the cells in {32P}orthophosphoric acid for about four generations . The cells were harvested in the logarithmic phase, resuspended in a buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37 degrees C in the presence of 3H-labelled amino acids . This procedure preferentially labels growing peptidyl chains . Polysomes were isolated, the fraction in the post-translocational state was assessed by a puromycin reaction and the tRNA content/70S ribosome was quantified in comparison to the amount of 5S rRNA determined after separation by gel electrophoresis . The data revealed that at least 75% of post-translocational ribosomes in isolated native polysomes carry a tRNA in their E site . The results are consistent with the allosteric three-site model for the elongation cycle but disagree with the two-site model.

Ann Surg, 1989 Aug, 210(2), 239 - 45
Peripheral blood leukocyte kinetics following in vivo lipopolysaccharide (LPS) administration to normal human subjects . Influence of elicited hormones and cytokines; Richardson RP et al.; Lipopolysaccharide (LPS, endotoxin) administration to human subjects elicits significant elevations in plasma cachectin/TNF, epinephrine, and cortisol . This study examined the temporal relationship between changes in blood leukocyte subsets and plasma mediators following endotoxin administration to normal human subjects . A five-minute intravenous infusion of purified LPS (20 units/kg Escherichia coli) was administered to 12 healthy volunteers . Blood samples were obtained at varying intervals after infusion and analyzed for differential cell counts and lymphocyte subsets (CD2, CD3, CD4, CD8, CD20, and HLA-DR) by flow microfluorimetry, and also assayed for plasma cachectin/TNF, epinephrine, and cortisol . Plasma cachectin/TNF was significantly elevated at 75 and 90 minutes after infusion with a peak concentration of 261 +/- 115 pg/ml noted 75 minutes after infusion . A significant plasma epinephrine elevation of 181 +/- 75 pg/ml was demonstrated one hour after infusion, while significant elevations in plasma cortisol were noted from one to five hours after infusion with a peak level of 34 +/- 3 micrograms/dl three hours after infusion . A profound monocytopenia (p less than 0.01) was noted one hour after infusion . Temporally associated with the rise in plasma cortisol was a reversal of the early granulocytopenia to a significant granulocytosis (p less than 0.01 versus preinfusion mean), whereas a marked lymphocytopenia (p less than 0.01) was observed from one to six hours after infusion . During the period of hypercortisolemia, CD2, CD3, and CD4 lymphocyte percentages were decreased (p less than 0.01) while CD20 and HLA-DR lymphocyte percentages were increased (p less than 0.01) . There was a small percentage decrease in CD8 lymphocytes from one to 24 hours after infusion (p less than 0.01), although relative to the one-hour nadir, there was a significant rise in the percentage during the time of elevated plasma cortisol concentrations . A six-hour infusion of epinephrine (30 ng/kg/min) administered to six healthy volunteers resulted in a monocytosis (p less than 0.05) and granulocytosis (p less than 0.01) without a change in lymphocyte number or lymphocyte subset percentage . Previous reports have shown that in vivo corticosteroid infusion causes a prominent granulocytosis, monocytopenia, and lymphocytopenia with a decrease in the percentages of CD3 and CD4 lymphocytes . The peripheral blood leukocyte dynamics documented in the current study are similar to patterns observed following in vivo corticosteroid administration . This study suggests that the acute adrenocortical response to endotoxemia primarily mediates the subsequent changes in leukocyte subsets.

J Bacteriol, 1989 Aug, 171(8), 4494 - 7
Saturation of mismatch repair in the mutD5 mutator strain of Escherichia coli; Damagnez V et al.; The mutD (dnaQ) gene of Escherichia coli codes for the proofreading activity of DNA polymerase III . The very strong mutator phenotype of mutD5 strains seems to indicate that their postreplicational mismatch repair activity is also impaired . We show that the mismatch repair system of mutD5 strains is functional but saturated, presumably by the excess of DNA replication errors, since it is recovered by inhibiting chromosomal DNA replication . This recovery depends on de novo protein synthesis.

J Bacteriol, 1989 Aug, 171(8), 4479 - 85
Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I; Weglenski P et al.; Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I (NRI), the product of glnG, were obtained by two different selection procedures . The mutant proteins were purified and characterized . The concentrations of mutant proteins needed to activate transcription at the glnAp2 promoter were three to four times lower than that of the wild-type NRI . The rate of phosphorylation of these proteins and the stability of mutant NRI phosphate were found to be similar to those of the wild-type NRI . In one of the mutants, the site of the mutation was localized in the DNA region specifying the central domain of NRI.

J Bacteriol, 1989 Aug, 171(8), 4457 - 65
Spermidine biosynthesis in Escherichia coli: promoter and termination regions of the speED operon; Xie QW et al.; Two enzymes, S-adenosylmethionine decarboxylase and spermidine synthase, are essential for the biosynthesis of spermidine in Escherichia coli . We have previously shown that the genes encoding these enzymes (speD and speE) form an operon and that the area immediately upstream from the speE gene is necessary for the expression of both the speE and speD genes . We have now studied the upstream promoter and the downstream terminator regions of this operon more completely . We have shown that the major mRNA initiation site (Ia) of the operon is located 475 base pairs (bp) upstream from the speE gene and that there is an open reading frame that encodes for a polypeptide of 115 amino acids between the Ia site and the ATG start codon for the speE gene . Downstream from the stop codon for the speD gene is a potential hairpin structure immediately followed by an mRNA termination site, t . An additional mRNA termination site, t', is present about 110 bp downstream from t and is stronger than t . By comparing our DNA fragments with those prepared from this region of the E . coli chromosome by Kohara et al., we have located the speED operon on the physical map of the E . coli chromosome . We have shown that the orientation of the speED operon is counterclockwise and that the operon is located 137.5 to 140 kbp (2.9 minutes) clockwise from the zero position of the E . coli chromosomal map.

J Bacteriol, 1989 Aug, 171(8), 4349 - 54
Sequence and overexpression of the menD gene from Escherichia coli; Popp JL; The menD gene of Escherichia coli codes for the first enzyme of menaquinone biosynthesis, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase . DNA sequence analysis of menD shows an open reading frame encoding a 52-kilodalton protein . Possible promoter and ribosome binding sites are present . Insertion of the menD gene into a tac promoter expression vector leads to nearly a 100-fold increase in the level of SHCHC synthase activity upon induction with isopropyl-beta-D-thiogalactoside (IPTG) . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of {35S}methionine-labeled proteins shows a 61-kilodalton protein produced upon induction of the menD-containing expression vector . This is the first reported sequence analysis of a men gene and the first significant amplification of any of the menaquinone biosynthetic enzymes.

J Bacteriol, 1989 Aug, 171(8), 4315 - 9
Isolation and mapping of Escherichia coli mutations conferring resistance to division inhibition protein DicB; Labie C et al.; Temperature-sensitive dicA mutants of Escherichia coli, dicA1(Ts), are blocked for cell division, owing to derepressed expression of a division inhibition gene, dicB . We isolated mutants which survived a high temperature in the dicA1 background and which survived induced expression of dicB carried by a high-copy-number plasmid . Most of the mutations conferred very slow growth on the cells . Two were mapped to the 90-min cluster of genes involved in translation and transcription, in or very close to gene rpoB . The majority of the other mutations were found to cause variable degrees of minicell formation and to map within or very close to the minB locus . Contrary to these mutations, the canonical min-1 mutation did not confer resistance to DicB.

J Bacteriol, 1989 Aug, 171(8), 4303 - 14
Actively replicating nucleoids influence positioning of division sites in Escherichia coli filaments forming cells lacking DNA; Mulder E et al.; The positioning of constrictions in Escherichia coli filaments pinching off anucleate cells was analyzed by fluorescence microscopy of dnaX(Ts), dnaX(Ts) sfiA, dnaA46(Ts), gyrA(Am) supF(Ts), and gyrB(Ts) mutants . In filaments with actively replicating nucleoids, constrictions were positioned close to the nucleoid, whereas in nonreplicating filaments, positioning of constrictions within the anucleate region was nearly random . We conclude that constriction positioning depends in an unknown way on nucleoid replication activity.

J Bacteriol, 1989 Aug, 171(8), 4241 - 7
Mercury operon regulation by the merR gene of the organomercurial resistance system of plasmid pDU1358; Nucifora G et al.; The structural basis for induction of the mercury resistance operon with inorganic mercury and with the organomercurial compound phenylmercuric acetate was addressed by DNA sequencing analysis and by lac fusion transcription experiments regulated by merR in trans from broad-spectrum-resistance plasmid pDU1358 (Hg2+ and phenylmercury responding) . The lac fusion results were compared with those from a narrow-spectrum-resistance (Hg2+ responding but not phenylmercuric responding) operon and the pDU1358 merR deleted at the 3' end . The nucleotide sequence of the beginning region of the broad-spectrum mer operon of plasmid pDU1358 was determined, including that of the merR gene, the operator-promoter region, the merT and merP genes, and the first 60% of the merA gene . Comparison of this sequence with DNA sequences of narrow-spectrum mer operons from transposon Tn501 and plasmid R100 showed that a major difference occurred in the 3' 29 base pairs of the merR gene, resulting in unrelated C-terminal 10 amino acids . A hybrid mer operon consisting of the merR gene from pDU1358, a hybrid merA gene (determining mercuric reductase enzyme), and lacking the merB gene (determining phenylmercury lyase activity) was inducible by both phenylmercury and inorganic Hg2+ . This shows that organomercurial lyase is not needed for induction by organomercurial compounds . A mutant form of pDU1358 merR missing the C-terminal 17 amino acids responded to inorganic Hg2+ but not to phenylmercury . Thus, the C-terminal region of the MerR protein of the pDU1358 mer operon is involved in the recognition of phenylmercury.

J Bacteriol, 1989 Aug, 171(8), 4207 - 16
Transcription mapping of the Escherichia coli chromosome by electron microscopy; French SL et al.; The distinctive double Christmas tree morphology of rRNA operons as visualized by electron microscopy makes them easy to recognize in chromatin spreads from Escherichia coli . On the basis of the pattern of nascent transcripts on nearby transcription units and the relative distances of the operons from one another and the replication origin, we are now able to specifically identify five of the seven rRNA operons in E . coli . The use of rRNA operons as markers of both position and distance has resulted in the morphological mapping of a significant portion of the E . coli chromosome; over 600 kilobase pairs in the 84- to 90-min and 72-min regions can now be recognized . Since individual rRNA operons could be identified, direct comparisons could be made of their transcriptional activities . As judged by the densities of RNA polymerases along the operons, rrnA, rrnB, rrnC, rrnD, and rrnE were all transcribed at similar levels, with one RNA polymerase every 85 base pairs . The ability to recognize individual operons and specific regions of the chromosome allows direct comparisons of various genetic parameters.

J Bacteriol, 1989 Aug, 171(8), 4170 - 7
Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis; Couto LB et al.; Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea . Following fixation of mutations in E . coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E . coli 6451 (recA Strr) . Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host . Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent . Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis . The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells . When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels . In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction . This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase . These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced . The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.

J Bacteriol, 1989 Aug, 171(8), 4112 - 20
Stabilization of the 3' one-third of Escherichia coli ribosomal protein S20 mRNA in mutants lacking polynucleotide phosphorylase; Mackie GA; Mutations which largely inactivate polynucleotide phosphorylase and which render RNase II thermolabile exert two effects on the metabolism of the two nested mRNAs which encode ribosomal protein S20 . (i) The lifetime of both mRNA species is extended 2.5-fold at 38 degrees C in a strain harboring both mutations . (ii) A relatively stable truncated fragment of these mRNAs accumulates to significant levels in strains lacking polynucleotide phosphorylase . The truncated RNA (Po RNA) is 147 to 148 residues long and is coterminal with the 3' ends of intact S20 mRNAs . Its 5' end appears to be generated by endonucleolytic cleavage to the 5' side of a G residue in the sequence AACCGAUC . The data are consistent with the hypothesis that S20 mRNAs can be degraded by alternative pathways . The normal pathway depends on functional polynucleotide phosphorylase and is concerted, since S20 mRNAs disappear without accumulation of detectable intermediates in the decay process . The slower alternative pathway is followed when polynucleotide phosphorylase is inactivated by mutation . This pathway is distinguished by segmental rather than concerted degradation of S20 mRNAs and involves at least one endonucleolytic cleavage . The 5' two-thirds of S20 mRNAs decays significantly more quickly than the 3' third in this latter mode of mRNA turnover.

Endocrinology, 1989 Aug, 125(2), 1097 - 9
Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor antibodies; Chang CS et al.; Complementary DNA segments that encode different domains of human and rat androgen receptors were fused to the Escherichia coli trpE gene using pATH expression vectors . Fusion proteins expressed by the bacteria were used to immunize rats and rabbits to obtain polyclonal antibodies to androgen receptors . Spleen cells of immunized rats were fused with myeloma cells to obtain stable hybridomas that produced monoclonal antibodies . Gradient centrifugation and immuno-precipitation assays indicated that the antibodies interacted with androgen receptors specifically.

Circ Res, 1989 Aug, 65(2), 502 - 14
Effects of recombinant human tumor necrosis factor alpha, lymphotoxin, and Escherichia coli lipopolysaccharide on hemodynamics, lung microvascular permeability, and eicosanoid synthesis in anesthetized sheep; Kreil EA et al.; We infused recombinant human tumor necrosis factor alpha (rhTNF alpha), lymphotoxin (rhLT), and Escherichia coli 0111:B4 lipopolysaccharide (LPS) into anesthetized sheep with a lung lymph fistula to compare their effects on systemic and pulmonary hemodynamics, lung lymph dynamics, and eicosanoid release . rhTNF alpha (25-150 micrograms/kg, n = 6 sheep), but not rhLT (25 micrograms/kg, n = 3), rapidly increased lung lymph and plasma levels of 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha) and caused profound systemic vasodilation and hypotension . Meclofenamate pretreatment (10 mg/kg) of three other sheep given 25 micrograms/kg rhTNF alpha prevented the increase of lymph and plasma 6-k-PGF1 alpha levels, systemic vasodilation, and the early (less than 2 hrs) but not the late (4-6 hours) hypotension caused by rhTNF alpha . LPS (1 micrograms/kg, n = 11) induced a briefer increase of lymph 6-k-PGF1 alpha levels than did rhTNF alpha while plasma 6-k-PGF1 alpha levels did not increase . LPS induced more gradual hypotension than did rhTNF alpha but did not cause systemic vasodilation . LPS and rhTNF alpha, but not rhLT, increased lymph thromboxane B2 (TXB2) levels during the first hour of study, whereas only LPS acutely increased plasma TXB2 levels . LPS caused acute pulmonary vasoconstriction and greater acute pulmonary artery hypertension than did either rhTNF alpha or rhLT . Whereas LPS-treated sheep required less fluid transfusion than rhTNF alpha-treated sheep to maintain mean systemic arterial pressure greater than 50 mm Hg, LPS infusion caused a greater increase of lung lymph protein clearance . rhTNF alpha caused minimal alterations of lung microvascular permeability . We conclude that eicosanoid mediators contribute importantly to differences of systemic and pulmonary hemodynamics caused by these agents in sheep . rhTNF alpha cannot account for all of the LPS-induced hemodynamic, lung lymph, and eicosanoid responses in sheep.

Cell Immunol, 1989 Aug, 122(1), 262 - 73
Molecular mimicry between uveitopathogenic site of retinal S-antigen and Escherichia coli protein: induction of experimental autoimmune uveitis and lymphocyte cross-reaction; Singh VK et al.; Experimental autoimmune uveitis (EAU) is caused by the immunization of microgram amounts of a soluble retinal protein, known as S-antigen, in susceptible animal strains including primates . The disease serves as an animal model of ocular inflammation . We induced EAU and pinealitis in Lewis rats with small synthetic peptides, corresponding to the amino acid sequence in Escherichia coli protein, which contains six consecutive amino acids identical to a uveitopathogenic site in human S-antigen (peptide M) . EAU and pinealitis induced in rats by synthetic peptide derived from E . coli was indistinguishable from those induced by native S-antigen or other uveitopathogenic synthetic peptides corresponding to the amino acid sequence of S-antigen . Furthermore, lymph node cells from animals immunized with either peptide M or peptide derived from E . coli protein showed significant proliferation in the presence of either peptide when tested in vitro for lymphocyte mitogenesis using {3H}thymidine . Thus, molecular mimicry, a process by which an immune response directed against a nonself protein cross-reacts with a normal host protein, may play a role in autoimmunity.

Infect Immun, 1989 Aug, 57(8), 2553 - 8
The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity; Pinkney M et al.; Site-directed mutagenesis of the TGC codon in a cloned streptolysin O (SLO) gene exchanged the single Cys residue in SLO for either Ala or Ser . The parent wild-type SLO (SLO.Cys-530) and the SLO.Ala-530 and SLO.Ser-530 mutant toxins, expressed in Escherichia coli, were purified and analyzed . Wild-type SLO.Cys-530 and the SLO.Ala-530 mutant showed no significant differences in their specific hemolytic activities, while the SLO.Ser-530 mutant had a reduced (ca . 25%), but still considerable, specific hemolytic activity as compared with that of wild-type SLO . The parent and mutant toxins extracted from lysed erythrocyte membranes had similar sedimentation profiles on sucrose density gradients, suggesting that the mutations did not affect the ability of SLO to form oligomers in membranes . These results show that the widely held assumption that the in vitro cytolytic activity of SLO requires an essential Cys residue is not true.

Acta Chir Scand, 1989 Aug, 155(8), 369 - 75
Effects of the beta-2 receptor agonist terbutaline on hemodynamics and gas-exchange in endotoxin shock; Sigurdsson GH et al.; The effects of the beta-2 receptor agonist, terbutaline, on hemodynamics and gas-exchange were evaluated in sheep exposed to endotoxin shock . Terbutaline was not given until signs of shock and lung injury had developed . Twenty sheep were anesthetized and ventilated without PEEP . After 90 min of stabilization (t = 0) all animals received E . coli endotoxin 10 micrograms/kg by i.v . infusion during 15 min . Thirty minutes later (t = 30) 10 animals (group TER) received i.v . infusion of terbutaline, 20 micrograms/kg/h, during 4 hours, while the other 10 served as controls (group S) . The endotoxin infusion resulted in marked increase in pulmonary artery pressure (PAP) and a significant decrease in mean arterial pressure (MAP), respiratory compliance, arterial oxygen tension (PaO2) and oxygen delivery index (DO2) in both groups (t = 15 and t = 30) . After 4 hours the PAP had decreased significantly in the terbutaline treated animals, but remained high in the controls (p less than 0.01) . Also, MAP, PaO2, DO2 and compliance improved significantly in the terbutaline treated animals . The wet to dry weight ratio of the lungs was 4.85 +/- 0.2 in the terbutaline treated and 5.35 +/- 0.5 in the controls (p less than 0.05) . It was concluded that terbutaline treatment improves gas-exchange and hemodynamics in sheep exposed to endotoxin shock.

Vet Microbiol, 1989 Aug, 20(4), 377 - 81
Evaluation of monoclonal antibodies to K88, K99, F41 and 987P fimbrial adhesins for the detection of porcine enterotoxigenic Escherichia coli in paraffin-wax tissue sections; Thorns CJ et al.; A panel of monoclonal antibodies against fimbrial adhesins of porcine enterotoxigenic Escherichia coli were evaluated for the detection of enteric colibacillosis in paraffin-wax embedded sections of piglet small intestine . Using the immunoperoxidase technique, monoclonal antibodies were used to detect epitopes on the K99 adhesin and on the a and c regions of the K88 adhesin . However, monoclonal antibodies to the F41 and 987P adhesins failed to react in sections with organisms colonising the intestine of gnotobiotic piglets monoinfected with strains bearing those adhesins, whereas corresponding polyclonal antisera gave positive results . In contrast to apparent expression of all K99 organisms, only a proportion of organisms were identified by monoclonal or polyclonal antibodies as expressing K88 . In some instances, failure of immunostaining was attributed to prolonged storage of tissue in formalin.

Am J Trop Med Hyg, 1989 Aug, 41(2), 220 - 3
Pilus colonization factors of enterotoxigenic Escherichia coli isolated from infantile diarrheal patients in Papua New Guinea; Honda T et al.; We examined the possibility of pilus colonization factor (or adhesin) production by 9 enterotoxigenic Escherichia coli isolated from infantile diarrheal patients in Papua New Guinea . By hydrophobicity, hemagglutination and bacterial agglutination with antisera against various known adhesins, none of the 9 strains produced known pilus adhesins . Three strains which produce heat-labile and heat-stable enterotoxins may produce a new type of pili which is not hydrophobic and differs from common (type 1) pili.

J Bacteriol, 1989 Aug, 171(8), 4281 - 9
Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells; Bilge SS et al.; A fimbrial adhesin, designated F1845, was found to be responsible for the diffuse HEp-2 cell adherence of a diarrheal Escherichia coli isolate . The genetic determinant of F1845 was cloned, and the order of the genes necessary for production of F1845 was determined by maxicell analysis . Five polypeptides with apparent sizes of 10, 95, 27, 15.5, and 14.3 kilodaltons (kDa) were found to be encoded in that order by the F1845 determinant . The nucleotide sequence of the 14.3-kDa subunit gene was determined and found to share extensive homology in its signal sequence with the gene encoding the structural subunit of the AFA-1 hemagglutinin of a uropathogenic E . coli strain (A . Labigne-Roussel, M.A . Schmidt, W . Walz, and S . Falkow, J . Bacteriol . 162:1285-1292, 1985) but not in the region encoding the mature protein . Southern blot hybridizations indicated that the F1845 determinants are of chromosomal origin . Hybridization studies using a probe from the region encoding the 95-kDa polypeptide indicated that related sequences may be plasmid associated in some strains and chromosomal in others . Additional hybridization studies of E . coli isolates possessing sequence homology to the F1845 determinant suggest that the sequences in the 5' region of the F1845 structural subunit gene are more highly conserved than sequences in the 3' region.

Infect Immun, 1989 Aug, 57(8), 2420 - 4
Influence of a glycine or proline substitution on the functional properties of a 14-amino-acid analog of Escherichia coli heat-stable enterotoxin; Waldman SA et al.; Analogs of Escherichia coli heat-stable enterotoxin (ST) differing in chain length or the presence of turn-forming residues were assessed for binding to receptors, activation of particulate guanylate cyclase, and stimulation of secretion in suckling mice . These analogs included the native 18-amino-acid peptide (ST), the 14-amino-acid carboxy terminus of this native peptide with a proline at position 12 (ST{5-18}proline), and the 14-amino-acid carboxy terminus in which the proline at position 12 was substituted with glycine (ST{5-18}glycine) . Each analog bound to the receptor in a dose-dependent fashion, completely displacing {125I}ST in competitive binding assays . However, their potencies differed significantly: ST demonstrated the highest affinity (inhibition constant {Ki}, 10(-9) M), followed by ST{5-18}proline (Ki, 10(-7) M) and ST{5-18}glycine (Ki, 10(-6) M) . Similarly, these peptides maximally activated particulate guanylate cyclase and stimulated intestinal secretion in suckling mice . Their rank order of potency in these assays was similar to that described for receptor binding: ST greater than ST{5-18}proline greater than ST{5-18}glycine . These data demonstrate that the full peptide structure is not absolutely required for pharmacological, biochemical, or biological activity . However, the four amino-terminal residues contribute significantly to the potency of these peptides . In addition, the turn imposed by the proline residue at position 12 is not absolutely required for receptor occupancy or activation of the biochemical cascade that results in intestinal secretion . However, it significantly increases the potency of the toxin . These data illustrate the importance of primary and secondary structures to the biochemical, pharmacological, and physiological activities of the ST produced by E . coli.

J Gen Microbiol, 1989 Aug, 135 ( Pt 8), 2319 - 28
Expression of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: export and processing of the apoprotein; Pollock WB et al.; The expression of cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) was examined in Escherichia coli transformed with either of two plasmids, pJ8 and pJ81 . The former has an 840 bp insert of D . vulgaris DNA, containing the structural gene for cytochrome c3 (387 bp) and its promoter region . Plasmid pJ81 was generated from pJ8 by deoxyoligonucleotide-directed mutagenesis to direct the synthesis of a protein with an altered signal peptidase cleavage site {Ala(-1)----Asp(-1)} . Synthesis of the 14 kDa precursor, which was partly processed to the 12 kDa mature protein, was observed in cells of E . coli TG2(pJ8) by SDS gel electrophoresis and Western blotting . Analysis of spheroplasts revealed that the processed polypeptide was present in the periplasm while the precursor was found only in the membrane/cytoplasmic fraction . No processing was observed in E . coli TG2(pJ81) cells, due to the mutation of the signal peptide cleavage site . No insertion of haem into the E . coli product could be detected in E . coli TG2(pJ8) cells by post-electrophoretic protohaem fluorescence analysis . The sensitivity of the cytochrome c3 synthesized in E . coli TG2(pJ8) to digestion by chymotrypsin also indicated that the apoprotein was formed . The results indicate that E . coli is capable of synthesizing and exporting the cytochrome c3 polypeptide, but fails to insert the haems.

J Biochem (Tokyo), 1989 Aug, 106(2), 274 - 81
Identification and characterization of inhibitory sequences in four repeating domains of the endogenous inhibitor for calcium-dependent protease; Kawasaki H et al.; We reported previously the cDNA cloning of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) and the expression of its fragments in Escherichia coli . The CANP inhibitor has four internal repeating domains each spanning about 140 amino acid residues . The inhibitory activity arises from these domains which have a well-conserved sequence, TIPPXYR, in their central positions . The inhibitory activities of various fragments expressed in E . coli suggest the involvement of the regions around the well-conserved sequences . In this report, we describe further detailed investigation on the interaction site of the CANP inhibitor with CANP by truncating inhibitor fragments and by using chemically synthesized peptides . The results clearly indicate that the sequence around the well-conserved sequence, TIPPXYR, is an interaction site . A peptide as short as 23 amino acid residues retained inhibitory activity, but a 9-residue peptide corresponding to the conserved sequence, VTIPPKYRE had none . The inhibitory sequence is suggested as LGXKDREXTIPPXYRXLL . The analysis of the competition between an inhibitor peptide and an irreversible inhibitor, E-64 for the reaction with the active site suggests no involvement of the active site cysteine residue of CANP in the inhibitory interaction between CANP and the CANP inhibitor . The high specificity of the CANP inhibitor to CANP arises from its interaction with residues other than the active site cysteine residue, possibly the subsite for substrate-binding of CANP.

Biochimie, 1989 Aug, 71(8), 903 - 15
The yeast ATP synthase subunit 4: structure and function; Velours J et al.; The structure of ATP synthase subunit 4 was determined by using the oligonucleotide probe procedure . This subunit is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass . Its relative molecular mass is 25 kDa . The ATP4 gene was isolated and sequenced . The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein 244 amino acids long . Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 kDa . Subunit 4 shows homology with the b-subunit of Escherichia coli ATP synthase and the b-subunit of beef heart mitochondrial ATP synthase . By using homologous transformation, a mutant lacking wild subunit 4 was constructed . This mutant is devoid of oxidative phosphorylation and F1 is loosely bound to the membrane . Our data are in favor of a structural relationship between subunit 4 and the mitochondrially-translated subunit 6 during biogenesis of F0.

Gene, 1989 Aug 1, 80(1), 21 - 8
Efficient cloning of a mutant adenylate-kinase-encoding gene from Escherichia coli; Liang P et al.; An optimized system has been developed for the transfer of a mutant gene from the Escherichia coli chromosome to a plasmid carrying the wild type (wt) allele . The wt allele was first cloned into a low-copy-number, self-transmissible plasmid with a single EcoRI, HindIII, and BamHI site . The plasmid was then transferred to a mutant strain that had been previously transformed with a high-copy-number plasmid carrying the recA+ gene to allow efficient homologous recombination . A 15% frequency of homogenotization was obtained during cloning of an adk gene that encodes a temperature-sensitive adenylate kinase (AK) . The mutant AK had decreased mobility on sodium dodecyl sulfate-polyacrylamide gels compared with the wt enzyme . This was due to a point mutation that changed leucine-107 in the wt enzyme to glutamine-107 in the mutant enzyme as determined by nucleotide sequencing.

EMBO J, 1989 Aug, 8(8), 2435 - 41
Evidence of a ter specific binding protein essential for the termination reaction of DNA replication in Escherichia coli; Kobayashi T et al.; Activity binding specifically to the 22 bp of the DNA replication terminus (ter) sequence on plasmid R6K and the Escherichia coli genome was detected in the crude extract of E . coli cells . This activity was inactivated by heat or by protease but not by RNase treatments . Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located . By mutagenesis of the 5.0 kb fragment on the plasmid with transposon Tn3 and subsequent replacement of the corresponding chromosomal region with the resulting mutant alleles, we isolated tau- mutants completely defective in ter binding activity . These mutants simultaneously lost the activity to block the progress of the DNA replication fork at any ter site, on the genome or the plasmid . It would thus appear that the ter binding protein plays an essential role in the termination reaction, at the ter sites.

EMBO J, 1989 Aug, 8(8), 2403 - 10
The Escherichia coli regulatory protein OxyR discriminates between methylated and unmethylated states of the phage Mu mom promoter; Bolker M et al.; Expression of the phage Mu mom gene is transcriptionally regulated by DNA methylation . Three GATC sites upstream of the mom promoter have to be methylated by the Escherichia coli deoxyadenosine methylase (Dam) to allow initiation of transcription . An E . coli dam strain was mutagenized with Tn5 in an attempt to isolate mutants which allow mom gene expression . Three independent Tn5 mutants were isolated, each mapped to a gene at 89.6 min which we designate momR . The wildtype gene was cloned and sequenced, it encodes a protein of 305 amino acids . The protein belongs to a group of related bacterial activators recently identified as the LysR family (Henikoff et al., 1988) . MomR protein was overproduced and purified . Expression of momR is autoregulated; MomR binds to a 43 bp region upstream of its coding sequence . In the mom promoter MomR protects a 43 bp region containing the three GATC sites . Specific binding to these sequences was observed only with unmethylated DNA . Fortuitously, we learned that MomR is identical to OxyR, a regulatory protein responding to oxidative stress . We discuss the implications of this control for Mu development.

Mol Gen Genet, 1989 Aug, 218(2), 358 - 60
Effect of base pair mismatches on recombination via the RecBCD pathway; Shen P et al.; The effect of base pair mismatches on recombination via the RecBCD pathway was studied in mutS and wild-type Escherichia coli, using substrates that contain single or multiple mismatches . Recombination between homologous DNA inserts in lambda phage and pBR322-derived plasmids forms phage-plasmid cointegrates that result from an odd numbers of crossovers . In the mutS host, when the sequence homology of a pair of 405 bp substrates decreased from 100% to 89%, the recombinant frequency decreased by about 9-fold, while in the wild-type host the decrease was about 240-fold . These results suggest that multiple mismatches can reduce recombinant frequencies by impeding the mechanism of recombination itself, and by provoking mismatch repair . Single mismatches in 31 bp substrates caused reductions in recombinant frequencies of 2- or 12-fold, depending on the location of the mismatch . However, unlike the reduction by multiple mismatches, the reduction of the recombinant frequencies by single mismatches was the same in both mutS and wild-type hosts . Thus a single mismatch is sufficient to impede recombination, and mismatch repair seems unable to act on single mismatches in very short homologies during recombination.

Mol Gen Genet, 1989 Aug, 218(2), 222 - 8
I elements of Drosophila melanogaster generate specific chromosomal rearrangements during transposition; Busseau I et al.; We report a detailed molecular analysis of three chromosomal rearrangements, which have been produced during I-R hybrid dysgenesis in Drosophila melanogaster . They all disrupt the yellow gene . One of them is a deletion; the other two are inversions, which may be interpreted as the results of recombination events between I elements inserted at their break points . These events appear to occur at the time of transposition and involve integrating rather than resident I elements . They are produced by a mechanism very similar to homologous ectopic recombination.

Cell Calcium, 1989 Aug-Sep, 10(6), 425 - 32
Alterations in binding of inositol 1,4,5-trisphosphate to subcellular structures of rat liver during chronic endotoxemia; Deaciuc IV et al.; Inositol 1,4,5-trisphosphate (IP3) binding to, and Ca2+ uptake and release by plasma membrane- and endoplasmic reticulum-enriched fractions of rat liver were measured after continuous Escherichia coli endotoxin (ET) administration in vivo . IP3 binding to both fractions was significantly reduced by ET treatment . This was associated with decreased Ca2+ uptake and impaired IP3-dependent Ca2+ release . A decrease of 5'-nucleotidase specific activity of plasma membrane-enriched fraction was also observed in ET treated rats . The results suggest that previously observed impairments in the ability of hepatocytes to mobilize Ca2+, to activate glycogen phosphorylase and to respond--when saponin permeabilized--by Ca2+ release upon IP3 addition during chronic endotoxemia are due to alterations in both IP3 binding to the subcellular fractions that are imputed to be targets of IP3, and a decrease in the size of IP3-sensitive pool of releasable Ca2+.

J Gen Virol, 1989 Aug, 70 ( Pt 8), 2037 - 49
Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein; Mason PW et al.; Expression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs) . All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge . We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA . To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E . coli following progressive Bal 31 exonuclease digestion of the 5' and 3' ends of the cDNA fragment . Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the 10 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid . Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs . Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6062 - 6
Recombination of knotted substrates by Tn3 resolvase; Droge P et al.; We studied the site orientation specificity for recombination by purified Tn3 resolvase . With standard plasmid substrates, resolvase acts only on directly repeated recombination sites . Knotting, however, makes inverted site substrates equally efficient . The structure of the knotted products of recombination shows that the DNA wrapped around resolvase in the synaptic intermediate has the same local geometry as the standard substrate but is reversed in topological sign . Similarly, the same strand exchange with the two substrates generates supercoils with opposite signs . Thus, DNA geometry rather than topology is critical for these features of recombination . The knotted inverse substrate like the direct site substrate must be (-) supercoiled under standard reaction conditions . However, under conditions in which supercoiling is not required, the structure of the knotted product is apparently the same . This indicates that the unique direction of strand exchange is determined by the structure of the synaptosome and not by (-) supercoiling of the substrate.

Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5908 - 12
Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda; Phadnis SH et al.; We constructed a derivative of transposon Tn5 called Tn5supF for insertion mutagenesis and sequencing DNAs cloned in phage lambda . This element carries a supF amber-suppressor tRNA gene . Its insertion into lambda can be selected by plaque formation by using nonsuppressing (sup0) Escherichia coli for amber mutant lambda phage and sup0 dnaB-amber E . coli for nonamber lambda phage . Tn5supF is just 264 base pairs long . It transposes efficiently and inserts quasi-randomly into DNA targets . The unique sequences near its termini can be used as primer binding sites for dideoxynucleotide DNA sequencing, thus permitting the direct sequencing of DNAs cloned in phage lambda without subcloning.

Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5683 - 7
Transmembrane signaling by a chimera of the Escherichia coli aspartate receptor and the human insulin receptor; Moe GR et al.; Since many receptors apparently contain only one or two membrane-spanning segments, their transmembrane topology should be similar . This feature suggests that these receptors share common mechanisms of transmembrane signaling . To test the degree of conservation of signaling properties, a chimeric receptor containing the ligand-binding extracellular domain of the Escherichia coli aspartate chemoreceptor and the cytosolic portion of the human insulin receptor was constructed . This chimeric receptor is active as a tyrosine kinase, and aspartate stimulates its activity . Some interesting differences are noted in the target proteins phosphorylated by the chimera compared to the wild-type insulin receptor . These results indicate that features of the signaling mechanisms used by these diverse receptors are conserved, but that interesting changes in the protein properties are caused by differences in the neighboring domains.

Mutat Res, 1989 Aug, 213(2), 149 - 56
Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3'----5' exonuclease (proofreading) activity; Isbell RJ et al.; Escherichia coli strains carrying the temperature-dependent dnaQ49 allele are strong mutators at 37 degrees C . Since the dnaQ49 gene encodes the epsilon subunit of DNA polymerase III, it is thought that the large number of errors results in part from impaired proofreading activity during DNA replication . We have examined dnaQ49-induced reversion patterns of defined trpA alleles to determine the kinds of errors produced by dnaQ49 at 30 degrees C and 37 degrees C . We found that at 37 degrees C dnaQ49 produced all types of base-pair substitutions in addition to frameshifts with transitions generally occurring more frequently than transversions . This generalized mutator activity is very similar to that displayed in rich medium by mutD5, another mutator allele at the dnaQ locus . However, when dnaQ49 strains were cultured at 30 degrees C, not only were reversion frequencies much lower than at 37 degrees C, but in addition, the spectrum was altered . Transversions became proportionally more prevalent in the reversion spectra at the lower temperature . We suggest the possibility that at 37 degrees C dnaQ49 results in defective proofreading and methyl-directed postreplicative mismatch repair, while at 30 degrees C mismatch repair is fully and proofreading partially restored.

J Cell Biol, 1989 Aug, 109(2), 593 - 605
Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis; Way M et al.; Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli . The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin . A number of deletion mutants have been expressed to explore the function of the three actin binding sites . Their design is based on the sixfold segmental repeat in the protein sequence . (These sites are located in segment 1, segments 2-3, and segments 4-6) . Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis . S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization . S4-6 binds a single actin monomer but only in calcium . These observations confirm and extend current knowledge on the properties of the two halves of gelsolin . Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites . S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment) . S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity . S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization . A model is presented for the involvement of the three binding sites in the various activities of gelsolin.

J Bacteriol, 1989 Aug, 171(8), 4498 - 500
Intracellular PPi concentration is not directly dependent on amount of inorganic pyrophosphatase in Escherichia coli K-12 cells; Kukko-Kalske E et al.; No correlation was observed between the level of inorganic pyrophosphatase (PPase) and the intracellular concentration of PPi in Escherichia coli cells . In exponentially growing cells the intracellular PPi concentration was in every case 1.5 nmol/mg (dry weight) or about 0.5 mM, even though the amount of PPase was varied from 15 to 2,600% of the control amount by mutation or by using a multicopy plasmid with an inserted gene (ppa) encoding PPase . The PPi concentration could, however, be increased or decreased from the control level under some stressful conditions.

J Bacteriol, 1989 Aug, 171(8), 4442 - 7
A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation; Skare JT et al.; We have developed a selection for mutations in a trpC-tonB gene fusion that takes advantage of the properties of the plasmid-encoded TrpC-TonB hybrid protein . The TrpC-TonB hybrid protein consists of amino acids 1 through 25 of the normally cytoplasmic protein, TrpC, fused to amino acids 12 through 239 of TonB . It is expressed from the trp promoter and is regulated by the trpR gene and the presence or absence of tryptophan . Under repressing conditions in the presence of tryptophan, the trpC-tonB gene can restore phi 80 sensitivity to a tonB deletion mutant, which indicates that TrpC-TonB can be exported and is functional . High-level expression of TrpC-TonB protein in the absence of tryptophan results in virtually immediate cessation of growth for strains carrying the trpC-tonB plasmid . By selecting for survivors of the induced growth inhibition (overproduction lethality), we have isolated a variety of mutations . Many of the mutations decrease expression of the TrpC-TonB protein, as expected . In addition, three independently isolated mutants expressing normal levels of TrpC-TonB protein result in a Gly----Asp substitution within the hydrophobic amino terminus of TonB . The mutant proteins are designated TrpC-TonBG26D . The mutations are suppressed by prlA alleles, known to suppress export (signal sequence) mutations . TrpC-TonB proteins carrying the Gly----Asp substitution accumulate in the cytoplasm . We conclude that the Gly----Asp substitution is an export mutation . TrpC-TonBG26D protein has been purified and used to raise polyclonal antibodies that specifically recognize both TrpC-TonB protein and wild-type TonB protein.

J Bacteriol, 1989 Aug, 171(8), 4362 - 9
Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli; Barbieri JT et al.; The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli . Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene . E . coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed . Extracts of E . coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity . Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E . coli recognized the pertussis toxin peptide leader sequence . The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E . coli leader peptidase correctly processed the pertussis toxin peptide leader sequence . Two single amino acid substitutions at residue 26 (C180I-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide . C180I-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide . In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction.

J Bacteriol, 1989 Aug, 171(8), 4290 - 7
Overproduction and identification of the ftsQ gene product, an essential cell division protein in Escherichia coli K-12; Storts DR et al.; ftsQ is an essential cell division gene in Escherichia coli . The ftsQ gene has been sequenced, and a presumptive open reading frame has been identified; however, no protein product has been observed (A.C . Robinson, D.J . Kenan, G.F . Hatfull, N.F . Sullivan, R . Spiegelberg, and W.D . Donachie, J . Bacteriol . 160:546-555, 1984, and Q.M . Yi, S . Rockenbach, J.E . Ward, Jr., and J . Lutkenhaus, J . Mol . Biol . 184:399-412, 1985) . The ftsQ gene was isolated on a 970-base-pair EcoRI-PvuII fragment of the E . coli chromosome and used to construct a trp-lac (Ptac) transcriptional fusion in plasmid pKK223-3 . The fused construct (pDSC78) complemented an ftsQ1(Ts) mutant strain in trans, restoring growth at 42 degrees C on low-salt medium . An ftsQ1(Ts) mutant strain transformed with pDSC78 appeared normal upon microscopic examination, with no indication of filamentation . The ftsQ gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis of radiolabeled, isopropyl-beta-D-thiogalactopyranoside-induced maxicell and normal cell extracts . As predicted from the nucleotide sequence, the 970-base-pair EcoRI-PvuII fragment encoded a polypeptide of approximately 31,400 daltons . Analysis of the data obtained from pulse-chase experiments in maxicells and normal cells suggests that the FtsQ protein is stable . Most of the radiolabeled FtsQ protein from maxicells was found in the inner membrane . On the basis of available information, the prior inability to detect FtsQ can be attributed to low levels of transcription or translation rather than to proteolysis.

J Bacteriol, 1989 Aug, 171(8), 4272 - 80
Expression of Escherichia coli dnaA and mioC genes as a function of growth rate; Chiaramello AE et al.; The synthesis of specific cellular components related to the initiation process of DNA replication was correlated with changes in growth rate . The concentrations of DnaA protein and mioC mRNA were determined for cells grown at six different growth rates; both increased relative to either total protein or total RNA, respectively, as the growth rate increased . Expression from the chromosomal mioC promoter, which contains a DnaA protein-binding site, was not repressed when the DnaA protein concentration was increased and was not derepressed in a dnaA46 mutant at 42 degrees C . The mioC transcript had a characteristic mRNA-type half-life of 1.51 min.

J Bacteriol, 1989 Aug, 171(8), 4248 - 53
A novel sigma factor is involved in expression of the rpoH gene of Escherichia coli; Wang QP et al.; The Escherichia coli rpoH gene encoding sigma 32, which is involved in the heat shock response, is transcribed from as many as four promoters . We have isolated a novel sigma factor of about 24 kilodaltons that allows core RNA polymerase to transcribe preferentially from one of these promoters, rpoH3p . This promoter is known to be regulated by DnaA protein . The sigma 24 factor was isolated from a preparation of RNA polymerase by electroelution from sodium dodecyl sulfate-polyacrylamide gels followed by renaturation . Expression of heat shock proteins is induced by treatments which include those that induce the stringent response . Under such conditions, decreased transcription from rpoH3p and no increase in transcription from other rpoH promoters were observed . This result suggests that induction of heat shock proteins by the stringent response is not mediated by increased transcription of the rpoH gene.

J Urol, 1989 Aug, 142(2 Pt 1), 399 - 402
Suppression of leukocyte function and intracellular content of ATP in hyperosmotic condition comparable to the renal medulla; Matsumoto T et al.; The effects of hyperosmolality induced by high concentrations of NaCl and urea on the functions of human polymorphonuclear leukocytes was examined . Phagocytosis, intracellular killing of bacteria and superoxide production were inhibited under hyperosmotic conditions . The mechanism of the inhibition of superoxide production was different for NaCl and urea . The inhibition by NaCl was indirect and abolished at 4C and the inhibition by urea was immediate, direct and not abolished at 4C . The intracellular ATP content of leukocytes under hyperosmotic conditions induced by NaCl was significantly reduced and this reduction was abolished at 4C . On the other hand, ATP content under such conditions induced with urea was not reduced . These results suggest that the inhibition of leukocyte function by NaCl is due to an exhaustion of energy source caused by an activation of Na-K pump, and the inhibition by urea is direct inactivation of leukocytes and not due to energy exhaustion.

Infect Immun, 1989 Aug, 57(8), 2481 - 8
Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis; Andersen AB et al.; Only a limited number of proteins from Mycobacterium tuberculosis have so far been shown to possess species-specific epitopes as defined by monoclonal antibodies . One such protein is protein antigen b (Pab) of molecular weight 38,000, which binds the monoclonal antibodies HYT 28, HAT 2, HBT 12, HGT 3, TB 71, and TB 72 . The gene encoding this protein was isolated from a lambda gt11 M . tuberculosis DNA library . The nucleotide sequence of the recombinant mycobacterial insert was determined, and an open reading frame of 374 amino acids was identified . The amino acid sequence exhibited 30% homology to a phosphate-binding protein, PstS, from Escherichia coli . The pab gene was subcloned into pBR322 in conjunction with the lacZ gene, and deletions were obtained from the 3' end . The anti-Pab monoclonal antibodies were used to probe crude protein lysates of E . coli transformed with the deletion plasmids . The monoclonal antibodies showed two reactivity patterns; one group of antibodies were dependent on the presence of the ultimate 91 amino acids of the protein, whereas another group of antibodies recognized an antigenic domain located on the middle portion of the molecule . None of the antibodies bound to the N-terminal 117-amino-acid peptide.

Yakugaku Zasshi, 1989 Aug, 109(8), 582 - 91
{In vitro replication of plasmid pKYM}; Okabe H et al.; The crude extract (fraction II) prepared from E . coli could replicate plasmid pKYM, only when the extract contained the rep protein which was produced by the plasmid and essential for its multiplication in vivo . The intermediate of replication was accumulated when a dideoxynucleotide triphosphate was added to the reaction mixture . By analyzing the intermediate, the initiation site of the deoxyribonucleic acid replication and the direction of replication could be determined . The replication initiated inside the ori region of pKYM and the direction was unidirectional . The analysis also suggested that the synthesis of the lagging strand stopped at almost the same site as the initiation site.

Biochem J, 1989 Aug 1, 261(3), 707 - 13
Delta-elimination in the repair of AP (apurinic/apyrimidinic) sites in DNA; Bailly V et al.; {5'-32P}pdT8d(-)dT7, containing an AP (apurinic/apyrimidinic) site in the ninth position, and {d(-)-1',2'-3H, 5'-32P}DNA, containing AP sites labelled with 3H in the 1' and 2' positions of the base-free deoxyribose {d(-)} and with 32P 5' to this deoxyribose, were used to investigate the yields of the beta-elimination and delta-elimination reactions catalysed by spermine, and also the yield of hydrolysis, by the 3'-phosphatase activity of T4 polynucleotide kinase, of the 3'-phosphate resulting from the beta delta-elimination . Phage-phi X174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: beta-elimination, delta-elimination, hydrolysis of 3'-phosphate, DNA polymerization and ligation . Spermine, in one experiment, and Escherichia coli formamidopyrimidine: DNA glycosylase, in another experiment, were used to catalyse the first and second steps (beta-elimination and delta-elimination) . These repair pathways, involving a delta-elimination step, may be operational not only in E . coli repairing its DNA containing a formamido-pyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites.

J Autoimmun, 1989 Aug, 2(4), 353 - 7
Molecular cloning of cDNAs expressing SS-B/La protein; Kohsaka H et al.; Using serum from a patient with Sjogren's syndrome containing a high titer of anti-SS-B/La antibody, cDNA clones (a representative clone was called pA158) were isolated from a human fibroblast cDNA library in lambda gt11 expression vector . After subcloning of pA158 cDNA into an expression plasmid vector pEX-2, a large amount of the recombinant fusion protein with cro-beta-galactosidase (called pA158EX) was obtained in E . coli culture containing the recombinant pEX-2 . Antibodies against pA158EX were purified from the patient serum by Sepharose 4B conjugated with the purified pA158EX protein . Immunofluorescent staining of HEp-2 cells with the anti-pA158EX antibodies showed a speckled nuclear staining . In immunoblot analysis, the anti-pA158EX antibodies reacted with 50 kDa protein that was compatible with SS-B/La protein . Immunoprecipitation of leukocyte lysate with the anti-pA158EX antibodies and the following RNA analysis showed that the antibody precipitated Y5 RNA . These findings indicate pA158 is a cDNA for SS-B/La protein . The purified fusion protein was used for enzyme-linked immunosorbent assay (ELISA) . Optical density values of anti-SS-B positive sera were high, but those of anti-SS-B negative sera and healthy donor sera were low . In the Northern blot using human RNA and pA158 cDNA, a single band about 1.8 kb was recognized . A full-length cDNA was further obtained by screening of pcD library using pA158 cDNA as a probe.

J Clin Invest, 1989 Aug, 84(2), 562 - 7
Role of antigen selectivity in autoimmune responses to the Ku (p70/p80) antigen; Reeves WH et al.; Levels of anti-Ku (p70/p80) antibodies were measured longitudinally in sera from four individuals with systemic lupus erythematosus or related disorders . Antibodies to the native Ku antigen (p70/p80 complex) varied over a range of up to 577-fold . Large fluctuations were also observed in the levels of autoantibodies to several distinct epitopes of the Ku (p70/p80) antigen . Levels of these individual autoantibody populations generally paralleled one another, suggesting that they are coordinately regulated . A similar pattern of anti-DNA antibody fluctuation was seen in some sera . To examine the possibility that these autoantibodies were generated by polyclonal B cell activation, the levels of anti-Ku (p70/p80) and anti-DNA antibodies were compared to the levels of antibodies to Escherichia coli proteins, tetanus toxoid, and bovine insulin, transferrin, cytochrome c, serum albumin, and thyroglobulin . In sera from the same individual, anti-Ku (p70/p80) antibodies were sometimes produced in the complete absence of polyclonal activation, and at other times were accompanied by increased polyclonal activation . Anti-DNA antibody levels more closely paralleled the level of polyclonal activation than did the anti-Ku (p70/p80) levels . These studies suggest that anti-Ku (p70/p80) antibodies are generated by an antigen-selective mechanism, but that polyclonal activation frequently, although not invariably, accompanies autoantibody production . This observation is consistent with the possibility that polyclonal activation might be secondary to autoantibody production.

J Bacteriol, 1989 Aug, 171(8), 4417 - 24
Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump; Shirvan MH et al.; The primary extrusion of Na+ from Mycoplasma gallisepticum cells was demonstrated by showing that when Na+-loaded cells were incubated with both glucose (10 mM) and the uncoupler SF6847 (0.4 microM), rapid acidification of the cell interior occurred, resulting in the quenching of acridine orange fluorescence . No acidification was obtained with Na+-depleted cells or with cells loaded with either KCl, RbCl, LiCl, or CsCl . Acidification was inhibited by dicyclohexylcarbodiimide (50 microM) and diethylstilbesterol (50 microM), but not by vanadate (100 microM) . By collapsing delta chi with tetraphenylphosphonium (200 microM) or KCl (25 mM), the fluorescence was dequenched . The results are consistent with a delta chi-driven uncoupler-dependent proton gradient generated by an electrogenic ion pump specific for Na+ . The ATPase activity of M . gallisepticum membranes was found to be Mg2+ dependent over the entire pH range tested (5.5 to 9.5) . Na+ (greater than 10 mM) caused a threefold increase in the ATPase activity at pH 8.5, but had only a small effect at pH 5.5 . In an Na+-free medium, the enzyme exhibited a pH optimum of 7.0 to 7.5, with a specific activity of 30 +/- 5 mumol of phosphate released per h per mg of membrane protein . In the presence of Na+, the optimum pH was between 8.5 and 9.0, with a specific activity of 52 +/- 6 mumol . The Na+-stimulated ATPase activity at pH 8.5 was much more stable to prolonged storage than the Na+-independent activity . Further evidence that two distinct ATPases exist was obtained by showing that M . gallisepticum membranes possess a 52-kilodalton (kDa) protein that reacts with antibodies raised against the beta-subunit of Escherichia coli ATPase as well as a 68-kDa protein that reacts with the anti-yeast plasma membrane ATPases antibodies . It is postulated that the Na+ -stimulated ATPases functions as the electrogenic Na+ pump.

FASEB J, 1989 Aug, 3(10), 2164 - 78
A perspective of the binding change mechanism for ATP synthesis; Boyer PD; An overview of research in the field of bioenergetics that led to the development of the binding change mechanism for ATP synthesis is presented, with emphasis on research from the author's laboratory . The text follows closely the Rose Award Lecture given at the 1989 meeting of the American Society for Biochemistry and Molecular Biology . Remarkable advances have revealed that the ubiquitous membrane-bound ATP synthase has unusual composition and properties . The enzyme complex has 1, 2, 3, or 9-12 copies of eight or more protein subunits . The catalytic sites are located on three copies of an approximately 55-kDa subunit . It has the strongest positive catalytic cooperativity known for any enzyme . Examples are given of selected experimental results that have provided insights into its mechanism . These include demonstration of the characteristics, location, and function of catalytic and noncatalytic adenine nucleotide binding sites and the incisive information provided by measurement of phosphate oxygen exchanges and distribution of 18(O) in ATP or Pi formed by catalysis . Research from various laboratories gives support to the binding change mechanism in which energy from proton translocation serves principally to promote release of tightly bound ATP, with sequential participation of three catalytic sites . Some speculative suggestions about a rotational catalysis and about the different forms assumed by the ATPase are included.

Plant Mol Biol, 1989 Aug, 13(2), 139 - 50
Isolation, characterization and sequence analysis of a full-length cDNA clone encoding NADH-dependent hydroxypyruvate reductase from cucumber; Greenler JM et al.; A full-length cDNA encoding NADH-dependent hydroxypyruvate reductase (HPR), a photorespiratory enzyme localized in leaf peroxisomes, was isolated from a lambda gt11 cDNA library made by reverse transcription of poly(A)+ RNA from cucumber cotyledons . In vitro transcription and translation of this clone yielded a major polypeptide which was identical in size, 43 kDA, to the product of in vitro translation of cotyledonary poly(A)+ RNA and subsequent immunoprecipitation with HPR antiserum . Escherichia coli cultures transformed with a plasmid construct containing the cDNA insert were induced to express HPR enzyme activity . RNA blot analysis showed that HPR transcript levels rise significantly in the first eight days of light-grown seedling development . This closely resembles the pattern seen for HPR-specific translatable mRNA . DNA blot analysis indicated that a single HPR gene is likely present per haploid genome . Nucleotide sequence analysis revealed an open reading frame of 1146 bases which encodes a polypeptide with a calculated molecular weight of 41.7 kDa . The derived amino acid sequence from this open reading frame is 26% identical and 50% similar to the amino acid sequence of the E . coli enzyme phosphoglycerate dehydrogenase, which catalyzes a similar reaction and functions in a related pathway . Statistical analyses show that this similarity is significant (z greater than 10) . The derived amino acid sequence for HPR also contains the characteristics of an NAD-binding domain.

Jpn J Cancer Res, 1989 Aug, 80(8), 747 - 53
Diversity of cellular molecules in human cells detected by monoclonal antibodies reactive with c-myc proteins produced in Escherichia coli; Naoe T et al.; Six clones of monoclonal antibodies, MYC-1 to -6, were prepared by using two kinds of truncated c-myc proteins, p23 and p42, produced in Escherichia coli as immunogens . Analysis with enzyme-linked immunosorbent assays and immunoblotting assays with peptides produced in Escherichia coli showed that 5 clones of monoclonal antibodies, MYC-1 to -4 and -6, were reactive with c-myc protein encoded by exon 2 . The remaining one clone, MYC-5, was reactive with the portion of c-myc protein encoded by exon 3 . All monoclonal antibodies were also reactive with phosphorylated c-myc protein produced by insect cells infected by the baculovirus expression vector with the human c-myc gene . With immunoblotting assays using cellular lysates, MYC-1 and -3 detected bands at the levels of 58 kDa and 60 kDa, MYC-5 detected a band at 56 kDa and MYC-6 detected bands at 68 kDa and 75 kDa . All of these bands were detectable in nuclear extracts of HL-60 and Colo320, both of which have amplified c-myc genes, and also the extract of RmycYl which is the c-myc gene transfectant into 3Yl rat cells . None of them was detectable in peripheral blood mononuclear cells and 3Yl, both of which lacked activated c-myc genes . This indicates that these nuclear proteins are either c-myc gene products or molecules closely related to the c-myc gene . The remaining two clones, MYC-2 and -4, detected a band at the level of 85 kDa in cytoplasmic extracts of all the above-mentioned cells independent of the presence of the c-myc gene . This suggests that 85 kDa protein might be irrelevant to the c-myc gene . The 56 kDa protein was detectable by MYC-5 in phytohemagglutinin-stimulated peripheral blood mononuclear cells as well as leukemic cells of some patients.

Protein Eng, 1989 Aug, 2(8), 621 - 5
Site-directed mutagenesis at the active site Trp120 of Aspergillus awamori glucoamylase; Sierks MR et al.; Trp120 of Aspergillus awamori glucoamylase has previously been shown by chemical modification to be essential for activity and tentatively to be located near subsite 4 of the active site . To further test its role, restriction sites were inserted in the cloned A.awamori gene around the Trp120 coding region, and cassette mutagenesis was used to replace it with His, Leu, Phe and Tyr . All four mutants displayed 2% or less of the maximal activity (kcat) of wild-type glucoamylase towards maltose and maltoheptaose . Michaelis constants (KM) of mutants decreased 2- to 3-fold for maltose and were essentially unchanged for maltoheptaose compared with the wild type, except for a greater than 3-fold decrease for maltoheptaose with the Trp120----Tyr mutant . This mutant also bound isomaltose more strongly and had more selectivity for its hydrolysis than wild-type glucoamylase . A subsite map generated from malto-oligosaccharide substrates having 2-7 D-glucosyl residues indicated that subsites 1 and 2 had greater affinity for D-glucosyl residues in the Trp120----Tyr mutant than in wild-type glucoamylase . These results suggest that Trp120 from a distant subsite is crucial for the stabilization of the transition-state complex in subsites 1 and 2.

Mol Biochem Parasitol, 1989 Aug, 36(1), 19 - 28
The 86-kilodalton antigen from Schistosoma mansoni is a heat-shock protein homologous to yeast HSP-90; Johnson KS et al.; We report the sequence of a cDNA clone encoding an 86-kDa polypeptide antigen (p86) from Schistosoma mansoni . Fusion proteins made in Escherichia coli are recognized by human infection sera . The reading frame of this antigen is highly homologous to those of the large heat-shock proteins of Saccharomyces cerevisiae (HSP90) and Drosophila melanogaster (HSP83) . mRNA encoding p86 increases in response to heat shock of adult worms, as does HSP70 . Comparisons of the sequences of HSP70 and HSP83 homologues show that these two families of heat-shock proteins are not significantly related except for the last four amino acid residues, which are Glu-Glu-Val-Asp in every case . This sequence is not found at the carboxy terminus of any other protein in the current databases.

Gene, 1989 Aug 1, 80(1), 129 - 36
Synthesis and sequence-specific proteolysis of a hybrid protein (colicin A::growth hormone releasing factor) produced in Escherichia coli; Geli V et al.; DNA constructs coding for human growth hormone (hGH)-releasing factor (hGRF) preceded by the specific recognition sequence for the activated blood coagulation factor X (FXa), fused in frame to the N-terminal 172-amino acid residues of colicin A, have been expressed in Escherichia coli . The construct was placed under the control of the inducible caa promoter in an operon containing a downstream gene coding for the cell lysis protein, Cal . Induction resulted in excretion of only the processed colicin A fragment . Replacement of Cal by the terminator from phage fd resulted in high expression of the hybrid protein, which was recovered as cytoplasmic aggregates . Enzymatic cleavage of the purified and renatured hybrid protein using FXa allowed the recovery of authentic hGRF.

Mol Gen Genet, 1989 Aug, 218(2), 361 - 3
A locus involved in kanamycin, chloramphenicol and L-serine resistance is located in the bglY-galU region of the Escherichia coli K12 chromosome; Lejeune P et al.; Spontaneous mutants of Escherichia coli K12 displaying an increased level of the kanamycin resistance conferred by plasmid pGR71 were selected . Several mutants obtained in this way apparently carry large chromosomal deletions extending into galU and/or bglY (27 min) . This positive selection of deletions allowed detection of a new locus located between galU and bglY . Deletions of this locus are responsible for increased resistance to kanamycin (Irk), decreased resistance to L-serine in minimal medium (Drs) and decreased resistance to chloramphenicol (Drc) when a cat gene is present in the bacteria.

Exp Cell Res, 1989 Aug, 183(2), 319 - 25
Expression of a mouse metallothionein-Escherichia coli beta-galactosidase fusion gene (MT-beta gal) in early mouse embryos; Stevens ME et al.; We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli beta-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos . A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages . We observed staining indicative of exogenous beta-galactosidase activity in 5-17% of DNA-injected embryos assayed at preimplantation stages after 16-24 h treatment with ZnSO4 . Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.

Br J Ophthalmol, 1989 Aug, 73(8), 587 - 90
Recombinant interferon-gamma induces HLA-DR expression on human corneal epithelial and endothelial cells in vitro: a preliminary report; el-Asrar AM et al.; The effect of interferon-gamma on the expression in situ of major histocompatibility complex (MHC) products in human corneas was studied in vitro . Incubation for four days with 5 or 50 mg/l of Escherichia coli-derived recombinant human interferon-gamma resulted in the appearance de novo of MHC class II or HLA-DR antigens on variable numbers of corneal epithelial cells as well as on corneal endothelium, whereas it had no effect on the expression of MHC class I or HLA-ABC antigens . These results may help to explain the mechanism underlying the expression of HLA-DR antigens on corneal and limbal epithelium in various inflammatory eye diseases.

Virology, 1989 Aug, 171(2), 331 - 41
Factors affecting long-term stability of Moloney murine leukemia virus-based vectors; Xu L et al.; We have examined the long-term functional and structural stability of retroviral vectors in infected murine cells . We have used Moloney murine leukemia virus-based vectors expressing human HPRT, firefly luciferase (luc), and Escherichia coli beta-galactosidase (lacZ) as reporter genes, and the human HPRT and the transposon Tn5 neomycin resistance (neo) gene as selectable markers . All vectors, whether single or double gene, yielded both stable and unstable clones . Stability of the proviruses was dependent on a number of factors, including the nature of the infected cell, the reporter gene, the integration site of the provirus, the relative positions of the component genes in multigene vectors, and the presence or absence of selection pressure . Selection pressure was helpful, but not universally effective, in maintaining provirus structural and functional integrity . Reporter gene expression from an internal promoter was likely to be unstable with or without selection for an upstream, LTR-driven neo gene . In some clones, loss of proviral gene expression was accompanied by deletions, while other inactive clones retained an apparently intact provirus . In the latter clones, treatment with 5-azacytidine failed to reactivate the reporter genes, but superinfection with helper virus resulted in the reappearance of transmissible vector, indicating a reversible epigenetic mechanism for proviral shutdown . The design of effective retroviral vectors and their possible use in vivo will require further characterization of these determinants of provirus stability.

Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5728 - 32
Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library; Sastry L et al.; Efficient generation of catalytic antibodies is uniquely dependent on the exact nature of the binding interactions in the antigen-antibody complex . Current methods for generation of monoclonal antibodies do not efficiently survey the immunological repertoire and, therefore, they limit the number of catalysts that can be obtained . We are exploring methods to clone and express the immunological repertoire in Escherichia coli . As the essential first step, we present here a method for the establishment of a highly diverse heavy chain variable region library . Consequently, it should now be possible to express and recombine the heavy and light chain variable region fragments to generate a large array of functional combining portions of the antibody molecule . This technology may provide an alternative to the hybridoma methodology for accessing the monoclonal antibody specificity of the immune system.

J Leukoc Biol, 1989 Aug, 46(2), 169 - 74
Change of membrane fluidity of rat neutrophils accompanying Escherichia coli inoculation; Masuda M et al.; Membrane fluidity of rat neutrophils was studied following Escherichia coli inoculation, and characteristic changes were observed . Membrane fluidity was assessed by the excimer-forming lipid technique using pyrenedecanoic acid and flow cytometry and expressed as the fluorescence intensity ratios of excimer and monomer pyrenedecanoic acid (IE/IM ratio) . High IE/IM ratios indicated high membrane fluidity . The IE/IM ratio of rat neutrophils (0.50 +/- 0.048) increased after E . coli inoculation, reaching a maximum of almost 1.00 after 10-20 min and then returning to its starting value . Intravenous injection of heat-killed E . coli or E . coli-conditioned culture supernatants into rats induced a rapid increase of IE/IM ratios, which returned to initial levels after 20 min . The effect on membrane fluidity of in vitro neutrophil incubation with E . coli, heat-killed E . coli, or E . coli-conditioned culture supernatants was similar to that observed in vivo . Addition of 5 mM ethylenediaminetetraacetic acid (EDTA) did not affect neutrophil membrane fluidity . Addition of either 5 micrograms/ml cytochalasin B or 10(-5) M colchicine did not directly affect neutrophil membrane fluidity but did block the change observed following incubation with bacteria.

Am J Respir Cell Mol Biol, 1989 Aug, 1(2), 89 - 93
Bleomycin induces the hsp 70 heat shock promoter in cultured cells; Moseley PL et al.; Bleomycin-induced lung disease is characterized by cell injury followed by fibroblast proliferation . Cells respond to injury by synthesizing a family of heat shock proteins . These proteins are critical to cell survival, and those of the 70,000 MW group (hsp 70) are essential for cell division and proliferation . To evaluate the effect of bleomycin on heat shock gene expression, we transfected a gene construct containing the hsp 70 heat shock gene promoter into fibroblasts . Doses of bleomycin, which have previously been shown to augment lung fibroblast proliferation, induce the hsp 70 heat shock promoter in the transfected cells . Bleomycin did not induce the expression of a non-hsp promoter placed in cells as a control of nonspecific gene activation . These observations suggest that bleomycin exposure may cause significant alterations in important DNA promoter regions such as the hsp 70 promoter and point to new ways to assess bleomycin-induced changes in cells.

Gene, 1989 Aug 1, 80(1), 13 - 9
Murine monoclonal antibodies which recognize active sites of Escherichia coli NusA protein and epitope mapping by gene fusion; Nakamura Y et al.; Seven hybridomas producing murine monoclonal antibodies reactive against NusA protein of Escherichia coli were prepared . Antigenic determinants of these monoclonal antibodies have been mapped by immunoblotting analyses using fusion proteins containing parts of NusA . The epitope of the N14 antibody maps in a hydrophobic amino acid (aa) cluster and consists of at least Ala-181 and Ser-183 residues . nusA1 and nusA11 mutations, which cause aa changes of these residues, abolish the antigenic reactivity to the N14 antibody . These antibodies react with intact NusA protein, indicating that the epitopes are exposed on the surface of NusA . Most of these epitopes cluster around the nusA1 and nusA11 mutation loci.

Agents Actions, 1989 Aug, 28(1-2), 103 - 7
Vasodepression ex vivo after administration of inflammatory mediators in vivo in the rat; Bekemeier H et al.; When rats were pretreated by intraplantar or i.v . injection of various inflammatory mediators, the vasopressor effect of i.a . norepinephrine in the subsequently isolated perfused hindlegs of the rats was found to be partly depressed . This vasodepression could also be detected if mediators were directly co-perfused in the isolated hindlegs . The vasodepressor effect was strongest following histamine pretreatment and co-perfusion, respectively, whereas endotoxin 0111:B4, PAF-acether, PGE1, and LTD4 were less effective . Only weak or no vasodepression could be induced by bradykinin, serotonin, lysolecithin, and substance P (1-11) . The significance of the results is discussed with respect to anaphylactic disorders.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6283 - 7
Immunogenicity of peptide fusions to hepatitis B virus core antigen; Stahl SJ et al.; Several gene fusions have been constructed in which coding sequences for antigenic regions of the pre-S sequences of hepatitis B virus, hepatitis B surface antigen, and the envelope protein of human immunodeficiency virus were linked to the 3' end of that for the first 144 residues of hepatitis B core antigen . The sequences were expressed efficiently in Escherichia coli to give stable products that assembled to form particles morphologically similar to hepatitis B core antigen itself . The products exhibited the antigenic and immunogenic characteristics of both the hepatitis B core antigen epitopes and the epitopes carried by the additional sequences, thus illustrating the value of such proteins as immunological reagents and potential vaccines.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6235 - 9
Structure and coding properties of Bs1, a maize retrovirus-like transposon; Jin YK et al.; We have sequenced Bs1, an insertion element isolated from a null allele of the Adh1 locus encoding alcohol dehydrogenase in maize . The Bs1 element is 3203 base pairs (bp) in length, has 302-bp identical long terminal direct repeats (LTRs), and created a 5-bp flanking direct duplication of target Adh1 DNA upon insertion . The 5' LTR is followed by a canonical primer binding site with homology to the plant initiator methionyl-tRNA, and the 3' LTR is directly preceded by a polypurine stretch like that observed in retroviruses and retrotransposons . Bs1 encodes two overlapping open reading frames specifying peptides of 740 and 168 amino acids . The longer open reading frame specifies a peptide with amino acid homology to the protease and nucleic acid binding moiety of retroviruses and retrotransposons . The deduced amino acid sequence encoded by Bs1 lacks convincing homology to the polymerase (reverse transcriptase) encoded by retroposons, despite the fact that this polymerase-encoding domain is routinely the most conserved region of any such element . The sequence and relatively small size of Bs1 suggest that this element is a deleted retrotransposon that inserted into Adh1 with the aid of a reverse transcriptase function provided in trans . In vitro transcribed Bs1 complementary RNA was translated in vitro to produce both a protein of 81 kDa representing open reading frame 1 (ORF1) and one of the 95-kDa size predicted for the frame-shifted fusion of ORF1 and ORF2 . As with many other retroposons, the efficiency of translational initiation at the AUG beginning ORF1 was not noticeably affected by the presence of one or more upstream, unproductive AUGs in the complementary RNA transcript.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6176 - 80
Large aqueous channels in membrane vesicles derived from the rough endoplasmic reticulum of canine pancreas or the plasma membrane of Escherichia coli; Simon SM et al.; Voltage clamp conditions were used to study the membrane permeability properties of rough microsomes (RM) derived from the rough endoplasmic reticulum of canine pancreas and inverted vesicles (InV) derived from the plasma membrane of Escherichia coli . Membrane vesicles of RM or InV were fused to a planar lipid bilayer that was formed in a hole of a partition separating two chambers . Fusion of a single RM vesicle yielded a single-step conductance increase . Some preparations yielded unitary conductances of 20, 55, 80, and 115 pS in 45 mM potassium glutamate . These channels were largely open at negative membrane potential on the cytoplasmic side of the RM membrane, mostly closed at positive voltages, permeable to amino acids, and slightly more selective for anions than cations . There was a dramatic increase in the number of open channels when 100 microM GTP was added to the cytoplasmic side of the fused RM, whereas 100 microM guanosine 5'-{gamma-thio}triphosphate caused closing of channels . ATP had no effect . A large channel of 115 pS at 45 mM potassium glutamate was also detected after the fusion of InV . As both RM and InV share the ability to translocate secretory proteins, it is possible that the 115-pS channel in both membranes represents a protein-conducting channel.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6143 - 7
In-plane phase transition of an integral membrane protein: nucleation of the OmpF matrix porin rectangular polymorph; Dorset DL et al.; A hexagonal polymorph (a = 79 A) of OmpF matrix porin from Escherichia coli spontaneously transforms to a rectangular form (a = 79 A, b = 137 A) after several months' storage in the refrigerator . Nucleation of this second polymorph is first disclosed by diffuse streaks in electron diffraction patterns or in computer-generated Fourier transforms of electron microscope images . With time, this streaking is resolved as an apparent superlattice, and eventually domains of orthorhombic polymorph are detected in the parent hexagonal lattice that can be oriented in either of three directions, depending on the polarity of the orthorhombic crystal growth . Models for this phenomenon based on protein trimer rotation successfully explain the progress of the phase transition and, if protein-protein interactions are the most important interactions between adjacent trimers in the lipid matrix, the transition is quite similar to what occurs with molecular crystals.

J Bacteriol, 1989 Aug, 171(8), 4105 - 11
A novel mutation, cog, which results in production of a new porin protein (OmpG) of Escherichia coli K-12; Misra R et al.; A mutant of Escherichia coli K-12 which produces a new outer membrane protein, OmpG, was isolated and genetically and biochemically characterized . The presence of OmpG allows growth on maltodextrins in the absence of the LamB maltoporin . The data obtained from in vivo growth and uptake experiments suggested that the presence of the OmpG protein results in an increase in outer membrane permeability for small hydrophilic compounds . In light of these findings, we suggest that OmpG is a porinlike protein . The mutation which results in the expression of OmpG has been termed cog (for control of OmpG) and mapped to 29 min on the E . coli chromosome . Diploid analysis shows that the mutant cog-192 allele is recessive for both the Dex+ and OmpG+ phenotypes . We propose that the cog mutation destroys a negative regulatory function and therefore derepresses ompG expression.

J Virol, 1989 Aug, 63(8), 3416 - 22
Semliki Forest virus E2 envelope epitopes induce a nonneutralizing humoral response which protects mice against lethal challenge; Grosfeld H et al.; Along the 422 amino acids of the Semliki Forest virus (SFV) E2 envelope glycoprotein, we identified 13 peptide cassettes (ranging in size from 15 to 25 amino acids and designated A through N) that contain hydrophilic sequences flanked by amino acid sequences conserved in the E2 envelopes of the alphavirus family . Six peptide blocks containing either a single cassette or two to three contiguous cassettes (A, BC, DE, FG, HIK, and LMN) were produced in Escherichia coli as recombinant proteins fused to the N terminus of beta-galactosidase . All of the SFV E2 recombinant polypeptides except A-beta-galactosidase were recognized on Western blots (immunoblots) by anti-SFV polyclonal antisera . In addition, these five recombinant proteins induced in mice antibodies that interacted specifically with SFV E2 protein on Western blots as well as with the intact virions in an enzyme-linked immunosorbent assay . The six hybrid proteins were used to vaccinate mice and were tested for the ability to confer resistance against lethal doses of SFV . Peptides BC and HIK, located at amino acid positions 114 to 149 and 216 to 288, respectively, of E2, protected partially (40 to 60%) against SFV challenge . A third peptide, LMN, located between amino acid positions 289 and 352, rendered mice totally resistant to an SFV challenge of 250 50% lethal doses . The partially protective effects of the BC and HIK cassettes and the high efficacy of the LMN cassette were consistently demonstrated, independent of the adjuvant (complete Freund or alum), immunization protocol, and strain of mice used . None of the antisera raised against any given cassette could neutralize the virus in an in vitro tissue culture assay or in a plaque reduction neutralization test . Nevertheless, passive transfer experiments demonstrated that in the case of LMN, the protective effect was mainly of a humoral nature.

Dev Biol, 1989 Aug, 134(2), 462 - 72
The S-locus specific glycoproteins of Brassica accumulate in the cell wall of developing stigma papillae; Kandasamy MK et al.; Self-incompatibility in Brassica oleracea is now viewed as a cellular interaction between pollen and the papillar cells of the stigma surface . In this species, the inhibition of self-pollen occurs at the stigma surface under the influence of S-locus specific glycoproteins (SLSG) . We used antibodies specific for a protein epitope of SLSG to study the subcellular distribution of these molecules in the stigmatic papillae . The antibodies have uncovered an interesting epitope polymorphism in SLSG encoded by subsets of S-alleles, thus providing us with useful genetic controls to directly verify the specificity of the immunolocalization data . Examination of thin sections of Brassica stigmas following indirect immunogold labeling showed that SLSG accumulate in the papillar cell wall, at the site where inhibition of self-pollen tube development has been shown to occur . In addition, the absence of gold particles over the papillar cell walls in the immature stigmas of very young buds, and the intense labeling of these walls in the stigmas of mature buds and open flowers, correlates well with the acquisition of the self-incompatibility response by the developing stigma.

FEBS Lett, 1989 Jul 31, 252(1-2), 139 - 43
The narK gene product participates in nitrate transport induced in Escherichia coli nitrate-respiring cells; Noji S et al.; The nucleotide sequence of the Escherichia coli narK gene, which is located in the upstream region of the narCHJI operon, was determined . The narK gene encodes a very hydrophobic protein with 463 amino acid residues (Mr 49,693) . A narK deletion mutant, under conditions for the induction of nitrate respiration, was unable to perform nitrate transport . Loss of transport activity was recovered by transforming the mutant with a narK+ plasmid . Thus, we conclude that the narK gene encodes a transmembrane protein participating in nitrate transport . In the narK promoter region, we defined a unique sequence that we designate as a 'nitrate box', functioning as a putative NarL-binding site, in addition to the consensus sequence of the 'anaero-box'.

Biochem Biophys Res Commun, 1989 Jul 31, 162(2), 892 - 9
Bluetongue virus-17 fusion protein ns1 expressed in Escherichia coli by pUC vectors; Wang LF et al.; The cDNA coding for the major nonstructural protein, NS1, of bluetongue serotype 17 (BTV-17) was cloned previously . Using pUC plasmids, we have successfully expressed the NS1 protein in Escherichia coli as a LacZ-NS1 fusion protein . The recombinant NS1 protein reacted with rabbit anti-BTV-17 antiserum, and was thus immunologically indistinguishable from the native BTV-17 NS1 protein . This was the first bluetongue viral protein to be produced in a bacterial system.

Biochem Biophys Res Commun, 1989 Jul 31, 162(2), 830 - 7
Mechanism of fibrin-specific fibrinolysis by staphylokinase: participation of alpha 2-plasmin inhibitor; Sakai M et al.; When the extent of plasminogen activation by staphylokinase (SAK) or streptokinase (SK) was measured in human plasma, SAK barely induced plasminogen activation, whereas SK activated plasminogen significantly . When the plasma was clotted with thrombin, the plasminogen activation by SAK was markedly enhanced, but that of SK was little enhanced . Similarly, in a purified system composed of plasminogen, fibrinogen and alpha 2-plasmin inhibitor (alpha 2-PI, alpha 2-antiplasmin), such a fibrin clot increased the activity of SAK significantly . However, when alpha 2-PI was removed from the reaction system, enhancement of the SAK reaction was not observed . In addition, SAK as distinct from SK, showed very little interference with the action of alpha 2-PI . Plasminogen activation by SAK is thus essentially inhibited by alpha 2-PI, but this reaction is not inhibited in fibrin clots . These results suggest that SAK forms a complex with plasminogen, which binds to fibrin and induces fibrinolysis.

Schweiz Med Wochenschr, 1989 Jul 29, 119(30), 1053 - 6
{Selective vitamin B 12 malabsorption in a 19-year-old patient}; Walser A et al.; Selective malabsorption of vitamin B12, or Imerslund-Grasbeck disease, is a rare congenital condition . The normal ileal uptake of intrinsic-factor-bound vitamin B12 is deficient . Inheritance is autosomal recessive . Megaloblastic anemia generally appears in infancy and is usually accompanied by mild proteinuria . The case is presented of a 19-year-old woman with megaloblastic anemia due to selective vitamin B12 malabsorption . As far as we know this is the only patient in whom the disease appeared so late . Of special interest are also the marginally low Schilling tests of the patient's parents . Further conditions which may lead to vitamin B12 deficiency are also discussed . A hypothesis is put forward concerning the late appearance of the disease in this particular case . Pathophysiologic, nephrologic and genetic aspects of selective vitamin B12 malabsorption are briefly reviewed.

Science, 1989 Jul 28, 245(4916), 404 - 7
The location of DNA in RecA-DNA helical filaments; Egelman EH et al.; The helical filament that the RecA protein of Escherichia coli forms around DNA is the active apparatus in protein-catalyzed homologous genetic recombination . The actual position of DNA within this complex has been unknown . Image analysis has been performed on electron micrographs of filaments of RecA on double-stranded DNA and on single-stranded DNA to visualize a difference that is consistent with one strand of the double-stranded DNA . This localization of the DNA gives additional information about the unusual structure of DNA in the complex with RecA protein.

Biochim Biophys Acta, 1989 Jul 27, 997(1-2), 1 - 8
Spin-labeled analogues of ATP, ADP and AMP: substitutes for normal nucleotides in biochemical systems; Ubom GA et al.; The different roles and effectiveness of adenosine monophosphate, diphosphate and triphosphate labeled at the 6 position of the purine ring with 2,2,6,6-tetramethylpiperidine-1-oxyl in reactions catalyzed by Escherichia coli glutamine synthetase (GS) have been investigated . Our results show that the spin-labeled ATP (Tempo-ATP) serves as a substrate in the glutamine synthesis reaction and in the adenylation of E . coli glutamine synthetase catalyzed by ATP: glutamine adenylyl transferase (ATase) with essentially the same effectiveness as normal ATP . In another reaction (gamma-glutamyltransferase), Tempo ADP serves as an effector with a Km of 9.4 . 10(-8) M compared to 1.2 . 10(-8) M for the normal ADP, while covalently bonded Tempo-AMP serves as a modifier on the catalytic properties of E . coli glutamine synthetase just as the covalently bonded normal AMP does . The dissociation constants between the labeled nucleotides, Mn2+, Mg2+ and Ca2+ are in the same order of magnitude as the binding constants for those cations and the corresponding normal nucleotides . Our findings indicate that the spin-labeled nucleotides are good substitutes for the normal nucleotides in the biochemical systems studied.

Biochemistry, 1989 Jul 25, 28(15), 6446 - 54
Structure of synthetic unmethylated 16S ribosomal RNA as purified RNA and in reconstituted 30S ribosomal subunits; Ericson G et al.; 16S ribosomal RNA was made by in vitro transcription of a cloned gene, and its structure was compared to authentic 16S ribosomal RNA . The comparison was made by subjecting the two types of 16S rRNA to chemical reagents that react specifically with unpaired bases and determining the extent of reaction by reverse transcription and gel electrophoresis of the cDNA . In solution, the rRNAs were indistinguishable in their pattern of reactivity, except for a difference at A1408 and many differences in the region between residues 470 and 562 . When the 16S rRNAs were reconstituted into 30S ribosomal subunits, these reactivity differences were absent . When the synthetic 16S rRNA was reconstituted into 30S subunits and then extracted and tested in solution, its pattern of chemical reactivity in the 470-562 region was the same as authentic 16S rRNA, but differences in the 1408-1410 region persisted . This study indicates that the synthetic 16S rRNA has a secondary structure in solution similar to a native secondary structure except in two regions, one apparently incorrectly folded during synthesis and the other in which nucleotides which are normally methylated in authentic 16S rRNA may be responsible for a structural difference.

Biochemistry, 1989 Jul 25, 28(15), 6310 - 7
Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli; Gollop N et al.; Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously . The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R{( 2KB}/{pyruvate}), was found to be 40 +/- 3 under a wide variety of conditions . Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme . At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration . On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions . In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids . 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate . However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism . The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 Jul 25, 28(15), 6269 - 75
Orientation, accessibility, and mobility of equilenin bound to the active site of steroid isomerase; Eames TC et al.; The fluorescent aromatic steroid equilenin, which contains a beta-naphthol moiety, is bound by 3-oxo-delta 5-steroid isomerase . The excitation and emission fluorescence spectra of equilenin when bound to the enzyme, as well as the fluorescence decay time, are indicative of ground-state ionization . In view of the high efficiency of tyrosine quenching, which approaches 100%, the beta-naphthol moiety of equilenin must be in proximity to all three tyrosines of steroid isomerase to account for the observed efficiency of radiationless energy transfer . From the observed response to an external quencher, it appears that enzyme-bound equilenin is largely shielded from solvent . Fluorescence anisotropy measurements indicate a high degree of immobilization of the bound ligand . These models are consistent with proposed models of the enzyme-substrate complex.

Biochemistry, 1989 Jul 25, 28(15), 6198 - 207
Discrimination between DNA sequences by the EcoRV restriction endonuclease; Taylor JD et al.; The EcoRV restriction endonuclease cleaves not only its recognition sequence on DNA, GATATC, but also, at vastly reduced rates, a number of alternative DNA sequences . The plasmid pAT153 contains 12 alternative sites, each of which differs from the recognition sequence by one base pair . The EcoRV nuclease showed a marked preference for one particular site from among these alternatives . This noncognate site was located at the sequence GTTATC, and the mechanism of action of EcoRV at this site was analyzed . The mechanism differed from that at the cognate site in three respects . First, the affinity of the enzyme for the noncognate site was lower than that for the cognate site, but, by itself, this cannot account for the specificity of EcoRV as measured from the values of kcat/Km . Second, the enzyme had a lower affinity for Mg2+ when it was bound to the noncognate site than when it was bound to its cognate site: this appears to be a key factor in limiting the rates of DNA cleavage at alternative sites . Third, the reaction pathway at the noncognate site differed from that at the cognate site . At the former, the EcoRV enzyme cleaved first one strand of the DNA and then the other while at the latter, both strands were cut in one concerted reaction . The difference in reaction pathway allows DNA ligase to proofread the activity of EcoRV by selective repair of single-strand breaks at noncognate sites, as opposed to double-strand breaks at the cognate site . The addition of DNA ligase to reactions with EcoRV made no difference to product formation at the cognate site, but products from reactions at noncognate sites were no longer detected.

Biochemistry, 1989 Jul 25, 28(15), 6150 - 6
Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta; Marion D et al.; The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) {15N}interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example . The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy . We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra . The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C alpha H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C alpha H(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom . Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence.

Nucleic Acids Res, 1989 Jul 25, 17(14), 5725 - 36
Isolation and characterization of the gene coding for Escherichia coli arginyl-tRNA synthetase; Eriani G et al.; The gene coding for Escherichia coli arginyl-tRNA synthetase (argS) was isolated as a fragment of 2.4 kb after analysis and subcloning of recombinant plasmids from the Clarke and Carbon library . The clone bearing the gene overproduces arginyl-tRNA synthetase by a factor 100 . This means that the enzyme represents more than 20% of the cellular total protein content . Sequencing revealed that the fragment contains a unique open reading frame of 1734 bp flanked at its 5' and 3' ends respectively by 247 bp and 397 bp . The length of the corresponding protein (577 aa) is well consistent with earlier Mr determination (about 70 kd) . Primer extension analysis of the ArgRS mRNA by reverse transcriptase, located its 5' end respectively at 8 and 30 nucleotides downstream of a TATA and a TTGAC like element (CTGAC) and 60 nucleotides upstream of the unusual translation initiation codon GUG; nuclease S1 analysis located the 3'-end at 48 bp downstream of the translation termination codon . argS has a codon usage pattern typical for highly expressed E . coli genes . With the exception of the presence of a HVGH sequence similar to the HIGH consensus element, ArgRS has no relevant sequence homologies with other aminoacyl-tRNA synthetases.

Nucleic Acids Res, 1989 Jul 25, 17(14), 5501 - 7
Translational reinitiation in the presence and absence of a Shine and Dalgarno sequence; Spanjaard RA et al.; The process of translational reinitiation in Escherichia coli was studied in a two cistron system where expression of the downstream reporter gene was dependent on translation of an upstream reading frame . The dependence was almost absolute . Upstream translation increased expression of the downstream gene by two to three orders of magnitude . This large difference allowed us to quantitate restarts in a meaningful manner . In the absence of a Shine and Dalgarno (SD) region reinitiation occurred but its efficiency was about 10% of that found in the SD carrying counterpart . We discuss three ways by which translational coupling between neighboring cistrons can be enforced.

J Biol Chem, 1989 Jul 25, 264(21), 12700 - 8
Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein; Lindberg G et al.; We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection . It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues . Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA . Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides . The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA . The binding involves displacement of cations from the DNA and anions from the protein . The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration . The degree of cooperativity (omega) is relatively independent of salt concentration . At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer . The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA . N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.

J Biol Chem, 1989 Jul 25, 264(21), 12695 - 9
In vitro requirements for N4 RNA polymerase II-specific initiation; Abravaya K et al.; The synthesis of coliphage N4 middle RNAs requires the activity of three phage-coded proteins, P7, P4, and P17, of molecular weights 40,000, 30,000, and 15,000, respectively . P4 and P7 constitute a heterodimeric RNA polymerase, N4 RNA polymerase II, which cannot transcribe or bind to native N4 DNA . Denatured templates are transcribed but without specificity . We present the development of an in vitro transcription system, composed of purified N4 RNA polymerase II and solubilized P17, which accurately utilizes in vivo sites of transcription initiation . Possible mechanisms for P17 action are discussed.

J Biol Chem, 1989 Jul 25, 264(21), 12520 - 5
Globotetraosylceramide is recognized by the pig edema disease toxin; DeGrandis S et al.; The pig edema disease toxin has been shown by a tlc glycolipid binding assay to bind specifically to globotetraosylceramide (Gb4, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer.) . Binding was reduced for globotriosylceramide (Gb3, Gal alpha 1-4Gal beta 1-4GlcCer) and more markedly for the Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer) . Paragloboside, blood group A glycolipids, glycolipids terminating in Gal NAc beta 1-4Gal-, and glycolipids in which globoside was present as an internal sequence did not bind the toxin . Isogloboside (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4GlcCer) was efficiently recognized . This toxin is genetically related to the verotoxin (or Shiga-like) family of toxins for which Gb3 has been shown to be the receptor . The difference in susceptibility of cell lines to the cytotoxicity of the pig edema disease toxin and the Shiga and Shiga-like toxins is consistent with the difference in receptor glycolipid binding.

J Biol Chem, 1989 Jul 25, 264(21), 12510 - 9
Lipid binding by Escherichia coli pyruvate oxidase is disrupted by small alterations of the carboxyl-terminal region; Grabau C et al.; Escherichia coli pyruvate oxidase is a membrane-associated flavoprotein dehydrogenase which is greatly activated by lipids and detergents . The carboxyl-terminal region of the protein has been shown to play a critical role in the interaction with lipids . We report mutations generated by chemical and oligonucleotide-mediated site-directed mutagenesis of the poxB gene which result in enzymes defective in lipid activation . Nine mutants were isolated which encode enzymes with point mutations in the carboxyl-terminal segment of the protein . Two mutant lesions introduced termination codons giving enzymes lacking the last nine or three amino acids of the protein which were unable to interact with detergents in vitro and were unable to function in vivo . Of the missense mutants isolated, two were most informative . One was the substitution of Glu-564 with proline in the PoxB16 oxidase . This residue lies in the center of a putative lipid-binding amphipathic alpha-helix (Arg-558 to Thr-568) located close to the carboxyl terminus . Strains producing the PoxB16 oxidase were devoid of oxidase activity in vivo, the enzymes could not be activated by Triton X-100, and were activated poorly by phospholipids in vitro . These results indicated that the PoxB16 oxidase lacked normal lipid-binding ability . Another mutant oxidase (PoxB15) in which proline was substituted for Asp-560 at the beginning of the amphipathic alpha-helix had normal oxidase activity . These findings indicate that the amphipathic alpha-helix structure plays an essential role in the activation and lipid-binding properties of the enzyme . The second informative missense mutation was the substitution of the carboxyl-terminal arginine with glycine . This enzyme showed normal activation in vitro by phospholipids and some detergents, and somewhat reduced activity in vivo . This mutant enzyme appeared to dissociate from detergent vesicles more readily than does the normal enzyme . A model for the membrane interaction of the carboxyl terminus based on the properties of these mutant proteins is presented.

J Biol Chem, 1989 Jul 25, 264(21), 12455 - 61
Phospholipid dependence of homogeneous, reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli; Scheideler MA et al.; A novel mixed micelle assay for the sn-glycerol-3-phosphate acyltransferase of Escherichia coli was developed using the nonionic detergent octaethylenegly-coldodecyl ether . The assay permitted investigation of the phospholipid dependence of enzyme activity at phospholipid/detergent ratios of 5:1 (w/w) to 2:1 depending on the phospholipid employed . The higher ratio yielded maximal activity when E . coli phospholipids were used; the lower ratio was observed with cardiolipin(E . coli) . Phosphatidylglycerol(E . coli) and phosphatidylethanolamine(E . coli) also restored enzyme activity . Activation by phosphatidylethanolamine(E . coli) was pH-dependent and relatively inefficient . The synthetic, disaturated (1,2-palmitoyl)phosphatidylglycerol reconstituted only 25% of the total enzyme activity as that observed with the monounsaturated (1-palmitoyl, 2-oleoyl) species . Full activation of enzyme was achieved with (1,2-dioleoyl)phosphatidylglycerol . Phosphatidylcholine and phosphatidic acid were unable to reconstitute enzyme activity . Chromatographic sizing of the sn-glycerol-3-phosphate acyltransferase, following reconstitution in cardiolipin(E . coli)/octaethyleneglycoldodecyl ether mixed micelles, suggested that the monomeric form of the enzyme was active.

J Biol Chem, 1989 Jul 25, 264(21), 12284 - 8
Regulation of basal level expression of the tryptophan operon of Escherichia coli; Roesser JR et al.; Mutations in prfB, encoding release factor 2 (UGA- and UAA-specific), increase transcription termination at the trp operon attenuator in Escherichia coli strains grown in the presence of tryptophan . The prfB mutations have no effect on basal level expression in strains in which the natural trp leader peptide stop codon UGA was replaced by either UAG or UAA . The effect of introducing prfB mutations into mutant strains containing altered trp leader regions that influence basal level transcription readthrough was also determined . Our findings support a model for basal level control of trp operon expression in which ribosome release from the leader peptide stop codon, formation of alternative transcript secondary structures, and the position of the transcribing RNA polymerase regulate expression.

J Biol Chem, 1989 Jul 25, 264(21), 12249 - 52
Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli; Aberg A et al.; The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues . In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis . The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol . The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site . Our classification of redox-active cysteines differs from the location of the active site cysteines in E . coli ribonucleotide reductase suggested previously (Lin, A.-N . I., Ashley, G . W., and Stubbe, J . (1987) Biochemistry 26, 6905-6909).

Biochemistry, 1989 Jul 25, 28(15), 6281 - 94
Partial unfolding of dodecameric glutamine synthetase from Escherichia coli: temperature-induced, reversible transitions of two domains; Shrake A et al.; Glutamine synthetase (GS), Mr 622,000, from Escherichia coli contains 12 active sites formed at heterologous interfaces between subunits {Almassy, R . J., Janson, C . A., Hamlin, R., Xuong, N.-H., & Eisenberg, D . (1986) Nature (London) 323, 304-309} . Temperature-induced changes in UV spectra from 3 to 68 degrees C were reversible with the Mn2+- or Mg2+-enzyme at pH 7.0 (50 degrees C) in 100 mM KCl . No dissociation or aggregation of dodecamer occurred at high temperatures . The thermal transition involves the exposure of approximately 0.7 of the 2 Trp residues/subunit (by UV difference spectroscopy) and 2 of the 17 Tyr residues/subunit (change in exposure from 4.7 to 6.7 Tyr/subunit by second-derivative spectral analysis) . Monitoring changes in Trp and Tyr exposure independently gives data that conform to a two-state model for partial unfolding with Tm values (where delta G unfolding = 0) differing by 2-3 degrees C at each level of {Mn2+} studied and with average delta HvH values of 80 and 94 kcal/mol, respectively . These observations suggest that two regions of the oligomeric structure unfold separately as independent transitions (random model) . However, the data can be fit equally with a sequential model in which the Trp transition occurs first upon heating . By fitting with either model, Tm values increase from approximately 47 to approximately 54 degrees C with increasing free {Mn2+} from 3.6 to 49 microM but decrease from approximately 54 to approximately 43 degrees C by further increasing free {Mn2+} from 0.05 to 10 mM; such behavior indicates that the high-temperature form of the enzyme binds Mn2+ more weakly but has more binding sites than the native enzyme . The high-temperature Mn-enzyme form is somewhat less unfolded than is the catalytically inactive apoenzyme, which undergoes no further Trp or Tyr exposure on heating and therefore is assumed to be the high-temperature form of divalent cation-free GS . Adding substrates {ADP, L-Met-(SR)-sulfoximine, Gln, Gln + NH2OH, or Gln + ADP} to Mn.GS increased Tm to varying extents by preferential binding to the folded form . Indeed, the transition-state analogue complex GS.(Mn2.ADP.L-Met-(S)-sulfoximine phosphate)12 was stable in the folded form to at least 72 degrees C . Moreover, an Arrhenius plot for gamma-glutamyl transfer activity was linear from 4 to 72 degrees C with Ea = 18.3 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1989 Jul 25, 28(15), 6145 - 50
G protein-effector coupling: interactions of recombinant inhibitory gamma subunit with transducin and phosphodiesterase; Griswold-Prenner I et al.; A bacterial expression vector for the inhibitory gamma subunit of retinal rod phosphodiesterase has been constructed by inserting a mouse gamma cDNA into pUC19 . Escherichia coli 222 transformed with this plasmid produces a 12-kDa recombinant protein consisting of 18 additional amino acids attached to the amino terminus of gamma . The fusion protein, designated beta-gal-gamma, has been refolded into an active form in formic acid and partially purified by gel filtration chromatography . Despite a large extended sequence at the amino terminus, beta-gal-gamma is able to inhibit the activity of trypsin-activated phosphodiesterase, bind tightly to the catalytic alpha beta subunits, and interact with the alpha subunit of transducin in a nucleotide-dependent manner . The availability of large quantities of active bacterial gamma, together with the ability to change its primary structure by site-directed mutagenesis, promises to provide considerable new information on the interaction between transducin and phosphodiesterase, as well as insights into the molecular mechanism of G protein-effector coupling.

Nucleic Acids Res, 1989 Jul 25, 17(14), 5517 - 27
Up-promoter mutations in the positively-regulated mer promoter of Tn501; Lund P et al.; Transcription from the mer promoter of transposon Tn501 is repressed by MerR (the product of the merR gene) in the absence of Hg2+, and activated by MerR in the presence of Hg2+ . In the absence of MerR, the mer promoter has weak constitutive activity . The DNA sequence of the mer promoter shows candidate -35 and -10 sequences at the unusually high spacing of 19 base-pairs . We have selected for spontaneous mutations in the mer promoter that confer an up-promoter phenotype . Four different mutants have been isolated . Three of these are single base-pair deletions between the -10 and -35 sequences . A fourth removes the -10 sequence entirely, and places a second potential -10 sequence 17 base-pairs from the -35 sequence . None of these mutant promoters are induced by MerR in the presence of Hg2+ . Two of them are repressed by MerR irrespective of the presence or absence of Hg2+ . Models for the mode of action of the MerR protein are discussed in the light of these results . Our data support a mechanism in which the MerR protein in the presence of Hg2+ acts to change the conformation of DNA in the mer promoter.

Biochemistry, 1989 Jul 25, 28(15), 6259 - 69
Bidirectional transcription footprinting of DNA binding ligands; White RJ et al.; An in vitro transcription assay has been developed to define the exact location of DNA binding ligands . The method employs two counterdirected Escherichia coli promoters separated by approximately 100 bp . Selective transcription from each promoter yields transcripts up to each ligand site . Nonsaturating levels of ligands result in fractional occupancy of ligand at each site, and hence a range of RNA transcript lengths . The bidirectional promoter system results in a transcription footprint which was derived from transcription from both promoters up to the 5' side of each occupied ligand site and defines the sequence specificity and binding site size of the DNA-bound ligand . The transcriptional footprint is precise to +/- 1 bp from the 5' and 3' ends of each binding site . Multiple ligand sites can be ranked in terms of relative fractional occupancy at each site, and the ranking is comparable from either transcription direction . The method was compared to classical DNase I footprinting with a series of DNA binding drugs {actinomycin D, echinomycin, bis(thiadaunomycin), mithramycin, nogalamycin, and an acridine-tripyrrole} . In all cases, specific binding sites were resolved more clearly by transcription footprinting than by DNase I footprinting . Because of the nature of the transcription assay, all occupied ligand sites were detected by this method, in contrast to DNase I footprinting where many sequences are not probed, and where ligand sites are often not accurately defined.

J Biol Chem, 1989 Jul 25, 264(21), 12717 - 29
Structural and enzymatic studies of the T4 DNA replication system . II . ATPase properties of the polymerase accessory protein complex; Jarvis TC et al.; In this paper we report a detailed enzymatic characterization of the interaction of the polymerase accessory protein complex of the T4 DNA replication system with the various nucleic acid cofactors that activate the ATPase of the complex . We show that the ATPase activity of the T4 coded gene 44/62 protein complex is stimulated synergistically by binding of DNA and T4 gene 45 protein and that the level of ATPase activation appears to be directly correlated with the binding of nucleic acid cofactor . Binding of any partially or completely single-stranded DNA to the complete accessory protein complex increases the catalytic activity (as measured by Vmax) while decreasing the binding affinity for the ATP substrate . While single-stranded DNA is a moderately effective cofactor, we find that the optimal nucleic acid-binding site for the complex is the primer-template junction, rather than single-stranded DNA ends as previously reported in the literature . Gene 45 protein plays an essential role in directing the specificity of binding to primer-template sites, lowering the Km for primer-template sites almost 1000-fold, and increasing Vmax 100-fold, compared with the analogous values for gene 44/62 protein alone . The most effective primer-template site for binding and enzymatic activation has the physiologically relevant recessed 3'-OH configuration and an optimal size in excess of 18 base pairs of duplex DNA . We find that the chemical nature of the primer terminus (i.e . 3'-OH or 3'-H) does not affect the extent of ATPase activation and that binding of the polymerase accessory protein complex to DNA cofactors is salt concentration dependent but appreciably less so when the activating DNA is a primer-template junction . Finally, we show that the gene 32 protein (T4 coded single-stranded DNA-binding protein) can compete with the polymerase accessory protein complex for single-stranded DNA but not for the primer-template junction activation sites . The implications of these results for the structure and function of the polymerase accessory protein complex within the T4 DNA replication system are discussed.

Biochemistry, 1989 Jul 25, 28(15), 6305 - 9
Active-site modification of mammalian DNA polymerase beta with pyridoxal 5'-phosphate: mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket; Basu A et al.; Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase beta . Kinetic studies indicate that the mechanism of PLP inhibition is complex . In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested . Reduction of the PLP-treated enzyme with sodium {3H}borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity . The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active . This protective effect is not observed in the absence of template-primer . Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification . Of these four sites, only one is protected by dNTP from pyridoxylation . Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation . These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate.

J Biol Chem, 1989 Jul 25, 264(21), 12394 - 401
The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion; Zahraoui A et al.; Seven cDNA clones corresponding to the rab1, rab2, rab3A, rab3B, rab4, rab5, and rab6 genes were isolated from a human pheochromocytoma cDNA library . They encode 23-25 kDa polypeptides which share approximately 30-50% homology and belong to the ras superfamily . The rab1, rab2, rab3A, and rab4 proteins are the human counterparts of the rat rab gene products that we have previously characterized . Comparison of the seven human rab proteins with the yeast YPT1 (YPT1p) and SEC4 (SEC4p) proteins reveals highly significant sequence similarities . H-rab1p shows 75% amino acid identity with YPT1p and may be therefore considered as its human counterpart . The other proteins share approximately 40% homology with YPT1p and SEC4p . The homology (approximately 30%) between these rab proteins and p21ras is restricted to the four conserved domains involved in the GTP/GDP binding . Human rab proteins were produced in Escherichia coli . Large amounts of rab proteins in soluble form can be extracted and purified without the use of detergents . All six proteins bind GTP and exhibit GTPase activities . A possible involvement of the rab proteins in secretion is discussed.

J Biol Chem, 1989 Jul 25, 264(21), 12226 - 31
Site-specific aflatoxin B1 adduction of sequence-positioned nucleosome core particles; Moyer R et al.; The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles . We have circumvented this problem by using nucleosomes that are homogenous in DNA sequence and hence in DNA-histone contact points . A cloned DNA fragment containing a sea urchin 5 S gene which precisely positions a histone octamer was employed . By using 32P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels . The small methylating agent dimethyl sulfate and the bulky alkylating agent aflatoxin B1-dichloride (AFB1-Cl2) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in the sequence-positioned core particle . We find dimethyl sulfate to bind with equal preference to naked or nucleosomal DNA . In contrast, AFB1-Cl2 binding is suppressed an average of 2.4-fold at guanyl sites within nucleosomes compared with AFB1-Cl2 affinity at the corresponding site in naked DNA . The DNA is more accessible in regions near the particle boundary . We observe no other histone-imposed localized changes in AFB1-Cl2 sequence specificity . Further, sites of DNase I cleavage or proposed DNA bending show neither enhanced nor reduced AFB1-Cl2 adduction to N7-guanine . Since AFB1-Cl2 binding sites lie in the major groove, nucleosomal DNA appears accessible to AFB1-Cl2 at all points of analysis but with an access which is uniformly restricted in the central 100 nucleotides of the core particle . The data available do not indicate further localized or site-specific perturbations in DNA interactions with the two carcinogens studied.

Biochim Biophys Acta, 1989 Jul 24, 983(1), 30 - 4
Studies on the binding substances on human erythrocytes for the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli; Sugii S et al.; To study the predominant binding substance for the heat-labile enterotoxin (LTc) isolated from chicken enterotoxigenic Escherichia coli, competitive binding assays were performed with neuraminidase-treated human type B erythrocytes and 125I-labeled B subunit of LTc (LTc-B) . Of all inhibitors used, the ganglioside GM1 was the most effective in inhibiting the binding of 125I-labeled LTc-B to the erythrocytes . The other gangliosides used as inhibitors, gangliosides GD1b, GD1a, GM2, GT1b and GM3, were about 24, 166, 250, 440 and at least 440 times less reactive than ganglioside GM1, respectively . With glycoproteins as inhibitors, on the other hand, hog A + H, porcine thyroglobulin and bovine salivary mucin were over 10(4) times less potent . No inhibition was obtained by other mono-, di- and polysaccharides at the highest concentrations used . These findings suggest that the predominant binding substance on neuraminidase-treated human type B erythrocytes for the LTc-B is ganglioside GM1 and that the combining site of LTc-B may be specific for the terminal disaccharide (galactose-N-acetyl-D-galactosamine)-linked portion of ganglioside GM1.

Science, 1989 Jul 21, 245(4915), 298 - 301
In vitro transcription enhancement by purified derivatives of the glucocorticoid receptor; Freedman LP et al.; Mammalian glucocorticoid receptors enhance transcription from linked promoters by binding to glucocorticoid response element (GRE) DNA sequences . Understanding the mechanism of receptor action will require biochemical studies with purified components . Enhancement was observed in vitro with derivatives of the receptor that were expressed in Escherichia coli, purified, and added to a cell-free extract from Drosophila embryo nuclei . Transcription from promoters linked to one or multiple GREs was selectively enhanced by as much as six times . The effect was weaker with only one GRE, and enhancement was abolished by a point mutation that inactivates the GRE in vivo.

J Mol Biol, 1989 Jul 20, 208(2), 327 - 39
Crystal structure of rat intestinal fatty-acid-binding protein . Refinement and analysis of the Escherichia coli-derived protein with bound palmitate; Sacchettini JC et al.; Rat intestinal fatty-acid-binding protein (I-FABP) is a small (15,124 Mr) cytoplasmic polypeptide that binds long-chain fatty acids in a non-covalent fashion . I-FABP is a member of a family of intracellular binding proteins that are thought to participate in the uptake, transport and/or metabolic targeting of hydrophobic ligands . The crystal structure of Escherichia coli-derived rat I-FABP with a single molecule of bound palmitate has been refined to 2 A resolution using a combination of least-squares methods, energy refinement and molecular dynamics . The combined methods resulted in a model with a crystallographic R-factor of 17.8% (7775 reflections, sigma greater than 2.0), root-mean-square bond length deviation of 0.009 A and root-mean-square bond angle deviation of 2.85 degrees . I-FABP contains ten antiparallel beta-strands organized into two approximately orthogonal, beta-sheets . The hydrocarbon tail of its single C16:0 ligand is present in a well-ordered, distinctively bent conformation . The carboxylate group of the fatty acid is located in the interior of I-FABP and forms a unique "quintet" of electrostatic interactions involving Arg106; Gln 115, and two solvent molecules . The hydrocarbon tail is bent with a slight left-handed helical twist from the carboxylate group to C-16 . The bent methylene chain resides in a "cradle" formed by the side-chains of hydrophobic, mainly aromatic, amino acid residues . The refined molecular model of holo-I-FABP suggests several potential locations for entry and exiting of the fatty acid.

J Mol Biol, 1989 Jul 20, 208(2), 217 - 23
Effect of ethylation of operator-phosphates on Gal repressor binding . DNA contortion by repressor; Majumdar A et al.; Gal repressor inhibits transcription by binding to two operators (OE and OI) in the gal operon . By ethylating DNA, we have identified 23 phosphate groups (11 on OE and 12 in OI) in the DNA backbone of gal operators that when ethylated interfere with repressor binding . By inference, either (1) such a phosphate is contacted or closely approached by Gal repressor, or (2) the structure of DNA generated by ethylation of such a phosphate, although not a site of direct contact, is not compatible with repressor binding . Within an operator, these phosphates are arranged with a perfect symmetry aligned with the operator dyad symmetry, indicating that each half-symmetry is contacted by a subunit of repressor dimer . Unlike in many other similar DNA-protein systems, the same phosphates in the gal operator are distributed around a B-form of DNA helix cylinder covering greater than 180 degrees . Models have been proposed to describe the disposition of the Gal repressor-operator complex, which would explain the layout of the participating phosphate groups around the surface of the DNA helix . We have discussed two ways by which Gal repressor can induce structural changes in DNA.

FEBS Lett, 1989 Jul 17, 251(1-2), 257 - 60
Synthesis of human endothelin-1 precursors in Escherichia coli; Watanabe T et al.; Endothelin, the most potent vasoconstrictor found in nature, is thought to be important in the regulation of blood pressure and/or local blood distribution . Human placenta cDNA fragment encoding preproendothelin-1 (preproET-1) and its carboxyl terminal mature precursor (C-matured precursor) was expressed in E . coli . These products were characterized by both enzyme immunoassay and Western blot analysis.

FEBS Lett, 1989 Jul 17, 251(1-2), 79 - 83
Human nuclear protein interacting with a conservative sequence motif of Alu-family DNA repeats; Tomilin NV et al.; Human retrotransposons, Alu-family DNA repeats (AFRs), have variable nucleotide sequence but conservative short elements, which may have important functions, are also present . In our previous reports we have described human nuclear DNA-binding protein interacting with AFRs and evidence was presented that the protein recognizes sequence motif 5'-GGAGGC-3' which is conserved in the spacer of RNA polymerase III promoter of AFRs and in the SV40 T-antigen-dependent replication origin of AFRs . In this study it was found that double-stranded synthetic oligonucleotides containing indicated conservative sequences of AFRs actually have high-affinity binding site for HeLa nuclear protein . The data suggest that non-infected human cells contain nuclear DNA-binding protein which recognizes the conservative sequence motif of AFRs - GGAGGC.

Gene, 1989 Jul 15, 79(2), 375 - 80
Molecular analysis of the pac gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger; Lacalle RA et al.; Nucleotide sequence of a 906-bp fragment of Streptomyces alboniger DNA containing the gene (pac), which encodes a puromycin N-acetyltransferase (PAC), has been determined . The pac gene contains a 600-nt open reading frame, starting with an ATG codon, which encodes a polypeptide of Mr 21,531; this is consistent with the 23 +/- 1.5 kDa size of the PAC enzyme . High-resolution S1 mapping indicates that transcription starts at or next to a C residue 35 bp upstream from the putative ATG start codon . A 263-bp DNA fragment from the 5' region of the pac gene has promoter activity in the promoter-probe plasmid pIJ486 . Its -35 and -10 regions show significant structural homology to the corresponding regions of the hyg gene promoter, but they are different from the promoter sequences of other Streptomyces and Escherichia coli genes.

Gene, 1989 Jul 15, 79(2), 345 - 54
Purification of human interleukin-4 produced in Escherichia coli; Jayaram B et al.; An interleukin-4 (IL4)-encoding cDNA isolated from human splenocytes was used to construct an expression plasmid that directs a high-level synthesis of mature IL4 protein in Escherichia coli . The expression was under the control of the major leftward promoter, pL, of phage lambda and the phage Mu ribosome-binding site . The IL4 protein was present as insoluble inclusion bodies in the bacterial extract . The IL4 could be solubilized in 5 M MgCl2 and was purified to homogeneity by several chromatographic steps . The yield of protein from bacteria ranged between 3 and 5 mg of IL4 protein per gram of wet cells . The specific activity of the recombinant human IL4 was about the same as that of the natural product.

Gene, 1989 Jul 15, 79(2), 309 - 24
A human tRNA gene heterocluster encoding threonine, proline and valine tRNAs; Shortridge RD et al.; A cluster of three tRNA genes encoding a tRNA(UGUThr), a tRNA(UGGPro), and a tRNA(AACVal), and two Alu-elements occur in a 6.0-kb human DNA fragment . The tRNA(Thr) gene is 2.7-kb upstream from the tRNA(Pro) gene, which is separated by 367 bp from the tRNA(Val) gene . One Alu-element actually overlaps the tRNA(Val) gene and is of opposite polarity to all three tRNA genes . All three tRNA genes are accurately transcribed in a homologous HeLa cell extract, since the ribonuclease T1 fingerprints of the tRNA transcripts are consistent with the nucleotide sequences of the tRNAs . The upstream region flanking the tRNA(Thr) gene has two tracts of alternating purine/pyrimidine residues potentially capable of adopting the Z-DNA conformation, and presumptive binding sites for two RNA polymerase II transcription factors . The tRNA(Thr) gene apparently has a substantially higher in vitro transcriptional efficiency than the other two tRNA genes in this cluster, and a tRNA(GCCGly) gene from another human DNA segment . Deletion constructs of the tRNA(Thr) gene retaining 272, 168, and 33 bp of original 5'-flanking DNA had about the same in vitro transcriptional efficiency, whereas that of the construct with only 2 bp of 5'-flanking human DNA was drastically reduced . The tRNA(Thr) gene constructs with 272 and 168 bp of original 5'-flanking DNA apparently reduce the transcriptional efficiencies of the proline and glycine tRNA genes, implicating the upstream region from the tRNA(Thr) gene as being crucial for its high transcriptional efficiency.

Biochem J, 1989 Jul 15, 261(2), 431 - 5
Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase; Nimmo HG et al.; Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process . Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit . The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site . Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys . The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1 . The Km of the enzyme for isocritrate declined over the same pH range.

Z Hautkr, 1989 Jul 15, 64(7), 596, 599 - 601
{Sexually transmitted diseases as causes of disorders of male fertility}; Krause W et al.; Infections with sexually transmitted germs may affect the male fertility in different ways . Possible consequences are impairment of the spermatogenesis, induction of auto-immune mechanisms, spermatodysfunction, and inflammatory occlusion of the ejaculatory duct . Only in high concentrations, bacteria (e.g . E . coli) may result in reducing the motility of spermatozoa . The germ counts observed under clinical conditions, however, do not come up with these high levels . The same is true for mycoplasmas . As a whole, sexually transmitted infections only play a minor role with regard to male infertility.

Eur J Biochem, 1989 Jul 15, 183(1), 19 - 24
Subunit association defects in Escherichia coli ribosome mutants lacking proteins S20 and L11; Gotz F et al.; The subunit association capacity of 30S and 50S ribosomal subunits from Escherichia coli mutants lacking protein S20 or L11 as well as of 50S subunits depleted of L7/L12 was tested by sucrose gradient centrifugation and by a nitrocellulose filtration method based on the protection from hydrolysis with peptidyl-tRNA hydrolase of ribosome-bound AcPhe-tRNA . It was found that the subunits lacking either S20 or L11 display an altered association capacity, while the 50S subunits lacking L7/L12 have normal association behavior . The association of S20-lacking 30S subunits is quantitatively reduced, especially at low Mg2+ concentrations (5-12 mM), and produces loosely interacting particles which dissociate during sucrose gradient centrifugation . The association of L11-lacking 50S subunits is quantitatively near-normal at all Mg2+ concentrations and produces loosely associating particles only at low Mg2+ concentrations (5-8 mM); the mechanism of their association with 30S subunits, however, or the structure of the resulting 30S-50S couples is altered in such a way as to cause the ejection of an AcPhe-tRNA molecule pre-bound to the 30S subunits in response to poly(U).

J Biol Chem, 1989 Jul 15, 264(20), 11989 - 94
TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin; Ziegelin G et al.; Transfer of plasmid RP4 during bacterial conjugation requires the plasmid-encoded TraJ protein, which binds to the transfer origin (Furste, J . P., Pansegrau, W., Ziegelin, G., Kroger, M., and Lanka, E . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 1771-1775) . As indicated by traJ mutants, the TraJ protein is a constituent of the relaxosome, the initiation complex of transfer DNA replication . The traJ gene maps adjacent to the transfer origin (oriT) . The structural gene consists of a 372-base pair sequence encoding a polypeptide of 122 amino acids (13,282 Da) . TraJ was purified from an Escherichia coli strain overproducing the protein . DNA footprinting experiments involving DNase I demonstrated that the purified protein binds to the right arm of a 19-base pair inverted repeat within oriT . Hydroxyl radical footprints of the DNA-protein complex revealed that TraJ protein is bound to only one side of the DNA helix.

J Biol Chem, 1989 Jul 15, 264(20), 11833 - 8
Both a short hydrophobic domain and a carboxyl-terminal hydrophilic region are important for signal function in the Escherichia coli leader peptidase; Zhu HY et al.; Leader peptidase, typical of inner membrane proteins of Escherichia coli, does not have an amino-terminal leader sequence . This protein contains an internal signal peptide, residues 51-83, which is essential for assembly and remains as a membrane anchor domain . We have employed site-directed mutagenesis techniques to either delete residues within this domain or substitute a charged amino acid for one of these residues to determine the important properties of the internal signal . The deletion analysis showed that a very small apolar domain, residues 70-76, is essential for assembly, whereas residues that flank it are dispensable for its function . However, point mutations with charged amino acid residues within the polar sequence (residues 77-82) slow or abolish leader peptidase membrane assembly . Thus, a polar region, Arg-Ser-Phe-Ile-Tyr-Glu, is important for the signal peptide function of leader peptidase, unlike other signals identified thus far.

J Biol Chem, 1989 Jul 15, 264(20), 11649 - 52
Peptide-specific antibody for the melibiose carrier of Escherichia coli localizes the carboxyl terminus to the cytoplasmic face of the membrane; Botfield MC et al.; The synthetic decapeptide NH2-Cys-Val-Gly-Ala-Val-Ser-Asp-Val-Lys-Ala-COOH (designated MBct10), which corresponds to the carboxyl terminus of the melibiose carrier of Escherichia coli, was synthesized and used to raise antibodies in a rabbit . Anti-MBct10 antibodies recognizes the normal melibiose carrier but not a truncated carrier lacking 14 carboxyl-terminal amino acids . Thus the antibodies are specific for the carboxyl terminus of the carrier and not for other domains of the protein . When right-side-out and inside-out membrane vesicles were probed with anti-MBct10 serum, only the inside-out vesicles bound antibody . The carboxyl terminus of the melibiose carrier protein is therefore exposed on the cytoplasmic surface of the membrane . The co-localization of both NH2- and carboxyl termini to the cytoplasmic surface dictates that the protein cross the membrane an even number of times . These data together with hydrophobicity analysis support a topological model for the melibiose carrier with 10 or 12 transmembrane domains.

J Biol Chem, 1989 Jul 15, 264(20), 11643 - 8
Carboxyl-terminal truncations of the melibiose carrier of Escherichia coli; Botfield MC et al.; The melibiose carrier of Escherichia coli is predicted to possess a short NH2 terminus, 11 transmembrane segments joined by short hydrophilic regions, and a 40-residue hydrophilic carboxyl terminus of unknown function . This paper describes truncations of the carboxyl terminus at eight locations using site-specific mutagenesis to introduce stop codons . Measurement of sugar transport and cation-coupling characteristics indicate that the carboxyl tail plays no direct role in substrate recognition or energy transduction . Thirty-six amino acids could be removed from the hydrophilic carboxyl domain without the loss of sugar specificity, facilitated diffusion, uphill transport, H+-coupling or Na+-coupling characteristics . These results are consistent with the hypothesis that the sugar/cation binding site is formed by the interaction of the transmembrane helices 3, 4, 6, 9, and 10 and does not involve the carboxyl-terminal portion of the protein . When truncations were made within the hydrophobic domain of transmembrane helix 11 (truncations of 41 or more residues), the carrier was no longer found in the membrane . This suggests that the carboxyl terminus may be involved in the membrane insertion process, stabilization of the carrier within the membrane following insertion, or protection of the inserted carrier from proteolytic scavenging . A new plasmid that expresses the temperature-resistant isoform of the melibiose carrier under inducible control of a tac promoter, designated pKKMB, is also described.

J Biol Chem, 1989 Jul 15, 264(20), 11621 - 5
Conformational stability and activity of ribonuclease T1 and mutants . Gln25----Lys, Glu58----Ala, and the double mutant; Shirley BA et al.; Ribonuclease T1 (RNase T1) and mutants Gln25----Lys, Glu58----Ala, and the double mutant were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli . The wild-type RNase T1 prepared from the cloned gene was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae . Urea and thermal unfolding experiments show that Gln25----Lys is 0.9 kcal/mol more stable and Glu58----Ala is 0.8 kcal/mol less stable than wild-type RNase T1 . In the double mutant, these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1 . For the double mutant, the dependence of delta G on urea concentration is significantly greater than for wild-type RNase T1 or the single mutants . This suggests that the double mutant unfolds more completely in urea than the other proteins . The activity of Gln25----Lys is identical with that of wild-type RNase T1 . The activities of Glu58----Ala and the double mutant are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in kcat), and 37% of wild-type when RNA hydrolysis is measured . Thus, Glu58 is important, but not essential to the activity of RNase T1.

Gene, 1989 Jul 15, 79(2), 269 - 77
Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5; Miyazaki J et al.; We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon . This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells . Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal) . Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10 . We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells . We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.

Gene, 1989 Jul 15, 79(2), 249 - 58
Purification of enzymatically active poliovirus proteinase 3C produced in Escherichia coli; Takahara Y et al.; A viral protein 3C of the poliovirus (PV) Sabin 2 strain, a possible core region of the viral proteinase, was expressed in Escherichia coli using a recombinant DNA technology . The protein was recovered as a soluble protein from the insoluble protein fraction of the bacterial lysate, and was purified by a simple procedure with column chromatography . The viral capsid precursor P1 (1ABCD) of the PV Sabin 3 strain, which had been similarly produced in E . coli, was mixed with the purified or crude recombinant 3C . Immunoblotting assay with monoclonal antibodies specific to capsid proteins 1C (VP3) and 1D (VP1) of the PV Sabin 3 strain revealed that the in vitro reaction products contained 1C (VP3), 1D (VP1) and 1ABC (VP0-VP3) . The data indicated that processing of the polyprotein P1 by the recombinant 3C proceeded properly in vitro, although an undigested product, 1ABC, is always detected in the reaction mixture . The results strongly suggest that, in addition to a protein 3CD, the 3C protein itself is also catalytically active in the processing of the viral capsid precursor polyprotein P1.

Gene, 1989 Jul 15, 79(2), 239 - 48
Cloning and expression of the major inner capsid protein of SA-11 simian rotavirus in Escherichia coli; Smith RE et al.; The major inner capsid protein (VP6) of SA-11 simian rotavirus has been expressed in Escherichia coli using a cloned cDNA derived from SA-11 double-stranded RNA segment 6 . The cloned gene was fused to the N-terminal coding sequence of lacZ resulting in the synthesis of a 44-kDa protein . Several smaller polypeptides were also observed, resulting predominantly from transcription and translation within the gene 6 coding sequence . The recombinant VP6 proved to be antigenic by immunoblot analysis using polyclonal serum against SA-11 rotavirus and by Western-blot analysis using monospecific serum derived from purified viral VP6.

Biochem J, 1989 Jul 15, 261(2), 437 - 43
The cytochromes of anaerobically grown Escherichia coli . An electron-paramagnetic-resonance study of the cytochrome bd complex in situ; Rothery RA et al.; The e.p.r . signals attributable to a cytochrome bd-type ubiquinol:O2 oxidoreductase (cytochrome b-558-b-595-d) were studied in a cytoplasmic membrane preparation of Escherichia coli that had been grown on glycerol with fumarate as respiratory-chain oxidant . Two major high-spin ferric haem signals were resolved on the basis of their potentiometric behaviour: a rhombic high-spin species (gx = 6.25, gy = 5.54) was assigned to haem b-595, and an axial high-spin (gx = 5.97, gy = 5.96) species was assigned to the haem d . These signals titrated with Em.7 values of 154 and 261 mV respectively, corresponding closely to optically determined values for haem b-595 and haem d . At high potentials (greater than 300 mV) the rhombic species attributable to haem b-595 underwent a partial transition to a second rhombic species with g-values of 6.24 (gx) and 5.67 (gy) . The high-spin ferric haem spectra were affected by O2, CO, cyanide and pH . A low-spin ferric haem signal was observed at g = 3.3 (gz), which titrated with an Em.7 of 226 mV, and this was assigned to haem b-558 . The data support a model for cytochrome bd with two ligand-binding sites, a single haem d and a single haem b-595.

Gene, 1989 Jul 15, 79(2), 369 - 74
Expression in Escherichia coli of the major outer capsid protein of infectious pancreatic necrosis virus; Lawrence WR et al.; The outer capsid polypeptide, VP2, represents the major neutralizing antigen of infectious pancreatic necrosis virus (IPNV) . A 926-bp viral cDNA, encoding an N-terminal truncated VP2, was cloned into the pWR590 expression plasmid family resulting in a C-terminal extension of a truncated Escherichia coli beta-galactosidase (beta Gal) under the control of the lac promoter . When cells transformed by in-phase hybrid plasmids were induced by isopropylthiogalactoside, high levels of the 100-kDa beta Gal-VP2 fusion protein accumulated within 4 h after induction . The fusion protein reacted in Western blots both with rabbit anti-beta Gal and with neutralizing mouse anti-VP2 monoclonal antibody . Sera of rabbits immunized with semipurified fusion protein reacted with the VP2 polypeptide in Western blots and with intact purified virus in ELISA and also neutralized IPNV infectivity in a plaque-reduction assay . Out-of-phase hybrid plasmids did not produce the fusion protein but expressed a small amount of structurally discrete VP2-specific sequences probably by internal initiation of translation at an in-phase AUG codon near the 5' end of the VP2 gene.

Science, 1989 Jul 14, 245(4914), 160 - 4
DNA mismatch correction in a defined system; Lahue RS et al.; DNA mismatch correction is a strand-specific process involving recognition of noncomplementary Watson-Crick nucleotide pairs and participation of widely separated DNA sites . The Escherichia coli methyl-directed reaction has been reconstituted in a purified system consisting of MutH, MutL, and MutS proteins, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, DNA ligase, along with ATP (adenosine triphosphate), and the four deoxynucleoside triphosphates . This set of proteins can process seven of the eight base-base mismatches in a strand-specific reaction that is directed by the state of methylation of a single d(GATC) sequence located 1 kilobase from the mispair.

Biochemistry, 1989 Jul 11, 28(14), 5821 - 6
Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease; Pierce JR et al.; Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both phi X174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated . We have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA . Using a piperidine cleavage method and DNA sequence analysis, we have found that all guanine residues can be modified by N-OH-AF and NAAAF . These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form . phi X174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-AF . However, we have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF . In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct . Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site . The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed.

Biochemistry, 1989 Jul 11, 28(14), 6127 - 32
Site-directed mutagenesis of tyrosine residues in the lac permease of Escherichia coli; Roepe PD et al.; By using oligonucleotide-directed, site-specific mutagenesis, each of the 14 Tyr residues in the lac permease of Escherichia coli was replaced with Phe, and the activity of each mutant was studied with respect to active transport, equilibrium exchange, and efflux . Ten of the mutations have no significant effect on permease activity . Of the four mutations that alter activity, replacement of Tyr26 or Tyr336 with Phe severely decreases all modes of translocation, and the binding affinity of the mutant permease for p-nitrophenyl alpha-D-galactopyranoside is markedly decreased (i.e., KD is increased) . In addition, the Phe336 mutant permease is inserted into the membrane to a lesser extent than wild-type permease, as judged by immunoblot experiments . Permease containing Phe in place of Tyr236 catalyzes lactose exchange approximately 40% as well as wild-type permease but does not catalyze active transport or efflux . Finally, permease with Phe in place of Tyr382 catalyzes equilibrium exchange normally, but exhibits low rates of active transport and efflux without being uncoupled, thereby suggesting that replacement of Tyr382 with Phe alters a kinetic step involving translocation of the unloaded permease across the membrane.

Biochemistry, 1989 Jul 11, 28(14), 5871 - 81
Enhancement of Escherichia coli RecA protein enzymatic function by dATP; Menetski JP et al.; The Escherichia coli recA protein has been shown to hydrolyze several nucleoside triphosphates in the presence of ssDNA . The substitution of dATP for rATP has significant effects on various recA protein biochemical properties . In the presence of dATP, recA protein can invade more secondary structure in native ssDNA than it can in the presence of rATP . The dATP-recA protein complex can compete more effectively with the E . coli ssDNA binding protein (SSB) for ssDNA binding sites compared with the rATP-recA protein complex . Finally, the rate of dATP hydrolysis stimulated by dsDNA is greater than the rate of rATP hydrolysis . These effects, in turn, are observed as alterations in the recA protein catalyzed DNA strand exchange reaction . In the absence of SSB protein, the rate of joint molecule and product formation in the DNA strand exchange reaction is greater in the presence of dATP than in the presence of rATP . The rate of product formation in the dATP-dependent reaction is also faster than the rATP-dependent reaction when SSB protein is added to the ssDNA before recA protein; the rate of rATP-dependent product formation is inhibited 10-fold under these conditions . This nucleotide, dATP, was previously shown to induce an apparent affinity of recA protein for ssDNA which is higher than any other NTP . These results suggest that the observed enhancement of enzymatic activity may be related to the steady-state properties of the high-affinity ssDNA binding state of recA protein . In addition, the data suggest that recA protein functions in NTP hydrolysis as a dimer of protein filaments and that the binding of ssDNA to only one of the recA filaments is sufficient to activate all recA protein molecules in the dimeric filament . The implications of this finding to the enzymatic function of recA protein are discussed.

Nucleic Acids Res, 1989 Jul 11, 17(13), 5361 - 75
Scission of RNA by the chemical nuclease of 1,10-phenanthroline-copper ion: preference for single-stranded loops; Murakawa GJ et al.; The scission of RNA by the chemical nuclease activity of 1,10-phenanthroline-copper (OP-Cu) has been studied using a lac mRNA fragment and tRNAphe as substrates . Since the chemical mechanism of scission involves oxidative attack on the ribose, scission is observed at all nucleotides including dihydrouridine and Y-bases . Specificity for single-stranded loop regions is apparent from the similarity of the reactivity of OP-Cu to the single-strand specific reagents dimethyl sulfate and diethyl pyrocarbonate using the fragment of lac mRNA as a substrate . Similar preference is observed in the reaction with tRNA although scission in the helical acceptor stem is also observed.

Nucleic Acids Res, 1989 Jul 11, 17(13), 4937 - 46
Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support; Hultman T et al.; Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support . The DNA is immobilized through selective incorporation of biotin into one of the strands . A single stranded template, suitable for sequencing, is obtained through strand-specific elution . Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies . The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions . The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.

Biochemistry, 1989 Jul 11, 28(14), 5728 - 34
Thiol and amino analogues as alternate substrates for glycerokinase from Candida mycoderma; Knight WB et al.; The kinetic and catalytic mechanism of glycerokinase from Candida mycoderma was examined with thiol and amino analogues of glycerol and with MgAMPPCP, an analogue of MgATP . (S)-1-Aminopropanediol was phosphorylated on nitrogen (Vmax 0.4% that of glycerol) while the R enantiomer was phosphorylated on oxygen (Vmax 0.7% that of glycerol) . (S)-1-Mercaptopropanediol was phosphorylated on oxygen (Vmax 3.5% that of glycerol), while the R enantiomer was phosphorylated on sulfur (Vmax 0.001% that of glycerol) . The hydroxyl group at C-2 thus orients the substrate in the active site, while that at the carbon remote from phosphorylation enhances both catalysis and binding of the substrate, presumably because of hydrogen-bonding interactions . The kinetic mechanism is random with a high degree of synergistic binding between the substrates, so that the mechanism appears ordered with glycerol adding first but equilibrium ordered with MgATP binding first with the amino analogues.

Biochemistry, 1989 Jul 11, 28(14), 5847 - 55
Probing the phosphates of the Escherichia coli ribosomal 16S RNA in its naked form, in the 30S subunit, and in the 70S ribosome; Baudin F et al.; Ethylnitrosourea is an alkylating reagent which preferentially modifies phosphates in nucleic acids . It was used to map phosphates in naked Escherichia coli 16S rRNA engaged in tertiary interactions through hydrogen bonds or ion coordination . Of the phosphates, 7% are found involved in such interactions, and 57% of them are located in loops or interhelical regions, where they are involved in maintaining local intrinsic structures or long-distance tertiary interactions . The other phosphates (43%) are found in helical regions . These phosphates often occur at the proximity of bulged nucleotides or in irregular helices containing noncanonical base pairs (and bulges) and are assumed to bind cations in order to neutralize negative charges and to stabilize unusual phosphate backbone folding . In the 30S subunit, ENU allowed mapping of phosphates in contact with proteins . The RNA is not uniformly engaged in RNA/protein interactions . Regions 1-51, 250-310, 567-612, 650-670, and 1307-1382 are particularly buried whereas the 3'-terminal domain and the 5'-proximal region (nucleotides 53-218) are exposed . The conformation of 16S rRNA is not drastically affected by protein binding, but conformational adjustments are detected in several defined regions . They are found in the 5' domain (region 147-172), in the central domain (region 827-872), in the 3' major domain (nucleotides 955-956, 994, 1054, 1181, 1257, and 1262-1263), and in the 3'-terminal domain (around 1400) . The 50S subunit shields clusters of phosphates located at the subunit interface . The most extensive protections are observed in the 3'-terminal domain (1490-1542), in the central region of the molecule (770-930), and in the upper 3' major domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 Jul 11, 28(14), 5814 - 20
Synthesis and characterization of 5-{(4-Azidophenacyl)thio}uridine 5'-triphosphate, a cleavable photo-cross-linking nucleotide analogue; Hanna MM et al.; The synthesis, isolation, and characterization of a new photo-cross-linking uridine 5'-triphosphate analogue are described . This nucleotide analogue, 5-{(4-azidophenacyl)thio}uridine 5'-triphosphate (5-APAS-UTP), contains an aryl azide group approximately 10 A from the uridine ring . The azide is photoactivated by irradiation at 300 nm, resulting in covalent attachment of the nucleotide to adjacent molecules . The nucleotide can be desulfurated with Raney nickel to cause molecular cleavage between the base and the aryl azide . Desulfuration yields uridine 5'-triphosphate and p-azidoacetophenone . If the analogue is cross-linked to another molecule, desulfuration leaves the analogue's acetophenone group attached to that molecule . This effectively leaves behind a molecular tag on molecules that interact with the uridine analogue either as monomeric nucleotide or as part of an RNA molecule . This nucleotide analogue can be incorporated into internal positions in RNA by transcription in vitro with Escherichia coli RNA polymerase . It can therefore be used to examine interactions between RNA and other molecules (e.g., proteins or nucleic acids) . Because the sulfur atom can be selectively removed, the covalent bonds formed between analogue-containing RNA and other molecules can be cleaved, when desired, to facilitate identification of the cross-linked molecules and RNA nucleotides in the cross-linked complex.

Nucleic Acids Res, 1989 Jul 11, 17(13), 5107 - 14
Alpha-DNA.IX: Parallel annealing of alpha-anomeric oligodeoxyribonucleotides to natural mRNA is required for interference in RNase H mediated hydrolysis and reverse transcription; Gagnor C et al.; ps- and aps-alpha anomeric oligodeoxyribonucleotides were designed to recognize in parallel (ps) or antiparallel (aps) orientation two different sites of a 1000 base-long mRNA . Northern blots experiments indicate that only ps-alpha-oligonucleotides were able to hybridize to the mRNA target . Furthermore, only ps-alpha-oligonucleotides were able, in a sequence specific way, to protect the mRNA target against RNase H mediated hydrolysis or to inactivate the priming capacity of beta-oligodeoxynucleotide probes in reverse transcription . Formation of parallel-stranded mRNA alpha-oligonucleotide miniduplexes which prevents hybridization of beta-oligonucleotide probes is the most likely mechanism accounting for these results . Use of alpha-oligonucleotides as potential gene control agents is discussed.

Biochim Biophys Acta, 1989 Jul 10, 982(2), 253 - 64
Galactoside-dependent proton transport by mutants of the Escherichia coli lactose carrier: substitution of tyrosine for histidine-322 and of leucine for serine-306; King SC et al.; The lac Y genes from two Escherichia coli mutants, MAB20 and AA22, have been cloned in a multicopy plasmid by a novel 'sucrose marker exchange' method . Characterization showed that the plasmids express a lactose carrier with poor affinity for lactose . Neither mutant carried out concentrative uptake with methyl beta-D-galactopyranoside, lactose, or melibiose as the substrate . Nor did the mutants catalyze counterflow or exchange with methyl beta-D-galactopyranoside . Both mutants did, however, retain the capacity to carry out facilitated diffusion with lactose or melibiose . DNA sequencing revealed that MAB20 (histidine-322 to tyrosine) and AA22 (serine-306 to leucine) have amino acid substitutions within the putative 'charge-relay' domain thought to be responsible for proton transport . Galactoside-dependent H+ transport was readily measured in both mutants . We conclude, therefore, that the presence of a histidine residue at position 322 of the lactose carrier is not obligatory for H+ transport per se.

Biochim Biophys Acta, 1989 Jul 7, 1008(2), 247 - 50
Cloning, sequence and expression in Escherichia coli of cDNA for ovine pregrowth hormone; Warwick JM et al.; cDNA prepared from mRNA from ovine anterior pituitary glands was cloned in Escherichia coli and the sequence of a clone encoding the full coding sequence of ovine pregrowth hormone (preGH) determined . The predicted sequence for ovine GH agrees with that determined previously on the protein, except that residue 99 is asparagine rather than aspartic acid . The cDNA sequence also accords with one of the two genomic sequences for the ovine GH gene that have been reported . Expression plasmids using trp and lac promoters were constructed which allowed expression at low levels of ovine preGH in E . coli, as detected by immunoblotting and immunoassay.

Biochim Biophys Acta, 1989 Jul 7, 1008(2), 146 - 56
Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives; Graifer DM et al.; A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues . Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site . Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex . The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27 . Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

Biochim Biophys Acta, 1989 Jul 7, 1008(2), 189 - 92
Expression of human erythrocyte NADH-cytochrome b5 reductase as an alpha-thrombin-cleavable fused protein in Escherichia coli; Shirabe K et al.; Recombinant fused protein containing human erythrocyte NADH-cytochrome b5 reductase (cytochrome b5 reductase, EC 1.6.2.2.) was produced in Escherichia coli, which was linked to the NH2 terminus of beta-galactosidase of the vector pUC13 via a recognition sequence of alpha-thrombin . Cleavage of purified fused protein with alpha-thrombin yielded the enzyme whose apparent molecular weight (32,000) was the same as the native enzyme . The amino-acid sequence from Phe-1 to Leu-10 was determined to be identical to that of the authentic enzyme . The purified enzyme showed an identical absorption spectrum and similar catalytic properties to the native enzyme . Establishment of the expression system would make it possible to determine the reaction mechanism of the enzyme.

Biochim Biophys Acta, 1989 Jul 6, 996(3), 260 - 2
Pressure-induced changes in the secondary structure of the Escherichia coli methionine repressor protein; Wong PT et al.; The effect of hydrostatic pressure on the conformational properties of the E . coli methionine repressor protein in aqueous solution was investigated by infrared spectroscopy . Changes in hydrostatic pressure produce dramatic changes in the spectral region of the conformation-sensitive amide I band . As the pressure is raised up to 18 kbar, the protein undergoes a rearrangement of alpha-helical segments into beta-type structures; after the pressure is released the beta-strands reconvert into less ordered alpha-helical or random segments.

J Mol Biol, 1989 Jul 5, 208(1), 199 - 205
Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis; Mitchell EJ et al.; Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains . MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay . Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively . Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus . Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues . These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains . Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels . Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.

J Mol Biol, 1989 Jul 5, 208(1), 57 - 64
Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA . IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA; Gray CW; Specimen-tilting in an electron microscope was used to determine the three-dimensional architecture of the helical complexes formed with DNA by the closely related single-stranded DNA binding proteins of fd and IKe filamentous viruses . The fd gene 5 protein is the only member of the DNA-helix-destabilizing class of proteins whose structure has been determined crystallographically, and yet a parameter essential to molecular modeling of the co-operative interaction of this protein with DNA, the helix handedness, has not been available prior to this work . We find that complexes formed by titrating fd viral DNA with either the fd or IKe gene 5 protein have a left-handed helical sense . Complexes isolated from Escherichia coli infected by fd virus are also found to be left-handed helical; hence, the left-handed fd helices are not an artefact of reconstitution in vitro . Because the proteins and nucleic acid of the complexes are composed of asymmetric units which cannot be fitted equivalently to right-handed and left-handed helices, these results rule out a previous computer graphics atomic model for the helical fd complexes: a right-handed helix had been assumed for the model . Our work provides a defined three-dimensional structural framework within which to model the protein-DNA and protein-protein interactions of two structurally related proteins that bind contiguously and co-operatively on single-stranded DNAs.

J Mol Biol, 1989 Jul 5, 208(1), 209 - 10
Crystallization of the DNA-binding Escherichia coli protein FIS; Choe HW et al.; The specific DNA-binding protein FIS (factor for inversion stimulation), which stimulates site-specific DNA inversion by interaction with an enhancer sequence, was purified from an Escherichia coli strain overproducing the protein . FIS was crystallized at room temperature by microdialysis against 1.2 to 1.5 M-sodium/potassium phosphate containing 10 mM-Tris.HCl, 0.5 to 1 M-NaCl and 1 mM-NaN3 at pH 8.0 to 8.2 . The crystals are stout prisms and suitable for X-ray diffraction study beyond 2.5 A resolution . They belong to the orthorhombic space group P2(1)2(1)2(1) . The unit cell has dimensions a = 47.57(4) A, b = 51.13(4) A, c = 79.83(6) A and contains one FIS dimer in the asymmetric unit.

J Mol Biol, 1989 Jul 5, 208(1), 207 - 8
Crystallization and preliminary X-ray diffraction study of the heat-labile enterotoxin B subunit produced by enterotoxigenic Escherichia coli; Tsuji T et al.; Heat-labile enterotoxin LT produced by enterotoxigenic Escherichia coli is composed of A and B subunits . The A subunit is enzymatically active; whereas, through the action of the B subunit, the toxin binds to the receptor, a GM1 ganglioside present on the cell surface . Crystals of the LT-B subunit were formed at room temperature by vapor diffusion with polyethylene glycol in the presence of the non-ionic detergent beta-octylglucoside . The crystals were characterized by X-radiation as orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions of a = 224.1 A, b = 65.3 A, c = 118.4 A . They diffract X-rays to a resolution of at least 2.5 A and are stable to X-rays.

J Biol Chem, 1989 Jul 5, 264(19), 11275 - 81
The beta subunit modulates bypass and termination at UV lesions during in vitro replication with DNA polymerase III holoenzyme of Escherichia coli; Shavitt O et al.; The cycling time of DNA polymerase III holoenzyme during replication of UV-irradiated single-stranded (ss) DNA was longer than with unirradiated DNA (8 versus 3 min, respectively), most likely due to slow dissociation from lesion-terminated nascent DNA strands . Initiation of elongation on primed ssDNA was not significantly inhibited by the presence of UV lesions as indicated by the identical distribution of replication products synthesized at early and late reaction times and by the identical duration of the initial synthesis bursts on both unirradiated and UV-irradiated DNA templates . When replication was performed with DNA polymerase III* supplemented with increasing quantities of purified beta 2 subunit, the cycling time on UV-irradiated DNA decreased from 14.8 min at 1.7 nM beta 2 down to 6 min at 170 nM beta 2, a concentration in which beta 2 was in large excess over the polymerase . In parallel to the reduction in cycling time, also the bypass frequency of cyclobutane-photodimers decreased with increasing beta 2 concentration, and at 170 nM beta 2, bypass of photodimers was essentially eliminated . It has been shown that polymerase complexes with more than one beta 2 per polymerase molecule were formed at high beta 2 concentrations (Lasken, R . S., and Kornberg, A . (1987) J . Biol . Chem . 262, 1720-1724) . It is plausible that polymerase complexes obtained under high beta 2 concentration dissociate from lesion-terminated primers faster than polymerase complexes formed at a low beta 2 concentration . This is expected to favor termination over bypass at pyrimidine photodimers and thus decrease their bypass frequency . These results suggest that the beta 2 subunit might act as a sensor for obstacles to replication caused by DNA damage, and that it terminates elongation at these sites by promoting dissociation . The intracellular concentration of beta 2 was estimated to be 250 nM (Kwon-Shin, O., Bodner, J . B., McHenry, C . S., and Bambara, R . A . (1987) J . Biol . Chem . 262, 2121-2130) and is 15-fold higher than the estimated intracellular concentration of DNA polymerase III holoenzyme (15 nM) . This high concentration of beta 2 may be responsible for the observation that very little (if any) bypass of pyrimidine photodimers occurred in vivo when the SOS system was not induced . Moreover, it predicts that bypass synthesis under SOS conditions might be associated with an altered form of the beta subunit.

J Biol Chem, 1989 Jul 5, 264(19), 11326 - 35
Activation of glutamate by gamma-glutamate kinase: formation of gamma-cis-cycloglutamyl phosphate, an analog of gamma-glutamyl phosphate; Seddon AP et al.; gamma-Glutamate kinase, the enzyme that catalyzes the first step in the pathway from glutamate to proline, has been postulated to convert glutamate to a gamma-activated form (possibly gamma-glutamyl phosphate), which is reduced by a NADPH-linked reductase to yield glutamate gamma-semialdehyde (in equilibrium with delta 1-pyrroline-5-carboxylate) . In the present work we found that the kinase, in the absence or presence of the reductase (and in the absence of NADPH), catalyzes stoichiometric formation of 5-oxo-L-proline and Pi from L-glutamate and ATP, but catalyzes hydroxamate formation at only about 10% of the rate of ATP-cleavage . A new substrate of the kinase was found; thus, cis-cycloglutamate (cis-1-amino-1,3-dicarboxycyclohexane), a glutamate analog which cannot cyclize to form an analog of 5-oxoproline, interacts effectively with the kinase . The trans form of cycloglutamate does not interact with the kinase; only the cis form can assume a diequatorial conformation equivalent to the extended conformation of glutamate . cis-Cycloglutamyl phosphate formation was shown and evidence was obtained for formation of an enzyme-ADP-cycloglutamyl phosphate complex . Although cis-cycloglutamyl phosphate is not a reducible substrate of the NADPH-dependent reductase, the findings indicate that it interacts with the reductase . These studies, which elucidate several aspects of the mechanism of the utilization of glutamate for formation of delta 1-pyrroline-5-carboxylate, support the hypothesis that the kinase and reductase function as an enzyme complex . A model is suggested in which gamma-glutamyl phosphate formed on the kinase interacts with the reductase to form a gamma-glutamyl-reductase complex, which is reduced by NADPH to yield glutamate gamma-semialdehyde.

J Biol Chem, 1989 Jul 5, 264(19), 11294 - 301
Glutamate overcomes the salt inhibition of DNA polymerase III holoenzyme; Griep MA et al.; Even though Escherichia coli can grow in media containing up to 1 M NaCl, one-fifth that amount of NaCl will completely inhibit the in vitro activity of DNA polymerase III holoenzyme . It has been established that the major intracellular ionic osmolytes are potassium and glutamate (Richey, B., Cayley, D . S., Mossing, M . C., Kolka, C., Anderson, C . F., Farrar, T . C., and Record, M . T., Jr . (1987) J . Biol . Chem . 262, 7157-7164) . We have found that holoenzyme catalyzes replication efficiently in vitro in up to 1 M potassium glutamate . Two salt effects on the replication of single-stranded DNA were observed . At low salt replicative activity was enhanced and at high salt there was anion-specific inhibition . We have found that DNA polymerase III holoenzyme tolerated 10-fold higher concentrations of glutamate than chloride . The ability of various anions to extend the useful range of salt concentrations followed the order: phosphate less than chloride less than N-Ac-glutamate less than acetate less than glycine less than aspartate less than glutamate . With the exception of phosphate, this order followed the Hofmeister series indicating that the anion-specific effects were due to anions interacting at the protein-water interface at weak anion binding sites . Glutamate did not reverse the inhibition by chloride . The low salt enhancement and high salt inhibition effects were additive for the two anions indicating that they competed for common anion binding sites . The major salt-sensitive step was holoenzyme binding to template rather than the subsequent elongation reaction.

J Biol Chem, 1989 Jul 5, 264(19), 10954 - 9
Amino acid sequence of the alpha and beta subunits of Methanosarcina barkeri ATPase deduced from cloned genes . Similarity to subunits of eukaryotic vacuolar and F0F1-ATPases; Inatomi K et al.; The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences . The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N . crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively . The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase . The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively . Furthermore, important amino acid residues identified genetically in the E . coli enzyme were conserved in the methanogenic enzyme . This sequence conservation suggests that vacuolar, F1, methanogenic, and S . acidocaldarius ATPases were derived from a common ancestral enzyme.

J Mol Biol, 1989 Jul 5, 208(1), 23 - 43
Identification and characterization of transcription termination sites in the Escherichia coli lacZ gene; Ruteshouser EC et al.; The Escherichia coli lacZ gene contains a series of latent transcriptional terminators that are responsible for the polar effects of certain mutations . We demonstrate, using gel electrophoretic size analyses and nuclease S1 mapping procedures, that RNA polymerase terminates RNA synthesis in the vicinity of five positions 180, 220, 379, 421 and 463 base-pairs downstream from the start point during transcription of lacZ DNA in vitro in the presence of rho factor . Termination at all but the 421 position depends on rho factor . In the in vitro assays with 0.05 M-KCl and excess rho (36 nM), the terminators are moderately effective, having efficiencies that range from about 8% at the 180 base-pair site to 56% at the 463 base-pair site . These termination stop points correspond to five of the 11 transcriptional pause sites between 180 and 463 base-pairs . Several stop points also correspond to 3' end points of lacZ mRNA isolated from cells containing the strongly polar lacZ-U118 mutation and from cells starved for serine, thus confirming that these latent terminators are responsible for the polar effect and demonstrating that they also function under a condition of physiological stress that prevents the transcription from being translated properly . Two other potential termination factors, NusA protein and cyclic AMP receptor protein have no effect in vitro on the efficiency of termination at the five lacZ sites.

J Biol Chem, 1989 Jul 5, 264(19), 11252 - 7
Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein; Fassina G et al.; Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein . One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence . Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column . Binding affinities were in the millimolar-micromolar range . Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence . Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer . Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism . Proteolytic degradation was retarded by the binding of these peptides . Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract.

Klin Wochenschr, 1989 Jul 3, 67(13), 682 - 6
{Acute interstitial nephritis following piperacillin}; Dorner O et al.; A 75-year-old woman developed fever, exanthema and nonoliguric renal failure 16 days after the beginning of Piperacillin treatment . Renal biopsy revealed lympho-plasma-cellular acute interstitial nephritis (AIN) . A lymphocyte-transformation-test showed significant stimulation of patient's lymphocytes by Piperacillin . Corticosteroid-therapy correlated to clinical and renal improvement . Nevertheless the patient died of foudroyant septicemia caused by E . coli . Our report describes the first immunologically documented case of AIN following Piperacillin treatment.

FEBS Lett, 1989 Jul 3, 250(2), 336 - 40
Antibodies against an inter-domain segment of polypeptide chain inhibit active-site coupling in the pyruvate dehydrogenase multienzyme complex; Radford SE et al.; A synthetic peptide, AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA, proved to be an efficient and specific immunogen in rabbits . The amino acid sequence of the peptide is identical to that of the inter-domain region (PEP3) linking the innermost of the three lipoyl domains to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli . Fab fragments from anti-PEP3 antibodies selectively inhibited active-site coupling in the complex without affecting the individual activities of the three component enzymes, highlighting the role of the inter-domain regions as flexible linkers in catalysis.

FEBS Lett, 1989 Jul 3, 250(2), 371 - 6
Production of plasmids giving high expression of recombinant DNA-derived ovine growth hormone variants in Escherichia coli; Wallis OC et al.; A method for the production of plasmids giving different levels of expression of ovine growth hormone (oGH) variants in E . coli is described . The cDNA sequence coding for mature oGH was inserted into the multiple cloning site of plasmid pUC8 and random deletions were then introduced 3' to the initiation codon . Clones producing GH (with varying N-terminal extensions) were identified by immunological screening . Levels of expression of GH-related protein, measured by immunoassay or on SDS-polyacrylamide gels, varied from over 20% to less than 0.05% of total cell protein . The coding sequence of plasmid pOGHe101, giving very high expression of variant oGH1, was determined.

FEBS Lett, 1989 Jul 3, 250(2), 161 - 5
Production and purification of a recombinant human 14 kDa beta-galactoside-binding lectin; Hirabayashi J et al.; The cDNA for a 14 kDa human beta-galactoside-binding lectin was inserted into a plasmid carrying a taq promoter, and the lectin protein was expressed in E . coli cells . The recombinant lectin was extracted from the cells and purified to apparent homogeneity by a single-step chromatography on an asialofetuin-agarose column . Subunit molecular mass (14 kDa), hemagglutinating activity and antigenicity were indistinguishable from those of the human placental lectin . Though the N-terminal of the placental lectin is blocked with an acetyl group, the recombinant lectin was found to have a free amino group . However, the N-terminal amino acid sequences were identical . The recombinant lectin was considered to have the same three-dimensional structure as the placental lectin.

FEBS Lett, 1989 Jul 3, 250(2), 153 - 6
Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia; Fukumori F et al.; Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose . A lambda gt11 expression library was constructed from A . aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL . An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained . The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids . The 5'-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1 . Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.

FEBS Lett, 1989 Jul 3, 250(2), 429 - 32
Regulation of colominic acid biosynthesis by temperature: role of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase; Gonzalez-Clemente C et al.; Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature . Evidence for the role of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E . coli grown at 20 and 37 degrees C . No activity was found in E . coli grown at 20 degrees C . CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20 degrees C were transferred to 37 degrees C and was halted when cells grown at 37 degrees C were transferred to 20 degrees C . These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.

FEBS Lett, 1989 Jul 3, 250(2), 139 - 41
3'-Fluoro-3'-deoxyribonucleoside 5'-triphosphates: synthesis and use as terminators of RNA biosynthesis; Mikhailopulo IA et al.; 3'-Fluoro-3'-deoxy-uridine, -cytidine, -adenosine and -guanosine have been synthesized by glycosylation of the corresponding silylated bases with 1-O-acetyl-2,5-di-O-benzoyl-3-fluoro-3-deoxy-D-ribofuranose in the presence of Friedel-Crafts catalysts and were converted to the 5'-triphosphates, NTP(3'-F) . It was shown that NTP(3'-F) are terminators of RNA synthesis catalyzed by DNA-dependent RNA polymerase from E . coli and may thus serve as tools for DNA sequencing.

Mol Gen Mikrobiol Virusol, 1989 Jul, (7), 8 - 10
{Formation of pAS8-1213 cointegrate with one of the plasmids of Azospirillum brasilense Sp245}; Matveev VIu et al.; Inheritance of the plasmid vector pAS8-1213 in Azospirillum brasilense Sp245 cells has been studied . The plasmid pAS8-1213 is shown to be uncapable of autonomous replication in the new host but able to integrate into the genetic structures of Azospirillum with high frequency . 90-95% of KmR-transconjugants of A . brasilense harbor pAS8-1213 cointegrated with the smaller host plasmid pAbSP245c(85Md) . The formed cointegrate can be transferred into Azospirillum spp . 75 and RecA- strains of E . coli (HB101 and DH1) and stably maintained in these cells . The IS21 element inherent of the plasmid pAS8-1213 is supposed to participate in pAS8-1213::pAbSP245c cointegrate formation.

J Biolumin Chemilumin, 1989 Jul, 4(1), 31 - 9
Bioluminescent click beetles revisited; Wood KV et al.; In studying beetle bioluminescence in the early 1960s, Dr . McElroy and his colleagues found that the Jamaican click beetle, Pyrophorus plagiophthalamus, was capable of emitting different colours of light . They further found that the luciferin substrate used by this beetle was the same as that in the firefly, demonstrating that the different colours of bioluminescence were due to differences in the structure of the luciferases . We have recently cloned cDNAs from this beetle species which code for at least four different luciferases . The luciferases are distinguishable by their different colours of bioluminescence when expressed in Escherichia coli . The sequence differences between these different luciferases are few, so the amino acids responsible for the different colours of emission must also be few . Through the construction of hybrid luciferases, by rearranging fragments of the original cDNA clones, we have identified some of these amino acid determinants of colour.

EMBO J, 1989 Jul, 8(7), 1927 - 34
Mutation in a heat-regulated hsp70 gene of Ustilago maydis; Holden DW et al.; Four genes (ums1, ums2, ums3 and ums4) representing an hsp70-related gene family were isolated from a genomic library of Ustilago maydis . All four genes are transcriptionally active during normal growth . Following a heat shock, the mRNA levels of ums1 and ums2 increase by approximately 5-fold, whereas the ums3 transcript becomes less abundant . The amount of ums4 mRNA remains relatively unchanged after heat treatment . The nucleotide sequence of the 5' non-coding and a portion of the ums2 coding region was determined . The sequence encoding the first 90 amino acids is 73% identical to corresponding regions of the Drosophila and yeast (SSA1) hsp70 genes . To investigate the effect of a mutation in ums2, a plasmid was constructed in which most of the transcriptional unit of ums2 was deleted and substituted with the Escherichia coli hygromycin B (hygB) phosphotransferase gene . Transcription of this gene is controlled by the ums2 promoter, allowing the expression of hygB resistance in Ustilago . The marker was introduced into diploid cells as a linear sequence with termini homologous to the 5' and 3' flanking regions of ums2 . In approximately 50% of transformants examined, one of the two wild-type ums2 alleles had been replaced by the mutated sequence, demonstrating the feasibility of using one-step gene disruption to create heterozygous diploids in Ustilago . The ums2/ums2::hygBr heterozygote produced teliospores after injection into corn plants, but only cells carrying functional ums2 were found among their meiotic progeny . Therefore ums2::hygBr segregates as a recessive lethal, which strongly suggests that ums2 is essential for growth in Ustilago.

Curr Genet, 1989 Jul, 16(1), 53 - 6
Improved transformation efficiency of Aspergillus niger using the homologous niaD gene for nitrate reductase; Campbell EI et al.; Aspergillus niger transformation frequencies of up to 1,176 transformants per micrograms DNA were achieved using the plasmid vector pSTA10 containing the A . niger nitrate reductase structural gene . Analysis of genomic endonuclease cleaved DNA from nitrate utilising transformants by DNA hybridisation, showed that most integration events are as a result of homologous recombination . The niaD transformation system was used successfully for the introduction of the unselected Escherichia coli fusion genes lacZ, encoding beta-galactosidase, and uidA, for beta-glucuronidase, as well as the Neurospora crassa tub-2 gene, for beta-tubulin . pSTA10 was also capable of transforming niaD mutants of other filamentous fungi such as A.nidulans, A . oryzae and Penicillium chrysogenum.

Thromb Res, 1989 Jul 1, 55(1), 87 - 97
Correlation between antigenic and functional expression of tissue factor on the surface of cultured human endothelial cells following stimulation by lipopolysaccharide endotoxin; Noguchi M et al.; Previous studies indicated that E . coli lipopolysaccharide (LPS) induces human umbilical vein endothelial cells (HUVEC) to express tissue factor activity . Using a radiolabeled anti-tissue factor monoclonal antibody to assess cell-surface tissue factor apoprotein antigen and a two-stage amidolytic assay to assess functional tissue factor activity, we have investigated the temporal relationship between antigenic expression and functional expression of tissue factor on the surface of LPS-stimulated HUVEC . Maximum tissue factor apoprotein antigenic expression on the surface of LPS-stimulated HUVEC was achieved in four hours after LPS treatment, while maximum functional tissue factor activity occurred after 6 hours . Specific binding of radiolabelled human factor VIIa to LPS-stimulated HUVEC paralleled the time course of the expression of tissue factor functional activity . Thus, these data indicate that the presence of of newly-synthesized tissue factor apoprotein antigen on the cell surface is insufficient by itself for maximal factor VIIa binding to occur, and provide presumptive evidence for the posttranslational processing of tissue factor apoprotein on the cell surface prior to its acquisition of ligand binding function.

Ann Rheum Dis, 1989 Jul, 48(7), 557 - 64
Characterisation of soluble and cell associated phospholipase A2 from rheumatoid synovial fluid; Gonzalez-Buritica H et al.; The hydrolysis of radiolabelled Escherichia coli phospholipids, and micellar dispersions of phosphatidylethanolamine and phosphatidylcholine, were used to characterise the phospholipase A2 activity in synovial fluid from patients with rheumatoid arthritis . Cell free fractions of synovial fluid contain a phospholipase A2 enzyme that preferentially releases {14C}oleic acid from E coli biomembranes (specific activity 291.3 (SEM 27.6) pmol/min/mg) . This enzyme requires calcium and is optimally active at neutral pH . Purified dispersions of phosphatidylethanolamine are also readily degraded by the soluble enzyme, but it is not active against phosphatidylcholine . The substitution of {14C}oleic acid by {3H}arachidonic acid for the labelling of E coli allowed differentiation between the soluble phospholipase A2 and the cell associated phospholipase A2 present in sonicates of mononuclear cells and neutrophils from peripheral blood and synovial fluid . The cell associated phospholipase A2 preferentially releases {3H}arachidonic acid from E coli cardiolipin . In this paper the phospholipid substrate specificity of phospholipase A2 from rheumatoid synovial fluid, the optimal assay conditions for its detection, and a standardised expression of activity in terms of pmol per minute per mg of protein are established.

Brain Res Mol Brain Res, 1989 Jul, 6(1), 1 - 10
Isolation of a novel retina-specific clone (MEKA cDNA) encoding a photoreceptor soluble protein; Kuo CH et al.; We have reported the isolation of clones which are candidates for retina-specific cDNAs . One of the cDNA clones, pCR-470, was further characterized . We found that mRNA corresponding to the pCR-470 was expressed only in the retina and encodes an unknown soluble protein whose molecular weight and pI are calculated to be 26,935 and 5.35, respectively . We designated it as a MEKA protein, because its amino acid sequence starts from M-E-K-A . It was found by in situ hybridization that MEKA mRNA was transcribed only in the photoreceptor cells and accumulated in the inner segments just like opsin mRNA . The MEKA cDNA was ligated with expression vector PEX 1, and a MEKA-fusion protein synthesized in E . coli was purified and used as an antigen . By the Western blot analysis anti-MEKA protein serum reacted with a soluble 32 kDa protein from bovine retina and 33 kDa for chick, but not with proteins from other tissues . Immunohistochemical study showed that anti-MEKA stained only the photoreceptor cells in bovine, chick, rat and mouse retinas.

Photochem Photobiol, 1989 Jul, 50(1), 75 - 84
Monofunctional angular furocoumarins: sequence specificity in DNA photobinding of 6,4,4'-trimethylangelicin and other angelicins; Miolo G et al.; The sequence specificity in the photoreaction (365 nm) of 6,4,4'-trimethylangelicin (TMA) with DNA fragments of the lac I gene of Escherichia coli was studied by using DNA sequencing methodology . In order to map the sites of TMA photoaddition, we took advantage of the (3'-5') exonuclease activity associated with T4 DNA polymerase, which is blocked by bulky adducts, such as furocoumarin photoadducts . A quantitative analysis of the sites of photoaddition is reported . TMA was demonstrated to photoreact with thymine and, to a lower extent, to cytosine . AT-rich sequences and TTT sites in a GC context are the most reactive sites towards TMA whereas TA, AT, CA, AC sites are weaker sites with similar reactivity . Cytosines in alternated CG sequences are also targets of TMA photobinding . We observed a less pronounced sequence specificity of TMA than that of other psoralen derivatives already studied (Sage and Moustacchi, 1987; Boyer et al., 1988) . A comparison with other furocoumarins 4,4'-dimethylangelicin (4,4'-DMA), 4'-methylangelicin (4'-MA), angelicin, 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) is also reported . The role of flanking sequence and consequently of the local conformation at the various sites of photoaddition is discussed . A preferential orientation of the TMA molecule during the intercalation in the dark is suggested . Hot alkali treatment of TMA-modified DNA did not reveal any DNA strand breakage due to photooxidized bases.

Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5478 - 82
Shuttle vectors for the archaebacterium Halobacterium volcanii; Lam WL et al.; Progress in archaebacterial molecular biology requires tools for genetic analysis . We describe vectors that can be selected and maintained in either Halobacterium volcanii or Escherichia coli . A genetic determinant for resistance to the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor mevinolin was isolated by "shotgun cloning" into a derivative of the endogenous H . volcanii plasmid pHV2, to form pWL2, which transforms sensitive H . volcanii to mevinolin resistance at high frequency . The resistance determinant, portions of pHV2, and an ampicillin- and tetracycline-resistance-conferring pBR322 derivative, pAT153, were ligated together to form the shuttle vectors pWL101 and pWL102 . We describe conditions for the use of these vectors and provide preliminary definition of regions essential for drug resistance and for plasmid replication and maintenance.

J Cell Biol, 1989 Jul, 109(1), 225 - 34
Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure; Stewart M et al.; We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated . An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E . coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared . A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin . Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate . These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm . Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm . More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat . The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly . Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.

Virology, 1989 Jul, 171(1), 264 - 6
The human immunodeficiency virus rev protein is a nuclear phosphoprotein; Cochrane A et al.; The human Immunodeficiency virus rev protein is one of several key regulatory proteins involved in the control of viral structural protein synthesis and replication . In this report, we identify the 20-kDa rev protein as a nuclear phosphoprotein . Post-translational modification was observed solely on serine residues . In vitro kinase reactions utilizing a derivative of Rev purified from Escherichia coli identified a nuclear kinase capable of phosphorylating exogenously added rev protein . Our results suggest that the nuclear kinase activity observed in vitro is likely to be responsible for the in vivo post-translational modification of Rev.

Crit Care Med, 1989 Jul, 17(7), 641 - 6
Effect of lipopolysaccharide on intestinal intramucosal hydrogen ion concentration in pigs: evidence of gut ischemia in a normodynamic model of septic shock; Fink MP et al.; We tested the hypothesis that lipopolysaccharide (LPS) leads to an imbalance between mesenteric oxygen delivery (DO2) and gut metabolic demand for oxygen, even when cardiac index (CI) is within the normal range . Two groups of pentobarbital-anesthetized pigs (13 to 17 kg) were studied . The first group (LPS; n = 9) was infused over 20 min with Escherichia coli LPS (100 micrograms/kg) and resuscitated with normal saline (1.2 ml/kg.min) . The second group (NS; n = 5) was not infused with LPS, but was resuscitated in the same way as the LPS group . Superior mesenteric arterial (SMA) blood flow and ileal intramucosal hydrogen ion concentration, {H+}, were determined using a Doppler-shift probe and a tonometric catheter, respectively . Infusing LPS did not affect CI, although mean arterial pressure and systemic vascular resistance were significantly reduced . SMA flow and mesenteric DO2 decreased significantly in the LPS group . Although mesenteric oxygen utilization was well preserved in both groups, ileal intramucosal {H+} was significantly higher in endotoxic animals . These data support the idea that mesenteric oxygen consumption is flow-limited in this clinically relevant porcine model of septic shock.

Biotechniques, 1989 Jul-Aug, 7(7), 722 - 8
Rapid generation of subclones for DNA sequencing using the reverse cloning procedure; Liu ZJ et al.; A fast and reliable procedure for generating subclones necessary for sequencing long stretches of DNA has been developed . The reverse cloning procedure involves cloning a fragment of DNA into a single-stranded plasmid or phage vector containing a polycloning region; synthesizing variable lengths of double-stranded DNA using a "Universal Primer"; isolating the double-stranded DNA; and force cloning the double-stranded DNA fragments into a complementary vector with the polycloning region in the reverse orientation . The resulting clones can be sequenced, using the same Universal Primer and T7 DNA polymerase, to provide overlapping DNA sequences . The reverse cloning procedure can be used to construct deletion mutations.

Mikrobiologiia, 1989 Jul-Aug, 58(4), 584 - 90
{Exchange of polyamines between the cell and the environment as one of the factors determining the growth of cultures of Escherichia coli with additional feeding}; Tkachenko AG et al.; Escherichia coli growth in a synthetic medium with glucose is accompanied by the interdependent change of putrescine content in the cell and its environment, which maintains a homeostatic equilibrium . The rate of putrescine efflux at the early stage of the culture growth is directly proportional to putrescine intracellular pool but the rate of putrescine uptake is inversely proportional to its value . The constitutive form of ornithine decarboxylase with the pH optimum 8.0 is responsible for putrescine synthesis . Glucose deficiency favours the utilisation of acidic glucose metabolites but the homeostatic system of putrescine makes its intracellular content remain at a high level, which stimulates constructive processes in the following growth.

J Gen Microbiol, 1989 Jul, 135 ( Pt 7), 1941 - 7
Interaction of the fluorescent dye 1-N-phenylnaphthylamine with Escherichia coli cells during heat stress and recovery from heat stress; Tsuchido T et al.; The fluorescent dye 1-N-phenylnaphthylamine permeated Escherichia coli cells after exposure to a heat stress at 55 degrees C in Tris/Mg2+ buffer, pH 8.0 . The rate of dye permeation increased with time during heat treatment and decreased gradually during subsequent incubation at 37 degrees C in a minimal medium . The initial level of rapid adsorption of the dye also increased with heating time, although it remained roughly constant during post-heating incubation . The results obtained suggest that the permeability barrier to the dye in the outer membrane was damaged by heat stress and was repaired after sublethal heating . RNA, protein and lipid syntheses, as well as an energy-yielding process, appeared to be necessary for the repair of impermeability to the dye.

Mikrobiol Zh, 1989 Jul-Aug, 51(4), 7 - 10
{The effect of the oligomerization of pBR322 on the level of transformation in Escherichia coli}; Fomin VV et al.; The ability of the known Escherichia coli strain JC3881 recB recC recF sbc15 to produce oligomeric and multimeric forms of pBR322 underlies the study presented . The individual oligomeric forms of pBR322 were isolated from the agarose gel . The plasmid forms were used for electron microscopic control and also introduced into the system of E . coli competent cells . The E . coli transformation level of different forms of plasmid DNA rose from monomers to pentamers . CCC forms of the plasmid possessed high efficiency of the E . coli cell transformation . The systems of the host recombination are to be significant in the process of plasmid oligomerization.

J Cell Sci, 1989 Jul, 93 ( Pt 3), 439 - 46
Aggregation of membrane-associated actin filaments following localized adherence of enteropathogenic Escherichia coli to HeLa cells; da Silva ML et al.; We have previously observed that enteropathogenic Escherichia coli (EPEC) adhere to HeLa cells in a localized manner, which we designated localized adherence as opposed to the diffuse pattern of adhesion . In this paper we have examined the effects of localized adherence of EPEC on the actin microfilament system of host HeLa cells . Centrifugation of bacteria onto HeLa cells improved the localized adherence and rapid rearrangements of actin filaments were detected by immunofluorescence and electron microscopy . Aggregation of microfilaments is consistently observed at the sites of localized adherence, and is abolished by cytochalasin D and low temperatures . Scanning electron microscopy indicates that these aggregates are surface microvilli entangled with attached EPEC.

Mol Biol (Mosk), 1989 Jul-Aug, 23(4), 996 - 1006
{A study of the intracellular fate of recombinant human interleukin 2 in Escherichia coli}; Meriin AB et al.; The expression of the human IL-2 recombinant gene in E . coli cells was studied . The processes which take place during thermo-induced expression and effect the state of the product were investigated . Experimental data on the membrane localisation of IL-2, the formation of aggregates (inclusion bodies) and polymers were obtained . It was determined that temperature significantly influence the kinetics of the processes of intracellular IL-2 production and IL-2 stability . It is supposed that the cell membrane state plays a determining role in these processes via temperature mediation . Thus, the formation of inclusion bodies described for a number of E . coli recombinant strains is probably stipulated not only by recombinant polypeptides properties, but also by cellular interactions.

Mol Biol (Mosk), 1989 Jul-Aug, 23(4), 1057 - 66
{Complementary addressed alkylation of 16s rRNA from Escherichia coli by 2',3'-0-{4-N-(2-chloroethyl)-N-methylamino}benzylidene derivatives of oligodeoxyribonucleotides . III . Segments of 16s rRNA interacting with the benzylidene derivative of d(pACCTTGTT)rA}; Zenkova MA et al.; 16S rRNA chain was cleaved by RNase H within covalent adduct with a 2',3'-0-{4-N-(2-chloroethyl)-N-methylamino}benzylidene derivative of d(pACCTTGTT)rA . It was shown that no less than 50% of cleavage occurs at the 1498-1506 site . The selectivity of alkylation and correspondingly the cleavage by RNase H, at this site practically does not increase when RNA was alkylated to a low extent, and also when a small excess of the reagent in respect to rRNA was used.

Prikl Biokhim Mikrobiol, 1989 Jul-Aug, 25(4), 558 - 64
{A fluorometric method of determining DNA in cells}; Ogievetskaia MM et al.; A convenient express method is proposed for cell lysis and DNA solubilization in the presence of sarcosyl and sodium citrate . The DNA content was determined in cell lysates by registration of fluorescence enhancement with Hoechst No 33258 ("Serva", FRG) . In 0.5 M NaCl solution the Hoechst-RNA fluorescence is negligible . An optimal molar ratio of DNA and Hoechst is in the range from 0.2 to 2.0 . Beyond the range the accuracy of DNA quantification become poor . An optimal range of DNA quantification in cells is 200-2000 ng/ml.

Mol Gen Mikrobiol Virusol, 1989 Jul, (7), 24 - 9
{UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells}; Golubovskaia VM et al.; Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E . coli strains . Uracil-containing DNA was prepared after growth of phage in an E . coli strain dut- ung- . The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase . Ung+ and ung- E . coli cells were transformed by DNA . In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50% . Absolute number of mutants was higher in ung+ cells . The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.

Biokhimiia, 1989 Jul, 54(7), 1075 - 81
{Level of Hu protein binding with DNA and the Hu/DNA ratio in Escherichia coli cells with various generation periods}; Kulyba NP et al.; The possibility of quantitative determination of protein HU in E . coli cell lysates was demonstrated, using enzyme immunoassay with monospecific polyclonal antibodies against HU and homogeneous protein HU . The protein HU/DNA ratio in two cultures of E . coli with different generation periods was found to be constant despite the differences in the levels of proteins HU and DNA . Protein HU was found to be represented by approximately 420.10(3) copies per fast growing . E . coli cell and by 320.10(3) copies per slowly growing cell . These amounts of the HU protein correspond to a ratio of one protein HU molecule per 44 +/- 8 base pairs of DNA and can provide for the nucleosome-like organization of approximately 30% of E . coli DNA length . The constant HU/DNA ratio which is similar to constant histones/DNA ratio provides additional evidence in favour of functional similarity of the HU E . coli DNA-binding protein to histones.

J Biolumin Chemilumin, 1989 Jul, 4(1), 351 - 6
Acridinium ester-labelled DNA oligonucleotide probes; Septak M; Chemiluminescent acridinium ester derivatives have been synthesized and covalently attached to suitably modified synthetic DNA oligonucleotides . Attachment of acridinium ester label to primary aliphatic amine group(s) present in the synthetic DNA probe molecule is rapid and efficient . Methods have been developed for efficient separation of acridinium ester-labelled DNA from unincorporated labelling reagent and underivatized DNA . The basic hydrogen peroxide detection reaction and photon counting conditions for measurement of chemiluminescence emission from acridinium ester-labelled DNA probes have been optimized . Under optimal conditions, the observed detection limit for the labelled DNA (1:1 mole ratio) is the same as for the free acridinium ester label, which is 2 attomole sensitivity in the best case studied.

Vet Immunol Immunopathol, 1989 Jul, 21(3-4), 261 - 78
Cloning and expression of the cDNA for bovine granulocyte-macrophage colony-stimulating factor; Leong SR et al.; A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library . This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence . This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E . coli . Periplasmic extracts contain a 14,500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells . The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF . Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall change . The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells . Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin . However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.

Vet Immunol Immunopathol, 1989 Jul, 21(3-4), 249 - 60
Sex-related differences in immune response and survival rate of broiler chickens; Leitner G et al.; In a comparison between male and female broiler chicks, the mortality rate of males was found to be significantly higher than that of females, starting from the second week of age until marketing at 7 or 8 weeks of age . The main causes of death during this period were various infectious diseases . This observation was explained by differences in the activity of humoral and cell-mediated immune responses between the sexes . In tests of antibody responses of young chicks to a variety of antigens (bacterial-E . coli, viral-Newcastle disease virus, and protein antigen-bovine serum albumin), females responded 24-72 h earlier than males and with higher peak antibody titers . In-vitro proliferation of T-lymphocytes to purified protein derivative and E . coli showed an earlier and greater response in females . The correlation between immune responsiveness and survival, as tested by challenging vaccinated chicks with pathogenic E . coli, showed a significantly higher mortality rate in vaccinated males, that was correlated with their lower antibody titer . We concluded, therefore, that sex-related differences in mortality rates of broiler chicks may result from a less efficient immune response in males.

Mol Microbiol, 1989 Jul, 3(7), 933 - 42
An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression; Bhriain NN et al.; The regulation of several genes in response to osmotic and anaerobic stress has been examined . We have demonstrated a clear overlap between these two regulatory signals . Thus, the osmotically induced proU and ompC genes require anaerobic growth for optimum induction while the anaerobically induced tppB gene is also regulated by osmolarity . Furthermore, normal expression of tppB and ompC requires the positive regulatory protein OmpR, yet this requirement can be partially, or even fully, overcome by altering the growth conditions . Finally, the pleiotropic, anaerobic regulatory locus, oxrC, is also shown to affect expression of the osmotically regulated proU gene . The oxrC mutation is shown to affect the level of negative supercoiling of plasmid DNA and its effects on gene expression can be explained as secondary consequences of altered DNA topology . We suggest that there is a class of 'stress-regulated' genes that are regulated by a common mechanism in response to different environmental signals . Furthermore, our data are consistent with the notion that this regulatory overlap is mediated by changes in DNA supercoiling in response to these environmental stresses.

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