Escherichia coli

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 nucleoside; the content was dependent on the dose of N4-aminocytidine . There was a linear relationship between the N4-aminocytosine content in DNA and the mutation frequency observed . These results constitute strong evidence for the view that the N4-aminocytidine-induced mutation in E . coli is caused by the incorporation of this agent into DNA as N4-aminodeoxycytidine . We also found that the major portion of radioactivity in DNA of cells that had been treated with {5-3H}N4-aminocytidine was in the deoxycytidine fraction . We propose a metabolic pathway for N4-aminocytidine in cells of E . coli . This pathway involves the formation of both N4-aminodeoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate; the deoxycytidine 5'-triphosphate formation is initiated by conversion of N4-aminocytidine into uridine . In support of this proposed scheme, a cytidine deaminase preparation obtained from E . coli catalyzed the decomposition of N4-aminocytidine into uridine and hydrazine.

J Bacteriol, 1988 Nov, 170(11), 5229 - 35
Thermosensitive omsA mutation of Escherichia coli that causes thermoregulated release of periplasmic proteins; Tsuruoka T et al.; A mutant of Escherichia coli with a thermosensitive defect, possibly in the outer membrane (omsA mutant), was isolated from E . coli K-12 by mutagenization and selection for thermosensitivity and beta-lactam supersensitivity of growth . The mutant also showed very high sensitivity to other antibiotics, such as macarbomycin, midecamycin, rifampin, and bacitracin . The mutation was recessive to the wild type and was mapped at about 4 min on the E . coli chromosome between fhuA and metD . The mutation caused rapid release into the medium of periplasmic enzymes such as RTEM penicillinase but practically no cytoplasmic enzyme when cells grown at 30 degrees C were transferred to 37 or 42 degrees C . Electron microscopic observations showed many large double-layered vesicles attached to the surface of cells incubated at 42 degrees C . We conclude that the mutant had a mutation that caused a temperature-dependent defect in the outer membrane structure or its assembly (named an oms mutation) . The omsA mutant may be useful for production of periplasmic proteins, which it releases into the culture medium on shift up of temperature.

J Bacteriol, 1988 Nov, 170(11), 5197 - 9
Enhanced acetohydroxy acid synthase III activity in an ilvH mutant of Escherichia coli K-12; Ricca E et al.; The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI and ilvH gene products . A missense mutation in ilvH (ilvH612), which reduced the sensitivity of the enzyme to the end product inhibition by valine, also increased its specific activity and lowered the Km for alpha-acetolactate synthesis . The mutation increased the sensitivity of acetohydroxy acid synthase III to dialysis and heat treatment and reduced the requirement for thiamine pyrophosphate addition to the assay mixture for activity . A strain carrying the ilvH612 mutation grew better than a homologous ilvH+ strain in the presence of leucine . The data indicate that this is a consequence of a more active acetohydroxy acid synthase III isozyme rather than the result of an alteration of the leucine-mediated repression of the ilvIH operon.

J Bacteriol, 1988 Nov, 170(11), 5185 - 91
Proline carrier mutant of Escherichia coli K-12 with altered cation sensitivity of substrate-binding activity: cloning, biochemical characterization, and identification of the mutation; Ohsawa M et al.; Two putP mutants of Escherichia coli K-12 that were defective in proline transport but retained the binding activities of the major proline carrier were isolated (T . Mogi, H . Yamamoto, T . Nakao, I . Yamato, and Y . Anraku, Mol . Gen . Genet . 202:35-41, 1986) . One of these mutations and three null-type mutations (K . Motojima, I . Yamato, and Y . Anraku, J . Bacteriol . 136:5-9, 1978) were cloned into a pBR322 putP+ hybrid plasmid (pTMP5) by in vivo recombination . Cytoplasmic membrane vesicles were prepared from the mutant strains and strains harboring pTMP5 putP plasmids, and the properties of the proline-binding reaction of the mutant putP carriers in membranes were examined under nonenergized conditions . The putP19, putP21, and putP22 mutations, which were mapped in the same DNA segment of the putP gene (Mogi et al., Mol . Gen . Genet . 202:35-41, 1986), caused the complete loss of proline carrier activity . The proline carriers encoded by the mutant putP genes, putP9 and putP32, and putP32 in pTMP5-32, which was derived from in vivo recombination with the putP32 mutation, had altered sodium ion and proton dependence of binding affinities for proline and were resistant to N-ethylmaleimide inactivation without changes in the specificities for substrates and alkaline metal cations . The nucleotide sequence of the putP32 lesion located on the 0.35-megadalton RsaI-PvuII fragment in the putP gene in pTMP5-32 was determined; the mutation changed a cytosine at position 1001 to a thymine, causing the alteration of arginine to cysteine at amino acid position 257 in the primary structure of the proline carrier . It was shown that this one point mutation was enough to produce the phenotype of pTMP5-32 by in vitro DNA replacement of the AcyI-PvuII fragment of the wild-type putP gene with the DNA fragment containing the mutated nucleotide sequence.

J Bacteriol, 1988 Nov, 170(11), 5153 - 60
Divergence of the aerobactin iron uptake systems encoded by plasmids pColV-K30 in Escherichia coli K-12 and pSMN1 in Aerobacter aerogenes 62-1; Waters VL et al.; Although the aerobactin-mediated iron uptake system has been characterized genetically in Escherichia coli, the siderophore aerobactin was chemically characterized after purification from culture supernatants of Aerobacter aerogenes 62-1, a member of the Klebsielleae . We have cloned and mapped the genes encoding the aerobactin system genes of A . aerogenes 62-1 and begun characterization of the relevant proteins and enzymatic activities of this plasmid-mediated aerobactin system . Published chemical data indicate that the siderophore aerobactin of E . coli is the same molecule as the aerobactin of Aerobacter aerogenes 62-1, but we have found that both the genes and the complement of proteins making up the biosynthetic enzymes in the two systems have diverged . In contrast, the outer membrane receptors for ferric aerobactin of the two systems showed immunologic cross-reactivity, were of the same molecular size (74 kilodaltons), and were encoded by homologous DNA sequences.

J Bacteriol, 1988 Nov, 170(11), 5134 - 40
Purification and characterization of the wild-type and mutant carboxy-terminal domains of the Escherichia coli Tar chemoreceptor; Kaplan N et al.; The carboxy-terminal half of the Escherichia coli Tar chemoreceptor protein was cloned into an overproducing plasmid with the transcription of the insert under the control of the strong hybrid tac promoter . Two dominant mutations in the tar gene, which result in "tumble-only" (tar-526) or "swim-only" (tar-529) phenotypes and which are postulated to produce proteins locked in specific signalling modes, were introduced separately onto the overproducing plasmid . After induction with isopropyl-beta-D-thiogalactopyranoside, cells containing the plasmids produced about 10% of their soluble cellular protein as the carboxy-terminal fragments . A scheme to purify the overproduced fragments was developed . Typical yields of pure fragment were 5, 30, and 20 mg per liter of induced culture for the wild type, 526 mutant, and 529 mutant, respectively . Fast-protein liquid chromatography-gel filtration analysis of the pure fragments showed that they all existed as oligomers (ca . 103,000 daltons), possibly trimers or tetramers (monomer size is 31,000 daltons) . However, the 529 mutant fragment showed an additional oligomeric form (240,000 daltons) corresponding approximately to an octamer . When chromatographed in the presence of 1% octylglucoside, all three fragments showed an identical single oligomeric size of about 135,000 daltons . Further differences between the fragments such as ion-exchange behavior and susceptibility to degradation were found . Taken together, these results suggest that conformational differences between the 529 mutant fragment and the other fragments exist and that these differences may correlate with the phenotypic effects of the tar-529 mutation.

J Bacteriol, 1988 Nov, 170(11), 5076 - 9
Evidence in vivo for autogenous control of the cyclic AMP receptor protein gene (crp) in Escherichia coli by divergent RNA; Okamoto K et al.; Control of crp expression in vivo was studied by using the cloned crp gene . The synthesis of the product of this gene, cyclic AMP (cAMP) receptor protein (CRP), was strongly reduced by exogenous cAMP . This regulation was completely abolished by the inactivation of a divergent promoter located within the crp promoter region . These data are consistent with our in vitro studies (Okamoto and Freundlich, Proc.Natl . Acad . Sci . USA 83:5000-5004, 1986), which showed that crp autoregulation is due to the inhibition of crp transcription by divergent (antisense) RNA produced by cAMP-CRP activation of the divergent promoter.

J Bacteriol, 1988 Nov, 170(11), 5067 - 75
Degradation of a signal peptide by protease IV and oligopeptidase A; Novak P et al.; The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC) . The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids . In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction . Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV . Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide . Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions . Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14) . Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond . Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate . These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.

J Bacteriol, 1988 Nov, 170(11), 5042 - 50
Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli; Yamagishi M et al.; Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation . When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60% . This demonstrates that the synthesis of r-proteins is repressed under normal conditions . The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis . Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded . Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions . In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene . Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein . These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly.

J Virol, 1988 Nov, 62(11), 4393 - 7
Processing of in vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV) type 1 by HIV proteinase generated in Escherichia coli; Krausslich HG et al.; We expressed the gag and proteinase regions of human immunodeficiency virus (HIV) type 1 by transcription and translation in vitro . A synthetic RNA spanning the gag and pro domains gave primarily the unprocessed capsid precursor pr53 . Efficient cleavage of this precursor was observed when the gag and pro domains were placed in the same translational reading frame, yielding equimolar amounts of the gag protein and of proteinase (PR) . Expression of HIV type 1 PR in Escherichia coli as a fusion protein gave rapid autocatalytic processing to an HIV-specific protein of approximately 11 kilodaltons . HIV PR generated in E . coli specifically induced cleavage of the HIV capsid precursor, whereas deletion of the carboxy-terminal 17 amino acids of the proteinase rendered it inactive . Inhibitor studies showed that the enzyme was insensitive to inhibitors of serine and cysteine proteinases and metalloproteinases and was inhibited only by a very high concentration (1 mM) of pepstatin A.

Crit Care Med, 1988 Nov, 16(11), 1128 - 31
Low dose ibuprofen reverses the hemodynamic alterations of canine endotoxin shock; Balk RA et al.; The dose response of the nonsteroidal anti-inflammatory drug, ibuprofen, was evaluated in order to determine the most efficacious dose in the treatment of canine endotoxin shock . Fifteen minutes after an infusion of Escherichia coli endotoxin, four groups of dogs were given a single iv dose of 1, 5, 10, or 20 mg/kg of ibuprofen . These groups were compared to endotoxin only and saline control groups . All ibuprofen doses significantly improved the systolic, diastolic, and mean systemic arterial BP . The improvement in systemic BP was accompanied by an increase in the systemic vascular resistance . Pulmonary vascular pressures and resistance also increased after ibuprofen administration . The lack of a dose response and the demonstrated beneficial effect of low dose ibuprofen in the reversal of the hypotension associated with experimental canine endotoxin shock lead us to recommend the use of low dose ibuprofen for future endotoxin and sepsis studies . Use of low dose ibuprofen might have less of an effect on renal perfusion and would therefore be more likely to produce the beneficial hemodynamic response without compromising renal function.

Proc Natl Acad Sci U S A, 1988 Nov, 85(21), 7882 - 6
Cleavage of the cII protein of phage lambda by purified HflA protease: control of the switch between lysis and lysogeny; Cheng HH et al.; The activity of the cII protein of phage lambda is probably the critical controlling factor in the choice of the lytic or lysogenic pathway by an infecting virus . Previous work has established that cII activity is regulated through the turnover of cII protein; the products of the hflA and hflB loci of Escherichia coli are needed for a degradative reaction, and lambda cIII functions in stabilizing cII . By using the cloned hflA locus, we have purified a cII-cleaving enzyme that we term HflA . Purified HflA contains three polypeptides; at least two of the subunits are products of the hflA region, and the third is probably a cleavage product of the larger of these two hflA-encoded polypeptides . The HflA protease activity cleaves cII to small fragments . We conclude that the switch between lambda developmental pathways involves regulated cleavage of cII by the specific protease HflA.

Am J Vet Res, 1988 Nov, 49(11), 1794 - 9
Characterization and purification of the F17 adhesin on the surface of bovine enteropathogenic and septicemic Escherichia coli; Lintermans PF et al.; The F17 antigen from bovine enterotoxigenic Escherichia coli strain (E coli 25KHO9), which adhered to calf intestinal villi, was isolated . An enterotoxin-negative derivative (25KHO9st) was used for further studies . Using an immunogold-labeling technique, the F17 antigen was characterized as a fimbrial protein . Pure fimbriae with a subunit molecular weight of 20,000 were obtained by homogenization and use of a sucrose gradient . The adhesion of E coli 25KHO9st was mediated by the F17 fimbriae, as both F17 antibodies and F17 protein blocked the adhesion of the strain 25KHO9st . The F17 fimbriae were serologically distinct from K88, K99, F41, and 987P fimbriae and did not agglutinate bovine, ovine, guinea pig, human, or chicken erythrocytes . Peptide fingerprint analysis revealed F17 and F(Y) adhesins to be homologous, if not identical.

Res Vet Sci, 1988 Nov, 45(3), 418 - 9
Fimbria-like adhesive factor (EAF 44) from verocytotoxigenic Escherichia coli of bovine origin; Yano T et al.; An adhesin with fimbria-like properties was present in four strains of verocytotoxigenic Escherichia coli isolated from diarrhoeic calves in Brazil.

Mol Cell Biol, 1988 Nov, 8(11), 4598 - 607
DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context; Hoey T et al.; The homeo box gene even-skipped (eve) encodes a 376-amino-acid protein that binds with high affinity to sequences located near the 5' termini of the eve and en genes . The 5' en sites are A + T rich and contain copies of the 10-base-pair (bp) consensus sequence T-C-A-A-T-T-A-A-A-T . In contrast, the 5' eve sites are G + C rich and contain the 9-bp sequence T-C-A-G-C-A-C-C-G . Among the five different homeo box proteins that have been tested for binding, eve is unique in that it shows virtually equal preference for the A + T-rich 5' en binding sites and the G + C-rich 5' eve sites . Most of the other proteins bind with a relatively higher affinity to the en sites than to the eve sites . In an effort to identify the regions of the eve protein that are responsible for its efficient binding to both classes of recognition sequences, we analyzed the DNA-binding properties of various mutant eve proteins . These studies suggest that the homeo domain of the eve protein is responsible for both binding activities . However, mutations in distant regions of the protein influenced the binding behavior of the eve homeo domain and caused a reduction in binding to the G + C class of recognition sites . We propose that the protein context of the homeo domain can influence its DNA-binding properties.

EMBO J, 1988 Nov, 7(11), 3331 - 6
Receptors compete for adaptors found in plasma membrane coated pits; Pearse BM; An affinity matrix of LDL receptor cytoplasmic tails binds the HA-II 100/50/16 kd complexes found in plasma membrane coated pits . Other receptors (or their cytoplasmic domains), which are localized in coated pits during endocytosis, inhibit this binding . This includes an 8 residue peptide containing tyrosine, corresponding to the cytoplasmic portion of a mutant influenza haemagglutinin . In contrast, the equivalent peptide lacking tyrosine (like the tail of the native haemagglutinin, a protein excluded from coated pits) does not compete . These results imply that the HA-II complex has a recognition site for a common signal, probably involving a tyrosine residue, carried by the LDL receptor and competing receptors also found in plasma membrane coated pits . The HA-II complex therefore fulfils the role of an 'adaptor', the name proposed for the structural units which mediate the binding of clathrin to receptors in coated vesicles . Another related complex, the HA-I adaptor, which is restricted to Golgi coated pits, probably does not recognize the 'tyrosine signal' on the LDL receptor tail . The HA-I adaptor is likely to contain a recognition site for a different signal carried by receptors, e.g . the mannose-6-phosphate receptor, which are found in Golgi coated pits.

Am J Physiol, 1988 Nov, 255(5 Pt 1), E629 - 35
Adrenergic blockade prevents endotoxin-induced increases in glucose metabolism; Hargrove DM et al.; Combined alpha- and beta-adrenergic blockade was used to investigate the role of catecholamines in endotoxin-induced elevations in glucose kinetics . Glucose kinetics were measured before and for 4 h after the injection of endotoxin {100 micrograms/100 g body wt iv, 30% lethal dose (LD30) at 24 h} . Adrenergic blockade was achieved by the bolus injection of phentolamine and propranolol followed by their continuous infusion . Endotoxin-treated rats exhibited a transient hyperglycemia and sustained (greater than 4 h) increase in plasma lactate concentration, as well as elevated rates of glucose appearance (Ra, 83%), disappearance (Rd, 58%), recycling (160%), and metabolic clearance (23%) . Adrenergic blockade prevented endotoxin-induced increases in plasma glucose concentration, Ra, Rd, and recycling but not glucose clearance . The increase in plasma lactate concentration was blunted by 35% . After 2 h, endotoxic animals infused with adrenergic antagonists developed hypoglycemia, which may have resulted from an increased plasma insulin concentration . The attenuation of elevated glucose turnover by adrenergic blockade in the endotoxin-treated animals was not due to a reduction in plasma glucagon level or differences in plasma insulin concentration . Administration of the alpha- or beta-adrenergic antagonists separately blunted but did not prevent endotoxin-induced changes in glucose kinetics, and therefore the efficacy of the adrenergic blockade could not be assigned to a single receptor class . These results indicate that catecholamines are important contributory factors to many of the early alterations in carbohydrate metabolism observed during endotoxemia.

J Bacteriol, 1988 Nov, 170(11), 5059 - 66
Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis; Knapp S et al.; Expression of virulence factors by Bordetella pertussis is altered by environmental signals (antigenic modulation) and is dependent on an activator encoded by a gene called vir . We have used TnphoA (Tn5 IS50L::phoA) gene fusions to define two sets of genes whose expression is either activated (vag loci) or repressed (vrg loci) by modulation signals . Both groups of genes appear to be regulated by the vir gene product in that, in the absence of modulators, null mutations in vir lead to the repression of vag gene fusions and derepression of vrg gene fusions . Mutants of B . pertussis were isolated that constitutively express virulence factors in the presence of the modulator MgSO4, nicotinic acid, or low incubation temperature . We designate the gene that carries such mutations mod (modulation) and have characterized one (mod-1) of these mod constitutive mutations . A method was developed for the insertional inactivation of the vir gene by using the integration of a suicide replicon . Inactivation of the vir gene in the mod-1 mutant, followed by transcomplementation with the cloned wild-type vir gene, gives the Mod-1 constitutive phenotype, showing that the mod-1 mutation defines a gene distinct from vir . The gene carrying the mod-1 mutation is linked to vir and was cloned on a recombinant cosmid (pLAF-C1) which transcomplements the vir-1::Tn5 mutation in B . pertussis 347 . Introduction of pLAF-C1 into vir mutant and vir+ B . pertussis strains also gives the Mod-1 constitutive phenotype, indicating that mod-1 is a dominant allele . These data suggest that the mod gene product could have sensory functions for the environmental signals that affect the expression of vir-regulated genes of B . pertussis . The mod constitutive strains and plasmids described here also have applications in pertussis vaccine development.

Int J Radiat Biol, 1988 Nov, 54(5), 709 - 22
Characterization of damage in gamma-irradiated and OsO4-treated DNA using methoxyamine; Liuzzi M et al.; Unlabelled and radiolabelled methoxyamine have been used to characterize DNA damage caused by gamma-rays or by the chemical reagent osmium tetroxide (OsO4) . Both treatments introduce in DNA a number of methoxyamine-binding sites proportional to the dose . Whereas the number of these sites remains constant after the OsO4 treatment it increases during postirradiation incubation; the postirradiation appearance of methoxyamine-binding sites is enhanced by the presence of methoxyamine . OsO4 treatment and gamma-irradiation also induce the formation of alkali-labile sites in DNA . Whereas the number of these sites remains constant after OsO4 treatment, it increases during postirradiation incubation and an alkaline medium accelerates their formation . A fraction of the alkali-labile sites found in gamma-irradiated DNA is methoxyamine-labile; by contrast, the OsO4-treated DNA is stable in the presence of methoxyamine.

Infect Immun, 1988 Nov, 56(11), 2979 - 83
Isolation and characterization of the localized adherence factor of enteropathogenic Escherichia coli; Scaletsky IC et al.; The binding factor of enteropathogenic Escherichia coli O111:H- responsible for localized adherence (LA) on HeLa cells was investigated . Inhibition of LA by carbohydrates and lectins showed that the reactive epitope on HeLa cells contains N-acetylgalactosamine units . Treatment of bacteria with EDTA for extraction of lipopolysaccharides eliminated these polymers as binding factors . Such treatment also caused a marked increase in adhesion suggesting steric hindrance by lipopolysaccharides of the LA factor binding capacity . Immunoblotting with rabbit antibodies showed a strong reaction with two components with approximate molecular sizes of 29 and 32 kilodaltons (kDa) present in the outer membrane preparations of bacteria . Both the absorbed rabbit immune serum and the outer membrane preparation of the bacteria inhibited bacterial adhesion by 100% . Outer membrane components were isolated from an N-acetylgalactosamine-agarose column by elution with KSCN, labeled with 125I, and immunoprecipitated with absorbed rabbit hyperimmune antiserum . The only component precipitated was the protein doublet at 29 to 32 kDa corresponding to the components detected by immunoblotting . The predominant component was always the 32-kDa polypeptide . We conclude that this component of the outer membrane is the best candidate for the LA factor in enteropathogenic E . coli.

Infect Immun, 1988 Nov, 56(11), 2918 - 24
Cloning and characterization of a new type of fimbria (S/F1C-related fimbria) expressed by an Escherichia coli O75:K1:H7 blood culture isolate; Pawelzik M et al.; The Escherichia coli blood culture isolate BK658 (O75:K1:H7) expresses F1A and F1B fimbriae as well as a third fimbrial type which reacts with anti-S-fimbrial antiserum but fails to show S-specific binding properties (i.e., agglutination of bovine erythrocytes) . To characterize these fimbriae, we cloned the respective genetic determinant in E . coli K-12 . The resulting recombinant clone HB101(pMMP658-6) expresses fimbriae of 1.2-micron length and a diameter of approximately 7 nm . The determinant codes for the fimbrillin subunit, a protein of 17 kilodaltons in size, and for at least five other proteins of 87, 31, 23, 14.3, and 13.8 kilodaltons . By restriction analysis and by DNA-DNA hybridization, it could be shown that the cloned fimbrial determinant of strain BK658 exhibits a high degree of sequence homology to the gene clusters coding for S fimbrial adhesins (sfa) and F1C fimbriae (foc) . By using the Western blot (immunoblot) technique and a quantitative enzyme-linked immunosorbent assay, it could be further demonstrated that the cloned fimbriae of BK658, S fimbriae, and F1C fimbriae share cross-reactive epitopes as well as antigenic determinants specific for each fimbrial type . No antigenic cross-reactivity with F1C fimbriae could be detected . The results indicate a genetical and serological relatedness of the cloned fimbriae to S fimbriae and F1C fimbriae . Therefore, this new type of fimbriae is preliminarily termed S/F1C-related fimbriae (Sfr).

Mol Biol (Mosk), 1988 Nov-Dec, 22(6), 1451 - 5
{Rapid isolation of phage lambda DNA}; Zabarovskii ER et al.; A modified procedure in two versions (micro, for 10 ml of phage lysate, and macro, for 200-500 ml) is described for preparing lambda phage DNA . The main advantage of the modified method is that it gives a possibility to isolate high-quality DNA from lambda phage lysates in 2-3 hrs . Only standard solutions (TE, NaCl, SDS, MgCl2, EDTA, RNAse A) were used throughout the whole protocol . Incubation with DNAse I and proteinase K was omitted and in microvariant concentration of the phage by PEG 6000 was excluded . Digestion by RNAse A was performed in solution with EDTA and SDS and leads to RNA degradation . The yields of DNA (0.5-2 micrograms per ml of L-broth) are similar to those obtained by other methods . DNA quality is better than in the samples of DNA prepared by other express-methods and practically the same as after CsCl centrifugation . DNA can be used for splitting by restriction enzymes, cloning and gene library construction.

J Clin Lab Immunol, 1988 Nov, 27(3), 127 - 32
Kinetics and parameters of the induction of interleukin 1 secretion by rat Kupffer cells; Shirahama M et al.; Rat Kupffer cells stimulated with bacterial lipopolysaccharide (LPS) produced high levels of interleukin 1 (IL-1), determined by assaying the thymocyte proliferating activity . The unstimulated Kupffer cells secreted no detectable activity . LPS-induced production of IL-1 activity was dose-dependent and as little as 0.1 micrograms/ml of LPS induced the IL-1 production . Thirty minutes were required for LPS to induce sufficient amounts of IL-1 activity . IL-2 activity was not detected in the Kupffer cell culture medium . Another soluble agent, N-acetylmuramyl-L-alanyl-D-isoglutamine, or phagocytable agents, silica and zymosan, also stimulated IL-1 production by Kupffer cells, whereas phorbol myristate acetate did not . Kinetic studies revealed a rapid increase in both intra- and extracellular IL-1 activity within 1 hr following the addition of LPS, with a peak at 6 hr . This IL-1 activity produced by Kupffer cells was heat labile, resistant to freezing and thawing and precipitated with 65% ammonium sulfate . Upon gel filtration, the activity was present in three peaks of approximately 110, 35 and 15 kd . The hepatocyte stimulating activity, when assayed by alpha 2-macroglobulin inducing activity, was detected in the first two peaks but not in the third peak . These studies suggest that LAF activity is distinct from hepatocyte stimulating activity at least in the molecular weight.

Biochemistry, 1988 Nov 1, 27(22), 8458 - 65
Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme; Frasca V et al.; Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) has been isolated from Escherichia coli B in homogeneous form . The enzyme is isolated in an inactive form with the visible absorbance properties of cob(II)alamin . The inactive enzyme exhibits an electron paramagnetic resonance (EPR) spectrum at 38 K that is characteristic of cob(II)alamin at acid pH, where the protonated dimethylbenzimidazole substituent is not coordinated with the cobalt nucleus (base-off cobalamin) . An additional, variable component of the EPR spectrum of the inactive enzyme has the characteristics of a cob(III)alamin-superoxide complex . Previous work by others {Taylor, R.T., & Weissbach, H . (1969) Arch . Biochem . Biophys . 129, 745-766 . Fujii, K., & Huennekens, F.M . (1979) in Biochemical Aspects of Nutrition (Yagi, K., Ed.) pp 173-183, Japan Scientific Societies, Tokyo} has demonstrated that the enzyme can be activated by reductive methylation using adenosylmethionine as the methyl donor . We present data indicating that the conversion of inactive to methylated enzyme is correlated with the disappearance of the EPR spectrum as expected for the conversion of paramagnetic cob(II)alamin to diamagnetic methylcobalamin . When the methyl group is transferred from the methylated enzyme to homocysteine under aerobic conditions, cob(II)alamin/cob(III)alamin-superoxide enzyme is regenerated as indicated by the return of the visible absorbance properties of the initially isolated enzyme and partial return of the EPR spectrum . Our enzyme preparations contain copper in approximately 1:1 stoichiometry with cobalt as determined by atomic absorption spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Mikrobiol Virusol, 1988 Nov, (11), 21 - 5
{Unpaired bases in the B--Z junction of the supercoiled plasmid}; Ulanov BP et al.; The restriction analysis has been used to establish that O-beta-diethylaminoethylhydroxylamine (OHA) produces modification of unpaired cytidines in the polylinker region adjacent to the Z-insert (dG-dC)10 . (dG-dC)10 in the negatively supercoiled plasmid pGC20 . The length of the transition region between B- and Z-portions of DNA is not less than 36 bps . The reaction of OHA with the unpaired cytidines in the B-Z junction is a fixing one and produces no secondary despiralling of the neighboring regions . The reaction with DNA proceeds much slower than the one with monomers and single-strand polynucleotides . The structural nonuniformity has been observed, which is manifested in the alternating B and "non-B" form DNA in the B-Z junction . It is suggested that these junctions may contain nucleotide sequences which are stable to violation of the B structure during the change in superhelical density of DNA.

Genetics, 1988 Nov, 120(3), 779 - 90
The mutation bronze-mutable 4 derivative 6856 in maize is caused by the insertion of a novel 6.7-kilobase pair transposon in the untranslated leader region of the bronze-1 gene; Klein AS et al.; The Ds-controlled allele, bz-m4 Derivative 6856 {bz-m4 D6856}, is reported to have an altered temporal- and tissue-specific pattern of gene expression . We have cloned this allele and have characterized it at the molecular level . The mutation was caused by the insertion of a complex transposon-like structure 36 base pairs downstream from the Bz mRNA cap site . The insert is 6.7-kbp long . Ds elements, each approximately 2 kbp in length, are at both ends of the insert . The sequence between the Ds elements is a partial duplication of flanking sequences from the 3' end of the Bz gene . These data suggest that Ds initially inserted near the 3' end of the gene and mobilized adjacent sequences as it transposed.

Genetics, 1988 Nov, 120(3), 645 - 50
Transposon Tn5 target specificity: preference for insertion at G/C pairs; Lodge JK et al.; The procaryotic transposon Tn5 inserts into many different sites within a single gene, but some sites (hotspots) are targeted repeatedly . Hotspots are not closely related in sequence, but most have G/C pairs at the ends of the nine base pairs duplicated by Tn5 insertion . In pBR322, the major hotspot coincides with the "-10 region" of the tet promoter . We mutated the G/C pairs at this hotspot and assayed for insertion into hotspot I, resistance to tetracycline, and plasmid supercoiling . We found that changing the G/C pairs to A/T pairs reduced the frequency of insertion into the hotspot by at least fivefold . The reduction in hotspot use caused by these G/C to A/T changes was not attributable to changes in plasmid supercoiling or tet promoter strength.

Genetics, 1988 Nov, 120(3), 621 - 3
Genetic applications of an inverse polymerase chain reaction; Ochman H et al.; A method is presented for the rapid in vitro amplification of DNA sequences that flank a region of known sequence . The method uses the polymerase chain reaction (PCR), but it has the primers oriented in the reverse direction of the usual orientation . The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle . This procedure of inverse PCR (IPCR) has many applications in molecular genetics, for example, the amplification and identification of sequences flanking transposable elements . In this paper we show the feasibility of IPCR by amplifying the sequences that flank an IS1 element in the genome of a natural isolate of Escherichia coli.

Mol Gen Genet, 1988 Nov, 214(3), 490 - 5
Characterization of the transposon carrying the STII gene of enterotoxigenic Escherichia coli; Hu ST et al.; The Escherichia coli enterotoxin STII gene is carried by Tn4521 . The terminal repeats of Tn4521 are composed of IS2 sequences; however, neither repeat is a complete IS2 . In order to determine how this seemingly defective transposon could transpose, mutations were generated within Tn4521 to determine the regions essential for transposition . The left terminal repeat region was found to be non-essential, but the right terminal repeat area was demonstrated to be crucial for transposition . Within the right terminal repeat area is an open reading frame (ORF), capable of encoding a 159 amino acid protein, which was shown by frameshift mutation analysis to be required for transposition . This protein may be the transposase of Tn4521 . A pair of 11 bp repeat sequences flanking the ORF was also pair of 11 bp repeat sequences flanking the ORF was also found to be important . The right 11 bp repeat is part of the left IS2 terminal sequence, and the left 11 bp repeat is located about 300 bp upstream from the right IS2 terminal sequence located within the right terminal repeat region . The results of this study suggest that Tn4521 is a functional transposon and that the sequence including this pair of 11 bp sequences plus the intervening sequence is a transposable element which may be responsible for Tn4521 transposition.

Mol Gen Genet, 1988 Nov, 214(3), 467 - 73
Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli; Foster PL et al.; Epsilon, a fidelity subunit of Escherichia coli DNA Polymerase III, is encoded by dnaQ+ . dnaQ49 is a recessive allele that confers temperature-sensitive and salt-suppressible phenotypes for both replication fidelity and viability . SOS mutagenesis in E . coli is regulated by LexA and requires activated RecA (RecA*) and the products of the umuDC operon . dnaQ49 strains with various recA, lexA and umuDC alleles were constructed to determine if activities induced as part of the SOS response influence epsilon activity . We found: (1) both UmuDC and RecA* independently enhance the dnaQ49 mutator phenotype, and (2) expression of RecA* activity in the absence of UmuDC function increases the temperature sensitivity for viability of dnaQ49 . These results support the hypothesis that RecA and one or both of the UmuDC proteins interact with the replication complex during SOS mutagenesis.

Mol Gen Genet, 1988 Nov, 214(3), 433 - 8
Insertion sequence IS5 contains a sharply curved DNA structure at its terminus; Muramatsu S et al.; It was demonstrated that insertion sequence IS5 contains a sequence-directed bent (sharply curved) DNA structure at its terminus, close to one of its 16 bp terminal repeats . The minimal number of copies of IS5 related sequences and the locations of the latter on the Escherichia coli K12 W3110 chromosome were determined . Evidence is presented of the occurrence of IS5 mediated translocation and duplication of a large DNA segment on the E . coli chromosome.

Mol Gen Genet, 1988 Nov, 214(3), 373 - 8
Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector; Hauser C et al.; The polypeptide encoded in the Activator (Ac) element of Zea mays L . has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) . Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert . Upon infection of S . frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells . This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies . Recombinant infected cells lack this protein . The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments . The Ac specific protein is a nuclear phosphoprotein and represents about 1%-2% of the newly synthesized protein.

Mol Microbiol, 1988 Nov, 2(6), 701 - 7
Inactivation of the FNR protein of Escherichia coli by targeted mutagenesis in the N-terminal region; Spiro S et al.; The FNR protein of Escherichia coli is a regulatory protein that activates the transcription of its target genes in response to oxygen limitation . Site-directed mutagenesis was used to show that a 28-residue N-terminal segment containing three cysteines is essential for normal FNR function . The cysteine residue which is centrally located in the three-cysteine cluster (Cys-Ala-Ile-His-Cys-Gln-Asp-Cys) was also shown to be essential for FNR activity . Possible mechanisms by which this cysteine residue might function in the response of FNR to anaerobiosis are discussed.

Genes Dev, 1988 Nov, 2(11), 1414 - 23
The Drosophila melanogaster suppressor of Hairy-wing protein binds to specific sequences of the gypsy retrotransposon; Spana C et al.; Mutations at the suppressor of Hairy-wing {su(Hw), 3-54.8} locus reverse the phenotype of second-site mutations induced by the gypsy transposable element in Drosophila melanogaster . This gene encodes a protein with a predicted molecular weight of 109,000 that contains an acidic domain and 12 copies of the DNA-binding 'Zn finger' motif . The su(Hw) protein was overexpressed in Escherichia coli and Drosophila cells, and partially purified . It was shown to interact specifically in vitro with a 367-bp DNA fragment that contains 12 copies of the sequence PyPuTTGCATACCPy located in the 5'-untranslated region of gypsy, between the 5' long terminal repeat (LTR) and the first ATG initiation codon . This sequence shows striking homology to some mammalian transcriptional enhancer elements, supporting a role for the su(Hw) protein in the control of gypsy transcription . In addition, the su(Hw) protein is present at approximately 100-200 sites on Drosophila polytene chromosomes, suggesting that it also interacts in vivo with DNA and might be involved functionally in the regulation of normal cellular genes.

EMBO J, 1988 Nov, 7(11), 3617 - 22
DNA deoxyribophosphodiesterase; Franklin WA et al.; A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli . The enzyme catalyses the hydrolytic release of 2-deoxyribose-5-phosphate from single-strand interruptions in DNA with a base-free residue on the 5' side . The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5'-phosphatase activity . The enzyme has a mol . wt of approximately 50,000-55,000 and has been termed DNA deoxyribophosphodiesterase (dRpase) . The protein presumably is active in DNA excision repair to remove a sugar-phosphate residue from an endonucleolytically incised apurinic/apyrimidinic site, prior to gap filling and ligation.

EMBO J, 1988 Nov, 7(11), 3553 - 7
Signal recognition particle (SRP) stabilizes the translocation-competent conformation of pre-secretory proteins; Sanz P et al.; When affinity-purified proOmpA was diluted out of 8 M urea into a sample of yeast microsomes, it was translocated and processed in the absence of any cytosolic factors; an intact membrane and ATP were the only requirements . The translocation competence of proOmpA was lost, however, during a 15-h incubation at 0 degrees C . The competence was retained when trigger factor and a yeast cytosolic extract were present during incubations at 0 degrees C . The same reactions were carried out with affinity-purified prepro-alpha-factor, and the same results were obtained with the exception that trigger factor was not required . When the various cytosolic factors were replaced with SRP, the addition of yeast microsomes after 15 h resulted in the translocation and processing (and glycosylation) of both proOmpA and prepro-alpha-factor . Pancreatic microsomes were also used in this type of assay, and it was found that proOmpA (but not prepro-alpha-factor) could be translocated when diluted out of urea . In this case, as with yeast microsomes, translocation competence was maintained by SRP . These results show that in addition to a recognition and targeting function, SRP can stabilize the translocation-competent conformation of pre-secretory proteins in vitro for translocation across eukaryotic membranes.

Eur J Biochem, 1988 Nov 1, 177(2), 267 - 71
Alterations in the extracellular domain of M13 procoat protein make its membrane insertion dependent on secA and secY; Kuhn A; The products of genes secA and secY (SecA and SecY) are putative components of a bacterial protein export machinery and are required for the export of many periplasmic and membrane proteins . Only a few proteins, among them the M13 procoat protein, insert independently of SecA and SecY . To investigate the reason why the procoat protein inserts independently of sec functions, various hybrid proteins were constructed . By in-frame gene fusions the central procoat region, which translocates across the membrane, was extended in size . Fragments of the ompA gene ranging from 522-294 bp were ligated with the procoat gene . The hybrid proteins were inserted into the membrane and processed normally, but only in the presence of functional SecA and SecY.

Virology, 1988 Nov, 167(1), 279 - 83
Beta-galactosidase as a marker in the peripheral and neural tissues of the herpes simplex virus-infected mouse; Ho DY et al.; We have inserted a modified Escherichia coli lacZ gene, placed under the control of herpes simplex virus alpha 4 or beta 8 regulatory signals, into the HSV-1 genome disrupting the viral thymidine kinase gene . Using beta-galactosidase as an in situ indicator of viral gene expression, we detected expression from these recombinant HSV in dermal and neural tissues of the BALB/c mouse . Our detection of beta-galactosidase expression in neuronal cells indicates that TK-deficient viruses are capable of invading mouse neuronal cells and expressing up to the beta class of gene product.

Virology, 1988 Nov, 167(1), 150 - 5
Characterization of two early promoters of cyanophage N-1; Schneider GJ et al.; Restriction fragments of cyanophage N-1 DNA, containing genes transcribed early in infection of Anabaena, were identified by hybridization with RNA prepared from infected cells . Two of these fragments were isolated by binding to Anabaena RNA polymerase on filters and were subsequently cloned . The start sites for transcription by Anabaena RNA polymerase were determined by run-off assays . The sequences of the two promoter regions thus localized were similar to each other in the -35 and -10 regions and nearly identical to the consensus sequence for promoters in Escherichia coli.

J Bacteriol, 1988 Nov, 170(11), 5389 - 91
Distinct mutation sites in prlA suppressor mutant strains of Escherichia coli respond either to suppression of signal peptide mutations or to blockage of staphylokinase processing; Sako T et al.; We have cloned and sequenced some prlA mutant alleles of the Escherichia coli secY gene . From the mutation sites determined, it is strongly suggested that distinct regions in the SecY (PrlA) protein are involved in the recognition of different structural features of a signal peptide as it functions.

J Bacteriol, 1988 Nov, 170(11), 5368 - 70
Synthesis and overproduction of the 5A protein of insertion sequence IS5; Chernak JM et al.; We have demonstrated both the synthesis and overproduction of the 5A protein encoded by the longest open reading frame of the bacterial insertion sequence IS5 . Expression was obtained in vitro and in Escherichia coli maxicells from plasmids containing IS5 in either orientation, as well as in vitro from a restriction fragment containing exclusively IS5 DNA . When IS5 was cloned in the appropriate orientation downstream of a strong tac promoter, production of the 5A protein was increased to 10 to 20% of the total protein synthesized in vitro.

J Bacteriol, 1988 Nov, 170(11), 5289 - 97
Identification and characterization of the Myxococcus xanthus bsgA gene product; Gill RE et al.; The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program . They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells . A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli . The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis . The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product . The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M . xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.

J Bacteriol, 1988 Nov, 170(11), 5117 - 24
Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product; Icho T; The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope . Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane . The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions . About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes . A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid . This overproduced protein also revealed the dual subcellular localization.

J Bacteriol, 1988 Nov, 170(11), 5110 - 6
Membrane-bound phosphatases in Escherichia coli: sequence of the pgpA gene; Icho T; One of the phosphatidyl glycerophosphate phosphatase genes of Escherichia coli, pgpA, was cloned, and its DNA sequence was determined . Its 507-base-pair open reading frame was consistent with the 18,000-molecular-weight product identified by a maxicell experiment . Between its possible promoter and methionine initiation codon, a repetitive extragenic palindromic sequence was found.

J Bacteriol, 1988 Nov, 170(11), 5080 - 5
Regulation of ompC and ompF expression in Escherichia coli in the absence of envZ; Forst S et al.; The expression of the genes encoding the major outer membrane porin proteins OmpF and OmpC in Escherichia coli is regulated by ompR, which encodes the transcriptional activator protein OmpR, and envZ, which encodes a receptorlike protein located in the inner membrane . To examine the role of EnvZ in the expression of the osmoregulated porin genes, we analyzed the production of OmpF and OmpC in cells that lack envZ function . We show that EnvZ is required for the maximal production of OmpC in cells grown in minimal medium but is not essential for the efficient induction of OmpC that occurs during a shift to a high-osmolarity medium . In contrast, the production of OmpF in cells that lack envZ function was similar to that of the parent strain, whereas OmpF repression during a shift to a high-osmolarity medium was incomplete in the absence of EnvZ . These results are discussed in the context of the putative role of EnvZ in the expression of ompF and ompC.

J Physiol, 1988 Nov, 405, 547 - 62
Effects of urethane, ambient temperature and injection route on rat body temperature and metabolism due to endotoxins; Bibby DC et al.; 1 . We have investigated the influence of environmental temperature, anaesthesia and route of administration on rectal temperature and other metabolic responses to two preparations of bacterial endotoxin in male adult Wistar rats . 2 . Urethane anaesthesia, environmental temperatures of 20 and 28 degrees C, subcutaneous (S.C.) and intraperitoneal (I.P.) routes of administration and butanol and trichloroacetic acid (TCA) extracts of E . coli endotoxin (1.2 mg/kg) were used . 3 . In addition to rectal temperature, serum zinc, albumin and urea concentrations and liver protein, RNA and zinc contents were measured . 4 . Fevers were produced by injections of both endotoxins, by either route at 28 degrees C . Butanol-extracted endotoxin produced a more rapid response than the TCA extract via the I.P . route whereas the TCA extract produced a higher temperature than the butanol extract when the S.C . route was used . 5 . Fevers were inhibited at an environmental temperature of 20 degrees C and by anaesthesia, while the former had no effect on compositional changes the latter inhibited the fall in serum zinc in response to subcutaneous doses of either endotoxin and the increase in liver zinc content in response to the butanol extract of endotoxin . 6 . At 20 degrees C a marked fall in rectal temperature occurred in conscious rats 2 h after receiving the TCA but not the butanol extract of endotoxin . Temperature depression was more severe when endotoxin was administered by the I.P . route . 7 . Serum urea was elevated in conscious rats by the TCA extract of endotoxin via both routes but only by the I.P . route for the butanol extract of endotoxin . In anaesthetized animals only the TCA extract of endotoxin raised serum urea concentration when given intraperitoneally . 8 . Serum albumin and liver protein and RNA were unaffected by endotoxin injections over the 7 h time course of the study . 9 . Rectal temperature responses to endotoxins were influenced in direction and magnitude by all variables employed in the study, while compositional changes were unaffected by environmental temperature but influenced to varying degrees by urethane anaesthesia and the route of administration employed.

Biochemistry, 1988 Nov 1, 27(22), 8421 - 8
Site-directed mutagenesis of the charged residues near the carboxy terminus of the colicin E1 ion channel; Shiver JW et al.; Colicin E1 was altered by oligonucleotide-directed mutagenesis at the site of three charged residues on the COOH side of the 35-residue hydrophobic segment in the channel-forming domain . Asp-509 is one of five conserved acidic residues in the channel domain of colicins A, B, E1, Ia, and Ib and is the first charged residue following the hydrophobic segment, followed by the basic residues Lys-510 and Lys-512 . Asp-509 and Lys-512 were changed to amber and ochre stop codons, respectively, while Lys-510 was mutated to a Met codon . Proteins truncated after residue 508 or 511, and missing the last 14 or 11 residues, were obtained from a nonsuppressing cell strain harboring the mutant plasmid while full-length colicin molecules with single residue changes at Asp-509 to Leu, Ser, and Gln, and Lys-512 to Tyr, were obtained by using appropriate suppressor strains . The truncated colicins displayed (i) a low cytotoxicity, approximately 1% of intact wild-type colicin, (ii) 10-fold less in vitro channel activity with liposomes, and (iii) reduced labeling of the colicin in liposomes by a phospholipid photoaffinity probe, showing that one or more of the residues following Asn-511 is necessary for both in vivo and in vitro activity and insertion into the bilayer . (iv) The truncated mutants also displayed an altered conformation at pH 6 that allowed greater binding and activity with liposomes at this pH relative to wild type . The cytotoxicity of single residue substitutions at Asp-509 showed a range of cytotoxicities, wild type greater than Ser-509 greater than Gln-509 greater than Leu-509, although none of these changes greatly affected the in vitro channel activity or pH dependence.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Nov 1, 27(22), 8302 - 6
Relative stability of parallel- and antiparallel-stranded duplex DNA; Germann MW et al.; We have recently shown that DNA containing homopolymeric A-T base pairs can form a parallel-stranded intramolecular duplex {van de Sande et al . (1988) Science (Washington, D.C.) 241, 551-557} . In the present paper, we demonstrate that parallel-stranded DNA can also be formed in unconstrained bimolecular DNA of appropriate sequence homology . Three deoxyoligonucleotides, a 21-mer {dCCCATATATATTTTTTTTCCC}, a ps-15-mer {dTATATATAAAAAAAA}, and an aps-15-mer {dAAAAAAAATATATAT}, have been synthesized . Annealing of 21-mer and aps-15-mer results in the formation of a conventional antiparallel duplex (aps); however, the combination of 21-mer and ps-15-mer forms a duplex in which the two strands are in a parallel orientation (ps) . The parallel-stranded structure was established from the following criteria: (i) The parallel-stranded structure shows a 1:1 stoichiometry of the constituent strands . (ii) Gel electrophoretic mobility of the ps and aps duplexes are similar under native conditions . (iii) Spectroscopic properties of the ps duplex are characteristics for a base-paired structure but are different from the aps structure . (iv) Both duplexes undergo a thermally induced helix to coil transition; however, the melting temperature for the ps duplex is 22 degrees C lower . (v) The minor groove binding drug Hoechst 33258 shows a reduced affinity for the ps compared to the aps duplex . (vi) The parallel-stranded duplex is not a substrate for DNA Escherichia coli polymerase I (Klenow fragment) or AMV reverse transcriptase . Parallel-stranded DNA can exist under normal solution conditions, but competition experiments show it to be thermodynamically less favorable than the conventional antiparallel form.

Jpn J Cancer Res, 1988 Nov, 79(11), 1168 - 73
A gag-env hybrid protein of human T-cell leukemia virus type I and its application to serum diagnosis; Kuga T et al.; A fused gene of the gag and env sequences of human T-cell leukemia virus type I (HTLV-I), the causative agent of adult T-cell leukemia, was constructed in vitro and expressed in Escherichia coli . The gag-env hybrid protein accumulated as insoluble granules with a yield of approximately 12% of the total proteins . In an enzyme-linked immunosorbent assay done with the use of the gag-env hybrid protein, all 57 seropositive sera gave positive signals, and none of the sera from normal persons did . This system can produce large quantities of the gag-env hybrid protein, which can be used for mass screening of human sera for HTLV-I infection.

Mol Gen Genet, 1988 Nov, 214(3), 503 - 8
Nucleotide sequence of the hemB gene of Escherichia coli K12; Echelard Y et al.; The hemB gene of Escherichia coli K12, coding for porphobilinogen synthase (PBG-S; syn., 5-aminolevulinic acid dehydratase, ALA-D), was cloned following insertion of an EcoRI fragment of plasmid F'13 into the mobilizable vector pCR1 . The hybrid plasmid carrying the hemB gene was able to complement a hemB mutant of E . coli K12: not only was the PBG-S activity of the mutant restored after the acquisition of the hemB gene, but it was about ten times higher than that of the wild type . Subcloning of the original EcoRI fragment (14.6 kb) enabled us to locate the hemB gene on an NruI-HpaI fragment of about 1.1 kb . The hemB promoter was located toward the NruI end of the fragment, as shown by the use of the pKO promoter-probe series of vectors . Sequencing of the hemB gene indicated the presence of an open reading frame (ORF) of 1051 nucleotides, which should correspond to the HemB protein . Primer extension experiments enabled us to identify the 5' end of the hemB mRNA, and to deduce the -10 and -35 regions of the hemB promoter . Protein synthesis performed by an in vitro coupled transcription-translation system, showed the presence of a protein of about 35 kDa . This is in agreement with the molecular weight of the HemB protein (35.6 kDa), as deduced from the nucleotide sequence of the gene . Comparison of the amino acid sequences of E . coli and human PBG-S allowed the detection of several regions of strong homology between the two proteins . Two of these regions correspond, as expected, to the putative zinc-binding and catalytic sites of the human PBG-S.

Mol Gen Genet, 1988 Nov, 214(3), 474 - 81
Transcription in the region of the replication origin, oriC, of Escherichia coli: termination of asnC transcripts; Gielow A et al.; Transcription from the asnC promoter was found to proceed through the replication origin, oriC, into the gidA gene of Escherichia coli . Between 10% and 20% of asnC transcripts reached oriC in vivo . Termination sites in the intergenic region between asnC and mioC and within mioC were determined in vivo and in vitro using S1 mapping . Only about 10% of the transcripts terminated at the asnC terminator in vivo . DnaA protein dependent termination was observed close to the binding site, dnaA box, for DnaA protein . In the in vitro replication system asnC transcripts did not reach oriC, suggesting that asnC transcripts are not involved in the initiation of replication, contrary to mioC transcripts . We suggest that oriC and mioC might have been transposed during evolution into an asnC regulation.

Mol Gen Genet, 1988 Nov, 214(3), 379 - 88
Mapping of the multiple regulatory sites for putP and putA expression in the putC region of Escherichia coli; Nakao T et al.; The effects of regulatory proteins on the expression of putP and putA were studied using put-lacZ fusion genes . The expression of the putP-lacZ gene was activated by the glnG gene product and the catabolite gene activator protein (CAP) . The putA gene product inhibited activation of putP-lacZ gene expression by CAP or the glnG gene product and its inhibition was greater in the absence of proline . The expression of the putA-lacZ gene was activated by CAP and repressed by the glnG gene product . The putA gene product acted as a repressor in the absence of proline, but not in its presence . Studies using put-lacZ fusion genes with upstream deletions showed that the region required for the activation of putP by CAP was within 234 bp upstream of the translational initiation site and that that for the activation of putA was within 107 bp upstream of the translational initiation site of the putA gene . This supported the suggested locations of CAP binding sites . The region required for induction of putP and putA expression by proline was located at the HpaI site 182 bp upstream of the translational starting site of putA, suggesting that a sequence of dyad symmetry located 1 bp to the left of the HpaI site is a candidate for the binding site of the putA gene product.

Scand J Immunol, 1988 Nov, 28(5), 547 - 51
Different origins of IgA antibodies with various antigen specificities appearing in rat bile; Nilsson K et al.; Eight lactating and seven non-lactating female rats were immunized in Peyer's patches (Pp) with Escherichia coli 06 carrying type 1 pili . Eight days later the thoracic duct was drained and bile was collected at the same time . During the lymph drainage in the lactating rats, the biliary IgA anti-pili antibodies decreased less than the anti-lipopolysaccharide (LPS) antibodies . The non-lactating rats showed only a very small difference in decrease between the anti-pili and the anti-LPS antibodies . Transfer of mesenteric lymph node cells from male donor rats immunized with the E . coli strain to syngeneic male recipients resulted in the appearance of both IgA anti-pili and anti-LPS antibodies in the bile . This is at variance with the results seen in lactating rats, where only biliary IgA anti-LPS is seen after similar cell transfer . In conclusion, the study shows that in lactating rats a larger proportion of biliary IgA anti-pili antibodies than anti-LPS antibodies is derived from extraintestinal sites, such as the mammary glands or the liver . Thus, this study confirms that the nature of the antigen influences the transfer of the secretory antibody response to different secretions.

Mol Cell Biol, 1988 Nov, 8(11), 4829 - 39
Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals; Denome RM et al.; We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region . The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region . This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection . All plasmids contained the SV40 origin of replication . Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays . In all constructs, all polyadenylation signals were used . Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal . Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal . Changing the distance between the 5' cap and first signal did not affect signal use . Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism . Four possible mechanisms of polyadenylation signal recognition are discussed.

EMBO J, 1988 Nov, 7(11), 3571 - 6
A model for the spatial arrangement of the proteins in the large subunit of the Escherichia coli ribosome; Walleczek J et al.; A three-dimensional model for the arrangement of 29 of the 33 proteins from the Escherichia coli large ribosomal subunit has been generated by interactive computer graphics . The topographical information that served as input in the model building process was obtained by combining the immunoelectron microscopically determined network of epitope-epitope distances on the surface of the large ribosomal subunit with in situ protein-protein cross-linking data . These two independent sets of data were shown to be compatible by geometric analysis, thus allowing the construction of an inherently consistent model . The model shows (i) that the lower third of the large subunit is protein-poor, (ii) that proteins known to be functionally involved in peptide bond formation and translocation are clustered in two separate regions, (iii) that proteins functionally interdependent during the self-assembly of the large subunit are close neighbours in the mature subunit and (iv) that proteins forming the early assembly nucleus are grouped together in a distinct region at the 'back' of the subunit.

J Med Virol, 1988 Nov, 26(3), 261 - 70
Natural antibodies to HIV-tat epitopes and expression of HIV-1 genes in vivo; Krone WJ et al.; The tat regulatory protein of HIV-1 was expressed as a fusion protein in E . coli and used as antigen to detect antibodies against HIV-tat (anti-tat) in the serum of HIV-1 infected children and adults . HIV-1-infected children showed a higher frequency (55%) of anti-tat than HIV-1-infected adults (36%) . Anti-tat were present in only 15% (3/20) of acutely infected individuals . Forty percent (10/25) of individuals with prolonged HIV-1 infection but without antigen were anti-tat positive . Only 13% (3/23) of HIV-1-antibody-positive individuals with prolonged HIV-1 antigenemia were anti-tat positive and titers of anti-tat antibodies declined with time . Pepscan analysis identified the amino terminus of HIV-tat as the major antibody-binding site . Antibodies to HIV-tat occurred as a harbinger of HIV-1 antigen expression and disappeared thereafter, possibly reflecting the transience of HIV-tat expression . Because of the low antigenicity of HIV-tat, antibodies to this regulatory protein are not a reliable marker for either early HIV-1 infection or subsequent disease progression.

Mol Pharmacol, 1988 Nov, 34(5), 702 - 6
Methyl mercury stimulates chain elongation by purified HeLa RNA polymerase II; Frenkel GD et al.; Methyl mercury (MeHg) inhibited the overall RNA synthetic reaction of HeLa RNA polymerase II . However, when RNA chain initiation was allowed to occur in its absence, MeHg stimulated the rate of the subsequent elongation stage of the reaction . Chain elongation with both double-stranded and single-stranded DNA templates was stimulated . This stimulatory effect was specific for MeHg; both p-hydroxymercuribenzoate and HgCl2 inhibited chain elongation (to about the same degree as they inhibited the overall reaction) . The stimulatory effect was also specific for the HeLa polymerase; with Escherichia coli RNA polymerase, MeHg inhibited elongation (to the same degree as it inhibited the overall reaction).

Proc Natl Acad Sci U S A, 1988 Nov, 85(22), 8668 - 72
Addition of a foreign oligopeptide to the major capsid protein of poliovirus; Colbere-Garapin F et al.; Insertion mutants of type 3 poliovirus (Sabin strain) were constructed that encode additional amino acid sequences at the level of residue 100 of the capsid polypeptide VP1 within the neutralization site 1, corresponding to a loop on the capsid surface . The addition of a tri- or hexapeptide did not hamper virus viability . The antigenic pattern of insertion mutants was only modified locally: all mutants lost reactivity of neutralization site 1 with the corresponding monoclonal antibodies, while the reactivity of sites 2 and 3 was unaffected by the insertion . We have shown for one of the mutants--vFG68--that the antigenic specificity of the neutralization site 1 was replaced by a new one . Although vFG68 differs from its parental Sabin strain only by the addition of three amino acids within VP1, neutralizing antibodies specific for vFG68 were induced by the native virion as well as by the heat-denatured mutated virions . Our results demonstrate that an oligopeptide of three or six amino acids can lengthen VP1 at the level of antigenic site 1 without affecting virus multiplication and that this foreign peptide is exposed on the virion surface.

J Bacteriol, 1988 Nov, 170(11), 5141 - 5
Nucleotide sequence of the nfo gene of Escherichia coli K-12; Saporito SM et al.; The nfo gene of Escherichia coli K-12 which encodes endonuclease IV has been sequenced . The predicted gene product has a molecular weight of 31,562, in good agreement with the size of the gene product estimated by maxicell analysis . The nfo promoter was mapped by primer extension of in vivo transcripts . Inspection of the nucleotide sequence revealed no regions of potential secondary structure corresponding to a transcriptional terminator downstream from the structural gene; however, there was a potential open reading frame immediately downstream from the nfo structural gene.

J Virol, 1988 Nov, 62(11), 4376 - 80
Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity; Levin JG et al.; The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli . A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme . The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase . The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively.

J Immunol, 1988 Nov 1, 141(9), 3128 - 34
A monoclonal anti-IgE antibody against an epitope (amino acids 367-376) in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23); Chretien I et al.; We have produced three different mAb specific for human IgE-Fc . Their binding pattern to either heat-denatured IgE or a family of overlapping IgE-derived recombinant peptides and their ability to affect interaction of IgE with its low affinity receptor Fc epsilon R2/CD23 demonstrate that they recognize distinct epitopes on the IgE molecule . All three mAb were able to induce basophil degranulation as measured by the induction of histamine release . mAb 173 recognizes a thermolabile epitope in the CH4 domain . It does not affect the binding of IgE to Fc epsilon R2/CD23 . mAb 272 recognizes a thermostable epitope that maps to a sequence of 36 amino acids (AA) spanning part of the CH2 and CH3 domain and it does not affect the binding of IgE to Fc epsilon R2/CD23 . mAb 27 recognizes a thermolabile epitope located on a 10 AA stretch (AA 367-376) in the CH3 domain . This area contains one N-linked oligosaccharide (Asn-371), but the antibody is not directed against carbohydrate because it binds to Escherichia coli-derived IgE peptides . mAb 27 inhibits the binding of IgE to Fc epsilon R2/CD23 but is still capable of reacting with IgE already bound to Fc epsilon R2/CD23 . These data suggest that upon binding to Fc epsilon R2/CD23, the IgE molecule engages one of two equivalent-binding sites close to the glycosylated area of the CH3 domain.

J Immunol, 1988 Nov 1, 141(9), 3072 - 7
Human recombinant IL-6/B cell stimulatory factor 2 augments murine antigen-specific antibody responses in vitro and in vivo; Takatsuki F et al.; The effects of human rIL-6/B cell stimulatory factor 2 (hrIL-6/BSF-2) from Escherichia coli on murine Ag, SRBC-specific antibody responses were examined in vitro and in vivo . HrBSF-2 was effective in augmenting the primary and the anamnestic plaque-forming cells response to SRBC in vitro . The augmentation of the primary response was apparent when B cell-enriched spleen cells (B cells) were cultured with BSF-2 in the presence of IL-2 . On the other hand, hrBSF-2 alone strongly enhanced the anamnestic response in a dose-dependent manner when spleen cells from SRBC-immunized mice were used . These effects of BSF-2 were abolished completely by anti-BSF-2 antibody, but not by normal rabbit Ig . Cell depletion experiments indicated that L3T4 (CD4)+ T cells, but not Lyt-2(CD8)+ T cells, and adherent cells (macrophages) have an important role in this BSF-2-induced augmentation of the response . In addition, kinetic studies showed that hrBSF-2 acts on B cells in the anamnestic response even when added relatively late in the culture . Finally, it was determined whether BSF-2 also could be active in modulating antibody responses in vivo . BSF-2 was shown to enhance the primary and secondary antibody responses in mice . The most apparent effect of BSF-2 was observed in the secondary response.

Philos Trans R Soc Lond B Biol Sci, 1988 Oct 31, 321(1207), 485 - 93
New approaches to the serotypic analysis of the epidemiology of Plasmodium falciparum; Forsyth KP et al.; Considerable antigenic heterogeneity of Plasmodium falciparum has been demonstrated in natural parasite populations . However, very little is known about the relative virulence, transmission efficiency and prevalence over space and time of parasites expressing different serotypes of variant antigens . The recent application of recombinant DNA techniques to express a wide range of P . falciparum antigens in Escherichia coli has led to a better understanding of the molecular basis of antigenic diversity of a number of parasite proteins including the precursor to the major merozoite surface antigen (PMMSA) and the heat-stable S-antigens . Highly specific reagents such as DNA probes, monoclonal antibodies and polyclonal antisera to either cloned antigens or synthetic peptides have become available for serotypic analysis of natural parasite populations . With these reagents important epidemiological questions can now be asked concerning the population biology of different serotypes of P . falciparum . The use of the polymorphic S-antigen system as a serotypic marker to analyse the transmission dynamics of P . falciparum in Madang, Papua New Guinea (PNG) is discussed . Results of serotyping studies with the S-antigen system highlight the complexities of malaria transmission, which require consideration in the design of malaria vaccine trials.

Biochem Biophys Res Commun, 1988 Oct 31, 156(2), 1054 - 60
Human placental endonuclease cleaves Holliday junctions; Jeyaseelan R et al.; A partially purified endonuclease from human placenta cleaves cruciform structures . The placental enzyme is active both on extruded cruciform structures from negatively supercoiled covalently closed circular plasmid DNA and on synthetic X-junctions formed by reannealing short oligonucleotides . Plasmids containing natural or cloned palindromes such as pBR322 and pHD101-3 were used as substrates . The synthetic X-junction tetramer DNA formed by reannealing short oligonucleotides, was converted into dimer form by the enzyme . This is the first report of an enzyme activity involved in resolution of recombination intermediates in higher eukaryotes and second report of a cellular enzyme.

Thromb Haemost, 1988 Oct 31, 60(2), 324 - 7
Induction of fibrinolysis by polyanions in human plasma; Klauser RJ; The fibrinolytic potency of several polyanions was comparatively investigated . Fibrinolytic activity was measured in a whole plasma assay using H-D-Val-Leu-Lys-pNA (S-2251) as chromogenic substrate and by a fibrin plate assay using plasminogen rich fibrin plates . In the chromogenic substrate assay potent fibrinolytic polyanions comprised dextran sulfate, GAGPS, pentosan polysulfate, polyanethol sulfate, l-carrageenan and i-carrageenan . Chondroitin sulfates A, B, C, keratan sulfate, ribonucleic acid, k-carrageenan and heparin were weakly fibrinolytic . Hyaluronic acid and lipopolysaccharide from E . coli were inactive . Similar results were obtained when fibrinolytic activity was measured by a fibrin plate assay . All polyanions except lipopolysaccharide produced lysis zones . Induction of fibrinolytic activity in human plasma was shown to be at least partially dependent on Hageman factor . In factor XII deficient plasma no fibrinolysis was induced by any of the polyanions when measured in the fibrin plate assay . In the chromogenic substrate assay corn Hageman factor inhibitor (CHFI) inhibited the activation of S-2251 cleaving enzyme by GAGPS, pentosan polysulfate, polyanethol sulfate, heparin, and ribonucleic acid near completely . The activation by dextran sulfate was inhibited by 45% . Heparin, pentosan polysulfate and GAGPS, three polyanions of therapeutic interest were separately compared . In both assays GAGPS proved the most potent activator, while pentosan polysulfate exhibited 83% and 44% and heparin 32% and 14% of GAGPS fibrinolytic activity in the chromogenic substrate test and the fibrin plate assay, respectively.

Gene, 1988 Oct 30, 70(2), 321 - 9
Use of transmissible plasmids as cloning vectors in Caulobacter crescentus; Schoenlein PV et al.; Cloning vectors for studies of Caulobacter crescentus genes should be transferrable between Escherichia coli and C . crescentus since a transformation system has not been developed for C . crescentus . We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C . crescentus . All of the plasmids tested were maintained in C . crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons . Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (SuR-SmR) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter . These data indicate that R300B does not contain sequences which would provide promoter function in C . crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression . To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon . In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7 . Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C . crescentus mutants.

Gene, 1988 Oct 30, 70(2), 415 - 7
Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli; Bukrinsky MI et al.; A new expression vector (pBB1) has been constructed for the regulated expression of genes in Escherichia coli . Based on the pUC plasmids, the pBB1 carries lacIts allele of the lac repressor gene . This makes it possible to control expression of cloned genes by shifting the temperature from 30 degrees C to 42 degrees C . Thus the vector combines advantages of the pUC plasmids with convenient regulation by temperature . Expression of a fragment of HIV-1 env gene was achieved with the help of this vector and shown by enzyme-linked immunosorbent assay and Western-blot analysis.

Gene, 1988 Oct 30, 70(2), 399 - 403
A new revision of the sequence of plasmid pBR322; Watson N; A revised sequence in the region immediately upstream from the rop gene of pBR322 is reported . Two base pairs in the accepted sequence do not exist in the plasmid DNA . Specifically, a TA base pair is missing at sequence coordinate 1893 {Sutcliffe, Cold Spring Harbor Symp . Quant . Biol . 43 (1979) 77-90} and an AT base pair is missing at position 1915, giving a total size for pBR322 of 4361 bp . These changes are in a potential translation initiation sequence and probably reflect errors in the original sequence rather than recent evolution of the plasmid.

Gene, 1988 Oct 30, 70(2), 393 - 7
Participation of hup gene product in ori2-dependent replication of fertility plasmid F; Wada M et al.; The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli . Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation . The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42 degrees C . These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.

Gene, 1988 Oct 30, 70(2), 387 - 92
Cloning and sequencing the HinfI restriction and modification genes; Chandrasegaran S et al.; The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322 . Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment . This subfragment was sequenced . Two large open reading frames (ORF's) are present . ORF1 codes for the methylase {predicted 359 amino acids (aa)} and ORF2 codes for the endonuclease (predicted 262 or 272 aa).

Gene, 1988 Oct 30, 70(2), 331 - 41
The cyclization of linear DNA in Escherichia coli by site-specific recombination; Sauer B et al.; The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1 . Linear plasmid molecules containing directly repeated loxP sites (lox2 plasmids) are cyclized in Cre+ E . coli strains after introduction either by transformation or by mini-Mu transduction . Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox2 plasmids after transformation . By use of E . coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox2 plasmids identical to those obtained with covalently closed circular plasmid DNA . Moreover, Cre+ E . coli recBC strains allow the efficient recovery of lox2 plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.

Gene, 1988 Oct 30, 70(2), 363 - 76
Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes; Romeo T et al.; The nucleotide sequences of the Escherichia coli genome between the glycogen biosynthetic genes glgB and glgC, and 1170 bp of DNA which follows glgA have been determined . The region between glgB and glgC contains an open reading frame (ORF) of 1521 bp which we call glgX . This ORF is capable of coding for an Mr 56,684 protein . The deduced amino acid (aa) sequence for the putative product shows significant similarity to the E . coli glycogen branching enzyme, and to several different glucan hydrolases and transferases . The regions of sequence similarity include residues which have been reported to be involved in substrate binding and catalysis by taka-amylase . This suggests that the proposed product may catalyze hydrolysis or glycosyl-transferase reactions . The cloned region which follows glgA contains an incomplete ORF (1149 bp), glgY, which appears to encode 383 aa of the N terminus of glycogen phosphorylase, based upon sequence similarity with the enzyme from rabbit muscle (47% identical aa residues) and with maltodextrin phosphorylase from E . coli (37% identical aa residues) . Results suggest that neither ORF is required for glycogen biosynthesis . The localization of glycogen biosynthetic and degradative genes together in a cluster may facilitate the regulation of these systems in vivo.

Gene, 1988 Oct 30, 70(2), 283 - 93
Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a gamma-actin; Fidel S et al.; The single actin gene from the filamentous fungus Aspergillus nidulans has been isolated and characterized . The only other organism reported to contain just one actin gene is another Ascomycete, the budding yeast Saccharomyces . The nucleotide sequence of the A . nidulans actin gene predicts a polypeptide containing the N-terminal sequence identifying the gamma-actin isotype . Until now this characteristic N terminus has only been reported to occur in vertebrate actin sequences . A monospecific anti-gamma-actin antiserum recognizes a single 42-kDa band in immunoblots of total Aspergillus protein . None of the six introns in the A . nidulans actin gene sequence aligns precisely with those found in other actin genes . One, unlike other known actin introns, is located in the 3'-untranslated region of the gene . The 5' and 3' ends of the gene have been characterized . The Aspergillus actin gene has a heterogeneous transcript size due to the presence of several different 3' termini . Of four characterized polyadenylated transcripts, only the longest contains a typical AATAAA polyadenylation signal near its 3' terminus . Using an integrative plasmid containing Aspergillus actin sequences and the pyr4 gene from Neurospora, the A . nidulans actin gene has been mapped to the first chromosome.

Gene, 1988 Oct 30, 70(2), 313 - 9
Novel eukaryotic expression vectors which permit single-stranded replication in Escherichia coli and in vitro translational analysis of cloned genes; Mongkolsuk S; The ability to express cloned genes transiently is an important technique in the study of eukaryotic gene expression . Numerous useful expression vectors have been constructed for this purpose although many of them share several common drawbacks . In this paper I describe the construction and characterization of novel expression vectors pSVKII and pSVKIII which have 13 and 8 unique restriction sites, respectively, suitable for cloning genes . These vectors have phage M13 ori, which permits them to produce and package ss DNA molecules in Escherichia coli host, thus facilitating the sequencing and site directed mutagenesis of a cloned gene . Expression vector pSVKIII has a T7 phage promoter located between the SV40 promoter and the multiple cloning sites, which permits efficient transcription and in vitro translation of the cloned gene prior to in vivo studies.

Presse Med, 1988 Oct 26, 17(37), 1914 - 6
{Pharmacokinetics of ceftazidime in the cerebrospinal fluid}; Decazes JM; A compound potentially effective in the treatment of meningitis should reach high concentrations in CSF . In animal experimental models, ceftazidime levels of 5-25 micrograms/ml were demonstrated during administration of doses generating serum levels similar to those achieved in humans . Human studies later confirmed CSF levels of 5-10 micrograms/ml in patients with meningitis . Ceftazidime CSF kinetic properties are adequate for use of this compound in patients with meningitis due to susceptible organisms.

Biochim Biophys Acta, 1988 Oct 26, 936(1), 74 - 80
The proton pore of the F0F1-ATPase of Escherichia coli: Ser-206 is not required for proton translocation; Howitt SM et al.; A series of experiments was carried out to investigate the role of some polar amino acids in the a-subunit of the ATP synthase of Escherichia coli . Site-directed mutagenesis resulted in the amino acid substitutions Ser-199----Ala, Ser-202----Ala, Ser-206----Ala, Arg-61----Gln or Asp-44----Asn . None of these amino acid substitutions affected the ability of the cells to carry out oxidative phosphorylation . It was concluded therefore that the effect of the substitution of leucine for Ser-206 reported previously (Cain, B.D . and Simoni, R.D . (1986) J . Biol . Chem . 261, 10043-10050) was due to the presence of the leucine rather than the absence of serine . Even though cells carrying the Asp-44----Asn substitution were able to carry out oxidative phosphorylation, membranes from such cells remained proton-impermeable after removal of the F1-ATPase . It appears likely that the proton pore of the F0 of the ATP synthase of E . coli consists of four amino acids, namely Arg-219, Glu-210 and His-245 of the a-subunit and Asp-61 of the c-subunit.

J Biol Chem, 1988 Oct 25, 263(30), 15506 - 12
Cloning and expression of a cDNA for human thioredoxin; Wollman EE et al.; Thioredoxin is the best representative enzyme of a group of proteins, widely distributed and possessing dithiol-disulfide oxidoreductase activity . We have constructed a cDNA library from messenger RNAs isolated from a lymphoblastoid B cell line (Epstein-Barr virus-immortalized normal human lymphocytes) . Screening of this library with synthetic oligonucleotide probes, constructed from the NH2-terminal amino acid sequence of a protein produced by this line, allowed us to identify a full-length cDNA clone coding for human thioredoxin . The open reading frame (315 nucleotides long) codes for a protein of 104 amino acids (excluding the initial methionine) . This protein possesses the highly conserved enzymatic active site common to plant and bacterial thioredoxins: Trp-Cys-Gly-Pro-Cys (amino acids 30-34) . These data provide for the first time the complete primary sequence of a thioredoxin of mammalian origin . Recombinant human thioredoxin, expressed in Escherichia coli, possesses a dithiol-reducing enzymatic activity as assayed on mammalian and plant substrates . It is able to reduce the interchain disulfide bridges of murine pentameric IgM and porcin insulin and also to activate vegetal NADP-malate dehydrogenase . Studies of human thioredoxin mRNA expression and regulation in immunocompetent cells of human origin indicate that the protein is weakly expressed in resting lymphocytes and monocytes, but the level of human thioredoxin mRNA transcription is quite important in activated monocytes and established dividing human cell lines.

J Biol Chem, 1988 Oct 25, 263(30), 15436 - 43
Cloning, nucleotide sequence, and expression of the flavodoxin gene from Desulfovibrio vulgaris (Hildenborough); Krey GD et al.; The gene coding for the flavodoxin protein from Desulfovibrio vulgaris (Hildenborough) has been identified, cloned, and sequenced . DNA fragments containing the flavodoxin gene were identified by hybridization of a mixed synthetic heptadecanucleotide probe to Southern blots of SalI-digested genomic DNA . The nucleotide sequences of the probe were derived from the published protein primary structure (Dubourdieu, M., LeGall, J., and Fox, J . L . (1973) Biochem . Biophys . Res . Commun . 52, 1418-1425) . The same oligonucleotide probe was used to screen libraries (in pUC19) containing size-selected SalI fragments . One recombinant, carrying a 1.6-kilobase (kb) insert which strongly hybridizes to the probe, was found to contain a nucleotide sequence which codes for the first 104 residues of the amino-terminal portion of the flavodoxin protein sequence but lacked the remainder of the gene . Therefore, a PstI restriction fragment from this clone was used as a probe to isolate the entire gene from a partial Sau3AI library in Charon 35 . Of the plaques which continued to hybridize strongly to this probe through repeated screenings, one recombinant, containing a 16-kb insert, was further characterized . The entire flavodoxin gene was localized within a 1.4-kb XhoI-SacI fragment of this clone . The complete nucleotide sequence of the structural gene for the flavodoxin protein from Desulfovibrio vulgaris and flanking sequences which may include promoter and regulatory sequences are reported here . The cloned flavodoxin gene was placed behind the hybrid tac promoter for overexpression of the protein in Escherichia coli . Modification to the 5'-end of the gene, including substitutions at the second codon, were required to obtain high levels of expression . The expressed recombinant flavodoxin protein is isolated from E . coli cells as the holoprotein with physical and spectral properties similar to the protein isolated from D . vulgaris . To our knowledge, this is the first example of the expression of a foreign flavodoxin gene in E . coli using recombinant DNA methods.

J Biol Chem, 1988 Oct 25, 263(30), 15277 - 81
Tyrosyl-tRNA synthetase from wheat germ; Quivy JP et al.; Tyrosyl-tRNA synthetase (TyrRS) was purified 5,000-fold from wheat germ extract by ultracentrifugation, precipitation with ammonium acetate, and column chromatography . Under denaturing conditions the enzyme ran as a single band on SDS-polyacrylamide electrophoresis with an apparent Mr of 55,000 . The native molecular weight determined by gel filtration was 110,000, suggesting a quaternary structure of an alpha 2 type for native TyrRS . Purified enzyme activity, based on the aminoacylation reaction, was studied in terms of Mg2+, ATP, pH, and KCl dependence . Optimum concentrations were 6 mM Mg2+, 4 mM ATP, and 200 mM KCl at pH 8 . The Km values for ATP, tyrosine, and tRNA were 40, 3.3, and 1.5 microM, respectively . The instability of the TyrRS activity and the methods used for stabilizing it are discussed . In wheat germ extract we found a second tyrosylating activity that works with Escherichia coli tRNA, but not with wheat germ tRNA . We believe that this enzyme is the mitochondrial tyrosyl-tRNA synthetase of wheat germ.

Nucleic Acids Res, 1988 Oct 25, 16(20), 9821 - 38
Arrangement of Dam methylation sites (GATC) in the Escherichia coli chromosome; Barras F et al.; The occurrence of GATC (Dam-recognition) sites in available E . coli DNA sequences (representing about 2% of the chromosome) has been determined by a simple numerical analysis . Our approach was to analyze the nucleotide composition of nine large sequenced DNA stretches ("cantles") in order to identify patterns of GATC distribution and to rationalize such patterns in biological/structural terms . The following observations were made: (i) In addition to oriC, GATC-rich regions are present in numerous locations . (ii) There is a wide variation in GATC frequency both between and within DNA cantles which led to the identification of a void-cluster pattern of GATC arrangement . The distance between two GATCs was never greater than 2 kb . (iii) GATC sites are found more frequently in translated regions than (in decreasing order) non-coding or non-translated regions . In particular, rRNA and tRNA encoding genes exhibit the lowest GATC content.

Nucleic Acids Res, 1988 Oct 25, 16(20), 9377 - 98
Mispair formation in DNA can involve rare tautomeric forms in the template; Strazewski P; The formation of pyridine-pyrimidine- and pyrimidine-pyrimidine base pairs after in vitro DNA replication with the large fragment of Escherichia coli DNA polymerase I indicates that Watson-Crick-like base pairing between pyrimidine bases can occur in the enzyme due to the presence of the rare tautomers of deoxycytidylate and thymidylate in the template strand . The implications to mispair formation in DNA, such as the difference between the structures of the mispairs during and after replication, are discussed and the possible action of mutagenic DNA protonating and deprotonating agents in vivo is considered.

J Biol Chem, 1988 Oct 25, 263(30), 15407 - 15
Structure and expression of the genes encoding the alpha and beta subunits of yeast phenylalanyl-tRNA synthetase; Sanni A et al.; The two genes FRS1 and FRS2 encoding, respectively, the large (alpha) and small (beta) subunits of cytoplasmic phenylalanyl-tRNA synthetase from bakers' yeast have been cloned and sequenced . The derived protein primary structures are confirmed by peptide sequences evenly distributed along the reading frames . These predict a subunit Mr of 67,347 for alpha and 57,433 for beta, in good agreement with earlier determinations carried out on the purified protein . These subunit sequences have been compared to those of Escherichia coli phenylalanyl-tRNA synthetase as well as to the small beta subunit of the corresponding yeast mitochondrial enzyme; limited but significant homology was found between the two alpha subunits on the one hand and between the three beta subunits on the other hand . The results suggest that these three enzymes, from E . coli, yeast cytoplasm, and yeast mitochondria, have strongly diverged from one another . The initiation sites of transcription have been determined for both yeast genes . Their 5'-upstream regions show no sequence similarities that would have indicated a coordinate control of gene expression at the transcriptional level . Measurements of steady-state levels of FRS-mRNAs in overproducing strains indicate that there is no restriction in mRNA synthesis . Therefore the control of gene expression, leading to a balanced synthesis of alpha and beta subunits, is likely to occur at the translational level.

J Biol Chem, 1988 Oct 25, 263(30), 15513 - 20
Increase of the DNA strand assimilation activity of recA protein by removal of the C terminus and structure-function studies of the resulting protein fragment; Benedict RC et al.; A proteolytic fragment of recA protein, missing about 15% of the protein at the C terminus, was found to promote assimilation of homologous single-stranded DNA into duplex DNA more efficiently than intact recA protein . This difference was not found if Escherichia coli single-stranded DNA binding protein was present . The ATPase activity of both intact recA protein and the fragment was identical . The difference in strand assimilation activity cannot be due to differences in single-stranded DNA affinity, since both the fragment and intact proteins bind to single-stranded DNA with nearly identical affinities . However, the fragment was found to bind double-stranded DNA more tightly and to aggregate more extensively than recA protein; both of these properties may be important in strand assimilation . Aggregation of the fragment was extensive in the presence of duplex DNA under the same condition where recA protein did not aggregate . The double-stranded DNA binding of both recA protein and the fragment responds to nucleotide cofactors in the same manner as single-stranded DNA binding, i.e . ADP weakens and ATP gamma S strengthens the association . The missing C-terminal region of recA protein includes a very acidic region that is homologous to other single-stranded DNA binding proteins and which has been implicated in DNA binding modulation . This C-terminal region may serve a similar function in recA protein, possibly inhibiting double-stranded DNA invasion . The possible role of the enhanced double-stranded DNA affinity of the fragment protein in the mechanism of strand assimilation is discussed.

J Biol Chem, 1988 Oct 25, 263(30), 15694 - 8
Mitochondrial ATP synthase . Overexpression in Escherichia coli of a rat liver beta subunit peptide and its interaction with adenine nucleotides; Garboczi DN et al.; The C-terminal two-thirds of the rat liver ATP synthase beta subunit has been overexpressed and exported to the Escherichia coli periplasm under the direction of the alkaline phosphatase (phoA) promoter and leader peptide . The processed soluble protein contains the 358 amino acids from glutamate 122 to the rat liver beta C-terminal serine 479, including all the regions that have been predicted by chemical and genetic modification studies to be involved in nucleotide, Pi, and Mg2+ binding . Through a simple sequence of Tris/EDTA/lysozyme treatment, osmotic lysis, and alkaline pH washes, the processed beta subunit fragment can be prepared in greater than 95% purity and at a yield of greater than 20 mg/liter of culture . It interacts with 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) which exhibits a strong enhancement of fluorescence upon binding . A similar enhancement is observed upon interaction with TNP-ADP . Enhancement observed with both TNP-nucleotides is markedly reduced by prior addition of either ATP or ADP and almost completely prevented by the ATP synthase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole . Both TNP-ATP and TNP-ADP bind at a stoichiometry of approximately 1 mol of nucleotide/mol of beta subunit fragment . Under the same conditions, TNP-AMP does not exhibit a fluorescence enhancement . This work demonstrates that, in the absence of interaction with other ATP synthase subunits, the rat liver beta subunit sequence from glutamate 122 to the C terminus exhibits no more than one readily detectable nucleotide binding domain . This success in producing a "functional" beta subunit fragment has significance for the pursuit of genetic and physical studies focused on the structure and function of the rat liver ATP synthase beta subunit.

J Biol Chem, 1988 Oct 25, 263(30), 15699 - 704
The nucleotide sequence and characterization of the relA gene of Escherichia coli; Metzger S et al.; The relA gene product of Escherichia coli is known to be responsible for the synthesis of guanosine 3',5'-bispyrophosphate (ppGpp) during the stringent response to amino acid starvation . This report presents the sequence of the relA gene region and assignment of its 743-codon open reading frame by the following criteria: 1) genetic complementation of ppGpp synthesis in a relaxed (relA1) mutant during the stringent response; 2) changes in 3-aminotriazole resistance during growth to mimic a relA+ phenotype; 3) verification of the presence of an amber codon at the normal carboxyl terminus of the relA gene; and 4) immunological assays of expression of the RelA protein . The apparent molecular mass of the cloned relA gene product is calculated to be 83,856 daltons and as visualized by immunoblotting is identical to that of the previously characterized protein . A promoter has been identified that directs relA gene transcription towards the pyrG gene, in a counterclockwise direction on the E . coli chromosome . Genomic Southern blot analyses verify that the relA regions cloned and subjected to nucleotide sequence analysis correspond to homologous regions on the E . coli chromosome.

FEBS Lett, 1988 Oct 24, 239(1), 99 - 104
Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA; Stob S et al.; Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo-CIDNP method . At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised . His-29 polarizations are weaker and broader in HP59 than in HP51 indicating that the C-terminal octapeptide in HP59 adopts a conformation that allows an interaction with His-29 . The photo-CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo-excited flavin . An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo-CIDNP effect of tyrosines 7 and 17 and abolishes the His-29 polarizations . The results are compared with earlier photo-CIDNP measurements on a complex of headpiece with poly{d(AT)} and with a model derived from a 2D NMR study on a lac headpiece-operator complex.

FEBS Lett, 1988 Oct 24, 239(1), 105 - 8
Thermodynamic parameters of the binding of the tight-binding I12X86 lac repressor to operator and non-operator DNA; Maurizot JC et al.; The thermodynamic parameters delta H and delta S corresponding to the binding of the tight-binding double mutant lac repressor I12X86 with operator and non-operator DNA fragments were determined using the nitrocellulose filter binding assay . In both cases the binding processes are entropically driven and accompanied by an unfavorable enthalpy variation . The differences between these parameters and those previously reported for the wild type lac repressor show that the strategy adopted by the mutant to interact with DNA is highly different from that of the wild type repressor and suggest more hydrophobic contacts between the mutant and DNA.

Science, 1988 Oct 21, 242(4877), 423 - 6
Single-chain antigen-binding proteins; Bird RE et al.; Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence . These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli . Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed . These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.

Cell, 1988 Oct 21, 55(2), 235 - 46
Functional expression of a sequence-specific endonuclease encoded by the retrotransposon R2Bm; Xiong YE et al.; A fraction of the 28S ribosomal genes in certain insect species is interrupted by the insertion elements R1 and R2 . These two elements from the silkworm Bombyx mori (R1Bm and R2Bm) are retrotransposons capable of transposing in a highly sequence-specific manner . We report here the functional expression in E . coli of the entire single open reading frame of R2Bm and show that it encodes a double-stranded endo-nuclease (integrase) that can specifically cleave the 28S gene at the R2 insertion site . The resulting cleavage is a 4 bp staggered 5' overhang . Deletion analysis of the 28S gene revealed that the DNA sequence required for specific cleavage is asymmetric with respect to the actual insertion (cleavage) site, with fewer than 10 bp required at one side and at least 24 bp at the other side of the site . A model is proposed based on these and previous data to account for the sequence-specific integration of the R2 retrotransposon.

J Mol Biol, 1988 Oct 20, 203(4), 949 - 59
Mutations in the Tn10 tet repressor that interfere with induction . Location of the tetracycline-binding domain; Smith LD et al.; Tetracycline induces transcription of the Tn10 tetracycline resistance gene (tetA) by binding to the tet repressor, thereby reducing the repressor's affinity for two operator sites that overlap the tet promoters . We characterized mutations in the tet repressor (tetRs mutations) that interfere with induction of tetA expression . The mutations were isolated on multicopy Tn10 tet plasmids by selecting for resistance to the inducer 5a,6-anhydrotetracycline . Under these conditions, maximal induction of tetA expression inhibits the growth of Escherichia coli K-12 . DNA sequence analysis of 25 spontaneous tetRs mutations identified amino acid changes at 13 different positions clustered near the middle of the 207 amino acid residue sequence of tet repressor . This region (residues 64 to 107) is distinct from the bihelical DNA-binding motif of tet repressor (residues 26 to 47) . The capacity of tetRs repressors to bind tet operator DNA and to respond to inducer was examined in vivo in tetA-lacZ fusion strains . In three cases, the capacity of tetRs repressors to bind tetracycline was examined in vitro using cell extracts enriched in repressor . Mutations 64Y (His64----Tyr) and 82H (Asn82----His) reduce the repressor's affinity for tetracycline more than 1000-fold and more than 100-fold, respectively, suggesting that His64 and Asn82 may be part of the inducer-binding site or directly involved in maintaining its conformation . Mutation 103I (Thr103----Ile) reduces the repressor's affinity for tetracycline less than tenfold, yet it interferes with induction to a greater extent than either 64Y or 82H, suggesting that 103I may also reduce the repressor's capacity to undergo a conformational change required for induction . The properties of tetRs mutants suggest that the region of amino acid residues 64 to 107 is involved in inducer binding and in signalling between the inducer-binding and operator-binding domains of the repressor.

Biochim Biophys Acta, 1988 Oct 20, 944(3), 321 - 8
A 2H-NMR study on the glycerol backbone of phospholipids extracted from Escherichia coli grown under high osmotic pressure: evidence for multiconformations of phosphatidylethanolamine; Yoshikawa W et al.; A glycerol-requiring auxotroph was isolated from mutagenized Escherichia coli K-12 UFAts cells . This auxotroph was used for the specific deuteration of E . coli phospholipids . The cells were grown under high osmotic pressure (in the presence of 2.0% KCl) . The membrane had a highly saturated fatty acid composition (76% phosphatidylethanolamine, 20% cardiolipin and 4% phosphatidylglycerol) . The deuterium magnetic resonance spectra of coarse liposomes of the extracted phospholipids with perdeuterated glycerol incorporated into them were measured . To obtain well characterized information, phospholipid mixtures reconstituted from the deuterated and nondeuterated components at the same ratios as in the case of the total extract were used . On the analysis of the spectra, the following conclusions were drawn . (1) The whole polar region of cardiolipin is dynamically symmetric and quite rigid in the presence of phosphatidylethanolamine . (2) Although the quadrupole splittings of the deuterons at the C-2 and C-3 positions of the glycerol backbone were similar to each other, those at the C-1 position for phosphatidylethanolamine and cardiolipin are different, even in the same bilayer . (3) Furthermore, each C-1 deuteron of phosphatidylethanolamine gave rise to a doublet, suggesting the presence of two backbone conformations, between which there is slow exchange . (4) The polar head group of phosphatidylethanolamine interacts with cardiolipin and phosphatidylglycerol in different ways, which could be responsible for the different osmotic properties of the vesicles composed of them.

Nature, 1988 Oct 20, 335(6192), 683 - 9
How eukaryotic transcriptional activators work; Ptashne M; A specific protein, bound to DNA, can activate transcription of a wide array of genes in many eukaryotes . Further analysis suggests a general outline for how eukaryotic transcriptional activators function and are controlled.

J Mol Biol, 1988 Oct 20, 203(4), 861 - 74
Biochemical basis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli; Lavery PE et al.; We compared the biochemical properties of the RecA441 protein to those of the wild-type RecA protein in an effort to account for the constitutive protease activity observed in recA441 strains . The two RecA proteins have similar properties in the absence of single-stranded DNA binding protein (SSB protein), and the differences that do exist shed little light on the temperature-inducible phenotype observed in recA441 strains . In contrast, several biochemical differences are apparent when the two proteins are compared in the presence of SSB protein, and these are conducive to a hypothesis that explains the temperature-sensitive behavior observed in these strains . We find that both the single-stranded DNA (ssDNA)-dependent ATPase and LexA-protease activities of RecA441 protein are more resistant to inhibition by SSB protein than are the activities of the wild-type protein . Additionally, the RecA441 protein is more capable of using ssDNA that has been precoated with SSB protein as a substrate for ATPase and protease activities, implying that RecA441 protein is more proficient at displacing SSB protein from ssDNA . The enhanced SSB protein displacement ability of the RecA441 protein is dependent on elevated temperature . These observations are consistent with the hypothesis that the RecA441 protein competes more efficiently with SSB protein for limited ssDNA sites and can be activated to cleave repressors at elevated temperature by displacing SSB protein from the limited ssDNA that occurs naturally in Escherichia coli . Neither the ssDNA binding characteristics of the RecA441 protein nor the rate at which it transfers from one DNA molecule to another provides an explanation for its enhanced activities, leading us to conclude that kinetics of RecA441 protein association with DNA may be responsible for the properties of the RecA441 protein.

J Mol Biol, 1988 Oct 20, 203(4), 927 - 38
Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III; Pinkney M et al.; Soluble extracts of Escherichia coli capable of carrying out replication of the mini-RK2 derivative pCT461 have been prepared from cells carrying this plasmid or from plasmid-free bacteria . The latter are dependent upon exogenously added plasmid-encoded replication protein (TrfA) and require additional DnaA protein for optimum activity . This dependence upon DnaA was confirmed by the failure of DnaA-deficient cell extracts to support replication of pCT461 in the absence of added DnaA protein . Replication is unidirectional and begins at or near oriV, the vegetative replication origin of RK2 . DNase I protection studies with purified TrfA indicate that this protein acts by binding to short (17 base-pairs) directly repeated DNA sequences present in oriV . The in vitro replication is resistant to rifampicin but can be abolished by antibodies against DnaG protein (E . coli primase) or DnaB protein (helicase) and by DNA gyrase inhibitors . Inhibition by arabinosyl-CTP suggests that DNA polymerase III is responsible for elongation of nascent DNA strands . These results are discussed in relation to the mechanism of RK2 replication and in the context of the host range of the plasmid.

J Mol Biol, 1988 Oct 20, 203(4), 885 - 93
The role of a template sugar-phosphate backbone in the ribosomal decoding mechanism . Comparative study of poly(U) and poly(dT) template activity; Potapov AP et al.; To study the role of a template sugar-phosphate backbone in the ribosomal decoding process, poly(U), poly(dT) and poly(dU)-directed cell-free amino acid incorporation was investigated under the influence of neomycin and high concentrations of Mg2+ . The specificity of a factor-dependent translation system of Escherichia coli was shown to change according to the principle: "either ribo- or deoxyribopolynucleotide messenger" . Poly(dT) is shown to be effectively translated in the absence of elongation factors, both at low (2 degrees C) and high (37 degrees C) temperature . Neomycin inhibits factor-free poly(dT) translation . Little or no poly(U) translation is observed in this system . A chromatographic analysis of the oligophenylalanine residues synthesized seems to show that translocation is the main step responsible for ribosome specificity to the ribo- or deoxyribopolynucleotide template in both factor-dependent and factor-free translation systems.

Biochemistry, 1988 Oct 18, 27(21), 8114 - 21
Photochemical cross-linking of yeast tRNA(Phe) containing 8-azidoadenosine at positions 73 and 76 to the Escherichia coli ribosome; Wower J et al.; The 3'-terminal -A-C-C-A sequence of yeast tRNA(Phe) has been modified by replacing either adenosine-73 or adenosine-76 with the photoreactive analogue 8-azidoadenosine (8N3A) . The incorporation of 8N3A into tRNA(Phe) was accomplished by ligation of 8-azidoadenosine 3',5'-bisphosphate to the 3' end of tRNA molecules which were shortened by either one or four nucleotides . Replacement of the 3'-terminal A76 with 8N3A completely blocked aminoacylation of the tRNA . In contrast, the replacement of A73 with 8N3A has virtually no effect on the aminoacylation of tRNA(Phe) . Neither substitution hindered binding of the modified tRNAs to Escherichia coli ribosomes in the presence of poly(U) . Photoreactive tRNA derivatives bound noncovalently to the ribosomal P site were cross-linked to the 50S subunit upon irradiation at 300 nm . Nonaminoacylated tRNA(Phe) containing 8N3A at either position 73 or position 76 cross-linked exclusively to protein L27 . When N-acetylphenylalanyl-tRNA(Phe) containing 8N3A at position 73 was bound to the P site and irradiated, 23S rRNA was the main ribosomal component labeled, while smaller amounts of the tRNA were cross-linked to proteins L27 and L2 . Differences in the labeling pattern of nonaminoacylated and aminoacylated tRNA(Phe) containing 8N3A in position 73 suggest that the aminoacyl moiety may play an important role in the proper positioning of the 3' end of tRNA in the ribosomal P site . More generally, the results demonstrate the utility of 8N3A-substituted tRNA probes for the specific labeling of ribosomal components at the peptidyltransferase center.

Biochemistry, 1988 Oct 18, 27(21), 8081 - 7
Differential scanning calorimetric study of the thermal denaturation of aspartate transcarbamoylase of Escherichia coli; Edge V et al.; The thermal denaturation of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of high-sensitivity differential scanning calorimetry (DSC) . As previously reported {Vickers, K.P., Donovan, J.W., & Schachman, H.K . (1978) J . Biol . Chem . 253, 8493-8498}, the denaturational endotherm consists of two peaks, the lower of which is due to denaturation of the three regulatory, r2, subunits while the upper involves the two catalytic, c3, subunits . The temperature of maximal excess apparent specific heat, tm, of the lower peak is raised from the value of 51.4 degrees C for the isolated subunit to 66.8 degrees C as a result of subunit interactions, whereas tm for the c3 peak is essentially the same in the isolated subunit and in the holoenzyme, indicating that the denatured r2 subunits do not interact with the c3 subunits . The total specific denaturational enthalpy for c6r6, 4.83 +/- 0.16 cal g-1, is significantly larger than the weighted mean, 4.08 cal g-1, of the enthalpies for c3 and r2 . The fact that no endotherm is observed when previously scanned protein is rescanned indicates that the denaturation is irreversible, as is also the case with the r2 and c3 subunits . Empirical justification for analyzing the data in terms of equilibrium thermodynamics is cited . The observed DSC curves can be expressed within experimental uncertainty as the sum of five sequential two-state steps . The value of t 1/2, the temperature of half-completion, for each step increases with increasing protein concentration, indicating that some dissociation of the protein takes place during denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Oct 18, 27(21), 8050 - 6
Interactive binding between the substrate and allosteric sites of carbamoyl-phosphate synthetase; Kasprzak AA et al.; The interaction between Escherichia coli carbamoyl-phosphate synthetase (CPS) and a fluorescent analogue of an allosteric effector molecule, 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP), has been detected by using fluorescence techniques and kinetic measurements . From fluorescence anisotropy titrations, it was found that epsilon-AMP binds to a single site on CPS with Kd = 0.033 mM . The nucleotide had a small activating effect on the rate of synthesis of carbamoyl phosphate but had no effect on the Km for ATP . To test whether epsilon-AMP binds to an allosteric site, allosteric effectors (UMP, IMP, and CMP), known to bind at the UMP/IMP site, were added to solutions containing the epsilon-AMP-CPS complex . With addition of these effector molecules, a progressive decrease of the fluorescence anisotropy was observed, indicating that bound epsilon-AMP was displaced by the allosteric effectors examined . From these titrations, the dissociation constants for UMP, IMP, CMP, ribose 5-phosphate, 2-deoxyribose 5-phosphate, and orthophosphate were determined . When MgATP, a substrate, was employed as a titrant, the observed decrease in anisotropy was consistent with the formation of a ternary complex (epsilon-AMP-CPS-MgATP) . The effect of ATP binding, monitored at the allosteric site, was magnesium dependent, and free magnesium in solution was required to obtain a hyperbolic binding isotherm . Solvent accessibility of epsilon-AMP in binary (epsilon-AMP-CPS) and ternary (epsilon-AMP-CPS-MgATP) complexes was determined from acrylamide quenching, showing that the base of epsilon-AMP is well shielded from the solvent even in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Oct 18, 27(21), 8007 - 14
Serine hydroxymethyltransferase: mechanism of the racemization and transamination of D- and L-alanine; Shostak K et al.; The reaction specificity and stereochemical control of Escherichia coli serine hydroxymethyltransferase were investigated with D- and L-alanine as substrates . An active-site H228N mutant enzyme binds both D- and L-alanine with Kd values of 5 mM as compared to 30 and 10 mM, respectively, for the wild-type enzyme . Both wild-type and H228N enzymes form quinonoid complexes absorbing at 505 nm by catalyzing the loss of the alpha-proton from both D- and L-alanine . Racemization and transamination reactions were observed to occur with both alanine isomers as substrates . The relative rates of these reactions are quinonoid formation greater than alpha-proton solvent exchange greater than racemization greater than transamination . The observation that the rate of quinonoid formation with either alanine isomer is an order of magnitude faster than solvent exchange suggests that the alpha-protons from both D- and L-alanine are transferred to base(s) on the enzyme . The rate of racemization is 2 orders of magnitude slower than the formation of the quinonoid complexes . This latter difference in rate suggests that the quinonoid complexes formed from D- and L-alanine are not identical . The difference in structure of the two quinonoid complexes is proposed to be the active-site location of the alpha-protons lost from the two alanine isomers, rather than two orientations of the pyridoxal phosphate ring . The results are consistent with a two-base mechanism for racemization.

Biochemistry, 1988 Oct 18, 27(21), 7984 - 90
Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase; Scott AI et al.; The active site of porphobilinogen (PBG)1 deaminase (EC 4.3.1.8) from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-224, one of the two cysteine residues conserved in E . coli and human deaminase . By use of a hemA- strain of E . coli the enzyme was enriched from {5-13C}ALA and examined by 1H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked head to tail and terminating in a CH2-S bond to a cysteine residue . Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I . The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-{2,11-13C2}PBG reveals that the aninomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the alpha-free pyrrole . NMR spectroscopy of the ES2 complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the alpha-free pyrrole position of the enzyme . A mechanistic rationale for deaminase is presented.

Biochemistry, 1988 Oct 18, 27(21), 8028 - 33
On the intermediacy of carboxyphosphate in biotin-dependent carboxylations; Ogita T et al.; In the ATP-dependent carboxylation of biotin that is catalyzed by most biotin-dependent carboxylases, a fundamental mechanistic question is whether the ATP activates bicarbonate (via the formation of carboxyphosphate as an intermediate) or whether the ATP activates biotin (via the formation of O-phosphobiotin) . We have resorted to three mechanistic tests using the biotin carboxylase subunit of acetyl-CoA carboxylase from Escherichia coli: positional isotope exchange, intermediate trapping, and 18O tracer experiments on the ATPase activity . First, no catalysis of positional isotope exchange in adenosine 5'-{( alpha, beta-18O, beta, beta-18O2}triphosphate) was observed when either biotin or bicarbonate was absent, nor was any exchange seen in the presence of both N-1-methylbiotin and bicarbonate . Second, the putative carboxyphosphate intermediate could not be trapped as its trimethyl ester, under conditions of incubation and analysis where the authentic triester was shown to be adequately stable . In the third test, however, we showed that the ATPase activity of biotin carboxylase that is seen in the absence of biotin, an activity that is known to parallel the normal carboxylase reaction when biotin is present, occurs with the transfer of an 18O label directly from {18O}bicarbonate into the product Pi . This result suggests that the bicarbonate-dependent biotin-independent ATPase reaction catalyzed by biotin carboxylase goes via carboxyphosphate and that the carboxylation of biotin itself may proceed analogously.

J Biol Chem, 1988 Oct 15, 263(29), 15050 - 5
Identification of the differentiation-associated p93 tyrosine protein kinase of HL-60 leukemia cells as the product of the human c-fes locus and its expression in myelomonocytic cells; Smithgall TE et al.; A differentiation-associated 93-kDa tyrosine kinase (p93) was purified previously from the human promyelocytic leukemia cell line HL-60 . The present study conclusively identifies p93 as the c-fes proto-oncogene product and shows that expression of p93c-fes and its associated tyrosine kinase activity are marked in mature granulocytes, monocytes, and human myeloid leukemia cell lines . Antisera to peptides obtained by expression of c-fes cDNA fragments in Escherichia coli reacted strongly with p93 purified from HL-60 cells . Western blots using one of these antisera demonstrated high levels of p93c-fes protein in normal human granulocytes and monocytes, as well as the cell lines KG-1, THP-1, HEL, and U-937, all of which can be induced to differentiate along the myelomonocytic pathway . Conversely, in cell lines resistant to myeloid differentiation, p93c-fes expression was either very low or absent . Expression of immunoreactive p93c-fes in these cell lines showed a strong positive correlation with p93c-fes tyrosine kinase activity, which was measured in cell extracts using a nondenaturing gel assay . Finally, the expression of p93c-fes, its tyrosine kinase activity, and the binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) were all coordinately increased in HL-60 cells treated with the granulocytic differentiation inducer dimethyl sulfoxide, while all three parameters were low in untreated or differentiation-resistant HL-60 cells . These results suggest that expression of p93c-fes tyrosine kinase activity may be an essential component of myeloid differentiation and responsiveness to granulocyte-macrophage colony-stimulating factor.

J Biol Chem, 1988 Oct 15, 263(29), 14629 - 33
Activation of the ompC gene by the OmpR protein in Escherichia coli . The cis-acting upstream sequence can function in both orientations with respect to the canonical promoter; Maeda S et al.; Expression of the ompC gene coding for an outer membrane protein of Escherichia coli is regulated by a transcriptional activation mechanism that requires the ompR gene product that acts on nucleotide sequences located upstream of the -35 and -10 regions of the ompC promoter . Using an ompC-lacZ fusion gene, the orientation of the cis-acting upstream sequence (OmpR-binding site) with respect to the canonical -35 and -10 regions (RNA polymerase-binding site) was changed . The ompC gene was activated in an OmpR-dependent manner even when the upstream sequence was in the reverse orientation with respect to the -35 and -10 regions, providing that these two DNA elements were aligned stereospecifically on the DNA helix . Evidence is presented that the upstream sequence can increase the transcription efficiency of the ompC promoter in a manner relatively independent of its orientation with respect to the -35 and -10 regions.

Gene, 1988 Oct 15, 70(1), 57 - 65
Expression of the cDNA for mouse beta-nerve growth factor protein in Escherichia coli; Hu GL et al.; The cDNA coding for the mature beta-nerve growth factor (beta-NGF) has been cloned into a plasmid expression vector, pAS1, and expressed in Escherichia coli . The cDNA fragment in pAS1 is under the control of strong phage transcriptional and translational initiation elements that provide for regulated expression of cloned genes in E . coli . The protein, produced in bacteria at a level of about 0.0005-0.1% of cell protein, was purified by ammonium sulfate precipitation and ion exchange chromatography . The recombinant NGF was biologically active in the PC12 neurite outgrowth assay, and formed a band at Mr of about 11,000 to 12,000, when electrophoresed on sodium dodecyl sulfate-polyacrylamide gel and Western-blotted.

Biochem J, 1988 Oct 15, 255(2), 737 - 40
The identity of Escherichia coli bacterioferritin and cytochrome b1; Smith JM et al.; Two different procedures were used to prepare pure samples of 'cytochrome b1' (column chromatography) and 'bacterioferritin' (immunoprecipitation) from Escherichia coli K-12 strain CA265 . Both were crystallized, and X-ray-crystallographic data were compared with those from the bacterioferritin of E . coli strain W3300 used as a standard . We conclude that 'cytochrome b1' and 'bacterioferritin' are identical.

Biochem J, 1988 Oct 15, 255(2), 581 - 8
Dihydropteridine reductase from Escherichia coli; Vasudevan SG et al.; A dihydropteridine reductase from Escherichia coli was purified to apparent homogeneity . It is a dimeric enzyme with identical subunits (Mr 27000) and a free N-terminal group . It can use NADH (Vmax./Km 3.36 s-1) and NADPH (Vmax./Km 1.07 s-1) when 6-methyldihydro-(6H)-pterin is the second substrate, as well as quinonoid dihydro-(6H)-biopterin (Vmax./Km 0.69 s-1), dihydro-(6H)-neopterin (Vmax./Km 0.58 s-1), dihydro-(6H)-monapterin 0.66 s-1), 6-methyldihydro-(6H)-pterin and cis-6,7-dimethyldihydro-(6H)-pterin (Vmax./Km 0.66 s-1) when NADH is the second substrate . The pure reductase has a yellow colour and contains bound FAD . The enzyme also has pterin-independent NADH and NADPH oxidoreductase activities when potassium ferricyanide is the electron acceptor.

Eur J Biochem, 1988 Oct 15, 177(1), 81 - 90
Mutants with base changes at the 3'-end of the 16S RNA from Escherichia coli . Construction, expression and functional analysis; Rottmann N et al.; The functionally important 3' domain of the ribosomal 16S RNA was altered by in vitro DNA manipulations of a plasmid-encoded 16S RNA gene . By in vitro DNA manipulations two double mutants were constructed in which C1399 was converted to A and G1401 was changed to either U or C and a single point mutant was made wherein G1416 was changed to U . Only one of the mutated rRNA genes could be cloned in a plasmid under the control of the natural rrnB promoters (U1416) whereas all three mutations were cloned in a plasmid under the control of the lambda PL promoter . In a strain coding for the temperature-sensitive lambda repressor cI857 the mutant RNAs could be expressed conditionally . We could show that all three mutant rRNAs were efficiently incorporated into 30S ribosomes . However, all three mutants inhibited the formation of stable 70S particles to various degrees . The amounts of mutated rRNAs were quantified by primer extension analysis which enabled us to assess the proportion of the mutated ribosomes which are actively engaged in in vivo protein biosynthesis . While ribosomes carrying the U1416 mutation in the 16S RNA were active in vivo a strong selection against ribosomes with the A1399/U1401 mutation in the 16S RNA from the polysome fraction is apparent . Ribosomes with 16S RNA bearing the A1399/C1401 mutation did not show a measurable protein biosynthesis activity in vivo . The growth rate of cells harbouring the different mutations reflected the in vivo translation capacities of the mutant ribosomes . The results underline the importance of the highly conserved nucleotides in the 3' domain of the 16S RNA for ribosomal function.

Eur J Biochem, 1988 Oct 15, 177(1), 153 - 8
Primary structures of Escherichia coli pyruvate formate-lyase and pyruvate-formate-lyase-activating enzyme deduced from the DNA nucleotide sequences; Rodel W et al.; The structural gene of pyruvate formate-lyase (pfl) and that of pyruvate-formate-lyase-activating enzyme were shown to be adjacent on the chromosomal map of Escherichia coli . DNA sequencing was performed along a stretch of 3592 nucleotides to obtain the amino acid sequences of both proteins . The derived primary structures (759 and 245 residues) were confirmed by partial structure analyses on the purified proteins . The open reading frames are separated by a 194-nucleotide stretch, and their flanking regions include signal elements that are compatible with separate control of protein synthesis from the two genes.

J Biol Chem, 1988 Oct 15, 263(29), 15226 - 36
The two-component, ATP-dependent Clp protease of Escherichia coli . Purification, cloning, and mutational analysis of the ATP-binding component; Katayama Y et al.; The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity . ClpA is a protein with subunit Mr 81,000 . It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP . The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C . ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR . Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene . The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA . The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E . coli by homologous recombination . The clpA gene was mapped to 19 min on the E . coli chromosome . Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity . clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E . coli proteins SulA, RcsA, and glutamate dehydrogenase . The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation . Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.

J Biol Chem, 1988 Oct 15, 263(29), 15196 - 204
Messenger RNA orientation on the ribosome . Placement by electron microscopy of antibody-complementary oligodeoxynucleotide complexes; Olson HM et al.; Messenger RNA orients on the small ribosomal subunit by base pairing with a complementary sequence in ribosomal RNA . We have positioned this ribosomal RNA segment and thus oriented the mRNA using a new technique--localization of an antibody-recognizable modified complementary oligodeoxynucleotide by electron microscopy . A synthetic oligodeoxynucleotide complementary to the message-positioning ribosomal RNA sequence was modified at either or both ends with different antigenic markers . Electron microscopy of subunit-oligodeoxynucleotide-antibody complexes allowed separate placement of each terminal marker of the oligodeoxynucleotide probe . The 5'-end of the complementary sequence contacts the subunit at the platform tip (rRNA nucleotide 1542) . The message then extends along the interior side of the platform to the level of the fork of the cleft separating the platform from the subunit body, and displaced slightly to the convex side of the platform (rRNA nucleotide 1531) . Based on our results and data from other laboratories, we propose a model for the positioning of messenger RNA on the 30 S subunit.

J Biol Chem, 1988 Oct 15, 263(29), 15166 - 75
In vivo and in vitro structural analysis of the rplJ mRNA leader of Escherichia coli . Protection by bound L10-L7/L12; Climie SC et al.; The rplJL-rpoBC operon of Escherichia coli is regulated in part at the level of translation by an autogenous mechanism (feedback regulation) that involves ribosomal protein L10-L7/L12 . Feedback regulation occurs as the result of L10-L7/L12 binding to a site on the untranslated leader region of the rplJ mRNA that is located more than 100 nucleotides upstream from the translation start site . Previous studies have indicated that the secondary structure of the rplJ leader region is important for efficient translation and feedback regulation . We have done chemical modification experiments to examine the secondary structure of approximately 200 nucleotides of the rplJ leader region, and we propose a secondary structure that is consistent with the experimental data . RNA structure was probed in vitro by treating samples of total cellular RNA with diethyl pyrocarbonate and in vivo by treating log-phase cultures with dimethyl sulfate . Modified bases were detected by primer extension using three different oligonucleotide primers . The proposed structure includes five double-stranded regions designated I to V, separated by single-stranded segments numbered 1 to 5 . We have also identified specific nucleotides in the rplJ mRNA leader that are protected by purified L10-L7/L12 from methylation by dimethyl sulfate in vitro . The protected bases are located within a bulge-loop of region IV, a portion of the mRNA that has been shown genetically to be necessary for feedback regulation.

J Biol Chem, 1988 Oct 15, 263(29), 15064 - 70
Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers; Knauf MJ et al.; We have examined the effects of a variety of chemical modifications to recombinant human interleukin-2 (rIL-2) on its pharmacokinetic behavior in rats . Unmodified rIL-2 is cleared from plasma with half-lives of 3 and 44 min for the alpha and beta phases . Modification of rIL-2 with monomethoxy polyethylene glycol or polyoxyethylated glycerol increased the half-lives as much as 20-fold, although the volume of distribution remained unchanged at 88 +/- 13 ml/kg . The clearance rates correlated with the effective molecular size of the modified protein determined by size exclusion chromatography . Clearance decreased rapidly as the effective molecular size increased from 19.5 to 70 kDa, whereas above 70 kDa the clearance decreased more slowly . This abrupt change at 70 kDa may be related to the permeability threshold of the kidney glomerular membrane which retains proteins larger than albumin in the plasma . Using the relationship between clearance and effective molecular size, the clearance rates of mixtures of modified rIL-2 could be predicted based on their average effective molecular size . Since the effectiveness of rIL-2 therapy is likely to be related to its pharmacokinetic behavior, the ability to design a molecule with a predictable time course in plasma provides a means to study this relationship.

J Biol Chem, 1988 Oct 15, 263(29), 15000 - 7
What is the role of the transit peptide in thylakoid integration of the light-harvesting chlorophyll a/b protein?
Viitanen PV, Doran ER, Dunsmuir P.
Whereas it is widely accepted that the transit peptide of the precursor for the light-harvesting chlorophyll a/b protein (preLHCP) is responsible for targeting this polypeptide to chloroplasts, the signals which govern its intraorganellar targeting appears to be transit peptide-mediated for plastocyanin (Smeekins, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P . (1986) Cell 46, 365-375) and several other nuclear-encoded, thylakoid luminal proteins . To determine whether a similar mechanism operates for LHCP (an integral thylakoid protein), we have used oligonucleotide-directed mutagenesis to delete the proposed transit sequence from a petunia precursor of this polypeptide . Intact preLHCP and the deletion mutant product have been expressed in vitro, and their abilities to integrate into purified thylakoids have been compared . We have found that both polypeptides insert into thylakoids correctly, provided the latter are supplemented with a membrane-free stromal extract and Mg.ATP . Our results clearly demonstrate that whereas the transit peptide is required for transport into chloroplasts, thylakoid integration of preLHCP is determined by mature portions of the polypeptide . In addition, we note that transit peptide removal has little effect on the apparent solubility of the in vitro translation products.

J Biol Chem, 1988 Oct 15, 263(29), 14900 - 5
Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12; Milner JL et al.; Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R . L., McClellan, D . J., Milner, J . L., and Wood, J . M . (1986) J . Bacteriol . 166, 253-259) . Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress . That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP . Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift . In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase . Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake . These results suggested that proline porter II was respiration-dependent and probably ion-linked . Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient . Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles . Thus, activation of proline porter II was reversible . It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift.

J Biol Chem, 1988 Oct 15, 263(29), 14859 - 67
Purification and properties of lipid A disaccharide synthase of Escherichia coli; Radika K et al.; Lipid A disaccharide synthase of Escherichia coli catalyzes the reaction 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2',3'-diacyl-GlcN (beta,1'----6)2,3-diacyl-GlcN-1-P + UDP (Ray, B . L., Painter, G., and Raetz, C . R . H . (1984) J . Biol . Chem . 259, 4852-4859) . Using a strain that overproduces the enzyme about 200-fold we have devised a simple purification to near homogeneity, utilizing two types of dye-ligand resins and heparin-agarose . The overall purification starting with membrane-free extracts was 54-fold (16,000-fold relative to wild-type extracts) with a 31% yield . The subunit molecular mass determined by sodium dodecyl sulfate gel electrophoresis is approximately 42,000 daltons, and the native enzyme appears to be a dimer . The amino-terminal sequence is (X)-(Thr)-Glu-Gln-(X)-Pro-Leu-Thr-Ie-Ala..., consistent with the results predicted from the DNA sequence, Met-Thr-Glu-Gln-Arg-Pro-Leu-Thr-Ile-Ala... . The purified enzyme displays a strong kinetic preference for sugar substrates bearing two fatty acyl moieties, but it is, nevertheless, very useful for the semisynthetic preparation of many lipid A analogs . Gel filtration studies demonstrate that the natural substrates (2,3-diacyl-GlcN-1-P and UDP-2,3-diacyl-GlcN) form micelles (n approximately equal to 300), rather than bilayers, under conditions used to assay the enzyme . Unlike most enzymes of glycerophospholipid synthesis, the lipid A disaccharide synthase does not require the presence of a detergent for catalytic activity . At 1 mM UDP-2,3-diacyl-GlcN the Vmax and Km values for 2,3-diacyl-GlcN-1-P are 14,028 +/- 513 nmol/min/mg and 0.27 +/- 0.02 mM . When 2,3-diacyl-GlcN-1-P is maintained at 1 mM, they are 12,368 +/- 472 nmol/min/mg and 0.11 +/- 0.01 mM for UDP-2,3-diacyl-GlcN.

J Biol Chem, 1988 Oct 15, 263(29), 1480 - 11
Induction of the manganese-containing superoxide dismutase in Escherichia coli is independent of the oxidative stress (oxyR-controlled) regulon; Bowen SW et al.; The synthesis of manganese-superoxide dismutase in response to hydrogen peroxide and to paraquat was examined in strains of Escherichia coli with different mutations in the oxyR gene . Hydrogen peroxide treatment did not induce manganese-superoxide dismutase, but did induce the oxyR regulon . Paraquat induced this enzyme in a strain compromised in its ability to induce the defense response against oxidative stress (oxyR deletion) as well as in a strain that is constitutive and overexpresses the oxyR regulon . Catalase (HPI), but not manganese-superoxide dismutase, was over-expressed under anaerobic conditions in a strain harboring a constitutive oxyR mutation . The data clearly demonstrate that the induction of manganese-superoxide dismutase is independent of the oxyR-controlled regulon.

J Biol Chem, 1988 Oct 15, 263(29), 14790 - 3
Retardation of folding as a possible means of suppression of a mutation in the leader sequence of an exported protein; Liu GP et al.; We have proposed (Randall, L . L., and Hardy, S . J . S . (1986) Cell 46, 921-928) that during export of protein from Escherichia coli, there is a kinetic partitioning between the pathway that leads to productive translocation and the pathway that leads to folding of precursors into a stable conformation that is incompatible with export . This model predicts that a decrease in rate along the productive pathway resulting from a defect in the leader sequence could be partially overcome by slowing the folding of the precursor and thereby increasing the time during which that polypeptide would be competent to enter the export pathway . Here it is shown that a change in the mature portion of maltose-binding protein that is known to suppress a mutation in the leader sequence (Cover, W . H., Ryan, J . P., Bassford, P . J., Jr., Walsh, K . A., Bollinger, J., and Randall, L . L . (1987) J . Bacteriol . 169, 1794-1800) also decreases the rate of folding of the precursor.

J Biol Chem, 1988 Oct 15, 263(29), 14784 - 9
Mutagenesis by transient misalignment; Kunkel TA et al.; Based upon a consideration of two mutational hot spots produced during DNA synthesis by a eukaryotic DNA repair polymerase, we suggested that certain base substitution errors result not from direct miscoding but from correct coding by a transiently misaligned template-primer (Kunkel, T . A., and Alexander, P . S . (1986) J . Biol . Chem . 261, 160-166) . This model, which we called dislocation mutagenesis, has been directly tested . Introducing a single, phenotypically silent G----A base change into the template switches the base substitution specificity at the immediately adjacent hot spot, a T residue, from T----G transversions to T----A transversions . The cumulative change in frequency, represented by the disappearance of the T----G events and the appearance of the T----A events, is greater than 300-fold . These data demonstrate that during DNA synthesis in vitro, a base at one position can code a mutation at another position . This mechanism can operate over greater distances to produce complex mutations as well . We present one example in which a 123-base deletion containing three base changes at one end of the deletion can be precisely explained by transient misalignment . It remains to be established whether mutagenesis by dislocation operates in vivo to produce biologically significant changes in genetic information.

J Biol Chem, 1988 Oct 15, 263(29), 14769 - 75
Characterization of the cyn operon in Escherichia coli K12; Sung YC et al.; Escherichia coli can overcome the toxicity of environmental cyanate by hydrolysis of cyanate to ammonia and bicarbonate . This reaction is catalyzed by the enzyme cyanase, encoded by the cynS gene . The nucleotide sequence of cynS has been reported (Sung, Y.-c., Anderson, P . M., and Fuchs, J . A . (1987) J . Bacteriol . 169, 5224-5230) . The nucleotide sequence of the complete cyn operon has now been determined . The cyn operon is approximately 2600 base pairs and includes cynT, cynS, and cynX, which encode cyanate permease, cyanase, and a protein of unknown function, respectively . Two cyanate-inducible transcripts of 1500 and 2500 nucleotides, respectively, were detected by Northern blot analysis . S1 nuclease mapping experiments indicated that two different cyn mRNAs have a common 5'-end and two different 3'-ends . One 3'-end was located within the coding region of cynX, whereas the other 3'-end includes the entire DNA sequence of cynX . The longer transcript contained 98 nucleotides complementary to lac mRNA produced by the predominant lac transcription termination sequence . Termination vectors were used to show that both 3'-ends were generated by sequences that caused transcriptional termination in vivo . Expression vectors were used to demonstrate that a protein corresponding to the expected size was synthesized from the DNA fragment containing the open reading frame designated cynX . The predicted amino acid sequence of cynX indicates that it is a very hydrophobic protein . The level of cynX expression was significantly less than that of cynT or cynS expression.

J Biol Chem, 1988 Oct 15, 263(29), 14724 - 31
DNA sequences of random origin as probes of Escherichia coli promoter architecture; Horwitz MS et al.; In order to better understand the role of the -35 sequence motif in transcription initiation by Escherichia coli sigma 70 RNA polymerase holoenzyme, -35 promoter elements of limited nucleotide composition have been selected from random DNA sequences . Functional promoter elements have been identified that are composed of just two nucleotide species (A,T; G,C; T,C; and T,G) and one nucleotide species (poly(dT)) . From this study of 81 promoter mutations, two conclusions can be made . First, given a population of random DNA sequences, there exist sequences that form stronger -35 promoter elements than the consensus sequence . These sequences lack some features of the consensus sequence and reveal unexpected homology in the ordinarily nonconserved spacer sequence . Second, the sequence requirements at the -35 site may suggest a possible role for DNA secondary structure in the recognition of promoters by RNA polymerase.

J Biol Chem, 1988 Oct 15, 263(29), 14661 - 8
Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin; Holland MM et al.; An expression plasmid containing a hybrid gene encoding a protein having the primary amino acid sequence of the adenylate cyclase from Escherichia coli was constructed . When the gene was induced, the adenylate cyclase could be expressed at high levels in a cya- strain of E . coli . The majority of the enzymatic activity and protein (having a molecular weight of 95,000) induced was insoluble . However, treatment of the insoluble fraction of cell lysates with trypsin resulted in both an increase in and solubilization of the total amount of adenylate cyclase activity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the soluble protein produced by treatment with trypsin revealed a polypeptide having a molecular weight of 30,000 . This soluble, catalytically active fragment of adenylate cyclase was purified and subjected to amino-terminal sequence analyses; two amino-terminal sequences were identified beginning at residue 82 and at residue 342 of the intact enzyme . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified fragment followed by either silver or Coomassie Blue staining revealed the presence of only a single polypeptide having a molecular weight of 30,000; a short oligopeptide associated with the amino terminus at residue 342 could not be detected . Site-directed mutagenesis was used to place a stop codon at residue 341; the truncated enzyme was catalytically active, so the short oligopeptide is not necessary for catalysis . The Km for ATP, the Ka for Mg2+, and the Vmax determined for the product containing the 30,000-dalton fragment were similar to the values reported for the intact enzyme from E . coli.

J Biol Chem, 1988 Oct 15, 263(29), 14621 - 4
Receptor-mediated gene delivery and expression in vivo; Wu GY et al.; A soluble DNA carrier system was used to target a foreign gene specifically to liver in vivo via asialoglycoprotein receptors . The DNA carrier was prepared consisting of a galactose-terminal (asialo-)glycoprotein, asialoorosomucoid (AsOR), covalently linked to poly-L-lysine . The conjugate was complexed in a 2:1 molar ratio (based on AsOR content of the conjugate) to the plasmid, pSV2 CAT, containing the gene for the bacterial enzyme chloramphenicol acetyltransferase (CAT) . Intravenous injection of {32P}plasmid DNA complexed to the carrier demonstrated specific hepatic targeting with 85% of the injected counts taken up by the liver in 10 min compared to only 17% of the counts when the same amount of {32P}DNA alone was injected under identical conditions . Targeted pSV2 CAT DNA was detected at a level of 1.0 ng/g liver by hybridization of a {32P}pSV2 CAT cDNA probe to rat liver DNA extracted 24 h after intravenous injection of AsOR-poly-L-lysine-DNA complex containing 1.0 mg of DNA . Homogenates of livers taken 24 h after injection of the complex revealed that the targeted CAT gene was functional as reflected by the detection of CAT activity (approximately 4 microunits/mg protein) . Livers from control animals that received individual constituents of the complex produced no CAT activity . Simultaneous injection of excess AsOR to compete with the AsOR-poly-L-lysine-DNA complex for uptake by the liver inhibited CAT gene expression . Assays for CAT activity in other organs (spleen, kidney, lungs) failed to demonstrate any activity in these organs . This new soluble DNA carrier system can permit targeted delivery of foreign genes specifically to liver with resultant foreign gene expression in vivo.

J Biol Chem, 1988 Oct 15, 263(29), 15094 - 103
Studies on the mechanism of Escherichia coli DNA polymerase I large fragment . Effect of template sequence and substrate variation on termination of synthesis; Abbotts J et al.; Termination of Escherichia coli DNA polymerase I large fragment after processive synthesis on natural and other well-defined template.primer systems has been examined . We found that after any given deoxynucleoside monophosphate incorporation termination occurs in a nonrandom manner with phi X174 DNA as template: Termination is much more likely at some nucleotide residues along the template than at others . Analysis of these stronger termination sites indicates that the template base:incoming nucleotide combination influences termination . Introduction of a double-stranded region along the phi X174 template induces termination, and reducing dNTP concentrations or substituting 2'-deoxynucleoside 5'-O-(1-thio)triphosphate substrates also increases termination . Observations with the phi X174 DNA template system were extended with a defined template containing 1 inosine residue in an otherwise d(T)n homopolymer . Termination at the I residue is modulated by dCTP and decreases as dCTP concentration increases . A similar relationship is seen with the dCTP (1-thio) derivative, but termination is higher at given concentrations of this derivative than with dCTP . Pyrophosphate decreases general processivity in this system, but does not counteract the effect of increasing dCTP . Hill plot analysis of the dCTP effect in the inosine-containing template system gave a linear plot with Hill coefficient of 0.34, suggesting that dCTP influences termination at several steps in the polymerase reaction scheme . Substituting a methylated template base for I also increased termination, producing very strong blocks to processive synthesis . The results are consistent with a model in which termination occurs with several enzyme forms that are in equilibrium in an ordered catalytic mechanism.

Gene, 1988 Oct 15, 70(1), 161 - 70
Insertional restoration of beta-galactosidase alpha-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes; Cronan JE Jr et al.; The assembly of synthetic genes from oligodeoxynucleotides can be an inefficient process . Upon ligation of a synthetic assembly into a plasmid vector and transformation of an Escherichia coli host, it is often found that only a minor fraction of the putative recombinant plasmids contains synthetic sequences . Moreover, the synthetic sequences cloned are often altered versions of those originally designed . We have designed a biological test to detect those plasmids that contain synthetic sequences of the proper length, termini and reading frame . The test is the reversal of the beta-galactosidase alpha-complementation (blue-to-white) test used to detect the insertion of DNA segments into the polylinker sequences of the phage M13 mp, plasmids pUC, and related vectors . We begin with a modified vector defective in alpha-complementation and use insertion of the synthetic DNA segment to restore alpha-complementation . The alpha-complementation activity of the original vector (e.g., pUC18) was first abolished by a frameshift or DNA insertion within the polylinker sequence of the lacZ' gene segment . The alpha-complementation was then restored by insertion of the synthetic DNA sequence between the cohesive ends generated by digestion of two polylinker restriction sites . Formation of blue colonies requires the insertion of a DNA segment of appropriate length and termini to reconstruct the lacZ' open reading frame and thus is much more selective than the usual insertional inactivation strategy . We show that this 'insertional restoration' screening method markedly enhances the proper assembly of synthetic genes and describe manipulations to readily and reliably frameshift various polylinker sequences.

J Biol Chem, 1988 Oct 15, 263(29), 15110 - 7
Use of psoralen-modified oligonucleotides to trap three-stranded RecA-DNA complexes and repair of these cross-linked complexes by ABC excinuclease; Cheng S et al.; A series of site-specifically cross-linked, three-stranded complexes has been prepared using the RecA protein, form I (covalently closed and negatively supercoiled) pUC19 plasmid, and eight psoralen-monoadducted oligonucleotides between 30 and 107 residues in length . Complexes formed much less efficiently if linearized pUC19 was used as the duplex substrate . Quantitative analysis indicates that although RecA is able to utilize a 30-mer in its homologous pairing reaction, incorporation at 50% efficiency requires a single-stranded substrate at least 50 residues long . These three-stranded complexes have been used to study the action mechanism for cross-link repair by ABC excinuclease, and the results are consistent with the recent model of Van Houten, B., Gamper, H., Holbrook, S . R., Hearst, J . E., and Sancar, A . (1986b) Proc . Natl . Acad . Sci . U . S . A . 83, 8077-8081.

J Biol Chem, 1988 Oct 15, 263(29), 15083 - 93
Operon structure of dnaT and dnaC genes essential for normal and stable DNA replication of Escherichia coli chromosome; Masai H et al.; dnaT and dnaC of Escherichia coli, whose products are components of the primosome, encode genes essential for both normal chromosomal replication and stable DNA replication induced by SOS signals . dnaT and dnaC together with an unknown gene (P-18), which encodes an 18-kDa polypeptide present downstream of dnaC, are cotranscribed from a promoter located 104 base pairs upstream of dnaT . A portion of the transcripts are processed 16 or 27 base pairs upstream of the dnaC coding region and 20% of the transcripts extending through dnaC are terminated at a terminator 103 base pairs downstream of dnaC . The rest of the transcripts pass through this terminator and are terminated downstream of the P-18 gene . The predicted amino acid sequence of the P-18 protein indicate the presence of a 26-amino acid signal peptide at its N terminus, suggesting that this protein may be secreted into the membrane or a periplasmic space . Another gene, which encodes a 14-kDa hydrophobic protein (P-14), was identified upstream of dnaT . The P-14 gene is transcribed from an upstream promoter, and its transcript extends through dnaT and dnaC . These results indicate that dnaT and dnaC constitute an operon with two unknown genes whose products may be associated with a membrane.

J Biol Chem, 1988 Oct 15, 263(29), 15016 - 23
Initiation of lagging-strand synthesis for pBR322 plasmid DNA replication in vitro is dependent on primosomal protein i encoded by dnaT; Masai H et al.; The role of the primosome assembly and protein n' recognition site in replication of pBR322 plasmid was examined . The following evidence indicates that the primosome is involved in lagging-strand synthesis of pBR322 plasmid replication in vitro . Early replicative intermediates with newly synthesized leading strand, approximately 1 kilobase pair long, immediately downstream of the replication origin accumulate in products synthesized in extracts from a dnaT strain that lacks primosomal protein i or in wild-type extracts supplemented with anti-protein i antibody . These intermediates are converted efficiently into full-length DNA by addition of purified protein i . Consistent with the previously proposed role of the primosome (Arai, K . and Kornberg, A . (1981) Proc . Natl . Acad . Sci . U . S . A . 78, 69-73), an n' site on the lagging strand, but not on the leading strand, is required for efficient replication of the plasmid in vitro . Plasmids lacking an n' site on the lagging strand replicate only to a limited extent in vitro and early replicative intermediates carrying nascent leading strands are accumulated, although a portion of the intermediates complete replication to yield full-length DNA . The latter reaction is completely inhibited by addition of anti-protein i antibody . Insertion of the n' site of phage phi X174 into pBR322 plasmids lacking lagging-strand n' sites restores the replicative ability of the mutant plasmid comparable to that of the wild-type plasmid . These results indicate that protein i is essential for lagging-strand synthesis of pBR322 plasmid in vitro and that it may play an important role in the priming events as a part of either an n' site-dependent primosome or an n' site-independent, as yet unidentified, priming complex.

J Biol Chem, 1988 Oct 15, 263(29), 15008 - 15
The effect of dnaA protein and n' sites on the replication of plasmid ColE1; Ma D et al.; The role of the dna A protein in the replication of plasmid ColE1 and its derivatives was examined . Wild-type and mutant ColE1 plasmids were compared as to their ability to replicate in an in vitro replication system supplemented with ammonium sulfate fractionated extracts from a dnaA-overproducing strain . Synthesis on plasmid templates containing the wild-type origin of replication was stimulated 1.3-fold by addition of the dnaA-overproducing extract . A larger effect was observed after deletion of the primosome assembly site, the n' site, on the leading strand . On the latter template, synthesis was only about one-half that observed with the wild-type templates, but synthesis could be restored to normal levels by addition of the dnaA-overproducing fractions . When the n' site on the lagging strand of pBR322 was deleted, synthesis in the in vitro replication system was reduced to less than 10% of levels seen with intact templates . dnaA-overproducing extract did not restore activity since the dnaA site was also deleted on these plasmids . To verify that the observed stimulation of wild-type and leading strand n' site mutants was due to the dnaA protein, dnaA protein was purified to greater than 50% homogeneity, and antiserum was prepared . The purified protein stimulated synthesis on the plasmid templates to the same extent as the overproducing extracts, and dnaA antiserum blocked stimulation both by extracts and by the purified protein . Thus, dnaA protein, and, by inference, the dnaA recognition site at the ColE1 origin of replication seem to be important for ColE1 replication . The effect of dnaA protein is enhanced when the n'site is defective, suggesting that the dnaA protein plays a role similar to that of the proteins i, n, n', and n'' in directing primosome assembly, as proposed by Seufert, W., and Messer, W . ((1987) Cell 48, 73-78).

J Biol Chem, 1988 Oct 15, 263(29), 14732 - 8
Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli; Johnson MK et al.; The subunit location of the {2Fe-2S}, {3Fe-4S}, and {4Fe-4S} clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits . The results indicate that both the {2Fe-2S} and {3Fe-4S} clusters are located entirely in the iron-sulfur protein subunit . Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit . While the results are not definitive with respect to the location of the {4Fe-4S} cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits . These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing {2Fe-2S}, {3Fe-4S}, and {4Fe-4S} centers.

J Biol Chem, 1988 Oct 15, 263(29), 14617 - 20
In vivo and in vitro autoprocessing of human immunodeficiency virus protease expressed in Escherichia coli; Giam CZ et al.; The viral-specific protease of human immunodeficiency virus (HIV) has been expressed as a lacZ-protease fusion protein . This fusion protein contains protease cleavage sites at the gag/protease and protease/reverse transcriptase junctions and undergoes autoprocessing in vivo when expressed in Escherichia coli . The purified lacZ-protease fusion protein precursors also exhibit autoproteolytic activity in vitro . One cleavage product of the autoprocessing reactions is a 10-kDa protein that cross-reacts with peptide antisera prepared against the putative protease sequence . Consistent with the notion that HIV protease is an acid protease, its autoproteolytic activity is inhibited in alkaline buffers and by pepstatin A . The in vivo and in vitro autocleavage assays for HIV protease together with the overproduction of the protease should facilitate design and testing of therapeutic agents that inhibit gag-pol polyprotein processing and HIV virion maturation.

Eur J Biochem, 1988 Oct 15, 177(1), 91 - 6
Differential inhibition of various deoxyribonucleic acid polymerases by Evans blue and aurintricarboxylic acid; Nakane H et al.; The inhibitory effects of two anionic compounds, Evans blue and aurintricarboxylic acid (ATA), on various kinds of polynucleotide-synthesizing enzymes were examined . Under the assay conditions, optimized for each enzyme species, both these compounds strongly inhibited the activities of the purified human DNA polymerases alpha, beta, gamma, and DNA primase as well as those of DNA polymerase I and RNA polymerase from Escherichia coli and Rauscher leukemia virus reverse transcriptase . ATA was particularly effective in inhibiting retroviral reverse transcriptase and cellular DNA polymerase alpha . Evans blue, which is a structural analogue of suramin, exerted its inhibitory action largely by competing with the template.primer for the same binding site of the enzyme . On the other hand, ATA inhibited most, if not all, of these enzyme activities noncompetitively with respect to either the template.primers or nucleoside 5'-triphosphate substrates . The inhibition constants for ATA were, in general, smaller than those for Evans blue.

Eur J Biochem, 1988 Oct 15, 177(1), 117 - 24
Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5:K4:H4, a fructose-containing polysaccharide with a chondroitin backbone; Rodriguez ML et al.; The chemical structure of the K4-specific capsular polysaccharide (K4 antigen) of Escherichia coli O5:K4:H4 was elucidated by composition, carboxyl reduction periodate oxidation methylation nuclear-magnetic-resonance spectroscopy and enzymatic cleavage . The polysaccharide consists of a backbone with the structure----3)-beta-D-glucuronyl-(1,4)-beta-D-N-acetylgalactosaminyl(1- to which beta-fructofuranose is linked at C-3 of glucuronic acid . Mild acid hydrolysis liberated fructose and converted the K4 antigen into a polysaccharide which has the same structure as chondroitin . The defructosylated polysaccharide was a substrate for hyaluronidase and chondroitinase . The serological reactivity of the K4 polysaccharide was markedly reduced after defructosylation.

Science, 1988 Oct 14, 242(4876), 240 - 5
Mutant Trp repressors with new DNA-binding specificities; Bass S et al.; Oligonucleotide-directed mutagenesis of the codons for glutamine-68 (Gln68), lysine-72 (Lys72), isoleucine-79 (Ile79), alanine-80 (Ala80), and threonine-81 (Thr81) of the Escherichia coli trpR (tryptophan aporepressor) gene was used to make mutant repressors with each of 36 different amino acid changes . Mutant repressors were tested for binding to each member of a set of 28 different operators closely related to the consensus trp operator . Of the 36 mutant repressors, 11 bind a subset of the 28 operators; 5 of these have new binding specificities . These new specificities indicate that the hydroxyl group of Thr81 makes a specific contact with one of the four critical base pairs in a trp operator half-site, and the methyl group of Thr81 determines specificity at a second, critical base pair . The Trp repressor does not use the first two amino acids of its "recognition alpha-helix," Ile79 and Ala80, to make sequence-specific DNA contacts, and interacts with its operator in vivo in a way fundamentally different from the way that phage lambda repressor, lambda Cro protein, and coliphage 434 repressor contact their respective binding sites.

Biochem Biophys Res Commun, 1988 Oct 14, 156(1), 417 - 23
Slipped DNA structures within the enhancer region of the Moloney murine leukemia virus; Gama Sosa MA et al.; We have examined the S1 nuclease sensitivity of supercoiled plasmids harboring the Moloney Murine Leukemia Virus (MoMuLV) long terminal repeat (LTR) . S1 sensitivity was found within the LTR enhancer direct repeats . Transformation of E . coli DH5 cells with a construct containing most of the MoMuLV LTR yielded the precise deletion of one direct repeat and loss of S1 sensitivity . The dependence of S1 sensitivity on the presence of both direct repeats, together with the exact excision of one direct repeat by E . coli, suggests the presence of slipped DNA within the enhancer . Such structures may represent targets for effector proteins which mediate vital functions during viral propagation.

Biochem Biophys Res Commun, 1988 Oct 14, 156(1), 246 - 54
Yeast KEX2 genes encodes an endopeptidase homologous to subtilisin-like serine proteases; Mizuno K et al.; Yeast Saccharomyces cerevisiae KEX2 gene previously isolated, was characterized as the gene encoding a calcium-dependent endopeptidase required for processing of precursors of alpha-factor and killer toxin . In this study, we report the amino acid sequence of the KEX2 gene product deduced from nucleotide sequencing . Our results indicate that the KEX2 gene contains a 2,442-bp open reading frame encoding a polypeptide of 814 amino acids . The deduced amino acid sequence contains a region extensively homologous to the members of subtilisin-like serine protease family near the N-terminus . A putative membrane-spanning domain near the C-terminus was also detected . These facts indicate that the KEX2-encoded protein may function as a membrane-bound, subtilisin-like serine protease.

Biochim Biophys Acta, 1988 Oct 13, 967(1), 103 - 9
The activity and purification of membrane-associated thioredoxin reductase from human metastatic melanotic melanoma; Schallreuter KU et al.; Electron spin resonance spectroscopy has been used to measure the reduction of nitroxide radicals on a spin-labeled quaternary ammonium substrate by plasma membrane-associated thioredoxin reductase (EC 1.6.4.5) at the surface of cutaneous and subcutaneous melanoma metastases from one patient (B.M.) . Enzyme activity in these metastases was shown to be hyperactive compared to normal skin and was subject to inhibition by calcium . From the remainder of the tissue (50.6 g), plasma membrane-associated thioredoxin reductase has been isolated and its molecular properties were compared with the same enzyme purified from the cytosol of rat liver and Escherichia coli . The enzyme from melanoma possessed an identical molecular weight to that from rat liver as determined by SDS-polyacrylamide gel electrophoresis (Mr 58,000) . Upon fluorescence spectroscopic examination, the enzyme from melanoma was shown to contain flavin adenine dinucleotide as previously shown in the enzymes from E . coli and rat liver . The increased activities in plasma membrane-associated thioredoxin reductase in metastases of malignant melanotic melanoma are discussed in terms of the cellular functions of this important enzyme.

Biochim Biophys Acta, 1988 Oct 12, 956(3), 307 - 12
Electron spin-echo spectroscopic studies of Escherichia coli fumarate reductase; Cammack R et al.; Electron spin-echo envelope modulation (ESEEM) spectroscopy was applied to the study of reduced Centre 1 of Escherichia coli fumarate reductase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1) . The ESEEM spectrum derived from stimulated (3-pulse) echo envelopes obtained at 8.8 GHz contained lines at 0.9, 2.1, 3.0 and 4.2 MHz in the g = 1.94 region . When studied at 11.4 GHz, these low-frequency components scale with magnetic field in a manner indicating interaction between the unpaired electron spin of the Fe-S cluster and a weakly coupled 14N nucleus . Spectral simulations of these ESEEM data yield nuclear quadrupole interaction parameters indicative of peptide nitrogen . For oxidized protein, the magnetic-field dependence of the linear electric-field effect (LEFE) for Centre 3 was measured, and the results confirm the presence of a {3Fe-4S} cluster in the protein.

Nucleic Acids Res, 1988 Oct 11, 16(19), 9215 - 31
The structure of the regulatory region of the rat L1 (L1Rn, long interspersed repeated) DNA family of transposable elements; Furano AV et al.; Here we report the DNA structure of the left 1.5 kb of two newly isolated full length members of the rat L1 DNA family (L1Rn, long interspersed repeated DNA) . In contrast to earlier isolated rat L1 members, both of these contain promoter-like regions that are most likely full length . In addition, the promoter-like region of both members has undergone a partial tandem duplication . A second internal region of the left end of one of the reported members is also tandemly duplicated . The propensity of the left end of rat L1 elements to undergo this form of genetic rearrangement, as well as other structural features revealed by the present work, is discussed in light of the fact that during evolution the otherwise conserved mammalian L1 DNA families have each acquired completely different promoter-like regions . In an accompanying paper {Nur, I., Pascale, E., and Furano, A . V . (1988) Nucleic Acids Res . 16, submitted}, we report that one of the rat promoter-like regions can function as a promoter in rat cells when fused to the Escherichia coli chloramphenicol acyltransferase gene.

Nucleic Acids Res, 1988 Oct 11, 16(19), 9177 - 84
A novel procedure for selective cloning of NotI linking fragments from mammalian genomes; Ito T et al.; A novel procedure has been developed for selective cloning of NotI linking fragments from mammalian genomes . Since the majority of the NotI sites in mammalian genomes are considered to be localized in so-called HTF (HpaII tiny fragment) islands, an HTF library was constructed as an initial step to enrich the NotI sites . The plasmid DNAs were isolated en masse from the HTF library and digested with NotI . Linearized plasmid DNAs derived from NotI linking clones were efficiently separated from undigested circular DNAs by an unique pulsed field polyacrylamide gel electrophoresis (PF-PAGE) . The linear DNAs were eluted from the gel, recircularized with T4 DNA ligase and introduced into E . coli cells . About 95% of the transformants were found to contain NotI linking fragments . The procedure will thus provide a simple and useful way of collecting NotI linking fragments for long range physical mapping of mammalian genomes.

FEBS Lett, 1988 Oct 10, 238(2), 347 - 52
Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity; Van Ostade X et al.; Each of the two highly conserved tryptophan residues in hTNF (positions 28 and 114) was converted into phenylalanine by site-directed mutagenesis and the mutant proteins were partially purified . A cytotoxicity assay on mouse L929 cells showed only a slight reduction in biological activity, strongly suggesting that neither of the two amino acids is involved in the active site.

FEBS Lett, 1988 Oct 10, 238(2), 231 - 4
The 'molten globule' state is involved in the translocation of proteins across membranes?
Bychkova VE, Pain RH, Ptitsyn OB.
Strong evidence exists that the translocation of proteins across a variety of membranes involves a non-native or denatured conformational states . On the other hand a compact state having secondary but not rigid tertiary structure and called the 'molten globule' state has been identified as being stable under mild denaturing conditions . A similar state has been shown to accumulate on the folding pathway of globular proteins . These states are compact though sufficiently expanded to include water, and they are internally mobile . It is proposed that these molten globule states may be suitable candidates for protein translocation across biological membranes.

FEBS Lett, 1988 Oct 10, 238(2), 307 - 14
Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA; Olsen J et al.; The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone . Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion . A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E . coli aminopeptidase N and contains Zn2+ ligands . Therefore these residues are part of the active site . However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development . It is speculated that this part carries the apical address.

Biochim Biophys Acta, 1988 Oct 7, 971(3), 325 - 31
The effect of heat-stable Escherichia coli enterotoxin, theophylline and forskolin on cyclic nucleotide levels and mucosal surface (acid microclimate) pH in rat proximal jejunum in vivo; McKie AT et al.; The normally acidic mucosal surface pH of 6.24 +/- 0.02(30) in rat proximal jejunum in vivo is effectively neutralised by 30 min exposure to heat-stable Escherichia coli (STa) enterotoxin (14 micrograms/ml) to 6.80 +/- 0.07 (n = 5) or to a forskolin/theophylline combination (1 mM:20 mM) to 7.10 +/- 0.07(7) while perfusion with Krebs-phosphate buffer alone without glucose left the mucosal surface pH unchanged at a pH of 6.21 +/- 0.02(9) . Forskolin alone had no effect, and 20 mM theophylline moderately elevated the surface pH to 6.52 +/- 0.03(5) . Theophylline, forskolin and their combination all elevated cAMP levels per mg tissue DNA above control values while STa enterotoxin was without effect . In contrast, all agents elevated cGMP levels per mg tissue DNA above control levels . These findings indicate that surface pH is only moderately affected by changes in cAMP levels and is affected to a much greater extent by altered cGMP levels.

J Mol Biol, 1988 Oct 5, 203(3), 817 - 20
Integration host factor binds specifically to sites in the ilvGMEDA operon in Escherichia coli; Tsui P et al.; Integration host factor (IHF) of Escherichia coli is a histone-like protein that is involved both in site-specific recombination and in regulating the expression of a number of phage and bacterial genes . We have shown previously that transcription of the ilvGMEDA operon in E . coli is greatly reduced in IHF mutants . We report here that IHF specifically protects two sites within the ilvGMEDA promoter-regulatory region against DNase I digestion . These sites are located upstream from the promoter and in the leader region just prior to the sequence that specifies the attenuator . The footprinting experiments and gel retardation assays show that these sites have strong affinity for IHF . These data and results with ilvGMEDA-lac promoter fusions suggest a direct role for IHF in expression of the ilvGMEDA operon.

J Biol Chem, 1988 Oct 5, 263(28), 14261 - 6
Affinity labeling of ras oncogene product p21 with guanosine diphospho- and triphosphopyridoxals; Ohmi N et al.; Purified v-rasH p21 overproduced in Escherichia coli was treated with guanosine diphospho- and triphosphopyridoxals (GP2- and GP3-PL), affinity labeling reagents specific to a lysyl residue located in the guanine nucleotide binding site . GP2-PL and GP3-PL inhibited {3H}GDP binding to p21 competitively . Incubation of p21 with GP2-PL and GP3-PL followed by reduction with NaBH4 resulted in 40 and 50% loss of {3H}GDP binding activity, respectively, whereas the addition of excess GDP completely protected p21 from the inactivation . The tryptic digest of p21 which was modified with GP2-PL or GP3-PL in the presence or absence of protective GDP and subsequently reduced by NaBH4 was analyzed by reverse phase high performance liquid chromatography . The profile of the effluent monitored by the fluorescence from the pyridoxyl moiety showed the existence of peptides which were specifically labeled only in the absence of GDP . Structural analyses of these peptides allowed us to identify the labeled residue as Lys-16 . These results suggest that Lys-16 is located in the guanine nucleotide binding site, close to the beta- or gamma-phosphate group of the nucleotide.

J Mol Biol, 1988 Oct 5, 203(3), 803 - 16
Crystallographic refinement by simulated annealing . Application to a 2.8 A resolution structure of aspartate aminotransferase; Brunger AT; Crystallographic refinement by simulated annealing with molecular dynamics has been applied to a 2.8 A (1 A = 0.1 nm) resolution X-ray structure of aspartate aminotransferase . Comparison of the refined structure and a structure obtained by combined restrained least-squares refinement and manual re-fitting shows a similar R factor, stereochemistry, and mean difference from the isomorphous replacement phase centroids . Crystallographic refinement by simulated annealing accomplished structural changes and improvements of the electron density maps that were not possible by using restrained least-squares refinement without manual re-fitting . Crystallographic refinement by simulated annealing can generate an ensemble of structures, each of which agrees with the diffraction information . Regions of large variations of the ensemble indicate either erroneously fitted or disordered segments of the macromolecule.

J Mol Biol, 1988 Oct 5, 203(3), 781 - 98
Comparison of the structure of myosin subfragment 1 bound to actin and free in solution . A neutron scattering study using actin made "invisible" by deuteration; Curmi PM et al.; The structure of subfragment 1 (S1) bound to F-actin has been compared to the structure of free S1 using neutron scattering . The F-actin was rendered "invisible" to neutrons by selective deuteration and solvent contrast matching . Highly deuterated actin was purified from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli . The properties of this actin were found to be similar to those of protonated actin . The neutron-scattering pattern of S1 bound to this "invisible" actin was compared to that of free S1 . At near-physiological ionic strength, a strong interference effect was observed, which arose from pairs of S1 molecules cross-linking actin filaments . However, at low ionic strength the only differences that could be observed were attributed to interference effects between neutrons scattered from S1s bound randomly to equivalent sites on an actin filament . These effects became negligible as the fraction of actin sites occupied by S1 approached zero . Thus, we conclude that the scattering by S1 attached to F-actin is identical with that of free S1, to a resolution of about 2.5 nm . The difference in apparent radii of gyration is less than 0.05 nm . Modeling calculations have been carried out to determine the sensitivity of neutron scattering to possible S1 deformations . The calculations showed that deformations of the structure of S1 that are large enough ultimately to produce a powerstroke of 5 nm or greater are only consistent with the data if they involve at most about 20% of the S1 mass . These results restrict the class of plausible models describing force generation in muscle contraction.

J Mol Biol, 1988 Oct 5, 203(3), 753 - 60
Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging; Wagenknecht T et al.; Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli . In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers . In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit . We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.

J Mol Biol, 1988 Oct 5, 203(3), 643 - 63
Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli; Wek RC et al.; The ilvC gene encodes acetohydroxy acid isomeroreductase (EC 1.1.1.89), the second enzyme in the parallel isoleucine-valine biosynthetic pathway . Expression of the ilvC gene is induced by acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate . This substrate induction is mediated by a positive activator encoded by an adjacent gene, ilvY . The ilvY and ilvC genes are transcribed in opposite directions from promoters that are overlapping . In this paper we characterize the in vitro DNA binding properties of the ilvY-encoded activator protein . The ilvY product binds to two adjacent operator sites located in the divergent-overlapping ilvY and ilvC promoter region . One of these operators, designated O1 contains regions of dyad symmetry centered at position +17 relative to the ilvY transcriptional start site, and the second site, designated O2, contains an homologous inverted repeat sequence centered about the -35 region of the ilvC promoter . Binding of the ilvY product at the O1 and O2 operator sites is co-operative and this ilvY protein-DNA complex in the presence of acetohydroxy acid isomeroreductase substrate is a prerequisite for RNA polymerase binding to the ilvC promoter as detected by DNase I protection experiments . Additionally, chromosomal galK transcriptional fusion assays were performed to characterize the regulation of the ilvY and ilvC promoters in vivo . Transcription of the ilvC gene is maintained at a basal level of activity which is elevated as much as 15-fold in the presence of ilvY product and acetohydroxybutyrate . The ilvY product represses ilvY transcription in a manner that does not appear to be dependent on acetohydroxy acid isomeroreductase substrate . We discuss models in which activation of ilvC transcription results from a direct interaction of ilvY protein with RNA polymerase or an ilvY-mediated alteration of the DNA conformation of the ilvC -35 promoter region . Additionally, we discuss the role of acetohydroxybutyrate and acetolactate in ilvY transcriptional regulation.

J Mol Biol, 1988 Oct 5, 203(3), 635 - 41
UmuC function is not essential for the production of all targeted lacI mutations induced by ultraviolet light; Christensen JR et al.; Up to a quarter or more of the normal yield of lacI- mutations could be induced by ultraviolet light in a uvrA6 umuC122:: Tn5 strain if they were detected by plating on 5-bromo-4-chloro-3-indolyl-beta-D-galactoside medium, where all surviving cells can form colonies . Using phenyl beta-D-galactoside selection, which curtails post-irradiation growth, only low yields of mutations were induced . Nucleotide sequence analysis of 134 spontaneous and 145 ultraviolet light-induced mutations shows that broadly similar kinds of mutations were induced in the umuC mutant and its uvrA6 umuC+ counterpart . In particular, these data offer no reason for believing that most of the mutations induced in the umuC mutant were other than normal, targeted events . We conclude that UmuC function, rather than being essential, facilitates recovery and specifically, following the model of Bridges & Woodgate, that it facilitates the prompt resumption of chain elongation.

J Mol Biol, 1988 Oct 5, 203(3), 619 - 33
Ultraviolet light induces different spectra of lacI sequence changes in vegetative and conjugating cells of Escherichia coli; LeClerc JE et al.; We have analyzed the nucleotide sequence changes responsible for mutations from lacIs to lacI- induced in ultraviolet light-irradiated, excision-deficient cells . Irradiated cells were either used as donors in the conjugational transfer of an F' lacIs plasmid to SOS-induced, excision-deficient recipients or allowed to continue vegetative growth . Although the types and proportions of premutagenic lesions are likely to have been very similar in these two circumstances, analysis of the sequence data shows that different spectra of mutations were induced . In vegetative cells there were about equal numbers of transitions and transversions, but transitions outnumbered transversions by about three to one in exconjugants . About 90% of the single nucleotide substitutions could be assigned to a bipyrimidine target sequence in both sets of data, but they differed with respect to the location of the substitution: more or less equal numbers were found at the 3' and 5' sites of the probable bipyrimidine target in vegetative cells, but in exconjugants over 80% were at the 3' site . It is also possible that mutations were targeted more commonly at T-C sequences in exconjugants than in vegetative cells, but the evidence for this is less secure . We conclude that these results reflect some dissimilarity between vegetative cells and exconjugants in the way damaged DNA is replicated or lesions tolerated, but the particular features of these processes responsible for the different mutational spectra have not yet been identified.

J Mol Biol, 1988 Oct 5, 203(3), 569 - 83
Positive and negative regulators for glucitol (gut) operon expression in Escherichia coli; Yamada M et al.; Expression of the glucitol (gut) operon in Escherichia coli is regulated by an unusual, complex system which consists of an activator (encoded by the gutM gene) and a repressor (encoded by the gutR gene) in addition to the cAMP-CRP complex (CRP, cAMP receptor protein) . The activator and repressor are predicted to possess 119 (Mr = 12,955) and 257 (Mr = 28,240) aminoacyl residues, respectively, as deduced from the nucleotide sequences of their structural genes . Both of the genes encoding the two regulators are located downstream from the other known gut structural genes . Reverse transcriptase mapping revealed that the gutM gene is a promoter-distal constituent of the gut operon . The gutR gene has its own promoter, but expression of this gene is primarily due to readthrough from the gut operon operator-promoter . Thus, the gut operon consists of at least five structural genes and has the following gene order: gutOPABDMR . Interestingly, synthesis of the mRNA, which initiates at the promoter specific to the gutR gene, occurs within the gutM gene . Expressional control of the gut operon appears to occur as a consequence of the antagonistic action of the products of the autogenously regulated gutM and gutR genes . An additional cistron of the gut operon, of unknown function, may follow the gutR gene.

J Biol Chem, 1988 Oct 5, 263(28), 14426 - 32
Expression in Escherichia coli of full-length and mutant rat brain calbindin D28 . Comparison with the purified native protein; Gross MD et al.; Studies of vitamin D-dependent 28-kilodalton calcium binding protein (calbindin D28) have been hindered by difficulties in purifying large amounts of the protein . In order to overcome this problem, we cloned and expressed a full-length rat brain calbindin D28 cDNA . In addition, we isolated and purified to homogeneity, native rat brain calbindin D28 . The isolated native protein has an apparent molecular mass of 27 kDa and properties similar to those of the well-characterized chicken calbindin D28 . It has an acidic isoelectric point (approximately 4.5), a high affinity for calcium, and an amino terminus blocked to Edman degradation . The properties of the native and the recombinant proteins were examined by gel electrophoresis, isoelectric focusing, protein sequencing, amino acid composition analysis, and calcium binding assays . We demonstrated that: (i) the authentic and the full-length recombinant proteins have similar molecular weights and isoelectric points; (ii) the proteins have the same amino acid composition; (iii) the proteins bind calcium in a similar manner; (iv) the absence of a blocking NH2-terminal group in the recombinant protein does not appreciably influence the binding of calcium . To further examine the calcium binding properties of this protein, we constructed deletion mutants lacking one or both of the two putative degenerated calcium binding sites (EF hand regions) . These deletions resulted in smaller proteins that still bound calcium . The ability to express and purify calbindin D28 and mutants thereof should allow the systematic elucidation of structure-function relationships in this class of calcium binding proteins.

J Biol Chem, 1988 Oct 5, 263(28), 14302 - 7
Chloroplast rpoA, rpoB, and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription; Little MC et al.; The purpose of this study was to determine the relationship between putative chloroplast RNA polymerase subunit genes and known chloroplast transcriptional activities . We have prepared fusion polypeptide genes from fragments of chloroplast DNA homologous to bacterial RNA polymerase subunit genes and expression vectors carrying portions of the anthranilate synthetase gene (trpE) . Fusion proteins for chloroplast homologs of the RNA polymerase alpha (rpoA), beta (rpoB), and beta' (rpoC) subunits were obtained from these genes . The fusion polypeptides synthesized by Escherichia coli in vivo were purified and used as antigens for production of rabbit polyclonal anti-RNA polymerase subunit-specific antibodies . The purified antibodies were able to immobilize chloroplast DNA-dependent RNA polymerases from spinach, pea, and Euglena gracilis . In addition, the soluble chloroplast RNA polymerase activity in tRNA and mRNA synthesis was strongly inhibited by these antibodies under conditions which had little effect on transcription by the chloroplast transcriptionally active chromosome that preferentially transcribed rRNA genes (Greenberg, B . M., Narita, J . O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K . A., and Hallick, R . B . (1984) J . Biol . Chem . 259: 14880-14887) . From these data we conclude that the chloroplast genes homologous to bacterial RNA polymerase subunit genes are expressed in vivo and that the protein products specify at least three of the components of the chloroplast RNA polymerase(s) involved in tRNA and mRNA transcription.

J Biol Chem, 1988 Oct 5, 263(28), 14251 - 5
Ribosome release modulates basal level expression of the trp operon of Escherichia coli; Roesser JR et al.; The leader peptide stop codon (UGA) of the Escherichia coli trp operon was replaced by UAA and UAG . The transcriptional behavior of the mutated leader regions in vitro and the extent of transcription termination observed with each in vivo were virtually identical to that of the wild type leader region . Introduction of a release factor 1 (UAA- and UAG-specific) mutation into strains with the different stop codons caused increased termination in strains with UAA and UAG, but not with UGA (in cells grown in the presence of tryptophan) . This finding provides evidence for the view that ribosome release from the leader peptide stop codon is an important event in setting the basal level of transcription readthrough at the trp attenuator.

J Biol Chem, 1988 Oct 5, 263(28), 14008 - 14
Characterization and conservation of the inner E2 core domain structure of branched-chain alpha-keto acid dehydrogenase complex from bovine liver . Construction of a cDNA encoding the entire transacylase (E2b) precursor; Griffin TA et al.; A cDNA clone encoding the entire transacylase (E2b) precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex has been constructed from two overlapping incomplete cDNA clones which were isolated from a lambda ZAP library prepared from bovine liver poly(A)+ RNA . Nucleotide sequencing indicates that this bovine E2b cDNA insert (bE2-11) is 2701 base pairs in length with an open reading frame of 1446 base pairs . The bE2-11 cDNA insert encodes a leader peptide of 61 residues and a mature E2b polypeptide of 421 amino acid residues with a calculated monomeric molecular mass of 46,518 daltons . The molecular mass of the native E2b component isolated from bovine liver is 1,110,000 daltons as determined by sedimentation equilibrium . This value establishes the 24-subunit octahedral model for the quaternary structure of bovine E2b . The amino-terminal sequences of two tryptic fragments (A and B) of the E2b protein have been determined . Fragment A comprises residues 175 to 421 of the E2b protein and is the inner E2 core domain which contains the transacylase active site . Fragment B, produced by further tryptic cleavage of fragment, comprises residues 205 to 421, but does not have transacylase activity . Both fragments A and B confer the highly assembled 24-mer structure . The primary structure of the inner E2 core domain of bovine E2b (fragment A) is very similar to those of three other E2 proteins (human E2p, Escherichia coli E2p, and E . coli E2k) . These similarities suggest that these E2 proteins are structurally and evolutionarily related.

J Biol Chem, 1988 Oct 5, 263(28), 14446 - 55
Multiple DNA secondary structures in perfect inverted repeat inserts in plasmids . Right-handed B-DNA, cruciforms, and left-handed Z-DNA; Blaho JA et al.; The capabilities of five recombinant plasmids, containing relatively long (approximately 60-100 base pairs) perfect inverted repeat (IR) inserts, to support supercoil stabilized non-B-DNA structures were studied in vitro . The IRs were also alternating purine-pyrimidine sequences, thus, each could form either left-handed Z-DNA or cruciforms . Single-strand specific endonucleases, restriction endonucleases and methylases, and OsO4 modifications were used to characterize the DNA structures . Two-dimensional gel electrophoretic studies indicated that three of the five IRs formed both cruciforms and Z-DNA . (C-G) containing inserts preferred to form Z-DNA, whereas (T-G) sequences favored cruciforms . In vivo supercoil relaxation experiments demonstrated the existence of cruciforms in Escherichia coli . The physiological significance of these structures is discussed.

J Biol Chem, 1988 Oct 5, 263(28), 14276 - 80
Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli; Kawakami T et al.; We isolated mutants of Escherichia coli which showed Li+-resistant growth on melibiose . The melibiose carrier of the mutants lost the ability to couple to H+, whereas it retained the ability to couple to Na+ . The mutated gene, melB, of the mutants was cloned, and the nucleotide sequence was determined . The nucleotide replacements caused the following substitutions of amino acid residues in the melibiose carrier: Pro-142 with Ser, Leu-232 with Phe, or Ala-236 with Thr or Val . These amino acid residues are located in slightly hydrophobic regions of the melibiose carrier . The results provide strong support for the idea that such regions or their vicinities which contain those amino acid residues play an important role in H+ (or Li+) recognition or H+ (or Li+) transport by the melibiose carrier.

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