Escherichia coli

Infect Immun, 1990 Sep, 58(9), 2815 - 20
Anti-idiotypic antibodies counteract the invasion inhibition capacity of antibodies to major epitopes of the Plasmodium falciparum antigen Pf155/RESA; Wahlin B et al.; Rabbits were immunized with the synthetic peptide EENVEHDA or (EENV)2, corresponding to a tandemly repeated sequence in the C-terminal part of the Plasmodium falciparum antigen Pf155/RESA, or with Escherichia coli-derived fusion proteins containing the corresponding repeats . For all sera, the capacity of the total immunoglobulin G fractions to inhibit P . falciparum merozoite invasion in vitro was similar and relatively low . Affinity purification of Pf155/RESA-specific antibodies on parasite-infected erythrocyte monolayers or on peptide columns increased the inhibitory capacity 50 to 5,000 times, whereas the immunofluorescence titers were increased only 10 times . The addition of small amounts of total immunoglobulin G to the affinity-purified antibodies gave a marked and dose-dependent reduction of the inhibitory capacity of the purified antibodies . However, this reduction was only seen in combinations where the immunoglobulin G fraction was from the same serum as the affinity-purified antibodies, suggesting that it was mediated by anti-idiotypic antibodies reacting with non-cross-reacting idiotopes of the invasion-inhibiting antibodies.

J Virol, 1990 Sep, 64(9), 4321 - 8
Template switching by reverse transcriptase during DNA synthesis; Luo GX et al.; The ability of reverse transcriptase to make template switches during DNA synthesis is implicit in models of retrovirus genome replication, as well as in recombination and oncogene transduction . In order to understand such switching, we used in vitro reactions with purified nucleic acids and enzymes . The assay system involved the use of an end-labeled DNA primer so as to allow the quantitation of elongation on a donor template relative to the amount of elongation achieved by template switching (by means of sequence homology) when an acceptor template RNA was added . We examined several variables that affected the efficiency of the reaction: (i) the reaction time, (ii) the relative amounts of acceptor and donor template, (iii) the extent of sequence overlap between the donor and acceptor templates, and (iv) the presence or absence of RNase H activity associated with the reverse transcriptase . The basic reaction, with RNA templates and normal reverse transcriptase, yielded as much as 83% template switching . In the absence of RNase H, switching still occurred but the efficiency was lowered . Also, when the donor template was changed from RNA to DNA, there was still switching; not surprisingly, this was largely unaffected by the presence or absence of RNase H . Finally, we examined the action of the RNase H on RNA templates after primary transcription but prior to template switching . We found that in most cases, both ends of the original RNA template were able to maintain an association with the DNA product . This result was consistent with the work of others who have shown that RNase H acts as an endonuclease.

Appl Microbiol Biotechnol, 1990 Sep, 33(6), 680 - 2
Cloning of a gene coding for phosphotransacetylase from Escherichia coli; Yamamoto-Otake H et al.; A phosphotransacetylase gene (pta) has been cloned from a genomic DNA library of Escherichia coli 1100, a derivative of strain K-12 . The phosphotransacetylase activities of pta+ plasmid-containing strains were amplified about 150-fold under control of the lac promoter . The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis . The pta gene was found to be downstream of ackA by a combination of restriction analysis and plasmid subcloning . It is located about 13 kb upstream of the purF-folC-hisT region of the chromosome.

Appl Microbiol Biotechnol, 1990 Sep, 33(6), 619 - 23
Effect of free-cell growth parameters on oxygen concentration profiles in gel-immobilized recombinant Escherichia coli; Huang J et al.; Oxygen concentration profiles in immobilized recombinant cells were measured using a microsensor . Experimental results showed that the final depth of oxygen penetration in the carrageenan gel was always in the range 80-100 microns for the strains and media used, although profiles of oxygen concentration during the early stages of cell growth depended on the strain and nutrient medium used . Variations in the oxygen profiles corresponded to differences in kinetic parameters measured for the same strains and media in free-cell experiments.

Appl Microbiol Biotechnol, 1990 Sep, 33(6), 611 - 8
Measurement of oxygen concentration gradients in gel-immobilized recombinant Escherichia coli; Hooijmans CM et al.; In this study, an oxygen microsensor was used to measure oxygen concentration profiles in carrageenan gel particles containing growing, immobilized Escherichia coli B (pTG201) . Profiles, which were measured at intervals during continuous culture of gel slabs and beads, became increasingly steep with time . The oxygen penetration depth in the gel decreased with time, eventually reaching a steady state value of approximately 100 microns for both gel beads and slabs . A reaction-diffusion model employing zero-order cell growth kinetics was found to provide an excellent fit to the experimental concentration data . Growth rates estimated from profiles obtained during the first few hours of culture were 0.24h-1 (gel slabs) and 0.18h-1 (beads), compared to a value of 0.30 h-1 measured in free-cell suspensions at 25 degrees C.

Biotechnology (N Y), 1990 Sep, 8(9), 849 - 53
Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli; Khosla C et al.; We have previously cloned the Vitreoscilla hemoglobin gene (VHb) and expressed the protein in Escherichia coli in its active form . Under oxygen-limited conditions the presence of VHb improves protein synthesis as indicated by both total protein content and the activity of an enzyme expressed from a cloned gene present on a multicopy plasmid . Measurements of nitrogen utilization rates corroborate the observation of enhanced protein synthesis; however, the rates of carbon consumption and acid synthesis remain unchanged . This suggests that the net effect of VHb in E . coli is to improve the efficiency, rather than the kinetics, of oxygen-limited aerobic metabolism . We propose two possible models for the mechanism of action of VHb: the facilitated diffusion hypothesis and the intracellular redox effector hypothesis . These suggest other systems in which cloned VHb may enhance bioprocess productivity.

Biotechnology (N Y), 1990 Sep, 8(9), 841 - 4
A new vector for cloning large eukaryotic DNA segments in Escherichia coli; Leonardo ED et al.; We investigated the relationship between plasmid size and electroporation efficiency in E . coli and found that even very large plasmids can be transfected efficiently . The efficiencies are well above the minimum required to construct representative libraries of complex eukaryotic genomes . To exploit this observation we constructed a novel mammalian-E . coli shuttle vector whose replication in E . coli is driven by the F sex factor episome origin . This new vector system should accept inserts well in excess of 100 kb, thus putting the cloning of mammalian genes by direct phenotypic complementation within reach.

Biotechnology (N Y), 1990 Sep, 8(9), 833 - 9
Inheritance and expression of chimeric genes in the progeny of transgenic maize plants; Fromm ME et al.; We obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells . Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E . coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation . When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants . With selection, transgenic plants were obtained from three of the other eight calli . One of the two initial lines produced 15 fertile transgenic plants . The progeny of these plants contained and expressed the foreign genes . Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film.

Biophys Chem, 1990 Aug 31, 37(1-3), 183 - 96
Amino acid substitutions which stabilize aspartate transcarbamoylase in the R state disrupt both homotropic and heterotropic effects; Newell JO et al.; We have used site-specific amino acid substitutions to investigate the linkage between the allosteric properties of arpartate transcarbamoylase and the global conformational transition exhibited by the enzyme upon binding active-site ligands . Two mutationally altered enzymes in which an amino acid substitution had been introduced at a single position in the catalytic polypeptide chain (Lys-164----Glu and Glu-239----Lys) and a third species harboring both of these substitutions (Lys-164:Glu-239----Glu:Lys) were constructed . Sedimentation velocity difference studies were performed in order to assess the effects of the amino acid substitutions on the quaternary structure of the holoenzyme in the absence and presence of various active-site ligands, including the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which has been shown previously to promote the allosteric transition . In the absence of ligand, two of the mutationally altered enzymes, Lys-164----Glu and Lys-164:Glu-239----Glu:Lys, existed in the R conformation, isomorphous with that of the PALA-liganded wild-type holoenzyme . These enzymes exhibited no conformational change upon binding PALA . The unliganded Glu-239----Lys enzyme had an average sedimentation coefficient intermediate between that of the unliganded and PALA-liganded states of the wild-type enzyme which could be accounted for in terms of a mixture of T- and R-state molecules . This mutant enzyme was converted to the fully swollen conformation upon binding PALA, phosphate or carbamoyl phosphate . The allosteric properties of the mutationally altered species were investigated by PALA-binding studies and by steady-state enzyme kinetics . In each case, the mutationally altered enzymes were devoid of both homotropic and heterotropic effects, supporting the premise that the allosteric properties of the wild-type enzyme are linked to a ligand-promoted change in quaternary structure.

J Chromatogr, 1990 Aug 31, 515, 483 - 94
Isolation and characterization of recombinant eel growth hormone expressed in Escherichia coli; Sugimoto S et al.; To obtain information about the microheterogeneity of recombinant protein, recombinant eel growth hormone II (EGH) analogues expressed in Escherichia coli were isolated and characterized . The modification was classified into three types: monodeamidation of Asn, oxidation of Met and N-terminal formylation . Monodeamidated EGH was isolated by ion-exchange chromatography . The major deamidation site (Asn 147) was determined by peptide mapping using the substrate specificity of trypsin . Oxidized EGH and N-terminal-formylated EGH were isolated by reversed-phase high-performance liquid chromatography . Oxidized EGH was identified by amino acid composition analysis and N-terminal-formylated peptide by mass spectrometry.

Science, 1990 Aug 31, 249(4972), 1044 - 6
Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase; Dean AM et al.; The isocitrate dehydrogenase of Escherichia coli is regulated by covalent modification at the active site rather than, as expected, at an allosteric site . As a means of evaluating the mechanism of regulation, the kinetics of the substrate, 2R,3S-isocitrate, and a substrate analog, 2R-malate, were compared for the native, phosphorylated, and mutant enzymes . Phosphorylation decreases activity by more than a factor of 10(6) for the true substrate, but causes minor changes in the activity of the substrate analog . The kinetic results indicate that electrostatic repulsion and steric hindrance between the phosphoryl moiety and the gamma carboxyl group of 2R,3S-isocitrate are the major causes of the inactivation, with a lesser contribution from the loss of a hydrogen bond.

Science, 1990 Aug 31, 249(4972), 1012 - 6
Regulation of an enzyme by phosphorylation at the active site; Hurley JH et al.; The isocitrate dehydrogenase of Escherichia coli is an example of a ubiquitous class of enzymes that are regulated by covalent modification . In the three-dimensional structure of the enzyme-substrate complex, isocitrate forms a hydrogen bond with Ser113, the site of regulatory phosphorylation . The structures of Asp113 and Glu113 mutants, which mimic the inactivation of the enzyme by phosphorylation, show minimal conformational changes from wild type, as in the phosphorylated enzyme . Calculations based on observed structures suggest that the change in electrostatic potential when a negative charge is introduced either by phosporylation or site-directed mutagenesis is sufficient to inactivate the enzyme . Thus, direct interaction at a ligand binding site is an alternative mechanism to induced conformational changes from an allosteric site in the regulation of protein activity by phosphorylation.

Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 169 - 74
Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin; Zenno S et al.; Aequorin is a photoprotein that emits light in the presence of Ca2+ ions . To develop a bioluminescent immunoassay based on the light emission property of aequorin, we have expressed the apoaequorin fusion protein with S . aureus protein A in E . coli by recombinant DNA techniques . The fusion protein expressed was purified by IgG-Sepharose affinity chromatography, gel filtration and HPLC procedures . The purified protein A-apoaequorin fusion protein has both the luminescent activity of aequorin and the IgG-binding ability of protein A . We compared results obtained using the protein A-aequorin fusion protein with those obtained using a protein A conjugated horseradish peroxidase based immunoassay, and found them to yield similar results.

J Chromatogr, 1990 Aug 31, 515, 563 - 72
High-performance liquid chromatography of transfer ribonucleic acids on spherical hydroxyapatite beads . II . Effects of pH and sodium chloride on chromatography; Yamakawa Y et al.; The effects of pH and sodium chloride on the high-performance liquid chromatography of transfer ribonucleic acids (tRNAs) on spherical hydroxyapatite beads were investigated . Binding of tRNAs on the beads was strengthened by increasing the pH of the mobile phase (phosphate buffer, pH 6.4-8.0), a phenomenon which is opposite to the retention of proteins on the beads . By using phosphate buffer of pH 8.0 instead of pH 6.8, the resolution of tRNAs was improved significantly; as many as ten times more theoretical plates were calculated with the use of the former buffer . Addition of sodium chloride to the phosphate buffer has a bifunctional effect on the retention of tRNAs: elution of tRNA(f-Met) and tRNA(Val) was retarded whereas that of tRNA(Phe) was facilitated.

Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 319 - 24
Properties of the exchange rate of guanine nucleotides to the novel rap-2B protein; Molina y Vedia L et al.; Rap-2B is a novel ras-related protein that is 89% identical to rap-2 at the amino acid level . Based on its amino acid sequence, it is anticipated that rap-2B binds guanine nucleotides . Here we show that purified, bacterially expressed rap-2B does bind both GTP and GDP in a Mg2(+)-dependent fashion . The relative affinity of rap-2B for GTP is higher than that for GDP, both at low and high concentrations of Mg2+ . This contrasts with N-ras p21 and could be of functional significance . Moreover, a polyclonal antiserum was raised against the recombinant rap-2B protein purified from E . coli lysates . This antiserum recognized a major protein of Mr approximately 21000 on Western blots of platelet membrane proteins, and immunoprecipitates rap-2B complexed with GTP or GDP.

Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 280 - 6
Cooperative mechanosensitive ion channels in Escherichia coli; Szabo I et al.; A patch-clamp investigation was carried out on giant Escherichia coli spheroplasts . The membrane exhibited stretch-induced as well as "spontaneous" activity, with similar characteristics, i.e., a large number of conductance values arising from the cooperative behavior of channels in functional clusters . It appears likely that the same molecular species are responsible for both stretch-induced and "spontaneous" current conduction; the channel multiplexes can either respond to membrane stretch or function in an activate state, presumably brought about by the previous application of the mechanical stimulus.

Biochemistry, 1990 Aug 28, 29(34), 7890 - 4
Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase; Hager DA et al.; A method for the purification of highly pure and active Escherichia coli RNA polymerase holoenzyme is described . This method is simple, reproducible, and can be performed at room temperature . The procedure involves the high-performance liquid chromatography of a partially purified RNA polymerase sample on a Mono Q ion-exchange column . Under the conditions used, RNA polymerase holoenzyme is well separated from the core RNA polymerase and other impurities . The purified RNA polymerase contains virtually no impurities as judged by SDS-polyacrylamide gel electrophoresis . The purified RNA polymerase holoenzyme contains the sigma 70 subunit in stoichiometric amounts and is at least 90% active.

Eur J Biochem, 1990 Aug 28, 192(1), 127 - 31
Expression of the chemically synthesized coding region for the cytotoxin alpha-sarcin in Escherichia coli using a secretion cloning vector; Henze PP et al.; The coding region for the cytotoxin alpha-sarcin from Aspergillus giganteus has been chemically synthesized by the ligation of 19 overlapping oligodeoxyribonucleotides . An Escherichia coli clone producing the cytotoxin was constructed by inserting the synthesized gene directly downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E . coli), using the secretion cloning vector pIN-III-OmpA2 . The enzyme encoded by the chemically synthesized gene expressed in E . coli displayed properties identical to those of native alpha-sarcin isolated from A . giganteus with respect to its chemistry, antigenicity and ribonucleolytic activity in qualitative assays.

Biochim Biophys Acta, 1990 Aug 28, 1046(1), 97 - 105
Characterization of FadL-specific fatty acid binding in Escherichia coli; Black PN; The product of the fadL gene (FadL) is a central component of the long-chain fatty acid transport system of Escherichia coli . When fatty acid activation is blocked by a mutation in the structural gene for acyl CoA synthetase (fadD) transport is inhibited allowing a FadL-specific fatty acid binding activity to be measured . This binding activity was 4- to 6-fold greater in the fadL+ fadD strain LS6928 when compared to the delta fadLfadD strain LS6929 . With long-chain fatty acids, this binding activity was saturable and it was estimated that there were approx . 35,000 FadL-specific oleic acid binding sites per cell in the fadL+ strain LS6928 . The FadL-specific fatty acid binding affinity was highest for oleic acid (18:1) and palmitic acid (16:0) giving apparent KD values of 2.3.10(-7) M and 8.8.10(-7) M, respectively . FadL-specific binding affinity of myristic acid (14:0) was nearly an order of magnitude less and no FadL-specific binding of decanoic acid (10:0) could be measured . Two lines of evidence suggest that FadL-fatty acid binding occurs by a hydrophobic interaction: (1) There was a preference for the long-chain substrates oleic acid and palmitic acid; and (2) oleic acid binding activity was not significantly changed over the pH range 5.0 to 8.0 . The FadL-specific binding of oleic acid in the fadL+ strain LS6928 could be blocked by preincubation with antisera raised against purified FadL providing a clear correlation between the activity and identity of FadL . The binding activity associated with FadL was measured in vesicles of the outer membrane following passage over the hydrophobic resin Lipidex 1000 . The KD of oleic acid binding attributable to FadL in outer membranes vesicles (6.0.10(-7) M) was in close agreement with that determined in whole cells . Overall, these studies demonstrated that FadL binds long-chain fatty acids with a relatively high affinity prior to their transport across the outer membrane.

Biochemistry, 1990 Aug 28, 29(34), 7882 - 90
RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage; Monforte JA et al.; We have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase . Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's or 3'-O-methylNTP's) . When the transcript can form the "hammerhead" structure it self-cleaves to give a truncated product . The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using {alpha-thio}ATP in place of ATP . We have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point . There are sequence-dependent as well as length-dependent effects . The results suggest that 12 +/- 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others . The results indicate that the "hammerhead" structure does not disrupt the hybrid . It appears that the RNA beyond the hybrid is not restrained by interactions with the enzyme, since the last stem of the self-cleaving structure forms as soon as the RNA composing it emerges from the DNA-RNA hybrid . Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes . The relevance of the results to models for transcription termination is discussed.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 93 - 7
Escherichia coli 30S mutants lacking protein S20 are defective in translation initiation; Gotz F et al.; The 30S ribosomal subunits derived from Escherichia coli TA114, a a temperature-sensitive mutant lacking ribosomal protein S20, were shown to be defective in two ways: (a) they have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits; (b) they are defective in functions which do not require their interaction with the large subunit (i.e., the formation of ternary complexes with aminocyl-tRNAs and templates, including the formation of 30S initiation complexes with fMet-tRNA and mRNA) . The 30S (-S20) subunits seem to interact normally with both template and aminoacyl-tRNA individually, but appear to be impaired in the rate-limiting isomerization step leading to the formation of a codon-anticodon interaction in the P site.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 84 - 92
The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex; Romby P et al.; The conformation of the E . coli initiator tRNA and of the 16S rRNA at different steps leading to the 30S.IF2.fMet-ARN(fMet).AUG.GTP complex has been investigated using several structure-specific probes . As compared to elongator tRNA, the initiator tRNA exhibits specific structural features in the anticodon arm, the T and D loops and the acceptor arm . Initiation factor 2 (IF2) interacts with the T-loop and the minor groove of the T stem of the RNA, and induces an increased flexibility in the anticodon arm . In the 30S initiation complex, additional protection is observed in the acceptor stem and in the anticodon arm of the tRNA . Within the 30S subunit, IF2 does not significantly shield defined portions of 16S rRNA, but induces both reduction and enhancement of reactivity scattered in the entire molecule . Most are constrained in a region corresponding to the cleft, the lateral protrusion and the part of the head facing the protrusion . All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG message . The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 8 - 13
The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques; Brimacombe R et al.; A large number of intra-RNA and RNA-protein cross-link sites have been localized within the 23S RNA from E . coli 50 S ribosomal subunits . These sites, together with other data, are sufficient to constrain the secondary structure of the 23 S molecule into a compact three-dimensional shape . Some of the features of this structure are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking techniques . A corresponding model for the 16S RNA within the 30 S subunit has already been described, and here a site-directed cross-linking approach is being used to determine the path followed through the subunit by messenger RNA.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 38 - 44
Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm; Wower J et al.; Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome . To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe . The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation . Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe . Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19 . Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material . Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 343 - 50
The translational regulation of threonyl-tRNA synthetase . Functional relationship between the enzyme, the cognate tRNA and the ribosome; Moine H et al.; The E . coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA . This region folds in four well-defined domains . The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors . The second site corresponds to a stable stem-loop structure located in domain IV . Both sites are separated by a large unpaired region (domain III) . In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process . The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site . tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 34 - 7
Dissection of the 16S rRNA binding site for ribosomal protein S4; Sapag A et al.; The ribosomal protein S4 from Escherichia coli is essential for initiation of assembly of 30S ribosomal subunits . We have undertaken the identification of specific features required in the 16S rRNA for S4 recognition by synthesizing mutants bearing deletions within a 460 nucleotide region which contains the minimum S4 binding site . We made a set of large nested deletions in a subdomain of the molecule, as well as individual deletions of nine hairpins, and used a nitrocellulose filter binding assay to calculate association constants . Some small hairpins can be eliminated with only minor effects on S4 recognition, while three hairpins scattered throughout the domain (76-90, 376-389 and 456-476) are essential for specific interaction . The loop sequence of hairpin 456-476 is important for S4 binding, and may be directly recognized by the protein . Some of the essential features are in phylogenetically variable regions; consistent with this, Mycoplasma capricolum rRNA is only weakly recognized by S4, and no specific binding to Xenopus laevis rRNA can be detected.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 337 - 42
Transcriptional organization of the S10, spc and alpha operons of Escherichia coli; Lindahl L et al.; We have investigated the transcription patterns at the inter-operon regions between the S10 and spc, and spc and alpha ribosomal protein operons of Escherichia coli . Newly synthesized transcripts were characterized by RNase T1 protection experiments, and accumulated transcripts were mapped with S1 nuclease . With both techniques we found that about 75% of the RNA polymerases transcribing the S10 operon terminated at the position of a typical rho-independent terminator . In contrast, most or all RNA polymerases transcribing the spc operon continued into the alpha operon . Nevertheless, we observed that about 30% of the transcripts of the alpha operon were initiated at the alpha operon promoter.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 328 - 36
Translational control of ribosomal protein S15; Portier C et al.; The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene . Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger . In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes . The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon . Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot . Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15 . A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 317 - 22
Mapping of two promoters for elongation factor Tu within the structural gene for elongation factor G; Zengel JM et al.; The str operon of Escherichia coli contains the genes for ribosomal proteins S7 and S12 as well as elongation factors G and Tu (EF-G, EF-Tu) . We have previously reported that there is a secondary promoter for expression of EF-Tu mapping within the upstream fus gene encoding EF-G (Zengel, J.M . and Lindahl, L . (1982) Mol . Gen . Genet . 185, 487-492) and have identified several potential promoter sequences within fus (Zengel, J.M., Archer, R.H . and Lindahl, L . (1984) Nucleic Acids Res . 12, 2181-2192) . We have now further characterized this promoter activity . Measurements of transcription rates from various regions of the str operon in cells carrying the fus gene and the beginning of the tufA gene on a high copy number plasmid confirmed that transcription was initiated within a 600 bp EcoRI fragment in the distal portion of the fus gene . Furthermore, T1 nuclease mapping studies identified two 5' ends within this region, one about 400 bases upstream of tufA, the other about 270 bases upstream, suggesting that there are two tufA promoters within the fus gene . Both of these promoters are active in the intact chromosomal str operon.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 302 - 6
Potential binding sites of the trans-activator FIS are present upstream of all rRNA operons and of many but not all tRNA operons; Verbeek H et al.; FIS, the Escherichia coli protein that stimulates the inversion of various DNA segments by binding to a recombinational enhancer, trans-activates a number of stable RNA operons and binds to the upstream activator sequence (UAS) of these operons (Nilsson et al . (1990) EMBO J . 9, 727) . In a search for potential FIS-binding sites we have compared UASs of other stable RNA operons with a consensus FIS-binding sequence, compiled by comparing recombinational enhancers . Such sites can thus be recognized upstream of all rRNA and 13 tRNA operons . Matching with the consensus sequence varied, suggesting that the affinity of FIS for the sites differed . Accordingly, FIS binding to an upstream sequence of the metY(nusA) operon was found to be weaker than that to the UAS of the thrU(tufB) operon . No FIS binding sites were found upstream three tRNA operons.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 259 - 62
Third position base changes in codons 5' and 3' adjacent UGA codons affect UGA suppression in vivo; Buckingham RH et al.; The base sequence around nonsense codons affects the efficiency of nonsense codon suppression . Published data, comparing different nonsense sites in a mRNA, implicate the two bases downstream of the nonsense codon as major determinants of suppression efficiency . However, the results we report here indicate that the nature of the contiguous upstream codon can also affect nonsense suppression, as can the third (wobble) base of the contiguous downstream codon . These conclusions are drawn from experiments in which the two Ser codons UCU233 and UCG235 in a nonsense mutant form (UGA234) of the trpA gene in Escherichia coli have been replaced with other Ser codons by site-directed mutagenesis . Suppression of these trpA mutants has been studied in the presence of a UGA nonsense suppressor derived from glyT . We speculate that the non-site-specific effects of the two adjacent downstream bases may be largely at the level of the termination process, whereas more site-specific or codon-specific effects may operate primarily on the activity of the suppressor tRNA.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 248 - 51
Growth rate dependence of global amino acid composition; Emilsson V et al.; The global amino acid composition of bacteria growing in different media has been studied . The data reveal significant changes in the amino acid composition in the growth rate range between 0.5 and 2.1 doublings per hour at 37 degrees C . The changes are consistent with a progressive simplification of the protein population and mRNA pools as the growth rates increase.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 235 - 40
Characterization of protein synthesis factors from rabbit reticulocytes; Merrick WC et al.; As part of our efforts to characterize eukaryotic translation factors, we have sequenced a number of them chemically and inferred sequences from cDNA clones . To our surprise, there appears to be extensive identity of amino acid sequence in most factors characterized to date in that within mammalian species, usually greater than 99% identity is observed . Extreme examples are rabbit EF-1 alpha which is 100% identical to human EF-1 alpha and rabbit eIF-4AI and eIF-4AII which are 100% identical to mouse eIF-4AI and eIF-4AII for those amino acids sequenced (398/406 and 156/407, respectively) . An extended analysis has been made of EF-1 alpha which in rabbit has three different post-translational modifications, dimethyllysine, trimethyllysine and glycerylphosphorylethanolamine . A comparison of the primary structure of EF-1 alpha to E . coli EF-Tu indicates an overall sequence identity of 33% . However, within the amino terminal 180 amino acids (the GTP-binding domain), there are found regions of much greater identity (50/85 = 59%).

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 226 - 9
The interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0; Abrahams JP et al.; The binding of Tyr-{AEDANS-s2C}tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E . coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation . The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM . These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system . The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis . The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin . These findings provide a clue for the observed increase in translational errors in strains carrying the mutations . Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 222 - 5
Ternary complexes of Escherichia coli aminoacyl-tRNAs with the elongation factor Tu and GTP: thermodynamic and structural studies; Ott G et al.; The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions . The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA . The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP . The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues . An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support . It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 14 - 7
Is there a special function for U.G basepairs in ribosomal RNA?
van Knippenberg PH, Formenoy LJ, Heus HA.
U.G basepairs are well-established elements of RNA structure . The geometry of this pair is different, however, from classical Watson-Crick basepairs . This leads to an unusual stacking of the basepair: overlap with the basepair at the 5' side of the U (and the 3' side of the G) is strong (stacked) while it is weak with the basepair on the other side (destacked) . The closure of an RNA helix by a U.G pair will be energetically unfavourable when the U residue occupies the 5' end . In transfer RNA there is a strong selection against a 'destacked' U.G pair at helix ends . In the 16S rRNA model of Escherichia coli there are 72 U.G pairs of which 36 or 22 occupy a helix end, depending on how such an end is defined . There is a slight preference for 'stacked' U.G's in these positions . It is remarkable, however, that of 13 very conserved U.G pairs in the 16S (-like) rRNA, 7 occur at helix ends and that 5 of these have the 'destacked' configuration . It is suggested that these pairs, if they exist at all in a hydrogen-bounded form, are stabilized by co-axial stacking with other helices or by interaction with a protein.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 110 - 8
Secondary structure at the 3' terminal region of RNA coliphages: comparison with tRNA; Adhin MR et al.; Secondary structure models for the 3' non-coding region of the four groups of coliphage RNA are proposed based on comparative sequence analysis and on previously published data on the sensitivity of nucleotides in MS2 RNA to chemical modification and enzymes . We report the following observations . (1) In contrast to the coding regions, the structure at the 3' terminus is characterized by stable regular helices . We note the occurrence of the loop sequences 5'-GUUCGC and 5'-CGAAAG, that are reported to confer exceptional stability to stem structures . These features are probably present to promote the segregation of mother and daughter strands during replication . (2) Comparison of homologous helices indicates that only those base pair substitutions are allowed that maintain the thermodynamic stability . (3) We have compared the structure of phage RNA with tRNA . Overall similarity is low, but one common element may exist . It is a quasi-continuous helix of 12 basepairs that could be the equivalent of the 12 basepair long coaxially stacked helix, formed by the T psi C arm and the aminoacyl acceptor arm in tRNA . As in tRNA, this structure element starts after the fourth nucleotide from the 3' end . (4) Phage RNA contains a large variable region of about 35 nucleotides bulging out from the quasi-continuous helix . We speculate that the variable loop in present-day tRNA could be the remnant of the variable region found in phage RNA . The variable region contains overlapping binding sites for the replicase enzyme and the maturation protein . This common binding site may serve as a switch from replication to packaging.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 45 - 50
Interaction of tRNA with domain II of 23S rRNA; Hill WE et al.; The interaction of tRNA with domain II of 23S rRNA in E . coli ribosomes has been probed using short, complementary DNA oligodeoxyribonucleotides . Specifically, cDNA oligomers to the region 801-811 of the 23S rRNA were used to ascertain the interaction of this region with tRNA . It was found that when tRNA was bound to the P site, considerable competition occurred between tRNA and the cDNA oligomers which base paired with the nucleotides 807-811 . However, A-site bound tRNA neither displaced, nor was displaced, by cDNA oligomers to this region . Additionally, the binding of tRNA lacking the CACCA nucleotides on the 3' terminus was unaffected by the presence a cDNA oligomer complementary to nucleotides 803-811, indicating that the cDNA-tRNA competition was dependent on the 3' terminal nucleotides of tRNA.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 193 - 6
Similarities and differences in the inhibition patterns of thiostrepton and viomycin: evidence for two functionally different populations of P sites when occupied with AcPhe-tRNA; Kutay UR et al.; According to the allosteric three-site model for the ribosomal elongation cycle, the reactions from the pre- to the post-translocational state and vice versa represent allosteric transitions which are catalyzed by elongation factor (EF)-G and EF-Tu, respectively . It has been shown recently that the non-related antibiotics thiostrepton and viomycin inhibit protein biosynthesis via a surprisingly similar mechanism . Both drugs primarily block the allosteric transitions in either direction (Hausner et al . (1988) J . Biol . Chem . 263, 13103-13111) . Here we show that the secondary effects of these antibiotics differ strikingly . When the P site of poly(U) programmed ribosomes is quantitatively filled with AcPhe-tRNA, thiostrepton stimulates the rate of the formation of AcPhe-puromycin 2-fold, whereas viomycin inhibits the puromycin reaction (up to 75% inhibition) . The thiostrepton-dependent stimulation is only observed when the drug is given before the P site is occupied; when thiostrepton is added after pre-filling the P site, the peptidyltransferase activity is not affected, in contrast to the translocation reaction, which is blocked irrespective of whether the drug is administered before or after tRNA is bound . The effects of both drugs became distinctly more pronounced when the P sites were saturated with AcPhe-tRNA as compared to half-saturated ribosomes . We conclude that roughly one half of the ribosomes, which first bind AcPhe-tRNA to the P site, carry this ligand in a different orientation to that of the second half of the ribosome population . These two populations probably reflect the P site in the pre- and post-translocational state, respectively.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 174 - 8
The translation system of mammalian mitochondria; O'Brien TW et al.; Oligoribonucleotides and mRNA were used to define properties of the bovine mitoribosomal mRNA binding site . The RNA binding domain on the 28 S subunit spans approx . 80 nucleotides of the template, based on ribosome protection experiments, but the major interaction with the ribosome occurs over a 30 nucleotide stretch . The binding site for E . coli IF3 is conserved in bovine mitoribosomes, but mitochondrial factors appear essential for proper interaction of mRNA with mitoribosomes . The small subunit of bovine mitoribosomes contains a high-affinity binding site for guanyl nucleotides, further indication of specialized mechanisms for initiation complex formation and function of mammalian mitochondrial ribosomes.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 307 - 11
Sequences upstream of the-35 hexamer of rrnB P1 affect promoter strength and upstream activation; Josaitis CA et al.; Transcription from Escherichia coli ribosomal RNA promoters is increased about 20-fold in vivo by a DNA sequence (the Upstream Activation Region, UAR) located upstream of the -35 conserved hexamer . The UAR stimulates transcription through two mechanisms: one which involves binding of the Fis protein to the UAR, and another mechanisms which functions in the absence of additional protein factors . We have previously constructed a collection of mutations in the region upstream of the -35 hexamer of rrnB P1 . Most of these mutations have either no effect on promoter activity or decrease activity 2-5-fold in vivo (Gaal, T., Barkei, J., Dickson, R.R., De Boer, H.A., De Haseth, P.L., Alavi, H . and Gourse, R.L.(1989) J . Bacteriol . 171, 4852-4861) . Two mutations leave both the -35 consensus hexamer and the Fis binding consensus sequence intact, yet have larger (14-50-fold) effects on transcription . One substitution just upstream of the -35 hexamer (a C to T change at position -37) primarily affects intrinsic promoter strength, leaving the UAR functional . On the other hand, a three base pair deletion (bases -38 through -40) severely reduces UAR-mediated activity . A substitution covering the three base pair deletion was constructed and found to be activated normally . UAR function appears dependent on its position relative to the RNA polymerase binding site, suggesting that a particular spatial geometry may be necessary for Fis-dependent and/or factor-independent activation to occur.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 215 - 21
Mutagenesis of the NH2-terminal domain of elongation factor Tu; Gumusel F et al.; Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity . The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain) . The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure . The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity . Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive . In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell . This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu . The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 209 - 14
Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu; Jurnak F et al.; Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures . The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed . Crystallization of the EF-Tu-GMPPNP complex is reported.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4883 - 90
Crosslinking of tRNA containing a long extra arm to elongation factor Tu by trans-diamminedichloroplatinum(II); Rasmussen NJ et al.; A tRNA containing a long extra arm, namely E . coli tRNA(Leu1) has been crosslinked to elongation factor Tu, with the crosslinking reagent trans-diamminedichloroplatinum(II) . The nucleotide involved in the crosslinking was identified to be a guanosine in the variable region at position 47F or 47G.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4677 - 82
The role of modified purine 64 in initiator/elongator discrimination of tRNA(iMet) from yeast and wheat germ; Kiesewetter S et al.; The role of 2'-ribosylated adenosine 64 in tRNA(iMet) from yeast in initiation/elongation discrimination was investigated . As measured by in vitro translation in rabbit reticulocyte lysate, the specific removal of the 2'-ribosylphosphate at adenosine 64 via periodate oxidation allows tRNA(iMet) to read internal AUG codons of the globine messenger RNA . Yeast Met-tRNA(iMet) lacking the modification of nucleoside 64 forms ternary complexes with GTP and elongation factor Tu from Escherichia coli . The lack of modification at position 64 does not prevent tRNA(iMet) from participating in the initiation process of in vitro protein synthesis . Wheat germ tRNA(iMet) has a 2'-ribosylated guanosine at position 64 . Removal of this modification from the wheat germ tRNA(iMet) enables it to read internal AUG codons of globine and tobacco mosaic virus messenger RNA in reticulocyte and wheat germ translation systems, respectively.

Ned Tijdschr Geneeskd, 1990 Aug 25, 134(34), 1651 - 3
{The pneumoportogram, a rare finding in septicemia due to an intra-abdominal abscess}; Leenen LP et al.; A man aged 73 is described with radiologically revealed air in the portal vein, a peumoportogram, on the basis of intra-abdominal sepsis . Characteristic of this finding are the air shadows extending far into the periphery of the liver in contrast to an air cholangiogram . It is an ominous sign, mostly secondary to severe intra-abdominal pathology such as ischaemic bowel necrosis, peritonitis or abscesses . Treatment should be aimed at these causes . Prognosis depends on the underlying pathology.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4659 - 64
Cloning and nucleotide sequence of the genes coding for the Sau96I restriction and modification enzymes; Szilak L et al.; The genes coding for the GGNCC specific Sau96I restriction and modification enzymes were cloned and expressed in E . coli . The DNA sequence predicts a 430 amino acid protein (Mr: 49,252) for the methyltransferase and a 261 amino acid protein (Mr: 30,486) for the endonuclease . No protein sequence similarity was detected between the Sau96I methyltransferase and endonuclease . The methyltransferase contains the sequence elements characteristic for m5C-methyltransferases . In addition to this, M.Sau96I shows similarity, also in the variable region, with one m5C-methyltransferase (M.SinI) which has closely related recognition specificity (GGA/TCC) . M.Sau96I methylates the internal cytosine within the GGNCC recognition sequence . The Sau96I endonuclease appears to act as a monomer.

J Biol Chem, 1990 Aug 25, 265(24), 14669 - 74
Contacts between the factor TUF and RPG sequences; Vignais ML et al.; The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres . We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments . As many as 10 base pairs appear to be engaged in factor binding . Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5 . Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13 . The derived consensus sequence was RCAAYCCRYNCAYY . A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1 . The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M . The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

J Biol Chem, 1990 Aug 25, 265(24), 14579 - 91
Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli; Polesky AH et al.; The Klenow fragment structure, together with many biochemical experiments, has suggested a region of the protein that may contain the polymerase active site . We have changed 7 amino acid residues within this region by site-directed mutagenesis, yielding 12 mutant proteins which have been purified and analyzed in vitro . The results of steady-state kinetic determinations of Km(dNTP) and kcat for the polymerase reaction, together with measurements of DNA binding affinity, suggest strongly that this study has succeeded in targeting important active site residues . Moreover, the in vitro data allow dissection of the proposed active site region into two clusters of residues that are spatially, as well as functionally, fairly distinct . Mutations in Tyr766, Arg841, and Asn845 cause an increase in Km(dNTP), suggesting that contacts with the incoming dNTP are made in this region . Mutations in the second cluster of residues, Gln849, Arg668, and Asp882, cause a large decrease in kcat, suggesting a role for these residues in catalysis of the polymerase reaction . The DNA-binding properties of mutations at positions 849 and 668 may indicate that the catalytic role of these side chains is associated with their interaction with the DNA substrate . Screening of the mutations in vivo for the classical polA-defective phenotype (sensitivity to DNA damage) demonstrated that a genetic screen of this type may be a reasonable predictor or kcat or of DNA binding affinity in future mutational studies.

J Biol Chem, 1990 Aug 25, 265(24), 14536 - 43
Molecular analysis of the Escherichia coli ferric enterobactin receptor FepA; Armstrong SK et al.; In Escherichia coli, the outer membrane protein FepA is a receptor for the siderophore complex ferric enterobactin and for colicins B and D . To identify protein domains important for FepA activity, the effects of deletion and linker insertion mutations on receptor structure and function were examined . In-frame internal deletion mutations removing sequences encoding up to 304 amino acid residues resulted in functionally defective FepA polypeptides, although most were translocated efficiently to the outer membrane . One exception, a derivative lacking 87 internal amino acid residues near the N terminus, showed an inability to transport ferric enterobactin but retained limited colicin receptor function . Analysis of cells carrying 3'-terminal fepA deletion mutations suggested that residues within the C terminus of FepA may be involved in secretion and proper translocation of the protein to the outer membrane . Introduction of the peptide Leu-Glu after FepA residues 55, 142, or 324 severely impaired receptor function for all three ligands, while the same insertion after residues 339 or 359 had virtually no detrimental effect on FepA function . Foreign peptides inserted after residues 204 or 635 restricted colicin B and D function only, leaving ferric enterobactin transport ability at near wild-type levels . The results presented in this study have identified key regions of FepA potentially involved in receptor function and demonstrate the presence of both shared and unique ligand-responsive domains.

J Biol Chem, 1990 Aug 25, 265(24), 14327 - 34
Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment; Mullen GP et al.; DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity . As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM) . All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons . The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM) . The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold . dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold . The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected . The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites . The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand . Mg2+ competes at this site with a KD of 100 microM . It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity . Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory . The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1990 Aug 25, 265(24), 14227 - 33
Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate; Stover P et al.; The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate . In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex . Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics . There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h . The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate . This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase . Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative . The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s . Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme . Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E . coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4867 - 76
Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A; Laquel P et al.; DNA primase has been partially purified from wheat germ . This enzyme, like DNA primases characterized from many procaryotic and eucaryotic sources, catalyses the synthesis of primers involved in DNA replication . However, the wheat enzyme differs from animal DNA primase in that it is found partially associated with a DNA polymerase which differs greatly from DNA polymerase alpha . Moreover, the only wheat DNA polymerase able to initiate on a natural or synthetic RNA primer is DNA polymerase A . In this report we describe in greater detail the chromatographic behaviour of wheat DNA primase and its copurification with DNA polymerase A . Some biochemical properties of wheat DNA primase such as pH optimum, Mn + 2 or Mg + 2 optima, and temperature optimum have been determined . The enzyme is strongly inhibited by KCI, cordycepine triphosphate and dATP, and to a lesser extent by cAMP and formycine triphosphate . The primase product reaction is resistant to DNAse digestion and sensitive to RNAse digestion . Primase catalyses primer synthesis on M13 ssDNA as template allowing E.coli DNA polymerase I to replicate the primed M13 single-stranded DNA leading to double-stranded M13 DNA (RF) . M13 replication experiments were performed with wheat DNA polymerases A, B, CI and CII purified in our laboratory . Only DNA polymerase A is able to recognize RNA-primed M13 ssDNA.

J Biol Chem, 1990 Aug 25, 265(24), 14195 - 201
Structure-function studies of adenine nucleotide transport in mitochondria . I . Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria; Lawson JE et al.; The gene encoding the major ADP/ATP carrier in yeast AAC2 (pet9; Lawson, J., and Douglas, M . (1988) J . Biol . Chem . 263, 14812-14818) has been disrupted (delta AAC2) by itself and in combination with a disruption of a second translocator gene AAC1 (delta AAC1) . Disruption of AAC2 like the pet9 mutation renders yeast unable to grow on a nonfermentable carbon source . The AAC1 AAC2 double disruption exhibits a phenotype identical to the AAC2 . This provides the host strain for the analysis of point mutations in the AAC protein . We have initiated this structure-function analysis by characterizing and confirming that the pet9 mutation is a G to A transition resulting in an arginine to histidine change at position 96 . Site-directed replacements at Arg96 confirm its essential function for growth on a nonfermentable carbon source . These data also suggest that in the absence of functional AAC1 and AAC2 gene products, adenine nucleotide transport across the mitochondrial inner membrane must occur by an as yet unidentified translocator or translocation mechanism or that within these cells separate intra- and extramitochondrial adenine nucleotide pools can exist to support growth.

J Biol Chem, 1990 Aug 25, 265(24), 14632 - 7
Rat liver mitochondrial ATP synthase . Effects of mutations in the glycine-rich region of a beta subunit peptide on its interaction with adenine nucleotides; Garboczi DN et al.; The beta subunit of the rat liver mitochondrial ATP synthase contains a glycine-rich amino acid sequence implicated in binding nucleotides by its similarity to a sequence found in many other nucleotide-binding proteins . A C-terminal three-quarter-length rat liver beta subunit fragment (Glu122 through Ser479), containing this homology region, interacts with adenine nucleotides (Garboczi, D.N., Hullihen, J.H., and Pedersen, P.L . (1988) J . Biol . Chem . 263, 15694-15698) . Here we directly test the involvement of the glycine-rich region in nucleotide binding by altering its amino acid sequence through mutation or deletion . Twenty-one mutations within the glycine-rich region of the beta subunit cDNA were randomly generated . Wild-type and mutant beta subunit proteins were purified from overexpressing Escherichia coli strains . The mutant proteins were screened for changes in their interaction with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), a fluorescent nucleotide analog . Only one mutant protein bearing two amino acid changes (Gly153----Val, Gly156----Arg) exhibited a fluorescence enhancement higher than that of the wild-type "control." Further analysis of this protein revealed a lower affinity for TNP-ATP (Kd = 10 microM) compared with wild type (Kd = 5 microM) . In addition, a mutant from which amino acids Gly149-Lys214 had been deleted was prepared . This mutant protein, which lacks the entire glycine-rich region, also displayed a marked reduction in affinity for TNP-ATP (Kd greater than 60 microM) . Prior addition of 0.5 mM ATP significantly reduced the binding of TNP-ATP to both the double and deletion mutants . The altered interaction of nucleotide with both glycine-rich region mutants points to the involvement of this region in the binding site . Further, this work shows that a beta subunit protein that lacks the glycine-rich homology region can still interact with nucleotide, indicating that one or more additional regions of this subunit contribute to the nucleotide binding site.

J Biol Chem, 1990 Aug 25, 265(24), 14606 - 11
Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli; Wilhelm OG et al.; The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli . The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity . {35S}Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1 . The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively . Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues . We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid . The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction . The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site) . Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4695 - 701
Identification and characterization of a Dictyostelium discoideum ribosomal protein gene; Szymkowski DE et al.; We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins . Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium . The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins . Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome . Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle . This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated . To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide . These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome.

Science, 1990 Aug 24, 249(4971), 884 - 91
De novo design, expression, and characterization of Felix: a four-helix bundle protein of native-like sequence; Hecht MH et al.; The protein Felix was designed de novo to fold into an antiparallel four-helix bundle of specific topology . Its sequence of 79 amino acid residues is not homologous to any known protein sequence, but is "native-like" in that it is nonrepetitive and contains 19 of the 20 naturally occurring amino acids . Felix has been expressed from a synthetic gene cloned in Escherichia coli, and the protein has been purified to homogeneity . Physical characterization of the purified protein indicates that Felix (i) is monomeric in solution, (ii) is predominantly alpha-helical, (iii) contains a designed intramolecular disulfide bond linking the first and fourth helices, and (iv) buries its single tryptophan in an apolar environment and probably in close proximity with the disulfide bond . These physical properties rule out several alternative structures and indicate that Felix indeed folds into approximately the designed three-dimensional structure.

Biochim Biophys Acta, 1990 Aug 24, 1027(2), 155 - 62
Lipid and peptide specificities in signal peptide--lipid interactions in model membranes; Demel RA et al.; The present data show the critical importance of the anionic lipid content in monomolecular layers for the interaction with PhoE signal peptide . At 37 degrees C and 100 mM NaCl the interaction is maximal at 30-40 mol% anionic lipid . The results correlate with the reduced translocation competence of Escherichia coli strain HD3122, which has a much lower anionic lipid content as compared to the wild-type strain SD12 (De Vrije et al . (1988) Nature 334, 173-175) . PhoE signal peptide analogs as N-formyl PhoE signal peptide, PhoE signal peptide +(1-7) and PhoE signal peptide Val-8----Trp-8 show the same lipid preference as PhoE signal peptide . On the other hand the affinity for an anionic lipid interface is strongly reduced for PhoE signal peptide Lys-19,-20----Asp-19,-20, which correlates with the less efficient translocation of PhoE protein carrying this signal sequence . At limiting anionic lipid concentrations there is a temperature and salt effect on the observed interaction, which is related to a conformational change of the peptide . Signal sequences show clearly conformational flexibility in responds to environmental conditions . Under the conditions used in this study FTIR spectra of PhoE signal peptide-DOPG monolayers show a high content of beta-structure and beta-turn.

Cell, 1990 Aug 24, 62(4), 733 - 43
Stringent spacing requirements for transcription activation by CRP; Gaston K et al.; The cyclic AMP receptor protein-cAMP complex (CRP-cAMP) binds at a variety of distances upstream of several E . coli promoters and activates transcription . We have constructed a model system in which a consensus CRP binding site is placed at different distances upstream of the melR promoter . CRP-cAMP activates transcription from melR when bound at a number of positions, all of which lie on the same face of the DNA helix . The two distances at which transcription is strongly activated correspond exactly to those at which CRP-cAMP binds upstream of the well-studied galP1 and lac promoters . Footprinting of the synthetic promoters reveals that RNA polymerase makes identical contacts with their -10 regions even though CRP-cAMP binds at a different distance in each case . Kinetic analysis in vitro indicates that CRP-cAMP activates transcription from these promoters in similar but distinct ways . A model is proposed to explain this two-position activation.

Cell, 1990 Aug 24, 62(4), 745 - 55
Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases; Cherniack AD et al.; Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns . Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E . coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs . This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing . Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation . Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.

Biochemistry, 1990 Aug 21, 29(33), 7715 - 27
Absolute action spectrum of E-FADH2 and E-FADH2-MTHF forms of Escherichia coli DNA photolyase; Payne G et al.; Escherichia coli DNA photolyase mediates photorepair of pyrimidine dimers occurring in UV-damaged DNA . The enzyme contains two chromophores, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolylpolyglutamate (MTHF) . To define the roles of the two chromophores in the photochemical reaction(s) resulting in DNA repair and the effect of DNA structure on the photocatalytic step, we determined the absolute action spectra of the enzyme containing only FADH2 (E-FADH2) or both chromophores (E-FADH2-MTHF), with double- and single-stranded substrates and with substrates of different sequences in the immediate vicinity of the thymine dimer . We found that the shape of the action spectrum of E-FADH2 matches that of the absorption spectrum with a quantum yield phi (FADH2) = 0.69 . The action spectrum of E-FADH2-MTHF is also in a fairly good agreement with the absorption spectrum with phi (FADH2-MTHF) = 0.59 . From these values and from the previously established properties of the two chromophores, we propose that MTHF transfers energy to FADH2 with a quantum yield of phi epsilon T = 0.8 and that 1FADH2 singlet transfers an electron to or from the dimer with a quantum yield phi ET = 0.69 . The chemical nature of the chromophores did not change after several catalytic cycles . The enzyme repaired a thymine dimer in five different sequence contexts with the same efficiency . Similarly, single- and double-stranded DNAs were repaired with the same overall quantum yield.

Biochemistry, 1990 Aug 21, 29(33), 7691 - 701
Structural consequences of effector binding to the T state of aspartate carbamoyltransferase: crystal structures of the unligated and ATP- and CTP-complexed enzymes at 2.6-A resolution; Stevens RC et al.; The crystal structure of Escherichia coli aspartate carbamoyltransferase complexed with adenosine 5'-triphosphate (ATP) has been solved by molecular replacement and has been refined to a crystallographic residual of 0.17 at 2.6-A resolution by using the computer program X-PLOR . The unit cell dimensions of this crystal form are a = b = 122.2 A and c = 143.3 A and the space group is P321 . Although the c-axis unit cell dimension is approximately 1 A longer than the corresponding dimension of the CTP-ligated P321 crystal form (c = 142.2 A), the ATP-ligated enzyme adopts a T-like quaternary structure . The base moiety of ATP interacts with residues Glu10, Ile12, and Lys60 while the ribose is near Asp19 and Lys60; the triphosphate entity is bound to Lys94, although His20 and Arg96 are nearby . We observe a higher occupancy for ATP in the allosteric site of the R1 regulatory chain in comparison to the occupancy of the R6 allosteric site . These crystallographically independent sites are related by a molecular 2-fold axis . There are other violations of the noncrystallographic symmetry that are similar to those observed in the refined CTP-ligated aspartate carbamoyltransferase structure . These infringements on the molecular symmetry might be the result of intermolecular interactions in the crystal . To ensure the most meaningful comparison with the ATP-ligated structure, we refined the previously reported CTP-bound and unligated structures to crystallographic residuals between 0.17 and 0.18 using X-PLOR . These X-PLOR refined structures are not significantly different from the initial structures that had been crystallographically refined by a restrained least-squares method . After making all possible comparisons between the CTP- and ATP-ligated and the unligated T-state structures, we find that the most significant differences are located at the allosteric sites and in small changes in the quaternary structures . At the allosteric site, the binding of CTP and ATP successively enlarges the nucleotide binding cavity, particularly in the vicinity of the base . The changes in the quaternary structure can be characterized by an increase in the separation of the catalytic trimers by approximately 0.5 A as ATP binds to the unligated T structure . On the basis of these structural studies, we discuss the relationships between the conformational differences in the allosteric site and the small changes in the quaternary structure within the T form to the possible mechanisms for CTP inhibition and ATP activation.

Biochemistry, 1990 Aug 21, 29(33), 7666 - 76
Inhibition of recA protein promoted ATP hydrolysis . 1 . ATP gamma S and ADP are antagonistic inhibitors; Lee JW et al.; ADP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) inhibit recA protein promoted ATP hydrolysis by fundamentally different mechanisms . In both cases, at least two modes of inhibition are observed . For ADP, the first mode is competitive inhibition . The second mode is manifested by dissociation of recA protein from DNA . These are readily distinguished in a comparison of ATP hydrolyses that are activated by (a) DNA and (b) high (approximately 2 M) salt concentrations . Competitive inhibition with a significant degree of cooperativity is observed under both sets of conditions, although the DNA-dependent activity is more sensitive to ADP than the high-salt reaction . The reaction in the presence of poly(deoxythymidylic acid) or duplex DNA ceases when about 60% of the available ATP is hydrolyzed, reflecting an ADP-mediated dissociation of recA protein from the DNA that is governed by the ADP/ATP ratio . In contrast, ATP hydrolysis proceeds nearly to completion at high salt concentrations . At high concentrations of ATP and ATP gamma S, ATP gamma S also acts as a competitive inhibitor . At low concentrations of ATP gamma S and ATP, however, ATP gamma S activates ATP hydrolysis . These patterns are observed for recA-mediated ATP hydrolysis with either high salt concentrations or a poly(deoxythymidylic acid) {poly(dT)} cofactor, although the activation is observed at much lower ATP and ATP gamma S concentrations when poly(dT) is used . ATP gamma S can also relieve the inhibitory effect of ADP under some conditions . ATP gamma S and ADP are antagonistic inhibitors, reinforcing the idea that they stabilize different conformations of the protein and suggesting that these conformations are mutually exclusive . The ATP gamma S (ATP) conformation is active in ATP hydrolysis . The ADP conformation is inactive.

Biochemistry, 1990 Aug 21, 29(33), 7563 - 71
An essential proline in lambda repressor is required for resistance to intracellular proteolysis; Reidhaar-Olson JF et al.; Pro78 is a solvent-exposed residue at the N-terminal end of alpha-helix 5 in the DNA binding domain of lambda repressor . Random mutagenesis experiments have suggested that Pro78 is essential {Reidhaar-Olson, J.F., & Sauer, R.T . (1990) Proteins: Struct., Funct., Genet . (in press)} . To investigate the requirement for proline at this position, we constructed and studied the properties of a set of ten position 78 mutant proteins . All of these mutants have decreased intracellular activities and are expressed at significantly lower levels than wild type . Pulse-chase experiments show that the mutant proteins are rapidly degraded in the cell; the mutants examined had half-lives of 11-35 min, whereas the wild-type protein has a half-life of greater than 10 h . The rapid degradation of position 78 mutants is not suppressed by mutations that affect known Escherichia coli proteases . The Pro78----Ala mutant could be overexpressed in a dnaJ- strain and was purified . This mutant has full DNA binding activity in vitro, suggesting that its folded structure and ability to form active dimers are similar to those of wild type . The PA78 mutant (Tm = 48 degrees C) is less thermally stable than wild type (Tm = 55 degrees C) . Double-mutant studies show that this instability contributes to but is not the main cause of its rapid intracellular degradation and also suggest that proteolysis proceeds from the denatured forms of proteins containing the PA78 substitution . The PA78 mutation does not appear to introduce a new cleavage site for cellular proteases, nor does the mutation enhance susceptibility to proteases such as thermolysin and trypsin in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Aug 21, 29(33), 7557 - 63
Mutation of essential catalytic residues in pig citrate synthase; Alter GM et al.; Two amino acid residues, His274 and Asp375, were replaced singly in the active site of pig citrate synthase (PCS) with Gly274, Arg274, Gly375, Asn375, Glu375, and Gln375 . The nonmutant protein and the mutant proteins were expressed in and purified from Escherichia coli, and the effects of these amino acid substitutions on the overall reaction rate and conformation of the PCS protein were studied by initial velocity and full time course kinetic analysis, behavior during affinity column chromatography, and monoclonal antibody reactivity . Native and mutant proteins purified similarly had a subunit molecular weight of 50,000 and were homologous when examined with 10 independent a-PCS monoclonal IgGs or with a polyclonal anti-PHCS serum . No activity was detected for Asn375 or Gln375 . The kcats of the other purified mutant proteins, however, were decreased by about 10(3) compared to the nonmutant enzyme activity . The Km for oxalacetate was decreased 10-fold in the Glu375 protein and was reduced by half in Gly274 and Arg274 PCSs, while the Km for acetyl-CoA was decreased 2-3-fold in Gly274, Arg274, and Gln375 PCSs . A mechanism is proposed that electrostatically links His274 and Asp375.

FEBS Lett, 1990 Aug 20, 269(1), 26 - 8
The influence of oligonucleotide-effector on the selectivity of sequence specific modification of 16 S rRNA; Zenkova MA et al.; The influence of duplex stabilizing oligonucleotide-effector (oligonucleotide, carrying N-(2-hydroxyethyl)phenazinium residues on both ends), on selectivity of site-directed modification of E . coli 16 S rRNA (1542 nucleotides in length) under the conditions of its secondary structure stability was studied . The constant of cooperative binding of the reagent and the oligonucleotide-effector with 16 S rRNA was determined . The accuracy of modification was shown to double in the presence of 50 microM effector at 5 microM concentration of the reagent.

J Mol Biol, 1990 Aug 20, 214(4), 937 - 48
Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate; Matthews DA et al.; The structure of the Escherichia coli thymidylate synthase (TS) covalent inhibitory ternary complex consisting of enzyme, 5-fluoro-2'-deoxyuridylate (FdUMP) and 5,10-methylene tetrahydrofolate (CH2-H4PteGlu) has been determined at 2.5 A resolution using difference Fourier methods . This complex is believed to be a stable structural analog of a true catalytic intermediate . Knowledge of its three-dimensional structure and that for the apo enzyme, also reported here, suggests for the first time how TS may activate dUMP and CH2-H4PteGlu leading to formation of the intermediate and offers additional support for the hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the substrate nucleotide and N-5 of the cofactor . By correlating these structural results with the known stereospecificity of the TS-catalyzed reaction it can be inferred that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the bond linking C-5 of dUMP to C-11 of the cofactor . The elimination itself may be catalyzed by proton transfer to the cofactor's 5 nitrogen from invariant Asp169 buried deep in the TS active site . The juxtaposition of Asp169 and bound tetrahydrofolate in TS is remarkably reminiscent of binding geometry found in dihydrofolate reductase where a similarly conserved carboxyl group serves as a general acid for protonating the corresponding pyrazine ring nitrogen of dihydrofolate.

J Mol Biol, 1990 Aug 20, 214(4), 923 - 36
Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2'-deoxyuridylate and 10-propargyl-5,8-dideazafolate; Matthews DA et al.; The crystal structure of an Escherichia coli thymidylate synthase (TS) ternary complex containing 5-fluoro-2'-deoxyuridylate (FdUMP) and 10-propargyl-5,8-dideazafolate (PDDF) has been determined and refined at 2.3 A resolution . Each of the two chemically identical subunits folds into a three-layer domain anchored by a large six-stranded mixed beta-sheet . The backside of one sheet is juxtaposed against the corresponding face of the equivalent sheet in the second protomer creating a beta-sandwich . In contrast to other proteins of known structure in which aligned beta-sheets stack face to face with a counterclockwise rotation, sheets in the TS dimer are related by a clockwise twist . The substrate-binding pocket is a large funnel-shaped cleft extending some 25 A into the interior of each subunit and is surrounded by 30 amino acids, 28 from one subunit and two from the other . FdUMP binds at the bottom of this pocket covalently linked through C-6 to the sulfur of Cys146 . Up-pointing faces of the pyrimidine and ribose rings are exposed to provide a complementary docking surface for the quinazoline ring of PDDF . The quinazoline inhibitor binds in a partially folded conformation with its p-aminobenzoyl glutamate tail exposed at the entrance to the active site cleft . Ternary complex formation is associated with a large conformational change involving four residues at the protein's carboxy terminus that close down on the distal side of the inhibitor's quinazoline ring, capping the active site and sequestering the bound ligands from bulk solvent.

J Mol Biol, 1990 Aug 20, 214(4), 845 - 64
Closely spaced and divergent promoters for an aminoacyl-tRNA synthetase gene and a tRNA operon in Escherichia coli . Transcriptional and post-transcriptional regulation of gltX, valU and alaW; Brun YV et al.; The transcription of the gltX gene encoding the glutamyl-tRNA synthetase and of the adjacent valU and alaW tRNA operons of Escherichia coli K-12 has been studied . The alaW operon containing two tRNA(GGCAla) genes, is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand . The valU operon, containing three tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN) genes, is adjacent to gltX and is transcribed from the opposite strand . Its only promoter is upstream from the gltX promoters . The gltX gene transcript is monocistronic and its transcription initiates at three promoters, P1, P2 and P3 . The transcripts from one or more of these promoters are processed by RNase E to generate two major species of gltX mRNA, which are stable and whose relative abundance varies with growth conditions . The stability of gltX mRNA decreases in an RNase E- strain and its level increases with growth rate about three times more than that of the glutamyl-tRNA synthetase . The 5' region of these mRNAs can adopt a stable secondary structure (close to the ribosome binding site) that is similar to the anticodon and part of the dihydroU stems and loops of tRNA(Glu), and which might be involved in translational regulation of GluRS synthesis . The gltX and valU promoters share the same AT-rich and bent upstream region, whose position coincides with the position of the upstream activating sequences of tRNA and rRNA promoters to which they are similar . This suggests that gltX and valU share transcriptional regulatory mechanisms.

J Mol Biol, 1990 Aug 20, 214(4), 825 - 43
Precise mapping and comparison of two evolutionarily related regions of the Escherichia coli K-12 chromosome . Evolution of valU and lysT from an ancestral tRNA operon; Brun YV et al.; Two tRNA operons have been found near the gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli K-12 . The alaW operon previously undetected from genetic data and containing two identical tRNA(GGCAla) genes is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand . The valU operon containing genes for three identical tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN), is adjacent to gltX and is transcribed from the opposite strand . Five open reading frames were also found in this region encoding putative polypeptides of 62, 105, 130, 167 and 294 amino acid residues . ORF294 is a new member of the lysR family of bacterial transcriptional activators . The possibility that this is the xapR gene is discussed . Comparison of the physical and linkage maps of the E . coli chromosome in the 52 minute region has permitted precise mapping of most of the 18 genes in this region with the order nupC-glk- less than (alaW beta-ala W alpha)-1 kb- less than gltX-0.3 kb-(valU alpha-valU beta-valU gamma-lysV = supN) greater than xapR-xapA- less than lig-1 kb-cysK greater than -0.4 kb-ptsH greater than -0.05 kb-pstI greater than -0.05 kb-crr greater than -cysM-cysA in the clockwise order (greater than and less than indicate the direction of transcription; kb, 10(3) bases) . The last two genes of valU (52 min) and lysT (16.5 min) are arranged in a similar fashion and a highly conserved region has been found in both operons . This suggests that the valU and lysT operons probably arose by a duplication of an ancestral tRNA operon . This is the first example of what may be two different tRNA operons from the same organism evolving from an ancestral tRNA gene . Comparison of the 16 and 52 minute regions of the E . coli K-12 chromosome suggests that these two regions could share a common ancestor.

FEBS Lett, 1990 Aug 20, 269(1), 96 - 100
SecE-dependent overproduction of SecY in Escherichia coli . Evidence for interaction between two components of the secretory machinery; Matsuyama S et al.; The secY and secE genes were individually cloned and placed under the control of the tac promoter on plasmids . Induction with isopropyl-beta-D-thiogalactopyranoside resulted in the overproduction of SecE, but not that of SecY . The simultaneous induced expression of both genes in the same cells resulted in the overproduction of SecY together with that of SecE . SecY and SecE thus overproduced were localized in the cytoplasmic membrane as those expressed at the normal levels were . It is suggested that SecY and SecE interact with each other in the cytoplasmic membrane . The numbers of the SecY and SecE molecules per cell were estimated.

FEBS Lett, 1990 Aug 20, 269(1), 113 - 6
A protein with sequence identity to Skp (FirA) supports protein translocation into plasma membrane vesicles of Escherichia coli; Thome BM et al.; We have purified to homogeneity a 15 kDa-protein from a ribosomal salt extract of Escherichia coli that compensates in vitro a defect of SecA but not of SecB . Removal of this protein from a cell-free transcription/translation system impairs translocation into plasma membrane vesicles of the precursors of LamB and to a lesser degree also of OmpA . These results suggest a role of the 15 kDa-protein in bacterial protein export . The NH2-terminal 35 amino acids were found to be identical to those of the skp (firA) gene product, to which several putative functions have previously been attributed.

J Mol Biol, 1990 Aug 20, 214(4), 885 - 96
Tropomyosin has discrete actin-binding sites with sevenfold and fourteenfold periodicities; Hitchcock-DeGregori SE et al.; Analysis of the periodic distribution of amino acids in tropomyosin has revealed the presence of seven or 14 quasi-equivalent actin-binding sites . We tested the hypothesis of periodic actin-binding sites by making deletions of chicken striated alpha-tropomyosin cDNA using oligonucleotide-directed mutagenesis . The deletions corresponded to one-half (amino acid residues 47 to 67), two-thirds (residues 47 to 74) and one actin-binding site (residues 47 to 88), on the basis of there being seven sites . The mutant cDNAs were expressed as fusion and non-fusion proteins in Escherichia coli and analyzed for actin binding and regulatory function . Fusion tropomyosin binds to actin with an affinity similar to that of muscle tropomyosin . Of the mutant fusion tropomyosins, only that with a full site deleted retained actin affinity and the ability to inhibit the actomyosin S1 ATPase, though it was less effective than wild-type . We conclude that an integral number of half-turns of the tropomyosin coiled-coil, and the consequential sevenfold periodicity, as well as the correct orientation of the ends with respect to each other, are important for actin binding . On the other hand, non-fusion tropomyosin binds well to actin only in the presence of troponin, and the binding is calcium-sensitive . Assay of non-fusion mutant tropomyosins showed that mutants with deletion of one-half and one actin binding site both had high affinity for actin, equal to or slightly less than wild-type . The ability of these two mutants to regulate the actomyosin or acto-S1 ATPase with troponin in the absence of calcium was indistinguishable from that of the wild-type . The normal regulatory function of the mutant with a 1/14 deletion (removal of a quarter turn or half a site) indicates that a 14-fold periodicity is adequate for regulation, consistent with the presence of two sets of seven alpha and seven beta quasi-equivalent actin-binding sites . An alternative explanation is that the alpha-sites are of primary importance and that proper alignment of the alpha-sites in every second tropomyosin, as when half a site is deleted, is sufficient for normal regulatory function . Deletion of a non-integral period (2/3 of a site) severely compromised actin-binding and regulatory function, presumably due to the inability of the mutant to align properly on the actin filament.

FEBS Lett, 1990 Aug 20, 269(1), 60 - 4
Characterization of a chemically synthesized RNA having the sequence of the yeast initiator tRNA(Met); Bratty J et al.; A 75-unit long oligoribonucleotide corresponding to the sequence of the Saccharomyces cerevisiae initiator tRNA was synthesized chemically . The crude RNA was purified, and the sequence was verified by RNA sequencing techniques . A particularly useful purification step involved hydrophobic chromatography on BND-cellulose . The purified RNA could be aminoacylated to 28% of a bona fide initiator tRNA(Met) sample and threonylated to 76% of the level observed with native tRNA(fMet) from E . coli.

Eur J Biochem, 1990 Aug 17, 191(3), 701 - 4
Fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli . Nature of bonds involved in the allosteric mechanism; Speranza ML et al.; The allosteric properties of the fructose-1,6-bis-phosphate-activated pyruvate kinase from Escherichia coli were examined in the presence of a number of fructose bisphosphate analogues, as well as of increased ionic strength (NaCl) and of the hydrogen-bond-breaking agent, formamide . Fructose 2,6-bisphosphate, ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate gave allosteric activation (additive to that of fructose 1,6-bisphosphate) . Formamide always decreased Vmax, but left unchanged the Km for phosphoenolpyruvate, while it decreased the concentration of fructose bisphosphate required to give half-maximal activity (K0.5) . NaCl increased the K0.5 for both phosphoenolpyruvate and fructose bisphosphate, leaving Vmax unchanged . These results are consistent with ionic binding of fructose bisphosphate through phosphates and with a critical role of hydrogen bonds in stabilizing both the inactive and the active enzyme conformers.

Eur J Biochem, 1990 Aug 17, 191(3), 743 - 53
Escherichia coli DNA polymerase I: inherent exonuclease activities differentiate between monofunctional and bifunctional adducts of DNA and cis- or trans-diamminedichloroplatinum(II) . An exonuclease investigation of the kinetics of the adduct formation; Bernges F et al.; {3H}dGMP-3'-labelled, activated salmon testis DNA and {32P}dGMP-5'-labelled open circular M13 DNA were reacted with cis-diamminedichloroplatinum(II), cis-diamminechloroaquaplatinum(II), cis-diamminediaquaplatinum(II) or trans-diamminechloroaquaplatinum(II) . The reaction was arrested after arbitrary times by adjustment to slightly alkaline solution conditions . The platinum-containing DNA was digested with Escherichia coli DNA polymerase I . The progress of nucleotide release was measured by acid precipitation of undigested DNA . Solubilized nucleotides and adducts were analyzed by HPLC . The 3'-5'-exonuclease activity liberated single-coordinated dGMP-platinum(II) adducts from both cis- and trans-platinum(II) treated salmon testis DNA and a small fraction of adducts of cis-platinum(II) that coordinated two molecules of dGMP . The bisadduct was derived from non-neighboring guanine residues probably located at or close to 3'-termini . This nuclease activity neither cut between nor after neighboring guanine residues crosslinked by cis-platinum(II) . No bisadduct was liberated for trans-platinum(II) . The 5'-3'-exonuclease activity did not liberate any nucleotide adducts from cis-platinum(II)-treated DNa . However, it removed single-coordinated guanine adducts of trans-diamminedichloroplatinum(II) . From the kinetics of the appearance of dGMP monoadducts and the inhibition of digestion, a reaction scheme is formulated for the reaction of platinum(II) complexes with DNA that confirms and extends the previously published one {W . Schaller, H . Reisner & E . Holler (1987) Biochemistry 26, 943-950} . The longevity of the dGMP monoadduct intermediate is discussed in the context of the efficiency of cis-diamminedichloroplatinum(II) as an antitumor drug.

Biochim Biophys Acta, 1990 Aug 17, 1035(2), 230 - 6
Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli; Hayashi M et al.; It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction . The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells . The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis . The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD . The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH . The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively . Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM . The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver . Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E . coli to partially alleviate the toxicity of menadione.

Science, 1990 Aug 17, 249(4970), 783 - 6
External guide sequences for an RNA enzyme; Forster AC et al.; Ribonuclease P (RNase P) from Escherichia coli or its catalytic RNA subunit can efficiently cleave small RNA substrates that lack the conserved features of natural substrates of RNase P if an additional small RNA is also present . This additional RNA must contain a sequence complementary to the substrate {external guide sequence (EGS)} and a 3'-proximal CCA sequence to ensure cleavage . The aminoacyl acceptor stem and some additional 5'- and 3'-terminal sequences of a precursor transfer RNA are sufficient to allow efficient cleavage by RNAase P, and the 2'-hydroxyl group at the cleavage site is not absolutely necessary for cleavage . In principle, any RNA could be targeted by a custom-designed EGS RNA for specific cleavage by RNase P in vitro or in vivo.

Biochem Biophys Res Commun, 1990 Aug 16, 170(3), 1319 - 24
Thyroid hormone binding protein contains glycosylation site binding protein activity; Kimura H et al.; Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the beta-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein . We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure . To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E . coli transformed with human thyroid hormone binding protein cDNA . The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum.

Biochem Biophys Res Commun, 1990 Aug 16, 170(3), 1294 - 300
Site-specific mutagenesis of PRD1 DNA polymerase: mutations in highly conserved regions of the family B DNA polymerase; Jung GH et al.; The PRD1 DNA polymerase is a small multifunctional enzyme containing three major conserved amino acid sequences shared by family B DNA polymerases . Thus, the PRD1 DNA polymerase provides an useful model system with which to study structure-function relationships of DNA polymerase molecules . In order to investigate the functional and structural roles of the highly conserved amino acid sequences, we have introduced mutations into each of the 3 conserved regions of the PRD1 DNA polymerase . Genetic complementation study as well as DNA polymerase assay indicated that each mutation inactivated DNA polymerase catalytic activity, but not the 3' to 5' exonuclease activity.

J Biol Chem, 1990 Aug 15, 265(23), 13618 - 22
Identification and initial characterization of translational initiation factor 2 from bovine mitochondria; Liao HX et al.; The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria . This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure . IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography . IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E . coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes . The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP . Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions.

Anal Biochem, 1990 Aug 15, 189(1), 138 - 41
An ultrafiltration assay for nucleotide binding to ribonucleotide reductase; Ormo M et al.; Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli . The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane . No effects were seen due to hyperconcentration of protein at the membrane surface . The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM . The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C.

Biochem J, 1990 Aug 15, 270(1), 141 - 8
The organization of open complexes between Escherichia coli RNA polymerase and DNA fragments carrying promoters either with or without consensus -35 region sequences; Chan B et al.; Transcription initiation at the Escherichia coli galP1 promoter does not depend on specific nucleotide sequences in the -35 region . Footprint analysis of transcriptionally competent complexes between E . coli RNA polymerase and DNA fragments carrying galP1 shows that RNA polymerase protects sequences as far upstream as -55, whereas sequences around the -35 region are exposed . In contrast, with galP1 derivatives carrying -35 region sequences resembling the consensus, RNA polymerase protects bases as far as -45, and the -35 region is fully protected . Taken together, our data suggest that the overall architecture of RNA polymerase-promoter complexes can vary according to whether or not consensus -35 region sequences are present; in the absence of these sequences, open complex formation requires distortion of the promoter DNA . However, the unwinding of promoter DNA around the transcription start is not affected by the nature of the -35 region sequence . With a galP1 derivative carrying point mutations in the spacer region that greatly reduce promoter activity, the protection of bases by RNA polymerase around the -10 sequence and transcription start site is reduced . In contrast, protection of the region upstream of -25 is unaffected by the spacer mutations, although sequences from -46 to -54 become hypersensitive to attack by potassium permanganate, indicating severe distortion or kinking of this zone . We suggest that, with this galP1 derivative, RNA polymerase is blocked in a complex that is an intermediate on the path to open complex formation.

J Biol Chem, 1990 Aug 15, 265(23), 14023 - 9
Physicochemical properties of the lipopolysaccharide unit that activates B lymphocytes; Takayama K et al.; We have examined the physical state of highly purified deep rough chemotype lipopolysaccharide (ReLPS) from Escherichia coli D31m4 as an aqueous suspension and as complexes with bovine serum albumin min (BSA) . The ReLPS suspension showed large ellipsoidal particles 12-38 nm wide and 40-100 nm long . The solubility of this form of ReLPS was determined by equilibrium dialysis experiments to be 3.3 x 10(-8) M at 22 degrees C and 2.8 x 10(-8) M at 37 degrees C in 150 mM Tris-KCl, pH 7.5; 3.0 x 10(-8) M at 37 degrees C in 0.75 mM Tris-KCl, pH 7.5 . The BSA-ReLPS complexes were fractionated on a Sephacryl S-200 column to yield peaks I and II with apparent masses of about 240 and 70 kDa, respectively . Peak II was a BSA monomer with estimated BSA:ReLPS molar ratios of 1:1-1:7 . The ReLPS suspension and the two complexes were compared as antigens in enzyme-linked immunosorbent assays using three select monoclonal antibodies to lipopolysaccharide . The results were consistent with the high state of disaggregation of the ReLPS in both peaks I and II . Since the ReLPS in these complexes were not visible by electron microscopy, they did not contain vesicles or large particles . All forms of ReLPS tested were capable of stimulating 70Z/3, a lipopolysaccharide-responsive murine pre-B cell line . However, peak II was consistently more stimulatory at very low concentrations than the other preparations . The maximally stimulatory concentration of ReLPS for 70Z/3 cells was 40 ng/ml (1.6 x 10(-8) M) for peak II and 70 ng/ml (2.8 x 10(-8) M) for the ReLPS suspension . As expected, the above concentrations were at or below the solubility of the ReLPS . These results suggested that the highly disaggregated form of ReLPS (possibly the monomer) is the active unit that stimulates the cellular response in 70Z/3 cells.

J Biol Chem, 1990 Aug 15, 265(23), 14016 - 22
Three-dimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution; Walter MR et al.; The three-dimensional structure of thymidine phosphorylase from Escherichia coli has been determined at 2.8 A resolution using multiple-isomorphous-replacement techniques . The amino acid sequence deduced from the deoA DNA sequence is also reported . Thymidine phosphorylase exists in the crystal as an S-shaped dimer in which the subunits are related by a crystallographic 2-fold axis . Each subunit is composed of a small alpha-helical domain of six helices and a large alpha/beta domain . The alpha/beta domain includes a six-stranded mixed beta-sheet and a four-stranded antiparallel beta-sheet . The active site has been identified by difference Fourier analyses of the binding of thymine and thymidine and lies in a cavity between the small and large domains . The central beta-sheet is splayed open to accommodate a putative phosphate-binding site which is probably occupied by a sulfate ion in the crystal.

J Biol Chem, 1990 Aug 15, 265(23), 13890 - 8
Recombinant HIV2 protease processes HIV1 Pr53gag and analogous junction peptides in vitro; Pichuantes S et al.; A synthetic DNA fragment encoding a protease precursor of the human immunodeficiency virus type 2 (HIV2) was cloned and expressed in bacteria and yeast . A recombinant plasmid encoding a hybrid polypeptide consisting of human superoxide dismutase and an HIV2 protease precursor of 113 amino acids was constructed for regulated intracellular expression in bacteria . Induction of this plasmid produced an autoprocessed form of the retroviral enzyme possessing the correct molecular weight . Overexpression and secretion of the protease from yeast was achieved with an expression vector encoding the yeast pheromone alpha-factor signal/leader sequence fused to a protease precursor of 115 amino acids . Amino-terminal sequence analysis confirmed that the viral enzyme exported from yeast was correctly processed from its precursor by cleavage of the predicted Ala-Pro peptide bond located at the NH2 terminus of the protease in the pol open reading frame . No additional amino acid residues were required at the COOH terminus of the protease for this autoproteolytic event . The HIV2 protease expressed in bacteria and yeast was active in an in vitro assay when tested on the HIV1 polyprotein precursor, myristylated Pr53gag . Two synthetic peptides representing junction sequences in the HIV1 gag-pol precursor were used to assay purified HIV2 protease . The enzyme exhibited a kcat/KM of 23.2 min-1 mM-1 on the HIV1 matrix-capsid junction peptide and a kcat/KM of 71.4 min-1 mM-1 on the protease-reverse transcriptase junction peptide . These rates show that the HIV2 enzyme is efficient at hydrolyzing the HIV1 peptide junctions, revealing the analogous nature of the substrate specificities of the two enzymes.

J Biol Chem, 1990 Aug 15, 265(23), 13878 - 87
The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I; Bebenek K et al.; The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading . We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease . Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position . Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides . Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others . The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6) . The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity . Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger . The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease . Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme . It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error . Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.

J Biol Chem, 1990 Aug 15, 265(23), 13767 - 75
Characterization and expression of the unique calmodulin gene of Aspergillus nidulans; Rasmussen CD et al.; Complete cDNA and genomic clones for the unique calmodulin (CaM) gene of the filamentous fungus Aspergillus nidulans have been isolated and characterized . The gene contains five introns, of which three are at unique positions relative to other CaM genes . The A . nidulans CaM gene is transcribed as a single, 0.85-kilobase mRNA species that encodes a predicted protein 84% identical (93% similar if conservative changes are considered) to vertebrate CaM . The complete cDNA was ligated into a lambda PL promoter-regulated bacterial expression vector to allow expression of A . nidulans CaM in Escherichia coli . The expressed protein was purified from bacterial lysates by phenyl-Sepharose chromatography and migrated as a single species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In the presence of Ca2+, A . nidulans CaM exhibited a shift in apparent Mr identical to vertebrate CaM . The bacterially synthesized protein activated vertebrate CaM-dependent phosphodiesterase, CaM-dependent protein kinase II, and myosin light chain kinase with kinetics similar to vertebrate CaM . Isolated conidia (G0 spores) were germinated to induce synchronous cell cycle re-entry and the levels of CaM mRNA and protein determined . Both CaM and its mRNA were regulated during cell cycle re-entry . Calmodulin mRNA levels increased 20-fold as germlings progressed through the G1 phase, while CaM levels increased 2-fold prior to the initiation of DNA synthesis . Messenger RNA levels decreased during S-phase while protein levels increased an additional 2-fold, peaking at the onset of mitosis followed by a subsequent decrease as cells completed mitosis . Disruption of the CaM gene by site-specific homologous recombination was lethal, indicating that CaM is essential for cell cycle progression.

J Biol Chem, 1990 Aug 15, 265(23), 13553 - 9
Expression, purification, and crystallization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli; Yang W et al.; Ribonuclease H (RNase H) from Escherichia coli is an endonuclease that specifically degrades the RNAs of RNA:DNA hybrids . The enzyme is a single polypeptide chain of 155 amino acid residues, of which 4 are methionines . To solve the crystallographic three-dimensional structure of E . coli RNase H by the multi-wavelength anomalous diffraction technique, we have constructed methionine auxotrophic strains of E . coli that overexpress selenomethionyl RNase H . MIC88 yields about 10 mg of selenomethionyl RNase H per liter of culture, which is comparable to the overexpression of the natural recombinant protein . We have purified both proteins to homogeneity and crystallized them isomorphously in the presence of sulfate . These are Type I crystals of space group P2(1)2(1)2(1) with the cell parameters a = 41.8 A, b = 86.4 A, c = 36.4 A, one monomer per asymmetric unit, and approximately 36% (v/v) solvent . Crystals of both proteins diffract to beyond 2-A Bragg spacings and are relatively durable in an x-ray beam . On replacement of sulfate with NaCl, crystals of natural RNase H grow as Type I' (very similar to Type I) at pH between 7.0 and 8.0; at pH 8.8, crystals of Type II are obtained in space group P2(1)2(1)2(1) with a = 44.3 A, b = 87.3 A, and c = 35.7 A . Type II crystals can be converted to Type I by soaking in phosphate buffer . RNase H crystals of Type II have also been reported by Kanaya et al . (Kanaya, S., Kohara, A., Miyakawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M . (1989) J . Biol . Chem . 264, 11546-11549).

J Biol Chem, 1990 Aug 15, 265(23), 13528 - 32
The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni . Further characterization and gene expression in Escherichia coli; Yuan L et al.; Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy . While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies . We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence . Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted . Therefore, we purified the HGPRTase from crude extracts of the adult worms . The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met . This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence . We next expressed the correct size cDNA of the S . mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E . coli signal peptide for secretion of expressed product into the periplasmic space . Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus . That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus . The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E . coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.

J Biol Chem, 1990 Aug 15, 265(23), 13560 - 5
Identification and characterization of large, complex forms of chloroplast translational initiation factor 2 from Euglena gracilis; Ma L et al.; Chromatography of partially purified preparations of Euglena gracilis chloroplast initiation factor 2 (IF-2chl) on gel filtration resins indicates that this factor is present in high molecular mass forms ranging from 200 to 700 kDa . The higher molecular weight complexes can be separated from the 200,000 Mr form of this factor by chromatography on DEAE-cellulose . Further purification indicates that the majority of the IF-2chl is present as dimeric, tetrameric, and probably hexameric complexes of polypeptides of 97,000-110,000 in molecular weight . In addition, one form consisting of subunits of about 200,000 Mr has been detected . All of these species are active in promoting fMet-tRNA binding to chloroplast 30 S subunits in a message-dependent reaction . Initiation complex formation promoted by IF-2chl requires the presence of GTP . Similar levels of binding are obtained when GTP is replaced by a nonhydrolyzable analog suggesting that IF-2chl is acting stoichiometrically rather than catalytically under the conditions used . The activity of this factor is stimulated by the presence of either Escherichia coli or chloroplast IF-3 . None of the forms of IF-2chl detected is active on E . coli ribosomes.

Thromb Res, 1990 Aug 15, 59(4), 735 - 47
Role of activated platelets in endotoxin-induced DIC in rats; Ito T et al.; To clarify whether activated platelets play an important role in the occurrence and exacerbation of disseminated intravascular coagulation (DIC), we investigated the effects of 4 anti-platelet drugs, a PGI2 analog (CS-570), a thromboxane synthetase inhibitor (dazoxiben), a thromboxane receptor antagonist (BM-13177), and ticlopidine, in an experimental DIC model in rats . Experimental DIC was induced by a continuous infusion of lipopolysaccharide (LPS derived from E . coli, 055 B5, 25 mg/kg/hr) for 4 hrs . In the time-course determination of the coagulation parameters and prostanoids, an abrupt increase in TxB2 (a stable metabolite of TxA2) and 6-keto-PGF1 alpha (a stable metabolite of PGI2) was followed by a decrease in platelet count, a prolongation of blood coagulation time, and an increase in fibrinogen/fibrin degradation products (FDP) . Four hours after the start of LPS infusion, the rats were considered to be in the state of DIC . The effects of the anti-platelet drugs were investigated 4 hrs after the start of LPS infusion . CS-570 and ticlopidine ameliorated DIC in a dose-dependent manner . CS-570 (10 micrograms/kg/min) improved DIC in the platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fbg), and FDP, without affecting TxB2 and 6-keto-PGF1 alpha formation . Ticlopidine (200 mg/kg, i.p.) prevented the exacerbation of DIC in such item parameters as platelet count, APTT, and FDP . Both dazoxiben and BM-13177 (30 mg/kg, i.p.) ameliorated DIC in following parameters as platelet count, APTT and FDP . Dazoxiben, but not BM-13177, significantly inhibited the increase in TxB2 concentration at 4 hr . These observations suggest that drugs which inhibit platelet activation by a TxA2-dependent route are effective in improving DIC induced by LPS, and that drugs which inhibit multiple platelet-activating routes improve DIC in more item parameters than drugs which inhibit only the TxA2-dependent activating route . Consequently, it is concluded that activated platelets might play an important role in the occurrence and exacerbation of DIC induced by LPS, and that one of the roles of TxA2 in DIC is to activate platelets.

J Biol Chem, 1990 Aug 15, 265(23), 14030 - 5
Protein-protein interactions facilitate DNA binding by the glucocorticoid receptor DNA-binding domain; Dahlman-Wright K et al.; We have studied the interaction of the DNA-binding domain of the glucocorticoid receptor with a glucocorticoid response element from the tyrosine aminotransferase gene . This response element consists of two binding sites (half-sites) for the glucocorticoid receptor DNA-binding domain . The sequences of these two half-sites are not identical, and we have previously shown that binding occurs preferentially to one of the half-sites (Tsai, S.-Y., Carlstedt-Duke, J., Weigel, N . L., Dahlman, K., Gustafsson, J.-A., Tsai, M.-J., and O'Malley, B . W . (1988) Cell 55, 361-369) . We show here that binding to the low affinity half-site is dependent on previous occupancy of the high affinity half-site . This facilitated binding is dependent on the distance between the two half-sites and their relative orientation but is not dependent on the integrity of the DNA backbone . This is consistent with a model where DNA binding is not only dependent on interactions between the protein and its DNA target sequence but is also influenced by interactions between the protein molecules bound.

Biochemistry, 1990 Aug 14, 29(32), 7459 - 67
Complexes of Escherichia coli adenylate kinase and nucleotides: 1H NMR studies of the nucleotide sites in solution; Vetter IR et al.; One- and two-dimensional nuclear magnetic resonance (NMR) studies, in particular substrate--protein nuclear Overhauser effect (NOESY) measurements, as well as nucleotide and P1,P5-bis-(5'-adenosyl) pentaphosphate (AP5A) titrations and studies of the temperature-dependent unfolding of the tertiary structure of Escherichia coli adenylate kinase (AKEC) were performed . These experiments and comparison with the same type of experiments performed with the porcine enzyme {Rosch, P., Klaus, W., Auer, M., & Goody, R . S . (1989) Biochemistry 28, 4318-4325} led us to the following conclusions: (1) At pH 8 and concentrations of approximately 2.5-3 mM, AKEC is partially unfolded at 318 K . (2) ATP.Mg2+ binds to the ATP site with a dissociation constant of approximately 40 microM under the assumption that ATP binds to one nucleotide site only . (3) AP5A.Mg2+ binds to both nucleotide sites and thus simulates the active complex . (4) The ATP.Mg2+ adenine in the AKEC.AP5A.Mg2+ complex is located close to His134 and Phe19 . (5) The AKEC "G-loop" with bound ATP.Mg2+ is structurally highly homologous to the loop region in the oncogene product p21 with bound GTP.Mg2+.

Biochemistry, 1990 Aug 14, 29(32), 7451 - 9
Structurally and catalytically important residues in the phosphate binding loop of adenylate kinase of Escherichia coli; Reinstein J et al.; Amino acids in the phosphate binding loop of adenylate kinase of Escherichia coli were mutated by site-directed mutagenesis . The mutant proteins with a Pro-9----Gly (P9G) and with a Lys-13----Gln (K13Q) exchange were overexpressed and purified . They were characterized by steady-state kinetics, fluorescence binding, and structural studies, together with the phosphate binding loop mutants P9L and G10V prepared earlier {Reinstein, J., Brune, M., & Wittinghofer, A . (1988) Biochemistry 27, 4712-4720} . The results obtained show that all these mutations change the structure of the protein as evidenced by NMR spectroscopy and temperature-stability studies . All the mutant proteins have increased dissociation constants for substrates and inhibitors, but their catalytic activity, except for K13Q, is not reduced . The results obtained with K13Q suggest that this lysine residue, which is conserved in all guanine and many adenine nucleotide proteins, might have an important role in catalysis.

Biochemistry, 1990 Aug 14, 29(32), 7440 - 50
Fluorescence and NMR investigations on the ligand binding properties of adenylate kinases; Reinstein J et al.; A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented . This system is based on the use of the fluorescent ligand alpha,omega-di{(3' or 2')-O-(N-methylanthraniloyl)adenosine-5'} pentaphosphate (mAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A) . It allows the determination of dissociation constants for any ligand in the range of 1 x 10(-9) to 5 x 10(-2) M . Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium . An analysis of the binding of bisubstrate inhibitors is proposed and applied to these data . The techniques are used to describe the properties of a mutant enzyme with Gln-28----His (Q28H) prepared by site-directed mutagenesis in comparison to those of wild-type AK from Escherichia coli . This newly introduced histidine is already present in most other adenylate kinases and was regarded to be important or even essential for the catalytic reaction of AK . Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure . However, steady-state catalytic studies and binding experiments showed that the affinities for substrates and inhibitors are elevated from 3-fold (AMP) to 5-fold (ATP) to 15-fold (AP5A) compared to those of the wild-type enzyme . Together with the results obtained by Tian et al . {Tian, G., Sanders, C . R., Kishi, F., Nakazawa, A., & Tsai, M.-D . (1988) Biochemistry 27, 5544-5552} on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E . coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity.

Biochemistry, 1990 Aug 14, 29(32), 7425 - 32
Sequential assignment of the 1H nuclear magnetic resonance spectrum of barnase; Bycroft M et al.; Two-dimensional nuclear magnetic resonance spectroscopy has been used to study the bacterial ribonuclease barnase (MW 12,382) . Resonance assignments have been made for protons in all of the 110 residues . Analysis of medium- and long-range contacts in NOESY spectra has demonstrated that the major elements of secondary structure in barnase in solution are essentially identical with those found in the crystal structure.

Cell, 1990 Aug 10, 62(3), 457 - 67
IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice; Tepper RI et al.; We have assessed the biologic role of IL-4 by fusing its gene to an immunoglobulin promoter/enhancer and introducing it into transgenic mice . By attenuating the transgene promoter through the insertion of E . coli lac operator sequences, we have created a series of animals that constitutively express varying amounts of IL-4 . Overexpression of IL-4 results in a marked increase in serum IgE levels and the appearance of an inflammatory ocular lesion (blepharitis) with characteristic histopathologic features seen in allergic reactions . In addition, expression of the IL-4 transgene in the thymus perturbs T cell maturation, reducing the population of immature CD4+CD8+ thymocytes and peripheral T cells while increasing the population of mature CD8+ thymocytes . These results demonstrate that deregulation of a single cytokine gene in vivo can induce a complex inflammatory reaction resembling that observed in human allergic disease.

J Theor Biol, 1990 Aug 9, 145(3), 295 - 318
Theoretical and experimental analysis of the phage lambda genetic switch implies missing levels of co-operativity; Reinitz J et al.; The behavior of the cro-repressor switch in phage lambda, a temperate phage of Escherichia coli, is described at the organismal level within a dynamical system framework . The molecular biology of the switch has been well characterized up to the level of the regulation of transcription initiation . In this paper we construct a description of a lysogen whose prophage is mutated in certain genes, so that the switch is functionally isolated from the rest of the phage genome . Such a lysogen has two stable epigenetic states, and comparison of the theory with measurements of intracellular cro concentration corresponding to one of these states suggest that additional levels of regulation, not included in the current description of the switch, should exist for this system . In addition, we suggest a new method for the measurement of intracellular concentrations.

Biochemistry, 1990 Aug 7, 29(31), 7339 - 47
Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism; Longstaff C et al.; The serine protease inhibitor chymotrypsin inhibitor 2 (CI2 or BSPI2) has been expressed in Escherichia coli with the pINIIIompA3 expression vector to produce 20-40 mg/L of culture . Recombinant CI2 purified from this system has been characterized and found to be identical with CI2 from barley . Slow-binding kinetics were observed for the interaction between CI2 and subtilisin BPN', with Ki = 2.9 x 10(-12) M . Analysis of slow-binding data indicates that binding of the inhibitor follows the simplest model of E + I = EI with no kinetically detectable intermediate steps or proteolytic cleavage of the reactive site bond in CI2 (Met-59-Glu-60) . This, in agreement with crystallographic data, indicates that the enzyme-inhibitor adduct is the Michaelis complex, which is not chemically processed by the enzyme . Three mutant CI2 molecules with new P1 residues have also been examined with a range of serine proteases, including a mutant subtilisin . In agreement with earlier studies, we find the P1 amino acid an important determinant of specificity . CI2 Met----Lys-59 was found to be a temporary inhibitor of subtilisin BPN' but an effective inhibitor of subtilisin Carlsberg and subtilisin BPN'(Glu----Ser-156) . The structural reasons for this are discussed in relation to mechanisms of inhibition of serine proteases.

Biochemistry, 1990 Aug 7, 29(31), 7251 - 9
Damage repertoire of the Escherichia coli UvrABC nuclease complex includes abasic sites, base-damage analogues, and lesions containing adjacent 5' or 3' nicks; Snowden A et al.; Using oligonucleotide synthesis, we demonstrate a rapid and efficient method for the construction of DNA duplexes containing defined DNA lesions at specific positions . These DNA lesions include apyrimidinic sites, reduced apyrimidinic sites, and base-damage analogues consisting of O-methyl- or O-benzylhydroxylamine-modified apyrimidinic sites . A 49 base pair DNA duplex containing these lesions was specifically incised by the UvrABC nuclease complex . The incision sites occurred predominantly at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to the lesion . Multiple incisions were observed 3' to the lesion . The extent of DNA incisions was base-damage analogues greater than reduced apyrimidinic sites greater than apyrimidinic sites . Introduction of 3' or 5' nicks at the site of a base-damage analogue by treatment of these substrates with either endonuclease III or endonuclease IV reduced, but did not abolish, subsequent incision by the UvrABC complex, whereas introduction of a 3' nick at an abasic site increased the incision efficiency of the UvrABC complex . These data demonstrate a convergence of base and nucleotide excision repair pathways in the removal of specific base damages.

Biochemistry, 1990 Aug 7, 29(31), 7160 - 73
X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis; Wang JM et al.; The 2.2-A X-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli {Fishel, L.A., Villafranca, J . E., Mauro, J . M., & Kraut, J . (1987) Biochemistry 26, 351-360}, has been solved, together with the structures of three specifically designed single-site heme-cleft mutants . The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution . Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53----Ile substitution in CCP(MI) . A Trp-51----Phe mutant remains pentacoordinated and exhibits only minor distal structural adjustments . The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron {Smulevich, G., Mauro, J.M., Fishel, L . A., English, A . M., Kraut, J., & Spiro, T . G . (1988) Biochemistry 27, 5477-5485} . The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol . A proximal Trp-191----Phe mutant that has substantially diminished enzyme activity and altered magnetic properties {Mauro, J . M., Fishel, L . F., Hazzard, J . T., Meyer, T . E., Tollin, G., Cusanovich, M . A., & Kraut, J . (1988) Biochemistry 27, 6243-6256} accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme . This relatively large (ca . 1 A) local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged . This structure rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant . Changing proximal Asp-235 to Asn results in two significant localized structural changes . First, the heme iron moves toward the porphyrin plane, and distal water 595 now clearly resides in the iron coordination sphere at a distance of 2.0 A . The observation of hexacoordinated iron for the D235N mutant is in accord with previous resonance Raman results . Second, the indole side chain of Trp-191 has flipped over as a result of the mutation; the tryptophan N epsilon takes part in a new hydrogen bond with the backbone carbonyl oxygen of Leu-177.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochim Biophys Acta, 1990 Aug 6, 1045(3), 285 - 90
On the mode of action of basic phospholipase A2 from Naja nigricollis venom; Chwetzoff S; A study has been made of the cytotoxic activity of basic phospholipase A2 of venom from Naja nigricollis on different types of cells and of the participation of esterase activity in this cytotoxic activity . It was previously shown that the cytotoxicity observed is not due to a contaminant, since the cytotoxic action vanished after immunoprecipitation of basic phospholipase A2 by specific monoclonal antibodies . All eukaryotic cells tested were sensitive to the cytotoxic action of basic phospholipase A2 . In contrast, Escherichia coli K-12 wild strain was resistant . Participation of cell membranes in the sensitive or resistant character of cells to the phospholipase A2 attack was investigated using E . coli K-12 membrane mutants . Some membrane mutants were sensitive and the sensitivity or resistance to basic phospholipase A2 was found to be related to a single mutation in the locus envA . The requirement for esterase activity of phospholipase A2 in cytotoxic attack has been shown to be dependent on the cell line tested . Indeed, when the esterase activity of basic phospholipase A2 was eliminated by treatment with p-bromophenacyl bromide, the enzyme retained a cytotoxic potency inducing necrosis of certain tumoral cells grown in vitro, but not other cells such as erythrocytes, for which concomitant esterase activity was also necessary . In vivo studies of toxicity showed that the loss of either cytotoxic potency or esterase activity eliminates the lethal character of basic phospholipase A2 . This leads us to propose that in vivo toxicity of phospholipase A2 depends on simultaneous expression of esterase activity and a non-enzymatic property, manifested by in vitro cytotoxic potency.

J Biol Chem, 1990 Aug 5, 265(22), 12790 - 5
Identification of a plastid-specific ribosomal protein in the 30 S subunit of chloroplast ribosomes and isolation of the cDNA clone encoding its cytoplasmic precursor; Johnson CH et al.; We describe the isolation and characterization of a chloroplast ribosomal protein and a clone of its cDNA . This protein has no homology to any Escherichia coli ribosomal protein or to any known proteins . Due to this novel finding we propose it be called PSrp-1, i.e . a plastid-specific ribosomal protein . The precursor form of PSrp-1, deduced from the cDNA sequence, is 302-amino acid residues long . The mature PSrp-1, identified by amino-terminal sequencing, is a protein of 236 residues . The NH2-terminal 66 amino acids form the transit peptide that targets PSrp-1 into the chloroplast . We show that PSrp-1 is a protein of the chloroplast 30 S ribosomal subunit by Western blotting and sequencing the excised protein after two-dimensional gel electrophoresis . The possible evolutionary origin of PSrp-1 from the nucleated host cell of the endosymbiont theory is discussed.

J Mol Biol, 1990 Aug 5, 214(3), 685 - 97
Deletion of sites for initiation of DNA synthesis in the origin of broad host-range plasmid R1162; Zhou HS et al.; The origin of replication of the broad host-range plasmid R1162 contains two, oppositely facing initiation sites for DNA synthesis . Either of these sites can be deleted from an R1162 plasmid derivative . However, the resulting plasmids are unstable, maintained at a lower copy-number in the cell, and form dimers and other recombinants that are required for propagation of the plasmid . In vitro, a derivative lacking one initiation site is deficient in synthesis of the strand normally initiated from that site . The properties of the intact origin are restored if it contains two oppositely facing sites; one initiation site may substitute for the other, and each site need not be in its original orientation . Overall, the results suggest that synthesis of each strand of R1162 DNA is initiated at a single site, and that there is no efficient system for initiation of lagging strand synthesis during transit of the replication forks.

J Mol Biol, 1990 Aug 5, 214(3), 641 - 2
Crystallization and preliminary X-ray studies of D-serine dehydratase from Escherichia coli; Obmolova G et al.; Single crystals of D-serine dehydratase from Escherichia coli complexed with 3-amino-2-hydroxypropionate have been obtained from ammonium sulfate solution (pH 7.0) by vapor diffusion . The crystals belong to the trigonal space group P3(1) or P3(2) with a = b = 81.3 A and c = 58.4 A . The asymmetric unit cell contains one protein molecule with Mr = 48,289 . The crystals diffract to at least 3.0 A resolution and are suitable for X-ray structure analysis.

J Biol Chem, 1990 Aug 5, 265(22), 13335 - 43
Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme; Smith AT et al.; A synthetic gene encoding horseradish peroxidase isoenzyme C (HRP C) has been synthesized and expressed in Escherichia coli . The nonglycosylated recombinant enzyme (HRP C*) was produced in inclusion bodies in an insoluble inactive form containing only traces of heme . HRP C* was solubilized and conditions under which it folded to give active enzyme were determined . Folding was shown to be critically dependent upon the concentrations of urea, Ca2+, and heme and on oxidation by oxidized glutathione . Purification of active HRP C* from the folding mixture gave a peroxidase, with about half the activity of HRP C . Glycosylation is thus not essential for correct folding and activity . The C-terminal and N-terminal extensions to HRP identified previously in cloned cDNA sequences are also not required for correct folding . However, Ca2+ appears to play a key role in folding to give the active enzyme . The overall yield of purified active enzyme was 2-3%, but this could be increased by reprocessing material that precipitated during folding.

J Biol Chem, 1990 Aug 5, 265(22), 13248 - 53
Deletion analysis of the human insulin receptor ectodomain reveals independently folded soluble subdomains and insulin binding by a monomeric alpha-subunit; Schaefer EM et al.; A series of 13 deletions within the extracellular domain of the human insulin receptor delineates the boundaries of subdomains that fold de novo into stable proteins that are efficiently secreted and retain the epitopes required for interaction with two conformation-specific monoclonal antibodies . While most of these proteins fail to bind insulin, a truncation that includes only the alpha-subunit is secreted as a monomer that binds the hormone with an affinity only slightly less than that of the complete heterotetrameric extracellular domain . These results thus demarcate landmarks within the primary sequence which will now guide further analysis of the structure and function of this complex domain of the receptor.

J Biol Chem, 1990 Aug 5, 265(22), 13174 - 80
Genetic reconstruction and characterization of the recombinant transacylase (E2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex . Implication of histidine 391 as an active site residue; Griffin TA et al.; Genetically altered transacylase (E2b) proteins of the bovine branched-chain alpha-keto acid dehydrogenase complex were overexpressed in Escherichia coli and characterized . Deletion by PstI or Bal31 digestion of the amino-terminal region of the inner-core domain (residues 175-421) beyond residue 209 resulted in a complete loss of transacylase activity . The enzyme assay was carried out using {1-14C}isovaleryl-CoA and exogenous dihydrolipoamide as substrates . The removal of 4 residues (Thr-Ile-Pro-Ile) (residues 175-178) from the amino terminus of the inner-core domain significantly reduced the level of transacylase activity . The results establish that the segment between residues 175 and 209 is an integral part of the active site of E2b . The residue His-391 in the recombinant inner-core domain (E2b delta 167) was changed to Asn or Gln by site-directed mutagenesis . The wild-type and the two mutant inner-core domains were assembled into 24-mers as determined by gel filtration . However, both Asn and Gln mutations were accompanied by a complete loss of the enzymatic activity . Titration of the natural branched-chain alpha-keto dehydrogenase complex from pH 8 to 6 sharply reduced transacylase activity . The above data support the hypothesis that a conserved histidine residue in E2 acts as a general base for the transacylation reaction by analogy with E . coli chloramphenicol acetyltransferases.

J Biol Chem, 1990 Aug 5, 265(22), 13124 - 9
In vitro gamma-carboxylation of a 59-residue recombinant peptide including the propeptide and the gamma-carboxyglutamic acid domain of coagulation factor IX . Effect of mutations near the propeptide cleavage site; Wu SM et al.; We report the expression in Escherichia coli of a fusion protein that contains the propeptide sequence and gamma-carboxyglutamic acid domain (residues -18 to 41) of human factor IX (FIXGla) . CNBr was used to release FIXGla from the fusion protein . The 59-amino acid peptide is an efficient substrate for in vitro gamma-carboxylation . Its Km,app (0.55 microM) is several thousand-fold lower than that of the commonly used substrate FLEEL and about 5 times lower than proPT28 or proFIX28, (Hubbard, B . R., Jacobs, M., Ulrich, M . M . W., Walsh, C., Furie, B., and Furie, B . C . (1989) J . Biol . Chem . 264, 14145-14150) . In addition, FIXGla is the first peptide substrate that is carboxylated in vitro to more than one gamma-carboxyglutamic acid/molecule (6-11 gamma-carboxyglutamic acids/molecule) . We created peptides with mutations identical to FIXSan Dimas or FIXCambridge as well as a peptide with both mutations in the propeptide sequence and examined the effect of the mutations on in vitro carboxylation . Enzyme kinetic studies revealed no significant difference in Vmax/Km values between normal and mutant substrates . Maximum carbon dioxide incorporation was achieved with the double mutant . From these data we conclude the following . 1) FIXGla and its mutants are excellent substrates for studying the mechanism of gamma-carboxylase . 2) Although arginines at positions -4 and -1 are highly conserved in the propeptide sequence of all the vitamin K-dependent proteins, neither is critical for gamma-carboxylation.

J Biol Chem, 1990 Aug 5, 265(22), 13000 - 6
Identification of key residues in the amino-terminal third of human interleukin-1 alpha; Yanofsky SD et al.; Two mutational approaches were used to perform a thorough structure-function analysis of the first 53 residues of the 159-residue cytokine human interleukin-1 alpha (hIL-1 alpha) . In this study, a total of 26 deletions, 97 multiple amino acid substitutions, and 46 single amino acid substitutions were examined . A synthetic hIL-1 alpha gene with many unique restriction sites was constructed to facilitate the molecular manipulations that were performed . The mutational methods employed include: Bal-31 exonuclease-generated deletions at unique restriction sites and combinatorial cassette mutagenesis via segment replacement with synthetic DNA . The mutant hIL-1 alpha proteins were expressed at high levels in Escherichia coli and were assayed for biological activity in a mouse T cell proliferation assay . We observed that the activity of hIL-1 alpha was extraordinarily sensitive to deletion mutations . Most internal deletions of as few as 1 or 2 residues substantially reduced biological activity . Combinatorial cassette mutagenesis on residues 13-53 of hIL-1 alpha identified 15 important residue positions . Of these, 8 displayed strong preferences for residues with hydrophilic side chains, and the remainder preferred hydrophobic side chains . We found that functional hIL-1 alpha had an absolute requirement for a basic residue (Arg, Lys, or His) at either position 15 or 16, and that Leu was preferred at position 40.

J Biol Chem, 1990 Aug 5, 265(22), 12828 - 35
Reaction of LexA repressor with diisopropyl fluorophosphate . A test of the serine protease model; Roland KL et al.; The LexA repressor of Escherichia coli modulates the expression of the SOS regulon . In the presence of DNA damaging agents in vivo, the 202-amino acid LexA repressor is inactivated by specific RecA-mediated cleavage of the Ala-84/Gly-85 peptide bond . In vitro . LexA cleavage requires activated RecA at neutral pH, and proceeds spontaneously at high pH in an intramolecular reaction termed autodigestion . A model has been proposed for the mechanism of autodigestion in which serine 119 serves as the reactive nucleophile that attacks the Ala-84/Gly-85 peptide bond in a manner analogous to a serine protease, while uncharged lysine 156 activates the serine 119 hydroxyl group . In this work, we have tested this model by examining the effect of the serine protease inhibitor diisopropyl fluorophosphate (DFP) on autodigestion . We found that DFP inhibited autodigestion and that serine 119 was the only serine residue to react with DFP . We also examined {3H}DFP incorporation by a number of cleavage-impaired LexA mutant proteins and found that mutations in the proposed active site, but not in the cleavage site, significantly reduced the rate of {3H}DFP incorporation . Finally, we showed that the purified carboxyl-terminal domain, which contains the proposed catalytic residues, incorporated {3H}DFP at a rate indistinguishable from the intact protein . These data further support our current model for the mechanism of autodigestion and the organization of LexA.

J Biol Chem, 1990 Aug 5, 265(22), 12978 - 86
The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli; Chepuri V et al.; The cytochrome o terminal oxidase complex is a component of the aerobic respiratory chain of Escherichia coli . This enzyme catalyzes the oxidation of ubiquinol-8 to ubiquinone-8 within the cytoplasmic membrane and the concomitant reduction of O2 to H2O . The hydropathy profiles of the deduced amino acid sequences suggest that all five of the gene products of the cyo operon contain multiple membrane-spanning helical segments . The goal of this work was to obtain experimental evidence for the topology of the five gene products in the cytoplasmic membrane by using the technique of gene fusions . A number of random gene fusions were generated in vitro encoding hybrid proteins in which the amino-terminal portion was provided by the subunit of interest and the carboxyl-terminal portion by one of two sensor proteins, alkaline phosphatase lacking its signal sequence or beta-galactosidase . Results obtained are self-consistent, and topological models are proposed for all of the five gene products encoded by the cyo operon . Based on the sequence similarities with subunits of the aa3-type cytochrome c oxidases, the experimental evidence obtained here can be used to infer topological models for the mitochondrial encoded subunits of the eukaryotic cytochrome c oxidases.

J Biol Chem, 1990 Aug 5, 265(22), 13060 - 5
Molecular cloning of subunits of complex I from Neurospora crassa . Primary structure and in vitro expression of a 22-kDa polypeptide; Videira A et al.; A lambda gt11 cDNA expression library was screened with antibodies directed against individual subunits of complex I from Neurospora crassa . Clones encoding cytoplasmically synthesized polypeptides with apparent molecular masses of 22, 29, 31, and 33 kDa were isolated . Northern blot analysis revealed that the corresponding genes are transcribed into mRNA species of about 0.85, 0.95, 1.3, and 1.4 kilobases, respectively . Further characterization of clones encoding the 22-kDa subunit was performed . A cDNA insert of 755 base pairs containing the complete coding sequence was used to express the polypeptide in vitro . A precursor of the protein is synthesized on cytoplasmic ribosomes without a cleavable signal sequence . Our data indicate that after import into the organelle and before assembly into complex I, the 22-kDa polypeptide forms intramolecular disulfide bridge(s) . Nucleotide sequencing revealed an open reading frame coding for a protein of 183 amino acids . A molecular mass of 20,828 daltons was calculated . The polypeptide is hydrophilic and contains no obvious membrane-spanning domains . Eight cysteine residues arranged in a regular pattern are found in the primary structure of the protein . Therefore, this subunit is a good candidate to bind at least one of the iron-sulfur centers present in complex I of the respiratory chain.

J Mol Biol, 1990 Aug 5, 214(3), 787 - 96
Phycobiliprotein methylation . Effect of the gamma-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome; Swanson RV et al.; The phycobiliproteins contain a conserved unique modified residue, gamma-N-methylasparagine at beta-72 . This study examines the consequences of this methylation for the structure and function of phycocyanin and of phycobilisomes . An assay for the protein asparagine methylase activity was developed using {methyl-3H}S-adenosylmethionine and apophycocyanin purified from Escherichia coli containing the genes for the alpha and beta subunits of phycocyanin from Synechococcus sp . PCC 7002 as substrates . This assay permitted the partial purification, from Synechococcus sp . PCC 6301, of the activity that methylates phycocyanin and allophycocyanin completely at residue beta-72 . Using the methylase assay, two independent nitrosoguanidine-induced mutants of Synechococcus sp . PCC 7942 were isolated that do not exhibit detectable phycobiliprotein methylase activity . These mutants, designated pcm 1 and pcm 2, produce phycocyanin and allophycocyanin unmethylated at beta-72 . The phycobiliproteins in these mutants are assembled into phycobilisomes and can be methylated in vitro by the partially purified methylase from Synechococcus sp . PCC 6301 . The mutants produce phycobiliproteins in amounts comparable to those of wild-type and the mutant and wild-type phycocyanins are equivalent with respect to thermal stability profiles . Monomeric phycocyanins purified from these strains show small spectral shifts that correlate with the level of methylation . Phycobilisomes from the mutant strains exhibit defects in energy transfer, both in vivo and in vitro, that are also correlated with deficiencies in methylation . Unmethylated or undermethylated phycobilisomes show greater emission from phycocyanin and allophycocyanin and lower fluorescence emission quantum yields than do fully methylated particles . The results support the conclusion that the site-specific methylation of phycobiliproteins contributes significantly to the efficiency of directional energy transfer in the phycobilisome.

J Biol Chem, 1990 Aug 5, 265(22), 13314 - 9
Structure-function analysis of the human interferon gamma . The COOH terminus is not essential for functional activity; Luk SK et al.; The structure-function relationships for the human interferon gamma (HuIFN-gamma) were studied using recombinant variants that had various deletions at the carboxyl terminus . Four COOH-terminal deletion variants were constructed that contained the amino-terminal 122, 117, 111, and 106 amino acid residues . These variants were constructed by specific DNA modifications and were expressed in Escherichia coli . The deletion of 21 amino acid residues resulted in only 2- and 3-fold reduction in the antiviral and antiproliferative specific activities, respectively . Thus, the carboxyl-terminal 21 residues are not directly involved in the function of the HuIFN-gamma . The level of intracellular accumulation was also decreased by 3-5-fold . Further deletions of 26, 32, and 37 residues from the COOH terminus resulted in the lack of detectable activity as well as in 50-100-fold reduction in the level of accumulation in the bacterial cell . However, each of the modified plasmids was found to have comparable efficiency in directing the production of the respective variant molecules relative to the full-length HuIFN-gamma molecule in an in vitro transcription-translation assay . Thus, the failure of some of the deletion variant molecules to accumulate in the E . coli cell is likely due to their instability in vitro . The loss of the COOH-terminal 21 amino acid residues of HuIFN-gamma also resulted in a substantial reduction in the ability of the molecule to be renatured in vitro from the treatment with chaotrophic agents, a method frequently used to extract and purify recombinant polypeptides from E . coli host . The latter result may account for some earlier reports which inferred the involvement of the COOH terminus in the functions of the HuIFN-gamma molecule due to their failure to detect activity upon deletions of only 11-18 residues.

J Biol Chem, 1990 Aug 5, 265(22), 12761 - 2
Lysine can replace arginine 67 in the mediation of covalent attachment of FAD to histidine 71 of 6-hydroxy-D-nicotine oxidase; Mauch L et al.; The requirements for FAD-attachment to His71 of 6-hydroxy-D-nicotine oxidase (6-HDNO) were investigated by site-directed mutagenesis . The following amino acid replacements were introduced into the sequence Arg67-Ser68-Gly69-Gly70-His71 of the 6-HDNO-polypeptide: 1) Arg67 was replaced with Ala (A1 mutant); 2) Ser68 was replaced with Ala (A2 mutant); and 3) Arg67 was replaced with Lys (K mutant) . The substitution in mutant A2 had no effect on flavinylation, measured as {14C}FAD incorporation into apo-6-HDNO . Replacement of Arg67 with Ala prevented, but replacement with Lys permitted the flavinylation of His71 . Mutant A1 showed no 6-HDNO activity, whereas the replacement of Ser with Ala in mutant A2 had only a slight effect on 6-HDNO activity . The substitution of Lys for Arg67, however, reduced the specific 6-HDNO activity in extracts of Escherichia coli cells expressing the mutant polypeptide from 50.3 to 17.5 milliunits/mg protein . It is concluded that a basic amino acid residue (Arg67 or Lys67) is required to mediate the attachment of FAD to His71, and while Lys can substitute for Arg67 in this function, it can only partially replace Arg67 in the enzyme reaction mechanism of 6-HDNO.

J Biol Chem, 1990 Aug 5, 265(22), 12895 - 902
Nucleotide sequence of Escherichia coli asnB and deduced amino acid sequence of asparagine synthetase B; Scofield MA et al.; The Escherichia coli asparagine synthetase B gene (asnB) has been cloned into a temperature-sensitive, low copy plasmid, pOU71, as shown by the complementation of an E . coli asparagine auxotroph, E . coli JE6279 . The nucleotide sequence of asnB and the flanking sequences were determined . The proposed coding region for the gene is 1662 nucleotides in length, and the deduced amino acid sequence of the coding region results in a protein that has a molecular weight of 62,666 and contains 554 amino acids . A promoter region is identified based on the transcription start site that was determined by primer extension experiments . Homology studies of the asnB protein sequence with the human asparagine synthetase and E . coli asparagine synthetase A protein show that there is a high degree of homology with only the human asparagine synthetase . A purF type glutamine amide transfer domain was identified upon inspection of the amino-terminal amino acid sequence of the asparagine synthetase B protein.

J Biol Chem, 1990 Aug 5, 265(22), 13029 - 35
Alteration of serpin specificity by a protein cofactor . Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties; Ehrlich HJ et al.; Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate . Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1') . In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III . In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties . The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells . The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin . In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1) . In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively . Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA . It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin . Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity . PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.

Nature, 1990 Aug 2, 346(6283), 480 - 2
Specific expression of a silk-encoding gene of Bombyx in the anterior salivary gland of Drosophila; Bello B et al.; Successful expression of genes transferred into distantly related species in which genetic functions have been maintained through evolution has been reported previously . In the case of the silkmoth Bombyx mori and the fruitfly Drosophila melanogaster, both of which produce chorions (eggshells), Bombyx chorion genes are correctly expressed in Drosophila despite their estimated 240-Myr phylogenetic divergence . Here we report that, although Drosophila does not produce silk, mechanisms regulating transcription have been conserved between the salivary gland of the fruitfly and the silk gland of the silkmoth larva.

Int J Microcirc Clin Exp, 1990 Aug, 9(3), 303 - 18
Whole body plasma extravasation in saline and Haemaccel loaded rats: effects of endotoxemia; van Lambalgen AA et al.; Endotoxin causes only a limited increase in plasma leakage in rats . This may be due to a concomitant fall in venous pressure . We therefore studied effects of increases in this pressure on plasma loss . After a 60 min . endotoxin (20 mg/kg . hr E coli 0127-B8) or saline (control) infusion anesthetized rats were volume loaded (3 ml/10 min . 100 g for 10 min.) at t = 120 min . with saline (shock group n = 8; control group n = 8) or Haemaccel (shock group n = 6; control group n = 8) . At t = 180 min . the experiments ended . We measured mean arterial and central venous pressure, heart rate, arterial lactate, blood volume (51Cr-labeled red cells), hematocrit (conductivity cell), macromolecular extravasation (125I-HSA) and fluid retention (calculated from change in plasma volume) . Whole body transcapillary filtration coefficient (Kf) and interstitial compliance (Ci) were obtained from saline retention curves . During volume loading central venous pressure increased, then fell again . At the end of saline loading (t = 130) 50 and 60% of infused volume remained intravascularly in control and endotoxin treated rats, respectively; at the end of Haemaccel infusion retention was 110 and 120%, respectively . Fifty minutes later (t = 180) it was 25 and 30% after saline loading and 50 and 60% after Haemaccel loading in control and endotoxin treated rats, respectively . Between control and endotoxin group filtration coefficients (Kf; .094 vs .111 ml/min.mmHg.100 g, respectively) and compliances (Ci; 1.90 vs 1.58 ml/mmHg.100 g, respectively) were not significantly different . No increase in leakage of 125I-HSA was found in either group . Increased venous pressure thus did not reveal an increase in macromolecular permeability in endotoxin treated rats.

Biochem J, 1990 Aug 1, 269(3), 651 - 8
Abortive intermediates in transcription by wheat-germ RNA polymerase II . Dynamic aspects of enzyme/template interactions in selection of the enzyme synthetic mode; de Mercoyrol L et al.; At constant enzyme concentration and with the full set of nucleotide substrates dictated by template sequence, the chain-length distribution of polymeric product varies with template concentration in reactions catalysed by wheat-germ RNA polymerase II . Under the same conditions, but in the presence of a single ribonucleoside triphosphate, the rate of condensation of the triphosphate substrate to a dinucleotide primer also exhibits a complex dependence with the template concentration . This effect is observed using poly{d(A-T)} as a template . For both reactions there are two extreme types of behaviour in each of which transcription appears to involve a single enzyme synthetic mode, characterized by either a high (at low template concentration) or a low (at high template concentration) probability of releasing the transcripts . A strong correlation is found between these two pathways, such that conditions favouring the abortive release of trinucleotide products in the single-step addition reaction are associated with the synthesis of short-length RNA species in productive elongation, and reciprocally . A model previously developed by Papanicolaou, Lecomte & Ninio {(1986) J . Mol . Biol . 189, 435-448} to account for the kinetics of polymerization/excision ratios with Escherichia coli DNA polymerase I, and by Job, Soulie, Job & Shire {(1988) J . Theor . Biol . 134, 273-289} for kinetics of RNA-chain elongation by wheat-germ RNA polymerase II provides an explanation for the observed behaviour with the plant transcriptase . The basic requirement of this model is a slow equilibrium between two states of the polymerization complex with distinct probabilities of releasing the product . In the presence of Mn2+, and under conditions allowing the synthesis of poly{r(A-U)}, one of these states is involved in the formation of oligonucleotides shorter than 15 bases, whereas the other catalyses the polymerization of chains longer than 40 bases.

Virology, 1990 Aug, 177(2), 764 - 7
Construction of an infectious genomic clone of porcine parvovirus: effect of the 5'-end on DNA replication; Casal JI et al.; The linear single-stranded DNA genome of the porcine parvovirus, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pUC18 using a simple and reliable method . These clones were stable during propagation in Escherichia coli JM109 . The recombinant clones of porcine parvovirus were infectious when transfected into monolayers of swine testes cells as identified by the development of cytopathic effect, indirect immunofluorescence with specific antiserum, and hemagglutination assays . DNA isolated from progeny virus arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis . Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogeneous, cloned DNA . The presence of the turn of the 5'-end loop seems to be necessary to get stable infectious clones.

Mol Cell Biol, 1990 Aug, 10(8), 4334 - 44
The Drosophila even-skipped promoter is transcribed in a stage-specific manner in vitro and contains multiple, overlapping factor-binding sites; Read D et al.; To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts . We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo . This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei . One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor . Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis . Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis . We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.

Bioorg Khim, 1990 Aug, 16(8), 1045 - 51
{Solid phase ligation of synthetic DNA fragments}; Filippov SA et al.; A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E . coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase . The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad) . Both ways gave equal results with some preference in the tetrad case . The reliability of E . coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.

Mol Microbiol, 1990 Aug, 4(8), 1381 - 91
The mobilization and origin of transfer regions of a Thiobacillus ferrooxidans plasmid: relatedness to plasmids RSF1010 and pSC101; Drolet M et al.; The components for the mobilization function of a plasmid DNA during conjugation include a cis-acting sequence (the origin of transfer, oriT) and a transacting sequence coding for mobilization (Mob) proteins . By genetic and deletion analysis, we have located the mobilization region of pTF1, a cryptic plasmid previously isolated from a Thiobacillus ferrooxidans strain . Within a 2797 bse-pair sequenced region, several open reading frames (ORFs) were predicted; two of the ORFs are divergently transcribed and they encode proteins of calculated molecular masses, 42.6kD (ORF2) and 11.4kD (ORF6) . Surprisingly, these protein sequences are substantially similar to two of the previously characterized mobilization proteins of the Escherichia coli IncQ plasmid, RSF1010 . Moreover, the pTF1 ORF2 (now designated MobL) sequence is also found to be similar to a presumed mobilization protein of plasmid pSC101 . Regions of sequence identity of plasmids pTF1, RSF1010 and pSC101 include their oriT sites . By alkaline agarose gel electrophoresis and DNA sequencing, we have established the location of the relaxation complex nick site within the oriT of pTF1 . An identical nick site, which is adjacent to a characteristic 10 base-pair inverted repeat sequence, is also found for plasmid RSF1010 . A recombinant plasmid containing a 42 base-pair synthetic piece of DNA encompassing the pTF1 inverted repeat and nick sequence was shown to be oriT-active.

Mol Microbiol, 1990 Aug, 4(8), 1363 - 9
Secretion of Ricinus communis glyceraldehyde-3-phosphate dehydrogenase by Escherichia coli; Hekman WE et al.; A plasmid, pWEH1, was constructed containing a fusion of the DNA encoding the signal sequence of the Escherichia coli outer-membrane protein A to the 5'-end of a glyceraldehyde-3-phosphate dehydrogenase cDNA from Ricinus communis . When expressed in E . coli, the fusion protein was secreted by the normal membrane-potential-dependent pathway . Processing by signal peptidase was inhibited by low concentrations of phenethyl alcohol . Quantitative cell fractionation was used to show that the mature plant protein was associated with the bacterial outer membrane . The protein could not be released from the membrane by washing with alkaline sodium carbonate . Radioactivity from {U-14C}-palmitate was incorporated into the heterologous protein . These results suggest that the sequence of this normally cytoplasmic enzyme contains a cryptic lipid-modification site, and the combination of a signal sequence plus a lipid-modification sequence results in specific targeting to the bacterial outer membrane.

Mol Microbiol, 1990 Aug, 4(8), 1319 - 27
Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli; Schlensog V et al.; Through complementation of a trans-acting regulatory mutation a gene has been cloned whose product is required for the formate induction of the anaerobic expression of the formate hydrogenlyase structural genes . By restriction analysis, and from the size of the encoded protein, the gene could be identified as being equivalent to fhlA described by Sankar et al . (1988) . The nucleotide sequence of the fhlA gene was determined and it was shown to code for a protein with a calculated Mr of 78,467 . Analysis of the derived amino acid sequence showed that the carboxy-terminal domain of FHLA shares considerable sequence similarity with NIFA and NTRC, which are the 'regulators' of two-component regulatory systems . Carboxy-terminal truncation of, and introduction of amino-terminal deletions in, the fhlA gene delivered inactive gene products . When overexpressed, FHLA mediates activation of expression of the formate dehydrogenase and hydrogenase structural genes in the presence of formate also under aerobic growth conditions . FHLA appears to bind to the upstream regulatory sequence (URS) in the 5' flanking region of the fdhF gene since activation of fdhF expression was dependent on the presence of the URS.

J Mol Recognit, 1990 Aug, 3(4), 149 - 55
Purines in tRNAs required for recognition by ATP/CTP:tRNA nucleotidyltransferase from rabbit liver; Spacciapoli P et al.; Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase from rabbit liver was studied using 12 tRNAs, previously treated with the chemical modifier diethylpyrocarbonate (DEP) . Such chemically modified tRNAs were labeled with 32P by nucleotidyltransferase, using alpha-{32P}ATP as a cosubstrate . A carbethoxylated purine at position 57 in the psi-loop interfered with recognition of the tRNA in all instances . DEP-modified purines at other positions (58 in the psi-loop, 52 or 53 in the psi-stem, and 71-73 in the acceptor stem), also interfered with the interaction, but in only a few tRNAs . The mammalian enzyme was more similar to the homologous enzyme from yeast than that from bacteria, in its requirements for chemically unmodified purines . The extent of exclusion of modified bases from 32P-labeled material diminished as the concentration of enzyme increased, demonstrating that interference was not due to the inability of the chemically altered tRNA to refold into a recognizable conformation . The degree of purification of the enzyme did not affect the identity of bases that inhibited the reaction when modified.

Mol Gen Genet, 1990 Aug, 223(1), 159 - 62
Iron(III)hydroxamate transport of Escherichia coli K12: single amino acid replacements at potential ATP-binding sites inactivate the FhuC protein; Becker K et al.; The mechanism of iron(III)hydroxamate transport appears to be of the periplasmic binding protein dependent transport (PBT) kind which is energized by ATP hydrolysis . The FhuC protein contains two domains typical of ATP-binding proteins . Lysine in domain I was replaced by glutamine and glutamate, and aspartate in domain II by asparagine and glutamate, resulting in FhuC derivatives which no longer transported ferrichrome and albomycin . FhuC inactivation by the aspartate-glutamate substitution is especially noteworthy since the negative charge thought to be involved in Mg2(+)-ATP binding remains the same and the two amino acid side chains differ in only a CH2 group . It is concluded that the two domains that represent consensus sequences among all peripheral cytoplasmic membrane proteins of PBT systems are involved in substrate transport.

Mol Gen Genet, 1990 Aug, 223(1), 121 - 33
Transcription and regulation of expression of the Escherichia coli methionyl-tRNA synthetase gene; Dardel F et al.; The DNA sequence and transcriptional organization around the Escherichia coli methionyl-tRNA synthetase gene, metG, were resolved . This gene can be transcribed in vivo and in vitro from two distinct promoters separated by 510 nucleotides . The upstream promoter is located within the coding sequence of a divergent gene expressing a protein of Mr 39 kDa of unknown function . Transcription originating from this upstream promoter is attenuated by a Rho-independent terminator before entering the structural gene . This leader RNA contains several potentially stable secondary structures, one of which shows striking similarity to tRNA(Met), but no methionine-rich coding sequence . The regulation of metG expression was investigated by means of fusions to the lacZ gene . Transcription of a metG::lacZ fusion is induced in a metG mutant and, reciprocally, repression is observed in a methionyl-tRNA synthetase overproducing strain . A model of metG expression control is proposed.

Genetika, 1990 Aug, 26(8), 1370 - 9
{Isolation of a spontaneous mutant of Escherichia coli superproducing proline and resistant to elevated concentrations of NaCl}; Neumyvakin LV et al.; We obtained a spontaneous mutant of Escherichia coli that was characterized by both proline superproduction and the resistance to osmotic stress . The selection of mutants was carried out among 2.5.10(5) clones survived upon plating strain SU1604 containing the sex factor F104, with a chromosome fragment carrying genes proB and proA, on solid modium with the proline analogue L-azetidine-2-carboxylate (AzT) . The obtained mutant AztR clones were used as donors in replica crossing with a pro- recipient, followed by subsequent selection of AztR Pro+ exconjugants . Analysis of growth of 456 exconjugants in liquid minimal medium with NaCl at a concentration of 0.6 M helped to identify 9 mutants with increased salt tolerance, as compared with control . From these, we selected one mutant (denoted as SU1604/F'104S) which demonstrated the highest salt tolerance correlating with higher production of proline . Analysis of the mutant's properties suggests that it belongs to the group of Osm mutants.

Bol Med Hosp Infant Mex, 1990 Aug, 47(8), 551 - 6
{Clinical and biochemical evaluation of the administration of growth hormone}; Mendoza-Morfin F et al.; Ten children with isolated growth hormone deficiency were treated for 1 year with 0.5 UI/kg week with Somatrem (recombinant human growth hormone), given as intramuscular injections three times weekly . Before treatment the children had a chronological age of 7-12.4 years (mean 10.4 years), with a bone age at least 25% below the chronological age . There was no radiological evidence of an intra or suprasellar mass in any child, and no response to provocative growth hormone tests (with exercise or arginine-insulin injection) . Informed written consent for treatment was obtained from the parents of each child . Clinical signs were registered every month; triiodothyronine, thyroxine, thyrotropine, glucose, urea, creatinine, blood cells count, and hemoglobine, glycosylated hemoglobine, glutamic-piruvic and glutamic-oxalacetic transaminases, alkaline phosphatase, anti-human growth hormone and, E . coli antibodies, insulin like growth factor 1, and bone age were assessed every 3 months . The mean height velocity was 0.27 +/- 0.1 cm/month before treatment, and increased throughout treatment to a value of 0.62 +/- 0.16 cm/month after 12 months . Within the first year eight of the 10 children had a height increase of 8.4 +/- 0.98 cm . The other two children showed no significant difference; one of them with a very low socioeconomic status, and the other developed typhoid fever . All of the children showed an advance in bone age, but none reached a bone age appropriate for their chronological age; without modifications in the laboratory parameters . Insulin like growth factor 1 increased in 9 children . Pain at the injection site was the only side effect reported.

Burns, 1990 Aug, 16(4), 286 - 90
An investigation of immunological subclass function in burned adults; Belcher HJ et al.; The serum concentrations of the immunoglobulin subclasses and their functional activity against pneumococcal polysaccharide (PPS) and E . coli antigens have been studied for 15 days in 12 adult burned patients, of whom six were randomly allocated to receive biosynthetic human growth hormone (somatropin) and six to form a control group . The concentrations of the major subclasses, IgG1 and IgG2, fell below their normal ranges as a result of injury by burning but had recovered significantly by the eleventh day of the study . There were no significant changes in the concentrations of the IgG3 and IgG4 subclasses, however, the relative proportions of the four subclasses remained unchanged and were comparable to those found in normal man . Significantly reduced functional activities, compared with normal volunteers, were observed in all the subclasses except IgG1 . The activities of the major subclasses during the study were correlated with their serum concentrations and when corrected for their concentrations were comparable to those of normal blood donors . There were no significant increases in concentrations or activity of the subclasses as a result of somatropin administration . It is concluded that patients with moderately sized burns have reduced immunoglobulin function which results from the changes in serum concentration rather than from any qualitative change and that somatropin administration results in no improvement in immunoglobulin function.

FEMS Microbiol Rev, 1990 Aug, 6(4), 383 - 98
A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli; Knappe J et al.; Anaerobically growing Escherichia coli cells contain the enzyme pyruvate formate-lyase which catalyses the non-oxidative cleavage of pyruvate to acetyl-CoA and formate . The enzyme is subject to interconversion between inactive and active forms . The active form contains an oxygen-sensitive organic free radical located on the polypeptide chain which is essential for catalysis . It affords a novel homolytic C-C bond cleavage of the pyruvate substrate . The radical is generated by an iron-dependent converter enzyme which requires reduced flavodoxin and adenosyl methionine as co-substrates and pyruvate as a positive allosteric effector . A second converter enzyme, also iron-dependent, accomplishes the removal of the radical . This post-translational interconversion cycle controls the activity state of pyruvate formate-lyase in the anaerobic cell . Anaerobic conditions also regulate pyruvate formate-lyase at the level of gene expression . Multiple promoters are responsible for effecting a twelve to fifteen fold induction and they are coordinately controlled in response to the oxygen and metabolic status of the cell by sequences which are located far upstream of the pfl coding region . The transcription factor Fnr has been identified as being responsible for part of the anaerobic control of pfl expression, probably through direct interaction with the upstream sequences . In contrast, the expression of the gene encoding the first iron-dependent converter enzyme is unaffected by anaerobiosis and is independent of the Fnr protein.

Bioessays, 1990 Aug, 12(8), 371 - 6
From cell membrane to nucleotides: the phosphate regulon in Escherichia coli; Torriani A; Most of the essential cellular components, like nucleic acids, lipids and sugars, are phosphorylated . The phosphate equilibrium in Escherichia coli is regulated by the phosphate (Pi) input from the surrounding medium . Some 90 proteins are synthesized at an increased rate during Pi starvation and the global control of the cellular metabolism requires cross-talk with other regulatory mechanisms . Since the Pi concentration is normally low in E . coli's natural habitat, these cells have devised a mechanism for synthesis of about 15 proteins to accomplish two specific functions: transport of Pi and its intracellular regulation . The synthesis of these proteins is controlled by two genes (the phoB-phoR operon), involving both negative and positive functions . PhoR protein is a histidine protein kinase, induced in Pi starvation and is a transmembrane protein . It phosphorylates the regulator protein PhoB which is also Pi starvation-induced . The PhoB phosphorylated form binds specifically to a DNA sequence of 18 nucleotides (the pho Box), which is part of the promoters of the Pho genes . The genes controlled by phoB constitute the Pho regulon . The repression of phoA (the gene encoding alkaline phosphatase) by high Pi concentrations in the medium requires the presence of an intact Pst operon (pstS, pstC, pstA, pstB and phoU) and phoR . The products of pstA and pstC are membrane bound, whereas the product of pstS is periplasmic and PstB and PhoU proteins are cytoplasmic . The function of the PhoU protein may be regulated by cofactor nucleotides and may be involved in signaling the activation of the regulon via PhoR.

Mol Biol Med, 1990 Aug, 7(4), 365 - 9
Sequence of a cDNA encoding human galactose-1-phosphate uridyl transferase; Flach JE et al.; We report a revised sequence of a cDNA that encodes a human galactose-1-phosphate uridyl transferase . The cDNA is 1295 bases in length and encodes a 43,000 Mr protein . The sequence was derived from a cDNA clone isolated from a transformed human lymphoblast cell line and amplified in a polymerase chain reaction . The revised sequence reveals a higher degree of amino acid conservation between the human enzyme and the homologous enzymes from Escherichia coli and yeast than was previously thought to exist.

J Biochem (Tokyo), 1990 Aug, 108(2), 321 - 6
UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing; Katsube T et al.; We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme . The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs . The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues . All the partial amino acid sequences determined protein-chemically {Nakano et al . (1989) J . Biochem . 106, 528-532} confirmed the primary structure of the enzyme . An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry . On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis . In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal . Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.

J Biochem (Tokyo), 1990 Aug, 108(2), 267 - 70
Evidence for in vivo synthesis of thiamin triphosphate by cytosolic adenylate kinase in chicken skeletal muscle; Miyoshi K et al.; We showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP) and ADP in addition to ATP formation from ADP {Shikata, H . et al . (1989) Biochem . Int . 18, 933-942} . To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching . Thiamin phosphate metabolism in chicken skeletal muscle was also studied . i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle . Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle . ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained . Both AK1 activity and TTP-synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum . iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant . In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1990 Aug, 108(2), 207 - 14
Presequence does not prevent folding of a purified mitochondrial precursor protein and is essential for association with a reticulocyte cytosolic factor(s); Murakami K et al.; Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) {EC 2.1.3.3} is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues, then is imported posttranslationally nto the mitochondrial matrix . We expressed rat pOTC in Escherichia coli, purified it in a denatured form, and showed that could be transported into isolated mitochondria in the presence of rabbit reticulocyte lysate {Murakami et al . (1988) J . Biol . Chem . 263, 18437-18442} . In order to compare the properties of the precursor and mature form of OTC, the rat mature OTC was synthesized in E . coli and purified . The recombinant OTC represented about 5% of the total bacterial protein and was present in both the supernatant and precipitate of the disrupted bacteria . The OTC, extracted from the precipitate with 8 M urea or 6 M guanidine.HCl, was essentially homogeneous, as judged by SDS-PAGE . When guanidine.HCl-denatured mature OTC was diluted and incubated at 0 degrees C for 40-60 h, it was reactivated to a specific activity of 170 mumol/min/mg protein at 37 degrees C (18% of that of the purified mature enzyme) . Guanidine.HCl-denatured pOTC was activated to a specific activity of 125 mumol/min/mg protein under similar conditions . The native and reactivated OTC sedimented with an s20.w value of 6.2S, whereas the activated pOTC sedimented with an s20.w of 5.2S . The activated pOTC was more unstable than the reactivated OTC at 50 degrees C . These observations indicate that the presequence does not prevent pOTC from folding into an enzymatically active trimeric form, although the pOTC trimer appears to be less compact than the mature trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Nippon Yakurigaku Zasshi, 1990 Aug, 96(2), 41 - 7
{Second messengers and protein phosphorylation in cellular signal transduction}; Kuno T; Protein phosphorylation has been recognized as a major mechanism by which cellular functions are controlled by neurotransmitters and hormones . In this review, applications of molecular biological techniques to the analyses of regulatory mechanisms of protein phosphorylation by four major second messengers, cAMP, cGMP, diacylglycerol, and Ca2+, are described . 1) Complementary DNA of the regulatory subunit of the cAMP-dependent protein kinase was cloned and expressed in E . coli . Point mutations were introduced in order to analyze functional domains of the subunit . 2) The soluble isoform of guanylate cyclase was purified, and a cDNA of its 70-KD subunit was cloned . Cyclic GMP binding to purified cGMP-dependent protein kinase was characterized using a rapid filtration assay . 3) Primary structure of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A) was determined and the presence of the second isoform of the enzyme was shown by the cDNA cloning technique . 4) The regulatory domain of the protein kinase C was expressed in E . coli . Analysis using site-directed mutagenesis revealed that a "zinc finger"-like structure is responsible for the binding of phorbol esters . In these studies, the molecular biological approach has proven to be useful for clarifying the molecular mechanisms of cellular signal transduction related to second messengers and protein phosphorylation.

Del Med J, 1990 Aug, 62(8), 1155 - 6, 1159-64
Response of tumors to thermodynamic stimulation of the immune system; Keen AR et al.; Cytokines such as interferon, interleukin and tumor necrosis factor are natural body defense proteins which have been used individually in recent years to produce a few complete responses of some tumors in a few patients but their overall effect has been limited . The hypotheses is that a biologic stimulus such as endotoxin will stimulate the immune system in a more natural way and hence will be more likely to have an effect especially in the presence of a naturally produced fever than treatment from just one lymphokine . One of the side effects of current lymphokine studies has been fever usually spiking in nature but there has been no obvious effort to relate the extent of the fever to any tumor responses . Clinical experience has shown that increased temperature can enhance the results of radiotherapy and chemotherapy in some situations . Therefore it is logical to put this hypothesis to a test clinically . It is surprising that the combined effect of fever and lymphokine stimulation has not been reported aside from Coley's work, and it is only in retrospect that fever has been suggested to be essential to the tumor control noted . Most of the literature, animal and human, has focused on the destructive effect of temperature on tumor cells and not as part of an immune response to control tumor growth . Endotoxin is the pyrogen on which most current information is available . More effective and more available pyrogens may be developed later.

Protein Eng, 1990 Aug, 3(8), 725 - 31
Improvement of nutritional value and functional properties of soybean glycinin by protein engineering; Kim CS et al.; Glycinin is one of the predominant storage proteins of soybean . To improve its functional properties (heat-induced gelation and emulsification) and/or nutritional value, the A1aB1b proglycinin subunit was modified on the basis of genetically variable domains suggested from the comparison of amino acid sequences of glycinin-type globulins from various legumes and nonlegumes and the relationships between the structure and the functional properties of glycinin . Thus, nucleotide sequences corresponding to each of the variable domains were deleted from the cDNA encoding the A1aB1b proglycinin, and a synthetic DNA encoding four continuous methionines was inserted into the cDNA region corresponding to each of the variable domains . Expression plasmids carrying the modified cDNAs were constructed and expressed in Escherichia coli strain JM105 . Some of the modified proteins were accumulated as soluble proteins in the cells at a high level and self-assembled . They exhibited functional properties superior to those of the native glycinin from soybean, which establishes the possibility of creating theoretically designed novel glycinins with high food qualities.

Protein Eng, 1990 Aug, 3(8), 713 - 9
Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha; Yamagishi J et al.; To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques . An expression plasmid for TNF-alpha was mutagenized by passage through an E . coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis . Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity . The cytotoxic activities of purified TNF-alpha muteins, e.g . TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha . These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity . These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146 . In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor . This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site.

J Trop Pediatr, 1990 Aug, 36(4), 176 - 9
Epidemiological survey of the enteropathogenic Escherichia coli isolated from children with diarrhoea; Regua AH et al.; Escherichia coli was isolated in 382 (94 per cent) of 406 children from 0 to 3 years of age who had been hospitalized for diarrhoea at the Hospital Municipal Salles Neto, Rio de Janeiro . Enteropathogenic Escherichia coli strains were isolated in 67 samples (18 per cent), distributed among the serogroups that were tested as follows: 0111 (33 per cent); 0125 (19 per cent); 0126, 0127, and 0142 (9 per cent); 0128 and 0119 (8 per cent); 055 (5 per cent); 0114 (2 per cent) . No strains of EPEC belonging to serogroups 086, 0126, and 0158 were found . Among the samples in which EPEC strains were isolated, 15.0 per cent were children living in dwellings which had piped systems of water supply and drain, whereas with regard to those living in houses without such facilities, this percentage raised to 24 per cent . Similar results were found when the availability of water supply of drainage were taken separately.

J Pathol, 1990 Aug, 161(4), 321 - 5
Role of endotoxaemia in the development of hepatic failure following hepatic artery occlusion; Shibayama Y; The present study was undertaken to examine whether endotoxemia relates to the development of hepatic failure following surgical ligation or embolization of the hepatic artery . A small amount of endotoxin was given immediately or 1 week after hepatic artery ligation in rats, and liver function tests and morphological examination of the liver were performed at 24 h after the administration . Hepatic artery ligation alone or endotoxin administration alone did not induce functional and morphological alteration of the liver . However, when endotoxin was given immediately after hepatic artery ligation, there was an increase in serum transaminase activity and focal hepatocellular necrosis developed . On the other hand, when endotoxin was given 1 week after hepatic artery ligation, the hepatic injury was not induced because of the development of hepatic arterial collaterals . These data suggest that endotoxaemia may be a cause of hepatic failure following hepatic artery occlusion, and that the risk may persist until the establishment of hepatic arterial collaterals.

Can J Ophthalmol, 1990 Aug, 25(5), 260 - 2
Acanthamoeba keratitis with two species of Acanthamoeba; Beattie AM et al.; We describe a case of Acanthamoeba keratitis related to soft contact lens wear . The patient presented with a 3-week history of severe uniocular pain, radial stromal infiltrates and subepithelial infiltrates with no epithelial defect . Acanthamoeba was cultured from the corneal biopsy specimen, contact lens and lens case . The corneal biopsy culture grew both A . castellani and A . polyphaga as well as Escherichia coli . The patient was treated with topical dibromopropamidine isethionate (Brolene) drops, neomycin and polymyxin B drops and fortified gentamicin drops . Gradual clinical improvement ensued.

Biophys J, 1990 Aug, 58(2), 403 - 11
Permanent dipole moment of tRNA's and variation of their structure in solution; Porschke D et al.; The structure of six different tRNA molecules has been analyzed in solution by electrooptical measurements and by bead model simulations . The electric dichroism measured as a function of the field strength shows that tRNA's are associated with substantial permanent dipole moments, which are in the range of 1 x 10(-27) cm(identical to 300 D; before correction for the internal directing field) . Rotational diffusion time constants of tRNA molecules in their native state at 2 degrees C show a considerable variation . A particularly large value found for tRNA(Tyr) (50 ns) can be explained by its nine additional nucleotide residues . However, remarkable variations remain for tRNA molecules with the standard number of 76 nucleotide residues (tRNA(Phe) {yeast} 41.6 ns, tRNA(Val) {Escherichia coli} 44.9 ns, tRNA(Glu) {E . coli} 46.8 ns; tRNA(Phe) {E . coli} 48.3 ns) . These variations indicate modulations of the tertiary structure, which may be due to a change of the L-hinge angle . Bead models are used to simulate both electric and hydrodynamic parameters of tRNA molecules according to the crystal structure of tRNA(Phe) (yeast) . The asymmetric distribution of phosphate charges with respect to the center of diffusion leads, under the assumption of a constant charge reduction to 15% by ion condensation, to a theoretical dipole moment of 7.2 x 10(-28) cm, which is in reasonable agreement with the measurements . The dichroism decay curve calculated for tRNA(Phe) (yeast) is also consistent with the measurements and thus the structure in solution and in the crystal must be very similar in this case . However, our measurements also indicate that the structure of some other tRNA's in solution is different, even in cases with the same number of nucleotide residues.

Int J Exp Pathol, 1990 Aug, 71(4), 433 - 40
Protection against endotoxin-induced foetal resorption in mice by desferrioxamine and ebselen; Gower JD et al.; Endotoxin was administered to mice on their 13th day of pregnancy at doses which caused the resorption of approximately 50% of the implanted foetuses . The iron chelator desferrioxamine was found to significantly inhibit the percentage of resorptions induced by endotoxin in a dose-dependent manner . The highest dose of desferrioxamine (5 mg) given intravenously 30 min prior to, immediately after, and 4 and 24 h after endotoxin inoculation, reduced the percentage of resorptions from 56.9 to 17.9% . Administration of the novel selenium-containing compound ebselen, which is both an antioxidant and an inhibitor of leukotriene synthesis, was also found to significantly protect against endotoxin-induced foetal resorptions, reducing the percentage of resorbed foetuses from 52.9 to 26.0% when given at a dose of 50 mg/kg (s.c.) at the time of endotoxin inoculation and 24 and 48 h following . Both these compounds also significantly reduced the increase in spleen weights observed when the mice were given endotoxin . These results provide evidence that the iron-catalysed production of hydroxyl radicals from other oxygen-derived species and the formation of leukotrienes play an important role in the mechanism by which endotoxin causes foetal resorptions in the mouse.

Anal Chem, 1990 Aug 1, 62(15), 1628 - 31
Hybrid biosensor for the determination of lactose; Svorc J et al.; Genetically manipulated bacteria Escherichia coli K-12 recombinant PQ-37 and glucose oxidase (EC 1.1.3.4) were used for the construction of a hybrid lactose sensor because of the hyperproduction of beta-galactosidase (EC 3.2.1.23) by E . coli effected by a genotoxic agent . The biocatalytic layer was prepared by coimmobilization of the E . coli cells with glucose oxidase on the nylon network via glutardialdehyde and fixed to the Clark oxygen electrode . The influence of pH, temperature, and concentration of activators of beta-galactosidase on the sensor response was tested . Analyses of milk products were completed without any special pretreatment of the samples . The contents of lactose determined by hybrid sensor agree with conventional photometric measurements . Standard relative deviation is less than 3% for all samples . The half-life of operational stability is 30 days.

Circ Shock, 1990 Aug, 31(4), 387 - 94
Dopamine beta-hydroxylase inhibition reveals a selective influence of endotoxin on catecholamine content of rat tissues; Arnold J et al.; Endotoxin is known to affect sympathetic nervous system (SNS) activity . However, it is unknown whether selective activation occurs in different tissues . The present work assessed SNS activity in several tissues of endotoxin-treated rats by measuring the accumulation of dopamine following dopamine beta-hydroxylase inhibition with disulfiram . Twenty-four hours following endotoxin injection (1 mg/kg), body weight and food intake were significantly reduced . The weight of spleen and liver was significantly greater in endotoxin-injected rats compared to either control animals or rats pair-fed the same quantity of food ingested by the endotoxin-treated rats . Norepinephrine (NE) content of heart, liver, and brown adipose tissue (BAT) was significantly lower in endotoxin-injected animals, but it was unchanged in spleen and gastrocnemius muscle . Endotoxin caused a significant elevation in the dopamine content of heart, liver, BAT, and gastrocnemius muscle, whereas it had no effect on spleen . The results suggest that SNS activity is increased in heart, liver, BAT, and gastrocnemius muscle 26 h after endotoxin injection.

Circ Shock, 1990 Aug, 31(4), 365 - 75
Lack of beneficial effects of milrinone in severe septic shock; Lindgren S et al.; The effects of milrinone were investigated in a porcine septic shock model . Septic shock was induced by i.v . infusion of live Escherichia coli . Anesthetized pigs were treated with milrinone before the E . coli infusion or were left untreated as septic controls . E . coli caused a significant reduction of cardiac index, renal blood flow, and portal blood flow . Pulmonary vascular resistance was significantly increased, as were the systemic and preportal vascular resistances . One of the seven septic control pigs died . In milrinone-treated septic animals cardiac index was maintained for a longer period of time but blood pressure was significantly reduced compared with the control pigs . Pulmonary vasoconstriction was little affected by pre-treatment with milrinone . Four out of seven milrinone-treated pigs died during the observation period . It is concluded that in the settings of the used porcine septic shock model milrinone treatment has no beneficial effect.

Magn Reson Med, 1990 Aug, 15(2), 201 - 10
T2 of endotoxin lung injury with and without methylprednisolone treatment; Shioya S et al.; NMR relaxation times (T1 and T2) and the water content (WC) of in vitro rat lungs were measured during the course of endotoxin lung injury in rats . Measurements of normal lungs, untreated endotoxin-injured lungs, and endotoxin-injured lungs treated with methylprednisolone (MPSL) were compared . The untreated endotoxin lungs showed prolongation of the fast and slow T2 components (T2f and T2s), but no significant changes in T1 or water content . Also, there was no correlation between 1/WC and relaxation rates or between T1 and T2 . MPSL treatment prevented T2f and T2s prolongation; however, the duration of MPSL effectiveness was limited . Animals which were treated with MPSL more than 7 h prior to measurements showed T2 prolongation . This study indicates that NMR relaxation times, particularly T2, can be useful in evaluating lung injuries and their treatments.

J Inorg Biochem, 1990 Aug, 39(4), 317 - 25
Silver accumulation and resistance in Escherichia coli R1; Starodub ME et al.; E . coli R1 contains at least 2 large plasmids (83 and 77 kb) while E . coli S1 was previously cured of the 83 kb plasmid . Transformation using artificial competence, high-voltage electroporation, and plasmid mobilization experiments with the mobilizing plasmid RP4, failed to ascertain the 83 kb plasmid was responsible for Ag(+)-resistance . Silver accumulation by an Ag(+)-sensitive E . coli S1 strain was 5-fold higher than an Ag(+)-resistant E . coli R1 strain . The Ag(+)-resistant E . coli R1 strain produced 33% more H2S and 32% more intracellular acid labile SH than the Ag(+)-sensitive E . coli R1 strain when grown in the absence of AgNO3 . Hydrophobic interaction chromatography revealed E . coli R1 displayed higher cell surface hydrophobicity than E . coli S1 . HPLC protein analysis of cell-free extracts also revealed differences between protein fractions in E . coli R1 and S1 strains.

J Gen Virol, 1990 Aug, 71 ( Pt 8), 1669 - 74
Cloning and sequencing the messenger RNA of the N gene of viral haemorrhagic septicaemia virus; Bernard J et al.; The mRNA transcribed from the N gene of viral haemorrhagic septicaemia virus (VHSV) of salmonids has been cloned in Escherichia coli and expressed . Fusion proteins were recognized by monoclonal antibody directed against the N protein from the viral particle . A 1212 bp long open reading frame (ORF) coding for 404 amino acids with a calculated Mr of 44590 was deduced from the nucleotide sequence . The ORF was preceded by a 93 bp segment including in position 42 the AACAC pentanucleotide which is presumed to be the start signal for transcription by analogy with other rhabdoviral mRNAs . The upstream 41 bp region could correspond to the covalently linked positive polarity leader RNA as also found on the N mRNA from infectious haematopoietic necrosis virus (IHNV) . This may be a characteristic of fish lyssaviruses . The AAACC sequence, which is part of the leader, was not found . Amino acids 44 to 359 from IHNV and 45 to 360 from VHSV are 45.3% homologous . A strong homology which could reflect functional importance was also found for potential phosphorylation sites and hydrophobic peaks despite the fact that the two viruses evolved on different continents.

Am J Vet Res, 1990 Aug, 51(8), 1180 - 3
Effects of preweaning exposure to a starter diet on enterotoxigenic Escherichia coli-induced postweaning diarrhea in swine; Sarmiento JI et al.; Experiments were conducted to evaluate the effect of restricted feeding of a starter diet to suckling pigs (creep feeding) in a model of postweaning colibacillosis . The hypothesis that restricted creep feeding primes an intestinal allergic reaction to starter diet ingested after weaning was tested . Twenty-eight suckling pigs were fed a starter diet for 3 h/d on days 7, 8, and 9 after birth (creep-fed) . Twenty-six suckling pigs were not fed the diet until 3 weeks of age (not creep-fed), when all pigs were weaned and given the starter diet . One day after weaning, 24 creep-fed and 22 not creep-fed pigs were inoculated with K88+ enterotoxigenic Escherichia coli, and 4 pigs in each group were kept as noninoculated controls . Among inoculated pigs (principals), 10 creep-fed and 12 not creep-fed pigs were found to be genetically resistant to K88+ E coli and remained healthy during the 6-day postinoculation period, as did the noninoculated controls . Eighteen (10 creep-fed and 8 not creep-fed) of the 24 genetically susceptible principals developed diarrhea after inoculation . There were no significant differences in the incidence and severity of diarrhea, amount of body weight loss, and mortality between creep-fed and not creep-fed susceptible principal pigs . Histologic examination of intestine from control pigs and principals that survived for 6 days after infection did not reveal any substantial morphologic difference between creep-fed and not creep-fed groups . In conclusion, creep feeding was not required for the production of diarrhea in this model . Creep feeding did not induce morphologic changes characteristic of an allergic reaction in the small intestine.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6436 - 40
Isolation and expression of rat liver sepiapterin reductase cDNA; Citron BA et al.; Sepiapterin reductase (7,8-dihydrobiopterin: NADP+ oxidoreductase, EC 1.1.1.153) catalyzes the terminal step in the biosynthetic pathway for tetrahydrobiopterin, the cofactor necessary for aromatic amino acid hydroxylation . We report here the isolation of a cDNA clone for rat liver sepiapterin reductase . The cDNA has been excised from a lambda vector and the DNA sequence was determined . The insert contains the coding sequence for at least 95% of the rat enzyme and is fused to the Escherichia coli beta-galactosidase N-terminal segment and the lac promoter . The N-terminal region of the clone contains an extraordinarily high G + C content . The amino acid sequence deduced from the clone is in agreement with the size and composition of the enzyme and was matched to several tryptic peptide sequences . The enzyme encoded by the cDNA insert was shown to have sepiapterin reductase activity after expression in E . coli . Structural similarities were identified between this protein and several enzymes that should contain similar nucleotide and pteridine binding sites.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6378 - 82
Peptides on phage: a vast library of peptides for identifying ligands; Cwirla SE et al.; We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors . Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage . The vector fAFF1, derived from the tetracycline resistance-transducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene III . A library of 3 x 10(8) recombinants was generated by cloning randomly synthesized oligonucleotides . The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of beta-endorphin (Tyr-Gly-Gly-Phe) . Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody . The striking finding is that all 51 contained tyrosine as the N-terminal residue and that 48 contained glycine as the second residue . The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 microM (Tyr-Gly-Phe-Trp-Gly-Met) to 8.3 microM (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7.1 nM for a known high-affinity ligand (Tyr-Gly-Gly-Phe-Leu) . These results show that ligands can be identified with no prior information concerning antibody specificity . Peptide libraries are also likely to be useful in finding ligands that bind to other classes of receptors and in discovering pharmacologic agents.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6087 - 91
Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli; Burnouf D et al.; The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP{d(ApG)} adducts, although they account for only 25% of the lesions formed, are approximately 5 times more mutagenic than the major GG adduct . We report the construction of vectors bearing a single cisDDP{d(ApG)} lesion and their use in mutagenesis experiments in Escherichia coli . The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells . In SOS-induced cells, mutation frequencies of 1-2% were detected . All these mutations are targeted to the 5' base of the adduct . Single A----T transversions are mainly observed (80%), whereas A----G transitions account for 10% of the total mutations . Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5' to the adduct occur at a comparable frequency (10%) . No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP{d(ApG)} adducts are not blocking lesions . The high mutation specificity of cisDDP{d(ApG)}-induced mutagenesis is discussed in relation to structural data.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6034 - 8
Molecular characterization of the GCN4-DNA complex; Gartenberg MR et al.; We report studies of the DNA complex formed by GCN4, a transcriptional activator of eukaryotic amino acid biosynthetic operons . The DNA thermodynamic binding domain, defined by primer extension analysis, spans at least 18 base pairs, a site much larger than the 9-base-pair consensus defined by homology with naturally occurring binding sites . Chemical modification experiments reveal multiple sites of protein-DNA contact: methylation of any guanine N-7 or adenine N-3, ethylation of any phosphate oxygen, or elimination of any nucleoside within a region spanning nearly one and a half turns of the double helix reduces the binding affinity of the complex measurably . Nevertheless, the protein yields no detectable hydroxyl radical footprint, implying that the minor groove is reagent-accessible in the protein-DNA complex . These chemical modification patterns indicate that GCN4 does not utilize any of the DNA-recognition motifs of paradigm DNA-binding proteins . Assays to detect DNA bending induced by truncated or intact GCN4 indicate that protein conformation and not a protein-induced bend is responsible for the anomalous electrophoretic behavior of GCN4-DNA complexes.

Biochem Pharmacol, 1990 Aug 1, 40(3), 473 - 9
Stimulation and inhibition of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced strand breaks and interstrand cross-linking in Col E1 plasmid deoxyribonucleic acid by polyamines and inorganic cations; Srivenugopal KS et al.; The influence of various polyamines and metallic cations on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced DNA single-strand breaks and DNA interstrand cross-linking was in Col E1 plasmid using electrophoretic techniques . Spermidine and spermine (0.4 to 1.5 mM concentration range) markedly stimulated BCNU-induced DNA nicking, whereas putrescine had no effect on the nicking process . In contrast to the polyamines, BCNU-induced DNA nicking was decreased by the three inorganic cations, Na+ (100 and 200 mM), Mg2+ (0.5 and 1.5 mM), and Co3+ (NH3)6 (0.2 and 0.4 mM), with the trivalent hexamminecobalt ions being most inhibitory . When the monofunctional N-methyl-N-nitrosourea (MNU) was used (instead of the bifunctionally active BCNU) to alkylate Col E1 DNA, nicking of the DNA was inhibited by spermidine . Furthermore, the ability of chloroethylated Col E1 DNA to form interstrand cross-links after treatment with BCU was inhibited by 0.5 mM spermidine and 0.5 mM spermine, both concentrations within the intracellular range . Putrescine at 3-6 mM only marginally stimulated DNA cross-linking . In comparison, the inorganic cations all enhanced Col E1 DNA cross-linking by BCNU, with the rank order of cross-link stimulation being Mg2+, Na+, and Co3+ (NH3)6 . These results provide evidence that polyamines can interact with DNA to modulate chloroethylnitrosourea-induced DNA damage and that the interaction is not only a function of the charge on the polyamine molecule but also of the chemical structure of the polyamine.

Surgery, 1990 Aug, 108(2), 370 - 4; discussion 374-5
Development and reversal of endotoxemia and endotoxin-related death in obstructive jaundice; Diamond T et al.; Gut-derived endotoxemia has been implicated in postoperative complications in patients with jaundice . It is thought that absence of bile in the gut predisposes to portal absorption of endotoxin and endotoxemia is reversed by oral bile salt replacement or internal biliary drainage and return of bile to the gut, but not by external drainage . We believe that the importance of gastrointestinal bile flow has been overestimated and biliary obstruction and the integrity of hepatocyte and Kupffer cell function are more important in the development and reversal of endotoxemia . In experiment 1, serum endotoxin concentrations were measured in control rats (n = 10) after choledochovesical fistula (n = 15) and bile duct ligation (n = 15) and after relief of biliary obstruction by internal drainage (choledochoduodenostomy; n = 8) and sterile external drainage (choledochovesical fistula; n = 8), with a quantitative limulus assay . In experiment 2, mortality rates were measured in similar groups 48 hours after administration of oral endotoxin (5 mg/100 gm) and intravenous lead acetate (5 mg/100 gm) . Bilirubin levels were elevated in bile duct ligation (192 +/- 13 mumols/L) compared with control animals and those with choledochovesical fistula, internal drainage, and external drainage (10.6 +/- 1.5 mumols/L) . In experiment 1, significant portal endotoxemia and systemic endotoxemia occurred in bile duct ligation (portal, 130.4 +/- 12.9 pg/ml; systemic, 91.8 +/- 11.0 pg/ml) but not in choledochovesical fistula (portal, 49.3 +/- 17.1 pg/ml; systemic, 27.2 +/- 11.5 pg/ml) . Relief of obstruction by both internal and external drainage reversed endotoxemia . In experiment 2, significant death occurred in bile duct ligation (13 of 15) but not in choledochovesical fistula (3 of 15), and relief of obstruction by both internal and external drainage prevented death . These results confirm that biliary obstruction is a more important factor than is gastrointestinal bile flow in the development and reversal of endotoxemia.

J Cell Physiol, 1990 Aug, 144(2), 190 - 6
Colony stimulating factor-1 is a negative regulator of the macrophage respiratory burst; Phillips WA et al.; Several cytokines have previously been shown to prime macrophages for enhanced release of oxygen radicals in response to subsequent stimulation . We now demonstrate that the presence of the macrophage-specific colony stimulating factor-1 (CSF-1) inhibits the priming of murine macrophages by a variety of agents including tumor necrosis factor alpha, granulocyte/macrophage colony stimulating factor, interferon-gamma, and bacterial lipopolysaccharide . CSF-1 is also able to reduce the respiratory burst in the absence of priming . Our results indicate that CSF-1 is a potent negative regulator of the macrophage respiratory burst which acts to oppose the priming (enhancing) action of macrophage activating agents . We propose that CSF-1 may have a potentially important and previously unrecognized, role as a physiological regulator which restricts or terminates the activation of macrophages in order to prevent an uncontrolled inflammatory reaction.

Am J Hum Genet, 1990 Aug, 47(2), 202 - 17
Mutations causing hemophilia B: direct estimate of the underlying rates of spontaneous germ-line transitions, transversions, and deletions in a human gene; Koeberl DD et al.; Spontaneous mutation provides the substrate for evolution on one hand and for genetic susceptibility to disease on the other hand . X-linked diseases such as hemophilia B offer an opportunity to examine recent germ-line mutations in humans . By utilizing the direct sequencing method of genomic amplification with transcript sequencing, eight regions (2.46 kb) of likely functional significance in the factor IX gene have been sequenced in a total of 60 consecutive, unrelated hemophiliacs . The high frequency of patient ascertainment from three regions in the midwestern United States and Canada suggests that the sample is representative of hemophiliacs of northern European descent . Twenty-six of the delineated mutations are reported herein, and the group of 60 is analyzed as a whole . From the pattern of mutations causing disease and from a knowledge of evolutionarily conserved amino acids, it is possible to reconstruct the underlying pattern of mutation and to calculate the mutation rates per base pair per generation for transitions (27 x 10(-10)), transversions (4.1 x 10(-10), and deletions (0.9 x 10(-10)) for a total mutation rate of 32 x 10(-10) . The proportion of transitions at non-CpG nucleotides is elevated sevenfold over that expected if one base substitution were as likely as another . At the dinucleotide CpG, transitions are elevated 24-fold relative to transitions at other sites . The pattern of spontaneous mutations in factor IX resembles that observed in Escherichia coli when the data are corrected for ascertainment bias . The aggregate data hint that most mutations may be due to endogenous processes . The following additional conclusions emerge from the data: (1) Although in recent decades reproductive fitness in individuals with mild and moderate hemophilia has been approximately normal, the large number of different mutations found strongly suggest that these levels of disease substantially compromised reproduction in previous centuries . (2) Mutations which putatively affect splicing account for at least 13% of independent mutations, indicating that the division of the gene into eight exons presents a significant genetic cost for the organism . In one individual a "silent" mutation at lysine 5 is likely to cause hemophilia by generating a perfect splice donor consensus sequence in exon b . (3) All the missense mutations occurred at evolutionarily conserved amino acids . As additional data are generated on the pattern of mutations caused by specific mutagens, it will be possible to utilize the pattern of spontaneous mutation to estimate the maximal contribution of that mutagen during the past century.

Proc Natl Acad Sci U S A, 1990 Aug, 87(15), 5940 - 4
Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription; Tsung K et al.; The OmpR binding sequence (OBS) in the upstream region of the ompF promoter of Escherichia coli was fused to 27 synthetic promoters . Transcription from a number of weak promoters, regardless of their sequences, was dramatically activated in the presence of OmpR, a transcriptional activator . In vivo DNA footprinting revealed that OmpR enhanced the binding of RNA polymerase to the promoters . This enhancement was essential for transcription of weak promoters, while OmpR binding to the OBS fused to a strong promoter was inhibitory for transcription . These results indicate that OmpR stabilizes the formation of an RNA polymerase-promoter complex, possibly a closed promoter complex, and that a transcription activator can serve not only as a positive but also as a negative regulator for gene expression.

J Bacteriol, 1990 Aug, 172(8), 4741 - 3
Stimulation of glutamine transport by osmotic stress in Escherichia coli K-12; Gehring K et al.; Osmotic stress produced by high concentrations of sucrose stimulated the high-affinity transport of glutamine in Escherichia coli cells . Glutamine transport via a low-affinity system was not affected . Osmotic stress produced by NaCl, in contrast, inhibited the transport of glutamine and some other amino acids . Maltose transport was strongly inhibited by osmotic stress.

J Bacteriol, 1990 Aug, 172(8), 4736 - 40
High-level expression of the FtsA protein inhibits cell septation in Escherichia coli K-12; Wang HC et al.; DNA fragments encoding the ftsA gene were subcloned into plasmids downstream of a lac promoter or a tac promoter . These plasmid constructs, when transformed into wild-type and mutant strains, inhibited normal cell septation, causing the formation of long nonseptate filaments . This phenotype is due to overproduction of the FtsA protein.

J Bacteriol, 1990 Aug, 172(8), 4696 - 700
Molecular cloning, nucleotide sequence, and expression of shl, a new gene in the 2-minute region of the genetic map of Escherichia coli; Leclerc G et al.; Cells of Escherichia coli that harbor supH (an allele of the wild-type gene serU) are sensitive to UV irradiation and temperature and appear to have an impaired cell division control mechanism . We found that a gene located at the 2-min region, designated shl, inhibited the growth of supH-harboring cells when carried by a high-copy-number plasmid, whereas the same plasmid had no visible effect when present in parental cells . The amino acid sequence predicted from the nucleotide sequence of the shl gene indicated a similarity to the GalR and LacI repressor proteins, suggesting it is a transcription regulator . The sequence between the promoter and the structural genes revealed the presence of a short open reading frame of 28 amino acid residues followed by a segment of 81 base pairs . These structural features suggest that a transcription antitermination mechanism may be involved in the regulation of expression of the shl gene . The possibility that shl is a regulator of serU is discussed.

J Bacteriol, 1990 Aug, 172(8), 4682 - 4
Isogenic variants of Escherichia coli with altered morphology have peptidoglycan with identical muropeptide composition; de Jonge BL; The peptidoglycan compositions of three isogenic morphological mutants of Escherichia coli were determined by high-pressure liquid chromatography analysis . The muropeptide compositions of the peptidoglycan of these mutants were the same, indicating that the shape of E . coli is not (solely) determined by the chemical composition of the peptidoglycan . Furthermore, it appeared that the muropeptide composition of the peptidoglycan was not affected by growth temperature.

J Bacteriol, 1990 Aug, 172(8), 4652 - 60
Cloning, expression, and characterization of the Escherichia coli K-12 rfaD gene; Pegues JC et al.; The rfaD gene encodes ADP-L-glycero-D-mannoheptose-6-epimerase, an enzyme required for the biosynthesis of the lipopolysaccharide precursor ADP-L-glycerol-D-mannoheptose . The precise localization of the rfaD gene on a 1.3-kilobase SspI-HpaI fragment is reported . The rfaD gene and the flanking regions were completely sequenced . The location of the rfaD gene on the physical map of the Escherichia coli chromosome was determined . Primer extension studies were used to define the regulatory region of the rfaD gene . The cloned rfaD gene directed the synthesis of a 37,000-dalton polypeptide in several in vivo and in vitro expression systems . N-terminal analysis of purified ADP-L-glycero-D-mannoheptose-6-epimerase confirmed the first 34-amino-acid sequence deduced from the nucleotide sequence of the rfaD gene coding region . The primary structure of the rfaD protein contains the sequence fingerprint for the ADP-binding beta alpha beta fold at the N terminus.

J Bacteriol, 1990 Aug, 172(8), 4631 - 40
Nucleotide sequence and analysis of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli; Moore RC et al.; The DNA sequence of a 3.23-kilobase fragment of the Escherichia coli chromosome encoding biosynthetic arginine decarboxylase (ADC) was determined . This sequence contained the speA open reading frame (ORF) as well as partial speB and metK ORFs . The ADC ORF is 1,974 nucleotides long; the deduced polypeptide contains 658 amino acids with a molecular size of 73,980 daltons . The molecular weight and predicted ADC amino acid composition are nearly identical to the amino acid analysis of purified ADC performed by Wu and Morris (J . Biol . Chem . 248:1687-1695, 1973) . A translational speA-lacZ fusion, pRM65, including 1,389 base pairs (463 amino acids) of the 5' end of speA was constructed . Western blots (immunoblots) with beta-galactosidase antisera revealed two ADC::beta-galactosidase fusion proteins in E . coli bearing pRM65: 160,000 and 156,000 daltons representing precursor and mature hybrid proteins, respectively . The predicted amino acid sequence of ADC contains a region of six amino acid residues found in two bacterial diaminopimelic acid decarboxylases and three eucaryotic ornithine decarboxylases . This conserved sequence is located approximately eight amino acids from the putative pyridoxal phosphate-binding site of ADC and is predicted to be involved in substrate binding.

J Bacteriol, 1990 Aug, 172(8), 4603 - 9
Intrinsic bends and integration host factor binding at F plasmid oriT; Tsai MM et al.; F plasmid oriT DNA extending from the F kilobase coordinate 66.7 (base pair {bp} 1 on the oriT sequence map) rightward to bp 527 was analyzed for intrinsic bends (by permutation assays) and for binding of integration host factor (IHF) (by gel retardation and DNase footprinting) . Intrinsic bending of the 527-bp fragment (bend center approximately at bp 240) was represented as a composite of at least two components located near bp 170 and near bp 260 . IHF bound primarily to a site extending from bp 165 to 195 and with lower affinity to a site extending from bp 287 to 319 . The intrinsic curvature and sequences to which IHF binds (IHF is known to bend DNA) may play a structural role in oriT function.

J Bacteriol, 1990 Aug, 172(8), 4587 - 92
Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli; MacInnes JI et al.; The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated . No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes . However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR . Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain . For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX . Four cysteine residues in the amino-terminal region are also conserved . The promoter region of hlyX is very similar to that of fnr . It has a putative -10 sequence which closely resembles the E . coli -10 consensus sequence . This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence . Functional homology between HlyX and FNR was also demonstrated . Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E . coli . These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E . coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.

J Bacteriol, 1990 Aug, 172(8), 4555 - 62
Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction; He B et al.; Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP . The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains . These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon . Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR . Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter . The purR product, purine repressor, was shown to bind specifically to each operator . Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression.

J Bacteriol, 1990 Aug, 172(8), 4441 - 7
Location of a P1 plasmid replication inhibitor determinant within the initiator gene; Muraiso K et al.; The P1 plasmid replication initiator protein, RepA, binds to its own promoter and represses transcription efficiently . There are only about 20 RepA dimers present per repA gene . A possible reason for this highly restrained expression became evident when repA expression was increased by using foreign promoters: with fivefold overexpression, the replication rate was diminished, and with 40-fold overexpression, replication was not detectable . The inhibition was P1 specific: growth of Escherichia coli and replication of pSC101, R6K, and mini-F plasmids were not affected . The activity is apparently not from RepA itself . Excess purified RepA did not inhibit replication in vitro . Mutations of the repA translation initiation codon reduced synthesis of the initiator but not the inhibitory activity . Deletion from either the N- or C-terminal ends of repA (28 and 69 codons, respectively, out of the 286-codon open reading frame) affected the initiator but not the inhibitory activity . Further deletions affected both the activities . These results demonstrate that the integrity of the initiator is not required for inhibition, but involvement of an unstable initiator fragment or of initiator mRNA cannot be ruled out.

J Bacteriol, 1990 Aug, 172(8), 4263 - 70
Characterization of the F-plasmid conjugative transfer gene traU; Moore D et al.; We characterized the traU gene of the Escherichia coli K-12 conjugative plasmid F . Plasmids carrying segments of the F transfer operon were tested for their capacity to complement F lac traU526 . The protein products of TraU+ clones were identified, and the nucleotide sequence of traU was determined . traU mapped between traW and trbC . It encodes a 330-amino-acid, Mr36,786 polypeptide that is processed . Ethanol caused accumulation of a precursor polypeptide; removal of ethanol permitted processing of the protein to occur . Because F lac traU526 strains appear to be resistant to F-pilus-specific phages, traU has been considered an F-pilus assembly gene . However, electron microscopic analysis indicated that the traU526 amber mutation caused only a 50% reduction in F-piliation . Since F lac traU526 strains also retain considerable transfer proficiency, new traU mutations were constructed by replacing a segment of traU with a kanamycin resistance gene . Introduction of these mutations into a transfer-proficient plasmid caused a drastic reduction in transfer proficiency, but pilus filaments remained visible at approximately 20% of the wild-type frequency . Like traU526 strains, such mutants were unable to plaque F-pilus-specific phages but exhibited a slight sensitivity on spot tests . Complementation with a TraU+ plasmid restored the wild-type transfer and phage sensitivity phenotypes . Thus, an intact traU product appears to be more essential to conjugal DNA transfer than to assembly of pilus filaments.

J Bacteriol, 1990 Aug, 172(8), 4214 - 21
A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene; Sohail A et al.; Deamination of 5-methylcytosine in DNA results in T/G mismatches . If unrepaired, these mismatches can lead to C-to-T transition mutations . The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand . Previously we have shown that a plasmid containing an 11-kilobase fragment from the E . coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair . We have now mapped the regions essential for the two phenotypes . In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa . The genes responsible for these phenotypes have been identified by DNA sequence analysis . The gene essential for cytosine methylation, dcm, is predicted to code for a 473-amino-acid protein and is not required for VSP repair . It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRII methylase . The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein . This gene, named vsr, is not essential for DNA methylation . Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm . The two genes appear to be transcribed from a common promoter but are in different translational registers . This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation.

J Med Chem, 1990 Aug, 33(8), 2157 - 62
2-(4-Amino-4-carboxybutyl)aziridine-2-carboxylic acid . A potent irreversible inhibitor of diaminopimelic acid epimerase . Spontaneous formation from alpha-(halomethyl)diaminopimelic acids; Gerhart F et al.; 2-(4-Amino-4-carboxybutyl)aziridine-2-carboxylic acid (3) (aziridino-DAP) was identified as the product of spontaneous hydrolysis of alpha-(halomethyl)diaminopimelic acids (alpha-halomethyl-DAPs) 2a-c . Under physiological conditions, 3 is an extremely potent irreversible inhibitor of the bacterial enzyme diaminopimelic acid epimerase (DAP-epimerase; EC 5.1.1.7) . This unusual mode of action of an alpha-halomethyl amino acid with a non-pyridoxal enzyme is investigated . Synthesis and characterization of 2a-c and 3, kinetics of spontaneous formation of 3 from alpha-halomethyl-DAPs, and kinetics of enzyme inhibition by both 3 and by alpha-halomethyl-DAPs are reported.

J Infect Dis, 1990 Aug, 162(2), 550 - 2
Challenge studies in volunteers using Escherichia coli strains with diffuse adherence to HEp-2 cells; Tacket CO et al.; Diffusely adherent (DA) Escherichia coli, a newly described category, has been associated with diarrhea in children in some but not all epidemiologic studies . To investigate its diarrheagenic potential, two strains of DA E . coli were fed to adult volunteers in doses of 10(5)-10(10) colony-forming units . None of 22 volunteers who received strain 57-1 or of 21 who received strain C1845 developed symptoms of diarrhea as defined for this study . All volunteers shed the challenge DA E . coli in their stools . Duodenal string cultures were positive in some volunteers only at the highest doses . Only 6 developed IgG or IgA antibodies to whole DA E . coli . Thus, the pathogenic potential of DA E . coli could not be shown in this model . Further studies are needed to characterize potential virulence factors of DA E . coli that might distinguish a pathogenic subset.

J Infect Dis, 1990 Aug, 162(2), 460 - 5
A novel chromosomal TEM derivative and alterations in outer membrane proteins together mediate selective ceftazidime resistance in Escherichia coli; Weber DA et al.; A clinical Escherichia coli isolate (MG32) resistant to ceftazidime but susceptible to other third-generation cephalosporins was previously examined and found to harbor TEM-1 and exhibit alterations in outer membrane proteins . Reexamination of this isolate revealed the additional presence of a novel TEM-1 derivative, now designated TEM-12 . Evaluation of ceftazidime and cefotaxime minimum inhibitory concentrations for isogenic derivatives demonstrated a major role for TEM-12 in the decreased susceptibility observed . This was selectively enhanced for ceftazidime resistance by the altered porins of E . coli MG32 . TEM-12 appeared identical to TEM-101, an in vitro TEM derivative, in both isoelectric point (pI 5.25) and substrate profile . Hybridization and cloning of the TEM-12 determinant revealed that, unlike other TEM derivatives, TEM-12 is chromosomally encoded, not plasmid-encoded.

J Infect Dis, 1990 Aug, 162(2), 448 - 53
Seroepidemiologic evaluation of anti-toxic and anti-colonization factor immunity against infections by LT-producing Escherichia coli in rural Bangladesh; Clemens JD et al.; To evaluate serologic immunity against clinical infections by heat-labile enterotoxin-producing Escherichia coli (LT-ETEC) in rural Bangladesh, 124 children and adult women with LT-ETEC diarrhea (cases) were compared with 347 age-matched community controls . In paired acute-convalescent sera from the cases, IgG anti-CFA I and anti-CFA II antibody titers increased eight-to ninefold after infection by LT-ETEC with the homologous CFA, and IgG anti-LT antibody titers increased fourfold for all LT-ETEC infections . Anti-CFA and anti-LT titers peaked in controls aged 12-23 months, the age group with the highest incidence of ETEC infections . However, antibody titers were similar in acute sera from cases and in sera from controls . Although serum IgG anti-CFA and anti-LT antibodies rose in response to LT-ETEC infections and paralleled the age-specific incidence of ETEC in the community, these antibodies were not associated with a lower risk of LT-ETEC diarrhea.

Mol Cell Biol, 1990 Aug, 10(8), 4139 - 45
Function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenza virus (H1 N1); Nath ST et al.; Polymerase basic protein 1 (PB1) of influenza virus (A/WSN/33), when expressed from cloned cDNA in the absence of other viral proteins, accumulates in the nucleus . We have examined the location and nature of the nuclear localization signal of PB1 by using deletion mutants and chimeric constructions with chicken muscle pyruvate kinase, a cytoplasmic protein . Our studies showed some novel features of the nuclear localization signal of PB1 . The signal was present internally within residues 180 to 252 of PB1 . Moreover, unlike most nuclear localization signals, it was not a single stretch of contiguous amino acids . Instead, it possessed two discontinuous regions separated by an intervening sequence which could be deleted without affecting its nuclear localization property . On the other hand, deletion of either of the two signal regions rendered the protein cytoplasmic, indicating that the function of both regions is required for nuclear localization and that one region alone is not sufficient . Both of these signal regions contained short stretches of basic residues . Possible ways by which this novel bipartite signal can function in nuclear localization are discussed.

Mol Cell Biol, 1990 Aug, 10(8), 4080 - 8
Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP; Vauti F et al.; We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction . Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria . A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs . This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.

Mol Cell Biol, 1990 Aug, 10(8), 3917 - 25
Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III; Van Houten JV et al.; Yeast autonomously replicating sequence (ARS) elements contain an 11-base-pair core consensus sequence (5'-{A/T}TTTAT{A/G}TTT{A/T}-3') that is required for function . The contribution of each position within this sequence to ARS activity was tested by creating all possible single-base mutations within the core consensus sequence of ARS307 (formerly called the C2G1 ARS) and testing their effects on high-frequency transformation and on plasmid stability . Of the 33 mutations, 22 abolished ARS function as measured by high-frequency transformation, 7 caused more than twofold reductions in plasmid stability, and 4 had no effect on plasmid stability . Mutations that reduced or abolished ARS activity occurred at each position in the consensus sequence, demonstrating that each position of this sequence contributes to ARS function . Of the four mutations that had no effect on ARS activity, three created alternative perfect matches to the core consensus sequence, demonstrating that the alternate bases allowed by the consensus sequence are, indeed, interchangeable . In addition, a change from T to C at position 6 did not perturb wild-type efficiency . To test whether the essential region extends beyond the 11-base-pair consensus sequence, the effects on plasmid stability of point mutations one base 3' to the T-rich strand of the core consensus sequence (position 12) and deletion mutations that altered bases 5' to the T-rich strand of the core consensus sequence were examined . An A at position 12 or the removal of three T residues 5' to the core consensus sequence severely diminished ARS efficiency, showing that the region required for full ARS efficiency extends beyond the core consensus sequence in both directions.

J Virol, 1990 Aug, 64(8), 3963 - 6
Hepatitis B virus X protein produced in Escherichia coli is biologically functional; Jameel S et al.; The hepatitis B virus X gene product trans activates transcription from a variety of viral and cellular regulatory elements . We expressed the complete, nonfused X protein in Escherichia coli and showed it to be active in trans activating a human immunodeficiency virus long terminal repeat-linked chloramphenicol acetyltransferase reporter gene.

J Virol, 1990 Aug, 64(8), 3938 - 47
Mutational analysis of a native substrate of the human immunodeficiency virus type 1 proteinase; Partin K et al.; Proteolytic processing of the gag/pol precursor by the human immunodeficiency virus type 1 proteinase is essential for the production of infectious viral particles . Although the sites of virus-specific cleavages have been determined, the primary amino acid sequences surrounding these sites are heterogeneous and the determinants that direct the cleavage specificity exhibited by human immunodeficiency virus type 1 proteinase remain largely undefined . We performed mutational analysis of the Tyr/Pro site, which produces the amino terminus of the viral capsid protein, and the Phe/Pro site, which produces the amino terminus of the proteinase . Mutations were made in a clone encoding a frameshift mutation that results in the expression of equimolar amounts of the substrate and proteinase in the form of a truncated gag/pol precursor . After single-amino-acid substitutions were made, their effects on proteolytic processing were examined by in vitro transcription and in vitro translation of the synthetic mRNA; translation products were then processed by exogenously added purified proteinase . Single-amino-acid substitutions yielded both substrates which were processed with wild-type efficiency and substrates on which processing was impaired . At the Tyr/Pro site in gag, processing was severely inhibited by substitutions within the P4, P2, P1, and P2' positions . The Phe/Pro site in pol, however, demonstrated far greater tolerance to amino acid substitution . These data suggest that the primary amino acid sequence around a scissile bond is more critical for cleavage of the Tyr/Pro site than the Phe/Pro site.

J Leukoc Biol, 1990 Aug, 48(2), 129 - 37
Neutrophil accumulation and plasma leakage induced in vivo by neutrophil-activating peptide-1; Colditz IG et al.; Neutrophil accumulation and plasma leakage induced in rabbit skin by neutrophil-activating peptide-1 (NAP-1, a 72 amino acid peptide produced by monocytes and a variety of tissue cells), E . coli endotoxin, and interleukin-1 (IL-1) were compared . Neutrophil accumulation at sites injected with NAP-1 was intense, rapid, and long-lasting; it reached a maximum rate during the first 30 min, continued at constant rate for 4-6 h, and remained detectable up to at least 8 h . In contrast, the neutrophil-attracting effect of endotoxin and IL-1 was slower in onset and more transient; it peaked in the first 2 h and declined to a very low level after 4 h . Plasma leakage induced by NAP-1 had a shorter time course than neutrophil accumulation and ceased after 6 h . Depletion of blood neutrophils by treatment with hydroxyurea prevented the plasma leakage induced by NAP-1 or endotoxin but not by histamine . Desensitization to NAP-1 was studied by restimulation of lesions . Following restimulation with NAP-1 after intervals from 6-10 h, there was diminished infiltration of neutrophils, while nearly normal responses were obtained after an interval of 24 h . Desensitization was dose dependent and affected both plasma leakage and neutrophil accumulation . In lesions initiated with NAP-1 there were normal responses following restimulation with endotoxin but marked desensitization to IL-1, suggesting that NAP-1 may contribute to inflammation induced by IL-1 but not by endotoxin . This study indicates that NAP-1 is a potent mediator of neutrophil accumulation in vivo, with characteristics similar to those reported for C5 fragments, but with a more protracted action.

Infect Immun, 1990 Aug, 58(8), 2701 - 5
The Chlamydia trachomatis hyp operon is homologous to the groE stress response operon of Escherichia coli; Morrison RP et al.; The Chlamydia trachomatis serovar A hyp operon was cloned, sequenced, and expressed in Escherichia coli . Two cotranscribed open reading frames, hypA and hypB, encoded polypeptides of 17 and 57 kilodaltons, respectively . The deduced amino acid sequences of serovar A HypA and HypB proteins were (respectively) 85 and 94% identical with HypA and HypB proteins of Chlamydia psittaci GPIC, and HypB was greater than 50% identical to 60-kilodalton stress response proteins from other procaryotes and eucaryotes . The sequence should be useful in defining the antigenic structure of the Chlamydia trachomatis HypB protein, a necessary step toward understanding the relationship between the immune response to this protein and the pathogenesis of human chlamydial diseases.

Infect Immun, 1990 Aug, 58(8), 2478 - 86
Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis; Lundemose AG et al.; The synthesis of early proteins from Chlamydia trachomatis serovar L2 was analyzed by two-dimensional gel electrophoresis . By pulse-label experiments, the synthesis of seven proteins was observed at 2 to 8 h postinfection before the major outer membrane protein was detected at 8 to 10 h after infection . The early proteins were synthesized throughout the 30-h period investigated, but the synthesis of three proteins of 75, 62, and 45 kilodaltons decreased from 26 to 30 h postinfection . Pulse-chase analysis showed that the signals from the same three proteins declined 26 to 30 h after infection . Three of the early proteins were identified as the S1 ribosomal protein, the GroEL-like protein, and DnaK-like protein, respectively.

EMBO J, 1990 Aug, 9(8), 2537 - 42
Multiple cDNA clones encoding nuclear proteins that bind to the tax-dependent enhancer of HTLV-1: all contain a leucine zipper structure and basic amino acid domain; Yoshimura T et al.; A trans-activator protein, p40tax, of human T cell leukemia virus type 1 (HTLV-1) activates its own promoter and cellular promoters of IL-2, IL-2 receptor alpha and GM-CSF genes . We isolated three cDNA clones encoding cellular proteins that bind to the p40tax-dependent enhancer of HTLV-1 by screening a lambda gt11 cDNA library of an HTLV-1 infected cell line . All three proteins, TREB5, TREB7 and TREB36, contained a leucine zipper structure and basic amino acid domain, which are conserved in FOS, JUN and CREB, and also had multiple potential phosphorylation sites . The proteins expressed in Escherichia coli bound to the p40tax-dependent enhancer of the 21 bp sequence, but not to an inactive mutant carrying a mutation in the CRE region . In DNase I footprint analysis, all three proteins protected the 21 bp sequences in the LTR; however, the patterns were not identical to each other . TREB7 and TREB36 protected all three repeats of the 21 bp, but TREB5 protected only the second repeat . TREB7 and TREB36 protected the 5' and middle portions of the 21 bp which are essential for p40tax-mediated trans-activation, whereas TREB5 and CREB1 protected a narrower part of the middle region of the second 21 bp repeat containing the CRE consensus sequence . These structural features and DNA binding properties suggest that TREB proteins are members of a CREB protein family and that some of them (i.e., TREB7 and TREB36) may be involved in p40tax-mediated trans-activation.

New Biol, 1990 Aug, 2(8), 739 - 46
Transfer and expression of the lacZ gene in rat brain neurons mediated by herpes simplex virus mutants; Chiocca EA et al.; Three mutants of herpes simplex virus type 1 (HSV-1) were used to deliver and express the Escherichia coli lacZ gene in cells of the rat central nervous system . Because the lacZ gene was inserted in place of the genes encoding one of the immediate-early viral proteins ICP0 or ICP4 or the early viral protein thymidine kinase, these mutants were compromised or defective in their ability to replicate . All mutant vectors exhibited reduced pathogenesis in animals as compared to the wild type HSV-1 strain KOS . In all cases lacZ was under the control of immediate-early or early viral promoters that are active in the early phase of infection . Expression of beta-galactosidase was observed in cortical neurons following stereotactic inoculation of mutant viruses into adult rat brains; distinct patterns of expression were observed with each mutant vector . Injection of the ICP0 mutant in the frontal cortex and caudate nucleus resulted in beta-galactosidase expression in a substantial number of cells around the inoculation site and at some distance from it for 14 days, with maximum expression after 3 days . The ICP0 vector appeared to have reached the ipsilateral and contralateral cingulate cortex by retrograde transport . Following inoculations of the ICP4 and thymidine kinase vectors into the same brain regions, only a few cells in areas immediately adjacent to the injection track expressed beta-galactosidase and they did so for only a few days . These herpes virus-derived vectors provide a means for the in situ delivery and expression of specific genes in neurons in the central nervous system with little adverse effect on animals.

J Med Virol, 1990 Aug, 31(4), 277 - 83
Recombinant proteins VP1 and VP3 of hepatitis A virus prime for neutralizing response; Gauss-Muller V et al.; Six overlapping genomic regions of capsid proteins VP1 and VP3 of hepatitis A virus (HAV) inserted into the expression vectors pBD or pUR respectively expressed beta-galactosidase-HAV fusion proteins . The recombinant proteins were poorly soluble so they were difficult to detect by human anti-HAV sera in radioimmunoassay, but the fusion proteins dissolved in sodium dodecyl sulfate reacted with human and rabbit anti-HAV-positive sera in immunoblots . Antisera against VP1 and VP3 recombinant proteins reacted with the respective structural proteins of HAV in immunoblots . Two recombinant proteins, one including the first 120 amino acids of the N-terminus of VP1 and the other containing all of VP1 except for the first 60 N-terminal amino acids, induced a transient neutralizing antibody response in rabbits . Antisera directed against other regions of VP1 and VP3 neither neutralized viral infectivity nor recognized native virus in a competitive radioimmunoassay . However, when immunized animals were challenged with a sub-immunogenic dose of HAV, all animals responded with stable virus-neutralizing antibodies.

Mol Gen Genet, 1990 Aug, 223(1), 152 - 5
Construction of an Azospirillum brasilense Sp7 recA mutant; Croes C et al.; Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant . Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A . brasilense recA gene on a 1.2 kb DNA fragment . One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A . brasilense by marker exchange . The resulting A . brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.

Vet Microbiol, 1990 Aug, 24(2), 105 - 12
Expression of fowl adenovirus type 10 antigens in Escherichia coli; Sheppard M et al.; In an attempt to construct a genetic map of the fowl adenovirus (FAV) and to determine which viral proteins are natural immunogens in chickens and hence may be relevant to protective immunity we have constructed an expression library of FAV type 10 DNA . The genomic DNA was partially digested with the restriction endonuclease Sau3A, and this DNA was inserted into the 3' terminal end of the beta-galactosidase gene in a plasmid vector . To date, approximately 600 clones have been identified that express FAV type 10 antigens as determined by immunological screening with rabbit antisera to purified virus, including one that has amino acid homology with the 100 kDa protein of human adenovirus type 5 . These antigen positive clones were found to contain DNA from FAV type 10 genome as determined by hybridisation to FAV DNA . These clones will allow the further characterisation of FAV and possibly the identification of potential vaccine molecules.

J Biochem (Tokyo), 1990 Aug, 108(2), 149 - 52
Combination of heteronuclear 1H-15N and 1H-13C three-dimensional nuclear magnetic resonance experiments for amide-directed sequential assignment in larger proteins; Nagayama K et al.; A new strategy for the sequential assignment of backbone proton resonances in larger proteins involving a unique combination of four types of heteronuclear three-dimensional (3D) NMR spectroscopies is reported . This method relies on the uniform labeling of amide nitrogens with 15N and of alpha-carbons with 13C . Heteronuclear 1H-15N TOCSY-HMQC and NOESY-HMQC experiments can reveal connections between cross-peaks arising from the NHi-C alpha Hi-1 and NHi-C alpha Hi connectivities in the finger-print region in in general . They also specifically reveal the sequential amide-amide connectivities among the amide cross-peaks for the alpha-helices . Heteronuclear 1H-13C HMQC-TOCSY and HMQC-NOESY experiments can reveal connections between cross-peaks arising from the NHi-C alpha Hi and NHi+1-C alpha Hi connectivities in the finger-print region in general . The combination of the two sets of results reveals the complete unambiguous sequential connection of cross-peaks for the proton resonances in the peptide backbone . The application of the new strategy is reported for a protein, ribonuclease H, with a molecular weight of 17.6 kDa.

Behring Inst Mitt, 1990 Aug, (85), 28 - 34
Development of an IgM antibody capture test using labelled fusion protein as antigen for diagnosis of B19 human parvovirus infections; Morinet F et al.; A new anti-B19 IgM ELISA was developed using the antibody-capture principle . Biotinylated fusion protein was used as antigen . The specificity of the test was analysed using sera IgM positive to rubella, hepatitis B core antigen, cytomegalovirus and Epstein Barr virus as well with sera positive for rheumatoid factors, antinuclear antibodies and with sera from normal blood donors . The specificity of the test was 96.18% . One hundred serum samples were tested by the new ELISA and the standard MACRIA tests for the presence of B19 IgM . Eighty-eight sera gave the same results with both tests . Fifty-three were negative and 35 were positive . Six sera were ELISA negative MACRIA positive and six MACRIA negative ELISA positive . In a clinical study with 725 sera from suspected B19 infections, 161 (22%) were recorded as positive by the ELISA test . The positive sera were from patients suffering from arthritis (35%), rash (35%), acute or chronic erythroblastopenia (21%), pancytopenia (5%), vascular purpura (2%) and lymphadenopathy (2%).

Genetics, 1990 Aug, 125(4), 691 - 702
Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12; Berg BL et al.; Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli . This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase . We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N . The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N . Our analysis indicates that fdnGHI is organized as an operon . We mapped the fdn operon to 32 min on the E . coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon) . Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate . This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI . We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

Genetics, 1990 Aug, 125(4), 683 - 90
Exchange of spacer regions between rRNA operons in Escherichia coli; Harvey S et al.; The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions . The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B) . We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type . Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA) . While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected . At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer . In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal . In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons . Two examples of inversion of one-half of the E . coli chromosome between rrnG and rrnH were observed . The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.

J Gen Virol, 1990 Aug, 71 ( Pt 8), 1767 - 74
Virus neutralizing activity induced by synthetic peptides of glycoprotein D of herpes simplex virus type 1, selected by their reactivity with hyperimmune sera from mice; Geerligs HJ et al.; Mice were immunized with synthetic peptides covering the first 56 amino acids of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) and a fusion protein, produced in Escherichia coli, containing the first 55 amino acid residues of gD . It was found that mice immunized with peptides composed of amino acid residues 1 to 13, 18 to 30 . 22 to 38 and 38 to 56 of gD were not significantly protected against a lethal challenge with HSV-1 . Immunization with peptide 9-21 and the gD fusion protein resulted in significant protection . Antisera, from mice immunized with HSV-1, were investigated for reactivity with a series of 57 overlapping gD peptides covering the entire amino acid sequence, except for the membrane-spanning region . All antisera reacted with peptides 9-21, 10-24, 151-165, 216-232, 282-301 and with peptide 340-354 located in the anchoring region of gD, and 15 other peptides were recognized by at least one antiserum . Twelve peptides (10-24, 151-165, 216-232, 244-267, 260-274, 270-284, 260-284, 282-301, 300-314, 340-354, 348-362 and 355-369) reacted most frequently with the hyperimmune sera from mice and were selected for further study . These were conjugated to bovine serum albumin and used to immunize rabbits . Only antisera against peptide 10-24, which covers the same epitope as peptide 9-21, neutralized HSV-1 in vitro.

J Gen Virol, 1990 Aug, 71 ( Pt 8), 1757 - 65
The protein kinase encoded in the short unique region of pseudorabies virus: description of the gene and identification of its product in virions and in infected cells; Zhang G et al.; We have cloned and determined the nucleotide sequence of a gene, pk, that lies immediately upstream from the gene encoding glycoprotein X in the short unique region of the alphaherpesvirus, pseudorabies virus (PRV) . The gene has the potential to encode a protein of 334 amino acids, and is related to gene US3 of herpes simplex virus type 1 (HSV-1), which has been shown to encode a protein kinase . The predicted amino acid sequence encoded by the PRV pk gene is homologous to the corresponding sequence encoded by the HSV-1 US3 gene in the C-terminal catalytic domain, but diverges markedly in the N-terminal domain . As with HSV-1, the mRNA for the pk gene appears to be 3' coterminal with that for the glycoprotein downstream . An antiserum was raised against a protein generated from the fusion of part of the PRV pk catalytic domain with Escherichia coli beta-galactosidase . This specifically reacted with a previously described physically homogeneous protein kinase, PRV-PK, isolated from hamster fibroblasts lytically infected with PRV . Although the majority of the PRV-PK is found in the cytoplasm, some was also detected in purified PRV virions by using the same antibody; a similar distribution was found for the HSV-1 protein kinase, using an antiserum raised against the corresponding HSV-1 fusion protein . When presented with heatinactivated virions, purified PRV-PK (in common with certain cellular protein kinases also present in the virion) was able to phosphorylate in vitro the major virion phosphoprotein phosphorylated in vivo.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6383 - 7
Escherichia coli helicase II (UvrD) protein initiates DNA unwinding at nicks and blunt ends; Runyon GT et al.; The Escherichia coli uvrD gene product, helicase II, is required for both methyl-directed mismatch and uvrABC excision repair and is believed to function by unwinding duplex DNA . Initiation of unwinding may occur specifically at either a mismatch or a nick, although no direct evidence for this has previously been reported . It has recently been shown that helicase II can unwind fully duplex linear and nicked circular DNA with lengths of at least approximately 2700 base pairs in vitro; hence, a flanking region of single-stranded DNA is not required to initiate DNA unwinding . In studies with uniquely nicked duplex DNA, we present EM evidence that helicase II protein initiates DNA unwinding at the nick, with unwinding proceeding bidirectionally . We also show that helicase II protein initiates DNA unwinding at the blunt ends of linear DNA, rather than in internal regions . These data provide direct evidence that helicase II protein can initiate unwinding of duplex DNA at a nick, in the absence of auxiliary proteins . We propose that helicase II may initiate unwinding from a nick in a number of DNA repair processes.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6272 - 6
DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase; Hollingsworth RE Jr et al.; The yeast Cdc7 protein is indispensable to initiation of nuclear DNA replication, based on the phenotype of the conditional, temperature-sensitive (ts) cdc7 mutants at the restrictive temperature . This protein has likewise been implicated in commitment to meiotic DNA recombination and induced mutagenesis, which may result from error-prone DNA repair . Our previous work revealed sequence similarity between the Cdc7 protein and known protein kinases . To determine whether it possesses kinase activity, we have immunoprecipitated the protein from Cdc7-overproducing yeast cells by using polyclonal antibodies raised against a nondenatured beta-galactosidase-Cdc7 fusion protein . In this report, we demonstrate that Cdc7 immune complexes are capable of phosphorylating mammalian histone H1 on serine and/or threonine residues . Immune complexes derived from cells harboring the cdc7-2 ts mutant gene on a high copy number plasmid possess a thermolabile kinase activity . Thus, we postulate that Cdc7 may regulate the various DNA metabolic pathways by phosphorylating one or more target substrates . Because Cdc7 kinase acts downstream of Cdc28/cdc2 kinase function at "start," the transition from G1 to S phase in the cell cycle may be the result of a cascade of protein phosphorylation.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6213 - 7
Probe mapping to facilitate transposon-based DNA sequencing; Strausbaugh LD et al.; A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers . For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed . We demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies . This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods . To test the efficiency of probe mapping, 49 insertions of the transposon gamma delta (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented . In addition, oligonucleotide primers specific for unique subterminal gamma delta segments were used to prime dideoxynucleotide double-stranded sequencing . These data provided an opportunity to rigorously examine gamma delta insertion sites . The insertions were quite randomly distributed, even though the target DNA fragment had both A + T-rich and G + C-rich regions; in G + C-rich DNA, the insertions were found in A + T-rich "valleys." These data demonstrate that gamma delta is an excellent choice for supplying mobile primer binding sites to cloned DNA and that transposon-based probe mapping permits the sequences of large cloned segments to be determined without any subcloning.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6092 - 6
In vitro maturation and encapsidation of the DNA of transposable Mu-like phage D108; Burns CM et al.; Mu and D108 are related, temperate, transposable coliphages with unusual modes of DNA replication (transposition) and virion DNA maturation . These double-stranded DNA genomes replicate intrachromosomally and are matured and encapsidated linked to DNA sequences flanking the dispersed, integrated phage genomes . We have developed an in vitro system that employs crude lysates prepared from cells late in the Mu lytic cycle and that is proficient for both maturation and encapsidation of D108 DNA . Different forms of phage DNA were packaged at different efficiencies, with a circular pSC101::D108cts10 plasmid being most efficient, linearized plasmid less so, and mature virion DNA a poor substrate . The addition of purified D108 Ner protein to the reaction had no effect, whereas D108 repressor (c protein) inhibited the reaction . Escherichia coli integration host factor and D108 transposase proteins exerted an inhibitory effect on circular DNA substrates but had little effect on linear DNA packaging . This in vitro system, coupled with that developed for transposition, can now be used to biochemically dissect the protein and substrate requirements of these phages' DNA maturation pathway and the nature of the molecular switch between DNA transposition and encapsidation.

J Bacteriol, 1990 Aug, 172(8), 4529 - 35
The leucine regulon of Escherichia coli K-12: a mutation in rblA alters expression of L-leucine-dependent metabolic operons; Tuan LR et al.; We have isolated and characterized a highly pleiotropic Escherichia coli mutant affected in the activity of a number of enzymes involved in different metabolic pathways, all of which are regulated by leucine . Selected for its ability to grow with L-serine as sole carbon source, the rbl-1::Tn10 mutant had high levels of L-serine deaminase activity (due to increased transcription of the structural gene) and of another amino acid-degrading enzyme, L-threonine dehydrogenase, and decreased transcription of the operons serA and ilvIH, coding for biosynthetic enzymes . The rbl mutation suppressed the slow growth of a metK mutant, deficient in S-adenosylmethionine synthetase . Furthermore, metK mutants spontaneously accumulated faster-growing rbl-like derivatives, and a commonly used metK strain, RG62, carries such a mutation . The rbl gene is located near 20 min on the E . coli genetic map . All phenotypes of the rbl mutant could be observed in rbl+ strains cultivated in the presence of L-leucine, and exogenous L-leucine had little further effect on the rbl strains . We propose that the rbl gene product is the regulator of a global response to leucine.

J Bacteriol, 1990 Aug, 172(8), 4386 - 91
A single DnaA box is sufficient for initiation from the P1 plasmid origin; Abeles AL et al.; The P1 plasmid replication origin requires the host DnaA protein for function . Two DnaA-binding boxes lie in tandem within the previously defined minimal origin, constituting its left boundary . Three more boxes lie 200 base pairs to the right of these, in the leader region for the P1 repA gene . We show that either set alone is active for origin function . One of the two origin boxes is relatively inactive . Constructs with just one of the five boxes are active for specific origin function as long as the box conforms exactly to the published consensus . This single consensus box is functional when placed either to the left or right of the core origin sequences . The flexibility shown by this system suggests that the boxes play a role different from those in the host oriC origin, where the number and position of boxes are critical.

J Bacteriol, 1990 Aug, 172(8), 4339 - 47
surA, an Escherichia coli gene essential for survival in stationary phase; Tormo A et al.; Mutations in genes not required for exponential growth but essential for survival in stationary phase were isolated in an effort to understand the ability of wild-type Escherichia coli cells to remain viable during prolonged periods of nutritional deprivation . The phenotype of these mutations is referred to as Sur- (survival) and the genes are designated sur . The detailed analysis of one of these mutations is presented here . The mutation (surA1) caused by insertion of a mini-Tn10 element defined a new gene located near 1 min on the E . coli chromosome . It was located directly upstream of pdxA and formed part of a complex operon . Evidence is presented supporting the interpretation that cells harboring the surA1 mutation die during stationary phase while similar insertion mutations in other genes of the operon do not lead to a Sur- phenotype . Strains harboring surA1 had a normal doubling time in both rich and minimal medium, but cultures lost viability after several days in stationary phase . Analysis of revertants and suppressors of surA1, which arose after prolonged incubation in stationary phase, indicates that DNA rearrangements (excisions and duplications) occurred in cultures of this strain even when the viable-cell counts were below 10(2) cells per ml . Cells containing suppressing mutations then grew in the same culture to 10(8) cells per ml, taking over the population . The implications of these observations to our understanding of stationary-phase mutagenesis are discussed.

Virology, 1990 Aug, 177(2), 570 - 7
Analysis of the binding sites for the varicella-zoster virus gene 51 product within the viral origin of DNA replication; Stow ND et al.; The C-terminal 322 amino acids of the varicella-zoster virus (VZV) gene 51 product were expressed in Escherichia coli and shown to bind to specific DNA sequences within the VZV origin of DNA replication . The gene 51 product and its herpes simplex virus type 1 homolog (the UL9 protein) are capable of recognizing identical DNA sequences but the arrangement of binding sites within the origin regions of the two viruses differs . Three binding sites for the VZV gene 51 protein were identified within the VZV origin region and these lie in the same orientation and on the same side of the origin palindromic DNA sequence . DNA replication assays in transfected tissue culture cells demonstrated that the site closest to the palindrome is essential for origin activity whereas the most distal site is dispensable . The middle binding site may play an auxiliary role in DNA synthesis.

J Leukoc Biol, 1990 Aug, 48(2), 123 - 8
Superoxide anion generation in the liver during the early stage of endotoxemia in rats; Bautista AP et al.; Infiltration of polymorphonuclear neutrophils (PMN) in the rat liver 3 hr after an intravenous (IV) injection of a sublethal dose of Escherichia coli lipopolysaccharide (LPS) was observed without any significant alteration in the total number of Kupffer and endothelial cells . Since previous studies have demonstrated that phagocytic cells in the liver were in a state of metabolic activation under similar experimental conditions, we investigated the in vitro generation of superoxide anion (O2-) by this cell type following the administration of LPS . Kupffer cells from normal rats did not release O2-, in contrast to those obtained from LPS-treated rats . The generation of O2- by Kupffer cells from endotoxic rats was elevated from 3.0 +/- 1.9 nmol/10(6) cells/60 min (mean +/- SD) in the absence of macrophage (M phi) activators, to 5.0 +/- 2.36, 11.33 +/- 5.40, and 4.33 +/- 0.90 in the presence of opsonized zymosan, phorbol myristate acetate (PMA), and the calcium ionophore A23187, respectively . Hepatocytes from normal or endotoxic rats did not produce detectable O2- . Endothelial cells from LPS-treated rats generated less than 0.8 nmol/10(6) cells in the presence of zymosan . PMN that accumulated in the livers of endotoxic rats released O2- only in the presence of zymosan (8.12 +/- 5.40), PMA (15.43 +/- 5.84), or A23187 (1.70 +/- 0.12) . The O2- generation by blood monocytes and PMN increased significantly after endotoxin administration and in the presence of activators . These results suggest that the hypermetabolic state of phagocytic cells in the liver shortly after LPS treatment may be correlated with the increased generation of O2- . The latter may subsequently contribute to the induction of hepatic injury in endotoxemia.

Infect Immun, 1990 Aug, 58(8), 2585 - 92
The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing; Szeto L et al.; The Legionella pneumophila major secretory protein (Msp) is a Zn2+ metalloprotease whose function in pathogenesis is unknown . The structural gene for the Msp protease, mspA, was isolated from an L . pneumophila genomic library . In Escherichia coli which contain plasmids with the mspA gene, Msp protein and activity are found in the periplasmic space and the cytoplasm . Transposon mutagenesis with Tn9 of an mspA-containing plasmid in E . coli yielded mutants which no longer expressed protease activity and others with increased protease activity . These results suggested that mspA expression might be regulated . Msp was shown to be produced at a much higher level in L . pneumophila grown in rich compared to semidefined media . A Tn9 insertion which abolishes Msp expression was introduced into the L . pneumophila genome . This mspA::Tn9 L . pneumophila strain showed no detectable production of Msp by immunoblot analysis, and it had less than 0.1% of the protease activity found in the wild-type strain . This mutant was fully capable of growing within and killing human macrophages derived from the HL-60 cell line.

Exp Cell Res, 1990 Aug, 189(2), 163 - 8
High-frequency transfection of cultured human epidermal basal cells that differentiate to form a multilayered tissue; Jensen TG et al.; Primary cultures of human keratinocytes form a multilayered tissue . By incubating the tissue cultures in Ca2(+)-free medium the differentiated cell layers can be stripped off leaving a basal cell monolayer . We have developed a method for high-frequency transfection of these epidermal basal cells with genes inserted into Epstein-Barr virus-based expression vectors . Using the Escherichia coli lac z gene as a marker gene, the transient and long-term expression and the fate of the transfected cells were studied . During regeneration of the multilayered tissue most of the transfected basal cells enlarge and undergo differentiation, but a minor population remains as basal cells . Incubation with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate results in an increase in the proportion of transfected keratinocytes that are small, suggesting a relative expansion of the immature cell pool.

Arch Biochem Biophys, 1990 Aug 1, 280(2), 416 - 20
Electron paramagnetic resonance measurements of the hydration of Mn(II) in ternary complexes with GDP and ras p21 proteins; Smithers GW et al.; Electron paramagnetic resonance (EPR) spectroscopy has been used to determine the hydration numbers of Mn(II) in complexes with GDP and three forms of ras p21 . EPR signals of Mn(II) in the GDP complex with viral-Harvey p21pRAS1 (Arg 12, Thr 59), p21EC (Gly 12, Thr 59), and p21EJ (Val 12, Thr 59) have narrow line-widths that permit ready observation of inhomogeneous broadening from unresolved superhyperfine coupling with the nuclear spin of 17O of directly coordinated oxygen ligands . Quantitative analysis of the lineshapes for the samples in H2 17O-enriched water indicates that four water ligands coordinate to the metal ion in the GDP complexes with all three proteins . The four solvent ligands, together with an oxygen from the beta-phosphate group of GDP, leave space for only one ligand from the protein . An X-ray diffraction-derived model for the MgII beta-gamma-imidoguanosine-5'-triphosphate complex with p21 shows coordination of Mg(II) to the beta- and gamma-phosphate groups of the nucleotide as well as to the hydroxyl groups of Thr 35 and Ser 17 (Pai, E.F., Kabusch, W., Krengel, U., Holmes, K . H., John, J., and Wittinghofer, A., 1989, Nature (London) 341, 209-214) . Thus, upon conversion of the nucleotide from a triphosphate to a diphosphate, solvent replaces both the gamma-phosphate of the nucleotide and one of the protein ligands . The EPR results are consistent with a recent X-ray crystallographic model for the p21-MgIIGDP complex (Milburn, M . V., Tong, L., DeVos, A . M., Brunger, A., Yamaizumi, Z., Nishimura, S., and Kim, S.-H., 1990, Science 247, 939-945) . EPR spectra of complexes with the three forms of ras p21 differ with respect to the intrinsic linewidths of the EPR signals . These subtle differences in linewidth appear to originate from slight differences in local disorder near the metal-nucleotide binding site.

Eur Cytokine Netw, 1990 Aug-Sep, 1(3), 169 - 76
Granulocyte-macrophage colony stimulating factor and interleukin-3 regulate the production of an interleukin-1 inhibitor by the differentiated AML-193 leukemic cell line; Kindler V et al.; Treatment of the AML-193 leukemic cell line with phorbol myristate acetate (PMA) resulted in the loss of their ability to proliferate in response to GM-CSF or IL-3 . This was not due to a change in number or affinity of GM-CSF receptors, but possibly resulted from an other cellular mechanism . The AML-193 differentiated cells acquired the ability to phagocytose glutaraldehyde-fixed E.coli in a similar fashion to mature macrophages . In addition the PMA-differentiated AML-193 cells now secreted a factor which specifically inhibited the binding of interleukin-1 (IL-1) to its receptor on the murine thymoma cell line EL-4.6.1C10 . The synthesis of this inhibitor was further increased by the addition of GM-CSF or IL-3 . Pulse labelling experiments showed that this activity was due to a 26 kDa protein that bound to the IL-1 receptor even in the presence of neutralizing antibodies against IL-1 alpha or IL-1 beta, and this binding was only antagonized by IL-1 alpha or IL-1 beta . In contrast, peripheral monocytes obtained from the blood of normal donors, when induced with either GM-CSF or IL-3, produced large quantities of inhibitor in the absence of PMA . This report clearly shows that a leukaemic cell line can respond to GM-CSF and IL-3 in different ways before and after in vitro differentiation.

Mol Microbiol, 1990 Aug, 4(8), 1233 - 40
Post-transcriptional control in the polycistronic operon environment: studies of the atp operon of Escherichia coli; McCarthy JE; Post-transcriptional control mechanisms assume special significance in polycistronic operons . Differential gene expression in the atp operon of Escherichia coli is primarily attributable to translational control and, to a lesser extent, to control of mRNA stability . At the same time, the polycistronic environment influences, to varying degrees, the relative importance of the different types of post-transcriptional control . The present article briefly reviews more recent results obtained through studies of the atp operon . Investigations of the pathway and kinetics of mRNA decay have yielded new information about the role of degradative mechanisms in the overall scheme of control . Moreover, translational coupling has been shown to feature as a major form of interaction between the atp genes . The relevance of these and other data is discussed in the wider context of the post-transcriptional control mechanisms generally available to E . coli.

Biochem J, 1990 Aug 1, 269(3), 709 - 15
Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2; Hayashi H et al.; Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro . Its actual role in vivo, however is unknown . We obtained and characterized five monoclonal antibodies to lipocortin I . Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro . Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine . L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro . L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E . coli membranes or to phosphatidylserine . These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E . coli membranes . Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.

J Bacteriol, 1990 Aug, 172(8), 4378 - 85
Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3; Schmellik-Sandage CS et al.; Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) . Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase . (i) In most cases, beta-galactosidase specific activity increased only two- to threefold . (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity . (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis . These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations . By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113 . The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.

Virology, 1990 Aug, 177(2), 829 - 32
Two forms of the major barley stripe mosaic virus nonstructural protein are synthesized in vivo from alternative initiation codons; Petty IT et al.; Barley stripe mosaic virus (BSMV) has a tripartite genome comprising RNAs designated alpha, beta, and gamma, which collectively encode seven polypeptides . We show here that an antiserum raised against an abundant disease-specific protein from BSMV-infected plants reacts specifically with the viral beta b gene product expressed as part of a beta-galactosidase fusion protein in Escherichia coli . Two predominant forms of the protein, beta b and beta b', are synthesized in vivo . Infectious in vitro transcripts derived from wild-type and mutant BSMV cDNA clones have been used to map the initiation site for translation of the beta b protein in vivo . The results of our mutagenesis experiments are consistent with a model in which translation of the beta b' protein is initiated by ribosomes that scan past the 5'-proximal beta b initiation site . A mutant which is able to synthesize only the shorter beta b' protein was indistinguishable from the wild-type with respect to all of the phenotypes tested . Thus, the beta b form of the protein is dispensable in planta, and whether the two forms of the protein have different functions in vivo is unclear at present.

Shigaku, 1990 Aug, 78(2), 208 - 32
{Chemical composition and biological activities of lipopolysaccharides extracted from Treponema denticola and Treponema vincentii}; Kurimoto T et al.; Lipopolysaccharides (LPSs) were isolated from Treponema denticola (T . denticola) and Treponema vincentii (T . vincentii) by the phenol/water (PW) and the phenol/chloroform/petroleum-ether (PCP) procedures . 1) T . denticola PW-LPS (LPS isolated by the PW procedure), PCP-sup-LPS (LPS isolated by the PCP procedure in the supernates of ultracentrifugation), PCP-ppt-LPS (precipitated LPS isolated by the PCP procedure, obtained after ultracentrifugation), and T . vincentii PCP-ppt-LPS were composed of carbohydrate, hexosamine, protein, fatty acid, and phosphorus . T . vincentii PW-LPS was contained major amount of carbohydrates and small amount of fatty acids . 2-keto-3-deoxyoctonic acid (KDD) was not detected in these LPSs . 2) The major fatty acids of T . denticola PW-LPS and PCP-sup-LPS were palmitic, stearic, oleic, and linoleic acids . The major fatty acids of T . vincentii PCP-ppt-LPS were palmitic, stearic, myristic, oleic, and linoleic acids . Hydroxy fatty acids were not detected . 3) Glucose, galactose, and mannose were comprised in T . denticola PW-LPS . Glucose, galactose, and arabinose were comprised in T . denticola PCP-sup-LPS . Glucose and galactose were comprised in T . vincentii PCP-ppt-LPS . 4) The Limulus amoebocyte lysate (LAL) clotting activity of T . denticola PW-LPS was 1/10, as compared with that of Escherichia coli (E . coli) UKT-B LPS standard . The LAL clotting activities of T . denticola PCP-sup-LPS, T . vincentii PW-LPS, and T . vincentii PCP-ppt-LPS were 1/100, as compared with that of E . coli LPS standard . 5) Five hundred micrograms/kg of T . denticola PCP-sup-LPS was pyrogenic in rabbits . Two thousand micrograms/kg of T . vincentii PCP-ppt-LPS was pyrogenic in rabbits . 6) T . denticola PCP-sup-LPS and T . vincentii PCP-ppt-LPS were capable of increasing or decreasing the release of lysosomal enzymes from human polymorphonuclear leukocytes.

Biochimie, 1990 Aug, 72(8), 609 - 16
Engineering aspartate transcarbamylase; Herve G et al.; Aspartate transcarbamylase from Escherichia coli is one of the most extensively studied regulatory enzymes as a model of cooperativity and allostery . Numerous methods are used to engineer variants of this molecule: random and site-directed mutagenesis, dissociation and reassociation of the catalytic and regulatory subunits and chains, construction of hybrids made from normal and modified subunits or chains, interspecific hybrids and construction of chimeric enzymes . These methods provide detailed information on the regions, domains, interfaces and aminoacid residues which are involved in the mechanism of co-operativity between the catalytic sites, and of regulation by the antagonistic effectors CTP and ATP . These effectors induce the transmission of intramolecular signals whose pathways begin to be delineated.

Biochimie, 1990 Aug, 72(8), 537 - 44
Yeast methionyl-tRNA synthetase: analysis of the N-terminal extension and the putative tRNA anticodon binding region by site-directed mutagenesis; Walter P et al.; Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme . We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame . The results show that 185 amino acids are dispensable for activity and stability . Removal of the next 5 residues affects the activity of the enzyme . The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step . The Km for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained . The Km for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected . There is, however, a great effect on the Vmax of the enzyme . Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft . It could, however, act at a distance by propagating a mutational alteration of the catalytic residues . The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized . By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA . Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.

Environ Health Perspect, 1990 Aug, 88, 133 - 9
Products of the fos and jun proto-oncogenes bind cooperatively to the AP1 DNA recognition sequence; Risse G et al.; The products of the proto-oncogenes c-fos and c-jun form a tight protein complex that is a major component of the transcription factor AP1 . To analyze the role of fos in the binding of this complex to the AP1 DNA recognition sequence and the mechanism of interaction in further detail, we have expressed a fos protein in E . coli using an expression vector containing the temperature-inducible lambda PL promoter and a synthetic translational start codon . The fos protein encoded by this construct (termed Baf) was enriched by biochemical purification techniques and was found to form a specific complex with c-jun obtained by in vitro transcription/translation . As shown in gel retardation assays, the baf/jun complex binds to the AP1 DNA recognition sequence with high affinity, while no significant binding was observed with either of the individual protein components, indicating cooperative DNA binding of the two proteins . The fact that the bacterial baf protein does not undergo glycosylation indicates that the post-translational modification of eukaryotic c-fos with N-acetylglucosamine is not required for the formation of a stable fos/jun/DNA complex.

J Interferon Res, 1990 Aug, 10(4), 425 - 33
Fever induced by Escherichia coli or intrahypothalamic prostaglandin E2 enhances interferon-gamma synthesis; Taylor MW et al.; We previously showed that hyperthermia induced in rhesus monkeys (Macaca mulatta) by forced passive heating "primes" the peripheral lymphocyte population for increased synthesis of interferon-gamma (IFN-gamma) . It was not clear whether these data could be extrapolated to the physiological response in naturally occurring fever . Therefore, in the current experiments, the temperature of rhesus monkeys was raised either by systemic injection of killed Escherichia coli or by intrahypothalamic administration of prostaglandin E2 . Mononuclear cells collected subsequently from such monkeys produced more IFN-gamma in response to stimulation with mitogens than cells from control monkeys . Direct administration of IFN-alpha, -beta, or -gamma to the hypothalamus did not affect the body temperature of rhesus monkeys.

J Biochem (Tokyo), 1990 Aug, 108(2), 175 - 84
Three-dimensional structures of aspartate aminotransferase from Escherichia coli and its mutant enzyme at 2.5 A resolution; Kamitori S et al.; The structure of Escherichia coli aspartate aminotransferase complex with the inhibitor 2-methylaspartate, and that of the mutant enzyme in which an arginine was substituted for a lysine residue thereby forming a Schiff base with the coenzyme pyridoxal 5'-phosphate, were determined at 2.5 A resolution, by the molecular replacement method using the known structure of pig cytosolic aspartate aminotransferase . The enzyme catalyzes the reversible transamination between L-aspartate and alpha-ketoglutarate, and forms a dimeric structure of two identical subunits . Each subunit comprises two domains, a small and a large one . Although, in general, the overall and secondary structure of E . coli enzyme are similar to those of higher animals, some differences of enzymatic action between the enzyme from E . coli and those from higher animals could be explained on the basis of the X-ray structures and molecular mechanics calculation based on them.

Kekkaku, 1990 Aug, 65(8), 507 - 17
{Molecular cloning and expression of Mycobacterium tuberculosis Aoyama B peptide antigen genes in Escherichia coli--a gene encoding 60 kD antigen (AT201) and immunological activity of recombinant peptides (15 and 60 kD)}; Tanaka-Hayashi T et al.; To obtain recombinant peptides related to PPDs, we constructed a genomic library from the DNA of Mycobacterium tuberculosis Aoyama B, a standard strain in Japan to manufacture PPDs, using plasmid vectors pUC18 series . Seven clones reacting the anti-PPDs-rabbit-serum on immunoblotting were obtained, and restriction map was analysed . A nucleotide sequence and a putative open reading frame (ORF) of pAT01, encoding 15kD peptide, as well as the mode of expression was reported previously) . In this study, nucleotide sequence of 60 kD peptide gene was determined, and the comparative database analysis (GENBANK) revealed a striking level of homology to so called mycobacterial heat shock protein . The expression mode of pAT201 encoding 60 kD, as well as pAT01 encoding 15 kD peptide, indicated that these peptides were not hybrid proteins with the lacZ gene product, but they were consisted of peptides only mycobacterial source . Therefore, 15 kD and 60 kD directly were subjected to immunological studies . The peptides were extracted from E . coli, carrying pAT01 or pAT201, purified through series of DEAE chromatography and followed by Detoxi-Gel to remove LPS . 15 kD peptide behaved almost similar to PPDs both in the DTH skin reaction and the lymphocyte proliferation response on guinea pigs or rats in respect to sensitivity . However, 60 kD was unique in that, and it behaved like a general mitogen . We discussed role of 60 kD peptide, comparing with the common antigen, generally found in most species of bacteria as the heat shock protein.

Neuron, 1990 Aug, 5(2), 187 - 97
Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control; Forss-Petter S et al.; To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E . coli lacZ gene . Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues . Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity . Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene . These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.

Surgery, 1990 Aug, 108(2), 254 - 9; discussion 259-61
Both cyclooxygenase-dependent and cyclooxygenase-independent pathways mediate the neuroendocrine response in humans; Michie HR et al.; The neuroendocrine response that occurs after operation, trauma, or infection is essential for maintaining homeostasis within the host . Corticotropin-releasing hormone, (CRH), arginine vasopressin (AVP), and products of the cyclooxygenase pathway have been proposed as possible mediators of the pituitary-adrenal response . We studied the relationship between the induction of these mediators and the onset of the neuroendocrine response in healthy male volunteers receiving one of two standard provocative stimuli: (1) Escherichia coli endotoxin (4 ng/kg intravenously) and (2) hypoglycemia induced by insulin (0.15 units/kg intravenously) . Additional subjects receiving these stimuli were pretreated with the cyclooxygenase inhibitor ibuprofen, 1600 mg orally . Serial measurements were made of circulating concentrations of CRH, AVP, adrenocorticotropic hormone, cortisol, and catecholamines . A sudden rise in circulating AVP (but not CRH) concentrations preceded the onset of all other responses after both stimuli . The prevention of this surge of AVP after endotoxin by ibuprofen was associated with blockade of the neuroendocrine response . These data demonstrate that two independent pathways are available for stimulation of the stress response . After both stimuli, peripheral AVP (but not CRH) levels may mirror alterations that occur within the central nervous system.

Lab Invest, 1990 Aug, 63(2), 221 - 32
Mono-hydroxyeicosatetraenoic acids during porcine endotoxemia . Effect of a platelet-activating factor receptor antagonist; Olson NC et al.; Infusion of endotoxin into domestic pigs induces an acute respiratory failure that has many similarities with the adult respiratory distress syndrome . We hypothesized that mono-hydroxyeicosatetraenoic acids (HETE) and platelet-activating factor (PAF) might be involved in this model of respiratory failure . Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized young pigs at 5 micrograms/kg the first hour, followed by 2 micrograms/kg/hour for 3 hours in the presence and absence of SRI 63-675, a specific PAF receptor antagonist . SRI 63-675 (10 mg/kg before endotoxin + 3 mg/kg/hour during endotoxemia) blocked or attenuated endotoxin-induced pulmonary hypertension, bronchoconstriction, hypoxemia, thrombocytopenia, increased permeability of the alveolar-capillary membrane, and the increases in plasma (at 3 and 4 hours) and bronchoalveolar lavage fluid concentrations of 5-, 12-, and 15-HETE . In a separate group of pigs, before treatment with SRI 63-675, ex vivo stimulation of whole blood with calcium ionophore (at -25 min) caused large increases in plasma concentrations of 5-HETE and, to a lesser extent, 12-HETE . At 4 hours, these increases were not significantly modified in blood derived from pigs treated with SRI 63-675 (10 mg/kg + 3 mg/kg/hour from 0 to 4 hours), indicating no direct inhibition of 5- or 12-lipoxygenase and suggesting that the in vivo effects were PAF receptor-mediated . We conclude that PAF contributes to the release of HETEs during endotoxemia and that this phenomenon could be important in the pathophysiology associated with endotoxin-induced lung injury in anesthetized pigs.

J Clin Endocrinol Metab, 1990 Aug, 71(2), 436 - 41
Trophoblast-derived interleukin-6 (IL-6) regulates human chorionic gonadotropin release through IL-6 receptor on human trophoblasts; Nishino E et al.; We examined the capacity of trophoblast-derived interleukin-6 (IL-6) to stimulate secretion of placental hormones, including hCG . IL-6 stimulated hCG secretion by trophoblasts to a level similar to that stimulated by a GnRH analog . The analog, however, released hCG by an IL-6-independent mechanism because PM-1, a monoclonal antibody specific for IL-6 receptors (R), failed to block GnRH-mediated responses, but completely blocked IL-6-mediated hCG secretion, suggesting the existence of two distinct regulatory pathways for hCG release . Immunohistochemical analysis with another IL-6-R-specific antibody, MT-18, showed that IL-6-R was located only on the trophoblast layer of the placenta . Our data revealed the existence of a local regulatory network by which trophoblast-derived IL-6 interacts with IL-6-R on the trophoblasts, resulting in hCG release . Thus, two different regulatory networks, an IL-6 and IL-6-R system and a GnRH and GnRH-R system, regulate hCG release by human trophoblasts independently.

Proc Natl Acad Sci U S A, 1990 Aug, 87(15), 5768 - 72
Residues in three conserved regions of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase are required for quaternary structure; Fitchen JH et al.; To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120 . These altered small subunits were cosynthesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli . Mutants were analyzed for effects on quaternary structure and catalytic activity . Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme . Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits . The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.

J Bacteriol, 1990 Aug, 172(8), 4719 - 20
Escherichia coli strains with multiple DNA repair defects are hyperinduced for the SOS response; Foster PL; Escherichia coli strains defective for the repair of apurinic/apyrimidinic sites and for the UvrABC excision repair pathway could be constructed if they also carried a mutation in ung, which encodes uracil glycosylase, or sulA, which encodes an SOS-inducible inhibitor of septation . The resultant strains were sensitive to alkylation damage and hyperinduced for the SOS response, but had unpredictable spontaneous mutation rates.

J Bacteriol, 1990 Aug, 172(8), 4563 - 70
The ilvIH operon of Escherichia coli is positively regulated; Platko JV et al.; The ilvIH operon of Escherichia coli (located near min 2) encodes acetohydroxyacid synthase III, an isozyme involved in branched-chain amino acid biosynthesis . A strain with lacZ fused to the ilvIH promoter was constructed . Transposon Tn10 was introduced into this strain, and tetracycline-resistant derivatives were screened for those in which ilvIH promoter expression was markedly reduced . In one such derivative, strain CV1008, beta-galactosidase expression was reduced more than 30-fold . The transposon giving rise to this phenotype inserted near min 20 on the E . coli chromosome . Extract from a wild-type strain contains a protein, the IHB protein, that binds to two sites upstream of the ilvIH promoter (E . Ricca, D . A . Aker, and J . M . Calvo, J . Bacteriol . 171:1658-1664, 1989) . Extract from strain CV1008 lacks IHB-binding activity . These results indicate that the IHB protein is a positive regulator of ilvIH operon expression . The gene that encodes the IHB protein, ihb, was cloned by complementing the transposon-induced mutation . Definitive evidence that the cloned DNA encodes the IHB protein was provided by determining the sequence of more than 17 amino acids at the N terminus of the IHB protein and comparing it with the nucleotide sequence . A mutation that prevents repression of the ilvIH operon by leucine in vivo and that alters the DNA-binding characteristics of the IHB protein in vitro was shown to be an allele of the ihb gene . The ihb gene is identical to oppI, a gene that regulates the oppABCDF operon (E . A . Austin, J . C . Andrews, and S . A . Short, Abstr . Mol . Genet . Bacteria Phages, p . 153, 1989) . Thus, oppI/ihb encodes a protein that regulates both ilvIH, an operon that is repressed by leucine, and oppABCDF, an operon involved in peptide transport that is induced by leucine . We propose that the designation lrp be used in the future instead of oppI or ihb and that Lrp (leucine-responsive regulatory protein) be used in place of IHB.

J Bacteriol, 1990 Aug, 172(8), 4288 - 94
A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli; Sumantran VN et al.; A novel L-threonine transport system is induced in Escherichia coli cells when incubated in amino acid-rich medium under anaerobic conditions . Genetic and biochemical analyses with plasmids harboring mutations in the anaerobically expressed tdcABC operon indicated that the tdcC gene product was responsible for L-threonine uptake . Competition experiments revealed that the L-threonine transport system is also involved in L-serine uptake and is partially shared for L-leucine transport; L-alanine, L-valine, and L-isoleucine did not affect L-threonine uptake . Transport of L-threonine was inhibited by the respiratory chain inhibitors KCN and carbonyl cyanide m-chlorophenylhydrazone and was Na+ independent . These results identify for the first time an E . coli gene encoding a permease specific for L-threonine-L-serine transport that is distinct from the previously described threonine-serine transport systems . A two-dimensional topological model predicted from the amino acid composition and hydropathy plot showed that the TdcC polypeptide appears to be an integral membrane protein with several membrane-spanning domains exhibiting a striking similarity with other bacterial permeases.

Mol Cell Biol, 1990 Aug, 10(8), 4221 - 32
Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase; Repetto B et al.; Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex . The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library . Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex . Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate . The expression of KGD2 was transcriptionally regulated by glucose . Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides . The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells . Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E . coli in episomal plasmids, and the resultant constructs were tested for expression of beta-galactosidase in wild-type yeast cells and in hap2 and hap3 mutants . Results of the lacZ fusion assays indicated that transcription of KGD2 is activated by the HAP2 and HAP3 proteins . The regulated expression of KGD2 was found to depend on sequences that map to a region 244 to 484 nucleotides upstream of the structural gene . This region contains two short sequence elements that differ by one nucleotide from the consensus core (5'-TN{A/G}TTGGT-3') that has been proposed to be essential for binding of the HAP activation complex . These data together with earlier reports on the regulation of the KGD1 and LPD1 genes for the alpha-ketoglutarate and dihydrolipoyl dehydrogenases indicate that all three enzyme components of the complex are catabolite repressed and subject to positive regulation by the HAP2 and HAP3 proteins.

Mol Cell Biol, 1990 Aug, 10(8), 4116 - 22
Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein; Matsui Y et al.; We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol . This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it . In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI . The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565 . This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively . The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity . smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus . smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi . The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable . This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.

Mol Cell Biol, 1990 Aug, 10(8), 3884 - 95
A cis-acting element present in multiple genes serves as a repressor protein binding site for the yeast CAR1 gene; Luche RM et al.; Induction of the arginase (CAR1) gene expression in Saccharomyces cerevisiae has previously been shown to require participation of a cis-dominantly regulated upstream repression sequence (URS) . Deletion of this element results in high-level expression of the CAR1 gene without inducer . To determine the structure of the CAR1 URS element, we performed a saturation mutagenesis . Results of the mutagenic analysis indicated that the CAR1 URS was a 9-base-pair palindromic sequence, 5'-AGCCGCCGA-3' . A DNA fragment containing this sequence was shown to bind one or more proteins by a gel shift assay . DNA fragments containing point mutations that completely eliminated URS function were not effective competitors in this assay, whereas those which supported URS function were effective competitors . Sequences in the 5'-flanking regions of 14 other genes were found to be homologous to the CAR1 URS . These sequences were shown to support varying degrees of URS function in the expression vector assay, to bind protein as demonstrated by the gel shift assay, and to compete with a DNA fragment containing the CAR1 URS for protein binding . These results indicate that the CAR1 URS element possesses the characteristics of a repressor binding site . Further, they are consistent with the suggestion that sites homologous to the CAR1 URS may be situated in the 5'-flanking regions of multiple unrelated yeast genes . The widespread occurrence of this element raises the possibility that it is the target site for one or more negatively acting general transcription factors.

Infect Immun, 1990 Aug, 58(8), 2659 - 63
Leukotriene and hydroxyeicosatetraenoic acid generation elicited by low doses of Escherichia coli hemolysin in rabbit lungs; Grimminger F et al.; Low doses of Escherichia coli hemolysin cause thromboxane-mediated hypertension and vascular leakage in blood-free perfused rabbit lungs (W . Seeger, H . Walter, N . Suttorp, M . Muhly, and S . Bhakdi, J . Clin . Invest . 84:220-227, 1989) . The recirculating buffer medium and bronchoalveolar lavage fluid from lungs exposed to hemolysin (2.5 hemolytic units per ml) in the presence of cyclooxygenase inhibitor were analyzed for leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) by reverse-phase and straight-phase high-pressure liquid chromatographic techniques combined with UV spectrum analysis and post-high-pressure liquid chromatography radioimmunoassay . A rapid release of large amounts of cysteinyl-LTs and leukotriene B4 (LTB4) into the intravascular space was noted (total sum, approximately 4 to 5 micrograms) . Similar quantities have hitherto been elicited only by high concentrations of the artificial calcium ionophore A 23187 . Moreover, a marked liberation of 5-HETE and 12-hydroxyheptadecatrienoic acid into the buffer medium occurred, whereas LTB4 represented the predominant compound in the lavage fluid . The hemolysin-induced burst of LT and HETE generation preceded the onset of vascular leakage . The outstanding capacity of E . coli hemolysin to produce the liberation of potent lipid mediators is probably relevant to the pathways of vascular injury and amplification of inflammatory events during severe infection with hemolytic E . coli strains.

Int J Biol Macromol, 1990 Aug, 12(4), 226 - 32
Analysis of patterns of twist angles in DNA double helix; Zhang CT et al.; We have analysed theoretically the patterns of twist angles of B-DNA by the Tung-Harvey model mainly . It is shown that for a sequence of twist angles a smaller twist angle tends to follow a larger one and vice versa . Therefore the sequence of twist angles always takes a gentle zig-zag form . For simplicity we convert the sequence of twist angles to a symbolic sequence consisting of L and S, where L or S represents a large or a small angle, respectively . The -10 and -35 regions of 112 well-defined promoters for E . coli RNA polymerase, which were compiled by Hawley and McClure, have been analysed in terms of LS sequences in detail . The results shows that the number of LS sequences for promoters is considerably limited and the promoter mutations do not change the patterns of LS sequences in most cases . Several new ideas, which are believed to be useful in the further study, have been presented.

Kobe J Med Sci, 1990 Aug, 36(3-4), 71 - 86
Possible binding proteins of ras p21 in human erythrocyte membrane; Tanimoto T; The direct binding protein(s) of ras p21 was investigated in the inside-out vesicles of human erythrocyte ghosts using the pure v-Kirsten (Ki)-ras p21 synthesized in E . coli . The bound ras p21 was detected immunochemically using an anti-v-Ki-ras p21 monoclonal antibody . ras p21 was overlaid on the vesicle proteins immobilized on a nitrocellulose sheet transferred from the gel of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . ras p21 bound to bands 4.2 and 6 . ras p21-binding to bands 4.2 and 6 was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein . Furthermore, when ras p21 was mixed with inside-out vesicles and then centrifuged, ras p21 was coprecipitated with the vesicles . Prior digestion of the vesicles with trypsin reduced this binding significantly . These results indicate that v-Ki-ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as fat as tested in an in vitro cell-free system.

Mol Microbiol, 1990 Aug, 4(8), 1311 - 8
Mannose-sensitive haemagglutination in the absence of piliation in Escherichia coli; Hultgren SJ et al.; The relationship between type 1 pilus structure and the mannose-sensitive adhesin was investigated by analysing the properties of an 11.2 kb fragment of DNA derived from the chromosomal pil region of a type 1 piliated uropathogenic strain of Escherichia coli . The recombinant plasmids pHA9 and pSJH9, containing the cloned fragment, conferred a mannose-sensitive haemagglutination (MSHA)-positive but non-piliated phenotype on recipient cells of ORN104 . Most of the DNA sequences homologous to the pilA and hyp genes were not present in the 11.2 kb insert, and the genetic information necessary for MSHA in the absence of piliation spanned a 6.5 kb region of the cloned fragment . The polypeptides expressed by pSJH9 were examined in minicells and Tn1000 insertions in three genes encoding proteins of molecular weights 90 kD, 29 kD and 17 kD abolished the MSHA phenotype.

J Gen Microbiol, 1990 Aug, 136 ( Pt 8), 1609 - 14
Binding of K99 fimbriae of enterotoxigenic Escherichia coli to pig small intestinal mucin glycopeptides; Lindahl M et al.; Binding of purified K99 fimbriae to cryostat sections of pig small intestine was detected . Binding sites were located in the mucus layer, but not in the submucosal connective tissue . High-Mr mucin glycopeptides from pig small intestine were found to bind to K99-fimbriated enterotoxigenic Escherichia coli, in contrast to non-fimbriated cells . Sialic acid specificity of K99 fimbriae was demonstrated by the significant reduction in binding upon desialylation of mucin glycopeptides . The binding was saturable and the dissociation constant was estimated to be 6 x 10(-7) M . Fimbriated bacteria were calculated to possess 2.3 x 10(3) binding sites per cell.

AIDS, 1990 Aug, 4(8), 791 - 7
Syncytium induction by fresh HIV isolates: quantitative analysis using a transactivation beta-gal assay; Emilie D et al.; We used a quantitative bioassay (the beta-gal assay) to visualize and quantify syncytium induction by fresh HIV isolates . This bioassay is based on the transactivation by tat of a chimeric gene comprising an HIV-1 long terminal repeat (LTR) fused to a modified lacZ gene of Escherichia coli . The chimeric gene encodes a beta-galactosidase which is translocated to the nucleus . It allows the enzymatic staining of all nuclei from HIV-induced syncytia . Using this unequivocal assay (the beta-gal assay), we could assess the syncytium-inducing properties of fresh HIV isolates after only 4 days of coculture of patient lymphocytes with activated normal lymphocytes . Syncytium-inducing HIV isolates were detected in 11 out of 40 seropositive patients studied . They were isolated mainly from AIDS patients: eight out of 17 grade IV (according to Centers for Disease Control criteria) patients were infected with syncytium-inducing strains . However, of 23 grade II and III patients tested, syncytium-inducing HIV strains were isolated from three cases . These three patients displayed no detectable p24 antigenaemia and had a CD4+ cell count of greater than 300 cells/microliter . The in vitro replication rate of HIV grown from 36 patient blood samples was then examined by sequential p24 antigen measurements in coculture supernatants . The 10 samples leading to syncytium formation also exhibited the highest replication rate . The possibility of unequivocally detecting syncytium-inducing strains after only a few days of coculture will make this detection routine and rapid . In addition, the limited period of amplification required is a significant advantage as it minimizes the emergence of HIV variants selected during long-term in vitro cultures.

Proc Natl Acad Sci U S A, 1990 Aug, 87(15), 5672 - 6
Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1; Lee SH et al.; In the presence of a single-stranded-DNA-binding protein (SSB), the elongation of primed DNA templates by DNA polymerase delta (pol delta) is dependent on ATP and two protein factors, activator 1 (A1) and proliferating cell nuclear antigen (PCNA) . We have examined the interaction of these proteins with (dA)4500.(dT)12-18 by measuring their ability to form stable complexes with this DNA . In the presence of ATP, A1, PCNA, and pol delta formed a stable complex with DNA that could be isolated by gel filtration . Incubation of the isolated complex with dTTP resulted in the synthesis of poly(dT) . While ATP was required for the formation of this complex, it was not required for the subsequent elongation of DNA . The temporal requirements for complex formation were determined . A1 was found to bind first, followed by the ATP-dependent addition of PCNA to the A1.DNA complex, while pol delta was added last . Each of these complexes could be isolated by gel filtration, indicating that they possessed a high degree of stability . The binding of PCNA to the A1-SSB-coated primed DNA occurred with adenosine 5'-{gamma-thio}triphosphate as well as ATP . However, the binding of pol delta to the PCNA.A1-DNA complex was observed only when the latter complex was formed in the presence of ATP . The complete complex was formed after incubation at 37 degrees C for 2 min, whereas no complex was detected after incubation at 0 degree C . These results indicate that these proteins act in a manner analogous to the accessory proteins that play critical roles in the elongation reaction catalyzed by T4 phage DNA polymerase and Escherichia coli DNA polymerase III.

J Infect Dis, 1990 Aug, 162(2), 442 - 7
Enterotoxins and adhesins of enterotoxigenic Escherichia coli: are they risk factors for acute diarrhea in the community?
Lopez-Vidal Y, Calva JJ, Trujillo A, Ponce de Leon A, Ramos A, Svennerholm AM, Ruiz-Palacios GM.
A cohort of 228 Mexican children less than 5 years old was followed during the enterotoxigenic Escherichia coli(ETEC) season . The incidence of ETEC diarrhea-associated and asymptomatic infections was determined, and E . coli strains isolated from stool samples were tested for heat-labile and heat-stable toxins and for expression of colonization factor antigens (CFA) . Of the children, 61% had at least one ETEC infection . Children with ETEC isolated from stools were more likely to have diarrhea than were ETEC-free age-matched control children (odds ratio {OR} = 4.5; 95% confidence interval {CI} = 2.9-7.0) . Strains carrying CFA/IV, CFA/I, or CFA/II were found in 23%, 18%, and 5% of ETEC infections, respectively . The risk of having diarrhea associated with a CFA-expressing versus a CFA-negative ETEC strain was the same (age-adjusted OR = 0.8; 95% CI = 0.4-1.6) . These data should be considered in the development of a diarrhea vaccine containing only CFAs.

Infect Immun, 1990 Aug, 58(8), 2710 - 4
Electron microscopic study of coexpression of adhesive protein capsules and polysaccharide capsules in Escherichia coli; Kroncke KD et al.; Escherichia coli 21535 (O21:K4:H4 with nonfimbrial adhesin NFA-6) and 21511 (O7:K98:H6 with nonfimbrial adhesin NFA-4) were analyzed by immunoelectron microscopy with a K98-specific antiserum and K4- and NFA-4-specific and NFA-6-reactive monoclonal antibodies . The bacteria were analyzed in ultrathin sections after stabilization of the capsules with specific antibodies by embedding in Epon 812 as well as in Lowicryl K4M . With the Lowicryl-embedded samples, the polysaccharide K antigens were labeled by the immunogold technique . It was found that with both strains all bacteria expressed the polysaccharide capsule, while in each case about 20% expressed the protein capsule in addition . Thus, in both invasive E . coli strains, bacteria are present which express composite capsules with the adhesin (recognition protein) at the cell-distal outer region and the K antigen (acidic polysaccharide) at the cell-proximal inner region . These findings are discussed with respect to the participation of the capsular compartments in unspecific host defense.

J Clin Lab Immunol, 1990 Aug, 32(4), 195 - 200
Purified recombinant 60 kD SS-A/Ro protein--an antigenic source for ELISA screening of patients with rheumatic diseases; Ben-Chetrit E et al.; A 60 kD SS-A/Ro recombinant protein was expressed in E . coli cells and purified by the centrifugation procedure . Since the substrate contained a substantial amount of bacterial proteins it was not sufficient for ELISA . Therefore the material obtained by centrifugation was resolved on SDS-gel and the 60 kD region was excised and electro-eluted . Two hundred and nine sera from normal donors and patients with rheumatic diseases were analyzed by ELISA using the purified recombinant 60 kD SS-A/Ro protein . A comparison with Ouchterlony and Western blot revealed that ELISA proved to be a sensitive and specific method, which could discriminate between anti 60 kD antibodies and other autoantibodies . The present study showed that recombinant 60 kD may provide a purified source of antigen for ELISA while this method may serve as a useful tool for screening and evaluation of anti 60 kD antibodies and their relevance to clinical manifestations.

J Pharmacobiodyn, 1990 Aug, 13(8), 487 - 92
Heterocycles related to nucleotides . XI . An organomercurial column chromatography for thiouridine; Sato E et al.; By the organomercurial column: chromagel or Sephadex coupled to p-aminophenylmercury chloride, 4- and 2-thiouridine were selectively separated from the mixtures with major nucleotides . Oligonucleotides containing 4-thiouridilic acid were also separated from the ribonuclease-T1 digests of unfractionated transfer ribonucleic acid of E . coli.

J Pharmacobiodyn, 1990 Aug, 13(8), 483 - 6
Heterocycles related to nucleotides . X . Reaction of thiouridine with 2-chloromercurio-4-nitrophenol; Sato E et al.; 2-Chloromercurio-4-nitrophenol reacts reversibly with thiouridine, this reaction was applied to a chemical modification of E . coli transfer ribonucleic acid.

Immunol Lett, 1990 Aug, 25(1-3), 143 - 8
The T cell reactivity against the major merozoite protein of Plasmodium falciparum; Crisanti A et al.; We have undertaken a systematic search for T cell epitopes within the sequence of the major merozoite surface antigen (GP190) of Plasmodium falciparum . Recombinant polypeptides expressed in E . coli were used to evaluate the reactivity of peripheral blood mononuclear cells (PBMC) from both inhabitants of a rural community of West Africa exposed to P . falciparum transmission and from German patients with diagnosis of acute malaria . Although the proliferative response of the PBMC was in most cases very low, several T cell clones could be established . Deletion analysis of each gp190-derived polypeptide allowed the identification of six different T cell epitopes . Epitopes could be mapped within the dimorphic region of gp190, which also contains the sequences most frequently recognized by sera from adult individuals living in endemic areas.

Mol Microbiol, 1990 Aug, 4(8), 1259 - 68
The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure-function relationships in the TraT protein; Taylor IM et al.; The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion . The structure and function of the TraT protein determined by plasmid R6-5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein . The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti-C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface . Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin-resistant oligomeric form characteristic of the wild-type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions . Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface-exclusion activity . Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild-type protein . Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed.

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