J Biol Chem, 1988 Nov 5, 263(31), 15857 - 9 Escherichia coli DNA topoisomerase I is a zinc metalloprotein with three repetitive zinc-binding domains; Tse-Dinh YC et al.; Escherichia coli DNA topoisomerase I catalyzes interconversions of different DNA topological isomers by the breakage and rejoining of DNA phosphodiester bonds . It has a crucial role in maintaining an optimal DNA superhelicity in E . coli . It is a single polypeptide of 864 amino acids . Analysis of the amino acid sequence reveals three tandem repeat units each containing two pairs of cysteines suggesting that each unit may form a zinc-binding domain . We have determined that each enzyme molecule contains three to four zinc atoms using inductively coupled plasma-atomic emission analysis . Modification of the cysteine residues and removal of the zinc from the enzyme result in loss of activity . Zinc ions are needed for full recovery of enzyme activity when the cysteine modification is reversed . Comparison with the zinc-binding domains of the sequence-specific DNA-binding proteins shows significant differences. J Mol Biol, 1988 Nov 5, 204(1), 79 - 94 What constitutes the signal for the initiation of protein synthesis on Escherichia coli mRNAs? Dreyfus M. Small DNA fragments (60 to 80 nucleotides), randomly obtained from a collection of 14 catabolic, biosynthetic or regulatory Escherichia coli genes, have been shot-gun cloned in place of the lacZ ribosome binding site . A total of 47 recombinants showing substantial beta-galactosidase synthesis (at least 1/30th of the wild-type) were isolated, and their newly acquired translational starts were characterized . Of these, 46 were found to carry a ribosome binding site from one of the original genes, and only one, a non-natural start . Moreover, 12 out of the 14 natural starts were found . The two that were not found are the only ones lacking a Shine-Dalgarno element . So, real starts are generally active in the lac mRNA, whereas the many sites (approx . 100 in this gene collection) that carry a Shine-Dalgarno element followed by AUG or GUG but are located in intra- or intergenic regions, or on non-transcribed strands, are inactive . I conclude that: (1) these "false" starts, being strongly discriminated against in the lac message, are presumably also inactive in their original mRNAs; (2) the discriminating information, being portable from one mRNA to another, must be contained within a small DNA region surrounding the starts . Indeed, I further show that it generally lies within a sequence of about 35 nucleotides bracketing real starts; and (3) this information must have a larger effect on initiation than the exact structure of the mRNA, because the discrimination persists despite a complete change of this structure . Previous statistical analysis has shown that real starts differ from false starts in having a non-random sequence composition from nucleotides -20 to +15 with respect to the start . To uncover whether these biases constitute the discriminating information or simply reflect coding constraints, translational starts were randomly searched in eukaryotic, largely non-coding, DNA . These "eukaryotic" starts all have an in-phase AUG or GUG, preceded by a typical Shine-Dalgarno sequence; outside these elements, the initiator region is strikingly rich in A, and poor in C . These biases match those found around real starts, demonstrating that they are indeed part of the initiation signal . Finally, I describe a simple procedure for introducing any DNA fragment in place of the lac operator site on the E . coli chromosome. J Mol Biol, 1988 Nov 5, 204(1), 51 - 60 Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA translation and decay; Cho KO et al.; In studies with a trpE promoter-strength measuring system we observed that constructs containing the Escherichia coli trp promoter and its adjacent transcribed region yielded lower levels of trpE protein than were expected . To analyze this observation we introduced mutational changes in the nucleotide sequence preceding the trpE Shine-Dalgarno region and examined their effects on trpE mRNA synthesis, translation and decay . We found that certain deletion, insertion and substitution mutations in the pre-Shine-Dalgarno region caused a two- to fivefold increase in trpE enzyme activity . These increases were accompanied by increases in steady-state levels of trpE mRNA . Pulse-chase analyses of trpE mRNA degradation revealed that the observed steady-state trpE mRNA levels correlated with changes in trpE mRNA stability . These findings are interpreted in terms of alternative models in which the primary effect of mutational changes that elevate trpE expression is to increase trpE mRNA translation, versus increasing trpE mRNA stability. Science, 1988 Nov 4, 242(4879), 765 - 8 Anticodon switching changes the identity of methionine and valine transfer RNAs; Schulman LH et al.; The anticodon has previously been shown to play a role in recognition of certain transfer RNAs by aminoacyl-tRNA synthetases; however, the extent to which this sequence dictates tRNA identity is generally unknown . To investigate the contribution of the anticodon to the identity of Escherichia coli methionine and valine tRNAs, in vitro transcripts of these tRNAs were prepared that contained normal and interchanged anticodon sequences . Transcripts containing wild-type tRNA sequences were excellent substrates for their respective cognate aminoacyl-tRNA synthetases and were effectively discriminated against by a variety of noncognate enzymes . The mutant tRNAs produced by switching the anticodon sequences lost their original tRNA identity and assumed an identity corresponding to the acquired anticodon sequence . These results indicate that the anticodon contains sufficient information to distinguish methionine and valine tRNAs with high fidelity. Nature, 1988 Nov 3, 336(6194), 83 - 6 The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements; Riabowol KT et al.; Transcriptional regulation of eukaryotic genes by cyclic AMP requires a cAMP-dependent protein kinase (A kinase) . Two hypotheses have been proposed to explain how the holoenzyme of the A kinase induces transcription . The regulatory subunits of the A kinase, which bind cAMP and DNA, and have amino-acid homology with the Escherichia coli catabolite activator protein could directly stimulate gene expression . Alternatively, phosphorylation by the catalytic subunits could induce transcription by activating proteins involved in gene transcription . To distinguish between these models, we microinjected purified preparations of the catalytic and regulatory subunits of A kinase into tissue culture cells and monitored expression of a stably integrated fusion gene containing a cAMP-responsive human promoter fused to a bacterial reporter gene, or of the endogenous c-fos gene . The catalytic subunit stimulated expression of these genes, whereas the regulatory subunit did not . These results indicate that the catalytic subunit of A kinase is sufficient to induce expression of two cAMP-responsive genes, without increasing levels of cAMP. Biochim Biophys Acta, 1988 Nov 2, 957(1), 143 - 51 Purification and properties of a novel recombinant human hybrid interferon, delta-4 alpha 2/alpha 1; Le HV et al.; The human interferon (huIFN) delta-4 alpha 2(5-62)/alpha 1(64-166) is a genetically engineered hybrid that consists of residues 5-62 of huIFN alpha 2 and residues 64-166 of huIFN alpha 1 . This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN alpha subtypes . This novel recombinant hybrid was purified from Escherichia coli to greater than 95% homogeneity . The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography . The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17,800 and 17,100, both of which exhibited antiviral activity . Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding . The purified protein exhibited a high specific antiviral activity of 7 x 10(7) units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on murine L929 cells . The level of antiproliferative activity of huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) on various cell lines of different histological origin appeared to be more comparable to that of huIFN alpha 1 than huIFN alpha 2 . The data suggest that huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) hybrid may be a useful tool for understanding huIFN structure-function relations. J Biochem (Tokyo), 1988 Nov, 104(5), 837 - 40 Site-specific mutagenesis of the human interleukin-1 beta gene: the role of arginine residue at the N-terminal region; Kamogashira T et al.; Using the expression system for site-specific mutagenesis in Escherichia coli, we have made deletion mutants at the N-terminal or C-terminal region of human interleukin-1 beta (IL-1 beta) consisting of 153 amino acids . The truncated mutants showed that at least 147 amino acids (numbers 4-150) in IL-1 beta are necessary for the exertion of biological activity . When we changed the arginine at the 4th position (Arg4) in IL-1 beta to other specific amino acids, there was a marked difference in the relative extent of biological and receptor binding activities among the mutants . The order of the mutants was Arg4 = Lys4 greater than Gln4 greater than Gly4 = des-Arg4 greater than Asp4 . Our results demonstrate that the arginine residue at the 4th position in IL-1 beta is important, but not essential, for IL-1 beta to exhibit its biological and receptor binding activities, and the positive charge at this site plays a key role for IL-1 beta to exert the activities. Arch Surg, 1988 Nov, 123(11), 1429 - 32 Effect of prostaglandin E on immune function in multiple animal models; Waymack JP et al.; The immunosuppression seen following major trauma and burns has long been attributed in part to prostaglandin E (PGE) . This has been due primarily to the demonstration that PGE levels are elevated following burns and that when PGE is added to leukocyte cultures, it impairs multiple types of leukocyte functions . We investigated the effect of a new long-acting PGE derivative, 16,16-dimethyl-PGE, on immune function in multiple animal models . The PGE derivative had no effect on mortality in burn sepsis models but improved mean survival times in an Escherichia coli peritonitis model . The PGE derivative impaired neutrophil migration into burn wounds at lower dosages . In a rat burn model, when PGE was administered parenterally, it failed to impair cell-mediated immunity at any dosage and improved lymphocyte function at certain dosages . These data indicate that PGE may not be as immunosuppressive in in vivo models as it has been shown to be in in vitro models. Plasmid, 1988 Nov, 20(3), 241 - 8 Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum; Leiting B et al.; An extrachromosomally replicating transformation vector for Dictyostelium discoideum has been constructed using sequences of the endogenous Dictyostelium plasmid Ddp2 . This transformation vector pnDeI (9.6 kb) replicates as a high copy number plasmid in Dictyostelium and is located in the nucleus . It has been constructed as shuttle vector containing the Escherichia coli vector pUC19 for replication and selection in E . coli and a part of the Tn903 transposon which confers resistance to G418 for selection in Dictyostelium . In order to show that the vector can be used for cloning and stable propagation of Dictyostelium DNA, a fragment of the Dictyostelium alpha-actinin gene that was marked with a synthetic oligonucleotide was cloned into pnDeI and found to be stably maintained in the extrachromosomal vector without undergoing noticeable recombination with the endogenous gene. Hinyokika Kiyo, 1988 Nov, 34(11), 2067 - 9 {The diffusion of enoxacin into the prostatic fluid in the chronic prostatitis patient}; Tanaka K et al.; To examine the diffusion of enoxacin into the prostatic fluid of patients with chronic prostatitis, the serum level and the expressed prostatic secretion (EPS) level of enoxacin were measured . Enoxacin was administered orally at a dose of 200 mg in 24 chronic prostatitis patients . One hour later, blood and EPS samples were taken . The level of enoxacin was measured by the bioassay using E . coli (Kp strain) . In 24 patients, the mean value of enoxacin was 1.24 micrograms/ml in the serum and 0.69 micrograms/ml in the EPS . The mean ratio of EPS/serum was 0.58 . These results indicated that enoxacin diffused well into the prostatic fluid of chronic prostatitis patients. J Chem Inf Comput Sci, 1988 Nov, 28(4), 194 - 210 Heuristic refinement method for the derivation of protein solution structures: validation on cytochrome b562; Brinkley JF et al.; A method is described for determining the family of protein structures compatible with solution data obtained primarily from nuclear magnetic resonance (NMR) spectroscopy . Starting with all possible conformations, the method systematically excludes conformations until the remaining structures are only those compatible with the data . The apparent computational intractability of this approach is reduced by assembling the protein in pieces, by considering the protein at several levels of abstraction, by utilizing constraint satisfaction methods to consider only a few atoms at a time, and by utilizing artificial intelligence methods of heuristic control to decide which actions will exclude the most conformations . Example results are presented for simulated NMR data from the known crystal structure of cytochrome b562 (103 residues) . For 10 sample backbones an average root-mean-square deviation from the crystal of 4.1 A was found for all alpha-carbon atoms and 2.8 A for helix alpha-carbons alone . The 10 backbones define the family of all structures compatible with the data and provide nearly correct starting structures for adjustment by any of the current structure determination methods. Microvasc Res, 1988 Nov, 36(3), 291 - 304 Effects of endotoxemia on systemic plasma loss and hematocrit in rats; van Lambalgen AA et al.; Endotoxemia in rats increases plasma extravasation but does not result in continuously rising hematocrit . These contradictory observations led us to design a study in anesthetized rats (C, control rats, n = 10; E, endotoxin rats, n = 10) in which we continuously measured in blood hematocrit (conductivity cell) and changes in concentration of 125I-HSA (human serum albumin) and 51Cr-labeled red cell (51Cr-RBC; multichannel analyzer) in an extracorporeal circuit . In two additional series of experiments we measured in blood samples changes in protein concentration (series II, C: n = 7, E: n = 7) and uptake of intraperitoneally injected 125I-HSA and 51Cr-RBC (reflecting lymph flow rate; series III, C: n = 6, E: n = 7) . Endotoxemia was induced by infusion (iv, 0.2 ml/100 g.hr) of Escherichia coli endotoxin (20 mg/kg) from t = 0 to t = 60 min; controls received saline . Experiments ended at t = 120 (series I and II) or 150 min (series III) . The endotoxemia resulted in a marked rise of serum lactate (by ca 500% at t = 120); heart rate increased and central venous pressure decreased (by ca 20 and -95% at t = 120, respectively) . All rats showed characteristic changes in hematocrit during endotoxemia: an increase from t = 20 to t = 45 (by ca 9%) followed by a decrease to preshock values or less at t = 120 . The 51Cr activity per microliter blood cells did not change, indicating that there was no red cell mobilization . Protein concentration and 125I-HSA activity also showed a temporary increase during endotoxemia, but 125I-HSA activity per gram protein was decreased . Peritoneal uptake of 125I-HSA and 51Cr-RBC was significantly increased during endotoxemia (by 200%) . We conclude that fluid extravasation during endotoxemia is temporary, mainly concerns plasma water, and is compensated by mechanisms like reabsorption and increased lymph flow, resulting in restoration of plasma volume. Immunology, 1988 Nov, 65(3), 433 - 6 Inhibition by glucocorticoids of mitogen-dependent histamine biosynthesis caused by histidine decarboxylase in cultured mouse spleen cells and peritoneal adherent cells; Oh C et al.; When spleen cells of C57BL/6 mice were cultured, their histidine decarboxylase (HDC) activity increased with a concomitant increase in the histamine concentration in the culture medium . The addition of concanavalin A (Con A) or E . coli lipopolysaccharide (LPS) to the culture enhanced the responses . Treatment with dexamethasone or corticosterone significantly inhibited both spontaneous and Con A-dependent induction of HDC and histamine biosynthesis . Progesterone and estradiol did not inhibit but rather enhanced the responses . Testosterone had little effect on HDC activity and the histamine level of the culture medium of spleen cells . Adherent cells obtained from glycogen-stimulated peritoneal exudates had the enzyme constitutively . Con A had no appreciable effect on the HDC activity and the histamine level of the culture of these adherent cells . However, the addition of conditioned medium of T + B lymphocytes that had been incubated in the presence of Con A rendered the adherent cells responsive to Con A, leading to an increase in the HDC activity and the histamine level of the culture of the cells . Treatment with dexamethasone largely abrogated the responses . The results suggest that HDC-dependent histamine biosynthesis by peritoneal adherent cells is inhibited by glucocorticoids, which act on the adherent cells per se. Circ Shock, 1988 Nov, 26(3), 297 - 309 Endotoxin-induced procoagulant activity in equine peripheral blood monocytes; Henry MM et al.; Increasing evidence has demonstrated the importance of monocyte procoagulant activity (PCA) in the pathogenesis of coagulopathies in a variety of diseases . Because endotoxin precipitated coagulopathies are common sequelae to intestinal ischemia/endotoxemia in the equine species, we investigated the ability of equine peripheral blood monocytes to express PCA . Monocytes isolated from five healthy adult horses were incubated in vitro with Escherichia coli endotoxin (10 micrograms), and the PCA was measured by the ability of cellular lysates to accelerate the clotting times of equine plasma in a modified one-stage recalcification assay . Equine monocyte PCA was identified as thromboplastin based on lack of clot formation in factor VII-deficient plasma . The induction of PCA occurred as early as 2 hr after endotoxin exposure, peaked at 6 hr (396% increase), and then gradually declined . The amount of PCA was proportional to the dose of endotoxin (0.01 to 100 micrograms) and the number of monocytes . Neither platelets nor neutrophils produced PCA, either in the absence or presence of endotoxin (1 microgram) . Lymphocytes at a concentration of 4 x 10(6)/ml RPMI did produce a significant amount of PCA, compared to the time-matched controls . Co-incubation of neutrophils or lymphocytes with monocytes did not alter the PCA, whereas coincubation of platelets and monocytes significantly enhanced the expression of PCA . This effect was further augmented by the addition of endotoxin (1 microgram). Arch Biochem Biophys, 1988 Nov 1, 266(2), 496 - 507 An NADP/thioredoxin system in leaves: purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach; Florencio FJ et al.; An NADP/thioredoxin system, consisting of NADPH, NADP-thioredoxin reductase (NTR), and its thioredoxin, thioredoxin h, has been previously described for heterotrophic plant tissues, i.e., wheat seeds and cultured carrot cells . Until now there was no evidence for this system in green leaves . Here, we report the identification of protein components of the NADP/thioredoxin system in leaves of several species . Thioredoxin h and NTR, which were both recovered in the extrachloroplastic fraction, were purified to apparent homogeneity from spinach leaves . This represents the first time that NTR has been characterized from a plant source . Similar to that from bacterial and mammalian sources, spinach leaf NTR was a flavoprotein (Mr 68,000) composed of two subunits of identical molecular mass (Mr 33,000) that resembled Escherichia coli NTR immunologically . Spinach thioredoxin h existed in two forms (Mr of 13,500 and 12,000) and was highly specific for plant NTR . Thioredoxin h and NTR partially purified from spinach roots showed properties similar to their counterparts from leaves . Spinach cytosolic thioredoxin h differed from chloroplast thioredoxin m or f from the same source but was similar to thioredoxin h from wheat seed in immunological properties. Am J Physiol, 1988 Nov, 255(5 Pt 2), H1106 - 13 Naloxone alters organ perfusion during endotoxin shock in conscious rats; Law WR et al.; Antagonism of endogenous opioids has been shown to improve survival time, increase blood pressure, and attenuate acidosis during endotoxin shock . However, some of the most severe problems associated with this condition arise from the circulatory disturbances that occur . We investigated the circulatory effects of naloxone during endotoxin shock as they relate to hemodynamic parameters in conscious, unrestrained rats . Blood flow and hemodynamic variables were measured in male, Sprague-Dawley rats (300-400 g) 24 h after surgical preparation . Rats were challenged with either 10 mg/kg Escherichia coli endotoxin (100% lethal dose) or intravenous saline . Measurements were made at 0, 10, 30, and 60 min postchallenge . Naloxone (2 mg/kg) or saline was given as a treatment (intravenous bolus) at 25 min postchallenge . Cardiac output and blood distribution (%CO) and flow were measured with radiolabeled microspheres . Cardiac output was depressed and total peripheral resistance was elevated 10 min into endotoxin shock . Naloxone treatment improved blood pressure significantly during endotoxin shock, as would be expected with the observed increase in total peripheral vascular resistance and no significant change in cardiac output . Improved perfusion of skeletal muscle is a likely explanation for lower serum lactate levels that have been reported to occur in this model after naloxone administration . Our data also indicate that naloxone may improve cardiac efficiency and does not interfere with maintenance of global cerebral blood flow . Collectively, these effects would contribute to the observed improved survival time after naloxone treatment.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1988 Nov, 85(22), 8497 - 501 Ligand binding to synthetic mutant myoglobin (His-E7----Gly): role of the distal histidine; Braunstein D et al.; Low-temperature flash photolysis with IR and visible spectroscopy was used to probe the influence of the distal histidine His-64(E7) of sperm-whale myoglobin (Mb) on the orientation of bound carbon monoxide (CO) and on the kinetics of CO rebinding . The synthesis and high-level expression of a sperm-whale myoglobin gene in Escherichia coli permits the efficient substitution of the distal histidine through site-directed mutagenesis . Substitution of His-E7 with glycine {GlyE7}Mb bound with CO (CO{GlyE7}Mb) results in one broad bound-CO IR stretch band, v(C-O), centered at 1973 cm-1 at 10 K, in contrast to three distinct bands for native and synthetic wild-type MbCO at 1966, 1945, and 1929 cm-1 . After flash photolysis at 10 K, the unbound state of CO{GlyE7}Mb exhibits two CO stretch bands, whereas MbCO has three . Fourier transform IR spectroscopy measurements of the linear dichroism after photoselective flash photolysis of CO bound to {GlyE7}Mb at 10 K reveals the bound CO to be oriented at an angle of alpha = 20 degrees +/- 2 degrees with respect to the heme normal . Flash photolysis data from 10 to 300 K provide evidence for a larger distal pocket and a smaller enthalpy barrier (by approximately 4 kJ/mol) for {GlyE7}MbCO as compared with wild-type MbCO . These results reinforce the notion that the dominant control of the binding step at the heme iron comes from the proximal side through the protein structure. Proc Natl Acad Sci U S A, 1988 Nov, 85(21), 7967 - 71 Translation of the sequence AGG-AGG yields 50% ribosomal frameshift; Spanjaard RA et al.; We have inserted the sequence 5'-AAG-GAGGU-3', which is complementary to the 3' terminus of Escherichia coli 16S rRNA, in a reading frame and analyzed its effect on the accuracy and overall rate of translation in vivo . Translation over the sequence yields a 50% ribosomal frameshift if the reading phase is A-AGG-AGG-U . The other two possible frames do not give shifts . The introduction of a UAA stop codon before (UAA-AGG-AGG-U) but not after (A-AGG-AGG-UAA) the AGG codons abolishes the frameshift . The change in the reading phase occurs exclusively to the +1 direction . Efficient frameshifting is also induced by the sequence A-AGA-AGA-U . The arginine codons AGG and AGA are read by minor tRNA . Suppression of frameshifting takes place when a gene for minor tRNA(Arg) is introduced on a multicopy plasmid . We suggest that frameshifting during translation of the A-AGG-AGG-U sequence is due to the erroneous decoding of the tandem AGG codons and arises by depletion of tRNA(Arg) . The complementarity of tandem AGG codons to the 3' terminus of 16S rRNA is a coincidence and apparently not related to the shift . Replacing the AGG-AGG sequence by the optimal arginine codons CGU-CGU does not increase the overall rate of translation. Mol Biochem Parasitol, 1988 Nov, 31(2), 117 - 25 Production of a recombinant antigen of Echinococcus multilocularis with high immunodiagnostic sensitivity and specificity; Vogel M et al.; A cDNA library derived from mRNA of metacestodes from Echinococcus multilocularis was constructed in the Escherichia coli expression vector lambda gt11 and screened with human patients' sera . The recombinant proteins of 11 positive phages were further evaluated for their potential as immunodiagnostic reagent . One isolated clone (from lysogen II/3) synthesized a fusion protein which was rapidly degraded . This intracellular degradation process provided two distinct polypeptides with Mr of about 31,000 and 33,000, both showing strong binding activity with specific patients' antibodies . The diagnostic value of the II/3-antigen was evaluated by mini-Western-blot with sera from 41 patients infected with E . multilocularis and sera from 77 patients with other helminthic infections, resulting in a diagnostic sensitivity of 98% and an over-all specificity of 96% . These results suggest the usefulness of the recombinant II/3-antigen for immunodiagnosis of human alveolar echinococcosis. Crit Care Med, 1988 Nov, 16(11), 1121 - 7 Effects of ibuprofen on neutrophil function and acute lung injury in canine endotoxin shock; Balk RA et al.; The role of the polymorphonuclear leukocyte in the development of acute lung injury has been the subject of much controversy . Experimental lung injury is associated with peripheral leukopenia and the intrapulmonary sequestration of leukocytes . We have previously shown that ibuprofen, a nonsteroidal anti-inflammatory drug, can improve the hemodynamic alterations of canine endotoxin shock . Ibuprofen has also been found to decrease leukocyte adherence . We investigated the dose response of ibuprofen on the increased neutrophil adherence and the extent of lung injury associated with canine endotoxin shock . Single doses of ibuprofen (1, 5, 10, and 20 mg/kg iv) were administered 15 min after Escherichia coli endotoxin . Endotoxemia resulted in leukopenia and an increased neutrophil adherence in both aortic and pulmonary artery blood . Endotoxin-treated animals exhibited increased neutrophils in the bronchoalveolar lavage fluid, a marker of lung injury . The 20-mg/kg ibuprofen dose decreased aortic granulocyte adherence at 30 min, while all ibuprofen doses decreased the aortic adherence at 120 min . The increased pulmonary artery neutrophil adherence was not affected by ibuprofen . Histologically, lung injury was manifested by intravascular leukostasis . Ibuprofen treatment did not affect the histologic or morphometric extent of the lung injury . The leukopenia and increased neutrophil adherence occur rapidly after endotoxemia and are associated with subsequent intravascular sequestration of leukocytes . Agents designed to prevent lung injury must either be given before the insult or be able to block the effects of the toxic products released by the activated granulocytes.(ABSTRACT TRUNCATED AT 250 WORDS) Ukr Biokhim Zh, 1988 Nov-Dec, 60(6), 75 - 8 {The use of ultrafiltration for concentration and purification of beta-galactosidase}; Gutsaliuk VM et al.; A possibility of concentration and purification of culture fluid of Escherichia coli by the ultrafiltration technique using Soviet cellulose acetate membranes were studied . It is established that cellulose acetate membranes may be used for purification from low molecular weight protein and concentration of preliminarily purified culture fluid of Escherichia coli . Membranes YAM-300 are most preferable . At the temperature of 18 degrees C, pressure 0.16-0.24 MPa and 20-fold concentration the yield of the enzyme at the ultrafiltration stage was 84.5%. JPEN J Parenter Enteral Nutr, 1988 Nov-Dec, 12(6), 615 - 8 Hepatic complications of total parenteral nutrition; Sax HC et al.; Elevations in serum hepatic enzyme levels and alterations in hepatic morphology have been noted in patients on total parenteral nutrition, in some cases progressing to fatal hepatic failure . Various factors such as toxins, inappropriate substrates, overfeeding, deficiency states, and gut hormone alterations have been implicated . It would appear that the tailoring of nutritional support to meet patient needs and the maintenance of normal gut integrity will be of increasing importance in reducing the incidence of this potentially fatal complication. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Nov, 270(1-2), 52 - 65 Studies on serum resistance in Escherichia coli; Kubens BS et al.; Serum-sensitive mutants and their serum-resistant smooth parental E . coli strains (Wf8, Wf26, and WF 52) have been investigated in respect to their binding of different complement components . These pairs consisting of a wild-type and its mutants represent a better model for the investigation of the mechanism of serum resistance than the comparison of unrelated strains . Both strains of a pair bind equivalent amounts of C3 . In binding assays using radiolabeled terminal components C6, C7, C8, and C9, the serum-sensitive strains do bind more late acting components than their resistant parental strains . An active membrane attack complex stably bound to the cell surface was found on the mutants, whereas with wild-type bacteria a complex could be isolated from the supernatant which is composed of the late acting complement components and S-protein . This complex is released from the surface of the wild-type bacteria without participation of C9. Mol Gen Genet, 1988 Nov, 214(3), 420 - 4 Regulation of nod gene expression in Bradyrhizobium japonicum; Banfalvi Z et al.; The best inducers of nod::lacZ translational fusions in Bradyrhizobium japonicum are isoflavones, primarily genistein and daidzein . Upstream of the nodABC genes in B . japonicum is a novel gene, nodY, which is coregulated with nodABC . Measurements of the activity of lacZ fusions to the nodD gene of B . japonicum show that this gene is inducible by soybean seed extract and selected flavonoid chemicals . The induction of the nodY ABC and nodD operons appears to require a functional nodD gene, indicating that the nodD gene product controls its own synthesis as well as other nod genes. Arch Biochem Biophys, 1988 Nov 1, 266(2), 351 - 68 Accurate measurement of psoralen-crosslinked DNA: direct biochemical measurements and indirect measurement by hybridization; Matsuo N et al.; This paper evaluates methods to measure crosslinkage due to psoralen plus light in total DNA and in specific sequences . DNA exposed in cells or in vitro to a bifunctional psoralen and near ultraviolet light accumulates interstrand crosslinks . Crosslinkage is the DNA mass fraction that is attached in both strands to a crosslink . We show here biochemical methods to measure psoralen photocrosslinkage accurately in total DNA . We also describe methods to measure photocrosslinkage indirectly, in specific sequences, by nucleic acid hybridization . We show that a single 4,5',8-trimethylpsoralen (TMP) crosslink causes at least 50 kbp of alkali-denatured DNA contiguous in both strands with it to snap back into the duplex form when the denatured preparation is returned to neutral pH . This process was so efficient that the DNA was not nicked by the single-strand nuclease S1 at 100-fold excess after snapping back . Uncrosslinked DNA was digested to acid-soluble material by the enzyme . Crosslinkage therefore equals the fraction of S1-resistant nucleotide in this kind of experiment . We alkali-denatured DNA samples crosslinked to varying degrees by varying TMP concentration at constant light exposure . We then measured crosslinkage by ethidium bromide (EtBr) fluorometry at pH 11.8; by EtBr fluorometry at neutral pH of S1 digests of the DNA; and by the fraction of radioactivity remaining acid insoluble in S1-digests of DNA labeled uniformly with {3H}deoxythymidine . These assays measure distinct physical properties of crosslinked DNA . Numerical agreement is expected only when all three measurements are accurate . Under optimum conditions, the three methods yielded identical results over the range of measurement . Using alkaline EtBr fluorescence in crude cell lysates, we detected crosslinks at frequencies in the range of 1.6 X 10(-7) per base pair . These levels were compatible with cell survival, attesting to the sensitivity of the measurement system . Crosslinkage affected hybridization as well . One crosslink prevented all alkali-denatured DNA contiguous in both strands with it from hybridizing to complementary DNA either on solid supports or in solution . Strand-length effects on crosslinkage and on reassociation caused solution hybridization levels to exceed those predicted by simple theory . In a quantitative, dot-blotting assay hybridization was linear up to membrane saturation by denatured, uncrosslinked DNA of any strand length.(ABSTRACT TRUNCATED AT 400 WORDS) Mutat Res, 1988 Nov, 202(1), 223 - 34 Effects of SOS and MucAB functions on reactivation and mutagenesis of M13 replicative form DNA bearing bulky lesions; Bennett CB et al.; We have previously determined the specificity of -1 frameshifts induced by aflatoxin-B1-2,3-dichloride (AFB1C12) in phage M13 double-strand replicative form (RF) DNA . The system consists of: (i) in vitro adduction of RF DNA of BK8, a lacZ + 1 frameshift derivative of phage M13mp8; (ii) transfection into unirradiated or UV-irradiated bacterial host cells; (iii) scoring and sequencing of revertants (i.e., -1 frameshifts) . The previous data had shown that induction of SOS functions enhanced mutagenesis . However, this increase in mutagenesis is not accompanied by enhanced survival in a majority of the strains tested . Here, we present evidence to show that the lack of SOS reactivation is a specific property of the RF DNA system rather than a specific property of the lesion . A model mechanism based on the replicative strategy of transfected RF DNA can account for these observations . In addition, we have calculated individual Weigle mutagenesis factors at 8 major mutagen induced sites reported previously . Analysis of these data indicates that, within a restricted subset of possible mutational events (i.e., -1 frameshifts), Weigle mutagenesis is affected by both the DNA sequence environment of the mutation site as well as the repair phenotype of the cell. Mutat Res, 1988 Nov, 202(1), 193 - 201 Structural requirement for the induction of the adaptive response in Escherichia coli among N-(substituted alkyl)-N-nitrosoureas; Takahashi K et al.; Using E . coli CSH26 transformed with a plasmid carrying an alkA'-lacZ' fused gene, a series of N-(substituted alkyl)-N-nitrosoureas were subjected to a colorimetric assay to evaluate their capacity to induce the adaptive response, an inducible DNA-repair network in E . coli . Some of these derivatives induced the response in greater or lesser degrees, while others did not . Several structural requirements for the induction were disclosed . The capacity of these derivatives to induce the SOS response, which is another inducible DNA-repair network, was also evaluated using E . coli transformed with a plasmid carrying a umuC'-lacZ' fused gene . Since all the derivatives induced the SOS response, the structural requirements for the adaptive response disclosed in this study are substantially related to the molecular mechanism involved in the adaptive response. J Bacteriol, 1988 Nov, 170(11), 5263 - 71 Alteration of the carboxyl-terminal domain of Ada protein influences its inducibility, specificity, and strength as a transcriptional activator; Shevell DE et al.; The ada gene of Escherichia coli K-12 encodes the regulatory protein for the adaptive response to alkylating agents . A set of plasmids carrying ordered deletions from the 3' end of the ada gene were isolated and characterized . These ada deletions encode fusion proteins that derive their amino termini from ada and their carboxyl termini from the downstream vector sequence that occurs before an in-frame stop codon . Several of these ada deletions encode Ada derivatives that constitutively activate ada transcription to very high levels . A second class of ada deletions encode Ada derivatives that are dominant inhibitors of the inducible transcription of ada but are inducible activators of alkA transcription . In addition, we found that two Ada derivatives containing the same ada sequences but fused to different vector-derived tails have strikingly different properties . One Ada derivative constitutively activates both ada and alkA expression to very high levels . In contrast, the other Ada derivative is an inducible activator of ada expression, like the wild-type Ada protein, but is not an inducible activator of alkA transcription . Our data suggest that the carboxyl terminus of the Ada protein plays a key role in modulating the ability of the Ada protein to function as a transcriptional activator. J Bacteriol, 1988 Nov, 170(11), 5018 - 26 Characterization of a cyanobacterial iron stress-induced gene similar to psbC; Laudenbach DE et al.; Recently we have reported that the flavodoxin gene from the cyanobacterium Anacystis nidulans R2 is transcribed as part of an iron stress-induced operon containing multiple mRNA species (D . E . Laudenbach, M . E . Reith, and N . A . Straus, J . Bacteriol . 170: 258-265, 1988) . Here we report that nucleotide sequence analyses of DNA located immediately upstream of the flavodoxin gene revealed an open reading frame of 1,026 bases (designated isiA; iron stress inducible) with a deduced amino acid sequence showing similarity to that of the psbC polypeptide of higher plants and cyanobacteria . Assuming proteolytic cleavage of the initial methionine residue, the open reading frame encodes a 341-amino-acid polypeptide with a molecular mass of 36,824 daltons . Amino acid sequence comparisons with known psbC polypeptides from spinach and A . nidulans R2 showed extensive similarity, especially in the proposed membrane-spanning regions . Mung bean nuclease mapping and primer extension experiments have localized a transcriptional start site to a position 19 bases upstream from the first methionine codon of the isiA gene product . The upstream region contains an Escherichia coli-like -10 sequence but lacks the typical -35 consensus sequence . Approximately 15, 25, and 150 bases upstream from the isiA transcription start site are 17 base sequences which resemble the operator sequences of iron-regulated genes of E . coli. Biotechniques, 1988 Nov-Dec, 6(10), 929 - 32 Lid lysates: an economical and rapid method for plasmid analysis; Hoekstra MF; A method for analyzing bacteria containing recombinant plasmids is described . It allows inexpensive and rapid manipulation and screening of a large number of clones without the need for extensive laboratory equipment. Postgrad Med J, 1988 Nov, 64(757), 897 - 8 Spontaneous pneumoretroperitoneum in a renal transplant recipient; Gleeson MJ et al.; A case of a spontaneous pneumoretroperitoneum in a 16 year old renal transplant recipient is reported . This fatal complication of immunosuppression has not been reported previously. J Gen Microbiol, 1988 Nov, 134 ( Pt 11), 3071 - 7 Effects of Ca2+ and a protonophore on growth of an Escherichia coli L-form; Onoda T et al.; The influence of Ca2+ ions on the growth of an L-form (NC7) derived from Escherichia coli K12 was investigated . In a medium containing NaCl as osmotic stabilizer 1 mM-Ca2+ was required for optimal growth of the L-form, while with KCl as osmotic stabilizer, in a medium containing 0.1 or 1.0 mM-Ca2+, optimium growth was observed at 32 and 37 degrees C, respectively . When the L-form, growing exponentially at 32 degrees C in medium containing KCl and 0.1 mM-Ca2+, was shifted to 37 degrees C growth was strikingly suppressed . In contrast, the suppression of growth in the presence of 1.0 mM-Ca2+ at 32 degrees C was relieved when the culture was shifted to 37 degrees C . When the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), at a final concentration of 10 microM, was added to a medium containing NaCl and sucrose as osmotic stabilizers, together with 10 mM-glucose, the parent strain could grow exponentially . In contrast, growth of the L-form was completely stopped by 10 microM-CCCP under the same conditions . In the presence of 20 microM-CCCP, the L-form accumulated more than twice as much 45Ca as in the absence of the protonophore . Thus, it is suggested that growth of the L-form NC7 is coupled to the protonmotive force . Possible mechanisms for the coupling of calcium to growth of the L-form are discussed. J Gen Microbiol, 1988 Nov, 134 ( Pt 11), 2965 - 75 The presence of two complete homologous meta pathway operons on TOL plasmid pWW53; Osborne DJ et al.; pWW53 is a 110 kbp catabolic plasmid which encodes the complete pathway for the utilization of toluene and the xylenes . The upper pathway operon xylCAB is located between two homologous but distinct meta pathway operons, xylDLEGF(I,J,K)H, which are in direct repeat . These have each been cloned on large HindIII restriction fragments HA (17.5 kbp) and HB (15.6 kbp), the restriction sites of which have been mapped . During growth of MT53 on benzoate, mutants which have lost the ability to grow on hydrocarbons such as m-xylene (Mxy-) but which retain the ability to grow on their carboxylic acid metabolites such as m-toluate (Mtol+) take over the culture before ultimately being displaced by plasmid-free strains which are Mxy- Mtol- . The plasmids in the Mxy- Mtol+ mutants are formed by a large deletion between homologous regions of the two duplicate meta pathway operons . This causes the loss of the intervening xylCAB operon and the formation of a hybrid xylDLEGF(I, J, K)H operon, starting with the genes originally on HA and terminating with the genes originally on HB. Mol Biol (Mosk), 1988 Nov-Dec, 22(6), 1623 - 31 {A study of the complex-formation of phenylalanyl-tRNA-synthetase from Escherichia coli using the tRNA-Phe method of small-angle x-ray scattering}; Tuzikov FV et al.; The method of small-angle X-ray scattering was employed to analyse the equilibrium enzyme-substrate complexes in solution . A new approach of analysis of the experimental data was developed . This type of analysis provides the determination of dissociation constants and structural parameters of enzyme-substrate complexes . The radius of gyration (Rg) and dimensions of half-axis of the equivalent prolonged ellipsoid (a, b) of E . coliphenylalanyl-tRNA synthetase and its complexes with one or two tRNA(Phe) molecules have been determined . The values of these parameters speak in favour of structural rearrangements due to the interaction of the enzyme with tRNA(Phe) . The thermodynamic characteristics of phenylalanyl-tRNA synthetase complexes with tRNA(Phe) testify to the negative cooperativity in binding of two tRNA molecules with the enzyme. West J Med, 1988 Nov, 149(5), 601 - 2 IUD appendicitis during pregnancy; McLaughlin DI et al.; PIP: Appendicitis caused by a misplaced IUD was found in a 29-year-old pregnant woman . The woman had had the device inserted 8 years before . About 5 months after placement and a severe experience of right lower quadrant pain, medical examination revealed that she was pregnant . Abdominal and pelvic X-ray films were thought to be consistent with IUD expulsion, a fairly common occurrence, with an estimated rate of 2-20% within 1 year of placement . Over the next 7 years, the woman continued to experience right lower quadrant pain, but the pain was mild until 20 weeks into her next pregnancy when she was hospitalized with nausea, anorexia, fever, and severe pain . Surgery revealed that her appendix and cecum were bound to an inflamed mass of tissue . During the course of an appendectomy, this tissue mass was found to contain a copper-coated IUD, which was removed by blunt dissection and gentle traction . The IUD had probably partially perforated the uterus on insertion; complete perforation followed in 2-3 months; and copper from the device caused inflammation that eventually involved the appendix . Several months after the appendectomy, it was discovered that the inflammatory mass had been replaced by dense adhesions . This case shows that abdominal and pelvic X-ray examinations may not be sufficient to locate a misplaced IUD in a pregnant woman . If a misplaced device is not clearly visible on X-ray films, further workup may be necessary to avoid the possibility of chronic abdominal pain and complications . Biol Chem Hoppe Seyler, 1988 Nov, 369(11), 1219 - 26 Reactivity of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli with pyridoxal 5'-phosphate; Valentini G et al.; The allosteric fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli was modified with pyridoxal 5'-phosphate in the presence and in the absence of phosphoenolpyruvate, fructose 1,6-bisphosphate, MgADP and MgATP . In all cases a time-dependent inactivation was observed, but the rate and the extent of inactivation varied according to the conditions used . The kinetic properties of the partially inactivated enzyme were differently modified by addition of substrates and effectors to the modification mixture, the parameters mostly affected being those concerning fructose 1,6-bisphosphate . Tryptic peptides obtained from fully inactivated pyruvate kinase in the different conditions have been separated . In all conditions three main 6-pyridoxyllysine-containing peptides were present, the amounts of which showed significant differences in the presence of fructose 1,6-bisphosphate and MgADP . The function of the labelled peptides and the evidence supporting the physical existence of different conformational states are discussed . The main conclusion concerns the involvement of one of the above peptides in the binding of the allosteric effector fructose 1,6-bisphosphate. Plasmid, 1988 Nov, 20(3), 266 - 70 A runaway-replication plasmid pSY343 contains two ssi signals; Bahk JD et al.; Taking advantage of the plaque morphology method, we detected two single-stranded initiation (ssi) signals in the plasmid pSY343; one was in the 170-nucleotide (nt) EcoRV-ThaI segment (170P), and the other was in the 93-nt DraI-FnuDII segment (93F), which were designated as ssiA and ssiB, respectively . We cloned the two ssi signals in the filamentous phage vectors M13 delta lac184 and flR199 . A conserved 7-nt consensus sequence involved in the n' recognition site for priming DNA initiation on single-stranded (ss) DNA templates (A . Van der Ende, R . Teerstra, H . Van der Avoort, and P.J . Weisbeek, 1983, Nucleic Acids Res . 11, 4957-4975) was found, three copies in 170P and one in 93F . These two ssi signals contain possible stem and loop structures . The 170P overlapped partly with the origin (ori) region of pSY343 and the 93F was away from the ori region . Growth of chimera phages such as M13 delta lac184/ssiA and M13 delta lac184/ssiB was 38- and 71-fold greater, respectively, than that of M13 delta lac184, 8 h after phage infection . The conversion efficiency in vivo of ss to replicative form (RF) DNA of these chimera phages carrying ssiA and ssiB was 1.9- and 2.2-fold greater, respectively, than that of M13 delta lac184, 50 min after infection. Plasmid, 1988 Nov, 20(3), 259 - 65 Location of the relaxation complex nick site within the minimal origin of transfer region of RK2; Guiney DG et al.; Transfer of plasmid DNA during bacterial conjugation begins at a specific site: the origin of transfer (oriT) . The oriT region of the broad host range plasmid RK2 is located on a 250 bp fragment . Deletions involving either end of this region reduce transfer function, indicating that an extended sequence is required for optimal oriT activity . The single-strand nick induced by the RK2 DNA-protein relaxation complex is located adjacent to the 19 bp inverted repeat within the minimal oriT sequence . These results provide strong evidence that the plasmid relaxation event induced in vitro represents the nicking reaction that initiates DNA transfer at oriT during conjugation. Plasmid, 1988 Nov, 20(3), 207 - 20 Stability of pBR322-derived plasmids; Chiang CS et al.; The stability of pBR322-derived plasmids was studied during growth of their Escherichia coli host in the absence of antibiotics . Plasmid pBR322, as well as its delta rom and delta bla derivatives, were lost from their host within 60 generations, but a number of delta tet derivatives were quite stable under the same conditions . An evaluation of the data indicated that primary plasmid loss due to random partitioning corresponds to the generation of a plasmid-free cell about every 10(4) divisions (probability P0; = "intrinsic" instability) . Secondary loss of plasmid-carrying cells resulted from a growth advantage of the plasmid-free cells when bacteria die, perhaps due to unrepaired lethal damage in the DNA, under conditions of stationary incubation (= "apparent" instability) . This cell death also occurred in the absence of plasmids but was accelerated by the presence of extra plasmid DNA in the cell and further accelerated by a functional tet gene . This was the reason for the differential apparent stabilities of delta bla and delta tet plasmids . There was no indication that an accumulation of plasmid multimers contributed to the plasmid instability, as has been suggested in the literature . The value of P0 = 10(-4) is 14 orders of magnitude greater than expected under the assumption of a random (Poisson) distribution of plasmid copy numbers in a population of cells. J Pediatr Surg, 1988 Nov, 23(11), 1048 - 50 Nonsurgical management of neonatal adrenal abscess; Mondor C et al.; The fifteenth case of neonatal adrenal abscess is reported . It is the first case successfully diagnosed with needle aspiration and treated with percutaneous drainage. Biochemistry, 1988 Nov 1, 27(22), 8338 - 43 Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase; Chaidaroglou A et al.; The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis . Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene . The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties . Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold . When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold . For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type . Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants . Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme . This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity {Daemen, F.J.M., & Riordan, J.F . (1974) Biochemistry 13, 2865-2871} . The data reported here suggest that although Arg-166 is important for activity is not essential . The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme . First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished. Biochemistry, 1988 Nov 1, 27(22), 8307 - 10 Site-directed mutagenesis of Pro327 in the lac permease of Escherichia coli; Lolkema JS et al.; By use of oligonucleotide-directed, site-specific mutagenesis, Pro327 in the lac permease of Escherichia coli has been replaced with Ala, Gly, or Leu . Permease with Ala at position 327 catalyzes lactose/H+ symport in a manner indistinguishable from wild-type permease . Permease with Gly at position 327, on the other hand, exhibits about one-tenth the activity of wild-type permease but catalyzes lactose accumulation to essentially the same steady-state level as wild-type permease . Finally, permease with Leu at position 327 is completely inactive . The results demonstrate that there is no relationship between permease activity and the helix-breaking (Pro and Gly) or helix-making (Ala and Leu) properties of the residue at position 327 . It is suggested that it is primarily a chemical property of the side chain at position 327 (i.e., bulk, hydropathy, and/or ability to hydrogen bond) that is critical for activity and that neither cis/trans isomerization of Pro327 nor the presence of a kink at this position is important. Bioorg Khim, 1988 Nov, 14(11), 1530 - 7 {Expression in Escherichia coli of DNA coding for human tumor necrosis factor}; Dobrynin VN et al.; The variants of expression in Escherichia coli of artificial DNA coding for human tumor necrosis factor, an important immune modulator with selective cytotoxic action on a number of transformed cell lines have been described . The DNA was placed under control of either phage M13 promoter of gene for main coat protein or tandem of pair of E . coli tryptophane promoters . It has been shown that E . coli cells harbouring plasmids described with artificial TNF gene provide good level of protein biosynthesis . The protein has been purified by anion exchange chromatography near to homogeneity and used for preparation of monoclonals . As result three hybridomas effectively produced high affinity monoclonal anti-TNF antibodies have been obtained and characterized. Microb Pathog, 1988 Nov, 5(5), 371 - 9 Two different mechanisms of serum resistance in Escherichia coli; Kubens BS et al.; Fifty-three serum-resistant strains of E . coli which were all able to grow in at least 50% normal human serum (NHS) were tested in respect to binding and consumption of C3b, factor H, C5, and C6 after incubation in pooled NHS . The results of immunofluorescence tests, hemolytic assays, and binding studies using radiolabeled components were comparable . The different binding patterns allowed us to divide the strains into three different groups . The main features of group I were the attachment of C3, C5, and C6 to the bacterial cells as well as consumption of C3 and C5, whereas factor H did not bind at all or only in small amounts . In addition, released MAC was detectable in the supernatant of reaction mixtures containing bacteria of a group I strain and NHS . In group II factor H was easily bound to the bacteria, but no C3, C5, and C6 binding or C5 consumption was detectable . In addition, strains of group III bound C3 and factor H and some strains also bound and consumed C5 . Because of the inhomogeneity of group III, this investigation was restricted to a comparison of groups I and II . From the results presented in this study we conclude that group I bacteria activate the whole complement cascade, whereas with bacteria of group II, complement activation is interrupted at the C3 level . These findings therefore indicate a second, alternative mechanism of serum resistance in E . coli. Microb Pathog, 1988 Nov, 5(5), 333 - 43 Hyperproduction of heat-stable enterotoxin (STA4) of Escherichia coli and analysis of the unusual electrophoretic behavior of reduced and alkylated forms of STAs; Rasheed JK et al.; The methanol-soluble heat-stable enterotoxin gene (estA4) of Escherichia coli (STA4) yielded 128-fold more toxin when expressed by a T7 RNA polymerase driven system than when driven by its own promoter . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vivo {35S}cysteine radiolabeled products of the cloned gene revealed an apparent molecular mass larger than that expected for a 19 amino acid polypeptide (mol . wt . 2049) . Purified {125I}radiolabeled enterotoxin, STA1 (mol . wt . 1979) showed an Mr of 3800 when reduced, 2000 when reduced and carboxylated, and 14,500 when reduced and carboxyamidated . Similar changes after carboxyamidation were obtained with two different chemically synthesized STAs . These unusual electrophoretic mobilities were shown to be common to all STAs studied . Alkylation of the reduced STA species occurred only at the six cysteine residues of the toxin . Upon gel filtration the native, reduced, and reduced and alkylated forms of STAs eluted from the column in close agreement to the molecular weight expected from the known amino acid composition of the peptides. J Biochem (Tokyo), 1988 Nov, 104(5), 777 - 84 Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties; Inoue K et al.; ilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor . As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E . coli W3110 . The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity . Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene . The enzyme existed in a hexamer with a subunit molecular weight of 34,000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C . et al . (1979) J . bacteriol . 139, 339-345) was supported by electron micrographs . The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm . The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm . The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm . There was no pH-dependent spectral shift . The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm . These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E . coli aspartate aminotransferase . Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate . A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme. Diabetes Care, 1988 Nov-Dec, 11 Suppl 1, 73 - 9 Role of advanced glycosylation products in complications of diabetes; Cerami A et al.; Glucose and other reducing sugars can react with proteins and nucleic acids, without the aid of enzymes, to form stable covalent adduct . These reactions, although studied by food chemists, have recently been found to occur in vivo . This has led to studies on the accumulation of these advanced glycosylation end products (AGE) and the role it plays in the aging of long-lived proteins and nucleic acids . In contrast to the Amadori product, which is in equilibrium with glucose, AGE is irreversibly attached to the proteins . The AGE moieties are brown, fluorescent chromophores that can cross-link proteins . We have identified and characterized two specific AGE glucose-derived cross-links in proteins 2-furoyl-4(5)-(2-furanyl)-1H-imidazole (FFI) and 1-alkyl-2-formyl-3,4-diglycosylpyrrole (AFGP) . By use of a radioimmunoassay for FFI identification, it has been possible to demonstrate the presence of FFI in situ in proteins that had been exposed to glucose in vitro and in vivo . Recently, we found that reducing sugars react with amino groups on DNA nucleotides in a manner analogous to the nonenzymatic glycosylation of amino groups on proteins . The AGE-DNA formed in this manner has spectral and fluorescent properties similar to those of AGE-proteins . We have observed that formation of AGE on DNA decreases the ability of the single-stranded virus f1 to transfect Escherichia coli . When the plasmid pBR322 containing ampicillin- and tetracycline-resistant genes is incubated with reducing sugars, specific mutations are observed . These mutations have been found to be caused by insertions and deletions of the DNA . Further studies are needed for measuring the amounts of AGE-DNA and proteins linked to DNA by AGE . Potential mechanisms for repair of AGE-DNA also needs to be explored further.(ABSTRACT TRUNCATED AT 250 WORDS) Genetics, 1988 Nov, 120(3), 657 - 65 Evidence for frameshift mutations in the hisH gene of Escherichia coli causing synthesis of a partially active glutamine amidotransferase; Pons FW et al.; Among eight strains carrying acridine-induced mutations in hisH, five which mapped at four different sites in the promoter-distal region of the gene showed His+ phenotypes on media containing a purine . By complementation analysis, hisH enzyme was shown to be required for growth on purines . Purine-sensitive His+ revertants of strains able to grow on purines carried second-site mutations which in one case could be shown to map in hisG . Strains able to grow on purines were able to grow on 2-thiazolyl-DL-alanine, too . We conclude that frameshift mutations in the promoter-distal part of the hisH gene of E . coli do not completely abolish the activity of the gene product. Genetics, 1988 Nov, 120(3), 651 - 5 Second-site revertants of Escherichia coli trp repressor mutants; Klig LS et al.; Second-site reversion studies were performed with five missense mutants with defects in the trp repressor of Escherichia coli . These mutants were altered throughout the gene . The same unidirectional mutagen used in the isolation of these mutants, hydroxylamine, was used in reversion studies, to increase the likelihood that the revertants obtained would have second-site changes . Most of the second-site revertants were found to have the same amino acid substitutions detected previously as superrepressor changes . These second-site revertant repressors were more active in vivo than their parental mutant repressors, in the presence or absence of exogenous tryptophan . Apparently superrepressor changes at many locations in this protein can act globally to increase the activity of mutant repressors. Curr Genet, 1988 Nov, 14(5), 519 - 28 Nucleotide sequences of cDNAs encoding four complete nuclear-encoded plastid ribosomal proteins; Gantt JS; The nucleotide sequences of four pea nuclear-encoded plastid ribosomal protein cDNAs have been determined . These cDNAs were shown to encode the complete precursor proteins . The transit sequences of the encoded proteins are similar to the transit sequences of other imported proteins being rich in serine and/or threonine and lacking aspartic and glutamic acid . The transit sequences do not, however, have any apparent amino acid sequence similarity with one another or with the transit sequences of other imported proteins . The derived amino acid sequences of the plastid ribosomal proteins were compared to the amino acid sequences of other ribosomal proteins . Significant amino acid sequence similarity was found between Escherichia coli ribosomal proteins L9 and L24 and two of the nuclear-encoded pea plastid ribosomal proteins. Curr Genet, 1988 Nov, 14(5), 425 - 9 Regulatory region of the Aspergillus nidulans argB gene; Goc A et al.; We have constructed a series of deletion plasmids which contain the Aspergillus nidulans argB gene for ornithine carbamoyltransferase (OTC) . These deletions comprise the 5' upstream sequence of the argB gene . The pro- arg- strain of A . nidulans was transformed with the above plasmids . Several arg+ transformants of integration types I and II, obtained using each of the deletion plasmids, were studied, and their ability to de-repress OTC level by proline starvation was compared . It was concluded that nucleotides located between -150 and -50 bp upstream of the argB gene are significant for its cross-pathway regulation . This regulatory region contains three copies of the TGACTC hexanucleotide which is a cis-acting regulatory sequence of general amino acid control in yeast. Anticancer Res, 1988 Nov-Dec, 8(6), 1307 - 11 Increased protective activity against acid precipitation of poly(U) in the serum of tumor-bearing mice; Kouretas D et al.; Transplantation of leukemia L1210 cells into DBA/2 mice and of Ehrlich ascites tumor cells into BALB/C mice resulted in a significant increase of protective activity against acid precipitation of poly (U) in the serum . The increase was observed as early as one day after the tumor transplantation and seems to be connected with cancer growth, since inoculation of L1210 cells into BALB/C mice did not affect the protective activity, evidently as a result of their well established inability to cause cancer in this strain . Furthermore, no increase of activity was observed when bacteria were inoculated into mice, or when the latter were partially hepatectomized . The results suggest that the protective activity against acid precipitation of poly (U) could prove to be a tumor marker for the early detection of cancer growth. Parasite Immunol, 1988 Nov, 10(6), 607 - 17 Immunization against Plasmodium falciparum with recombinant polypeptides produced in Escherichia coli; Holder AA et al.; Two proteins produced in recombinant Escherichia coli and containing amino acid sequences from the Plasmodium falciparum precursor to major merozoite surface antigens (PMMSA) have been partially purified . These proteins, together with a preparation of merozoites, have been used to immunize animals . The antibody response and the degree of protection were compared . Animals immunized with merozoites produced antibodies reacting with many P . falciparum proteins, whereas a response specific for PMMSA was detected in those receiving the recombinant material . Incomplete protection was conferred to both groups and there was no apparent correlation between antibody levels and protection. Mol Gen Genet, 1988 Nov, 214(3), 588 - 91 Chloroplast ribosomal protein L-18 in Chlamydomonas reinhardtii is processed during ribosome assembly; Liu XQ et al.; Chloroplast ribosomal protein L-18 is made in the cytoplasm as a precursor, imported into the chloroplast, and processed to the mature form in two steps . We report here that the intermediate produced following the first processing step associates specifically with a ribosomal complex migrating with the chloroplast ribosome large subunit peak in sucrose gradients, and is then processed into mature L-18 . This processing event is slowed down in mutant cells deficient in synthesis of non-ribosomal proteins in the chloroplast . Thus the second processing step of L-18 occurs during ribosome assembly, depends on one or more nonribosomal proteins made in the chloroplast, and may be required for the maturation of the 50 S ribosome subunit . The mature L-18 protein shows extensive sequence homology at its amino-terminus to Escherichia coli ribosomal protein L27, which is located at the interface between 30 S and 50 S subunits and is involved in the formation of the peptidyl-tRNA binding site. Mol Gen Genet, 1988 Nov, 214(3), 574 - 8 Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli; Claverys JP et al.; The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites . This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation . In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut+) and deficient (mutHLS-) strains . In mut+ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold . Removal of the GATC site distal to the cat- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site . The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation . In the absence of a GATC site, mutL- and mutS- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH- and mut+ strains . This indicates that 50%-70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands . Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair. Mol Gen Genet, 1988 Nov, 214(3), 553 - 61 The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes; Ludwig A et al.; The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA . We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats . The resulting mutant proteins were perfectly stable in E . coli and were secreted with the same efficiency as the wild-type HlyA . HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca2+ for activity, as compared to the wild-type haemolysin . Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca2+ concentrations . The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca2+ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca2+ . These data indicate that the repeat domain of haemolysin is responsible for Ca2+-dependent binding of haemolysin to the erythrocyte membrane . A model for the possible functional role of Ca2+ in haemolysis is presented. Mol Gen Genet, 1988 Nov, 214(3), 460 - 6 Mutation spectra of N-ethyl-N'-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli; Richardson KK et al.; DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target . We observed that 22/30 of the ENNG-induced mutations were GC----AT transitions, 4/30 were AT----GC transitions, 3/30 were AT----TA transversions, and 1/30 was an AT----CG transversion . We observed that 37/40 HENU-induced mutations were GC----AT transitions and that the remaining 3/40 were AT----GC transitions . A majority of the GC----AT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5'-GG(A or T)-3'; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . A DNA strand preference for the G----A changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG . The AT----GC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5'-NTC-3' . A strand preference was not apparent for these mutations. Mol Gen Genet, 1988 Nov, 214(3), 451 - 5 Replication of ColE2 and ColE3 plasmids: two ColE2 incompatibility functions; Tajima Y et al.; We have identified and localized two incompatibility determinants (IncA and IncB) within a 1.3 kb segment of ColE2 sufficient for autonomous replication . The IncA determinant is localized in a region shorter than 250 bp and expresses incompatibility against both ColE2 and ColE3 . The region which determines sensitivity to the IncA determinant seems to overlap with the region specifying the IncA determinant . The expression of the trans-acting factor(s) specifically required for replication of ColE2 interferes with expression of the IncA determinant against ColE2 but not against ColE3 . The IncA determinant might be at least partly responsible for the copy number control of the plasmid . The IncB determinant is localized in a 50 bp region (origin) which is sufficient for initiation of replication in the presence of the trans-acting factor(s) . The IncB determinant is specific for ColE2 and seems to be due to titration of the trans-acting essential replication factor(s) by binding. Mol Gen Genet, 1988 Nov, 214(3), 389 - 95 PsiB polypeptide prevents activation of RecA protein in Escherichia coli; Bailone A et al.; We further characterize a novel plasmid function preventing SOS induction called Psi (Plasmid SOS Inhibition) . We show that Psi function is expressed by psiB, a gene located at coordinate 54.9 of plasmid R6-5 and near oriT, the origin of conjugal transfer . Deletions and amber mutations of the psiB gene permitted us to demonstrate that PsiB polypeptide (apparent molecular weight, 12 kDa) is responsible for Psi function . PsiB protein prevents recA730-promoted mutagenesis and intra-chromosomal recombination but not recombination following conjugation . Overproduction of PsiB protein sensitizes the host cell to UV irradiation . We propose that PsiB polypeptide has an anti-SOS action by inhibiting activation of RecA protein, thus preventing the occurrence of LexA-controlled functions. Mol Microbiol, 1988 Nov, 2(6), 807 - 11 Functional domains of colicin A; Baty D et al.; A large number of mutations which introduce deletions in colicin A have been constructed . The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed . The receptor-binding properties of each protein were also analysed . From these results, we suggest that the NH2-terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane . The central region of colicin A (residues 173 to 336) contains the receptor-binding domain . The COOH-terminal domain (residues 389 to 592) carries the pore-forming activity. Mol Microbiol, 1988 Nov, 2(6), 785 - 95 Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli; Bilous PT et al.; The nucleotide sequence of a 6.5 kilobasepair chromosomal DNA fragment encoding the anaerobic dimethylsulphoxide (DMSO) reductase operon of Escherichia coli has been determined . The DMSO reductase structural operon was shown to consist of three open reading frames, namely dmsABC, encoding polypeptides with predicted molecular weights of 87,350, 23,070, and 30,789 Daltons, respectively . The DMS A polypeptide displayed a high degree of amino acid sequence homology with the single-subunit enzyme, biotin sulphoxide reductase (bisC) and with formate dehydrogenase (fdhF), suggesting that the active site and molybdopterin cofactor binding site that is common to these enzymes is located in the DMS A subunit . A comparison of the predicted N-terminal amino acids of the dmsA gene product to those of the 82,600 subunit of purified DMSO reductase indicated that post-translational processing of a 16 amino acid peptide at the amino terminus of DMS A had occurred . The DMS B polypeptide contains 16 cysteine residues organized in four clusters, two of which are typical of 4Fe-4S binding domains . The DMS C polypeptide is composed of eight segments of hydrophobic amino acids of appropriate length to cross the cytoplasmic membrane, suggesting that this subunit functions to anchor the enzyme to the membrane. Mol Microbiol, 1988 Nov, 2(6), 777 - 83 Phylogeny of metabolic pathways: O-acetylserine sulphydrylase A is homologous to the tryptophan synthase beta subunit; Levy S et al.; The cysK gene of Escherichia coli K-12 encoding O-acetylserine sulphydrylase A, was cloned and its nucleotide sequence, together with that of the flanking regions, was determined . The deduced amino acid sequence of the carboxy-terminal moiety of O-acetylserine sulphydrylase A shows significant similarity to the amino acid sequence of tryptophan synthase beta chain from several organisms . This sequence similarity is likely to reflect the structural homologies of substrates shared by both enzymes . This may indicate that these proteins, although catalysing different reactions in different metabolic pathways, have evolved from a common ancestral gene. Mol Microbiol, 1988 Nov, 2(6), 767 - 75 Nucleotide sequence of the ugp genes of Escherichia coli K-12: homology to the maltose system; Overduin P et al.; The nucleotide sequence of the ugp genes of Escherichia coli K-12, which encode a phosphate-limitation inducible uptake system for sn-glycerol-3-phosphate and glycerophosphoryl diesters, was determined . The genetic organization of the operon differed from previously published results . A single promoter, containing a putative pho box, was detected by S1-nuclease mapping . The promoter is followed by four open reading frames, designated ugpB, A, E and C, which encode a periplasmic binding protein, two hydrophobic membrane proteins and a protein that is likely to couple energy to the transport system, respectively . The sequences of the proteins contain the characteristics of several other binding protein-dependent transport systems, but they seem to be particularly closely related to the maltose system. Mol Microbiol, 1988 Nov, 2(6), 719 - 26 Suppression of IIIGlc-defects by enzymes IINag and IIBgl of the PEP:carbohydrate phosphotransferase system; Vogler AP et al.; The Enzymes II of the PEP:carbohydrate phosphotransferase system (PTS) specific for N-acetylglucosamine (IINag) and beta-glucosides (IIBgl) contain C-terminal domains that show homology with Enzyme IIIGlc of the PTS . We investigated whether one or both of the Enzymes II could substitute functionally for IIIGlc . The following results were obtained: (i) Enzyme IINag, synthesized from either a chromosomal or a plasmid-encoded nagE+ gene could replace IIIGlc in glucose, methyl alpha-glucoside and sucrose transport via the corresponding Enzymes II . An Enzyme IINag with a large deletion in the N-terminal domain but with an intact C-terminal domain could also replace IIIGlc in IIGlc-dependent glucose transport . (ii) After decryptification of the Escherichia coli bgl operon, Enzyme IIBgl could substitute for IIIGlc . (iii) Phospho-HPr-dependent phosphorylation of methyl alpha-glucoside via IINag/IIGlc is inhibited by antiserum against IIIGlc as is N-acetylglucosamine phosphorylation via IINag . (iv) In strains that contained the plasmid which coded for IINag, a protein band with a molecular weight of 62,000 D could be detected with antiserum against IIIGlc . We conclude from these results that the IIIGlc-like domain of Enzyme IINag and IIBgl can replace IIIGlc in IIIGlc-dependent carbohydrate transport and phosphorylation. EMBO J, 1988 Nov, 7(11), 3595 - 9 The assembly of the major outer membrane protein OmpF of Escherichia coli depends on lipid synthesis; Bolla JM et al.; Cerulenin, a drug which specifically blocks lipid synthesis, prevented both the trimerization of OmpF monomers and their assembly into the outer membrane of Escherichia coli B cells . A monoclonal antibody directed against a surface-exposed epitope of the trimer was used to probe the assembly of OmpF in the presence or absence of the drug . An inhibition level of 80% was reached 16 min after the addition of cerulenin . The accumulated monomeric form could not be assembled even after lipid synthesis was restored . Instead, it was slowly degraded . It was further shown that the inhibition of assembly resulted in a rapid inhibition of OmpF synthesis . These data demonstrate that there is a direct relationship between the synthesis of lipid (most likely lipopolysaccharide) and the correct export of OmpF . This coupling is required to promote the trimerization of the porin monomer and its assembly into the outer membrane. EMBO J, 1988 Nov, 7(11), 3589 - 94 The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli; Beauclerk AA et al.; Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of 'domain IV' of 23S rRNA . In particular, oligonucleotides derived from the sequence 1757-1935 were isolated and shown to rebind specifically to protein L2 in vitro . Other L2-protected oligonucleotides, also derived from domain IV (i.e . from residues 1955-2010) did not rebind to protein L2 in vitro nor did others derived from domain I . Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme. EMBO J, 1988 Nov, 7(11), 3577 - 87 The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA; Vester B et al.; The peptidyl transfer site has been localized at the centre of domain V of 23S-like ribosomal RNA (rRNA) primarily on the basis of a chloramphenicol binding site . The implicated region constitutes an unstructured circle in the current secondary structural model which contains several universally conserved nucleotides . With a view to investigate the function of this RNA region further, four of these conserved nucleotides, including one indirectly implicated in chloramphenicol binding, were selected for mutation in Escherichia coli 23S rRNA using oligonucleotide primers . Mutant RNAs were expressed in vivo on a plasmid-encoded rRNA (rrnB) operon and each one yielded dramatically altered phenotypes . Cells exhibiting A2060----C or A2450----C transversions were inviable and it was shown by inserting the mutated genes after a temperature-inducible promoter that the mutant RNAs were directly responsible . In addition, a G2502----A transition caused a decreased growth rate, probably due to a partial selection against mutant ribosome incorporation into polysomes, while an A2503----C transversion produced a decreased growth rate and conferred resistance to chloramphenicol . All of the mutant RNAs were incorporated into 50S subunits, but while the two lethal mutant RNAs were strongly selected against in 70S ribosomes, the plasmid-encoded A2503----C RNA was preferred over the chromosome-encoded RNA, contrary to current regulatory theories . The results establish the critical structural and functional importance of highly conserved nucleotides in the chloramphenicol binding region . A mechanistic model is also presented to explain the disruptive effect of chloramphenicol (and other antibiotics) on peptide bond formation at the ribosomal subunit interface. EMBO J, 1988 Nov, 7(11), 3539 - 45 A novel function of RNase P from Escherichia coli: processing of a suppressor tRNA precursor; Nomura T et al.; The leuX gene of Escherichia coli codes for a suppressor tRNA and forms a single gene operon containing its own promoter and Q-independent terminator . An analysis of the in vitro processing of leuX precursor revealed that the processing of the 5' end took place in a single-step reaction catalysed by RNase P while the 3' processing involved two successive reactions . The endonucleolytic cleavage activity of the 3' precursor sequence was found to copurify with RNase P . Heat inactivation of thermosensitive RNase P from two independent E . coli mutants abolished the cleavage activity of both the 5' and 3' ends . These results altogether suggest that RNase P carries the activity of 3' end cleavage as well as that of 5' processing . In the presence of Mg2+ alone, the leuX precursor was found to be self-cleaved at a site approximately 13 nt inside from the 5' end of mature tRNA . The self-cleaved precursor tRNA was no longer processed by the 3' endonuclease, suggesting that the 3' endonuclease recognizes a specific conformation of the precursor tRNA for action. Br Heart J, 1988 Nov, 60(5), 452 - 4 Echocardiographic demonstration of Escherichia coli endocarditis restricted to the pulmonary valve; Murray NH et al.; A 25 year old man with no history of heart disease presented with sweats and rigors . Echocardiography showed a large vegetation on the pulmonary valve and blood cultures grew Escherichia coli . Because of recurrent pulmonary emboli a large vegetation on the anterior leaflet of the pulmonary valve was excised . He recovered after a full course of antibiotics. Am Rev Respir Dis, 1988 Nov, 138(5), 1300 - 7 Granulocyte depletion prevents tumor necrosis factor-mediated acute lung injury in guinea pigs; Stephens KE et al.; To examine the role of polymorphonuclear neutrophils (PMN) and other granulocytes in the pathogenesis of acute lung injury caused by tumor necrosis factor alpha (TNF), we compared the permeability edema and pulmonary histopathology in normal (granulocyte sufficient) guinea pigs and in granulocytopenic guinea pigs treated with TNF . Circulating granulocytes were depleted with cyclophosphamide . Two groups of normal animals were treated with either saline (PMN+/Control) or 1.4 x 10(6) U/kg recombinant human TNF (PMN+/TNF) . Three granulocytopenic groups were treated with either saline (PMN-/Control), TNF (PMN-/TNF), or intravenous infusion of 2 x 10(9) E . coli strain J96 (PMN-/Sepsis) . We measured the amount of 125I-labeled albumin in bronchoalveolar lavage (BAL) fluid and whole lung tissue and the wet/dry lung weight ratio to assess pulmonary transvascular protein flux and edema . We also quantified PMN in BAL fluid and fixed lung tissue . There were no statistically significant differences in any of these parameters between the PMN+/Control, PMN-/Control, or PMN-/TNF groups, except that the PMN+/Control predictably had more PMN/alveolus than the PMN- groups . However, both the PMN+/TNF and the PMN-/Sepsis groups had increased amounts of 125I-labeled albumin in BAL fluid and lung tissue (p less than 0.01) and increased wet/dry lung weight ratios (p less than 0.05), compared to all other groups . Histopathologically, capillary congestion and moderate inflammation were seen in the PMN+/TNF group, and acute inflammation and gross alveolar hemorrhage were seen in the PMN-/Sepsis group.(ABSTRACT TRUNCATED AT 250 WORDS) APMIS, 1988 Nov, 96(11), 991 - 6 Effects of endotoxin on the pancreatic ultrastructure; Florholmen J et al.; Intravenous administration of Escherichia coli endotoxin caused a state of shock with increased serum cationic trypsin-like immunoreactivity (CTLI) in a porcine model . The pancreatic acinar cells revealed focal changes, including intracellular oedema, appearance of membrane-bound vacuoles and breaks in the plasma membrane . In the micro circulatory vessels, there was swelling of endothelial cells . Similar changes have been observed in hemorrhagic and cardiogenic shock . This study demonstrates severe ultrastructural changes in the pancreas during E . coli endotoxin shock. Eur J Biochem, 1988 Nov 1, 177(2), 319 - 25 Phosphoenolpyruvate-dependent flavinylation of 6-hydroxy-D-nicotine oxidase; Nagursky H et al.; The reaction leading to the flavinylation of apo-6-hydroxy-D-nicotine oxidase was investigated in cell-free extracts of Eschericia coli carrying the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene on the expression plasmid pDB222 . It was demonstrated that the reaction required phosphoenolpyruvate (P-pyruvate) in addition to FAD . When {32P}P-pyruvate or {14C}P-pyruvate were used in the reaction with apo-6-HDNO, no phosphorylated or pyruvylated apo-protein could be detected, however . In order to drive the reaction to completion, FAD and P-pyruvate had to be present simultaneously in the reaction mixture . When apo-6-HDNO, highly purified by affinity chromatography, was used in the reaction with P-pyruvate and FAD, no additional protein fraction was required . A possible reaction scheme for the formation of holoenzyme from 6-HDNO is discussed. Am J Physiol, 1988 Nov, 255(5 Pt 1), E617 - 20 Involvement of sympathetic nervous system and brown fat in endotoxin-induced fever in rats; Jepson MM et al.; The object of this study was to assess the role of brown adipose tissue (BAT) and the sympathetic nervous system in the rise in heat production associated with endotoxin-induced fever . Oxygen consumption (VO2) was found to be significantly increased (28%) over a 4-h period after two doses of endotoxin (Escherichia coli lipopolysaccharide, 0.3 mg/100 g body wt) given 24 h apart . Injection of a mixed beta-adrenoceptor antagonist (propranolol) reduced VO2 by 14% in endotoxin-treated rats, whereas the selective beta 1- (atenolol) or beta 2- (ICI 118551) antagonists suppressed VO2 by 10% . These drugs did not affect VO2 in control animals . BAT thermogenic activity assessed from measurements of in vitro mitochondrial guanosine 5'-diphosphate (GDP) binding was elevated by 54% in interscapular BAT and by 171% in other BAT depots . Surgical denervation of one lobe of the interscapular depot prevented these responses . Endotoxin failed to stimulate GDP binding in rats fed protein-deficient diets . This may have been because BAT thermogenic activity was already elevated in control rats fed these diets or because endotoxin caused a marked suppression of food intake in the protein-deficient animals . The results indicate that sympathetic activation of BAT is involved in the thermogenic responses to endotoxin and that these can be modified by dietary manipulation. Am J Pathol, 1988 Nov, 133(2), 204 - 10 Tumor cell-endothelial interactions . Increased adhesion of human melanoma cells to activated vascular endothelium; Rice GE et al.; The authors examined the adhesion of seven human melanoma cell lines to cultured human umbilical vein endothelial cells (HEC) that were activated by cytokines or bacterial endotoxin . The adhesion of Hs 294T and MEL-24 cells was markedly increased (approximately 2 to 12-fold) after pretreatment of HEC monolayers for 6 hours with tumor necrosis factor, interleukin-1, or endotoxin . Smaller increases were noted with the cell lines RPMI 7951, HT 144, Malme-3M, MEL-2, and no significant increase was observed with MEL-5 . Cytokine and endotoxin effects on melanoma-HEC adhesion were concentration- and time-dependent, with onset by 2 hours, peak at 6-8 hours and maintenance through 48 hours . Cytokine induction of increased HEC adhesiveness for melanoma cells was blocked by actinomycin-D or cycloheximide, suggesting the requirement for RNA and protein synthesis . Interaction of melanoma cells with subendothelial matrix did not appear to play a primary role because: 1) phase contrast and electron microscopy revealed direct contact between tumor cells and endothelial cells in standardized monolayer adhesion assays; 2) increased adhesion (rosette formation) of tumor cells to activated HEC was also observed after nonenzymatic resuspension of HEC, and 3) the matrix peptide GRGDSP partially blocked (approximately 45%) Hs 294T cell adhesion to subendothelial matrix, but had little or no effect on adhesion to activated HEC monolayers . Taken together, these data suggest that inducible HEC surface changes may mediate the adhesion of certain melanoma cells, thereby exerting an active influence over the metastatic process. Proc Natl Acad Sci U S A, 1988 Nov, 85(22), 8678 - 82 Secretion of functional antibody and Fab fragment from yeast cells; Horwitz AH et al.; We have constructed yeast strains that secrete functional mouse-human chimeric antibody and its Fab fragment into the culture medium . For chimeric whole antibody, cDNA copies of the chimeric light-chain and heavy-chain genes of an anti-tumor antibody were inserted into vectors containing the yeast phosphoglycerate kinase promoter, invertase signal sequence, and phosphoglycerate kinase polyadenylylation signal . Simultaneous expression of these genes in yeast resulted in secretion of properly folded and assembled chimeric antibody that bound to target cancer cells . Yeast chimeric antibody exhibited antibody-dependent cellular cytotoxicity activity but not complement-dependent cytotoxicity activity . For production of Fab fragments, a truncated heavy-chain (Fd) gene was created by introducing a stop codon near the codon for the amino acid at which papain digestion occurs . Simultaneous expression of the resulting chimeric Fd and light-chain genes in yeast resulted in secretion of properly folded and assembled Fab fragment that bound to target cancer cells. Proc Natl Acad Sci U S A, 1988 Nov, 85(22), 8410 - 4 Repair of 4,5',8-trimethylpsoralen monoadducts and cross-links by the Escherichia coli UvrABC endonuclease; Jones BK et al.; Using an oligonucleotide model substrate, we observed two unusual mechanisms of UvrABC endonuclease in the repair of 4,5',8-trimethylpsoralen monoadducts and crosslinks . (i) UvrABC endonuclease usually incises a psoralen monoadduct only on the damaged strand . However, for one of the monoadducts we studied, incision on the complementary undamaged strand was also observed at a very low frequency, as though the adduct were on the thymine across from the damaged strand . Although the details of the erroneous incision are not yet known, such erroneous incision is potentially mutagenic . (ii) In cross-link repair, we observed that the UvrABC endonuclease incises the cross-linked DNA on either the furan side strand or the pyrone side strand . The incisions are not equally efficient . These data suggest that the structure of a psoralen cross-link, as seen by a repair enzyme, varies with the DNA sequence. Proc Natl Acad Sci U S A, 1988 Nov, 85(21), 8141 - 5 Frequency and spectrum of mutations produced by a single cis-syn thymine-thymine cyclobutane dimer in a single-stranded vector; Banerjee SK et al.; We have constructed a single-stranded vector that contains a uniquely located cis-syn T-T cyclobutane dimer by ligating a synthetic oligomer containing this UV photoproduct into M13mp7 viral DNA linearized with EcoRI . In the absence of SOS induction, transfection of a uvrA6 mutant of Escherichia coli with this vector gave very few progeny plaques, and the data imply that a single dimer blocks replication in at least 99.5% of the molecules . In vitro photoreactivation completely abolished this inhibition . Transfection of cells irradiated with UV at 4 J.m-2 to induce the SOS response gave 27% of the number of plaques found with a dimer-free control . Nucleotide sequence analysis of 529 progeny phage showed that translesion synthesis was usually accurate: the normal sequence was found in 93% of them . Where mutations occurred, all were targeted single-nucleotide substitutions, with approximately 90% being targeted at the 3' nucleotide of the lesion: of a total of 26 mutations, 15 were 3' T----A, 8 were 3' T----C, and 3 were 5' T----C . No T----G mutations were found . In addition to these results with the normal construct, data were also obtained from vectors in which the M13mp7 cloning site, which forms a hairpin in single-stranded DNA, was present 4 nucleotides on the 3' side of the T-T dimer . These hairpin-containing vectors gave a very similar mutation frequency (8% versus 7%) but altered mutation spectrum: all 12 mutations detected were 3' T----A transversions, a difference from the previous set of data that is significant (P = 0.03). Proc Natl Acad Sci U S A, 1988 Nov, 85(21), 8126 - 30 Mechanisms of mutagenesis in the Escherichia coli mutator mutD5: role of DNA mismatch repair; Schaaper RM; To investigate the mechanisms of spontaneous mutation in the Escherichia coli mutD5 mutator strain, 502 mutations generated in this strain in the N-terminal part of the lacI gene were sequenced (i-d mutations) . Since the mutator strength of this strain depends on the medium in which it grows, mutations were analyzed in both minimal medium (moderate mutator activity) and rich medium (high mutator activity) . In either case, 95% of all mutations were base substitutions and 5% were single-base deletions . However, the nature and site distribution of the base substitutions differed dramatically for the two conditions . In minimal medium (mutation frequency, 480-fold above background), a majority (62%) were transversions, notably A.T----T.A at three 5'-GTGG-3' sequences . Most (64%) of the transitions under this condition occurred at specific sequences that are suggestive of a "dislocation" type of mutagenesis . In rich medium (mutation frequency, 37,000-fold above background), 90% of the base substitutions were transitions . These observations suggest that different modes of mutagenesis operate under the two conditions . mutD5 cells have been reported to be defective in exonucleolytic proofreading during DNA replication . The present data suggest that mutD cells in rich medium also suffer a defect in mutHLS-encoded mismatch correction . This hypothesis was confirmed by the direct measurement of mismatch repair in mutD5 cells by transfection of M13mp2 heteroduplex DNA. Proc Natl Acad Sci U S A, 1988 Nov, 85(21), 8037 - 41 Growth inhibition of human breast carcinoma and leukemia/lymphoma cell lines by recombinant interferon-beta 2; Chen L et al.; Recombinant human interferon-beta 2 was produced in Escherichia coli by direct expression of cDNA encoding the mature protein sequence . At concentrations that stimulate DNA synthesis and growth in B-cell hybridomas and plasmacytomas, the cytokine was found to exert a strong inhibition on the growth of a number of carcinoma and leukemia/lymphoma cell lines . This antigrowth effect was observed in clonogenic assays and by measurements of cell number and thymidine incorporation in growing cultures . The effect was blocked by antibodies to a synthetic peptide from the N terminus of the molecule . Normal diploid fibroblasts were inhibited at concentrations higher than those needed for breast carcinoma cells. Proc Natl Acad Sci U S A, 1988 Nov, 85(21), 7907 - 11 Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase; Szewczyk B et al.; The virion-associated RNA polymerase and the structural nucleoprotein of influenza A virus were separated by sodium dodecyl sulfate/PAGE, electroblotted to a polyvinylidine membrane, and eluted with good recovery from the membrane . After renaturation by incubating with Escherichia coli thioredoxin, these proteins were active in a reconstituted in vitro transcription reaction with purified genomic RNAs . All four proteins (i.e., the three subunits of the RNA polymerase as well as the structural nucleoprotein) were required for activity . The RNA products were plus-strand, mRNA-sized species. Proc Natl Acad Sci U S A, 1988 Nov, 85(21), 7902 - 6 Holliday junctions in FLP recombination: resolution by step-arrest mutants of FLP protein; Jayaram M et al.; The FLP "recombinase" of the 2-micron circle yeast plasmid can resolve synthetic FLP site-Holliday junctions . Mutants of the FLP protein that are blocked in recombination but are normal in substrate cleavage can also mediate resolution . The products of resolution by these mutants are almost exclusively nicked molecules with a protein-bound 3' end . There is no significant asymmetry in strand cleavage (top versus bottom) by the mutants in linear or in circular FLP substrates; nor is there a bias in resolution (toward parentals or toward recombinants) of Holliday junctions (corresponding to top- or to bottom-strand exchange) by wild-type FLP . During normal FLP recombination, a small amount of the expected Holliday intermediate can be detected. Proc Natl Acad Sci U S A, 1988 Nov, 85(21), 7892 - 6 Messenger RNA structure and gene regulation at the translational level in Escherichia coli: the case of threonine:tRNAThr ligase; Moine H et al.; Previous work showed that the expression of the Escherichia coli threonine:tRNAThr ligase (EC 6.1.1.3)-encoding gene (thrS) is negatively autoregulated at the translational level and that a region called the operator that is located between 10 and 50 base pairs upstream of the translation initiation codon of the thrS gene is directly involved in that control . The conformation of an in vitro synthesized RNA fragment extending over the thrS regulatory region has been investigated using chemical and enzymatic probes . This study shows that the RNA folds into four well-defined secondary-structure domains, one of them displaying structural similarities to the anticodon arm of tRNAThr . The conformation of three constitutive mutants containing single base changes in the operator region leading to the loss of the regulatory control was also investigated . The replacement of a base in the anticodon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in the regulatory process . However, single mutations in or close to the anticodon-like stem result in a partial or complete reorganization of the structure of the operator region . These rearrangements should affect the binding of the ligase to the operator, leading to loss of the regulatory process. Mutat Res, 1988 Nov, 202(1), 35 - 43 Frameshift mutagenesis in Escherichia coli by reversible DNA intercalators: sequence specificity; Rene B et al.; The mutagenic potency of the simple reversible intercalators isopropyl-OPC (iPr-OPC) and 9-aminoacridine (9-AA) is assessed in E . coli using reversion assays based on plasmids derived from pBR322 carrying various frameshift mutations within the tetracycline resistance gene in repetitive sequences: +/- 2 frameshift mutations within alternating GC sequences; +/- 1 frameshift mutation at runs of guanines . The results obtained show that iPr-OPC and 9-AA have a sequence specificity for mutagenesis: they revert +1 and -1 frameshift mutations within runs of monotonous G:C base pairs . The precise determination of the size of a small restriction fragment which contains the mutation allowed us to demonstrate that reversion occurred by -1 deletions for the +1 frameshift mutations and by +1 additions for the -1 frameshift mutations . The possible relations of this specific reversion with the base sequence specificity of the mutagenesis are briefly discussed. Mutat Res, 1988 Nov, 194(3), 171 - 81 An analysis of the mutagenicity of 1,2-dibromoethane to Escherichia coli: influence of DNA repair activities and metabolic pathways; Foster PL et al.; The mutagenicity of 1,2-dibromoethane (EDB) to Escherichia coli was reduced by the UV light-induced excision repair system but unaffected by the loss of a major apurinic/apyrimidinic site repair function . At high doses, 70-90% of the EDB-induced mutations were independent of SOS-mutagenic processing and approximately 50% were independent of glutathione conjugation . The SOS-independent mutations induced by EDB were unaffected by the enzymes that repair alkylation-induced DNA lesions . EDB-induced base substitutions were dominated by GC to AT and AT to GC transitions . These results suggest that EDB-induced premutagenic lesions have some, but not all, of the characteristics of simple alkyl lesions. J Surg Res, 1988 Nov, 45(5), 472 - 80 Activation of phospholipases A1 and A2 in heart, liver, and blood during endotoxin shock; Liu MS et al.; The effects of endotoxin shock on the activities of phospholipases A1 and A2 were studied in canine heart sarcolemma, liver plasma membrane, and blood serum . Results obtained from control dogs indicate that phospholipases A1 and A2 were equally active in cardiac sarcolemma . In liver plasma membrane the activity of phospholipase A1 was twice that of phospholipase A2, while in blood serum phospholipase A1 activity was only half that of phospholipase A2 . Results obtained 4 hr after endotoxin administration (0.5 mg/kg, iv) demonstrate that phospholipase A1 activities were activated by 117% in cardiac sarcolemma, 188% in liver plasma membrane, and 150% in blood serum, while phospholipases A2 activities were stimulated by 71% in cardiac sarcolemma, 175% in liver plasma membrane, and 31% in blood serum . The in vitro incubation of all three enzyme preparations from control dogs with endotoxin (5-15 micrograms/0.5 ml) stimulated both phospholipase A1 and A2 activities and the stimulation was concentration dependent . These data suggest that endotoxin may have a direct stimulatory effect on phospholipases A1 and A2 activities in the cell membranes of heart and liver, and also in blood serum . Since phospholipases A play an important role in the regulation of membrane structure and function, the observed activation of phospholipases A1 and A2 may have a physiological significance in the pathogenesis of cellular dysfunction in endotoxin shock. J Bacteriol, 1988 Nov, 170(11), 5405 - 7 mutM, a second mutator locus in Escherichia coli that generates G.C----T.A transversions; Cabrera M et al.; We used strains carrying specific lacZ alleles to identify a new mutator locus in Escherichia coli which generates only G.C----T.A transversions among base substitutions . The locus, mutM, mapped near the cysE locus, which is at 81 min on the genetic map. J Bacteriol, 1988 Nov, 170(11), 5382 - 4 Localization of the kdsA gene with the aid of the physical map of the Escherichia coli chromosome; Woisetschlager M et al.; The isolation and analysis of two recombinant plasmids containing the kdsA gene from Escherichia coli chromosomal gene libraries is reported . The subfragments obtained from the inserts correspond to the fragment pattern around coordinate 1,282 kilobases of the physical map of the E . coli chromosome (Kohara et al . Cell 50:495-508, 1987) . The kdsA gene has been located at coordinates 1,282 through 1,283 kilobases, corresponding to min 26.7 in the classical map coordinates . The kdsA gene is transcribed from this position toward the nearby nar gene. J Bacteriol, 1988 Nov, 170(11), 5378 - 81 Conditionally lethal and recessive UGA-suppressor mutations in the prfB gene encoding peptide chain release factor 2 of Escherichia coli; Kawakami K et al.; Strains carrying mutations in the prfB gene encoding peptide chain release factor 2 of Escherichia coli were isolated . prfB1, prfB2, and prfB3 were selected as suppressor mutations of a lacZ (UGA) mutation at 37 degrees C, one of which, prfB2, is temperature sensitive in growth . A prfB286 strain was selected as a conditionally lethal mutant which grows at 32 but not at 43 degrees C and was shown to have UGA-suppressor activity . All the mutations are recessive UGA-suppressors . These data indicate that release factor 2 is essential to E . coli growth and that all mutants isolated here trigger suppression of the UGA codon. J Bacteriol, 1988 Nov, 170(11), 5371 - 4 An in vivo complex with DNA photolyase blocks UV mutagenesis targeted at a thymine-cytosine dimer in Escherichia coli; Ruiz-Rubio M et al.; UV mutation frequency responses for two types of Escherichia coli prototrophic mutant were measured . Only the response associated with a mutation targeted by a thymine-cytosine pyrimidine dimer was reduced in the dark in cells with amplified DNA photolyase . This specific reduction is attributed to the interruption of mutational DNA synthesis by a photolyase complex at the targeting dimer. J Bacteriol, 1988 Nov, 170(11), 5317 - 24 Molecular cloning, expression, and analysis of the genes of the homoprotocatechuate catabolic pathway of Escherichia coli C; Jenkins JR et al.; The molecular cloning and fine-structure analysis of the homoprotocatechuate (hpc) catabolic pathway genes of Escherichia coli C are described . The genes were located in two operons, hpcBCDEF and hpcGH, that were very closely linked . A regulatory gene, hpcR, involved in the expression of both operons was also identified . Various subclones isolated in the study were useful in the production of chemical intermediates of the pathway . The availability of one such compound facilitated the discovery of a previously unrecognized isomerase involved in the catabolic sequence. J Bacteriol, 1988 Nov, 170(11), 5272 - 8 Chromosomal genes essential for stable maintenance of the mini-F plasmid in Escherichia coli; Niki H et al.; We have isolated mutants of Escherichia coli which do not support stable maintenance of mini-F plasmids (delta ccd rep+ sop+) . These host mutations, named hop, were classified into five linkage groups on the E . coli chromosome . Genetic analyses of these hop mutations by Hfr mating and P1 transduction showed their loci on the E . coli genetic map to be as follows: hopA in the gyrB-tnaA region, hopB in the bglB-oriC region, hopD between 8 and 15 min, and hopE in the argA-thyA region . Kinetics of stability of the sop+ and delta sop mini-F plasmids in these hop mutants suggest that the hopA mutants are defective in partitioning of mini-F rather than in plasmid replication . The hopB, hopC, and hopD mutants were partially defective in replication of mini-F . The physical structure of the plasmid DNA was normal in hopA, B, C, and D mutants . Large amounts of linear multimers of plasmid DNA accumulated in mutants of the fifth linkage group (hopE) . None of the hop mutations in any linkage group affected the normal growth of cells. J Bacteriol, 1988 Nov, 170(11), 5257 - 62 Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli; Negishi K et al.; N4-Aminocytidine, a nucleoside analog, is strongly mutagenic to various organisms including Escherichia coli . Using E . coli WP2 (trp), we measured the incorporation of {5-3H}N4-aminocytidine into DNA and at the same time measured the frequency of reversion of the wild type, thereby attempting to correlate the incorporation with mutation induction . First, we observed that N4-aminocytidine uptake by the E . coli cells was as efficient as cytidine uptake . High-pressure liquid chromatographic analysis of nucleoside mixtures obtained by enzymatic digestion of isolated cellular DNA showed that the DNA contained {3H}N4-aminodeoxycytidine, corresponding to 0.01 to 0.07% of the total nucle